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NATURE AT WORK: ONGOING SAGA OF EVOLUTION

Publication of The National Academy of Sciences, India

123

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CONTENTS Title

Page No.

Foreword Preface

v vii

Prof. Asis Datta, President, The National Academy of Sciences, India

Legacy Continuum 1.

The descent of humans and the Darwinian unification of all life P. Dayanandan

2.

Ida: A link to human evolution V.P. Sharma

21

3.

Darwin’s theory of evolution: Survival of nature’s fit! Veena Tandon and Gaurangi Maitra

33

4.

The life and research of JBS Haldane in India with special reference to Charles Darwin Krishna R. Dronamraju

5.

Charles Darwin: A driving force for humanity toward agnosticism Amit Sharma

1

51 59

Viruses, Microbes and Fungi 6.

Phylogeographic evolution of plant viruses Anupam Varma and Shelly Praveen

75

7.

Evolution of HIV-1 in India Pradeep Seth

93

8.

Darwin and microbial evolution T.K. Adhya and M. Patra

9.

Evolutionary relationships among cyanobacteria, algae and plants: Revisited in the light of Darwinism Radha Prasanna and B.D. Kaushik

10.

Biodiversity, phylogeny and evolution of fungi C. Manoharachari, I.K. Kunwar and S. Vishnuvardhan Reddy

103

119 141

Insects: Evolution in Action 11.

The origin of reproductive isolating mechanisms is an important event in the process of speciation: Evidences from Drosophila B.N. Singh

159

12.

Adaptive radiation and insects V.V. Ramamurthy and Asha Gaur

175

13.

Insights from mosquito evolution: Patterns, tempo and speciation Karamjit Singh Rai

197

14.

The saga of pollination biology Rajesh Tandon and H.Y. Mohan Ram

219

Genomics 15.

Darwinian evolution and post developments in genomics Arun Kumar Sharma

16.

Pathogen pressure and molecular evolutionary genetics of innate immunity genes in humans Partha P. Majumder

241

249

Unique Case Studies 17.

Macroevolution in relation to the drift models of the Indian plate Ashok Sahni and R.S. Loyal

18.

Testing the melanism-desiccation hypothesis: A case study in Darwinian evolution Ravi Parkash

279

Origin and evolution of human malaria parasite, P. falciparum and P. vivax Nidhi Datta and V.S. Chauhan

307

19.

267

20.

Evolutionary trends in soil-inhabiting Mahlaqa Choudhary and M. Shamim Jairajpuri

21.

Evolution of the cerebral cortex in amniotes: Anatomical consideration of neuronal types U.C. Srivastava and R.C. Maurya

329

Medicinal and aromatic plants: A case example of evolving secondary metabolome and biochemical pathway diversity Suman P.S. Khanuja, Tripta Jhang and Ajit Kumar Shasany

355

Conservation of Himalayan bioresources: An ecological, economical and evolutionary perspective Lok Man S. Palni and Ranbeer S. Rawal

369

22.

23.

Color Images Section

319

395

Foreword Charles Robert Darwin was born on 12th February, 1809 in Shrewsbury, England. Darwin shares his birthday with U.S. President Abraham Lincoln. Both were crusaders against slavery: Darwin disliked slavery and Lincoln abolished it. Darwin was a born naturalist and showed keen interest in nature from the very beginning. A breakthrough came when he was selected as a naturalist on the H.M.S. Beagle ship. His five year voyage on the Beagle started in 1931 and was completed in 1936. This was followed by publication of his research findings that challenged creationist views of the church. Darwin conducted a study of fossils and geological records and concluded rightly, that all life forms emerged over millions of years of evolution through the force of natural selection. In 1959 Darwin published his work on evolution in a book titled “On the Origin of Species by Means of Natural Selection or the Preservation of Favored Races”. The book was received as a scientific bomb shell and has since changed the human understanding of life forever. Today Darwin’s ideas on evolution provide foundation to modern biology. Darwin died of a heart attack on the 19th April 1882 and was buried in Westminster Abbey near the grave of Sir Isaac Newton. The scientific community is celebrating Darwin’s bicentenary worldwide in honor of his ingenuity, scientific thought, conviction and courage. Coinciding with the bicentenary celebrations are three major discoveries in paleontology, namely Darwinius masillae from Germany, skeleton of winged Dinosaur from China and fossilized Dinosaur eggs from India. The National Academy of Sciences, India (in short NASI) at Allahabad is paying tribute to British naturalist Charles Darwin by compilation of articles titled “Nature at Work: Ongoing Saga of Evolution”. Twenty three contributory articles bring out many facets of evolution and human understanding in this book. Articles begin with the Darwin’s legacy of the descent of man, ‘Ida’ the link to human evolution, theory of evolution, Haldane in India, and agnosticism. The second section describes the evolution of microbes, viruses and fungi affecting health and environment worldwide. Next section deals with the evolution of insects for understanding basic biology, adaptations, speciation, and interplaying with plants in pollination. The section on Genomics brings out the post-Darwinian developments in biology providing proof of unification at the molecular level and the discoveries of the cause of many genetic disorders. Finally there are seven unique case studies representing various facets of macro and micro Darwinian evolution taking examples from malaria, nematodes, amniotes, v

and implications for health and well being, hunger and food, bio-resources and conservation of biodiversity. In summary the book brings out the universality of Darwin’s concept of evolution ending in unification of life without exception from gross to molecular basis of the “Tree of Life”, a continuum. Many friends have been of great help in the preparation of this book. I wish to acknowledge the help and suggestions inter alia, 2009 Council of the NASI, Prof. M.G.K. Menon, Prof. H.Y. Mohan Ram, Prof. K.S. Rai, Prof. Anupam Varma, Dr. Amit Sharma, Prof. Veena Tandon, Prof. P. Dayanandan, Prof. R.C. Sobti, Dr. Mrs. Manju Sharma, and of course all the authors and co-authors for their excellent work on evolution. I am particularly grateful to the team of Springer India Pvt. Ltd. for their commitment and hard work that made it possible to bring out this excellent book on Darwin’s bicentenary. Finally, on my behalf and the NASI, I wish to thank Prof. Ananda Chakrabarty, distinguished Professor at the University of Illinois College of Medicine at Chicago, for the release of this book on 14th December 2009 during the 79th Annual Session of the Academy hosted by Calcutta University, Kolkata. Vinod P. Sharma CRDT, IIT, Delhi - 110016 Dated: 17th October, 2009

vi

Preface It is a pleasure that the National Academy of Sciences, India (NASI) Allahabad is bringing out a book titled “The Nature at Work: Ongoing Saga of Evolution” to celebrate bicentenary of Charles Robert Darwin. The book is edited by Dr. V.P. Sharma, former President, NASI. Charles Darwin was an English naturalist who realized and presented compelling evidences that all species of life have developed over time from common ancestors. Darwin’s scientific discoveries are the unifying theories of the life sciences. The theory of evolution really does explain everything in biology. The phenomena that Darwin understood in broad brush strokes can now be accounted for in the precise language of DNA. He established himself as an eminent geologist/biologist through his observations, theories and ideas and became famous as a popular author. This book is primarily for those who want to understand various theories of Charles Darwin. We have selected the most important parts of his writings and have added learned annotations. These annotations are highly precious and useful, especially for academics. This book is divided into five themes each clearly written by the eminent dignitaries/scientists of India. The text is well written and factual without being overwhelming. I found the information very interesting and well presented. We desire to express our gratitude to all the contributors who were kind enough to provide us relevant information on highly specialized areas of modern understanding of evolution and unification of life. I also believe that the book would provide an impression of his theories along with some new ideas which will serve as an inspiration and challenge for the professionals and enthusiast of science, young and old alike. A pressus book for personal possession, the libraries and an important item of gift of lasting value. Asis Datta President, NASI, New Delhi Dated: 14th October, 2009

vii

1 The descent of humans and the Darwinian unification of all life P. Dayanandan Department of Botany, Madras Christian College, 1/2 First Cross, MES Road, Tambaram, Chennai - 600 059 [email protected]

Abstract: The biological revolution that Darwin initiated has given us a view of an earth that was once devoid of life and where life originated and evolved from simpler pre-existing forms over immense periods of time. Darwin saw human beings as an integral part of this evolutionary history. How, when and where life originated remains an inscrutable challenge to science. However, discoveries in every branch of biology since the time of Darwin have affirmed the essential unity of life, including the 30 million or more extant species and more than 600 million extinct species. Darwin’s graphic representation of relationships between organisms is now employed to construct phylogenetic trees at all levels of biological organization. A Neanderthal fossil was the only hominin remain known at the time of Darwin. Today paleoanthropologists work with more than 20 species of hominins. The study of these fossil ancestors combined with the molecular dating techniques based on the analysis of mutations in mitochondrial and Y-chromosomal DNA have vindicated Darwin’s worldview of the origin of humans in Africa, as well as his suggestion that the great apes are the closest living ancestors of Homo sapiens. The discovery that all human beings on earth are descendants of one woman, the African ‘Eve’ who lived not more than 200,000 years ago has extended the Darwinian biological worldview into a unifying philosophical perspective. It is now possible to determine the mitochondrial and Y-chromosomal branch (haplogroup) of any human being with comparative ease. This helps in tracing the possible time and route of migration of our ancestors. The emerging consensus that our ancestors left Africa as recently as 70,000 years ago has profound implications for history, culture, linguistics, psychology and human predicament. The unification of all life, including human life is perhaps the most enduring contribution of Darwin’s evolutionary theory. Human beings are yet to fully comprehend the significance of this unification. Perhaps there is a lesson in Darwinism for how we humans should deal with each other and treat other life and nature with which we have an organic and primeval connection. Keywords: African ‘Eve’, Darwin, Evolution, Grand unification, Haplogroups, Human phylogeny, Mitochondrial DNA, Out of Africa, Tree of life, Y-chromosome

V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

2

Nature at work: Ongoing saga of evolution

Introduction One hundred and fifty years ago Darwin concluded the Origin of Species with his magnificent view of life on earth as one of organic relationship developed over immense time scales from one or a few forms of life: “There is grandeur in this view of life .... from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved.”1 Charles Wallace certainly shared an identical and independently developed evolutionary view of life. However, Wallace could not reconcile himself with Darwin’s views on the origin of human beings and how natural selection could account for the mental capacities and moral sensibilities of humans. Darwin formulated his theory based on extensive field work, meticulous observations and documentation and relentlessly pursued his views for nearly 50 years from the beginning of his exploration on the Beagle. Meanwhile the Origin of Species, first published in 1859, went through six editions. In the Origin of Species all that Darwin revealed about human evolution was this cryptic statement: “light will be thrown on the origin of man and his history.” Four years later in 1863, Thomas Huxley, who was a friend and ardent supporter of Darwin, applied the concept of evolution by natural selection to humans in his book Evidence as to Man’s Place in Nature.2 In his 1871 Descent of Man, the second most influential of his numerous publications, Darwin revealed that human beings too, were an unequivocal product of evolution.3 This in essence was a grand unification of all life. Implicit in the evolutionary theory is the still grander unification of life with nonliving matter. The origin of life was an abominable mystery then, and it continues to be no less a daunting challenge today in spite of the 1953 Miller-Urey experiment.3,4 During the past 150 years the best of biology has provided answers to the two fundamental questions that Darwin struggled with, namely the mechanism of inheritance and the process by which heritable variations occur. In turn biologists have come to assert with Dobzhansky that: “Nothing in biology makes sense, except in the light of evolution.”5 In this chapter we focus on how science has updated this extraordinary story of life on earth and the place and history of the human species; a story that was first narrated so eloquently by Darwin. In essence this updated story tells us that: life has a history measured in millions and billions of years; all species have descended in a branching pattern from common ancestors; populations can change over time and evolve into species; biodiversity is a product of evolution; all life, including the estimated 30 million living species and 600 million extinct species are related to each other; human beings who share some 500 genes with all living things are genetically related to all other life; modern humans evolved as recently as 250,000 to 200,000 years ago and all people living today share common descent from one woman, the ‘most recent maternal common ancestor’, who lived perhaps as recently as 200,000

The descent of humans and the Darwinian unification of all life

3

years ago. There is indeed grandeur in this view of life. Perhaps there can be no better tribute to Darwin in this bicentenary year than what paleoanthropologists and human evolutionary biologists and molecular geneticists are accomplishing to unravel human origins and their migratory routes by peering into fossil bones and DNA of chromosomes and mitochondria.

Darwin’s trees and unification of life Darwin made the first attempts at drawing ‘tree of life’ diagrams that initiated a powerful method of visualizing the very concept of evolution. As early as 1837/1838 Darwin drew trees (Figs.1–3) to clarify to himself the branching pattern of evolutionary divergence and extinction of species. Twenty years later another sketch of a ‘tree of life’ was the only illustration that appeared in the Origin of Species (Fig. 4). As the pre-Darwinian artificial systems of classification were replaced by classifications based on explicitly evolutionary principles, a variety of phylogenetic trees were constructed. As the available characters expanded from morphological and anatomical to biochemical and molecular data, sophisticated analytical tools such as computer assisted statistical methods of parsimony, maximum likelihood, least squares, neighbor-joining and Bayesian inference are now used in cladistics

Figures 1–3. Darwin drew these diagrams 20 years before the publication of the Origin of Species. In Figures 1 and 2 the branches represent living species and the dotted lines the extinct ancestors. Darwin noted that this tree of life should perhaps be “called the coral of life” with dead branches representing transitional forms that cannot be seen. Darwin’s comments for Figure 3 emphasized the importance of the extinction of species: “I think case must be that one generation then should be as many living as now. To do this and to have many species in same genus (as is) requires extinction. Thus between A and B immense gap of relation. C and B the finest gradation, B and D rather greater distinction” (From Darwin’s Notebook B: Transmutation of species, 1837–1838. http://darwin-online.org.uk/)

4


Figure 4. This diagram of the divergence of taxa is the only diagram that appeared in all six editions of the Origin of Species in the chapter on ‘Natural Selection’. The branching trees were used to explain how eleven species (A–L) of a genus can evolve and produce living lineages (A and I). Species F has remained unaltered as a living fossil. All other lineages have gone extinct. Darwin’s concept of evolutionary timescales was immense. He wrote: “The intervals between the horizontal lines in the diagram may represent each a thousand generations; but it would have been better if each had represented ten thousand generations”

and molecular systematics to construct evermore instructive trees. The legacy of Darwin’s “ever branching and beautiful ramifications” is all too evident in the new generation of trees of life such as the hyperbolic and cladogenetic trees and trees based on genome size.6 All modern phylogenetic trees of life, as the one shown in Figure 5, stand testimony to Darwin’s grand unification of all life. Trees such as this are now constructed as specialists working on various groups of organisms cooperate and pool their findings based on classical and molecular data. Darwin’s basic idea of branches diverging from common ancestors characterizes all such trees. In Figure 5 all life is shown to emerge from a common ancestor that first gave rise to Bacteria and the common ancestor of Archaea and Eucarya. The common ancestor of plants, protists, fungi and animals lived in the distant past. Plants share a more recent common ancestor with the characean green algae. Surprisingly, recent studies reveal that fungi and animals shared a common ancestor and hence fungi are more closely related to animals than plants.


5

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Figure 5. Phylogeny of living organisms including major endosymbiotic events. (Courtesy: Dr. J. Peter Gogarten, Department of Molecular and Cell Biology, University of Connecticut)

Most biologists now consider that endosymbiosis7 was a powerful mechanism responsible for the evolution of some major groups during early periods. Darwin, of course, was not aware of this evolutionary force. The diversification of all eukaryotes became possible only after a common ancestor of Eucarya acquired a Proteobacterium-like cell as the mitochondrial symbiont. The acquisition of cyanobacterial photosynthetic endosymbionts was responsible for further evolution and diversification of protists and plants. Interestingly, green and red algal cells became secondary plastids in at least six different endosymbiotic episodes. We now have a view of life where Darwin’s unification is seen to be further fortified by endosymbiotic exchanges that unite many millions of species, including humans. In effect, all eukaryotes are chimeras. In a surprising historical twist, a billion years after our ancestral eukaryotes acquired mitochondria, scientists are now probing the DNA of these bacteria-turned-mitochondria to trace human origins and the routes of migration from the African homeland.

Africa and the great apes Darwin looked towards Africa for the progenitors of humans. In his 1871 Descent of Man Darwin reasoned: in any great region living species are closely related to extinct forms; chimpanzee and gorilla are the nearest allies of Homo sapiens;

6


Africa is where these great apes are living today; Africa must have been formerly inhabited by extinct apes related to the chimpanzee and gorilla; therefore “it is somewhat more probable that our early progenitors lived on the African continent than elsewhere.”8 The only human ancestral fossil known when Darwin published the Descent of Man was a Neanderthal skull discovered in 1856 in the Neander Valley, Germany. Some earlier finds from Belgium were later identified as Neanderthal fossils. Darwin died in 1882. Later discoveries confirmed that Neanderthals were indeed members of human lineage and they were once widespread in Europe. This prompted a search for fossil hominins leading to the discovery of Homo erectus in Java (1890) and China (1920s) and Australopithecus africanus in Zambia (1924). Since the 1960s when Louis and Mary Leakey made initial discoveries at Olduvai in Northern Tanzania, increasingly abundant fossils are being unearthed. Darwin would not be surprised if he were to see this array of fossils linking modern humans to ancestral forms in Africa. With more than 20 species of hominins already described and more that are likely to be unearthed, Darwin can have a hearty laugh at his caricature as a missing link! It is of interest to learn that recent molecular studies of hominoids (humans, gibbons, and the great apes - orangutan, chimpanzee and gorilla) have led to reassessment of affinities and proposals for new classification schemes.9 Indeed, some have argued for considering the chimp (Pan) as a member of the genus Homo. Studies on single nucleotide polymorphism reveal that human DNA is about 98.4% identical to that of chimpanzees. In traditional classification schemes only humans and their fossil ancestors are included in the family Hominidae and are referred to as hominids. The discovery of closer genetic affinity between humans and chimps has broadened the concept of hominids to include lineages of humans and the great apes (chimps, gorillas and orangutans) as members of the family Hominidae. The lineages of humans and the African great apes (but not orangutans) are all members of a subfamily Homininae. In this new classification scheme humans (Homo) and related bipedal fossil genera in human lineage such as Ardipithecus, Australopithecus, Paranthropus and Kenyanthropus, and possibly the early genera Sahelanthropus and Orrorin are members of the tribe Hominini, hence the common name hominins. Human beings are members of the primates group. In this bicentenary year of Darwin a 47 million-year-old fossil, Darwinius masillae, nicknamed Ida, is on display and reveals the deep ancestry of humans tracing all the way back to prosimians such as lemurs (See chapter 2 by Prof. V.P. Sharma in this volume). As shown in Figure 6 primate evolution began about 55 million years ago (mya), in early Eocene epoch. Humans and apes are tailless and share similar brain and body organization as well as complex social life. Several fossils collectively referred to as Proconsul that lived 27–17 mya in Africa and presumed to be early apes or their ancestors suggest that the Old World monkeys and the apes diverged from


7

a common ancestor some 20-18 mya, in the early Miocene epoch. It was during the terminal Miocene epoch that the ape lineages separated from each other and the evolution of hominins accelerated. Molecular studies of hominoids suggest that the gibbons (Family Hylobatidae) and great apes (Family Hominidae) lineages diverged about 18–12 mya. The African great ape hominid lineage diverged from the orangutan hominid lineage about 12 mya. The Indian fossil Ramapithecus is considered to be an early genus in orangutan ancestry. Some believe that Pierolapithecus catalaunicus that lived about 13 mya might have been a common ancestor of the great apes and humans. The fossils Nakalipithecus from Kenya and Ouranopithecus from Greece appear to resemble the common ancestor of gorilla, chimpanzees and

Figure 6. A simplified family tree of primate evolution

humans. The human lineage emerged between 8 and 5 mya when first the gorilla and then the chimpanzee lineages separated from the hominin tribe. The earliest hominins might have been Sahelanthropus (7 mya) and Orrorin (6 mya). While chimpanzees and humans are closest living relatives, humans did not evolve from extant chimpanzees; both lineages had evolved independently over the past 6–7 million years. Further discussion in this chapter is devoted to a brief summary of evolution of the hominins leading to the emergence of modern humans Homo sapiens.

Descent of humans Although primates were present in Africa, Eurasia and the New World, the evolution of hominins was confined to Africa. The idea of a separate creation of human ‘races’ – known as polygeny – with its racist overtones is now discredited.

8


Some anthropologists still subscribe to a related concept of multiregionalism according to which speciation of different ‘races’ occurred after human-like species migrated out of Africa and settled in different regions of the world.10 We follow the emerging consensus views of ‘monogenesis’ and ‘Out of Africa’ according to which human evolution, including the emergence of anatomically modern humans (H. sapiens) occurred in Africa. During this period a few species of Homo (H. ergaster, H. erectus, H. antecessor and H. heidelbergensis) did move out of Africa within the last 2 million years but did not contribute to any further evolution of humans. The Out of Africa model is synonymous with the Recent Single-Origin Hypothesis, Replacement Hypothesis and Recent African Origin. Our own species evolved in Africa and a small group migrated out of Africa and colonized the rest of the world. The tree shown in Figure 7 is a graphic representation of the evolutionary relationship among the hominin fossils described so far. Paleoanthropology is one of the most active fields of research in biology, and specialists offer different interpretations of evolutionary relationships.11 Taxonomy of fossil hominins varies as some specialists tend to split and others to lump the different taxa. The 7 million-year-old Sahelanthropus tchadensis (Toumai), first described in 2001 from Chad, might have been a bipedal hominin. Some claim this to be a possible common ancestor of human and chimpanzee lineages. Our own genus, Homo, appeared about 5 million years later. Three other genera are known from Africa during the in-between period, namely Orrorin (6 mya) followed by Ardipithecus (5.5–4.4 mya) and Australopithecus (4–2 mya). A. afarensis, first described from Hadar, Ethiopia in 1973 might have been the ancestor of our genus Homo. Homo habilis, first discovered in 1962 in the Olduvai Gorge in Tanzania, lived from 2.4–1.4 mya in East and South Africa, and closely resembled Australopithecus. Nicknamed ‘handy man’, H. habilis was the first hominin to have made and used stone tools known as ‘choppers’ in the early Paleolithic period, more than 2 mya. Evolution of hominins continued in Africa eventually resulting in the emergence of anatomically modern H. sapiens, about 200,000 years ago. Yet H. sapiens was not the first hominin to leave Africa. H. ergaster (1.8 mya) closely followed by H. erectus and later H. antecessor and H. heidelbergensis (=H. rhodesiensis) left Africa bringing the Paleolithic technology to Eurasia. H. heidelbergensis and H. neanderthalensis with a thick skull, prominent brow ridges and non-prominent chin but with brain size (1200–1400 cc) similar to modern humans are sometimes referred to as “archaic H. sapiens”. Darwin’s insightful prediction that our earliest progenitors would be found in Africa turned out to be true. It might surprise us that in a matter of 4 million years the continent witnessed the rapid evolution of four genera (Australopithecus, Kenyanthropus, Paranthropus and Homo) and nearly 20 species of hominins; and


9

Figure 7. An interpretative tree of relationship among hominins with time-scale. The connecting lines indicate well-supported relationship between the species

more will surely be discovered. Darwin also reasoned that the absence of any human remains in older fossil strata must indicate that the evolution of humans must have occurred recently and exceptionally rapidly. Several factors seem to have influenced human evolution. Climate change that resulted in the conversion of tropical forests into woodlands and savannah grasslands might have influenced the Miocene ancestral apes to descend from trees and become bipedal, thereby changing their foraging habits and freeing their forelimbs to make tools. Ice ages during the past 2 million years might have acted as selective agents in human physiological and physical adaptations and influenced the patterns of migration as hominins moved in search of food plants and animals. Brain development and the extraordinary ability to shape tools with hands and the mutual influence between brain development and the need to respond to the changing environment have all contributed to unique human qualities such as planning, innovation, abstract thinking and symbolic behavior. It appears that during the past 10,000 years humans have been evolving 100 times faster than in the past. The Neolithic revolution, which resulted in excessive consumption of starch and milk, urbanization, poor sanitation, epidemic diseases and changing food habits all seem to have operated and continue to operate as selective agents.12 Homo neanderthalensis lived until about 30,000 years ago in Europe while the small-sized H. floresiensis, discovered in 2004 in Indonesia, might have become extinct as recently as 15,000 years ago. Since the Neanderthals and modern humans

10


were contemporaries in parts of Europe, researchers have wondered if there were gene exchanges between them. Recently the mitochondrial DNA (mtDNA) from fossil bones of H. neanderthalensis were extracted and compared with the DNA sequence of H. sapiens. It appears that the sequences are very different and the two remained as separate species ever since they evolved from a common ancestor, some 660,000 years ago until the Neanderthals went extinct about 30,000 years ago.13 The only genus and species of hominin that is living today and the only species that ever occupied all the continents is H. sapiens. During the past 20 years molecular geneticists have revealed a remarkable story of the origin and spread of humans, a story of unification of all human beings through a bacterial endosymbiont, the mitochondrion, and recluse Y-chromosome!

Humanity unified - the mitochondrial African mother Human beings, all 6.5 billion now living and the billions more that will be added, are remarkably like each other with 99.9% genetic similarity. A mere 0.1% difference is responsible for the diversity we observe. We are not a highly variable species. This similarity is due to our recent origin in Africa and the presumed small size of the population that left Africa and colonized the rest of the globe. Within the last 20 years a remarkable and unexpected story of our ancestry has emerged and is now profoundly affecting the way we think of ourselves. In an important paper published in 1987, Cann, Stoneking and Wilson reported their analysis of mtDNA samples of 147 people from five geographic populations. Using restriction mapping techniques to detect polymorphism and parsimony methods to infer a common ancestor they came to the conclusion: “All these mtDNAs stem from one woman who is postulated to have lived about 200,000 years ago, probably in Africa. All the populations examined except the African population have multiple origins, implying that each area was colonized repeatedly.”14 This ancestral woman is now popularly known as ‘African Eve’ and ‘Mitochondrial Eve’. Subsequent studies15 have confirmed the findings of Cann et al. although the estimated age of this ‘mother’ is reported to be in the range of 150,000–250,000 years before present. Since the mtDNA now found in all human mitochondria do not undergo recombination, theoretically they all can be traced back to a single root, the so-called ‘coalescent point’. This point represents the Mitochondrial Eve. The implications of these studies are: modern humans evolved in Africa; about 200,000 years ago an ancestral woman in East Africa passed on her mitochondria to her offspring; transmitted to every generation only through the female line to both sons and daughters, her mtDNA is now present in all people on earth; this woman is the matrilineal most recent common ancestor (MRCA) of all people who ever lived since then, and the more than 6 billion people who are living today in all continents, including Africa. The matrilineal MRCA (= African Eve, Mitochondrial Eve) was not the only woman living at that time in Africa. For various reasons the


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mtDNAs of other women were not passed on to subsequent generations; perhaps these other women did not beget daughters or their female lineages died out. It is likely that through their sons their genes still occur in the nuclear DNA of people living today. The mtDNA of our matrilineal MRCA is found in people outside of Africa also because it was carried by her female descendants when some people left Africa about 100,000 years later to colonize all other continents.

And a Y-chromosomal African father While a mitochondrion is inherited only through the mother, the Y-chromosome is passed on to the next generation only through the father. The difference between the two is that while both men and women receive mitochondria from their mothers, the Y-chromosome is passed on only to males. As with mtDNA it is possible to trace back the root – the coalescent point – of all Y-chromosomes found in males since most of Y-chromosomal DNA (YDNA) does not recombine during meiosis. A study16 of YDNA polymorphism in a worldwide sample of men using methods similar to the analysis of mtDNA revealed that the root of the human male family tree was placed in Africa, just like the female root. However, the date of the putative patrilineal MRCA, the ‘Y-chromosomal Adam’, was just 60,000 years. Again this does not imply that there were no other men around; only that the Y-chromosome of this male MRCA is now found in all men, in all continents. The more recent date (60,000–90,000 years) for the patrilineal MRCA is explained on the basis of human sexual behavior where the variance of reproductive success is higher for men. Unlike women, who all have more equal opportunities to have children, a few men can mate with more women and pass on their genes. The Y-chromosomal lineages of men who lived before the MRCA were lost. The mitochondria of one woman and the Y-chromosomes derived later from one man in Africa were passed on to people who spread throughout Africa, and through a group that migrated out of Africa to all other people in non-African continents. One would expect greatest genetic diversity in the DNA of mitochondria, Y-chromosomes, as well as other chromosomes in Africa, as indeed is shown by several studies.17

Wandering humans and their DNA trees and haplogroups The circular mtDNA inherited through mothers and the male-specific YDNA both carry mutations that are used for understanding human origins and for constructing trees from which the geographical origin and migration of human populations could be inferred.18 Since the mutations occur at a fairly regular rate it is possible to estimate the time when populations branched off using molecular dating techniques. mtDNA and most of YDNA do not recombine and hence genes are not shuffled as they are in autosomal chromosomes. mtDNA offers the advantage of small size of only 16,569 base pairs and 37 genes. These advantages are now widely

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exploited to discover an ever increasing number of polymorphic sites. Single nucleotide polymorphism (SNP) due to point mutations can be easily identified with restriction mapping. Contemporary human populations have accumulated a large number of mutations in mtDNA since the time of Mitochondrial Eve, the MRCA.19,20 Mitochondria also possess short Hyper Variable Regions (HVR1, 2 and 3) which have rates of mutation 100 times greater than that of the nuclear genome. The sequence of nucleotides in these regions (e.g. HVR1: 16,001–16,569) can be compared with a Cambridge Reference Sequence to determine specific markers that help assign individuals to unique mitochondrial haplogroups. Haplogroups are branches on the family tree of groups of people who share a set of genetic markers. All members of a haplogroup share a marker that first appeared in the MRCA of that group. Y-chromosome polymorphism is now increasingly used to understand the origin and migration of human populations. YDNA consists of many millions of nucleotides and provides scope for examining thousands of mutations. Besides SNPs the Y-chromosomes also carry non-coding microsatellites or Short Tandem Repeat (STR) regions consisting of repetitive DNA sequences. These repetitions lead to copying errors resulting in addition or subtraction of repeat units. Each STR is designated by a DYS number (DNA Y-chromosome Segment number). Analysis of markers on YDNA provides necessary information to assign individuals to Y-chromosomal haplogroups. Mitochondrial and Y-chromosomal haplogroups

Figure 8. A simplified tree of mitochondrial haplogroups. Haplogroups L4, L5, L6 and L7 are not shown. Haplogroups M, N and R can be considered as ‘Super haplogroups’ since they have many subclades. Some of the defining markers are shown. Marker mutations may occur as SNPs in coding regions or in HVR1 and HVR2 regions. The most ancient haplogroups L0–L3 are African; only during the colonial period people with these markers have been moved to non-African continents


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are now routinely analyzed in several laboratories around the world. A number of private testing companies also analyze DNA samples and provide information on genealogies and geographic origins. The Genographic Project21 is an ambitious worldwide project undertaken by the National Geographic Society with geneticist Spencer Wells to map the human family tree and trace the migratory routes of our ancestors.22 The Y-chromosome Consortium23 at the University of Arizona carries out collaborative studies on YDNA variations and nomenclature of Y haplogroups. Family trees constructed using mitochondrial markers provide a graphic picture of deep human matrilineal ancestry (Fig. 8). Three main groups, designated by letters L, M and N, and many subgroups are known. The earliest branching of the tree occurred in Africa about 200,000 years ago when the L0/L1 split occurred somewhere in East Africa. Today L0 is widespread among the San and Mbuti people in Africa. L1 and its branch L2 are also confined to Africa. Only after M and N macrohaplogroups branched off from L3 was there any major migration of the female ancestor out of Africa. Thus the woman in whose mtDNA a mutation first occurred, to found the L3 haplogroup branch, is known as the ‘Eurasian Eve’ since her descendants were the first to move out of Africa. Today the M group derived from L3 is found in East Africa, Asia, Melanesia, Australia and the Americas. The

Figure 9. Y-chromosomal haplogroups showing the defining markers. Haplogroups A and B are African branches with most diverse lineages dating back to 60,000–50,000 years. Major haplogroups C, F and K occur in India especially as exclusive subclusters of H, L and R

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N group, also born in Africa, later migrated out of Africa and moved northwards spreading to many parts of Asia, Europe, India and the Americas. Haplogroup R that branched off from N is now found in East and West Eurasian populations, accounting for nearly 75% of the lineages. The YDNA family tree also reveals ancient splits that occurred in our male lineage in Africa (Fig. 9). Haplogroup A defined by the marker M91 is perhaps the deepest of our branches, now confined to Africa and prevalent among the Khoisan, Ethiopians and Nilotes. Haplogroup B with marker M60 is also confined to Africa mostly among the Pygmies and Hadzabe. Descendants of B with a marker M168 (‘Eurasian Adam’) were perhaps the first males to leave Africa more than 60,000 years ago. A man born to this migrant group had the marker M130 defining the haplogroup C. His descendants are considered to have moved along the coast into India, and moved beyond to Southeast Asia and finally into Australia. M130 is now found in 1%): Manipur,

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Nagaland, Andhra Pradesh, Tamil Nadu, Karnataka and Maharashtra. Certain districts in Goa and Gujarat have also reported high prevalence. Sexual transmission is driving the AIDS epidemic in India. This route accounts for about 86% of HIV infections in the country. Remaining 14% are accounted for by other routes namely blood transfusion, mother-child transmission and IV drug use, particularly in North-east India. Over one-third of all HIV infections occur in young people in the age group 15–24 years. Early studies have indicated the presence of both HIV-1 and HIV-2 in India.18,19 Subsequent studies further emphasized a predominance of subtype C strains in India, which were found to cluster with South African isolates.20,21 Other HIV-1 subtypes, A and B, have been reported in India between 1980s and early 1990s among the recipients of blood and blood products and IV drug users, respectively, suggesting multiple introductions of HIV-1 in this country. Subtype A strains were found to be related to Central and East African subtype and subtype B strains obtained from Manipur were related to subtype B sequences circulating in Thailand.21 However, recent studies have clearly shown that subtype C strains have displaced subtype B in the IV drug users in that part of India.22,23 The trends across the country show that there is no explosive HIV epidemic in India as a whole. However, there are serious sub-national epidemics in various parts of the country with rapid spread and evidence of high prevalence of HIV among both sexual transmitted infections (STI) and antenatal clinic attendees in different sites located in States of Andhra Pradesh, Maharashtra, Tamil Nadu, Gujarat, Pondicherry, Assam, Bihar, Chhatisgarh, Delhi, Haryana, Himachal Pradesh, Kerala, Orissa, Goa and Manipur. In high prevalence states the epidemic appears to be spreading gradually from urban to rural areas and from high-risk behavior groups to the general population. The epidemic continues to shift towards women with an estimated 39% of the infected being women17. An explosive epidemic driven by intravenous drug use has unfolded in the state of Manipur (North-east India) bordering Myanmar and is close to the Golden Triangle composed of Thailand, East Myanmar and West Laos and is the hub of international drug trafficking. A recent study documents that two-thirds HIV infections in this region of India are caused by subtype C and subtype B (Thai B) accounts for 20% of infections.22 The presence of multiple subtypes circulating in Manipur suggests the likelihood of recombinant viruses evolving in this region. Indeed, this has been corroborated by a recent study which reported presence of B/C recombinants from this region.24 Apart from north-eastern states there are also sporadic reports of the presence of A/C and B/C recombinants from West and South India.25,26 The occurrence of HIV-1 recombination in nature is borne out by the identification of genomes that are recombinants between different HIV-1 subtypes.27 Some of these recombinant viruses have become fixed in the human

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population and are referred to as circulating recombinant forms (CRFs), and in at least a few cases CRFs have become the predominant strain in specific geographic areas of infection such as A/E recombinants in Thailand and B/C recombinants in parts of Southeast Asia and China. HIV-1 recombinants are estimated to contribute to 10–40% and 10–30% of the infections in Africa and Asia, respectively. The identification of subtypes and CRFs provides a means of tracking dissemination of the pandemic worldwide. To delineate the molecular features of HIV-1 strains circulating in India, Phylogenetic analyses of sequences of Indian subtype C isolates along with a small number of subtype sequences from other countries revealed that almost all sequences from India form a distinct lineage within subtype C (CIN). Overall CIN lineage sequences were more closely related to each other (level of diversity, 10.2%) than to subtype C sequences from Botswana, Burundi, South Africa, Tanzania and Zimbabwe (range 15.3 to 20.7%). Suggesting thereby, much of the current Indian epidemic is descended from a single introduction into the country.28 In an assessment of the Phylogenetic relationships among subtype C sequences from eleven different countries including India, an overall star-like phylogeny was observed.29 Unlike sequences from South Africa and Botswana, which are scattered in numerous lineages, almost all sequences from India formed a monophyletic lineage, which is lying close to the sequences. Sequences from India generally clustered together more than sequences from other countries. Genetic characterization of the virus during the early seroconversion stage is crucial as the virus isolated is closely related to the transmitted strain and hence immunologically naïve. Phylogenetic analyses of Indian subtype C envelope sequences obtained from early seroconverts indicated that the Indian sequences not only clustered within the C clade but also clustered away from the African subtype C sequences.30 Moreover, a recent study demonstrating lower diversity within immunodominant epitopes and a tight clustering of Indian isolates29 suggested that production of a vaccine particularly against Indian subtype C may not be an unattainable and daunting task.

References 1.

Simon F, Mauclere P, Roques P, Loussert-Ajaka I, Muller-Trutwin MC, Saragosti S Georges-Courbot MC, barre-Sinoussi F, Brun-Vezinet F (1998) Identification of a new human-immunodeficiency virus type 1 distinct from group M and group O. Nat Med 4:1032–1057

2.

Korber B, Theiler J, Wolinsky S (1998) Limitation of a molecular clock applied to considerations of the origin of HIV-1. Science 280:1868–1871

3.

Gao F, Vidal N, Li Y, Trask SA, Chen Y, Kostrikis LG, Ho DD, Kim J, Oh MD, Choe K, Salminen M, Robertson DL, Shaw GM, Hahn BH, Peeters M (2001) Evidence of two distinct sub-subtypes within the HIV-1 subtype A radiation. AIDS Res Hum Retroviruses 17:675–688

100


4.

Bobkov AF, Kazennova EV, Selimova LM, Khanina TA, Ryabov GS, Bobkova MR, Sukhanova AL, Kravchenko AV, Ladnaya NN, Weber JN, Pokrovsky VV (2004) Temporal trends in the HIV-1 epidemic in Russia: Predominance of subtype A. J Med Virol 74: 191–196

5.

Jeeninga RE, Hoogenkamp M, Armand-Ugon M, de Baar M, Verhoef K, Berkhout B (2000) Functional differences between the long terminal repeat transcriptional promoters of human immunodeficiency virus type 1 subtypes A through G. J Virol 74:3740–3751

6.

Gao F, Robertson DL, Carruthers CD, Morrison SG, Jian B, Chen Y, Barré-Sinoussi F, Girard M, Srinivasan A, Abimiku AG, Shaw GM, Sharp PM, Hahn BH (1998) A comprehensive panel of near-full-length clones and reference sequences for non-subtype B isolates of human immunodeficiency virus type 1. J Virol 72:5680–5698

7.

Salemi M, de Oliveira T, Soares MA, Pybus O, Dumans AT, Vandamme AM, Tanuri A, Cassol S, Fitch WM (2005) Different epidemic potentials of the HIV-1B and C subtypes. J Mol Evol 60:598–605

8.

UNAIDS (2004) AIDS Epidemic Update. www.unaids.org

9.

Thomson MM, Najera R (2001) Travel and the introduction of human immunodeficiency virus type 1 non-B subtype genetic forms into Western countries. Clin Infect Dis 32: 1732–1737

10.

Couturier E, Damond F, Roques P, Fleury H, Barin F, Brunet JB, Brun-Vézinet F, Simon F (2000) HIV-1 diversity in France, 1996-1998. The AC 11 laboratory network. AIDS 14:289–296

11.

Salminen MO, Johansson B, Sönnerborg A, Ayehunie S, Gotte D, Leinikki P, Burke DS, McCutchan FE (1996) Full-length sequence of an Ethiopian human immunodeficiency virus type 1 (HIV-1) isolate of genetic subtype C. AIDS Res Hum Retroviruses 20:1329–1339

12.

McCormack GP, Glynn JR, Crampin AC, Sibande F, Mulawa D, Bliss L, Broadbent P, Abarca K, Pönnighaus JM, Fine PE, Clewley JP (2002) Early evolution of the human immunodeficiency virus type 1 subtype C epidemic in rural Malawi. J Virol 76: 12890–12899

13.

Neilson JR, John GC, Carr JK, Lewis P, Kreiss JK, Jackson S, Nduati RW, Mbori-Ngacha D, Panteleeff DD, Bodrug S, Giachetti C, Bott MA, Richardson BA, Bwayo J, Ndinya-Achola J, Overbaugh J (1999) Subtypes of human immunodeficiency virus type 1 and disease stage among women in Nairobi, Kenya. J Virol 73:4393–4403

14.

Vidal N, Peeters M, Mulanga-Kabeya C, Nzilambi N, Robertson D, Ilunga W, Sema H, Tshimanga K, Bongo B, Delaporte E (2000) Unprecedented degree of human immunodeficiency virus type 1 (HIV-1) group M genetic diversity in the Democratic Republic of Congo suggests that the HIV-1 pandemic originated in Central Africa. J Virol 74:10498–10507

15.

Yu XF, Chen J, Shao Y, Beyrer C, Lai S (1998) Two subtypes of HIV-1 among injectiondrug users in southern China. Lancet 351:1250

16.

Tatt ID, Barlow KL, Clewley JP, Gill ON, Parry JV (2004) Surveillance of HIV-1 subtypes among heterosexuals in England and Wales, 1997–2000. J Acquir Immune Defic Syndr 36:1092–1099

17.

National AIDS Control Organization. website. www.naco.nic.in

18.

Dietrich U, Grez M, von Briesen H, Panhans B, Geissendörfer M, Kühnel H, Maniar J, Mahambre G, Becker WB, Becker ML, et al. (1994) HIV-1 strains from India are highly divergent from prototypic African and US/European strains, but are linked to a South African isolate. AIDS 7:23–27

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101

19.

Grez M, Dietrich U, Balfe P, von Briesen H, Maniar JK, Mahambre G, Delwart EL, Mullins JI, Rübsamen-Waigmann H (1994) Genetic analysis of human immunodeficiency virus type 1 and 2 (HIV-1 and HIV-2) mixed infections in India reveals a recent spread of HIV-1 and HIV-2 from a single ancestor for each of these viruses. J Virol 68:2161–2168

20.

Voevodin A, Crandall KA, Seth P, al Mufti S (1996) HIV type 1 subtypes B and C from new regions of India and Indian and Ethiopian expatriates in Kuwait. AIDS Res Hum Retroviruses 12(7):641–643

21.

Maitra A, Singh B, Banu S, Deshpande A, Robbins K, Kalish ML, Broor S, Seth P (1999) Subtypes of HIV type 1 circulating in India: Partial envelope sequences. AIDS Res Hum Retroviruses 15:941–944

22.

Mandal D, Jana S, Bhattacharya SK, Chakrabarti S (2002) HIV type 1 subtypes circulating in eastern and northeastern regions of India. AIDS Res Hum Retroviruses 18:1219–1227

23.

Gupta RM, Prasad VVSP, Rai A, Seth P (2005) Analysis of HIV type 1 subtype C full-length gag gene sequences from India: Novel observations and plausible implications. AIDS Res Hum Retroviruses 21:1066–1072

24.

Tripathy SP, Kulkarni SS, Jadhav SD, Agnihotri KD, Jere AJ, Kurle SN, Bhattacharya SK, Singh K, Tripathy SP, Paranjape RS (2005) Subtype B and subtype C HIV type-1 recombinants in the northeastern state of Manipur, India. AIDS Res Hum Retroviruses 21:152–157

25.

Lole KS, Bollinger RC, Paranjape RS, Gadkari D, Kulkarni SS, Novak NG, Ingersoll R, Sheppard HW, Ray SC (1998) Full-length human immunodeficiency virus type 1 genomes from subtype C-infected seroconverters in India, with evidence of intersubtype recombination. J Virol 73:152–160

26.

Siddappa NB, Dash PK, Mahadevan A, Desai A, Jayasuryan N, Ravi V, Satishchandra P, Shankar SK, Ranga U (2005) Identification of unique B/C recombinant strains of HIV-1 in the southern state of Karnataka, India. AIDS 19:1426–1429

27.

Malim MH, Emerman M (2001) HIV-1 sequence variation: Drift, shift and attenuation. Cell 104:469–472

28.

Shankarappa R, Chatterjee R, Learn GH, Neogi D, Ding M, Roy P, Ghosh A, Kingsley L, Harrison L, Mullins JI, Gupta P (2001) Human immunodeficiency virus type 1 env sequences from Calcutta in eastern India: Identification of features that distinguish subtype C sequences in India from other subtype C sequences. J Virol 75:10479–10487

29.

Khan IF, Vajpayee M, Prasad VV, Seth P (2007) Genetic diversity of HIV type 1 subtype C env gene sequences from India. AIDS Res Hum Retroviruses 23:934–940

30.

Kalpana A, Srikanth T, Abhay J, Sushama J, Swarali K, Ramesh P (2004) gp120 sequences from HIV type 1 subtype C early seroconverters in India. AIDS Res Hum Retroviruses 20:889–894

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8 Darwin and microbial evolution T.K. Adhya and M. Patra Central Rice Research Institute, Cuttack - 753006, Orissa, India [email protected]

Abstract: In the 200th year of Charles Darwin’s birth anniversary and 150th year of the publication of the ‘Origin of Species’, biologists are trying to re-evaluate the concept of the evolution as propounded by Darwin in the light of the knowledge gathered through recent developments in genomics and proteomics. Since no technique existed for the isolation of bacteria and the tremendous diversity of the microbial world was still largely unknown, at the time Darwin wrote his masterpiece, concept of microbial evolution was not widely discussed. This was further compounded by the lack of a theoretical basis of the concept of species in prokaryotes as microbial lineages show substantial variation in population structure, ranging from essentially clonal to nearly sexual. Comparative genomic studies have revealed that prokaryotic genetic material are under continuous selective pressure that have profoundly influenced its organization indicating that the gene pool available in the microbial world is far larger than previously thought. As per established knowledge of microbial genetics, local DNA sequence changes, segment-wise rearrangement of genomic DNA sequences by recombinational reshuffling and horizontal gene transfer are the evolutionary processes shaping the genome of the prokaryotes. Modern knowledge of the molecular biology of the genes and their expression has provided a strong input in understanding the genome-wide map of selection, linking gene variation to phenotype and ecology. Keywords: Prokaryotes, Microbial speciation, Natural selection, Microbial evolution, Molecular evolution, DNA rearrangement, Horizontal gene transfer

Introduction The year 2009 marks the 200th anniversary of Charles Darwin’s birth and the 150th anniversary of his most influential publication ‘On the Origin of Species by Means of Natural Selection’1 (Fig. 1). Darwin’s ideas have influenced biology ever since and transformed the biological sciences in a way much similar that of Copernicus, Galileo and Newton, who centuries earlier transformed the physical science. Darwin’s thinking on how species evolved, his ideas on ‘survival of the fittest’ and selection during evolution are the guiding principles of nearly all V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Figure 1. Title page of The Origin of Species

aspects of life science. The concept of natural selection – as the guiding principle of adaptive evolution – is Darwin’s seminal contribution. It provides a worldly explanation of nature’s operations that contrasted sharply with the traditional beliefs of supernatural interventions that predominated before ‘The Origin’. Darwin is deservedly credited as the originator of modern theory of evolution. In ‘Origin of Species’, he gathered the best available evidences for evolution. He explained how natural selection works, discussed the role of hereditary variation, the mechanisms of which were not well understood in Darwin’s time, and also considered the possible objections to his theory. Darwin’s clear elucidation of natural selection launched a revolutionary dimension in biology wherein organismal traits could be studied and interpreted as products of natural forces amenable of rational scientific scrutiny. Scientific studies of natural selection are now more popular and powerful than ever and they have revealed the evolutionary origins of numerous biological features and taxa. A major limitation in Darwin’s characterization of evolution concerned the hereditary mechanism, an aberration that the modern biology began to rectify early in the 20th century by incorporating Mendelian genetics and population genetics in the dictum of evolutionary synthesis.2 Today, in the era of genomics, biologists routinely extend studies of natural selection and evolution of features to the level of DNA. Genomic investigations are also providing fresh insights into the ancient mystery of the origin of species. Characteristically, ‘The Origin’ mentions little detail about the evolution of reproductive barriers, which

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under the modern biological species concept are keys to the understanding of speciation process.

Darwin and microbiology It is commonly assumed that Darwin had nothing to say about microbes and was ignorant of or at best indifferent to microbes and microbial evolution. 3 Microbiology was a nascent science when Darwin published his ‘Origin of Species’. No techniques existed for the isolation of bacteria and the tremendous diversity of the microbial world was still largely unknown. The ‘germ theory’, microorganisms as the causal agent for diseases, would not be developed until many years later. Although Darwin did not draw heavily on the microbial world for observational data in his own work, he used his and others’ knowledge of microorganisms to sustain his arguments about natural selection. Three main themes emerged from these observations: (a) biogeographical patterns and processes, (b) distinction between evolution through natural selection and evolution as progressive complexification, and (c) continuous nature of evolution operating on all life-forms. Microbial biogeography Biogeography had formed the insight of Darwin’s evolutionary theories and he considered the biogeography of microbial life to be very similar to that of macroorganisms. Natural selection eventually became the ultimate explanation of the biogeographic distribution of different life-forms. The idea of microbial diversity was used by Darwin as evidence for his argument about dispersability which he saw as central to the geographical distribution of species.4 He preferred ‘dispersal’ explanations to those involving geological disruption, such as break-up of land bridge due to ‘continental shift’. Darwin made several observations and inferences about the dispersal mechanisms of plants and animals and successfully worked them into his all-encompassing explanation of natural selection. Microbes and their distribution were just as explicitly adjoined by this thesis. Evolution of microbes by natural selection Very little was known about microbial physiology when Darwin developed his theory of natural selection. Nevertheless, taxonomy of microbes existed and these began to grow systematically after the establishment of Ferdinand Cohn’s scheme of classification.5 However, the inherent handicap of largely invisible and always confusing microbial diversity made it less pressing to explain the microbial component than the abundant new discoveries of obvious diversity of plants, animals and the fungi. These visible entities formed the primary factual

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basis for Darwin’s theory of natural selection albeit his intention of covering all life forms in his theory of natural selection. In fact, in responding to criticisms that the simplicity of microorganisms disproved his theory of natural selection, Darwin added an argument about the adaptiveness of microbial organization to the third edition of ‘The Origin’.6 Continuity of evolved microbial complexity Darwin’s argument was that an evolutionary record could be detected in the more simply organized life-forms, such as microbes, and also their simplicity must not confuse the natural historians to their organization complexity. The complexity, at least in the functional level that has been confirmed with the current knowledge of biology, led Darwin to speculate on the continuous theory of evolution and the common ancestry of all living organisms. In other words, Darwin saw in today’s microbes a record of the very early life-forms. Suggesting this link between early and current life from bacteria onwards, was an inevitable consequence of accepting descent with modification.

Darwin’s views on the evolution of microbes In his discussions on the microbial world, Darwin made at least three major evolutionary claims: (1) that all living entities, no matter how different they seem from animals and plants, do undergo natural selection; (2) that microbes demonstrate that evolution is not a progression from simple to complex; and (3) that microbes and their adaptive capacities are very important biological phenomena to understand if the history of evolution on Earth is to be understood. These dicta are crucial to the understanding of the evolutionary theory put forth by Darwin. Today, in the background of huge explosion in knowledge in molecular biology and genomics, Darwin’s basic principles still holds sway. Darwin’s discussion of microbes thus is the, testing of a fundamental framework to see whether it can accommodate all forms of life and all the life’s history. In this context, Darwin’s investigations of microbes gave his theory cardinal support and the widest possible application. To establish his theory as firm as possible with the largest of audience, Darwin focused instead upon the visible and the obvious.

Concept of speciation in prokaryotes Unlike in the reproduction of complex eukaryotes, cell fusion and recombination are not the necessary steps in the reproduction of prokaryotes. As a result, early models for understanding evolution and speciation in prokaryotes often focused on clonality and periodic selection. According to such models, all individuals within a species resemble each other because they descend from a single


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ancestor that bested its siblings by virtue of some beneficial mutation(s) fixing not only the favored mutation but the entire genome in which it first occurred. In practice, bacterial lineages show substantial variation in population structure, ranging from essentially clonal (e.g. Salmonella) to nearly sexual (e.g. Neisseria gonorrhoeae). The basis of the taxonomic hierarchy is the species. However, the concept of a prokaryote species still lacks a theoretical basis, and all existing definitions are pragmatic ones, such as, for example: ‘A species consists of an assemblage of individuals (or, in micro-organisms, of clonal populations) that share a high degree of phenotypic similarity, coupled with an appreciable dissimilarity from other assemblages of the same general kind’, or: ‘a collection of strains showing a high degree of overall similarity, compared to other, related groups of strains’.7 Ward (1998)8 called for the establishment of a ‘natural’ species concept for the prokaryotes, based on ‘evolutionary species’, being defined as lineages evolving separately from others and with their own unitary evolutionary roles and tendencies. The species definition is so extremely subjective because one cannot accurately determine and define such concepts as ‘a close resemblance’, ‘essential features’, or how many ‘distinguishing features’ are sufficient to create a species. In-depth essay on the current thoughts on the species concept for prokaryotes was recently published.9 The species should ideally be described as ‘a category that circumscribes a (preferable) genomically coherent group of individual isolates/strains sharing a high degree of similarity in (many) independent features, comparatively tested under highly standardized conditions’ (the phylo-phenetic species concept). Our present species concept is, to a large extent, based on the recommendations published in 1987 by a committee of experts,10 recently confirmed and extended by a new ad hoc committee.11 Species classification is based on a combination of diagnostic phenotypic features and genomic properties. The questions of whether clusters of strains observed in the bacterial world share the dynamic properties attributed to the eukaryote species, and whether the mechanisms driving the origin of new species in bacteria might be shared with the eukaryotes, were addressed.12 He concluded that, despite basic differences in the nature of the genetic exchange between bacteria and eukaryotes, bacterial species share many of the fundamental properties of eukaryotic species. In case of the prokaryotes, recombination depends on vectors such as bacteriophagemediated transduction or plasmid mediated conjugation, and is therefore limited by the host ranges of the respective vectors. Moreover, restriction endonuclease activity can greatly reduce the rate of recombination. Cohan12 further assessed the potential of the formation of new species within the prokaryote world as a result of the formation of new ‘ecotypes’. On the basis of multilocus sequence typing he defined an ecotype as a set of strains using the same or very similar ecological niches, such that an adaptive mutant from within the ecotype outcompetes all other

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strains of the same ecotype. The potential for speciation is very large indeed for the following reasons: ● Speciation in prokaryotes requires only ecological divergence (versus both reproductive and ecological divergence in sexual eukaryotes). ● The extremely large population size of prokaryotes makes rare mutation and recombination events much more accessible to a population than is the case for macro-organisms. ● A prokaryote species is open to gene transfers from many taxa, even those distantly related, so it can take up existing adaptations from anywhere in the prokaryote world. ● Recombination events have a localized nature, as only a small fraction of the donor’s genome is integrated. This allows for transfer of a useful adaptation without co-transfer of other donor segments that would be deleterious for the recipient. This is in contrast with the processes of meiosis and fertilization in eukaryotes, which yield hybrids that are a 1:1 mix of both genomes. The physiology of bacteria may thus be more modular than is the case for macro-organisms in that a new adaptation might be accommodated with very little fitness cost.12 Direct molecular analyses of natural microbial populations reveal patterns that should compel microbiologists to adopt a more natural species concept that has been known to biologists for decades. The species concept can be utilized to address the larger issue of delineating the concept of microbial evolution as had already been done for eukaryotes.

Microbial evolution in the present context As compared to the diploid nature of mostly higher organisms, prokaryotic microorganisms, archaea, eubacteria and their viruses, are usually haploid and they rapidly reveal phenotypic consequences of genetic modification. This and the fast propagation of these micro-organism facilitate genetic variation which is a prerequisite for biological revolution. Indeed, mixed populations of parental forms and different genetic variants are the substrate for natural selection that is exerted by the living conditions encountered by the organisms. Aided by recent developments in the understanding of genomics, prokaryotes certainly provide an interesting opportunity to study the mechanisms of evolution as (i) they harbor a previously unsuspected diversity even within species and populations,13 (ii) they are found in small to very large population sizes,14 (iii) they can survive or prosper in most inhospitable environments, and (iv) they frequently acquire and use genetic material from distantly related organisms.


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Universality and diversity of prokaryotic genomes Comparative genomic studies have revealed that prokaryotic genetic material are under continuous selective pressure that have profoundly influenced its organization. Processes of replication, transcription and the regulation of gene expression all impact how genes are arranged along the genome. It has long been known that the asymmetric manner in which the prokaryotic genetic material is replicated, with a leading and a lagging strand, is correlated with many evolutionary features, such as differential bias between the two strands and location of essential genes. Interestingly, the intensity of these biases shows a strong phylogenetic inertia. The replication of prokaryotic genetic material is oriented from the origin (Ori) to the terminus (Ter). This is also correlated with several evolutionary features like (a) in many genomes, genes near the terminus of replication tend to have lower G+C content and also exhibit higher rate of evolution;15 (b) also, as a result of the replication process, genes located close to the Ori can be significantly amplified in dividing cells, in comparison to those closer to the terminus. Not only these genes are over-represented in the Ori region but the genomes of fast-dividing bacteria show evidence that rearrangements that would disrupt this association are counter-selected.16 In spite of these common principles of genome organization, comparative genomics has revealed a previously unexpected degree of diversity among prokaryotic genomes. One of the most remarkable examples of this diversity is the comparison of gene contents within and between species. All forms of life seem to share only a handful of genes, ~60,17 and these are mostly dedicated to translation. The genes for other fundamental factions, such as DNA replication, transcription or basic metabolism seem to be more sporadically spread in the tree of life. More interestingly, this diversity of genome content can be seen at every phylogenetic scale. It has been estimated that the core genome of13 -proteobacteria contains 4,000 protein coding genes, these genomes share 80% shorter than their orthologs in other E. coli strains and are therefore likely to be pseudogenes.21 The most frequent causes of gene disruption are frame-shifts and truncations but some recent pathogens such as Yersinia pestis or Shigella flexneri exhibit a high proportion of pseudogenes due to introduction of ‘insertion sequence’ (IS) elements, probably as a result of relaxed selection pressure. Nevertheless, both gene loss and acquisition imparts variability in the prokaryotic population subjecting them to natural selection and further evolution. Local sequence change Local sequence changes might theoretically represent individual steps in the development of new biological functions. However, the mutational generation of a completely novel biological function may be extremely rare as long as natural selection cannot exert its pressure on an at least primitive function displayed by the product of the DNA sequence. The situation may change in the stepwise improvement of already available biological functions on which natural selection exerts its pressure. This is applicable to functional domains, entire genes as well as functional systems composed of different genes Evolutionary clocks used by the scientists to estimate evolutionary distances are based on local sequence changes often within a specific gene, for example a ribosomal RNA gene. Within functional genes lethal and heavily contra-selective mutations will of course not be maintained. Also, local sequence changes are known to affect different DNA regions with different efficiency. These reasons limit the precision of evolutionary clocks, but the method has its merits for the relative comparison of the evolutionary relations between different species. DNA rearrangements Gene conversion can also result from the intragenomic reshuffling of DNA sequences, if related sequences carried in the genome undergo recombination. Another often innovative and thus important effect of the DNA rearrangement strategy is the novel combination of available capacities by the fusion of different functional domains. Genomics reveal that different functional genes often share related sequence motifs and functional domains. Occasional DNA reshuffling is a good source for such fusion genes. The same holds true for the regrouping of expression control signals with different reading frames serving in protein

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production. DNA rearrangements can also bring about the duplication of DNA segments which may serve as substrates for further evolution. Similarly, deletion of DNA segments can also result from DNA rearrangement processes and can help in conjunction with natural selection to discharge the genome from inessential sequences. Bacterial population geneticists have known for some time that prokaryotic genomes do sometimes recombine,22 although early estimates suggested that rates of recombination were sufficiently low to be ignored when considering periodic selection events. Recent work has helped clarify the relationship between recombination and positive selection.23 Recombination and positive selection were both quantified in the Streptococcus core genome, and it was concluded that genes under positive selection are frequently recombined, a result also supported in a study of Listeria genome. Specifically, 78% of genes under positive selection in the Streptococcus pyogenes core genome were also inferred to be recombinant. Yet, of the genes identified as recombinant within this species, only a small fraction experienced positive selection. Therefore, although positively selected genes are frequently recombined, a substantial amount of within-species recombination shows no evidence of direct adaptive value. In other words, recombination within a species could be largely neutral while not so for recombination between species. Critical analysis of comparison between-species and within-species evolutionary events indicate 81% of all genes recombined between species also experienced positive selection, whereas only 4% of genes recombined within species also experienced positive selection. DNA acquisition The strategy of DNA acquisition allows microorganisms to share the evolutionary success of others. This strategy is very efficient and can result in essential new capabilities in a single step of acquisition. This is of particular relevance for accessory genes. Such genes may also open the possibility for a microorganism to populate new habitats. The evolutionary contribution of this strategy is thus considerable. This strategy can also affect functions of essential housekeeping genes by conversion upon horizontal transfer of DNA segments. In bacteria, horizontal gene transfer (HGT) is widely recognized as the mechanism responsible for the widespread distribution of antibiotic resistance genes, gene clusters encoding biodegradative pathways and pathogenecity determinants. HGT is also considered responsible for speciation and sub-speciation in bacteria Analysis of complete genome sequences is helping determine what types of genes are most prone to transfer. Recent studies using phylogenetic analysis of many genes conclude that genes which interact with many other genes (informational genes) are less prone to gene transfer than those with fewer interactions (operational


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genes). This was referred to as the ‘complexity hypothesis’.24 It is still not clear if complexity of interactions is the key factor here. For example, it is possible that sequence conservation of genes between species also influences the likelihood of transfer, which is probably what allows even rRNA genes to be transferred. In addition, because informational genes are more likely to be required for survival, they are probably less likely to be lost from a species. Therefore, there will be little opportunity to replace them with a homologous gene. In addition, some genes may even facilitate their own transfer. For example, genes could avoid the need for amelioration by carrying the features needed for their own expression. Possible examples include self-regulatory transcription factors (which could bind to their own promoter), amino-acyl tRNA synthetases (which have been shown to be prone to transfer and could help avoid codon usage problems), splicing factors, recombinases, and DNA polymerases. HGT is frequently invoked for those genes that have best matches to supposedly distant species. Lateral gene transfer can result in the addition of novel genes in a particular species — thus, in theory, gene transfers could be identified by detecting genes with uneven distribution patterns (e.g. if a particular gene is present in gram-positive bacteria and all plants but not other bacteria or eukaryotes). Some selected examples are: Thermophilic bacteria and archaea Analysis of the complete genomes of the bacteria Aquifex aeolicus and Thermotoga maritima and comparison with other complete genomes has led to the suggestion that large numbers of genes have been transferred between thermophilic bacteria and Archaea. In both cases, best-match methods have revealed that a high percentage of each proteome was most similar to Archaeal genes rather than bacterial genes, ~20% for A. aeolicus and 25% for T. maritime25. Transfers involving pathogen genomes A variety of studies have suggested the occurrence of extensive HGT in the history of many of the pathogen species for which complete genome sequences are now available. Analysis of nucleotide composition and codon usage has identified many regions of possible HGT in E. coli (up to 18% of the genome). Genome analysis suggests that a large percentage of the Xylella fastidiosa genome (~7%) appears to be derived from l-like phage. The finding that only a modest proportion of the genes in the Campylobacter jejuni genome had matches to genes from other -Proteobacteria, led to the suggestion that this species may have acquired many genes by HGT.

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Transfers involving whole genetic elements (small chromosomes, megaplasmids, plasmids) Analysis of complete genomes is revealing that many of the smaller genetic elements in microbes may have separate origins from the main chromosomes. For example, the linear plasmids of Borrelia appear to be prone to HGT among strains or species. Other proposed cases of discordant evolution of smaller genetic elements versus chromosomes include the Xylella fastidiosa plasmid, the megaplasmids and plasmid of D. radiodurans and the smaller chromosome of V. cholerae. The source of these smaller elements in each species remains unclear. Interestingly, phylogenetic analysis suggests that the megaplasmids of Rhizobia are only transferred among closely related strains or species and not across long distances. Antibiotic resistance: The immediate response The dissemination of antibiotic resistance genes among human and animal bacterial pathogens and associated commensal populations is the paradigm for HGT on a global scale. This is the best known example of very rapid adaptation of bacterial populations to a strong selective pressure. What has been learned is that this adaptation occurred (and still occurs if bacteria are challenged with a novel antibiotic) not by mutation of the menaced populations, but by acquisition and dissemination of, in most cases, simple antibiotic resistance genes by mobile genetic elements (Table 1). Nonetheless, the basis of antibiotic resistance development is formally simple: mobile genetic elements such as plasmids and transposons efficiently distributed the resistance determinants, singly or in clusters, among many genera and species of bacteria. Intergeneric boundaries were not respected, particularly the long-standing but only apparent distinction between Gram-positive and Gram-negative bacteria. Biodegradation pathways: delayed opportunistic response Based on studies with antibiotic resistance and its associated genes (e.g. genes for the metabolism of a number of sugars), it is not surprising that HGT has also led to the dissemination of gene clusters (operons) involved in the catabolism of xenobiotics in polluted environments. The selective pressures are similar to those of antibiotic resistance, but the process of degrading xenobiotic compounds requires more complex genetic systems, usually operons of ten or more genes, or even regulons of several operons with accompanying control circuits. An example of this can be found in the Sphingomonas aromaticovorans plasmid pNL1. This large conjugative plasmid contains 15 gene clusters directly associated with the catabolism or transport of aromatic compounds, allowing the host bacteria to


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Table 1. Mobile genetic elements involved in HGT in bacteria Mobile element

General features

Examples

Gene cassette

Mobility of ‘simple genes’ IntI1 Immediate constitutive IntI4 expression Extremely broad host range

Transposon

Mobility of genes and operons Very broad host range Loci screening for best performance

Tn21 Tn4651

Plasmid

Mobility of operons and regulons Immediate regulated expression Very broad host range

pNRG234a Nodulation plasmid of pNL1 rhizobium pCD1 Degradation of aromatic compounds Virulence determinants of Yersinia

CTX Bacteriophage Mobility of regulons ‘Maturation in chromosomes (via lysogeny) Narrow host range

Phenotype of specific element Antibiotic resistance (simple) Gene ‘maturation’ in Vibrio

Antibiotic resistance (complex) Toluene degradation

Virulence phage of Vibrio cholera

degrade compounds such as biphenyl, naphthalene, xylene and cresol. Although the dissemination of biodegradation pathways has not been analyzed to the same extent as that of antibiotic resistance, the available data clearly indicate that the genetic mechanisms responsible are likely to have been the same: plasmids and transposons distributed catabolic operons among a wide range of bacteria.26 It can be assumed that the degradation of xenobiotics by bacteria has intensified in the past century, as a result of the increased use of chemicals in industry and the waste disposal habits of humans, but the basic processes could have evolved much earlier. Significantly, degradative operons are also found integrated in the bacterial chromosome. In these cases, adjacent remnants of phage or plasmid genes remind us of the once-mobile nature of these operons. Armed with the awareness that HGT is so important in bacterial speciation, it is now possible to examine completely sequenced chromosomes in a new light and assess the extent of chromosomal DNAs of obscure lineage. Using E. coli and Salmonella as a model system, it has been shown that 17% of their genomes (i.e. ~800 kb), appears to have been acquired by HGT during the past 100 million

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years (the housekeeping genes of both organisms are ~90% identical). As the majority of this DNA was recruited recently, it is apparent that considerable genetic flux is occurring: the 234 detectable HGT events that have persisted are probably the tip of the iceberg of the thousands of mobile sequences that have entered and left any particular E. coli strain. Similarly, when the members of a well known collection of E. coli strains (the ECOR collection) were compared, they were found to be quite variable in the size and macro-organization of their chromosomes and plasmids. In summary, a significant proportion of the genome of any strain of a single bacterial species comprises fragments of DNA from various origins which, properly ‘nurtured’, can give rise to new bacterial species. The role of HGT seems to be crucial but one should not consider that gene exchanges have been so profound as to preclude the reconstruction of the history of life, in the sense of understanding how genomes have evolved to what they are. More integrative approaches combining information from species phylogenies, gene histories, ecology and cellular networks will be necessary to tell the chronicles of contemporary genomes. Involvement of non-genetic factors in the generation of genetic variants Nature also takes advantage of non-genetic factors for the production of genetic variants. Both intrinsic structural features of biological macromolecules and external influences, such as those by mutagens interacting with DNA are important. This is not limited to the strategy of local sequence changes but can also affect the other strategies of generation of variation. Another non-genetic factor influencing the production of genetic variations is the chance of random encounter. This plays its role in the interaction of enzymes with their substrate such as a site of recombination on DNA molecule. Random encounter is also clearly involved in the DNA acquisition strategy. During transformation, DNA molecule must encounter recipient bacteria, in transduction, the bacteriophage must encounter a host bacterium, and in conjugation, donor and recipient bacteria must encounter each other.

Conclusion Biological constituents of the process of biological evolution are likely to undergo biological evolution themselves. Identifying the signature of natural selection in microbial genomes can help to explore the unknown world of microbes. Which techniques can be used to identify positive selection depends on the rates and bounds of recombination in microbial populations. The first step in any study of natural selection in bacteria is to quantify the extent of recombination within a population before moving on to sequence-based tests. Once recombinant portions


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of the genome are identified, they can be tested for evidence of positive selection using diversity-based methods. Meanwhile, the non-recombinant clonal frame can be identified and tested for convergent evolution or excessive rates of functional substitutions. If possible, all tests should be performed genome-wide to estimate and control for demographic effects that might otherwise provide spurious evidence of positive selection. The modern knowledge of genomics and proteomics have provided a strong handle to the microbiologists and molecular biologists to understand the genomewide map of selection, linking gene variation to phenotype and ecology. Darwin was right in saying that many variations are minor but subtly alters protein structure or expression, but cumulatively, they leave a trail of footprints, which will finally lead us to a better understanding of the microbial world. Acknowledgment: This work was part of an in-house discussion-series held under the AMAAS project (theme: Microbial diversity and identification), funded by Indian Council of Agricultural Research, New Delhi. The senior author thanks Prof. Anupam Varma, for enthusing him to compile the stray thoughts in the form of this chapter.

References 1.

Darwin C (1859) In: On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life. 1st edition, John Murray, London

2.

Dobzhansky T (1937) In: Genetics and the Origin of Species. Columbia University Press

3.

Sapp J (2005) The bacterium’s place in nature. In: Microbial Phylogeny and Evolution: Concepts and Controversies. Sapp J (Ed.), Oxford University Press

4.

O’Malley MA (2007) The nineteenth-century roots of ‘everything is everywhere’. Nat Rev Microbiol 5:647–651

5.

Drews G (2000) The roots of microbiology and the influence of Ferdinand Cohn on microbiology of the 19th century. FEMS Microbiol Rev 24:225–249

6.

Darwin C (1861) In: On the Origin of Species by Means of Natural Selection, or the Preservation of Favoured Races in he Struggle for Life. 3rd edition, John Murray, London

7.

Colwell RR, Clayton RA, Ortiz-Conde BA, Jacobs D, Russek-Cohen E (1995) The microbial species concept and biodiversity. In: Microbial diversity and ecosystem function. Allsopp D, Colwell RR, Hawksworth DL (Eds.), CAB International Wallingford

8.

Ward DM (1998) A natural species concept for prokaryotes. Curr Opin Microbiol 1:271–277

9.

Gevers D, Cohan FM, Lawrence JG, Spratt BG, Coyene T, Feil EJ, Stackebrandt E, Van de Peer Y, Vandamme P Thompson FL, Swings J (2005) Opinion: re-evaluating prokaryotic species. Nat Rev Microbiol 3:733–739

10.

Wayne LG, et al. (1987) Report of the ad hoc committee on reconciliation of approaches to bacterial systematic. Int J Syst Bacteriol 37:463–464

118


11.

Stackebrandt E, et al. (2002) Report of the ad hoc committee for the re-evaluation of the species definition in bacteriology. Int J Syst Bacteriol 52:1045–1047

12.

Cohan FM (2001) Bacterial species and speciation. Syst Biol 50:513–524

13.

Binnewies TT, Motro Y, Hallin PF, Lund O, Dunn D, La T, Hampson DT, Bellgard M, Wassenaar TM, Ussery DW (2006) Ten years of bacterial genome sequencing: comparative genomics based discoveries. Funct Integr Genomics 6:165–185

14.

Lynch M, Conery JS (2003) The origins of genome complexity. Science 302:1401–1404

15.

Daubin V, Perriere G (2003) G+C structuring along the genome: a common feature in prokaryotes. Mol Biol Evol 20:471–483

16.

Couturier E, Rocha EP (2006) Replication-associated gene dosage effects shape the genomes of fast-growing bacteria but only for transcription and translation genes. Mol Microbiol 59:1506–1518

17.

Koonin EV (2003) Comparative genomics, minimal gene-sets and the last universal common ancestor. Nat Rev Microbiol 1:127–136

18.

Lerat E, Daubin V, Moran NA (2003) From gene trees to organismal phylogeny in prokaryotes: the case of -proteobacteria. PLoS Biol 1(1):e19

19.

Tettelin H, et al. (2005) Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: implications for the microbial “pan-genome”. Proc Natl Acad Sci USA 102:13950–13955

20.

Arber W (2000) Genetic variation: molecular mechanisms and impact on microbial evolution. FEMS Microbiol Rev 24:1–7

21.

Lerat E, Ochman H (2005) Recognizing the pseudogenes in bacterial genomes. Nucleic Acids Res 33:3125–3132

22.

Guttman DS, Dykhuizen DE (1994) Clonal divergence in Escherichia coli as a result of recombination, not mutation. Science 266:1380–1383

23.

Lefebure T, Stanhope MJ (2007) Evolution of the core and pan-genome of Streptococcus: positive selection, recombination, and genome composition. Genome Biol 8:R71

24.

Jain R, Rivera MC, Lake JA (1999) Horizontal gene transfer among genomes: the complexity hypothesis. Proc Natl Acad Sci USA 96:3801–3806

25.

Nelson KE, et al. (1999) Evidence for lateral gene transfer between Archaea and bacteria from genome sequence of Thermotoga maritima. Nature 399:323–329

26.

Lal R, Dogra C, Malhotra S, Sharma P, Pal R (2006) Diversity, distribution and divergence of lin genes in hexachlorocyclohexane-degrading sphingomonads. Trends Biotech 24:121–130

9 Evolutionary relationships among cyanobacteria, algae and plants: Revisited in the light of Darwinism Radha Prasanna* and B.D. Kaushik** *Division of Microbiology, Indian Agricultural Research Institute, New Delhi - 110012, India [email protected] **Netaji Subhas Institute of Technology, Dwarka, Sector 3, New Delhi - 110078, India [email protected]

Abstract: Algae represent an ancient group of plant like organisms, comprising a heterogeneous, polyphyletic assemblage of prokaryotes and eukaryotes, which have been primarily classified on the basis of their pigments, storage food material and cell wall characteristics. They have been playing a critical role in shaping the Earth’s atmosphere, recycling major elements such as C and N and mediating key biogeochemical processes and were the first to colonize land. Algae have been often conceptually and systematically linked to the land plants because they include some forms – such as seaweeds/kelps, which look much like plants, and as with plants – they are commonly sessile (attached/ rooted/occurring in mixed communities) and oxygenic photosynthesizers. DNA evidence suggests that the first eukaryotes (green plants) evolved from prokaryotes (through endosymbiotic events) between 2500 and 1000 million years ago. Among algae, blue green algae or cyanobacteria represent an ancient group of photosynthetic prokaryotes, whose ubiquity, metabolic flexibility and adaptive abilities have made them a subject of research worldwide. Their biological significance, especially as the prokaryotic interface between the primeval photosynthetic cell and present day plants is also well recognized. Darwin’s Theory of Evolution and Natural Selection have shaped much of our understanding of the evolution of this cosmopolitan group of algae and land plants. However, in the current scenario, with the advent of molecular phylogenetics and the expanding knowledge regarding the evolution of life through the use of bioinformatics, a thorough re-evaluation of algae and interrelationships with plants is needed. This compilation attempts to link Darwinism, Neo-Darwinism and Systemic Darwinism, with the rapidly generated information through modern molecular tools for a better understanding of evolution of land plants, as mediated through algae and endosymbiotic events combined with horizontal and lateral gene transfer processes. Keywords: Algae, Cyanobacteria, Endosymbionts, Evolution, Mitochondria, Plastids, Photosynthesis, rDNA V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Introduction Algae represent a morphologically and biochemically diverse assemblage of organisms, including cyanobacteria - which are amenable to a range of growth environments, culturing techniques and have applications in agriculture, industry and environmental management. This broad terminology includes both prokaryotes and eukaryotes, which exhibit tremendous diversity in metabolism, structure and habitat. Their ecological amplitude combined with their dynamic survival and adaptation strategies as primary colonizers and producers in the food chain make them one of the most significant contributors to the stability, fertility and maintenance of various ecosystems.1 It was more than three billion years ago, that the advent of primordial cyanobacteria marked a decisive turning point in the evolution of our planet by introduction of oxygen into a previously anoxygenic biosphere and bringing about global changes in the ionic composition of ancient water bodies.2 Their role in reclamation of wastewaters/wastelands, as pioneers in denuded/ degraded soils and as bioinoculants in rice based cropping systems is well established.3 In the present scenario, they have become prime targets for screening programs in search for novel compounds of potential pharmaceutical or diagnostic value and invaluable as tools for better understanding of diverse metabolic and evolutionary processes. Microalgal biotechnology is increasingly attracting attention in national and international research programs, as these organisms represent an unique and underexploited bioresource. Darwin’s Theory of Evolution is the widely held notion that all life is related and has descended from a common ancestor: the birds and the bananas, the fishes and the flowers – all related. Darwin’s general theory presumes the development of life from non-life and stresses a purely naturalistic (undirected) ‘descent with modification’, i.e. complex creatures evolve from more simplistic ancestors naturally over time. In a nutshell, as random genetic mutations occur within an organism’s genetic code, the beneficial mutations are preserved because they aid survival - a process known as ‘natural selection’. Such beneficial mutations are passed on to the next generation; over time, beneficial mutations accumulate and the result is an entirely different organism (not just a variation of the original, but an entirely different creature). For Darwin,4 the primary engine of evolutionary change was natural selection: “Furthermore, I am convinced that Natural Selection has been the main but not exclusive means of modification”. However, in the current scenario, Darwin’s Theory of Evolution seems to be a ‘theory in crisis’ in light of the tremendous advances made in molecular biology, biochemistry and genetics over the past fifty years. We now know that there are in fact tens of thousands of irreducibly complex systems on the cellular level. As molecular biologist Michael Denton wrote, “Although the tiniest bacterial

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cells are incredibly small, weighing >10-12 grams, each is in effect a veritable micro-miniaturized factory containing thousands of exquisitely designed pieces of intricate molecular machinery, made up altogether of one hundred thousand million atoms, far more complicated than any machinery built by man and absolutely without parallel in the non-living world”. Specified complexity therefore, pervades the microscopic biological world and Darwinism needs to be explored in the context of the rapidly generated information on genomics.5 This compilation therefore, focuses on the modern day significance of Darwinism in understanding algal evolutionary trends and interlinking with the evolution of land plants.

Concept of Darwinism Ancient Greek philosophers such as Anaximander postulated the development of life from non-life and the evolutionary descent of man from animal. While Darwin’s Theory of Evolution is a relatively young archetype; the evolutionary worldview itself is as old as antiquity. Charles Darwin simply brought something new to the old philosophy – a plausible mechanism called ‘natural selection.’ Natural selection acts to preserve and accumulate minor advantageous genetic mutations. Suppose a member of a species developed a functional advantage (it grew wings and learned to fly), its offspring would inherit that advantage and pass it on to their offspring. The inferior (disadvantaged) members of the same species would gradually die out, leaving only the superior (advantaged) members of the species. Natural selection is the preservation of a functional advantage that enables a species to compete better in the wild and can be considered the naturalistic equivalent to domestic breeding. Over the centuries, human breeders have produced dramatic changes in domestic animal populations by selecting individuals to breed. Breeders eliminate undesirable traits gradually over time. Similarly, natural selection eliminates inferior species gradually over time. Darwin’s Theory of Evolution is a slow gradual process. Darwin wrote “Natural selection acts only by taking advantage of slight successive variations; she can never take a great and sudden leap, but must advance by short and sure, though slow steps.” Thus, Darwin conceded that, “If it could be demonstrated that any complex organ existed, which could not possibly have been formed by numerous, successive, slight modifications, my theory would absolutely break down.” Such a complex organ would be known as an ‘irreducibly complex system’. The common mousetrap is an everyday non-biological example of irreducible complexity. It is composed of five basic parts: a catch (to hold the bait), a powerful spring, a thin rod called ‘the hammer,’ a holding bar to secure the hammer in place, and a platform to mount the trap. If any one of these parts is missing, the mechanism will not work. Each individual part is integral. The

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mousetrap is irreducibly complex and so are the present day biological systems which could not have evolved slowly, piece by piece. Darwin repeatedly wrote about the laws of variation, reproduction, correlation, and heredity and even about the laws of natural selection, the struggle for life, divergence of character, and extinction. The last sentence of On the Origin of Species established an analogy between Darwin’s evolutionary view of life and Newton’s ‘fixed laws of gravity’. Furthermore, in another well known passage he noted: “It is interesting to contemplate on entangled bank, clothed with many plants of many kinds, with various birds singing on the bushes, with various insects flitting about, and with worms crawling through the damp earth, and to reflect that these elaborately constructed forms, so different from each other, and dependent on each other in so complex a manner, have all been produced by laws acting around us”. (Darwin4 On the Origin of Species by means of Natural Selection, or the Preservation of Favoured Races in the Struggle for Life, Harvard Univ Press, Cambridge, MA.)

Modern day significance of Darwinism and the advent of Systemic Darwinism A major area of research on evolutionary biology focuses on individuality and adaptation across levels of selection and what ideally should we call as the unit of Darwinism and what should be its characteristics. The proper definition of a Darwinian individual constitutes one of the very fascinating and challenging areas of biology. In this context, two major clarifications have greatly abetted the understanding and fruitful expansion of the theory of natural selection and defining characteristics that define a Darwinian unit, in recent years. Firstly, the acknowledgement that interactors, not replicators, constitute the casual unit of selection; and the recognition that interactors are Darwinian individuals, and that such individuals exist with potency at several levels of organization (genes, organisms, demes and species in particular), thus engendering a rich hierarchical theory of selection in contrast with Darwin’s own emphasis on the organismic level.6 Some of the criteria that an entity must manifest to represent a Darwinian individual are as follows. The biological individual must exhibit a distinct birthpoint, distinct deathpoint and sufficient stability, followed by reproductive ability and interactive ability with the environment – these constitute the three basic properties of a Darwinian individual. Organisms, species and genes have been thoroughly investigated for their appropriateness in this context.


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The gene represents the most appropriate unit for marking evolutionary change for two reasons; firstly, the gene operates as a maximally faithful replicator across generations and as bookkeepers can therefore record the evolutionary history of populations as changing relative frequencies of these stable items. Secondly, genes become preferred units of bookkeeping because they represent the lowest-level individuals subject to clear and accurate recording in a genealogical hierarchy of inclusion. However, genes do not manifest sufficient stability or interactive ability as compared to organism or species; although they fulfil the other criteria. Organisms, on the other hand, although score well in terms of boundedness and functionality, but they have evolved powerful mechanisms to suppress selection at lower levels than itself. On defining Darwinian individuality across scales of size and time, the species prove most effective as Darwinian individuals in evolution, as compared to organisms or genes. The species individual, by not suppressing selection at lower levels, emerges as the suitable choice as a Darwinian unit because it maintains an excellent reservoir of traits and gains enormous flexibility that provides emergent fitness. This is very aptly illustrated by the studies of Tomitani et al.7 who integrated molecular phylogenetic, physiological, paleontological and geochemical data of cyanobacterial species and provided not only a calibration point, but also, very informative data which can further illuminate molecular evolutionary studies of deep nodes in the Tree of Life and thereby evolution of early organisms.

Systemic Darwinism Darwin’s 19th century, evolutionary theory of descent with modification through natural selection opened up a multidimensional and integrative conceptual space for biology. A significant amount of 21st century research focuses on systems (e.g. genomic, cellular, organismic, and ecological/global); systemic Darwinism8 is emerging in this context. It follows a ‘compositional paradigm’ according to which complex systems and their hierarchical networks of parts are the focus of biological investigation. Through the investigation of systems, Systemic Darwinism promises to reintegrate each dimension of Darwin’s original logical space. Moreover, this ideally and potentially unified theory of biological ontology coordinates and integrates a plurality of mathematical biological theories (e.g. self-organization /structure, cladistics/history and evolutionary genetics/function). Integrative Systemic Darwinism requires communal articulation from a plurality of perspectives. Although, it is more general than these, it draws on previous advances in Systems Theory, Systems Biology, and Hierarchical Theory. Systemic Darwinism would greatly further bioengineering research and would provide a significantly deeper and more critical understanding of biological reality. The concluding sentences of Darwin’s On the Origin of Species by Means of Natural

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Selection, or the Preservation of Favoured Races in the Struggle for Life (hereafter referred to as On the Origin of Species) sums this very effectively: “There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that whilst this planet has gone cycling on according to the fixed laws of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved”. Darwin’s ‘view of life’ as a conceptual space can be interpreted as consisting of three dimensions: explanatory pattern, levels of selection, and degree of difference among units of the same type. Each respective dimension is defined by a pair of poles: law and narrative explanations, organismic and hierarchial selection, and variational and essential thinking. Darwin’s integrative theory linking biological structure, history, and function was nursed and reared within this logical space. During the 20th century, the poles of each pair came to be seen as mutually exclusive. Systemic Darwinism, emerging at the beginning of the 20th century, promises to reintegrate Darwin’s view of life. Based on the theories of Darwin and the rapidly increasing documentation of fossil records vis a vis gene sequences, the evolution of algal lineages can provide useful indicators.

Evolution and paleontology of algae From tiny single-celled species one micrometer in diameter to giant seaweeds over 50m long, algae are abundant and ancient organisms which can be found in virtually every ecosystem in the biosphere. Algal taxonomists or phycologists, who specialize in their identification, naming and classification, believe that there exist 36,000–50,000 and possibly more than 10 million species of algae.9 Algae can best be defined as aquatic organisms (with exceptions) that are photosynthetic, oxygenic autotrophs, typically smaller (except kelps and few other genera) and less structurally complex than land plants. This rather inelegant definition allows the inclusion of cyanobacteria and other protists, which are otherwise distinguished by their prokaryotic structure. Fossil records show that algae were the first photosynthetic cellular organisms and all cytogenetic groups of plants and ultimately flowering plants may have arisen from algae. Chloroplasts are the right size to be descended from bacteria, reproduce in the same manner, by binary fission, and have their own genome in the form of a single circular DNA molecule. The enzymes and transport systems found on the folded inner membranes of chloroplasts are similar to those found on the cell membranes of modern cyanobacteria, as are their ribosomes. These similarities between cyanobacteria and chloroplasts suggest an evolutionary link between the two, and can be explained by the theory of endosymbiosis.


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The diversity of modern algal groups, and particularly of their chloroplasts, suggests that these endosymbiotic events were not unusual. Algal lineages: Linking the past with the future The oversimplified version of the story of algal evolution is tied to how they acquired their photosynthetic organelles or plastids. The first plants evolved from the engulfing of a photosynthetic prokaryote by an aerobic eukaryote. From this initial event two major plant groups evolved - the Green Algae and the Red Algae. After this primary endosymbiotic union, secondary and even tertiary endosymbioses occurred - algal cells themselves getting engulfed to give rise to other algal groups (Fig. 1). It is interesting to note that Chlamydia contain several plant-like genes which are attributed to horizontal transfer. On the other, Plasmodium and Trypanosoma represent apicomplexans, with residual or no plastids. The type of chlorophyll pigments together with the common algal carbon reserve materials, form a major basis for classifying algae. However, in recent times, cellular architecture, along with molecular sequences or genome architecture information has been utilized for classifying into several phyla (divisions). Modern algae comprise a range of organisms with very different structures but identical photosynthetic pigments. This suggests that very different host organisms have formed a symbiosis with the same photosynthetic cells. That is, the algal groups must have evolved through separate endosymbiotic events, i.e. polyphyletic, and the group as a whole is identified on the basis of a similar level of structure, rather than on its evolutionary origins.

Figure 1. Schematic outline of endosymbiotic events and evolution of diversity of life

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One of the several recent classification schemes divides algal species among eleven phyla, which includes Cyanophyta and Prochlorophyta, both containing oxygen–producing autotrophic prokaryotes and nine eukaryotic phyla.10 However, phylogenies based upon ribosomal RNA gene sequences suggest that the chlorophyll a and b are distributed among cyanobacterial taxa, rather than representing a coherent group, which suggests that, they should not be separated into different phyla.11 Based on monophyly (descent from a single ancestor) and molecular evidence, nine phyla have been distinguished – Cyanobacteria Glaucophyta, Euglenophyta, Cryptophyta, Haptophyta, Dinophyta, Ochrophyta, Rhodophyta and Chlorophyta. Modern ultrastructural and molecular studies have provided important information that has led to a reassessment of the evolution of algae.12 In addition, the dearth of fossil records for some groups of algae has hindered evolutionary studies, and the realization that some algae are more closely related to protozoa or fungi than they are to other algae came late, producing confusion in evolutionary thought and delays in understanding the evolution of the algae. Fossils of eukaryotes that resemble living brown algae have been found in sedimentary rocks from China that are 1700 million years old, while possibly the oldest photosynthetic eukaryote Grypania, and come from rocks 2100 million years old. Stromatolites, which are formed by cyanobacteria, provide living and fossil evidence of cyanobacteria going back 2700 million years. The order of algae with the best fossil record are the Dasycladales, which are calcified unicellular forms of elegant construction dating back at least to the Triassic Period. Some scientists consider the red algae, which bear little resemblance to any other group of organisms, to be very primitive eukaryotes that evolved from the prokaryotic blue-green algae.13 Evidence in support of this view includes the nearly identical photosynthetic pigments and the very similar starches among the red algae and the blue-green algae. Many scientists, however, attribute the similarity to an endosymbiotic origin of the red algal chloroplast from a blue-green algal symbiont. Other scientists suggest that the red algae evolved from the Cryptophyceae, with the loss of flagella, or from fungi by obtaining a chloroplast. In support of this view are similarities in mitosis and in cell wall plugs, special structures inserted into holes in the cell walls that interconnect cells. Some evidence suggests that such plugs regulate the intercellular movement of solutes. Ribosomal gene sequence data from studies in molecular biology suggest that the red algae arose along with animal, fungal, and green plant lineages.14 The fossil record for the algae is not nearly as complete as it is for land plants and animals. Red algal fossils are the oldest known algal fossils. Microscopic spherical algae (Eosphaera and Huroniospora) that resemble the living genus Porphyridium are known from the Gunflint Iron Formation of North America (formed about 1.9 billion years ago). Fossils that resemble modern tetraspores


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are known from the Amelia Dolomites of Australia (formed some 1.5 billion years ago). The best characterized fossils are the coralline red algae represented in fossil beds since the Precambrian time. Fossil Dinophyceae date from the Silurian Period (430 million years ago). Some workers consider at least a portion of the acritarchs, a group of cyst like fossils of unknown affinity, to be Dinophyceae, but few scientists agree with that view. The acritarchs occurred as early as 700 million years ago. The Chromophyta have the shortest fossil history among the major algal groups. Some scientists believe that the group is ancient, whereas others point out that there is a lack of data to support this view and suggest that the group evolved recently, as indicated by fossil and molecular data. The oldest chromophyte fossils, putative brown algae, are approximately 400 million years old. Coccolithophores, coccolith-bearing members of the Prymnesiophyceae, date from the Late Triassic Epoch (230 to 200 million years ago), with one reported from approximately 280 million years ago. Coccolithophores were extremely abundant during the Mesozoic Era, contributing to deep deposits such as those that constitute the white cliffs of southeast England. Most species became extinct at the end of the Cretaceous Period (65.5 million years ago), along with the dinosaurs, and indeed there are more extinct species of coccolithophores than there are living species. The Chrysophyceae, Bacillariophyceae, and Dictyochophyceae date from about 100 million years ago, and despite the mass extinctions 65.5 million years ago, many species still flourish. In Lompoc, California, US, their siliceous remains have formed deposits of diatomite almost 0.5 km (0.3 mile) in depth, while at Mývatn in Iceland, the lake bottom bears significant amounts of diatomite in the form of diatomaceous ooze, many meters in depth. Some of the green algal classes are also very old. Organic cysts resembling modern Micromonadophyceae cysts date from about 1.2 billion years ago. Tasmanites formed the Permian ‘white coal,’ or tasmanite, deposits of Tasmania and accumulated to a depth of several feet in deposits that extend for miles. Similar deposits in Alaska yield up to 150 gallons of oil per ton of sediment. Certain Ulvophyceae fossils that date from about one billion years ago are abundant in Paleozoic rocks. Some green algae deposit calcium carbonate on their cell walls, and these algae produced extensive limestone formations. The Charophyceae, as represented by the large stoneworts (order Charales), date from about 400 million years ago. The oospore, the fertilized female egg, has spirals on its surface that were imprinted by the spiraling protective cells that surrounded the oospore. Oospores from before about 225 million years ago had right-handed spirals, whereas those formed since that time has had left-handed spirals. The reason for the switch remains a mystery. The green algal classes are evolutionarily related, but their origins are unclear.2 Most consider the class Micromonadophyceae to be the most ancient group, and some fossil data support this view. The class

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Ulvophyceae is also ancient, whereas the classes Charophyceae and Chlorophyceae are more recent. The Euglenophyceae are believed to be an ancient lineage of algae that includes some zooflagellate protozoa, which is supported by ultrastructural and molecular data. Some scientists consider the colourless euglenophytes to be an older group and believe that the chloroplasts were incorporated by symbiogenesis more recently. The class Dinophyceae is of uncertain origin. During the 1960s and ’70s the unusual structure and chemical composition of the nuclear DNA of the Dinophyceae were interpreted as somewhat primitive features. Some scientists even considered the Dinophyceae to be mesokaryotes (intermediate between the prokaryotes and the eukaryotes); however, this view is no longer accepted. Their peculiar structure is considered as a result of evolutionary divergence, perhaps about 300 or 400 million years ago. The Dinophyceae may be distantly related to the chromophyte algae, but ribosomal gene sequence data suggest that their closest living relatives are the ciliated protozoa. It is likely that the Dinophyceae arose from nonphotosynthetic ancestors and that later some species of Dinophyceae adopted chloroplasts by symbiogenesis and thereby became capable of photosynthesis, although many of these organisms still retain the ability to ingest solid food, similar to protozoa. The origin of the chromophyte algae also remains unknown. Ultrastructural and molecular data suggest that they are in a protistan lineage that diverged from the protozoa and aquatic fungi about 300 to 400 million years ago. At that time, chloroplasts were incorporated, originally as endosymbionts, and since then the many chromophyte groups have been evolving. Fossil, ultrastructural, and ribosomal gene sequence data support this hypothesis. The Cryptophyceae are an evolutionary enigma. They have no fossil record, and phylogenetic data are conflicting. Although some workers align them near the red algae, because both groups possess phycobiliproteins in their chloroplasts, most scientists suggest that independent symbiotic origins for the red or blue colour of their chloroplasts could explain the similarity. Cryptophytes have flagellar hairs and other flagellar features that resemble those of the chromophyte algae; however, the mitochondrial structure and other ultrastructural features are distinct and argue against such a relationship. The Xanthophyceae may be even more recent, with fossils dating from about 20 million years ago, while fossil records of the remaining groups of algae, notably the Euglenophyceae and the Cryptophyceae, which lack mineralized walls, are negligible. Owing to their long history, cyanobacteria constitute an unusually diverse and heterogeneous group of prokaryotes, yet they form a coherent systematic group, which is distinct from other bacteria in many features. Cyanobacteria share a close evolutionary relationship with eukaryotes as they have the same photosynthetic


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pigments as the chloroplasts of algae and land plants. They comprise a remarkable group of organisms, whose ubiquity, antiquity and adaptability is unparalleled in biology. Their diverse morphologies coupled with independence for carbon, and nitrogen (in many forms), and resilience to extreme environmental conditions, underlies their widespread significance in the environment and in the evolution of life. These structurally simple organisms combine in themselves interesting facets of plant and bacterial metabolism, which is amenable to genetic exploitation, especially in enhancing our knowledge regarding phylogenetic and evolutionary interrelationships among prokaryotes and eukaryotes, and thereby evolution of life on this planet. Cyanobacteria are found everywhere - in marine, freshwater and terrestrial environments and as symbionts e.g. lichen - and contribute up to 50% of the atmosphere’s oxygen. To date, 17 cyanobacterial total genome-sequencing projects have been completed or are still in progress. From the 11 total cyanobacterial genome projects for which information is available, five were on marine cyanobacteria - and four of these were for extremely similar species - the Prochlorococcus, Synechococcus group of marine pico-phytoplankton. Such analyses have provided valuable information about the dynamics of microbial and plant genome evolution, biochemistry, physiology and ecology of photoautotrophic organisms. Cyanobacterial genomes reveal a complex evolutionary history, which cannot be represented by single strictly bifurcating tree for all genes, with all genes, belonging to any functional groups subject to transfer, without any specific bias. Interestingly, the proportion of metabolic gene transfers in interphylum transfers are more as compared to informational genes. Estimates have shown that about 4500 (18%) of all nuclear genes of extant land plants could derive from the cyanobacterial endosymbionts.7 The first complete genome sequences of marine cyanobacteria indicate unprecedented flexibility and dynamics and created a new level of insight into the ecology and molecular biology of these organisms.15 In this context, it may be interesting to re-visit the endosymbiotic theory in order to understand the evolution of plants from algae.

Endosymbiosis Chloroplasts, the sites of photosynthesis within plant cells, comprise a prominent and well-known class of plastids, sub cellular organelles with diverse, specialist functions in plant and algal cells.16 Mereschkowsky’s 1905 hypothesis which was initially greeted with skepticism or even derision, gained support from electron microscopical and biochemical studies which showed that plastids contain DNA, RNA and ribosomes, supplying a structural and biochemical basis for nonMendelian, cytoplasmic inheritance of plastid-related characters. Subsequent

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molecular genetic studies have demonstrated the ubiquity of plastid genomes and confirmed that their replication, transcription and translation closely resemble those of (eu) bacteria.17 The hypothesis that certain organelles of eukaryotic cells, in particular, the plant chloroplasts, evolved from bacteria is not that much younger than the Origin (Darwin 1895). It was proposed by several researchers in the late 19th century on the basis of microscopic study of plant cells that revealed conspicuous structural similarity between chloroplasts and cyanobacteria (then known as blue green algae) and was presented in a coherent form by Mereschkowsky in the beginning of 20th century. For the first two-thirds of the 20th century, this hypothesis of endosymbiosis remained a fringe speculation. However, this perception changed shortly after the appearance of the seminal 1967 publication of Sagan (Margulis) who summarized the available data on the similarity between certain organelles and bacteria, in particular, the striking discovery of organellar genomes, and came to the conclusion that not only chloroplasts but also the mitochondria evolved from endosymbiotic bacteria. Subsequent work, in particular, phylogenetic analysis of both genes contained in the mitochondrial genome and genes encoding proteins that function in the mitochondria and apparently were transferred form the mitochondrial to the nuclear genome, turned the endosymbiosis hypothesis into a well established fact. Moreover, these phylogenetic studies convincingly demonstrated the origin of mitochondria from a particular group of bacteria, the ά-proteobacteria. The major evolutionary role assigned to effectively unique events like endosymbiosis is, of course, incompatible with both gradualism and uniformitarianism.18,19 Chloroplasts are descendents of a cyanobacterial endosymbiont, but many chloroplast protein genes of endosymbiont origin are encoded by the nucleus.20 The chloroplast–cyanobacteria relationship is a typical target of orthogenomics, an analytical method that focuses on the relationship of orthologous genes. A pilot study of functional orthogenomics, combining bioinformatic and experimental analyses, was undertaken by Masayuki et al.21 to identify nuclear-encoded chloroplast proteins of endosymbiont origin (CPRENDOs). Phylogenetic profiling based on complete clustering of all proteins in 17 organisms, including eight cyanobacteria and two photosynthetic eukaryotes, was used to deduce 65 protein groups that are conserved in all oxygenic autotrophs analyzed but not in non-oxygenic organisms. With the exception of 28 well-characterized protein groups, 56 Arabidopsis proteins and 43 Synechocystis proteins in the 37 conserved homolog groups were analyzed. Green fluorescent protein (GFP) targeting experiments indicated that 54 Arabidopsis proteins were targeted to plastids. Expression of 39 Arabidopsis genes was promoted by light. Among the 40 disruptants of Synechocystis, 22 showed phenotypes related to photosynthesis. Arabidopsis mutants in 21 groups, including those reported previously, showed


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phenotypes. Characteristics of pulse amplitude modulation fluorescence were markedly different in corresponding mutants of Arabidopsis and Synechocystis in most cases, which led to the conclusion that physiological functions of orthologous genes may be different in chloroplasts and cyanobacteria. The gene sequences of the enzymes involved in the major steps of the carotenoid biosynthetic pathway in algae (Cyanobacteria, Rhodophyta and Chlorophyta) have been analyzed. Phylogenetic relationships among proteincoding DNA sequences were reconstructed by neighbor-joining (NJ) analysis for the respective carotenoid biosynthetic pathway genes (crt) in algae.22 The phylogenetic trees showed that the taxa of some genera have a closer evolutionary relationship with other genera in some gene sequences, which suggests a common ancient origin and that lateral gene transfer has occurred among unrelated genera. The phylogenetic analysis also revealed that lateral gene transfer may have taken place across algal genomes and the dN values suggest that most of the early crt genes are well conserved compared to the later crt genes.23 It is well established that the modern day chloroplasts were once free living cyanobacteria that became endosymbionts but molecular studies have yet to link plastids robustly with any particular group of contemporary cyanobacteria, leaving the precise lineage of cyanobacteria that gave rise to plastids unknown. Previous work has shown that many gene transfers to the nucleus have occurred during plastid evolution but estimates for the total number of genes that were transferred have been elusive. But the genomes of contemporary plastids encode only 5–10% as many genes as those of their free living cousins, indicating that many genes were either lost from plastids or transferred to the nucleus during the course of plant evolution.24,25 A comparison of 24,990 proteins encoded in the Arabidopsis genome to the proteins from three cyanobacterial genomes, 16 other prokaryotic reference genomes, and yeast was undertaken. Of 9,368 Arabidopsis proteins sufficiently conserved for primary sequence comparison, 866 detected homologues only among cyanobacteria and 834 other branched with cyanobacterial homologues in phylogenetic trees. Extrapolating from these conserved proteins to the whole genome, the data suggest that ≈ 4,500 of Arabidopsis protein-coding genes (= 18% of the total) were acquired from the cyanobacterial ancestor of plastids. These proteins encompassed all functional classes, and the majority of them are targeted to cell compartments other than the chloroplast. Phylogenetic analysis of proteins from Arabidopsis, three cyanobacterial genomes [Synechocystis sp. PCC6803, Prochlorococcus marinus, Nostoc punctiforme] other prokaryotic reference genomes and yeast, in addition to the phylogeny of 15 sequenced chloroplast genomes provided interesting information.24 Previous estimates have suggested that between 800 and perhaps as many as 2,000 genes in the Arabidopsis genome might come from cyanobacteria, but genome wide phylogenetic surveys that could provide direct estimates of this

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number are lacking.26,27 Analysis of 15 sequenced chloroplast genomes revealed 117 nuclear-encoded proteins are also still present in at least one chloroplast genome.24 A phylogeny of chloroplast genomes inferred from 41 proteins and 8,303 amino acid sites indicated that at least two independent secondary endosymbiotic events have occurred involving red algae and that amino acid composition bias in chloroplast proteins strongly affects plastid genome phylogeny. Therefore, it was concluded that the evolutionary process that transformed the cyanobacterial symbiont into a contemporary organelle involved both inheritance and invention. Such inheritances included photosynthesis, 70S ribosomes, cell division proteins, and, in some primitive plastids, a peptidoglycan wall. Important inventions included the protein import machinery, which permits the plastid to import nuclear-encoded proteins and hence to donate genes to the nucleus over evolutionary time. Contemporary chloroplast genomes encode between 60–200 proteins in various photosynthetic lineages and have thus undergone a process of severe genome reduction during the course of endosymbiosis, because contemporary cyanobacteria encode several thousand proteins. But plastids contain roughly just as many proteins as their free-living cyanobacterial cousins, current estimates suggesting that between 1,000 and 5,000 proteins in higher plants are targeted to plastids.

Evolutionary biology in the age of genomics The collection of completely sequenced genomes that is available on Darwin’s 200th anniversary consists of thousands of viral genomes, close to 1000 genomes of bacteria and Archaea, and close to 100 eukaryotic genomes.8,28,29 The importance of massive amounts of sequences for comparison is obvious because this material allows researchers to investigate mechanisms and specific events of evolution with the necessary statistical rigor and to reveal even subtle evolutionary trends. In addition, it is worth emphasizing that collections of diverse complete genomes are enormously useful beyond the sheer amount of sequence data. Although, certainly not all major taxa are adequately represented, this rapidly growing collection increasingly satisfies the demands of both micro evolutionary and macro evolutionary research. Complementary to the advances of traditional genomics is the more recent accumulation of extensive metagenomic data. Although metagenomics typically does not yield complete genomes, it provides invaluable information on the diversity of life in various environments and a means to understand life as it evolved. Horizontal gene transfer, the network of evolution and the forest replacing of the TOL (Tree of Life) Even long before the genomic era, microbiologists realized that bacteria had the capacity to exchange genetic information via horizontal gene transfer (HGT), in


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some cases, producing outcomes of major importance, such as antibiotic resistance (Raymond and Blankenship, 2003).30 Multiple molecular mechanisms of HGT have been elucidated including plasmid exchange, transduction (HGT mediated by bacteriophages) and transformation. These discoveries not withstanding, HGT was generally viewed as a minor phenomenon that is important only under special circumstances and, in any case, was not considered to jeopardize the concept of the TOL that could be reconstructed by phylogenetic analysis of rRNA and other conserved genes. This fundamental belief was challenged by early results of genome comparisons of bacteria and Archaea which indicated that, at least, in some prokaryotic genomes, a major fraction of genes were acquired via demonstrable HGT. The pathogenicity islands and similar symbiosis islands that comprise over 30% of the genome in many pathogenic and symbiotic bacteria are the prime case in point.31 Moreover, comparative analysis of the genomes of hyperthermophilic bacteria and Archaea suggested that even interdomain HGT can be extensive given shared habitats. The rate of HGT substantially differs from different genes depending on the gene functions, in part, according to the so called complexity hypothesis which posits that barriers might exist for HGT of genes encoding subunits of protein complexes because dosage imbalance and mixing of heterologous subunits resulting from such events could be deleterious. However, phylogenetic analyses indicate that even such genes, for instance, those for ribosomal proteins and RNA polymerase subunits, are not immune to HGT. The high prevalence of HGT in prokaryotes might, in part, explain the persistence of the organization of many operon hypotheses. This presents a notable case of a combination of selective (co regulation) and neutral (HGT) forces contributing to the evolution of a major aspect of genome organization. Eukaryotes are different from prokaryotes with respect to the role played by HGT in genome evolution. In multicellular eukaryotes, where germ line cells are distinct from the soma, HGT appears to be rare although not impossible. Under certain special circumstances, such as persistence of endosymbiotic bacteria in animals, transfer of large segments of bacterial genomes to the genome of the host is indeed common. Unicellular eukaryotes do seem to acquire bacterial genes between themselves on relatively frequent occasions. Far more crucial, however, is the major contribution of the genomes of the endosymbionts to the gene complements of all the eukaryotes.32 The discovery of mitochondria –like organelles and genes of apparent mitochondrial origin in all thoroughly characterized unicellular eukaryotes, essentially, ascertain that the last common ancestor of the extant eukaryotes already possessed the mitochondrial endosymbiont. In terms of their apparent phylogenetic affinities, eukaryotic genes that possess readily identifiable prokaryotic orthologs are sharply split into genes of likely archaeal origin (primarily, but not exclusively, components of information processing systems) and those of likely bacterial origin (mostly, metabolic enzymes and

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components of various cellular structures). It is often assumed on general grounds that the majority of ancestral ‘bacterial’ genes in eukaryotes are of mitochondrial origin but this is hard to demonstrate directly because in phylogenetic analysis, these genes cluster with diverse groups of bacteria. The symbiogenetic scenario according to which the α proteobacteria, ancestor of mitochondria invaded an archaeal host, and this event triggered eukaryogenesis including the formation of the signature structural features of the eukaryotic cells such as the endomembrane system, the cytoskeleton and the nucleus may be of relevance. Regardless of the exact role played by eukaryogenesis, there is no reasonable doubt that the gene complement of the eukaryotes is a chimera comprised of functionally distinct genes of archaeal and bacterial descents.28 Moreover, endosymbiosis apparently made substantial contributions to the gene complements of some of the individual major groups of eukaryotes. Thus, strong evidence was presented of massive HGT of thousands of genes from a cyanobacterial endosymbiont (the chloroplast) to the host (plant) genomes. Similarly, genes of apparent algal origin were detected in chromalveolates that engulfed a red alga in an act of secondary endosymbiosis.31 The observations of HGT as extensive, ubiquitous and occurring via multiple routes outline above lead to a fundamental generalization: the genomes of all life forms are collections of genes with diverse evolutionary histories. The corollary of this generalization is that the TOL concept must be substantially revised or abandoned because a single tree topology or even congruent topologies of trees for several highly conserved genes cannot possibly represent the history of all or even the majority of genes. Certainly, this conclusion is not to be taken as an indication that the concept of evolutionary tree introduced by Darwin4 should be abandoned altogether. First, trees have the potential to accurately represent the evolution of individual gene families. Secondly, there exist, beyond doubt, expansive part of life’s history for which congruent trees can be obtained for large sets of orthologous genes, and accordingly, the consensus topology of these tree qualifies as a species tree. Evolution of major groups of eukaryotes, such as animals or plants, is the most obvious case in point but tree like evolution seems to apply also to many groups of prokaryotes at relatively shallow phylogenetic depths. Evolution of life in its entirety is best depicted as: (i) A consensus tree of highly conserved genes that represents a ‘central trend’ in evolution, with HGT events, including massive ones associated with endosymbiosis, comprising horizontal connections between the tree branches (ii) A complex network where phases of tree-like evolution (with horizontal connections) are interspersed with ‘Big Bang’ phases of rampant horizontal exchange of genetic information that cannot be represented as trees in principle. In summary, comparative genomics and metagenomics reveal a vast,


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dynamic, interconnected world of selfish replicons that interact with genomes of cellular life forms and, over long spans of evolution, makes major contributions to the composition of chromosomes. In prokaryotes, the interaction between the bacterial and archaeal chromosomes and selfish replicons is so intensive, and the distinction between chromosomes and mega plasmids is blurred to such an extent that chromosomes are, probably, best viewed as ‘islands’ of relative stability in the turbulent ‘sea’ of mobile elements. In eukaryotes, especially, in multicellular forms that evolved the separation between the germ line and soma, the distinction between chromosomes and selfish replicons is sharper. Nevertheless, intragenomic mobility of selfish transposable elements is extensive, and intergenomic mobility, at least, within a species, is actually facilitated by sex, with bursts of transposable element propagation likely marking evolutionary transitions. The central role of mobile elements in genome evolution further undermines the TOL concept, although phylogenetic trees of individual hallmark genes can be highly informative for the reconstruction of the evolution of the selfish elements themselves. Modern genetic studies of plastid genomes have revealed that they encode only 60–200 proteins, while more than 5000 nuclear encoded gene products are targeted to plastids, and many chloroplast lacking genomes also reveal a great degree of similarity in their genome architecture.24,33 Therefore, the cyanobacteria have much greater implications in evolution, beyond plants. Conservation vis-a-vis fluidity of genomes A large body of information has been generated on the evolutionary conservation of gene sequences and structure versus the fluidity of gene composition and genome architecture. A fundamental observation supported by the entire body of evidence amassed by evolutionary genomics is that the sequences and structures of genes encoding proteins and structural RNAs are, generally, highly conserved through vast evolutionary spans. With the present collection of sequenced genomes, orthologs in distant taxa are found for the substantial majority of proteins encoded in each genome. For instance, recent genome sequencing of primitive animals, sea anemone and Trichoplax, revealed extensive conservation of the gene repertoire compared to mammals or birds, with the implication that the characteristic life span of an animal gene includes (at least) hundred millions of years. The results of extensive comparative analysis of plant, fungal and prokaryotic genomes are fully compatible with this conclusion.30 The striking conservation of gene sequences and structures contrasts the fluidity of the gene composition of genomes of all forms of life that is revealed by comparative genomics and evolutionary reconstruction. The (nearly) universal genes make up but a tiny fraction of the entire gene universe: altogether, this central core of cellular life consists of, at most, –70 genes that is, no more than 10% of the genes in even the smallest of the genomes of cellular life forms, but typically, closer to 1% of the genes or

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less. Although in each individual genome, the majority of the genes belong to a moderately conserved genetic ‘shell’ that is shared with distantly related organisms, within the entire gene universe, the core and shell genes (or more precisely, set of orthologous genes) are a small minority.34 Given this distinctive structure of the gene universe, evolutionary reconstruction inevitably yield a dynamic picture of genome evolution, with numerous genes lost and many others gained via HGT (mostly in prokaryotes), and gene duplication.35 Gene and genome duplication: The principal route of genomic innovation Gene duplications occurs throughout the evolution of any lineage but the rate of duplication is not uniform on large evolutionary scales, so that organizational transitions in evolution seem to be accompanied by bursts of gene duplication, conceivably, enabled by weak purifying selection during population bottlenecks. Perhaps, the most illustrative case in point is the emergence of eukaryotes that was accompanied by a wave of massive duplication, yielding the characteristic many-to-one co-orthologous relationship between eukaryotic genes and their prokaryotic ancestors. At the level of general concepts of evolutionary biology primarily concerned here, genomic studies on gene duplication lead to, at least, two substantial generalizations. First, the demonstration of the primary evolutionary significance of duplications including duplications of large genomes regions and whole genomes is a virtual death knell for Darwinian gradualism: even single gene duplication hardly qualifies as an infinitesimally small variation where WGD qualifies as a bonafide salutatory event. Secondly, the primacy of gene duplication with the subsequent (sometimes, rapid) diversification of the paralogs as the route of novel gene origin reinforces the metaphor of evolution as tinkerer: evolution clearly tends to generate new functional devices by tinkering with the old ones after making a backup copy rather than create novelty from scratch. Genome-wide quantification of selection and junk DNA: Distinct evolutionary regimes for different genomes There are major differences in the genome layouts between different lines of life evolution. Prokaryotes and, especially, viruses have ‘wall-to-wall’ genomes that consist, mainly, of genes encoding proteins and structural RNAs, with non coding regions comprising, with a few exceptions, no more than 10–15% of the genomic DNA. The genomes of unicellular eukaryotes have lower characteristic gene densities but, on the whole, do not depart too far from the prokaryotic principles, with most of the DNA dedicated to protein-coding, despite the distinct, exon-intron gene architecture. The genomes of multicellular eukaryotes are drastically different in that only a minority (a small minority in vertebrates) of the genomic DNA is comprised of sequences encoding proteins or structural


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RNAs. Generally, across the entire range of life forms, there is a notable negative exponential dependence between the density of protein-coding genes and genome size although significant deviations from this overall dependence are seen as well, particularly, in prokaryotes. Evolutionary genomics effectively demolished the straightforward concept of the TOL by revealing the dynamic, reticulated character of evolution where HGT, genome fusion, and interaction between genomes of cellular life forms and diverse selfish genetic elements take the central stage. In this dynamic worldview, each genome is a palimpsest, a diverse collection of genes with different evolutionary fates and widely varying likelihoods of being lost, transferred, or duplicated. So the TOL becomes a network, or perhaps, most appropriately, the Forest of Life that consists of trees, bushes, thickets of lianas, and of course, numerous dead trunks and branches. Whether the TOL can be salvaged as central trend in the evolution of multiple conserved genes or this concept should be squarely abandoned for the Forest of Life image remains as an open question. All the classical concepts have undergone transformation, turning into much more complex, pluralistic characterizations of the evolutionary process. Depicting the change in the widest strokes possible, Darwin’s paramount insight on the interplay between chance and order (introduced by natural selection) survived, even if in a new, much more complex and nuanced form, with specific contributions of different types of random processes and distinct types of selection. By contrast, the insistence on adaptation being the primary mode of evolution that is apparent in the Origin, but especially in the Modern Synthesis, became deeply suspicious if not outright obsolete; making room for a new worldview that gives much more prominence to non-adaptive process. The variability of the genome architectures presents an interesting dilemma to evolutionary biologists: do organisms possess unique genome architectures that are specifically adapted to satisfy unique functional demands of the respective organisms, or is evolution of genome architecture a mostly neutral process? Although local clustering of functionally related genes and other patterns suggestive of functionally relevant gene co-expression were repeatedly observed, these trends are relatively weak and by no means ubiquitous. Thus, the dominant factor in the evolution of genome architecture appears to be random, non-adaptive rearrangement rather than purifying or positive selection.

Conclusions The fundamental principles of molecular evolution have been established, and many specific observations of major importance and impact on the fundamentals of neo-Darwinism were made in the pre-genomic era, the rRNA based phylogeny being the premier case in point. However, the advent of full fledged genome

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sequencing quantitatively changed the entire enterprise of evolutionary biology and the availability of the treasure trove of genomic, metagenomic and post –genomic data has much altered our evolutionary concepts, especially in relation to Darwinism; and in some cases served to strengthen the significance of his views. The species-individual has emerged, rather than the organism, as a Darwinian interactor in selection at its own level, operating largely with cross-level exaptations arising from unsuppressed evolution of subparts (primarily organisms) at lower levels within itself. Such nonsuppression acts as a source of power by permitting species to draw upon a wider pool of features than organisms can access (for the organismic style of individuality enjoins active suppression of most selection at lower levels within itself). This can have wide ranging implications in understanding the evolution of land plants from algal ancestors. Beyond genomics and metagenomics, one of the hallmarks of the first decade of the new millennium is the progress of research in functional genomics and systems biology. These fields now yield high quality, genome-wide data on gene expression, genetic and protein-protein interactions, protein localization within cells, and more, opening new dimensions of evolutionary analysis, what is sometimes called Evolutionary Systems Biology. This new field of research has the potential to insights into the genome-wide connections between sequence evolution and other variables, such as the rate of expression, and often illuminate the selective and neutral components of the evolution of these aspects of genome functioning and the next decade may prove the most exciting in terms of evolutionary biology. The majority of the sequences in all genomes evolves under the pressure of purifying selection or, in organisms with the largest genomes, neutrally, with only a small fraction of mutations actually being beneficially and fixed by natural selection as envisioned by Darwin. Furthermore, the relative contributions of different evolutionary forces greatly vary between organismal lineages, primarily, owing to differences in population structure. Two centuries after Darwin’s birth, 150 years after the publication of his ‘Origin of Species’ and 50 years after the consolidation of the Modern Synthesis, comparative analysis of hundreds of genomes from many diverse taxa offers unprecedented opportunities for testing the conjecture of (neo) Darwinism and deciphering the mechanisms of evolution. Comparative genomics revealed a striking diversity of evolutionary processes that was unimaginable in the pre-genomic era. In addition to point mutations that can be equated with Darwin’s ‘infinitesimal changes’, genome evolution involves major contributions from gene and whole genome duplications, large deletions including loss of genes and entire genomic regions, various types of genome rearrangements, and interaction between genomes of cellular life forms and diverse selfish genetic elements. The emerging landscape of genome evolution includes the classic, Darwinian natural


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selection as an important component but is by far more pluralistic and complex than entailed by Darwin’s straightforward vision that was solidified in the Modern Synthesis. The key role played by algae, particularly cyanobacteria has been beyond doubt established in the mosaic of plant genomes. As Raven and Allen16 pointed out ‘What have the cyanobacteria not done for plants’ which implies subtly the significance of algae in the evolution and sustenance of life on Earth. Acknowledgements: The authors thank the ICAR funded projects and authorities of the Division of Microbiology, IARI, New Delhi, for providing necessary facilities for undertaking this study.

References 1.

Prasanna R, Kaushik BD (2005) Algal diversity from morphology to molecules. In: Microbial Diversity: Current Perspectives and Potential Applications. Satyanarayana T, Johri BN (Eds.), IK International, New Delhi

2.

Darley WM (1982) In: Algal Biology: A Physiological Approach. Blackwell Scientific, Oxford, UK

3.

Borowitzka MA (1998) Tubular photo bioreactors for large-scale algal culture. In: Cyanobacterial Biotechnology. Subramanian G, Kaushik BD, Venkatamaran GS (Eds.), Oxford & IBH Publishing Co., New Delhi

4.

Darwin C (1895) In: On the Origin of Species. Murray, London

5.

Pace NR (1997) A molecular view of microbial diversity and the biosphere. Science 276:734–740

6.

Gould SJ, Lloyd EA (1999) Individuality and adaptation across levels of selection: How shall we name and generalize the unit of Darwinism. Proc Nat Acad Sci USA 96:11904–11909

7.

Tomitani A, Knoll AH, Cavanaugh CM, Ohno T (2006) The evolutionary diversification of cyanobacteria: Molecular-phylogenetic and paleontological perspectives. Proc Nat Acad Sci USA 103:5442–5447

8.

Winther RG (2008) Systemic Darwinism. Proc Nat Acad Sci USA 105:11833–11838

9.

John DM, Maggs CA (1997) In: Species: The Units of Biodiversity. Claridge MF, Dawah HA, Wilson MR (Eds.), Chapman and Hall, London

10.

Hoek van den C, Mann DG, Jahns HM (1995) In: Algae: An introduction to Phycology. Cambridge University Press, Cambridge

11.

Ali AB, Baere RD, Auwera GVD, Wachter RD, Peer YVD (2001) Phylogenetic relationships among algae based on complete large-subunit rRNA sequences. Int J Syst Evolu Microbiol 51:737–749

12.

Koonin EV, Mushegian AR, Rudd KE (1996) Sequencing and analysis of bacterial genomes. Curr Biol 6:404–416

13.

Nozaki H, Matsuzaki M, Misumi O, Kuroiwa H, Hasegawa, M, Higashiyama T, Shin-I T, Kohara Y, Ogasawara N, Kuroiwa T (2004) Cyanobacterial genes transmitted to the nucleus before divergence of red algae in the chromista. J Molecul Evolut 59:103–113

14.

Kumar S, Rzhetsky A (1996) Evolutionary relationships of Eukaryotic kingdoms. J Molecul Evolut 42:183–193

15.

Shi T, Falkowski PG (2008) Genome evolution in cyanobacteria: The stable core and the variable shell. Proc Nat Acad Sci USA 105:2510–2515

140


16.

Raven JA, Douglas AE (2003) Genomes at the interface between bacteria and organelles. Proc R Soc Lond B Biol Sci 358:5–18 Mereschkowsky C (1905) Uber Natur und Ursprung der Chromatophoren im Pflanzenreiche. Biologia Centralbl 25:593–604 Woese CR, Fox GE (1977) Phylogenetic structure of the prokaryotic domain: The primary kingdoms. Proc Nat Acad Sci USA 74:5088–5090 Woese CR (1987) Bacterial evolution. Microbiol Rev 51:221–271 Gray MW (1992) The endosymbiont hypothesis revisited. Int Rev Cytol 141:233–357 Masayuki I, Makoto F, Kintake S, Naoki S (2009) Orthogenomics of photosynthetic organisms: Bioinformatic and experimental analysis of chloroplast proteins of endosymbiont origin in arabidopsis and their counterparts in synechocystis. Plant Cell Physiol 50(4): 773–788 Liang C, Zhao F, Wei W, Wen Z, Qin S (2006) Carotenoid biosynthesis in cyanobacteria: Structural and evolutionary scenarios based on comparative genomics. Int J Biol Sci 2(4):197–207 Ochman H, Lawrence JG, Groisman EA (2000) Lateral gene transfer and the nature of bacterial innovation. Nature 405:299–304 Martin W, Rujan T, Richly E, Hansen A, Cornelsen S, Lins T, Leister D, Stoebe B, Hasegawa M , Penny D (2002) Evolutionary analysis of Arabidopsis, cyanobacterial and chloroplast genomes reveals plastid phylogeny and thousands of cyanobacterial genes in the nucleus. Proc Nat Acad Sci USA 99:12246–12251 Mulkidjanian AY, Koonin EV, Makarova KS, Mekhedov SL, Sorokin A, Wolf YI, Dufresne A, Partensky F, Burd , Kaznadzey D, Haselkorn R, Galperin Y (2006) The cyanobacterial genome core and the origin of photosynthesis. Proc Nat Acad Sci USA 103:13126–13131 Lopez-Juez E (2007) Plastid biogenesis, between light and shadows. J Exper Bot 58: 11–26 Zhang Y, Chen M, Zhou BB, Jerminn LS, Larkum WD (2007) Evolution of the inner light-harvesting antenna protein family of cyanobacteria, algae, and plants. J Mol Evol 64:321–331 Dagan T, Martin W (2006) Getting a picture of microbial evolution en route to a network of genomes. Philosophical Transactions of the Royal Society (London). B Biological Sciences 364:2187–2196 Koonin EV (2009) Darwinian evolution in light of genomics. Nucleic Acids Res 37: 1011–1034 Raymond J, Blankenship RE (2003) Horizontal gene transfer in eukaryotic algal evolution. Proc Nat Acad Sci USA 100:7419–7420 Gogarten JP, Townsend JP (2005) Horizontal gene transfer, genome innovation and evolution. Nat Rev Microbiol 3:679–687 Palenik B (2002) The genomics of symbiosis: Hosts keep the baby and the bath water. Proc Nat Acad Sci USA 99:11996–11997 Raven JA, Allen JF (2003) Genomics and chloroplast evolution: What did cyanobacteria do for plants? Genome Biol 4:209 Zhaxybayeva O, Gogarten JP, Charlebois RL, Doolittle WF, Papke T (2006) Phylogenetic analyses of cyanobacterial genomes: Quantification of horizontal gene transfer events. Genome Res 16:1099–1108 Lynch M (2007) In: The Origin of Genome Architecture. Sinauer Associates, Sunderland, MA

17. 18. 19. 20. 21.

22.

23. 24.

25.

26. 27.

28.

29. 30. 31. 32. 33. 34.

35.

10 Biodiversity, phylogeny and evolution of fungi C. Manoharachary*, I.K. Kunwar** and S. Vishnuvardhan Reddy*** Botany Department, Osmania University Hyderabad - 500 007, India *[email protected], **[email protected], ***[email protected]

Abstract: India is rich in biodiversity. One-third of fungal diversity of the globe exists in India. The early fossil record of the fungi is poor, as unlike other organisms, fungal structures do not fossilize well. Thus, theories on phylogeny of fungi are based on the morphological features of the extant fungi. From the beginning of the 20th century fungi were proposed to be monophyletic, assuming that all the fungi were derived from an algal ancestor that lost its ability to photosynthesize. This gave rise to the flagellate fungi, from which rest of the fungi evolved. The loss of flagella and the evolution of zygospore gave rise to the Zygomycotina. The uninucleate zygospores of the Edogonales gave rise to the Ascomycota. The link between them being a fungus resembling Dipodascopsis (Ascomycota). The unicellular yeasts and complex filamentous Ascomycota members having extended dikaryotic stage evolved from it, believed to be similar to modern day Taphrina. This Taphrina-like ancestor was believed to have given rise to the ancestral Basidiomycota. Monophyletic origin of fungi was followed by most of the mycologists till 1960s. But, some mycologists proposed polyphyletic origin, with red algae as origin of Ascomycota. In late 1960s Oomycota was separated from the fungi. Slime molds were also separated into a different kingdom as well. Fungi were separated into two kingdoms: All the flagellate fungi were placed in kingdom Protista (including Chytridiomycota and slime molds) and the remainder in kingdom Myceteae. Hypothesis of phylogeny of fungi has changed radically with the advent of molecular techniques, ultrastructural and biochemical studies. On the basis of these studies in the late 1980s chytridiomycetes, zygomycetes, ascomycetes and basidiomycetes were included in kingdom Fungi. Oomycota, hyphochytrids, labyrinthulids, thraustochytrids and slime molds were accommodated in pseudo fungi. In 2007 a new classification of kingdom fungi-based on recent molecular phylogenetic analysis and morphotaxonomy was proposed, having one subkingdom – Dikarya and seven phyla. Recently in the 10th edition of Dictionary of the Fungi (2008) three kingdoms are accepted viz. Chromista, Fungi and Protozoa. True fungi belong to kingdom fungi having six phyla – Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, Microsporidia and Zygomycota. The review contributes to the understanding of phylogenetic hypotheses, evolutionary relationships and circumscription of the fungi. Keywords: Origin, Eukaryotes, Monophyletic, Polyphyletic, Molecular characters V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Introduction Fungi are highly specialized, non-chlorophyllous, heterotrophic, eukaryotic organisms with cell wall made up of chitin and β-glucans, nutrition absorptive, the absence of chlorophyll has enforced them to become saprophytes, parasites and symbionts. Thallus holocarpic or eucarpic, reproduce both asexually and sexually by producing spores characteristic of each group. The sexually produced fruit bodies are formed both in Ascomycotina and Basidiomycotina. Macroscopic fruit bodies are formed in mushrooms, puff balls, stink horns, bracket fungi, etc. Some fungi like mycorrhiza enter into symbiotic relationship with plants. Fungi are ubiquitous and widely distributed in nature. They are found growing on a variety of substrates having some organic matter and moisture. They are widely distributed in water, soil, on decaying plant and animal parts, etc. Fungi colonize, grow, multiply and survive on/in diversified habitats including extreme environments. Fungi are not only beautiful, but play a significant role in the daily life of human beings besides their utilization in industry, agriculture, medicine, food industry, textiles, bioremediation, natural cycling as biofertilizers and in many other ways. Fungal biotechnology has become an integral part of the human welfare.1 Biodiversity, the extent of biological variation on earth, has come to the fore as a key issue in science and politics since 1990s.2 This subject gained momentum in the 21st century in the light of global gene resources and environmental problems such as global climate change caused by the increase of CO2, loss of biodiversity, loss of tropical rain forests, acid rain, desertification, ozone depletion caused by freon gas and chemical pollution.3

Diversity spectrum The variety and galaxy of fungi and their natural beauty occupy prime place in the biological world and India has been the cradle for such fungi. Only a fraction of total fungal wealth has been subjected to scientific scrutiny and mycologists have to unravel the unexplored and hidden wealth. A conservative estimate provides 1.5 million fungal taxa in the world,2 and among these less than a tenth of the taxa in the kingdom fungi have been discovered.4 Recently Kirk et al.5 in the 10th edition of The Dictionary of Fungi gave a figure of 97,861 species which have been inventoried and described. One-third of fungal diversity of the globe exists in India. The number of fungi recorded in India is around 28,000 species, the largest biotic community after insects.1,6–8 About 350 new genera have been described from India, of which ≈32% were discovered by C.V. Subramanian of the University of Madras.5

Biodiversity, phylogeny and evolution of fungi

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Fossil history and evolution of fungi The fossil record of fungi dates back to the early Proterozoic Eon some 1,200 million years ago. The existence of fossil fungi indicates their evolutionary significance besides solving certain phylogenetic complexities. The early fossil record of the fungi is meager. For much of the Paleozoic Era, the fungi appear to have been aquatic, and consisted of organisms similar to the extant chytrids in having flagellum-bearing spores. The fungi probably colonized the land during the Ordovician. Fungal fossils were common in the early Devonian, when they were abundant in the Rhynie chert. All modern classes of fungi were present in the late Carboniferous (Pennsylvanian Epoch). For some time after the PermianTriassic extinction event, a fungal spike, originally thought to be an extraordinary abundance of fungal spores in sediments formed shortly after this event, suggesting that they were the dominant life form at this time – in nearly 100% of the fossil records available from this period. However, the relative proportion of fungal spores relative to spores formed by algal species is difficult to assess, the spike did not appear worldwide, and in many places it did not fall on the PermianTriassic boundary. Charles Darwin (1809–1882) was an English naturalist whose theory of evolution through natural selection is one of the greatest contributions ever made to science in his book The Origin of Species.9 It forms the basis of modern evolutionary theory. In another book The Descent of Man10 he applied his theory to the evolution of man from a primitive monkey-like animal. Both the books aroused worldwide controversy but, scientists still accept his basic idea. The fungi are of great importance agronomically, bioindustrially, medically and biologically. In spite of such significance attached, our knowledge is poor about phylogeny and evolution within the fungi and between fungi and other organisms, as well as the taxonomic inventory of fungal species diversity. Bruns et al.11 opined their simple and frequently convergent morphology, their lack of a useful fossil record, and their diversity have been major impediments to progress in this field. In the past, phylogenetic speculation of the fungi was based mainly on comparative analyzes of morphological, ontogenetical and biochemical data.12–21 Kimura’s neutral theory of molecular evolution impacted studies of the phylogeny and evolution.22 The development of molecular biological techniques (particularly gene cloning, nucleic acid sequencing and polymerase chain reaction), proliferation of high performance computers, and improvement of molecular evolutionary analysis programs have extended studies on relatedness, phylogeny and evolution of organisms, including fungi, at the molecular level.23–25 Analyzes using molecular phylogenetics support a monophyletic origin of the fungi. The taxonomy of the fungi is in a state of constant flux, especially due to recent research based on DNA comparisons. These current phylogenetic analyzes

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often overturn classifications based on older and sometimes less discriminative methods based on morphological features and biological species concepts obtained from experimental matings.

Phylogenetic hypotheses, evolutionary relationships and circumscription of the fungi The monophyletic origin of “fungi” is the dogma that made mycologists to explore the fungi and their relationships.20,26–28 In this theory, it is thought that all the fungi were derived from an algal ancestor that lost its ability to photosynthesize and gave rise to the flagellate fungi. This ancestor was thought to be a member of the Chytridiomycota, which was morphologically similar to their modern counterparts and produced posteriorly, uniflagellated zoospores. Like many of its modern counterparts, this ancestral chytrid was aquatic, but over time, became more adapted to the terrestrial environment. The loss of flagella and the evolution of the zygospore gave rise to the Zygomycota whose members were believed to be morphologically similar to Mucor or Rhizopus, which produce large, single, multispored, columellate sporangia. As this group evolved, the number of sporangiospores in a sporangium became reduced and the columella was lost, thereby the sporangiole was evolved, with the most advance members producing single-spored sporangioles, e.g. Cunninghamella. This led to the evolution of the conidium, an asexual spore typically produced by the Ascomycota and Basidiomycota where an anamorph stage is produced. Another line that arose within the Zygomycota was that which included the Endogonales. The zygospores produced in this line were uninucleate. This line was believed to have given rise to the Ascomycota. The link between the Zygomycota and Ascomycota was made with a fungus that resembled Dipodascopsis (Ascomycota). The Taphrina-like ancestor gave rise to other members of Ascomycota and ancestral Basidiomycota, which diverged into two lines. One line produced microcyclic rusts, which produced only teliospores and basidiospores and led to the present-day rusts. The other line produced the ancestor of the present day Auriculariales, which had a poorly differentiated basidiocarp. This line gave rise to the remaining basidial and basidiocarp types in the Basidiomycota. Bessey29 believed that the Zygomycota were derived from the Oomycota rather than Chytridiomycota, based on similarities in morphology of their sporangia and that both phyla produced coenocytic mycelium. Bessey,29 Sachs,30 Denison and Carroll31 believed that the Ascomycota were derived from the Floridean red algae (Rhodophyta). Although Kohlmeyer,32 Kohlmeyer and Kohlmeyer33 did not advocate the direct derivation of the Ascomycota from the Rhodophyta, they postulated a hypothetical, common ancestor that gave rise to both phyla.


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Demoulin15 had a similar hypothesis, but believed that both the Ascomycota and Basdiomycota were derived from a red algal ancestor. During this old era, characteristics not previously incorporated into determining fungal phylogeny and new tools that became available brought forth new theories on the interrelationship of the different fungal taxa. Some new criteria that were considered in fungal phylogeny included: ● Cell biological data on mitochondrial cristae, organelle distribution, flagellation, mitosis. ● Biochemical data on cell wall constituents, amino acid synthesis and enzymology. This indicated that the Chytridiomycota should be classified in the kingdom Myceteae, with the fungi; the Oomycota should be in the kingdom Stramenopila with the Chrysophyta and Phaeophyta as proposed by Pringsheim34 and Kreisel.35 Although their theory did turned out to agree with this modern interpretation in phylogeny, they based their classification of the Oomycota only on superficial morphological similarities. The Hyphochytriomycota were also first classified in this group at this time. The different phyla of slime molds were also shown to be more distantly related than once believed, and also, it is noteworthy that the Rhodophyta is unrelated to the Ascomycota. Although phylogeny in the higher taxa of fungi seem to be more resolved, the lower taxa now seem to be more confused. DNA sequence analysis does not support the Dipodascopsis-like hypothetical ancestor that was once thought to link the Zycomycota to the Ascomycota. Thus, we no longer have a link between the two phyla. Another Ascomycota dogma that has been demonstrated to be incorrect is the monophyletic origin of the ascosporogenous yeasts. Members of Saccharomycetales no longer appear to be monophyletic and are more distantly related to other ascosporogenous yeasts. Phylogeny within the Basidiomycota has also become more confused. The relatedness of taxa within this phylum, based on the morphology of the basidium, which was the main character used to classify fungi in this phylum, is not in agreement with DNA sequence analysis. Instead, it appears that the septal pore apparatus is a better indicator of relationship between members of the Basidiomycota. Thus, with the exception of the Tremellales, all of the various basidial types, that are formed in basidiocarps now appear to be more closely related than once believed, i.e. Agaricales, Gasteromycetes, Auriculariales, Dacrymycetales, etc. are now believed to form a monophyletic group with the Tremellales as a sister group. The Ustilaginomycetes and Uredinomycetes are believed to be more distantly related.

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Recent aspects Among earlier phylogenetic speculations concerning fungi and related organisms that have been made during the past 20 years, Cavalier-Smith’s 36 theory is noteworthy. He provided a framework for a taxonomic system and phylogeny for the fungal kingdom that was based mainly on cell wall chemistry, biosynthetic pathways for lysine, type of motile cells, cellular ultrastructure and 5S rRNA sequence divergence. He included only the chytridiomycetes, zygomycetes, ascomycetes and basidiomycetes in the kingdom fungi. These four fungal groups are characterized by chitinous cell walls12 and the aminoadipic acid (AAA) lysine biosynthetic pathway.37 The oomycetes, hyphochytrids, labyrinthulids, thraustochytrids, and slime molds, which are cellulosic 12 and have the (diaminopimelic acid; DAP) lysine biosynthetic pathway,37 were excluded from the fungi. These first four major groups were accommodated by Cavalier-Smith36 in the pseudo fungi and the slime molds in the kingdom Protozoa.38 He also assumed that fungi and animals had a common ancestor, a choanociliate (choanoflagellte) protozoan. He concluded that the kingdom fungi is monophyletic and also that all major eufungal taxa in the Endomycota, Ascomycota and Basidiomycota, evolved from the Entomophthorales from a chytridiomycete ancestor by loss of cilia (flagella). Evidence from 18S rDNA sequence divergence put an end to a debate on the circumscription of the kingdom Fungi, i.e. whether oomycetes, hyphochytrids and chytrids were the “true” fungi, and confirmed the extent of “true” fungi.11,39 The “true” fungi are chytridiomycetes, zygomycetes, ascomycetes and basidiomycetes. The AAA lysine pathway and the presence of chitin in the cell walls strongly support the 18S rDNA sequence-based phylogeny. In general, the “true” fungi are hyphal, have cell walls through most or all of their life cycle and are exclusively absorptive in their nutrition. These “true” fungi form a monophyletic group, though below the ~95 level-based on bootstrap analysis, and are distinguished phenotypically from cellular slime molds (Acrasiomycetes), plasmodial slime molds (Myxomycetes) and the oomycetes (Oomycota).39 In this phylogenetic tree, the two groups of slime molds diverged separately, prior to the terminal radiation of eukaryotes. This is consistent with many differences between slime molds and fungi in form, function and life cycle. The oomycetes (Achlya, Lagenidium and Phytophthora), hyphochytrids (Hyphochytridium) and labyrinthulids (Thraustochytrium and Ulkenia) form a cluster with brown algae and diatoms. These organisms have heterokont flagella, contain chlorophylls a and c, and are classified within the Chromista sensu CavalierSmith,38 or the Stramenopila40 segregated from the Chromista. The labyrinthulids appear to be basal to other heterokont algae and oomycetes and hyphochytrids within the Chromista.41,42 Maximum likelihood phylogentic analysis of the 18S


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rDNA suggests that Plasmodiophora brassicae, a severe pathogen of crucifers, may be more closely related to the alveolates than to any of the fungi.43 Evolutionary relationships between the three kingdoms of complex eukaryotes, fungi, plants and animals, are still controversial, because of lack of solid fossil evidence. However, recent 18S rDNA phylogenies and amino acid sequence of elongation factor show that the closest relatives to fungi are animals, not plants.44–46 Nikoh et al.47 concluded that the kingdom Animalia is closely related to the kingdom Fungi and is distantly related to the kingdom Plantae. In contrast, on the basis of the ribosomal protein peptide and nucleotide sequences, Plantae and Animalia are sister clades and Fungi form a more distinct clade to them.48 More data on informative molecular characters are needed to solve these evolutionary problems. It is believed that the chytrids are the most primitive fungi within the “true” fungi, because they are zoosporic and gave rise to the Zygomycota and other higher fungi. Molecular phylogenetic studies of the chytridiomycetes and zygomycetes are limited because their economic interest is relatively low, pure cultures have not been achieved for many species, and even detection of some species is very difficult. However, such studies are indispensable to fill the gaps in knowledge of fungal evolution; rRNA sequence comparisons answered the debate as to whether flagellated fungi were “true” fungi. Early phylogenetic analysis of 5S rRNA sequences by Walker49 indicated that Basidiobolus was closely related to the chytrids. The Harpellales, an order of the Trichomycetes, was proposed to have a close relationship with the Kickxellales (Zygomycota) based on similarities in septal pore ultrastructure, cell wall structure, asexual reproductive apparatus, and serological affinity,50 but this interpretation conflicted with Walker’s49 5S rRNA sequence comparison. Gunderson et al.51 revealed that the 18S rRNA sequence-based phylogeny supported exclusion of the oomycetes. The sequence analysis of 18S rRNA genes suggested that the chytrid Blastocladiella emersonii and the higher fungi shared a common ancestor,52 and that the rumen anaerobic fungus Neocallimastix spp. should be assigned to the chytridiomycetes.53 Numerous hypotheses on phylogeny and evolution of the higher fungi, i.e. the ascomycetes, basidiomycetes and their anamorphs, have been proposed.20,23,54,55 Among these, Savile’s20 phylogenetic considerations of higher fungi have attracted the attention of a lot of mycologists. He suggested that Taphrina was the closest survivor of the common ancestor of the higher fungi. He suspected that two major lineages evolved from “Prototaphrina” a common ancestor. One major lineage led to the present day Taphrina and the ascomycetes, whereas other major lines led to the basidiomycetes (the Uredinales line and the parasitic Auriculariaceae line) through a “Protobasidiomycete.” In this phylogenetic scheme Taphrina was considered to be the most important fungus.

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Phylogenetic trees inferred from 18S rDNA sequence divergence indicate the existence of the two divisions, Ascomycota and Basidiomycota, among the higher fungi.38,56-59 Blackwell et al.60 presented phylogenetic tree diagram of members of kingdom fungi, based on the phylogenetic studies of a number of mycologists with the goal of producing a stable higher-level phylogenetic classification of fungi. Mushroom fossils were reported by Hibbett et al.61,62 the impact from these fungal fossil records is great to fungal phylogenetics and evolution but the complete connections are rare. On the other hand, DNA is the macromolecule that harbors evolutionary information as well as genetic information.63 Evolution, at the molecular level, is observable as nucleotide (or base) changes in the DNA and amino acid changes in proteins.64 The 18S rRNA gene divergence has been an especially useful molecular character to elucidate the identity of fungal taxa and their evolutionary relationships. If there are conflicts between the molecular and morphological characters, both molecular and morphological data should be re-examined, but often the morphological data have been misinterpreted. The anamorphic yeast Saitoella complicata and the phytopathogen Mixia osmundae provide examples. Recent 18S rDNA sequence analyzes indicate the existence of two divisions within the higher fungi, the sister groups Ascomycota and Basidiomycota. This relationship suggests that the ascus and the basidium, as meiosporangium are phylogenetically meaningful. The molecular phylogenies do not support the existence of the deuteromycetes as a distinct higher taxon. Instead they integrate both anamorphs and teleomorphs into a molecular phylogenetic tree. The genome of Saccharomyces cerevisiae has been sequenced (see Saccharomyces Genome Database, http:www.stanford.edu/Saccharomyces/). Fungal sequence of mitochondrial (chytrids, zygomycetes, dikaryomycetes, Oomycota)65 and nuclear genomes (Saccharomyces, Candida, Aspergillus, Magnaporthe, Neurospora) (see http://fungus.genetics.uga.edu:5080/main.html) provide new nucleic acid targets for phylogenetics and population genetics.66

Evolution of fungi within the groups Bessey,13,29 Cain14 and Savile20 summarized their own ideas, as well as those of others, about fungal evolution. Since then, enhanced evolutionary studies brought a number of systematists to study the diverse array of organisms grouped as protists. The resulting consideration of all of the protists led to more inclusive comparisons necessary to define monophyletic lineages. One notable example of this wider view resulted in the exclusion of Oomycota from fungi and the inclusion of this group in Stramenopila. The phylogenetic tree is based largely on DNA sequence analysis and ultra structural features.11,39,46,67 Several points of the hypothesis are important to emphasize that: (1) The acrasid slime molds (Acrasiomycota) diverged early in


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the eukaryotic lineage along with certain ameba. These groups are characterized in part by their discoidal mitochondrial cristae. (2) Plasmodial slime molds (Myxomycota) and dictyostelid slime molds (Dictyosteliomycota) are additional taxa with amoeboid stages that diverged independently of each other at a later time. Both groups have tubular mitochondrial cristae. (3) Another lineage of interest, Stramenopila, is viewed as a kingdom of morphologically diverse organisms, usually having a heterokont flagellar condition. Oomycota, Hyphochytriomycota and Labyrinthulomycota, as well as several groups of algae with chlorophylls a and c are in a monophyletic lineage. (4) The red algae diverged in a lineage distinct from any group ever considered to be fungi. This is important as evolution of Ascomycota from a red algal ancestor has been proposed on several occasions. (5) Fungi (Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota) are restricted to a monophyletic lineage with a close relationship to animals through a choanoflagellate-like ancestor. (6) Little information is available for Plasmodiophoromycota; however the group may share some features of the infection apparatus and basal body structure with certain ciliate protozoans. (7) The recognition of the polyphyly of organisms that previously have been grouped in the kingdom protista. The use of phylogenetic analysis methods in conjugation with DNA sequences is undoubtedly important for mycology. For example, Oomycota and Chytridiomycota had already been distinguished on the basis of the flagellation type, biosynthetic pathways, wall chemistry and the genetic code. However, the inclusion of a wide range of taxa in phylogenetic analyzes has certainly improved the hypothesis by showing the degree of divergence between these groups. Another lineage of interest the monophyletic fungi (Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota) is supported as monophyletic by sequence analysis.11,39,52 Many of the same morphological and biochemical characters that were used to separate fungi from Oomycota are shared among all the fungal phyla. The hypotheses of relationships within fungi are less well supported. There is general agreement of the sister group relationship of Ascomycota and Basidiomycota, however, the position of some taxa remains problematical.11,39 Few species of Chytridiomycota and Zygomycota have been included in sequencing studies to allow generalizations on the group as a whole, and few morphological or biochemical characters are known to support each as a monophyletic group. Chytridiomycota has been distinguished from the other fungi by the presence of a single posterior flagellum. However, when this character is considered in the context of studies employing phylogenetic analysis, the flagellate condition is recognized as a primitive character and cannot be used to support a monophyletic lineage of this group. In addition, another character, the zygospore used to distinguish Zygomycota, may not be unique to the group but may be shared with some Chytridiomycota.

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Oomycota diverged within the monophyletic group of Stramenopila, an idea suggested much earlier34 because of similarities in sexual reproduction but with little other evidence. The presence of flagellar hairs, tubular mitochondrial cristae, and β-1,3- or β-1,6-linked glucans as storage product all serve to strengthen the hypotheses.67 Oomycota, Hyphochytriomycota and Labyrinthulomycota have been included among Stramenopila on the basis of flagellation and DNA sequence analysis.41,42 Many types of characters may provide evidence of relationships. Thus far at the higher taxonomic levels, nucleotide characters have been very important because they extend across all of the taxa of interest.

Chytridiomycota The Chytridiomycota are commonly known as chytrids. These fungi are ubiquitous with a worldwide distribution. Chytrids produce zoospores that are capable of active movement through aqueous phases with a single flagellum, leading early taxonomists to classify them as Protists. Molecular phylogenies, inferred from rRNA sequences in ribosomes, suggest that the chytrids are a basal group divergent from the other fungal divisions, consisting of four major clades with suggestive evidence for paraphyly or possibly polyphyly.

Blastocladiomycota The Blastocladiomycota were previously considered a taxonomic clade within the Chytridiomycota. Recent molecular data and ultrastructural characteristics, however, place the Blastocladiomycota as a sister clade to the Zygomycota, Glomeromycota and Dikarya (Ascomycota and Basidiomycota). The Blastocladiomycetes are fungi that are saprotrophs and parasites of all eukaryotic groups and undergo sporic meiosis unlike their close relatives, the chytrids, which mostly exhibit zygotic meiosis.

Neocallimastigomycota The Neocallimastigomycota were earlier placed in the phylum Chytridiomycota. Members of this small phylum are anaerobic organisms, living in the digestive system of larger herbivorous mammals and possibly in other terrestrial and aquatic environments. They lack mitochondria but contain hydrogenosomes of mitochondrial origin. As the related chrytrids, neocallimastigomycetes form zoospores that are posteriorly uniflagellate or polyflagellate.

Zygomycota The Zygomycota, commonly known as ‘sugar’ and ‘pin’ molds, reproduce sexually with meiospores called zygospores and asexually with sporangiospores. Black


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bread mold (Rhizopus stolonifer) is a common species that belongs to this group; another is Pilobolus capable of ejecting spores several meters through the air. Medically relevant genera include Mucor, Rhizomucor and Rhizopus. Molecular phylogenetic investigations have shown Zygomycota to be a polyphyletic phylum with evidence of paraphyly within this taxonomic group.

Ascomycota The Ascomycota, commonly known as sac fungi or ascomycetes, constitute the largest taxonomic group within the Eumycota. These fungi form meiotic spores called ascospores, which are enclosed in a special sac-like structure called an ascus. This division includes morels, a few mushrooms and truffles, single-celled yeasts (Saccharomyces, Kluyveromyces, Pichia and Candida), and many filamentous fungi living as saprotrophs, parasites and mutualistic symbionts. Prominent and important genera of filamentous ascomycetes include Aspergillus, Penicillium, Fusarium and Claviceps. Many ascomycete species have only been observed undergoing asexual reproduction (called anamorphic species), but analysis of molecular data has often been able to identify their closest teleomorphs in the Ascomycota. Because the products of meiosis are retained within the sac-like ascus, ascomycetes have been used for elucidating principles of genetics and heredity (e.g. Neurospora crassa).

Basidiomycota Members of the Basidiomycota, commonly known as the club fungi or basidiomycetes, produce meiospores called basidiospores on club-like stalks called basidia. Most common mushrooms belong to this group, as well as rust and smut fungi, which are major pathogens of cereals. Other important basidiomycetes include the maize pathogen Ustilago maydis, human commensal species of the genus Malassezia, and the opportunistic human pathogen, Cryptococcus neoformans.

Glomeromycota Members of the form arbuscular mycorrhizae with higher plants and reproduce asexually. The symbiotic association between the Glomeromycota and plants is ancient, with evidence dating to 400 million years ago. The fungal symbionts of arbuscular mycorrhiza form a monophyletic group phylum Glomeromycota in the true fungi.68 The only member of this clade known to form a different type of symbiosis is Geosiphon pyriformis, which associates with cyanobacteria. Because none of these fungi has been cultivated without their plant hosts or cyanobacterial partners, progress in obtaining multigene

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phylogenies has been slow and the nuclear-encoded rDNA genes have remained the only widely accessible molecular markers, rDNA phylogenies have revealed considerable polyphyly of some glomeromycotan genera that have been used to reassess taxonomic concepts. Environmental studies using phylogenetic methods for molecular identification have recorded an amazing diversity of unknown phylotypes, suggesting considerable cryptic species diversity. Protein gene sequences that have become available recently have challenged the rDNA supported sister group relationship of the Glomeromycota with Ascomycota/ Basidiomycota.68 However the number of taxa analyzed with these new markers is still too small to provide a comprehensive picture of intraphylum relationships.

Are the fungi animals? Fungi are more closely related to animals than plants. The evolutionary origin of fungi is important in determining the phylogenetic relationships between fungi, animals and plants, and in questioning a previous view of the origin of life, which stated that photosynthetic organisms were the first to evolve since they were utilized by heterotrophs as a food source. The key evidence in support of a fungi-animalia clade includes analysis of protein sequences biosynthetic pathways, cytochrome systems, mitochondrial genetic material, biochemical and structural cellular features, glycoproteins, mode of nutrition, and storage of nutritive materials. The hypothesis that fungi evolved from algae, the ancestor of photosynthetic plants is not well supported, the hypothesis that fungi evolved independently of both plants and animals is also not supported. Fungi are most closely-related to animals than plants. The biochemical evidence supporting the hypothesis of a fungi-animalia clade is particularly striking. The protein sequences of fungi show homology to those of animals. Fungi also display similarities to animals in the biosynthesis of polyunsaturated fatty acids. Fungi also show similarities to animals in their cytochrome systems. Fungi share common morphological and structural cellular features with animals. Fungi lack chloroplasts, as do animals. The evidence that was shown in support of hypothesis regarding RNA and the mitochondrial genome particularly the translation of UGA as tryptophan codon, as well as the similarities in biosynthetic pathways of animals and fungi are especially convincing. There certainly is a need for further research, particularly regarding the composition of the cell wall, which is an evolutionary novelty for fungi since it contains both chitin and cellulose.

Recent classifications In the early 20th century, the importance of nucleus was recognized and based on its presence or absence the microorganisms were grouped as prokaryotes and eukaryotes under protista. Whittaker69 proposed five kingdoms in the classification


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Figure 1. Five kingdoms of living organisms proposed by Whittaker69 (from Whittaker69)

of living organisms viz. monera, protista, fungi, plantae and animalia (Fig. 1). Woese et al.70,71 classified life on this planet in three “domains”- Bacteria, Archaea, and the Eucarya, each containing two or more kingdoms. Fungi were placed in Eucarya along with animalia, plantae and others (not defined). Hibbett et al.72 proposed a new classification of kingdom fungi-based on recent molecular phylogenetic analysis and morphotaxonomy (Fig. 2). It was subdivided with one subkingdom – Dikarya and seven phyla – Chytridiomycota, Neocallimastigomycota, Blastocladiomycota, Glomeromycota, Microsporidia, Basidiomycota and Ascomycota, having ten subphyla, 35 classes, 12 subclasses and 129 orders. The clade containing Ascomycota and Basidiomycota is classified as the subkingdom Dikarya reflecting the putative synapomorphy of dikaryotic hyphae. The most dramatic changes in the classification with relation to earlier works are in the groups that have been traditionally included in the Chytridiomycota and Zygomycota. The Chytridiomycota is retained in a restricted sense, with Blastocladiomycota and Neocallimastigomycota also as segregate phyla of flagellated fungi. Taxa earlier included in Zygomycota

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Figure 2. Phylogeny and classification of fungi (from Hibbett et al.72)

are now placed among Glomeromycota, and several subphyla incertae sedis including Mucoromycotina, Entomophthoromycotina, Kickxellomycotina and Zoopagomycotina. Microsporidia are included in the fungi, but no further subdivision of the group is proposed.72 In the 10th edition of Dictionary of the Fungi by Kirk et al.5 three kingdoms are accepted viz. Chromista, Fungi and Protozoa. True fungi belong to kingdom fungi having six phyla – Ascomycota, Basidiomycota, Chytridiomycota, Glomeromycota, Microsporidia and Zygomycota; 36 classes, 140 orders, 560


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families, 8,283 genera (+5,101 synonyms) and 97,861 species. Unlike the earlier editions of the dictionary Deuteromycotina is not accepted as a formal taxonomic category. They are not a monophyletic unit, but are fungi which have either lost a sexual phase or which are anamorphs of the other phyla (mainly Ascomycota, some Basidiomycota). With modern molecular or ultra structural techniques such fungi can be assigned to existing taxa.5 Acknowledgements: The authors are grateful to CSIR and Ministry of Environment and Forests for financial support.

References 1.

Manoharachary C, Sridhar K, Singh R, Adholeya A, Suryanarayana, TS, Rawat S, Johri BN (2005) Fungal biodiversity: Distribution, conservation and prospecting of fungi from India. Curr Sci 89(1):58–71

2.

Hawksworth DL (1991) The fungal dimension of biodiversity: magnitude, significance, and conservation. Mycol Res 95:641–655

3.

13.

Domoto A, Iwatsuki K (1997) Threats of global warming to biological diversity. Tshukiji Shokan, Tokyo (In Japanese) Hawksworth DL (1994) Fungal diversity and its implications for genetic resource collections. Stud Mycol 50:9–18 Kirk PM, Cannon PF, Minter DW, Stalpers JA (Eds.) (2008) In: Dictionary of the Fungi. 10th edition, CABI Bilgrami KS, Jamaluddin S, Rizwi MA (1991) Fungi of India - List and References. Today and Tomorrow’s Print and Publ., New Delhi Sarbhoy AK, Varshney JL, Agarwal DK (1996) Fungi of India (1982–92). CBS Publ and Distributors, New Delhi Jamaluddin, Goswami MG, Ojha BM (2004) Fungi of India - 1989–2001. Scientific Publ., Jodhpur, India Darwin CR (1859) On the origin of species by means of natural selection. John Murray Albemarle Street, London Darwin CR (1871) The descent of man and selection in relation to sex. Vol. I, II. John Murray Albemarle Street, London Bruns TD, White TJ, Taylor JW (1991) Fungal molecular systematics. Ann Rev Ecol Syst 22:525–564 Bartnicki-Garcia S (1987) The cell wall: a crucial structure in fungal evolution. In: Evolutionary biology of the fungi. Rayner ADM, Brasier CM, Moore D (Eds.), Cambridge University Press, Cambridge Bessey EA (1942) Some problems in fungus phylogeny. Mycologia 34:355–379

14.

Cain RF (1972) Evolution of the fungi. Mycologia 64:1–14

15.

Demoulin V (1974) The origin of Ascomycetes and Basidiomycetes, the case for a red algal ancestry. Bot Rev 40:315–345

16.

Gäumann EA (1952) The fungi, a description of their morphological features and evolutionary development. Hafner Publ., New York

17.

LéJohn HB (1971) Enzyme regulation, lysine pathways, and cell wall structures as indicators of major lines of evolution in fungi. Nature 231:164–168

4. 5. 6. 7. 8. 9. 10. 11. 12.

156


18.

LéJohn HB (1974) Biochemical parameters of fungal phylogenetics. Evol Biol 7:79–125

19.

Ragan MA, Chapman DJ (1978) A biochemical phylogeny of the protists. Academic Press, New York

20.

Savile DBO (1968) Possible interrelationships between fungal groups. In: The fungi, an advanced treatise. Ainsworth GC, Susmann AS (Eds.), Academic Press, New York

21.

Vogel HJ (1964) Distribution of lysine pathways among fungi: evolutionary implication. Am Naturalist 98:435–446

22.

Kimura M (1983) The neutral theory of molecular evolution. Cambridge University Press, Cambridge

23.

Sugiyama J (1998) Relatedness, phylogeny, and evolution of the fungi. Mycoscience 39:487–511

24.

Sugiyama J, Nishida H (1995) The higher fungi: their evolutionary relationships and implications for fungal systematics. In: Biodiversity and evolution. Arai R, Kato M, Doi Y (Eds.), The National Science Museum Foundation, Tokyo

25.

Sugiyama J, Nagahama T, Nishida H (1996) Fungal diversity and phylogeny with emphasis on 18S ribosomal DNA sequence divergence. In: Microbial diversity in time and space. Colwell RR, Simidu U, Ohwada K (Eds.), Plenum Press, New York

26.

Atkinson GF (1915) Phylogeny and relationships in the ascomycetes. Ann Missouri Botan Gardens 2:315–376

27.

Gäumann EA, Dodge CW (1928) Comparative morphology of fungi. McGraw Hill, New York

28.

Luttrell ES (1955) The ascostromatic ascomycetes. Mycologia 46:511–532

29.

Bessey EA (1950) Morphology and Taxonomy of Fungi. The Blakiston Co., Philadelphia

30.

Sachs J (1874) Lehrbuch der Botanik. 4th edition, Engelmann, Leipizig

31.

Denison WC, Carroll GC (1966)The Primitive Ascomycete: A New Look at an Old Problem. Mycologia 58:249–269

32.

Kohlmeyer J (1973) Fungi from marine algae. Botanica Marina 16:201–215

33.

Kohlmeyer J, Kohlmeyer E (1979) Marine Mycology, the Higher Fungi. Academic Press, San Francisco

34.

Pringsheim N (1858) Beiträge zur Morphologie und Systematik der Algen. II. Die Saprolegnieen. Jahrb Wiss Bot 1:284–304

35.

Kreisel H (1969) Grundzüge eines natürlichen systems der pilze. Cramer Lehre, Germany

36.

Cavalier-Smith T (1987) The origin of fungi and pseudofungi. In: Evolutionary biology of the fungi. ADM Rayner, CM Brasier, D Moore (Eds.), Cambridge Univ Press, Cambridge

37.

Vogel HJ (1965) Lysine biosynthesis and evolution: fungi, gymnosperms and angiosperms. In: Evolving genes and proteins. Bryson V, Vogel HJ (Eds.), Academic Press, New York

38.

Cavalier-Smith T (1993) Kingdom Protozoa and its 18 phyla. Microbiol Rev 57:953–994

39.

Bruns TD, Vilgalys R, Barns SM, Gonzalez D, Hibbett DS, Lane DJ, Simon L, Stickel S, Szaro TM, Weisburg WG, Sogin ML (1992) Evolutionary relationships within the Fungi: Analyses of nuclear small subunit rRNA sequences. Mol Phylogenet Evol 1:231–241 (1996) In: Introductory Mycology. 4th edition, John Wiley and Sons, New York

41.

Van der Auwere G, de Baere R, Van der Peer Y, de Rijk P Van der Broeck I, de Wachter R (1995) The phylogeny of the Hyphochytriomycota as deduced from ribosomal RNA sequences of Hyphochytrium catenoides. Mol Biol Evol 12:671–678


157

42.

Leipe DD, Wainright PO, Gunderson JH, Porter D, Patterson DJ, Valois F, Himmerich S, Sogin ML (1994) The stramenopiles from a molecular perspective: 16S-like rRNA sequences from Labyrinthuloides minuta and Cafeteria roenbergenis. Phycologia 33:369–377

43.

Castlebury LA, Domier LL (1998) Small subunit ribosomal RNA gene phylogeny of Plasmodiophora brassicae. Mycologia 90:102–107

44.

Baldauf SL, Palmer JD (1993) Animals and fungi are each other’s closest relatives: Congruent evidence from multiple proteins. Proc Nat Acad USA 90:11558–11562

45.

Sogin ML, EIwood HJ, Gunderson JH (1989) Phylogenetic meaning of the kingdom concept: an unusual ribosomal RNA from Giardia lamblia. Science 243:75–77

46.

Wainright PO, Hinkle G, Sogin ML, Stickel SK (1993) Monophyletic origins of the Metazoa: an evolutionary link with fungi. Science 260:340–342

47.

Nikoh N, Hayase N, Iwabe N, Kuma K, Miyata T (1994) Phylogenetic relationships of the kingdoms Animalia, Plantae, and Fungi inferred from 23 different protein species. Mol Biol Evol 11:762–768

48.

Veuthey AL, Bittar G (1998) Phylogenetic relationships of Fungi, Plantae, and Animalia inferred from homologous comparison of ribosomal proteins. J Mol Evol 47:81–92

49.

Walker WF (1984) 5S ribosomal RNA sequences from Zygomycotina and evolutionary implications. Syst Appl Microbiol 5:448–456

50.

Moss ST, Young TWK (1978) Phyletic considerations of the Harpellales and Asellariales (Trichomycetes, Zygomycotina) and the Kickxellales (Zygomycetes, Zygomycotina). Mycologia 70:944–963

51.

Gunderson JH, Elwood H, Ingold A, Kindle K, Sogin ML (1987) Phylogenetic relationships between chlorophytes, chrysophytes, and oomycetes. Proc Natl Acad Sci USA 84: 5823–5827

52.

Forster H, Coffey MD, Elwood H, Sogin ML (1990) Sequence analysis of the small subunit ribosomal RNAs of three zoosporic fungi and implications for fungal evolution. Mycologia 82:306–312

53.

Bowman BH, Taylor JW, Brownlee AG, Lee J, Lu SD, White TJ (1992) Molecular evolution of the fungi: Relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes. Mol Biol Evol 9:285–296

54.

Kramer CL (1987) The Taphrinales. In: The expanding realm of yeast-like fungi. de Hoog GS, Smith MT, Weijman ACM (Eds.), Elsevier, Amsterdam

55.

Tehler A (1988) A cladistic outline of the Eumycota. Cladistics 4:227–277

56.

Gargas A, Taylor JW (1995) Phylogeny of discomycetes, and early radiations of apothecial Ascomycotina inferred from SSU rDNA sequence data. Exp Mycol 19:7–15

57.

Nishida H, Sugiyama J (1993) Phylogenetic relationships among Taphrina, Saitoella, and other higher fungi. Mol Biol Evol 10:431–436

58.

Nishida H, Sugiyama J (1994) Archiascomycetes: detection of a major new lineage within the Ascomycota. Mycoscience 35:361–366

59.

Nishida H, Blanz PA, Sugiyama J (1993) The higher fungus Protomyces inouyei has two group I introns in the 18S rRNA gene. J Mol Evol 37:25–28

60.

Blackwell M, Hibbett DS, Taylor JW, Spatafora JW (2006) Research Coordination Networks: a phylogeny for kingdom Fungi (Deep Hypha). Mycologia 98(6):829–837

61.

Hibbett DS, Grimaldi D, Donoghue MJ (1995) Cretaceous mushrooms in amber. Nature 377:487

158


62.

Hibbett DS, Grimaldi D, Donoghue MJ (1991) Fossil mushrooms from Miocene and Cretaceous ambers and the evolution of homobasidiomycetes. Am J Bot 84:981

63.

Hillis DM, Moritz C, Mable C (Eds.) (1996) In: Molecular Systematics. 2nd edition, Sinauer Associates, Sunderland, Massachusetts

64.

Paquin B, Laforest MJ, Forget L, Roewer I., Wang Z, Longcore J, Lang BF (1997) The fungal mitochondrial genome project: evolution of fungal mitochondrial genomes and their gene expression. Curr Genet 31:380–395

65.

Ridley M (1996) In: Evolution. 2nd edition, Blackwell Science, Cambridge, Massachusetts

66.

Taylor JW (1998) Molecular systematics and evolution. (Abstract) 6th Intern Mycol Congr (IMC6), Jerusalem, Israel

67.

Barr DJ (1992) Evolution and kingdoms of organisms from the perspective of a mycologist. Mycologia 84:1–11

68.

Redecker D, Raab P (2006) Phylogeny of the Glomeromycota (arbuscular mycorrhizal fungi): recent developments and new gene markers. Mycologia 98:885–895

69.

Whittaker RH (1969) New concepts of kingdoms of organisms. Science 163:150–160

70.

Woese CR, Fox GE (1977) Phylogenetic structure of the prokaryotic domain: the primary kingdoms. Proc Natl Acad Sci USA 74(11):5088–5090

71.

Woese CR, Kandler O, Wheelis ML (1990) Towards a natural system of organisms: proposal for the domains Archaea, Bacteria, and Eucarya. Proc Natl Acad Sci USA 87(12): 4576–4579

72.

Hibbett DS, Binder M, Bischoff JF, Blackwell M, Cannon PF, et al. (2007) A higher-level phylogenetic classification of the Fungi. Mycol Res 111:509–547

11 The origin of reproductive isolating mechanisms is an important event in the process of speciation: Evidences from Drosophila B.N. Singh Genetics Laboratory, Department of Zoology, Banaras Hindu University, Varanasi - 221005, India [email protected]

Abstract: In the origin and maintenance of race and species, isolation is an indispensable factor and its role and importance have been recognized for a long time. Even Lamarck and Darwin pointed out that interbreeding of different populations may result in swampimg of the differences acquired during the process of evolutionary divergence. During the process of speciation the diverging populations must acquire some means of isolation so that the genes from one gene pool are prevented from dispersing freely into foreign gene pool. Reproductive isolating mechanisms prevent exchange of genes between Mendelian populations by genetically conditioned mechanisms which are intrinsic to the organisms themselves. Thus, the origin of reproductive isolating mechanisms is an important event in the process of cladogenesis (speciation). Drosophila characterized by rich species diversity (over 1500 described species) has been utilized in genetical, behavioral and evolutionary studies since the beginning of the last century as it is very good material for such studies. Different types of reproductive isolating mechanisms have been studied in Drosophila such as gametic isolation, ethological (sexual or behavioral) isolation, hybrid inviability and hybrid sterility. Among these four types of reproductive isolating mechanisms, ethological isolation is widespread in the genus Drosophila. Various techniques have been used by different investigators to test the degree of ethological isolation. In such studies, both types of ethological isolation, interspecific as well as intraspecific have been tested. In intraspecific sexual isolation tests, wild type and mutant strains of the same species have been utilized. The pattern of isolation as well as the degree of isolation has often been used to discuss the phylogenetic relationship among the species and also to elucidate the direction of evolution among closely related species of different species groups. There are different types of stimuli involved in mating behavior of Drosophila which provide basis for ethological isolation. There are interesting cases reported by Indian workers regarding the pattern of ethological isolation and the direction of evolution. Mechanisms of origin of reproductive isolating V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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mechanisms and also the models which have been proposed to predict the direction of evolution based on the mode of mating preference have been discussed while mentioning numerous examples of both kinds of incomplete sexual isolation, intra- and inter-specific. While considering different forms of post-zygotic reproductive isolation, hybrid sterility is the most common form and it is of special interest as its genetic basis may provide information about the mechanism of speciation. Key words: Reproductive isolating mechanisms, Drosophila, Phylogenetic relationship, Evolutionary sequence, Origin of isolating mechanisms

Introduction Even after 150 years of publication of Charles Darwin’s ‘On the Origin of Species by Means of Natural Selection’, his theory of evolution is important.1 Darwin’s theory of evolution has two components: (i) Descent with modification-all species living and extinct have descended from one or a few original forms of pre-existing species, and (ii) natural selection as causal agent of evolutionary change. Darwin also recognized that species not only evolve but also divide. Infact Darwin was one of the very few nineteenth century evolutionists whose major interest lay in the causal rather than the historical problems. The importance of isolation has been recognized for a long time. Lamarck and Darwin pointed out that interbreeding of different populations results in swamping of differences which they have acquired during the process of evolutionary change.2 Darwin who devoted so much of his life to the systematics of species, fully appreciated the significance of this level, as he made clear in choice of title for his great evolutionary classic ‘On the Origin of Species’.3 The idea of sympatric speciation was of course Darwin’s - an important process of biological diversity. Darwin saw sympatric speciation as the main if not the only engine of diversity. Darwin also thought that the species could arise either sympatrically, allopatrically or parapatrically.4 Dobzhansky realized that Darwin’s notion of speciation was implausible or at least incomplete, since ecologically distinct forms can not coexist without barriers to gene exchange. Dobzhansky then stressed the importance of reproductive isolating mechanisms. In his view, the problem of speciation was not the occupation of new niches but the origin of reproductive isolation. This problem was discussed in his seminal work Modern Synthesis, Dobzhansky’s Genetics and the Origin of Species.4 While discussing speciation and adaptation from Darwin to Dobzhansky, Schluter5 has pointed out that appreciation of the connection between adaptation and speciation began with Darwin when morphological concept of species largely prevailed. Darwin understood the importance of reproductive barriers between species but the study of speciation after the publication of his book focused mainly on the evolution of species differences, particularly of morphological traits but also of behavioral and

The origin of reproductive isolating mechanisms is an important event in the process of speciation: Evidences from Drosophila

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other phenotypic traits.5 Dobzhansky remarked that without isolation evolution is impossible. Role of isolation in evolution was stressed by Wagner6 and in a modern form by Jordan.7 Races and species differ from each other in many genes and gene combinations. If these genetically different populations interbreed, these will breakdown and the differences between them will be swamped off. The gene pool of every species is discretely isolated from that of any other.8,9 Therefore, during the process of species formation the diverging populations must acquire some mechanisms of isolation so that the genes from one gene pool are prevented from dispersing freely into a foreign gene pool. Dobzhansky (1937, cited in Dobzhansky2) coined the term “isolating mechanisms” for different factors which alone or in combination prevent the gene flow between different Mendelian populations. Among different kinds of isolation, two types are easily distinguished: geographic (spatial) and reproductive isolation. In spatial isolation, species and races are allopatric and females and males of these populations which are separated by certain forms of geographical barriers do not mate hence mating is prevented. Such allopatric populations may or may not show reproductive isolation. However, Mendelian populations separated by geographical barriers may develop contact zone and gene exchange and gene deffusion may occur if the two populations have not developed certain mechanisms to keep them as separate gene pools. Reproductive isolating mechanisms prevent the exchange of genes by means of genetically conditioned factors which are intrinsic to the organism’s themselves.2 The different Mendelian populations may coexist sympatrically and they must be isolated reproductively other wise their identity could not be maintained due to gene flow between them and would merge into a single gene pool. Dobzhansky2,8 and Mayr3 have classified reproductive isolating mechanisms into different kinds supported by numerous suitable examples from variety of animal groups. There are two major classifications of reproductive isolating mechanisms: pre-mating and post-mating isolating mechanisms. Some authors have also used the terms pre-zygotic and post-zygotic isolating mechanisms (see Strickberger10; Hartle and Hallgrimsson11). According to Mayr,3 isolating mechanisms are the most important attributes a species has. Drosophila has proved to be very suitable material for studies on evolution and population genetics.8 With the development of modern genetics in Drosophila, interest has been focused on the question of role of isolating mechanisms in evolution. Different types of reproductive isolating mechanisms have been studied in the genus Drosophila. These are: ethological isolation, gametic isolation, hybrid inviability and hybrid sterility. Numerous examples are available in Drosophila which shows the operation of these kinds of isolating mechanisms. 2–4,12–16 Ethological (behavioral or sexual) isolation constitutes the most important class of reproductive isolation in animal species. It is a premating barrier to gene exchange

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in which the opposite sexes of different populations fail to mate due to behavioral incompatibility. Thus, it becomes a primary mechanism of reproductive isolation in the process of speciation. Genetic basis of sexual isolation in Drosophila has been demonstrated.17,18 The mate recognition system has been considered important species attribute which acts as cohesive factor19 and genetic events may be responsible for the appearance of different mate recognition systems. Ethological isolation has been studied extensively in the genus Drosophila and has been found to be wide spread.12–15 Many of the species tested show complete isolation. However, numerous cases of incomplete sexual isolation among closely related species have been found. When isolation is incomplete preference for homogamic matings occurs in most cases. In certain cases, males and females of different species may mate randomly. Different populations or stocks of the same species may show incipient isolation. The degree and pattern of ethological isolation have often been used to discuss the phylogenetic relationship between the species and also their evolutionary sequence. The present review summarizes briefly the different types of reproductive isolating mechanisms in Drosophila with more emphasis on the results of sexual isolation studies (inter- and intra-specific) with particular reference to the pattern of isolation and the models proposed on the basis of one-sided ethological isolation to predict the direction of evolution. In Drosophila, to test ethological isolation between species/strains, various techniques used are also described. Further, the mechanisms of origin of isolating mechanisms and the types of stimuli involved in mating behavior of Drosophila are also discussed. Further, hybrid sterility which is a common form of post-zygotic reproductive isolation in Drosophila is also discussed.

Types of reproductive isolating mechanisms In the genus Drosophila, various kinds of reproductive isolating mechanisms have been studied by numerous investigators beginning from Sturtevant, Dobzhansky, Mayr, Crow, Patterson, Stone, Mainland and others. These isolating mechanisms are: ethological isolation, gametic isolation, hybrid inviability and hybrid sterility. Ethological (behavioral or sexual) isolation In ethological barrier to gene exchange, females and males of different species/ populations do not mate as there is lack of mutual attraction due to behavioral incompatibility. It has been extensively studied in Drosophila and found to be widespread. With the development of modern genetics in Drosophila, interest has been focused on the evolutionary significance of isolating mechanisms. Although


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many species show complete isolation, there are numerous cases of incomplete sexual isolation, both between the species and within the species (for references see Dobzhansky2; Patterson and Stone12; Mayr3; Spieth and Ringo13; Chatterjee and Singh14; Singh15; Coyne and Orr4). There are a considerable number of studies that have demonstrated the existence of genetic variation for sexual isolation among conspecific populations either by direct observation or by selection for enhanced or reduced homogamic matings.17 Genetic basis of courtship songs which is the basis of sexual isolation has been demonstrated in D. pseudoobscura and D. persimilis.20 Mayr3 said “if we were to rank the various reproductive isolating mechanisms of animals according to their importance, we would have to place behavioral isolation far a head of all others”. Thus sexual isolation constitutes the most important class among different types of reproductive isolation in animal species and plays a key role in speciation4. In studies of sexual isolation, different techniques have been employed by researchers: (i) Multiple-choice: In this method, both sexes are given choice to mate with either their own strain or species or with the other. Females and males of both types are placed together hence both the types of matings, homogamic and heterogamic, are possible. Under this method, there are four combinations of matings - two homogamic and two heterogamic. This method is the most appropriate method because it gives the best approach to natural conditions. (ii) Male-choice: Here only males are given choice. Both the types of females are kept with one type of males. (iii) In this technique, the females are given choice. One type of females is confined with both types of males. (iv) No-choice: In this method, no choice is given to the flies. One type of males is confined with one type of females. Mating success can be scored by different methods. Nowadays, frequently direct observation method is used in which Elens and Wattiaux21 mating chamber is used. To measure the degree of sexual isolation, either isolation index or isolation estimate is calculated. Sexual isolation is caused due to assortative (non-random) mating between males and females of the same species or strains One-sided or asymmetrical isolation in may Drosophila species groups where preferential mating occurs in one direction only where as in the opposite cross males and females mate randomly. Thus, isolation may be symmetrical (both sided) or asymmetrical (one-sided). Ethological isolation is based on the production and reception of stimuli by male and female. On the basis of main sense organs involved, these stimuli have been classified into different types: visual, auditory, and chemical. A male Drosophila provides the female a set of stimuli during courtship. Based on these stimuli, the female is able to discriminate between its own and alien males and is stimulated for copulation. Role of visual stimuli in mating has been detected in different species of Drosophila. All species court during day light and some also court in darkness. It has been shown that presence

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or absence of light has pronounced effect on mating of Drosophila. Various species exhibit varying degrees of light dependency in their mating behavior. Auditory stimuli play more important role in isolating mechanisms than visual stimuli. Songs, calls and other acoustic signals have precision and specificity. Females may not become receptive for copulation until stimulated by appropriate wing vibration of conspecific males. Males of most Drosophila species produce wing vibrations which cause the production of sound termed as male courtship songs. With respect to these songs, there is variation in different species. The antennae of females are the main organs to receive auditory signals. The male sex songs are species specific. The inter pulse interval of male pulse songs is most important and is discriminated by females. Thus, it forms the basis of behavioral isolation. When incipient isolation occurs between different strains of the same species, variation in the male pulse songs has been observed in certain cases. Chemical stimuli which act at a distance (olfactory) or on contact often serve as isolating mechanisms. It has been shown that the olfactory stimuli in the form of pheromones are involved in mating behavior of Drosophila. In both sexes, receptors for olfactory stimuli are present on the third antennal segment. During courtship, the physical contacts between the male and female also results in the transmission of chemostimuli. Chemostimuli are exchanged during tapping of female by the male (see Chatterjee and Singh,14 1989). Recently, evidences for temporal, mechanical and habitat isolation between certain species pairs have been reported: temporal isolation between D. melanogaster and D. simulans, mechanical isolation between D. mauritiana female and D. simulans male and habitat isolation between D. sechellia and D. simulans (see Coyne and Orr4). Gametic isolation In gametic isolation, spermatozoa of one species are not attracted to the eggs or are poorly viable in sexual ducts of another species. Gametic isolation which is post mating as well as prezygotic barrier has been reported to occur in different species groups of Drosophila.22–25 In cross insemination, the mobility of sperm in ventral receptacle of females of foreign species is rapidly lost so fertilization does not take place. In certain cases, females become sterile due to insemination reaction in interspecific matings. Hybrid inviability Hybrid inviability is a post-mating and post-zygotic intrinsic reproductive barrier in which hybrids suffer developmental defects causing full or partial inviability4. A very good example of this type of isolation involves D. melanogaster and D. simulans, two closely related and sibling species.26 When D. melanogaster females are crossed with D. simulans males, only hybrid females appear because


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hybrid males die during larval to pupal transition. In the opposite cross, only hybrid males are produced and the F1 females die as embryo26.This shows that hybrid inviability may appear at different stages of development and may affect one sex only or both and it may be complete or partial4. Hybrid sterility The problem of hybrid sterility goes back to Aristotle who discussed at length the sterility of mule. Hybrid sterility is biologically not very efficient mechanism of reproductive isolation as it causes wastage of gametes of both species and of hybrids.2 While considering different forms of reproductive isolation, hybrid sterility is the most common form of post-zygotic reproductive isolation in Drosophila. Further, it is of special interest because its genetic basis may provide information about the mechanism of speciation. Since males in Drosophila are heterogametic as is the case in mammals, hybrid sterility is of special interest. Hybrid male sterility appears very quickly after species divergence as demonstrated by a large number of reports.27 The rapid appearance of hybrid sterility in males accounts for the majority of cases which follow Haldane’s rule. Sterility in hybrid males represents a well defined developmental system (namely, spermatogenesis) for genetic analysis. The study of evolutionary relationship among the species is not complete without testing hybridization among them. The first review of interspecific hybridization studies in animals was published by Haldane28 who catalogued all the case known at that time in which artificially performed crosses produced hybrids with a distortion of sex-ratio or fertility. Haldane also suggested that in hybrids when one sex is absent, rare or sterile that sex is heterogametic sex. This suggestion has come to be known as Haldane’s rule. However, exceptions to this generalization have been found.29 The total number of hybridization (interspecific) reported in the genus Drosophila is 266 involving a total of 191 different species.18,30 There is also variation in the results when successful hybridization occurs. It has been observed that when two species hybridize, larvae may be produced in F1 which die before pupation, development may proceed till pupal stage but adults are not produced or F1 adults are produced showing varying degree of fertility or sex-ratio distortion. Further, hybridization may occur in one direction or both directions. Only those species which could be cultured in the laboratory were used in hybridization studies. There are some reports of natural hybridization in Drosophila.31 In total, there are eight cases of natural hybridization in Drosophila.30 Thus natural interspecific hybridization in Drosophila is rare. The species which could be hybridized in laboratory remain completely isolated in nature. Hybridization could be achieved among closely related species belonging to the same species groups but no case of interspecific hybridization has been reported between the species belonging to different species group.18 Although there is variation in the results in different species groups, the most common finding is

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the occurrence of hybrids of both sexes in F1 and males becoming sterile. This observation in Drosophila supports Haldane’s rule because male is heterogametic. The different species groups of Drosophila in which hybrid sterility is known are repleta, virilis, melanica, funebris, guarani, mesophragmatica, cardini, willistoni, melanogaster, saltans, and quinaria (for references, see Singh16,18). There are six semi species of D. paulistorum which is regarded as a cluster of species status nascendi32 and these semi-species show varying degree of reproductive isolation from one another. Male sterility has been found in hybrids of these semi species. It is interesting to note that these semi-species of D. paulistorum are isolated by different degrees of pre-mating (behavioral) as well as post-mating reproductive barriers. Thus there are numerous cases of hybrid sterility particularly in males of Drosophila which is an important type of post mating or post-zygotic reproductive isolating mechanism. Generally, the species formation is marked by the appearance of reproductive isolating mechanism which is based on those genetic changes that differentiate incipient species up to the level when the exchange of genes is completely prevented. Since there are a large number of intercrossable species pairs generating sterile hybrids, hybrid sterility is potential force for cladogenesis. Drosophila has been very favorable material for the study of speciation genetics. A number of studies carried out on the genetics of hybrid sterility in Drosophila have shown that there are genetic loci involved in determining sterility in hybrids of closely related species. However, the results of different studies differ with respect to the number of loci involved and the pattern of interaction of X-chromosome, Y-chromosome and autosomes determine hybrid male sterility in different species pairs (see Singh18; Mishra and Singh33). One-sided sexual isolation and the direction of evolution There are numerous cases of clear cut one-sided sexual isolation between closely related species of Drosophila as well as between geographic strains/races of the same species. These findings are of much evolutionary significance and have often been used to discuss their phylogenetic relationships. Two models which are opposite to each other have been proposed based on behavioral asymmetry to predict the direction of evolution elucidating ancestral and derived nature of species/strains. Kaneshiro’s Model: Kaneshiro34 studied sexual isolation among different members of the planitibia subgroup of Hawaiian species of Drosophila. He found asymmetrical isolation among these species. Based on behavioral asymmetry, he suggested that ancestral females strongly discriminate against the derived males but the converse situation does not hold true. Kaneshiro predicted the direction of evolution for the planitibia subgroup as D. differens→ D. planitibia→ D. silvestris. Kaneshiros’ model was also applied to interpret the evolutionary sequence of different geographic strains of D. adiastola and D. silvestris based on asymmetrical


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mating preference.35,36 The phylogenetic relationship between D. takahashii and D. pseudotakahashii, two closely related allopatric species has been discussed by Dwivedi et al.37 on the basis of behavioral asymmetry and it was suggested by these authors that D. takahashii is ancestral to D. pseudotakahashii which agrees with Kaneshiro’ model. Ranganath and his students38,39 studied sexual isolation among D. nasuta nasuta and D. nasuta albomicans and also among the parental races and newly evolved cytoraces. They have also found behavioral asymmetry and have discussed their results in the light of different models to predict the evolutionary significance. Powell40 observed one-sided behavioral isolation in laboratory populations of D. pseudoobscura subjected to flush-crash cycles and the results were consistent with Kaneshiro’s model. Founder principle of Mayr41 has been hypothesized as the main factor to explain inter-island speciation of Hawaiian Drosophila.42 Kaneshiro’s model is also based on founder principle (via genetic drift). Several facets of male courtship behavior are lost due to genetic drift which occurs during a new invasion of previously uninhabited locality by a small number of migrants.34 Due to the loss of certain elements of courtship in males of new species, females of ancestral species show strong discrimination against the males of derived species since they contain only a part of total courtship pattern of conspecific males. On the other hand, the females of new species readily accept the males of older species because they identify the courtship pattern of these males since they contain all the elements of courtship pattern in the conspecific males. However, the original concept of loss of certain courtship elements in males of derived species has been modified43,44 by suggesting that courtship requirements of derived species females are simplified during early stage of founder events when the size of population is drastically reduced. Giddings and Templeton45 have redefined the model of Kaneshiro by suggesting that it would apply under specific conditions. Watanabe and Kawanishi’s model: Watanabe and Kawanishi46 studied sexual isolation among D. melanogaster, D. simulans and D. mauritiana. They also used the published data of the D. virilis group.23,47 Based on the asymmetrical isolation, they suggested that it is the females of derived species which do not mate with the males of ancestral species and the species whose females readily accept the courtship overtures of other is the ancestral species. Thus the direction of evolution is just opposite to that of Kaneshiro’s model. It is interesting to note that the direction of evolution among the species of virilis group based on the model of Watanabe and Kawanishi agrees well with the phylogenetic relationship suggested on the basis of cytological, morphological and electrophoresis studies. Watanabe and Kawanishi argued that every subpopulation is always exposed to the danger of invasion by migrants of the ancestral population. If the gene flow from the ancestral population to the new subpopulation is restricted, the new population can survive against the danger of contamination with genes from the

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original population and can retain its identity. Thus, the first step for the initiation of speciation is the failure of mating between derived females and ancestral males which is the most important change for the creation of new species.46 Thus there is fundamental difference between the two models: Kaneshiro’s model is based on allopatric speciation but the Watanabe and Kawanishi’s model is based on sympatric speciation. Ancestral and derived status of the species has been discussed on the basis of behavioral asymmetry in virilis group and D. arozonensis and D. mojavensis species pairs and the direction of evolution agrees with Watanabe and Kawanishi’s model. Evidence in favor of Watanabe and Kawanishi’s model also comes from the phylogeny of willistoni group established on the basis of electrophoresis studies.48 Behavioral asymmetry observed between D. bipectinata and D. parabipectinata by Singh et al. 49 also lends support to Watanabe and Kawanishi’s model. D. bipectinata is considered ancestral to D. parabipectinata. Singh and Chatterjee50 observed asymmetrical isolation among geographic strains of D. ananassae and the results obtained by them also support Waranabe and Kawanishi’s model. Thus, both models having predictable value regarding the direction of evolution are supported by the results of different studies. However, Markow51 suggested that the evolutionary relationship proposed in these models are not the general concomitant of the evolutionary process although a consistent relationship having predictable value might exist in certain species groups. The relationship may depend on ecological and evolutionary history of a group and evolutionary events may occur differently in different groups. These models have also been criticized by Wasserman and Koepfer.52 Ehrman and Wasserman53 suggested that mode of mating preference alone can not be used to predict the direction of evolution as there may be more than one mechanism for producing asymmetrical mode of mating preference. Origin of reproductive isolating mechanisms According to the biological species concept, advocated by the founders of Modern Synthesis, ‘the question how new species evolve’ can be substituted by a more answerable question’ how reproductive isolation is established between populations’.54 Different theories have been proposed to explain how reproductive isolating mechanisms originate between populations. Muller55 suggested that reproductive barriers to gene exchange appear as a side effect of genetic divergence because populations adapted to different environments acquire genetic differences which lead to reproductive isolation. Thus, isolating mechanisms might originate as a consequence of random genetic drift or an accidental byproduct of genetic divergence in allopatric populations as they adapt to different environments.


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Since many genes are pleiotropic, such divergent populations might also exhibit behavioral differences. According to Carson,42 there is role of random genetic drift through founder effect in the origin of reproductive isolation which serves as barrier to gene flow. Thus reproductive isolation may originate allopatrically (or peripatrically) as a byproduct of reorganization of gene pool. Based on this principle, Cason42 has explained the evolution of Hawaiian species of Drosophila. It is interesting to note that there are more than 500 species of Drosophila found on Hawaiian Islands and their evolution has occurred through founder principle of Mayr41. Powell40 conducted laboratory experiments in D. pseudoobscura and based on his results, he extended evidence in support of flush-crash speciation theory of Carson.56 Strong assortative matings developed in cage populations of D. pseudoobscura which were passed through successive bottlenecks in population size. These studies also support Muller’s model regarding the origin of isolating mechanisms. Dobzhansky8,57 gave more importance to natural selection acting on appropriate genetic variability when the allopatric populations that has an incipient isolation become sympatric. When two allopatric populations become sympatric, natural selection acts to develop and strengthen the barriers to gene exchange between these populations whose hybridization results in reproductive wastage. Thus there is reinforcement of isolation by natural selection in sympatry. There are a number of examples available in Drosophila which support these models. As suggested by Dobzhansky, selection for premating isolation occurs in sympatry, it is expected that the degree of isolation would be stronger in sympatry than between allopatric populations of related species. A number of studies have been reported in Drosophila which corroborate Dobzhansky’s model. For example, results reported by Ehrman58 in semi-species group of D. paulistorum and by Wasserman and Koepfer59 in D. arizonensis and D. mojavensis. Sexual isolation appeared as a byproduct of natural selection between a population of D. mojavensis and its sibling species D. arizonensis.60 Sexual isolation has been induced by artificial selection between ebony and vestigial mutants of D. melanogaster.61 The degree of ethological isolation between different species/strains may be altered by artificial selection in the laboratory (see Chatterjee and Singh14). On the other hand, there are reports which extend evidence in favor of Muller’s model. There are cases which show that degree of behavioral isolation is stronger in allopatric populations than sympatric ones. This does not support the hypothesis of reinforcement of sexual isolation by natural selection. Similarly, results in this regard have been reported by different investigators.34,35,62 Singh and Chatterjee50 found high degree of isolation in isofemale lines than mass culture stocks of D. ananassae which also demonstrated that isolation in isofemale lines was caused due to random genetic drift resulting from founder effect.

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Conclusions Although Drosophila melanogaster was described in scientific literature in 1830 by German Entomologist JW Meigen63 and these flies were known to Ancient Greeks, they were used for the first time in experimental studies by FW Carpenter64 in 1905 to test reaction of these flies to light, gravity and mechanical stimulation and by W Castle65 in 1906 for studies on effects of inbreeding on fecundity. Results of these studies showed that it was suitable organism for experimental work and brought it to the attention of TH Morgan who used these flies for the first time for genetical studies and proposed the theory of linkage.66 In the experiments conducted by Sturtevant,67 non-random mating was detected due to differences in the vigor of mutant and wild type flies extending evidence for sexual isolation.68 These observations suggest that Drosophila was used for behavioral, genetical and evolutionary studies about 100 years ago. The researches carried out during the past 100 years concerning behavior, genetics and evolution in Drosophila support the Darwin’s theory of evolution which was main thesis of his book “On the Origin of Species” published 150 years ago. The importance and role of reproductive isolation in the process of cladogenesis was emphasized by Darwin which include temporal, habitat and behavioral barriers. This is evident from the following lines from his book: Finally, then I suppose that a large number of closely allied or representative species, now inhabiting open and continuous areas, were originally formed in parts formerly isolated; or that varieties became in fact isolated from haunting different stations, disliking each other, breeding at different times, and so as not to cross (see Stauffer69). Thus the origin of reproductive isolating mechanisms is an important event in the process of speciation which is evident from the work done in Drosophila. The foremost architect of synthetic theory of evolution and leading evolutionary geneticist of his time, Theodosius Dobzhansky70 remarked that “Nothing in biology makes sense except in the light of evolution”.

References 1. 2. 3. 4. 5. 6.

Darwin CR (1859) On the Origin of Species by Means of natural selection or the Preservation of favored races in the struggle for life. J Murray, London Dobzhansky T (1951) In: Genetics and the Origin of Species. 3rd edition, Columbia Univ Press, New York Mayr E (1963) In: Animal Species and Evolution. Belknap Press, Cambridge, MA Coyne JA, Orr HA (2004) In: Speciation. Sinauer Associates, Inc., Publishers, Sunderland, MA, Schluter D (2009) Evidence for Ecological Speciation and Its Alternative. Science 323: 737–741 Wagner M (1889) In: Die Entstehung der Arten Durch Raumliche Sonderang. Beno Schwalbe, Basel


171

7.

Jordan K (1905) Der Gegesatz zwischen geographischer und nichtgeographischer variation. Zeits Wiss Zool 83:151–210

8.

Dobzhansky T (1970) In: The Genetics of the Evolutionary Process. Columbia University Press, New York

9.

Mayr E (1970) In: Population, Species and Evolution. Harvard University Press, Cambridge, MA

10.

Strickberger MW (2000) In: Evolution. 3rd edition, Jones and Bartlett Publishers, Sudbury, MA

11.

Hartle BK, Hallgrimsson B (2008) In: Strickberger’ Evolution. 4th edition, Jones and Burtlett Publishers, Sudbury, MA

12.

Patterson JT, Stone WS (1952) Evolution in the genus Drosophila. Macmillan Company, New York

13.

Spieth HT, Ringo JM (1983) Mating behavior and sexual isolation in Drosophila. In: The Genetics and Biology of Drosophila. Ashburner M, Carson HL, Thompson JN (Eds.), Academic Press, New York

14.

Chatterjee S, Singh BN (1989) Sexual isolation in Drosphila. Ind Rev Life Sci 9:101–135

15.

Singh BN (1997) Mode of mating preference and the direction of evolution in Drosophila. Ind J Exp Biol 35:111–119

16.

Singh BN (2007) Reproductive isolation in Drosophila. Proc Natl Seminar evolutionary biology and biotechnology, Kolkata. Zoological Survey of India 87–100

17.

Zouros E (1989) Advances in genetics of reproductive isolation in Drosophila. Genome 31:211–220

18.

Singh BN (1994) Hybrid sterility and its genetic basis in Drosophila. Ind Rev Life Sci 14:3–20

19.

Paterson HE (1980) A comment on mate recognition system. Evolution 34:330–331

20.

Ewing AW (1969) The genetic basis of sound production in Drosophila pseusoobscura and D. persimilis. Anim Behav 17:555–560

21.

Elens AA, Wattiaux JM (1964) Direct observation of sexual isolation. Dros Inf Serv 39:118

22.

Patterson JT (1947) The insemination reaction and its bearing on the problem of speciation in the mulleri subgroup. Univ Texas Publ 4720:44–47

23.

Patterson JT, McDonald LW, Stone WS (1947) Sexual isolation between members of the virilis group of species. Univ Texas Publ 4720:7–31

24.

Dobzhansky T (1947) Effectiveness of intraspecific and interspecific matings in Drosophila pseudoobscura and D. persimilis. Am Nat 81:66–71

25.

Miller DD (1950) Mating behavior of Drosophila affinis and D. algonquin. Evolution 4:123–134

26.

Sturtevant AH (1920) Genetic studies on Drosophila simulans I. introduction. Hybrids with D. melanogaster. Genetics 5:488–500

27.

Wu CI (1992) A note on Haldane’s rule: Hybrid inviability versus hybrid sterility. Evolution 46:1584–1587

28.

Haldane JBS (1922) Sex ratio and unisexual sterility in hybrid animals. J Genet 12: 101–109

172


29.

White MJD (1973) In: Animal Cytology and Evolution. 3rd edition, Cambridge University Press, New York

30.

Bock IR (1984) Interspecific hybridization in the genus Drosophila. Evol Biol 18:41–49

31.

Bock IR (1978) The bipectinata complex: A study in interspecific hybridization in the genus Drosophila (Insecta: Diptera). Aust J Biol Sci 31:197–208

32.

Dobzhansky T, Spassky B (1959) Drosophila paulistorum, a cluster of species in status nascendii. Proc Natl Acad Sci USA 45:419–428

33.

Mishra PK, Singh BN (2005) Why hybrid males are sterile in Drosophila? Curr Sci 89:1813–1819

34.

Kaneshiro KY (1976) Ethological isolation and phylogeny in the planitibia subgroup of Hawaiian Drosophila. Evolution 30:740–745

35.

Arita LH, Kaneshiro KY (1979) Ethological isolation between two stocks of Drosophila adiastola Hardy. Proc Hawaiian Ent Soc 23:31–34

36.

Ahearn JN (1980) Evolution of behavioral reproductive isolation in a laboratory stock of Drosophila silvestris. Experientia 36:63–64

37.

Dwivedi YN, Singh BN, Gupta JP (1982) One-sided sexual isolation between Drosophila takahashii and D. pseudotakahashii. Experientia 38:318

38.

Ramachandra NB, Ranganath HA (1987) Mating preference between Drosophila nasuta nasuta and D. n. albomicans. Ind J Exp Biol 25:55–57

39.

Ramachandra NB, Ranganath HA (1994) Pattern of sexual isolation between parental races (Drosophila nasuta nasuta and D. n. albomicans) and the newly evolved races (Cytorace I and II). Ind J Exp Biol 32:98–102

40.

Powell JR (1978) The founder-flush speciation theory: An experimental approach. Evolution 32:464–474

41.

Mayr E (1942) In: Systematics and the Origin of Species. Columbia University Press, New York

42.

Carson HL (1971) Speciation and the founder principle. Stadler Genet Symp 3:51–70

43.

Kaneshiro KY (1971) Sexual isolation, speciation and the direction of evolution. Evolution 980(34):437–444

44.

Kaneshiro KY. Sexual isolation, and the direction of evolution in the biosystematics of Hawaiian Drosophilidae. Ann Rev Entomol 28:161–178

45.

Giddings LV, Templeton AR (1983) Behavioral phylogenies and the direction of evolution. Science 220:372–378

46.

Watanabe TK, Kawanishi M (1979) Mating preference and the direction of evolution in Drosophila. Science 205:906–907

47.

Patterson JT, Stone WS (1949) The relationship of novamexicana to the other members of the virilis group. Univ Texas Publ 4920:7–17

48.

Ayala FJ, Tracey ML, Barr LG, Ehrenfield JG (1974) Genetic and reproductive differentiation of the subspecies, Drosophila equinoxialis caribbensis. Evolution 28:24–41

49.

Singh BN, Dwivedi YN, Gupta JP (1981) Sexual isolation among three species of the Drosophila bipectinata species complex. Ind J Exp Biol, 19:898–900

50.

Singh BN, Chatterjee S (1985) Symmetrical and asymmetrical sexual isolation among laboratory strains of Drosophila ananassae. Can J Genet Cytol 27:405–409

51.

Markow TA (1981) Mating preferences are not predictive of the direction of evolution in experimental populations of Drosophila. Science 213:1405–1407


173

52.

Wasserman M, Koepfer HR (1980) Does asymmetrical mating preference show the direction of evolution? Evolution 34:1116–1124

53.

Ehrman L, Wasserman M (1987) The significance of asymmetrical sexual isolation. Evol Biol 21:1–20

54.

Sawamura K (1999) The origin of reproductive isolation: Biological mechanisms of genetic incompatibility. In: The Biology of Biodiversity. Kato M (Ed.), Springer-Verlag, Tokyo

55.

Muller HJ (1942) Isolating mechanisms, evolution and temperature. Biol Symp 6:71–125

56.

Carson HL (1975) Genetics of speciation. Am Nat 109:83–92

57.

Dobzhansky T (1940) Speciation as a stage in evolutionary divergence. Am Nat 74: 312–321

58.

Ehrman L (1965) Direct observation of sexual isolation between allopatric and between sympatric strains of different Drosophila paulistorum races. Evolution 19:459–464

59.

Wasserman M, Koepfer HR (1977) Character displacement for sexual isolation between Drosophila mojavensis and D. arizonensis. Evolution 31:812–823

60.

Zouros E, D’Entremont CJ (1980) Sexual isolation among populations of Drosophila mojavensis: response to pressure from a related species. Evolution 34:421–430

61.

Crossley S (1974) Changes in mating behavior produced by selection for ethological isolation between ebony and vestigial mutants of Drosophila melanogaster. Evolution 28:631–647

62.

Carracedo MC, Casares P, Sanmiguel E (1987) Sexual isolation between Drosophila melanogaster females and D. simulans males II influence of female receptivity on hybridization. Genome 29:334–339

63.

Meigen JW (1830) Systematische Beschreibung der bekannten europaischen zweiflugeligen insekten. Sechster Theil Schulz, Hamm

64.

Carpenter FW (1905) The reaction of the pomace fly (Drosophila ampelophila) to light, gravity and mechanical stimulation. Am Nat 39:157–171

65.

Castle WE (1906) Inbreeding, crossbreeding and sterility in Drosophila. Science 23:153

66.

Morgan TH (1911) Random segregation versus coupling in Mendelian inheritance. Science 34:384

67.

Sturtevant AH (1915) Experiments on sex recognition and the problem of sexual selection in Drosophila. J Anim Behav 5:351–366

68.

Parsons PA (1973) Behavioral and ecological genetics: A study in Drosophila. Clarendon Press, Oxford

69.

Stauffer RC (1975) Charles Darwin’s natural selection, being the second part of his big species book written from 1856 to 1858. Cambridge University Press, Cambridge

70.

Dobzhansky T (1973) Nothing in biology makes sense except in the light of evolution. Am Biol Teach 35:125–129

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12 Adaptive radiation and insects V.V. Ramamurthy* and Asha Gaur** Division of Entomology, Indian Agricultural Research Institute, New Delhi - 110012, India *[email protected], **[email protected]

Abstract: The fundamental aspect of principle of divergence has emerged in different spheres of evolutionary biology as propounded by Charles Darwin. Adaptive radiation is an aspect of evolutionary biology encompassing microevolution and macroevolution, explaining the principles of divergence, taking into account the theories of natural selection and struggle for life. In addition to the classical example of Darwin’s finches in the Galapagos Islands, there are many others, of which the phytophagous insects are a prominent one that could be examined in detail to explain the different paradigms of adaptive radiation. With this example the evolutionary process of natural selection can be explained in its totality when one ancestral species gives rise to two or more species with different kinds of feeding characteristics and other core biological phenomena. There are many criteria that can be adopted to establish that adaptive radiation has taken place and all these focus toward aspects of microevolution and macroevolution. Many times the ecological, reproductive, stratigraphic, barriers due to abiotic factors are the ones that are focused toward establishing the facts on adaptive radiation. There are evidences that pluralistic diversity in insects and their species, genetic and ecological diversity is mainly due to successful adaptive radiation. The macroevolutionary patterns on insect host plant evolution are enormously varied due to the special features of wings and metamorphosis in addition to characteristics of migration and dispersal. The inordinate fondness for beetles and other phytophagous insects exhibited by nature and discussed in detail is mainly due to adaptive radiation as explicit through sister group analysis done in the large insect groups. It is also established that the evolutionary rates in these insect groups are much enormous as exhibited by the Chrysomeloidea and Curculionoidea. There are examples like the cotton stem weevil, other gall-inducing Coleopterans and other insects wherein the coevolution on the insect plant surface had been brought out and integrated with molecular phylogenetic studies to establish the bridge between microevolution and macroevolution as envisioned by Charles Darwin. These are shedding light on the underlying causes of the patterns of diversification in phytophagous insects. It is very clear that such efforts are providing the ultimate appraisal of Darwin’s bridge between microevolution and macroevolution as components of adequate radiation. All these finally testify that the treatise ‘On the Origin of Species’ by Darwin is a living document that contains wealth of ideas, which if subjected to modern synthesis and innovative appraisal can provide multiple answers to many intriguing questions in V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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evolutionary biology, especially those relating to ‘inordinate fondness for beetles’ and the adaptive radiation in the phytophagous insects. Keywords: Adaptive radiation, Coleopterans, Coevolution, Criteria, Insects, Phytophagy, Sister group analysis

Introduction The fundamental truth of Charles Darwin’s principle of divergence has emerged in different facets of evolutionary biology. There are many evolutionary processes in the form of character displacement or some models of sympatric speciation, which had been discovered independently in different contexts over a century after the publication of ‘On the Origin of Species’ by Darwin.1 There is compelling evidence for biotic interactions in at least some extinction events, and close relationship between divergence and extinction. These find enough support for Darwin’s proposals especially as a viable link between microevolution and macroevolution. Understanding of microevolution and the macroevolution, and the bridge between these is possible only through integration of ecology, evolution and the role of history in shaping the diversification or decline of lineages. There is a need for increased integration between these fields to build a bridge between microevolution and macroevolution, to reinvent the Darwin’s mechanism of the turnover of species resulting from their evolution and interactions. The historical patterns of diversification of lineages which can now be mined from molecular phylogenies, are shedding light on the underlying causes of these patterns. There are compelling evidences that Darwin has to be revisited again to understand the evolution and the adaptive radiation. Needless to state the insects, in particular, the phytophagous ones will provide the optimum examples and case studies for such discoveries.

Adaptive radiation Adaptive radiation is an evolutionary process by which a single ancestral species evolves into several new species within a relatively short time in a specific geographic domain. The new species that evolve have evolutionary advantages to occupy an ecological niche and even gets specialized for a definite role in a habitat. Thus adaptive radiations describe the process by which a group of organisms adapts to diverse variety of situations. Naturally this involves coevolution with other organisms in the geographical domain focused toward their feeding and other essential life processes. This also encompasses the changes that are envisaged in the changed ecological environments, other life processes triggering dispersal, processes of natural selection, competition and struggle for survival. According

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to Darwin,1 the varieties of forms that are observed arose because “As many more individuals of each species are born than can possibly survive; and as consequently, there is a frequently recurring struggle for existence, it follows that any being’s if it vary however slightly in any manner profitable to itself, under the complex and sometimes varying conditions of life, will have a better chance of surviving and thus naturally selected. From the strong principle of inheritance, any selected variety will tend to propagate its new and modified form”. This explains the adaptive radiation very well even in the current contexts.

Evolution and adaptive radiation An evolutionary process by which new species emanate through natural selection, encompasses changes in the traits of organisms or their populations over time so that the modified forms are successful and establish themselves in an environment. These changes occur as a result of genetic mutations leading to the changes getting passed on to surviving generations. The populations or groups of populations result in new species with better advantage in the specific environment leading to a successful adaptive radiation. The mechanisms which lead to this kind of adaptive radiation include the divergence due to geographic isolation or due to other causes including inherent, genetic and or extraneous and other environmental factors. The divergence, when exhaustive and the barriers becoming exclusive, leads to the process of speciation being successful and adaptive radiation leading to modifications takes place. These modifications are endured as a result of the need for survival and struggle for existence in an ecological niche. Further these are focused toward profitability to a species under the complex and varying conditions of life through a process of natural selection as explained by Darwin.1 Also such an adaptively radiated species tends to propagate itself in its modified form for its sustainability, and the latter is mainly through its efforts for coevolution with other organisms in its selected habitats.

Criteria for adaptive radiation The modifications in the genetic, biological and ecological characteristics leading to the adaptive radiation have their own criteria to establish that it has taken place. There are many criteria that can be followed and these include the factors and barriers leading to reproductive isolation and ecological isolation. In any study resorting to establish that adaptive radiation has taken place, an insight into the critical areas of genetic or other kinds of reproductive isolation, in particular those of the barriers that result in the isolation, is essential. The changes in the genetic, reproductive and ecological aspects of populations and species in an ecological niche and the degree to which these changes elicit a suitable response in the biological phenomena and characteristics will be the yardstick for evaluating the essential criteria.

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The exploitation of ecological niches and their extent is an important factor toward adaptive radiation. Especially islands or isolated pockets that have specific sets of environmental conditions will trigger this kind of exploitation and the populations or groups of species that are successful in this endeavour will undergo adaptive radiation. The physical features such as water and other stratigraphic factors will lead to barriers, isolation of populations, and their adaptive radiation. In addition, there will be dispersal barriers due to factors like mountains, which are likely to radiate the populations at or near the limits of the range of dispersal. The rapid genesis of new species through sympatric or allopatric speciation is yet another criterion that can be adopted to evaluate species radiations and their divergence. In case of phytophagous species, the coevolution, in particular, in terms of pollination relationships becomes important. In species with broad geographical ranges, and wide dispersal abilities in view of large and wide habitat range and growth forms, the adaptive radiation can be gauged through the habitat ranges and their extent. Many biotic factors within the range of populations of a species can also be used as criteria. Especially when these factors relating to biology, development, growth and reproduction, and variations in feeding and other behavioral characteristics, which are crucial for the life of an organism, get altered, there is an upheaval in the biological aspects leading to radiation of the important biological processes. Hence, an insight into these characteristics and their dynamics will be important criteria for evaluating adaptive radiation. These biological changes will imply changes in the ecological aspects, and the biotic and abiotic factors when examined will provide the criteria for evaluating the origin and maintenance of adaptive radiation.

Adaptive radiation and Charles Darwin Adaptive radiation as implied by Charles Darwin through his classical studies on the South American finch like birds at the Galapagos Islands is a concept that can be applied more usefully. It is a concept defining the way in evolution of variety of species taking place from a single ancestral species. It is a process by which variety of species develop from their ancestral species wherein each gets adapted to requirements of new way of life. Most of the examples like the one explained by Darwin are over relatively short periods of time, when a closely related group of species has radiated in areas recently colonized by the ancestors of the group. The examples include Darwin’s finches on the Galapagos Islands, the honey creepers of the Hawaiian Islands, the cichlids of the eastern African lakes and the fruit flies of the Hawaiian Islands. Explaining one such example will elaborate the adaptive radiation in its entirety and the Darwin’s finches is a classical one (Fig. 1). About 1000 km west


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Figure 1. Few Darwin’s finches in the Galapagos Islands

of Ecuador is the Galapagos archipelago, which is known for surfacing a few million years ago. This archipelago was colonized by South American finch like birds probably 0.5 to 1.5 million years ago. This ancestral species gave rise to 13 duly recognized modern species of birds on the Galapagos. These 13 modern species have been tested by showing that all these are derived and closely related to one another than to any related species outside the islands. These members of the modern bird community have thus evolved from a single ancestral species rather than from multiple invasions of the island, with one species getting derived from each invasion. It has been found that these 13 species are adapted to eat different food, with related variations in their beak forms. Some of these have larger beaks while others have smaller, those which have them larger are as a result of modification required for cracking nuts, while those with smaller beaks are as a result of modifications required for gathering seeds with their beaks. One of these is a “woodpecker” finch, which lacks a true woodpecker’s powerful beak. Its beak performs the functions similar to that of a true woodpecker through using small sticks to probe for insects in a tree hole. Thus one of its species has individually adapted for a specific type of feeding and food through behavioral, functional and structural adaptations, and thereby behaves like a true woodpecker. Five of the 13 Galapagos finches are insectivorous, one is a vegetarian tree finch, three are ground dwelling seed eaters, two feed on cacti and two are warbler like. All these can be revealed through their modifications in their beaks and attributing a food or feeding behavior is easily possible in view of adaptation to different foods hence it is called adaptive radiation in terms of concept.

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Principle of divergence A single ancestral species evolves into a variety of forms through adaptations to different types of food, that too in the shortest possible time. Naturally, there are extreme similarities among these modern species which are fundamentally unique, owing to their evolution from a common ancestor resulting in a homologous group of organisms in a habitat as that existing in Galapagos archipelago. Thus in the process of adaptive radiation, the new species are evolving, and these evolve apart from each other, and the evolution is through divergence of characters. While diverging due to the new requirements, the ancestral species splits into two or more modern, derived species. This lineage splitting is a unique phenomena as regards speciation and it is opposite of convergence. The splitting leads to divergence of species and evolutionary lines, necessitated mainly due to the change in the requirements of food and feeding, or behavioral, ecological and other biological characters. The major cause is thus achieving less competition for food, and derives an advantage through a process of natural selection. Charles Darwin refers to this as “the principle of divergence” and this concept explains the tree of life that is the fundamental of evolutionary classification.

Adaptive radiation and insect biodiversity The biodiversity comprising of species, genetic and ecological diversity when analyzed indicates that insects are the most diverse and are the characteristic examples explaining adaptive radiation. This adaptive radiation stems from the fundamental fact that the insects and their host plants represent the major part of terrestrial diversity. Insect plant coevolution is the most discussed of all examples of diversification of living organisms, as phytophagous insects account for approximately 40% of all described insects. By far the most influential model of insect plant diversification is based on the hypothesis of insect plant coevolution, leading to interpretations of adaptive radiation as a life phenomenon in insects.2 The macroevolutionary patterns on the insect host plant evolution especially toward plant defense and host affiliations provide a model for explanations on the adaptive radiation. This is absolutely empirical as insects and plants have diversified over roughly the same intervals in the geological eras in which the flowering plants have evolved in consonance with the insects, especially toward pollination, seed dispersal and other activities. The diversification of insects, as in the case of Darwin’s finches in the Galapagos Islands, had happened due to the requirements of adaptation and coevolution consequent upon changes in herbivory and phytophagous feeding behavior. It has been documented that rather than accumulating herbivores at a rate proportional to their geographic area of distribution or biomass, some plant


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groups pose apparent chemical barriers to potential herbivore colonists, and seem accessible to relatively few insect lineages, possibly preadapted by use of chemically similar or related host plants.3 It has also been observed that longterm success of such coevolutionary associations and adaptive radiations leading to these patterns are influenced by ecological conditions in a long term. The inordinate fondness for beetles as explained by the well-known biologist JBS Haldane is a case to be discussed as it has been seen that diversity of insects and plants especially in tropics is an extraordinary example of successful adaptive radiation. This can be seen from the data on the world totals of described living species in the different orders of insects4 (Table 1). Some details of diversification in insects, with a focus on the phytophagous groups, are presented herein.

Metamorphosis and wings in adaptive radiation of insects The fundamental aspects of transition in insects and their adaptation have hovered around the differences in metamorphosis and wings in addition to score of other specific characters. In case of insects with complete metamorphosis, this transition has been focused toward resource partition wherein an insect will base its different components of life, namely the ones required for growth and development separated from those concerned with reproduction. The ability of the holometabolous insects with larval stages entirely differing from the adults in both structure and habits is one such adaptive radiations leading to successful and efficient utilization of resources especially food and shelter. In fact the development of tunneling behavior as an adaptive radiation in these insects is an ecological advantage and specialization toward an exclusive habitat. The development of wings and associated modifications in the legs is an additional feature which adds to the triggering of adaptive radiation toward their dispersal and migration in occupying specific ecological niches and to survive the process of natural selection. The evolution of plants has presented a new adaptive zone for insects to exploit. As insects have evolved means to exploit plants as food, through their varied mouth parts and their elaborate modifications, plants have evolved counter measures which led to greater diversification of plants. When this diversification has happened to the plants in the same geological era in which the insects have also undergone their diversification, the processes had got related and it had become mutualistic in terms of coevolution through adaptive radiation. In fact the successful pollination and seed dispersal of many flowering plants has coevolved along with insects as successful pollinators and movers. Despite the plants evolving defenses the insects have learnt to coexist through physiological adaptations for feeding on plants protected by toxic secondary compounds. The insects like tobacco aphid and the tobacco hornworm are some such examples, where there had been long-term effects of coevolution

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Table 1 World totals of described living species of Class Insecta Order20

Described Species4

Archaeognatha

504

Zygentoma

527

Ephemeroptera

3046

Odonata

5680

Dermaptera

1967

Notoptera

39

Plecoptera

3497

Embiodea

458

Zoraptera

34

Phasmatodea Orthoptera

2853 23,616

Mantodea

2384

Blattaria

4565

Isoptera

2864

Psocoptera

5574

Phthiraptera

5024

Thysanoptera

5749

Hemiptera

100,428

Coleoptera

359,891

Raphidioptera

225

Megaloptera

337

Neuroptera

5704

Hymenoptera Mecoptera

144,695 681

Siphonaptera

2048

Strepsiptera

603

Diptera

152,244

Trichoptera

12,868

Lepidoptera

156,793

TOTAL

1,004,893

as a result of adaptive radiation taking place rather more swiftly, and in a subtle manner. It has been demonstrated that phytophagy is an evolutionary innovation especially in beetles, when famous biologist JBS Haldane had stated that God


Figure 2. Adaptive radiation in major phytophagous insect orders

183

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has an inordinate fondness for beetles. There had been multiple processes of coadaptation and cospeciation in these coevolutionary phenomena, taking place at the microevolutionary and macroevolutionary aspects. These aspects lead to a variety of forms from a single ancestral stock often after colonizing an island group or restricted specialized habitats or through entering a new adaptive zone.

Adaptive radiation and phytophagy The adaptive radiation in phytophagous insects if explored will indicate that plants initially represented a new unexploited adaptive zone for insects. With the flowering plants getting diversified in the paleozoic era, this new unexploited adaptive zone became the foreground for the transformation of current day highly evolved insects especially the endopterygotes which have acquired a tunneling behavior for feeding or other essential biological purposes in their larval stages. Also, the resources got partitioned for the purpose of growth and development and delineated from those required for their reproduction in the adult stage. In fact, in these insects which have a pupal and transition stage, this delineation gets demarcated and the adaptive zone put forth by the flowering plants get well exploited toward the diversity. There are evidences that accelerated diversification across many independent insect groups have occurred and adaptive shifts had been repeatedly associated with phytophagous insect groups, in particular the Chrysomeloidea and Curculionoidea, which are the most successful of the herbivorous groups.

Sister group analysis and adaptive radiation Examples of adaptive radiation are enormous in insects and sister group analysis of adaptive radiation had proved this. When a lineage moves from an ancestral adaptive zone to a new one, adaptive shifts occur and such shifts are enormous in the most dominant phytophagous insect groups especially the coleopterans. It has been proved through analysis of sister groups that the differences in diversity in these could be used as a yardstick for evaluating adaptive radiation. If the sister group that has undergone adaptive shift is consistently more diverse than the original sister group that remains in the original adaptive zone, it can be confirmed that the adaptive radiation has been successful in causing divergence. It had been shown that higher plant feeding is found in nine orders of insects and it has probably arisen at least 50 times in just the extant forms with known habits (Fig. 2). The present phylogenetic identification allows identification of 13 pairs of sister groups, one of which feeds on higher plants and the other which does not5 (Table 2). It has also been conclusively established that the phytophagous lineage is more diverse than its presumed nonphytophagous sister group. Thus the phytophagous feeding habit is associated with increased diversification due to the


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Figure 3. Darwin’s view of microevolution and macroevolution1

processes of cospeciation and a coadaptation representing the macroevolutionary and microevolutionary processes. Most plant and insect ecologists believe that plant secondary compounds have evolved as defense against herbivory by phytophagous insects and that insects in turn have evolved behavioral and physiological adaptations to counter the effects of toxic secondary plant compounds. Such coadaptive responses between insects and plants also may be reflected in the extent of cospeciation of these groups.

Microevolution, macroevolution and adaptive radiation The relationship between microevolution and macroevolution must be understood to explain adaptive radiation. The microevolution or adaptation is always linked with macroevolution or speciation, phylogeny of the organisms above species level and the development of complex organs to effectively reach the diversification and achieve divergence. Many times this linkage or bridge is never understood by the evolutionary biologists as it cannot be explored in one’s lifespan and hence hypotheses are only possible. Hence, it is a major source of conflict between science and religious beliefs.6 Biologists often forget that Charles Darwin offered a way of resolving this issue, and his learned views hold good now and are required to be revisited. For Darwin, the interactions that define the struggle for existence and shape how organisms evolve were diverse, including competition, predation, parasitism, disease and pollination.

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Figure 4. Cotton stem weevil Pempherulus affinis (a) damage; (b) gall; (c) grub feeding on the internal tissues; (d) grub; (e) pupa; (f) adult

All these hold good for the phytophagous insects and as cause for their adaptive radiation Darwin illustrated the combined action of his principle of descent with modification, the principle of divergence and extinction in his ‘On the Origin of Species’ and this explains the link between microevolution and macroevolution1 (Fig. 3). Many of the resultant organisms go extinct, but some persist, becoming modified and improved descendants as in the case of many phytophagous insect groups of the present day. These always strive for their perpetuation so that they drive some of their close relatives to extinction and to fill the unoccupied insect plant interface, leading to cladogenesis where the splitting of one ancestral species leads to more than one descendant. Darwin had rightly propounded that the processes of such diversification and extinction can explain the gaps that are seen among the living species.


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Table 2 Sister group diversity comparisons on independent phytophagous insect lineages5 Primitively phytophagous lineage Taxon

Sister group

Approximate Taxon no.of species (phytophages only)

Hemiptera

Joppeicidae

Tingidae (? + Thaumastocoridae)

1,800 15 ≤1,815

Miridae

10,000 Isometopidae

Trichophora

>5,000 Aradidae

Approximate no. of species (excluding phytophages)

Sign of difference in diversity 1

+

60

+

1,000

+

7 1,200 190 ≤1,397

+

400

+

Coleoptera Elateridae + Cebrionidae

Scarabaeidae sensu lato, minus Geotrupinae Aphodiinae, and Scarabaeinae

9,000 Cerophytidae and/ 170 or 9,170 Eucnemidae and/or Throscidae 14,000 Geotrupinae or Aphodiinae/ Scarabaeinae

3,200

Languriinae

410 Lobarinae+ Erotylidae–

200 1,500 1,750

Epilachninae

700 Coccinellini

250

+

≤10,000

+

10,000 Panorpida

80,000

_

140,000 Trichoptera

7,000

+

80

+

1,733

+

200

+

Phytophaga Hymenoptera (Symphyta only) Lepidoptera

130,000 non-phytophagous Cucujoidea

Diptera Chloropidae Oscinellinae +Chloropinae

1,350 Siphonellopsinae 860 2,210

Tephritidae sensu stricto

4,000 All or part of nonphytophagous Tephritidae sensu lato

Agromyzidae

2,000 Clusiidae

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Figure 5. Adaptive radiation through host plants – cotton stem weevil case study

Adaptive radiation and evolutionary rates Phytophagous insects, which could be used as a classical model for explaining the adaptive radiation, are also unique models that can be utilized to explain evolutionary rates. In case of well-known phytophagous insect groups namely Coleoptera, in particular Chrysomeloidea, it has been shown that there are consistently different rates of evolutionary changes involving different kinds of characters of biological and ecological significance. These affect the cospeciation and coadaptation in relation to a particular host plant and also these changes become consistent with passage of time. Depending upon the rates of host use, the rates of evolutionary change varies and in fact this variation on the rates of change leads to diversification and divergence.7 Specificity on host tissues are well known in plant feeding insects, particularly in the diverse orders Lepidoptera,8,9 Coleoptera,10 and Hymenoptera.11,12 It has also been shown that this specificity extends to the whole host species leading to specialization and this has been widely accepted. There are many such examples of host specializations due to the specific behavioral and physiological adaptations.13,14

Gall-inducing Coleoptera and adaptive radiation Cotton stem weevil – a case study An ideal example that could be discussed as a case study in the context of biogeography and adaptive radiation in gall-inducing Coleoptera is that of cotton


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Figure 6. The diversity of larval stages in Coleoptera16

stem weevil Pempherulus affinis (Fig. 4). This species is widely distributed in India, from Kerala in the far south to Rajasthan in the north, and from Tamil Nadu and Andhra Pradesh in the east to the Gujarat in the west, as far as the Indian subregion goes. P. affinis is also known from adjacent countries, viz. Thailand and Myanmar, extending up to the Philippines, indicating its resourcefulness in terms of populations specializing on various species and varieties of gossypium cultivated in these areas and countries. It has also been documented that this species is adapting to other Malvales specific to this region namely Malvastrum spp., Sida spp., and other such weed plants found in the cotton agroecosystems. P. affinis is a typical example for a gall-inducing Coleoptera, which has diversified itself adapting to adverse conditions in the absence of its most preferred host plant viz., cotton, the gossypium spp., in spite of its specialized gall-inducing

190


Table 3 Contrasts of coleopterans associated with gymnosperms vs angiosperms10 Contrasts Primitively gymnosperm associated taxon 1 Nemonychidae

2 3

OxycoryninaeAllocoryninae AseminaeSpondylinae

4

Palophaginae

5

OrsodacninaeAulacoscelidinae

Diversity Primitively angiosperm associated taxon 85 AttelabinaeRhynchitinae, Apioninae, CurculionidaeRhynchophoridae 30 Belinae 78 Lepturinae, LamiinaeCerambycinae 3 MegalopodinaeZeugophorinae 26 Remaining Chrysomelidae

Diversity

44002

150 25000

400 33400

behavior. Detailed explorations in the region extending up to Thailand in the east and beyond Gujarat in the west may reveal its distribution extending toward other regions of the Orient and the adjoining Palearctic regions. This is due to the fact that their documented host plants have an extensive distribution in these regions (Fig. 5). Coleoptera and angiosperms feeding While indexing 22 species of Buprestidae and 15 species of Cerambycidae it was concluded that gall induction occurs almost exclusively in the most advanced superfamilies Chrysomeloidea and Curculionoidea.15 As far as Curculionoidea are concerned, gall-inducing behavior is restricted to the phanerognathous groups only, which have their feeding and oviposition behavior characterized by scooping, digging, burrowing or mining activities. The evolutionary trends discussed indicate that gall-inducing habit increases in the course of evolution of the Coleoptera, and has become established in three phyletic lineages namely Buprestoidea, Chrysomeloidea, and Curculionoidea.15 The phylogeny of the Phytophaga, the largest and oldest radiation of herbivorous beetles was reconstructed, using molecular data.10 The results of these analyses have been used to interpret the role of angiosperms in beetle diversification. It has been shown that repeated origins of angiosperm feeding beetle lineages are associated with enhanced rates of beetle diversification, indicating a series of adaptive radiations. Collectively


191

these radiations represent nearly half of the species in the order Coleoptera and a similar proportion of herbivorous insect species. These inferences will apply equally to gall inducing Coleoptera of the Orient and eastern Palearctic. It was concluded that there has been disproportionate rise in the diversity of post Cretaceous phytophagous beetles reflecting the likelihood of exponential rise in angiosperm diversity.10 Five independent contrasts of Coleoptera groups associated with gymnosperms versus angiosperms when analyzed have yielded a positive relationship in favour of the hypothesis that angiosperm feeding is associated with enhanced diversity (Table 3). It has also been shown that the addition of remaining (mostly weevils) subfamilies, not yet sequenced, will bring the total number of species to 135,000. The current affiliations of the oldest beetle lineages with preangiospermous seed plants support the hypothesis that these lineages retain affiliations that were formed early in the Mesozoic, before the diversification of flowering plants. The diversification of phytophagous beetles, thus, is consistent with the coevolutionary models that ascribe differences in the present diversity of insect and plant groups to evolutionary changes or characters (which affect their ecological interactions).2,3 Combined evidences from phylogenetic estimates and from the fossil record show a pronounced conservation in the evolution of beetle plant associations, which is important for the implication that plants might escape herbivory via key innovations. Thus gall inductions or other associations of coleopterans with plant galls might be concluded as such innovations toward coevolution. Curculionidae and Chrysomelidae proliferation of life history traits Correlated with angiosperm feeding is the proliferation of life history traits, in the Curculionidae and Chrysomelidae. In contrast with the strobilus feeding of conifer and cycad associated ancestors, diversification of the subfamilies that attack flowering plants has been accomplished by larval folivory, leaf mining and seed and root feeding in addition to gall induction, which exemplify the concept of adaptive radiation. The diversity of the larval stages of the coleopterans is a testimony to this fact16 (Fig. 6). This fact can be discussed in the light of the propositions that most of the pollen and nectar feeders are generalists, whereas gall-inducing insects are specialists and remain tied closely to their food plants.17 The specialized gall-inducing habit among insects is considered to have arisen from leaf mining Diptera and Microlepidoptera, which, over time, are purported to have sought ‘new’ food sources in concealed, and thus, ‘protected’ environments. This proposition derives support from the fact that gall-inducing habit originated from the behaviour of the tunneling Lepidoptera and Coleoptera. The success of gall-inducing Coleoptera like the other non gall inducing Coleoptera seems to have been enabled by the rise of flowering plants,

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and through many different evolutionary routes, in addition to maintaining the conservative host plant associations. These propositions hold good for the gall inducing and gall associated Coleoptera found in the Oriental and eastern Palearctic regions as well. Further exploration of the ecology and behavior of gall inducing Coleoptera will no doubt provide new insights into the evolution of this taxon, in particular throwing light on the coevolution between the gall inducers and their host plants, in these biogeographic regions. Gall-inducing coleopterans and their coevolution Taking into account the correlation between the richness of gall inducing insects and their host plants, the current estimated number of gall inducing insect species ranges between 21,000 and 210,000, with an average of 132,930 species. Gall inducers represent a gap in the taxonomic knowledge especially among insects in the tropical regions of the Orient and eastern Palaearctic. Although there had been several attempts to bridge this gap, it has not been achieved in its entirety, especially with regard to Coleoptera, which is one of the largest insect groups. Its members have interactions other than gall induction too, unlike other insects, due to their specialized feeding and reproductive behaviours. Under many situations it is difficult to delineate their specific roles and understand them, which contribute to widening of the gaps in the knowledge. Also, there are many species which are economically important in view of the proliferation by way of gall induction. Many species interact with other gall inducing organisms, forming an ecological community. Due to this complexity, many attempts to interpret the evolution of gall inducing Coleoptera, especially at the higher taxonomic levels have remained inconclusive. Chrysomeloidea and Curculionoidea: evolutionary trends Gall induction occurs almost exclusively in the most advanced Chrysomeloidea and Curculionoidea. The evolutionary trends indicate that gall inducing habit seems to increase in the course of evolution of the Coleoptera, and became established in three phyletic lineages namely Buprestoidea, Chrysomeloidea and Curculionoidea. Elucidation of the role of angiosperms in beetle diversification have shown that repeated origins of angiosperm feeding beetle lineages are associated with enhanced rates of beetle diversification. Groups associated with gymnosperms versus angiosperms, when analyzed yielded a positive inference in favour of the hypothesis that angiosperm feeding is associated with enhanced diversity. These inferences equally apply to the gall inducing coleopterans. The diversification of phytophagous beetles is consistent with the coevolutionary models which ascribe differences in the present diversity of insect and plant groups to their ecological interactions. Also there is a pronounced conservation


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in the evolution of beetle plant associations. This is important for the implication that plants might escape herbivory via key innovations. Gall inductions as coevolutionary innovations Gall inductions or other associations of coleopterans with plant galls might be concluded as innovations toward coevolution. The specialized gall inducing habit among insects is considered to have arisen from leaf mining Diptera and Microlepidoptera which over time have adapted themselves toward achieving efficiency in resource utilization or partition. This proposition derives support from the fact that gall inducing habit could have originated from tunneling behaviour as a novelty in the metamorphosis of a holometabolous insect. The success of gall inducing coleopterans thus seems to have been enabled by the rise of flowering plants, and through many different evolutionary routes, in addition to maintaining the conservative host plant associations. The information accumulated on the Coleoptera as gall inducers and interactors in galls has shown that such evolutionary behaviour is true with regard to the fauna of the Oriental and eastern Palearctic regions too. The little information that has been brought out by the present contribution indicates that tremendous scope exists for such explorations on the gall inducing coleopterans. No doubt such attempts will provide new insights into the evolutionary information, in particular toward the coevolution between the gall inducers and their host plants.

Conclusions The concept of reproductive isolation being the central part for the biological species derives its merit from the principle of divergence propounded by Darwin. Especially in case of phytophagous insects and their adaptive radiation, the fundamental truth of his principle of divergence has emerged in different aspects of their diversification at the insect plant interface as has been demonstrated in the phytophagous Coleoptera and in the case of gall inducing coleopterans and other insects by different workers.10,17,18 The compelling evidences now emerging in terms of molecular ecology in case of insect groups like Chrysomeloidea and Curculionoidea by the far the largest of the herbivorous groups of animals as explained in terms of their ecological interactions and fossil evidences on coevolution have proved that Darwin’s principle of divergence can be discovered independently in different contexts, especially in these large insect groups. These interactions leading to evolutionary changes in the form of microevolution and macroevolution and coupling of information gathered through fossil evidences on insects and plants finds enough support for Darwin’s ideology and provides the missing link between the cospeciation and coadaptation, and also convincingly explain the diversification as a result of adaptive radiation.

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The information about the historical aspects of adaptive radiation in insects in terms of insects and flowering plants diversification in the different geological eras through fossil evidences and coupling of these with historical patterns of diversification of lineages mined now through molecular phylogenies are shedding light on the underlying causes of the patterns of diversification in phytophagous insects. It is very clear that such efforts are providing the ultimate appraisal of Darwin’s bridge between microevolution and macroevolution as components of adequate radiation. All these finally testify that the ‘On the Origin of Species’ by Darwin is a living document that contains wealth of ideas, which if subjected to modern synthesis and innovative appraisal can provide multiple answers to many intriguing questions in evolutionary biology especially those relating to ‘inordinate fondness for beetles’ and the adaptive radiation in the phytophagous insects.

References 1.

Darwin C (1859) On the origin of species by means of natural selection or the preservation of favoured races on the struggle for life. John Murray, London

2.

Ehrlich PR, Raven PH (1964) Butterflies and plants: a study in co-evolution. Evolution 18:586–608

3.

Farrell BD, Mitter C (1994) Adaptive radiation in insects and plants: time and opportunity. Am Zool 34(1):57–69

4.

Foottit RG, Adler PH (2009) Insect biodiversity. In: Science and Society. John Wiley and Sons (Eds.), West Sussex, UK

5.

Mitter C, Farrell B, Weigmann BM (1988) The phylogenetic study of adaptive zones: Has phylogeny promoted insect diversification? Am Nat 132:107–128

6.

Reznick DN, Ricklefs RE (2009) Darwins bridge between microevolution and macroevolution. Nature 457:837–841

7.

Farrell BD, Sequeria AS (2004) Evolutionary rates in the adaptive radiation of beetles of plants. Evolution 58:1984–2001

8.

Powell JA (1980) Evolution of larval food preferences in micro Lepidoptera. Ann Rev Entomol 25:133–159

9.

Powell J, Mitter C, Farrell BD (1988) Evolution of larval food preferences in Lepidoptera: In: Handbook of Zoology. Evolution, systematics and biogeography. Kristensen N (Ed.), De Gruyter, New York

10.

Farrell BD (1998) Inordinate fondness explained: why are there so many beetles? Science 281:555–559

11.

Shaw SR (1998) Euphorine phylogeny: The evolution of diversity in host utilization by parasitoid wasps (Hymenoptera: Braconidae). Ecol Entomol 13:323–335

12.

Belshaw R, Quicke DLJ (2002) Robustness of ancestral state estimates: evolution of life history strategy in ichneumonoid parasitoids. Syst Biol 51:450–477

13.

Bernays EA (2001) Neural limitations in phytophagous insects: implications for diet breadth and evolution of host affiliation. Ann Rev Entomol 46:703–727


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14.

Via S (2001) Sympatric speciation in animals: the ugly duckling grows up. Trends Ecol Evol 16:381–390

15.

Korotyaev BA, Konstantinov AS, Lingafelter SW, Mandelshtham MY, Volkovitsh MG (2005) Gall inducing Coleoptera. In: Biology, Ecology and Evolution of Gall-Inducing Arthropods. Raman A, Schaefer CW, Enfield WM (Eds.), Science Publishers Inc., New Hampshire

16.

Boving AG, Craighead FC (Eds.) (1931) In: An Illustrated Synopsis of the Principal Larval Forms of the Order Coleoptera. The Entomological Society, Brooklyn, New York

17.

Raman A (2007) Insect induced plant galls of India: unresolved questions. Curr Sci 92:748–756

18.

Raman A, Burckhardt D, Harris KM (2009) Biology and adaptive radiation in the gall inducing Cecidomyiidae on Mangifera indica in the Indian subcontinent. Trop Zool 22:27–56

19.

Ramamurthy VV (2007) Faunistic, ecological, biogeographical and phylogenetic aspects of Coleoptera as gall inducers and associates in plant galls in the Orient and eastern Palearctic. Orient Ins 41:93–119

20.

Grimaldi DA, Engel M (Eds.) (2005) In: The Evolution of Insects. Cambridge University Press, Cambridgess

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13 Insights from mosquito evolution: Patterns, tempo and speciation Karamjit Singh Rai Biological Sciences, University of Notre Dame, IN, USA, and Zoology, School of Life Sciences, Guru Nanak Dev University, Amritsar, India [email protected]

Abstract: A great deal of information has accumulated on chromosome morphology and evolution, heterochromatin distribution and differentiation, and molecular structure, organization and evolution of genomes in the mosquito family Culicidae. Whereas numerically the haploid chromosome number (n = 3) has remained virtually unchanged, extensive variation exists at different levels of genomic structure and organization. A number of trends in genome evolution emerge when these data are considered in light of cladistic phylogenies of Culicidae and its sister families. Anophelinae have heteromorphic sex chromosomes, a small genome size and repetitive elements are distributed in a longperiod interspersion pattern. In contrast, Culicinae have homomorphic sex chromosomes and repetitive DNA is organized in a short-period interspersion pattern. There has been a general increase in genome size during the evolution of culicine taxa. The most likely hypothesis for the evolution of sex chromosomes and genome organization in Culicidae would be that homomorphic sex chromosomes and a long-period interspersion pattern are ancestral in lineages leading to Toxorhynchitinae and Culicinae. Larger genomes developed in subsequent culicine lineages through accumulation of short-period interspersed repetitive elements. Heteromorphic sex chromosomes evolved early in the evolution of Anophelinae and a long-period interspersion pattern was retained. An alternative route may be that Culicidae arose from a Chaoborid Mochlonyx-like ancestor with heteromorphic sex chromosomes and possibly short-period interspersion. This would require the loss of heteromorphic sex chromosomes in the lineage leading to Toxorhynchitinae and Culicinae and ‘shedding’ of repetitive elements in the lineage leading to Anophelinae. Several interesting patterns have emerged from studies of C-banding and the distribution of heterochromatin in Culicidae and phylogenies derived from these studies are supported by the modern cladistic analyses. Intensive multi-point linkage map studies suggest that recombination frequencies/ genome have remained relatively constant over the course of culicid evolution such that Anophelinae with relatively small genome size has a linkage map of similar size to Aedini. As a consequence, taxa in Anophelinae have a higher amount of recombination V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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per haploid genome size than Culicinae. Undoubtedly, family Culicidae represents one of the best studied systems of genome evolution in animals, and to ascertain the mechanics of sympatric v/s allopatric speciation in closely related group of species. Extensive variation in vector competence to arboviral and other pathogens exists within and between species. Although the genetic basis of susceptibility or refractoriness of mosquito populations to certain parasites has been known for more than half a century, the underlying molecular mechanisms controlling such differential expression have been resolved relatively recently. Apparently, arboviral susceptibility in mosquitoes is under polygenic control. However, major genes for susceptibility of mosquito vectors to malaria have been identified and mapped. Keywords: Mosquito evolution, Zoogeography, Chromosomal differentiation, Genome structure, Organization and evolution, Reproductive isolation, Speciation, Vector competence

Introduction Charles Darwin published his seminal work ‘On the Origin of Species’ in 1859.1 A century later, in the Spring quarter of 1959, as part of the Darwin Centennial Celebration, the University of Chicago, USA offered an interdisciplinary Advance Graduate Level course/Symposium (Anthropology 425-Zoology 425) involving a galaxy of leading biologists, geneticists, evolutionists, anthropologists, theologians and philosophers of the time to discuss, dissect and reconcile Darwin’s monumental theory of Natural Selection with what till then were largely disparate worlds of Science and the Christian church. Among other luminaries, the invited participants included renowned British evolutionist, Sir Julian Huxley, F.R.S., the American Noble Laureate geneticist, Herman J. Muller, Ernst Mayer (Harvard), George Gaylord Simpson (Amer. Museum of Natural Hist.), Theodosius Dobzhansky (Columbia U.), G. Ledyard Stebbins (UC, Davis), E.B. Ford, F.R.S. (Oxford), Noble Laureate ethologist, Nikolaas Tinbergen (Leiden and Oxford) and the Naturalist Marston Bates (Rockefeller Foundation). I had joined the Department of Botany, University of Chicago as Charles Hutchinson Fellow working for my Ph.D. in the previous fall quarter of 1958. I recall with considerable pride enrolling and participating in this admittedly thought provoking and high decibel Course/Symposium in the Spring Quarter of 1959. Personally, this resulted in a clearer understanding of the issues involved from the formal presentations, and the very vocal give and take discussions. At the end of the week, I thought all the stakeholders including the theologians, by and large, began to accept the reality of the evolutionary theory. Half a century later to have the opportunity to revisit this theme is, therefore, highly gratifying and nostalgic. This presentation briefly highlights the patterns and tempo of geological, taxonomic, genomic and ethological differentiation and speciation within and

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among populations of selected taxa of mosquitoes, in the context of their often distinctly contrasting geographical distribution, observed by members of my research laboratory during this period at the University of Notre Dame, USA, located some 100 miles east of my Alma mater, the University of Chicago. The family Culicidae is comprised of more than 3,400 mosquito species, many of which are major vectors of human disease. In view of their importance as vectors, many mosquito genera and species have been the subject of intensive cytological, genetic, evolutionary and molecular investigations over the last half a century.2-5 The purpose of this review is to highlight the salient features of the following studies as they relate to the patterns, pathways and tempo of intraand inter-specific differentiation at various levels of genetic organization and evolutionary relationships of various mosquito taxa: (1) Mosquito systematics employing cladistic analyses of morphological and molecular characters to estimate phylogenetic relationships among sister families to Culicidae and among Culicidae subfamilies, tribes, genera, subgenera and species. (2) Karyotypes and their evolution emphasizing that the number of chromosomes has remained at a constant 2n = 6 despite a relatively ancient origin of Culicidae, the evolution of both homomorphic and heteromorphic sex chromosomes and evidence of extensive chromosomal repatterning in the speciation process. (3) Molecular genome size and organization in Culicidae and considered in light of current phylogenetic relationships. Genome evolution is also reviewed in the context of extensive data on heterochromatin distribution and in terms of the linkage maps established through various recent intensive genome mapping protocols in Culicidae. (4) Ethological differentiation, reproductive isolation and speciation in sympatry versus allopatry, and (5) Brief observations concerning variation in and genetics of vector competence.

Mosquito systematics, zoogeography and speciation The family Culicidae, which includes all mosquitoes, is divided into three subfamilies, Anophelinae, Toxorhynchitinae, and Culicinae.6-10 Anophelinae includes three genera, the Neotropical Chagasia (4 species), the Australasian Bironella (9 species in 3 subgenera), and the nearly cosmopolitan Anopheles with some 422 species grouped in 6 subgenera. Toxorhynchitinae includes a single genus, Toxorhynchites with 76 species. Culicinae is by far the largest subfamily subdivided into 10 tribes, 33 genera, 117 subgenera and includes about 2,925

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described species. The total number of genera, subgenera and species in Culicidae currently stand at 37, 129, and 3,436, respectively.10 The genus Aedes, which includes some 962 species grouped in 43 subgenera, is one of the best studied and has been the primary focus of the author’s laboratory.3,4,5 Based on the available fossil record, and zoogeographic evidence involving past intercontinental connections and faunistic composition, it has been suggested that mosquitoes had evolved by the Jurassic, approximately 210 million years ago (MYA).11 This is about the time continental drift began.12 The continental break up led to fragmentation and geographical isolation of populations. This may have been accompanied by great ecological flux that promoted rapid speciation.13 Ross14 proposed that a burst of Culicinae lineages arose approximately 120 MYA. By the end of the Cretaceous, some 65 MYA, the generic composition of family Culicidae was well established. New Zealand has been in its present position of isolation for approximately the last 50 million years.17 With the exception of three species, Aedes notoscriptus, Aedes australicus, and Culex quinquefasciatus the present day mosquito fauna of New Zealand is relict and endemic. This provides circumstantial evidence that the genus Aedes existed prior to the island’s separation from Australia and that it was probably widely dispersed during the Cretaceous which began 145 MYA.15 Fossils of family Culicidae (Culex, Aedes) and its sister family Chaoboridae are well known from the Eocene (Tertiary) and Oligocene which began 60 and 55 MYA respectively.16 The phylogenetic relationship of Culicidae relative to other nematocerous dipteran families has been evaluated using modern cladistic analysis.5 Munstermann and Conn18 have reviewed the impact of molecular biology and cladistic analysis on systematics of selected taxa of Culicidae with particular emphasis on Aedes and Anopheles species. Phylogenies have been estimated with morphological characters19 and nucleotide sequences from the 18S and 5.8S nuclear ribosomal DNA (rDNA)20 and 28S rDNA.21 In summary, data from these analyses consistently support Chaoboridae-Corethrellidae as sister taxa to Culicidae. All analyses support Anophelinae as the basal clade in Culicidae and are consistent in placing Toxorhynchitinae as basal to the Culicinae. Within Culicinae, the tribe Sabethini is basal to Culicini and Aedini. All datasets support a monophyletic relationship between Culicini and Aedini.

Comparative karyotypes, chromosome morphology and evolution Chromosomal karyotypes have been established for a relatively large sample involving some 20 genera, 35 subgenera, and more than 300 species in family Culicidae.4,22-25 One of the most remarkable findings of this survey is that despite the ancient origin of the group and despite extensive repatterning of the genome


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involving translocations and inversions, 2,26 the basic chromosome number (2n = 6) has remained unchanged. The only exception, Chagasia bathana (2n = 8) of the subfamily Anophelinae, possesses three autosome pairs and a heteromorphic pair of sex chromosomes.28 All other anophelines possess two pairs of generally metacentric chromosomes of unequal size and one pair of heteromorphic sex chromosomes that often show extensive polymorphism in overall length and of the quantity and quality of heterochromatin differentiation among various species.23 The position of the centromeres in the heteromorphic X and Y chromosomes in Anophelinae varies from subtelocentric or acrocentric to submetacentric and metacentric.2931 In contrast, species of the subfamilies Toxorhynchitinae and Culicinae all possess three pairs of homomorphic metacentric and/or slightly submetacentric chromosomes: a pair of small chromosomes, a pair of large chromosomes and a pair of intermediate-sized chromosomes.3,22,24,32 In culicine mosquitoes, sex is determined by a gene at a single locus. Females are homozygous recessive at this locus while males are heterozygous for a dominant allele.33,3 In species in which linkage group-chromosome correlations have been made, the shortest chromosome contains the sex-locus and is therefore sex-determining.32,35,36 Differences clearly exist in overall lengths and arm ratios of individual chromosomes, both within and between species.3,37 Total chromosomal length among various genera varies almost five-fold, from 8.2 microns in Anopheles quadrimaculatus to 39.3 u in Aedes alcasidi. Within the genus Aedes, there is a three-fold variation in chromosome length.5 Genetic mapping data show that groups of allozyme loci have remained linked and collinear, particularly around the centromere regions, in a variety of culicine taxa but that these linkage groups have translocated and are inverted extensively involving the arms of the three culicine chromosomes.26,38 The extensive variation in chromosome number in most Diptera taxa belies the extreme conservation found in Culicidae. For example, chromosome number ranges from n = 3 to 7 in the genus Drosophila,39 and from n = 3 to 8 in the genus Glossina.40 In family Muscidae, most species possess six pairs of chromosomes; however six species have only five pairs each.41 Nevertheless, certain other Dipteran families such as Simulidae42 and Sarcophagidae also show extensive conservation of chromosome number, although some exceptions do occur.39 No logical explanation exists for the extraordinary conservation of the haploid chromosome number in Culicidae. The evolution of sex chromosomes Current dogma suggests that heteromorphic sex chromosomes evolved from virtually identical homologues. Both the theory43 and considerable experimental evidence suggest that it is the gradual accumulation of repetitive sequences on the

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Y chromosome followed by loss of recombination between the heteromorphic pair that leads to the differentiation of X and Y chromosomes. Theory predicts eventual loss of function and extinction of the Y chromosome.44-47 This directionality is generally referred to as the ‘Rise and Fall of the Y Chromosome’.45 Evolution of a heteromorphic Y-chromosome may have occurred only once or possibly may have been reversed in the evolution of sex chromosomes in Culicidae. The primitive Nematocera families Tipulidae and Dixidae possess homomorphic sex chromosomes. However, the sister families Chaoboridae-Corethrellidae contain genera with homomorphic (Eucorethra, Corethrella, Chaoborus) and heteromorphic (Mochlonyx) sex chromosomes.24 If homomorphy was ancestral in Culicidae then it was retained in the lineages leading to Toxorhynchitinae and Culicinae while heteromorphy probably evolved early in the evolution of Anophelinae and was retained in all taxa. This scenario is in conformity with the current dogma concerning the evolution of sex chromosomes.47 Alternatively, if as proposed by Rao and Rai (1987a), Culicidae arose from a Mochlonyx-like ancestor, then Anophelinae retained heteromorphic sex chromosomes, while homomorphic sex chromosomes evolved through euchromatinization or loss of the Y in Toxorhynchitinae and Culicinae.24

Hetrochromatin: Localization, variation, and expression The application of Giemsa C-and other banding procedures to somatic and meiotic chromosomes has provided important insights concerning linear differentiation and evolution of chromosomes in Culicidae. Studies have been conducted in 36 species belonging to seven genera of Culicinae (Aedes, Mansonia, Culiseta, Armigeres, Sabethes, Wyeomyia, Toxorhynchites) including 28 Aedes species,24,48,49 three species of Culex50 and several Anopheles species.29-31, 51-53 C-banding patterns were also studied in representative species of Tipulidae, Dixidae, and Chaoboridae in order to examine how chromosomes have evolved in these families.24 These studies established that the distribution of heterochromatin is markedly different in anopheline and culicine mosquitoes particularly in the heteromorphic sex chromosomes. All species showed the presence of heterochromatin around the centromeres of the autosomes although there are often large inter- and intraspecific differences in amounts of the same. Using different banding techniques, three types of heterochromatin were identified on the basis of staining characteristics in the pericentromeric regions in the Culicini species Culiseta longiareolata.53,54 In addition to centromeric bands, the autosomes in certain species such as Aedes bahamensis,24 and the long arms of the sex chromosomes in An. atroparvus55 possess telomeric C bands also. The organization of heterochromatin is markedly different among the two homologues of the sex chromosome pair in most Aedes species as well as between


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anopheline and culicine mosquitoes. Motara and Rai48,49 reported two distinct types, constitutive and facultative heterochromatin, in Aedes mosquitoes. The former is present around the centromere region of all the three chromosome pairs and the latter in an interstitial position on one of the arms of the female-determining (m) chromosome in most Aedes species. The intercalary band is located proximal to the centromere in Ae. annandalei and in telomeric position on both the male and female determining chromosomes in Ae. vittatus. Aedes mascarensis, Ae. katherinesis, Ae. excrucians, Ae. stimulans, Ae. cinereus and Ae. triseriatus lack the intercalary band. The fact that these species belong to three different subgenera suggests that heterochromatinization of particular segments is species specific. The male-determining chromosome (M) in Ae. aegypti lacks even the centromeric heterochromatin. The constitutive and facilitative heterochromatin replicate at different times in the cell cycle.56 Unlike Aedes, the intercalary heterochromatin is not present on the femaledetermining chromosome in Armigeres subalbatus or Toxorhynchites splendens but on an arm of one of the autosomes (chromosome II in the former and chromosome III in the latter).24 Rai et al.3 suggested a possible evolutionary derivation of the various heterochromatin patterns observed in Aedes species. The overall patterns observed among various genera are also suggestive of the role chromosome repatterning played in genome evolution. The expression of the intercalary C-band on the sex chromosome in a particular species varies as a function of the genetic background in which it is placed. This was revealed by Giemsa C-banding of the F1 hybrids and progeny of certain backcrosses between two closely related species, Aedes aegypti and Ae. mascarensis.48 Crosses involving Ae. aegypti females and Ae. mascarensis males produced F1 progeny in which the expression of the distal intercalary C-band on the female-determining (m) chromosome of Ae. aegypti was suppressed in both the males and the females. This indicated that the distal region of the femaledetermining (m) chromosome represented by the heterochromatic C-band was derepressed and that it became euchromatic. When F1 males from this cross were backcrossed to Ae. aegypti females, a proportion of the sons developed into intersexes and differed from normal males in their C-banding pattern. Thus, it was possible to relate abnormal sexual development of adult males in the backcross progeny to a selective activation of a discrete chromosomal locus on the male determining chromosome of their fathers.48 Reciprocal crosses (Ae. mascarensis females X Ae. aegypti males) gave expected results. The reversible genetic regulation of the facultative C-band apparently represents selective control of a chromosomal segment of one species (e.g. Ae. aegypti) through genetic interaction with another, Ae. mascarensis.48 Such genetic regulation, which was also observed in progeny of crosses involving Ae. katherinensis and Ae. hebrideus,24 may be widespread among Aedine mosquitoes and may help protect species integrity.

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In anopheline species, the heteromorphic chromosomes often show extensive differences in the amount, distribution and types of heterochromatin. The Y chromosome may be entirely heterochromatic in most Anopheles species while the X-chromosomes may be heterochromatic from < 1/2 to > 3/4 of their length even among closely related species. Furthermore, several of these species, for example, the Hyrcanus group (subgenus Anopheles), the maculatus group (subgenus Cellia) and others are polymorphic for the size of the X chromosome and for the amount of heterochromatin.29,30,31 Such differences are diagnostic and allow unambiguous identification of species whose polytene chromosome banding pattern is virtually homosequential.57 Presumably, different densities of the Giemsa bands on the X and the Y chromosomes in these species reflect different types of constitutive heterochromatin.58 Four satellite DNAs defined on Hoechst 3325S CsCl density gradients are similarly reflective of the presence of different types of heterochromatin in the An. stephensi genome.59 In conclusion, there seems little doubt that changes in amounts, types, and location of heterochromatin are associated with mosquito speciation, particularly in the subfamily Anophelinae and Culicinae. Dynamics of highly repetitive DNA sequences Intraspecific and interspecific variation in the sequence and abundance of eight highly repeated DNA sequences obtained from a population of Ae. albopictus was determined by dot-blot hybridization with genomic DNA of three species each of the Ae. albopictus and Ae. scutellaris subgroups in the Ae. albopictus group.60 The copy number differed widely from 0 (absent) to 810,000 both within and among species, and without any correlation with genome size. Extensive sequence divergence was also observed. The results suggest rapid and asynchronous turnover and evolution of these highly repeated DNA elements. In situ chromosomal localization of four cloned, repetitive DNA fragments (H-76, 61, H-19 and H-85) indicated that they are dispersed throughout the lengths of the three haploid chromosomes in all Aedes species examined.61,62 Although the sequences homologous to these cloned repetitive DNA fragments are present in other culicid genera, Haemagogus equinus, Tripteroideres bambusa and Anopheles quadrimaculatus, significant differences in their abundance and distribution were observed.61,62 Unlike such dispersed pattern in Aedes, Satellite 1 was localized to the heterochromatic arms of the X and the Y chromosomes and the centromere regions of chromosome 3 in Anopheles stephensi.59 Similarly, a highly repetitive DNA clone isolated from Aedes albopictus (H115) was shown to be located at an intercalary position on chromosome 1 in all Aedes species examined. 63 Southern hybridization of this DNA fragment with genomic DNA of An. quadrimaculatus on the other hand showed a dispersed pattern.


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An important difference in chromosome organization with regard to heterochromatin distribution between anophelines and most culicines may be critical in determining whether polytene chromosomes can be easily mapped. There is generally a good resolution of individual bands on each of the euchromatic chromosome arms in the anophelines, while culicines are largely refractory to this type of analysis. In anophelines, apparently much of the heterochromatin is clustered around the centromeres of each of the three pairs of chromosomes resulting in the formation of a chromocenter in polytene chromosome preparations. Of the eight mosquito genera in which polytene chromosome morphology has been studied, Anopheles alone possesses a chromocenter. All other genera (Aedes, Culex, Mansonia, Toxorhynchites, Orthopodomyia, Wyeomyia, Sabethes) lack a distinct chromocenter. Nevertheless, Orthopodomyia pulcripalpis64 and Sabathes cyaneus65 have yielded well resolved polytene chromosomes. This suggests that these taxa have long-period interspersion and may be more basal in culicid evolution. Furthermore, as indicated above, repetitive DNA constitutes a large proportion of the genome in culicine mosquitoes. Since this DNA undergoes latereplication during the S period,56 such dispersed sequences may conceivably act like micro-chromocenters thereby preventing effective separation of individual chromosomes. In examining karyotypes and C-banding patterns in species of Tipulidae, Dixidae, Chaoboridae, and Culicidae, Rao and Rai (1987a) 24 concluded that Culicidae arose from a chaoborid Mochlonyx-like ancestor and that the Anophelinae and Culicinae evolved along separate lineages from a common ancestral stock. The Chagasia karyotype was considered to be primitive for Anophelinae while the Toxorhynchites karyotype was considered primitive for Culicinae. The cladistic analyses discussed above support this proposal.

Genome size and general genome organization Interspecific variation and genome organization Considerable effort has been expended in recent years to determine haploid nuclear DNA amounts in the superfamily Culicoidea.66-69 This has been done through quantitative cytophotometry of Feulgen-stained primary spermatocytes and in a few cases through analyses of renaturation kinetics of nuclear DNA.69-71 As a result, haploid genome sizes have been established for 44 species belonging to 13 genera of mosquitoes and related Culicoidea families. Genome size is generally small in Anophelinae (0.23–0.29 pg/haploid genome).66,71,79 The single species, Toxorhynchites splendens examined in subfamily Toxorynchitinae possesses intermediate size genome of 0.62 pg as do Sabethes cyaneus and Wyeomyia smithii (Sabethini). The haploid genomes of Culex species examined ranged from 0.54 to 1.02 pg and those of Culiseta species (Culicini)

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from 0.92 to 1.25 pg. Armigeres subalbatus and Haemagogus equinus (Aedini) contained 1.24 pg and 1.12 pg, respectively. At the generic level, the cosmopolitan genus, Aedes showed more than three-fold variation in nuclear DNA amounts with the Polynesian species Ae. pseudoscutellaris and Aedes cooki (belonging to the Ae. scutellaris subgroup in the subgenus Stegomyia) possessing the lowest genome size of 0.59 pg while Ae. zoosophus (subgenus Protomacleaya) possessed the highest genome size of 1.9 pg among the 23 species examined.25,68 Placed in the context of phylogenetic relationships discussed earlier, these values suggest a general increase in genome size during the evolution of Culicidae. Black and Rai69 demonstrated that all classes of repetitive DNA sequences increased linearly in amount with total genome size. Furthermore, linear regression analysis of a fairly large data set involving 28 species belonging to 11 genera of the superfamily Culicoidea showed a highly significant positive correlation between total chromosomal length and haploid genome size.25 Nevertheless, eight-fold variation in haploid genome size was accompanied by only an approximate fivefold variation in the total chromosomal length indicating that DNA amounts have increased almost twice as much as the increase in chromosomal size. Studies using reassociation kinetics have provided information on genome organization in Anophelinae and Culicinae.69-71 Genome organization refers to the amounts, complexity and dispersion of repetitive elements in a genome. Two basic forms of genome organization have been described in eukaryotes.72 The first type is termed short-period interspersion and describes a pattern wherein single copy sequences, 1,000–2,000 bp in length, alternate regularly with short (200–600 bp) and moderately long (1,000–4,000 bp) repetitive sequences. This characterizes genome organization in the majority of animal species and was found in the culicine species Culex pipiens, Aedes aegypti, Ae. albopictus and Ae. triseriatus.74 The second type of genome organization is termed long-period interspersion and describes a pattern of long (> 5,600 bp) repeats alternating with very long (> 13,000 bp) uninterrupted stretches of unique sequences. Repeats in Anopheles quadrimaculatus69 and An. gambiae71 follow a long-period interspersion pattern. Genome organization is of the long-interspersion type in Chironomus tentans73 but has not been determined in sister families Chaoboridae-Corethrellidae. However, haploid DNA amounts of 0.47, 0.55 and 0.40 picograms (pg) were observed in the three principal genera Corethrella, Mochlonyx and Chaoborus respectively.68 In insects, long period interspersion is characteristic of most species with small genome sizes (0.1–0.5 pg/haploid genome) while short period interspersion tends to be associated with larger genomes with larger amounts of repetitive DNA.74 It is difficult to predict genome organization in ChaoboridaeCorethrellidae based on genome size because they fall into the upper limit for long interspersed species. Thus there remain two competing hypotheses for ancestral


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genome evolution in Culicidae. It is possible that long period interspersion was ancestral in Culicidae and was retained in the lineage leading to Anophelinae while larger genomes developed through accumulation of short period interspersed repetitive elements in Culicinae. The alternative hypothesis is that Culicidae arose from a short period interspersed species and, while that organization was retained in the Culicinae, repetitive elements were shed and became organized into a long period interspersion pattern in the Anophelinae. This is the scenario considered by Rao and Rai68 who proposed a phylogeny of the superfamily Culicoidea based on haploid chromosome numbers and genome sizes. They suggested that the line that possibly gave rise to Anophelinae from Mochlonyx-like ancestor underwent many deletions of highly repetitive DNA. However, there is little corroborating, empirical evidence for this. These arguments suggest that long-period interspersion may be ancestral in Culicidae. However, the hitherto unknown genome organization in Chaoboridae or Corethrellidae must be determined to test this hypothesis. Furthermore, analysis of genome organization in Sabethini and Toxorhynchitinae is imperative to determine when short-period interspersion arose in culicine evolution. Intraspecific genome size variation Studies of intraspecific variation in genome size in the author’s laboratory revealed unequivocally that DNA amounts are not fixed within species.67,75,80 An analysis of 47 geographic populations of Ae. albopictus from 18 countries showed a 2.5-fold variation in DNA amounts ranging from 0.62 pg in the Koh Samui population from Thailand to 1.66 pg in a population introduced to the continental United States from Houston, TX. Furthermore, extensive variation existed among and within populations from contiguous geographic locations. Genome size was independent of geographic origin in the various populations examined75. Using DNA-reassociation kinetics, Black and Rai 69 showed that the intraspecific variation in DNA content in two strains of Ae. albopictus was due mainly to highly repetitive DNA sequences. Further, MacLain et al.76 showed that populations of Ae. albopictus that were significantly different in DNA content also varied in the frequency of different classes of highly repetitive DNA. Thus, the variation in DNA content among populations of Ae. albopictus appears to be due mainly to repetitive DNA sequences that are under rapid change. This suggests that the amount of repetitive DNA is dynamic in Ae. albopictus and probably other mosquito species as well. Significant variation in haploid DNA content has also been observed among several species of invertebrates77 and vertebrates.78 Genome size also varies with the duration of generation time80 and body size.77 Cavalier-Smith (1985) proposed that variation in DNA amount is subject to natural selection and plays an adaptive

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role.79 Although exact function(s) of highly repetitive DNA have long been debated, biologically significant roles have been ascribed in various species. For example, it has been suggested that the proportion of repetitive DNA in Plasmodium berghei may be directly correlated with mosquito infectivity.81 Saturated linkage maps In recent years, simultaneous use of hundreds or thousands of molecular genetic markers in one or a few crosses has allowed a virtual revolution in construction of the so-called saturated linkage maps simultaneously. This technology has allowed a number of different laboratories to construct multi-point linkage maps of entire mosquito genomes. This was first accomplished by constructing a linkage map of Aedes aegypti using 50 RFLP markers from 42 random cDNA clones, 3 random genomic clones and 5 cDNAs of known origin.82 The lengths of chromosomes I, II and III were 49 cM, 60 cM, and 56 cM respectively (165 cM total). Antolin et al. (1996) constructed a linkage map of Ae. aegypti using Single Strand Conformation Polymorphism (SSCP) analysis of 94 RAPD markers.83 The lengths of linkage graphs I, II and III were 52 cM, 58 cM and 57 cM, respectively (168 cM total), remarkably similar to the cDNA map. Mutebi et al. (1997) constructed a linkage map of Ae. albopictus using SSCP analysis of 68 RAPD markers.84 The lengths of chromosomes I, II and III were 54 cM, 67 cM, and 104 cM respectively (225 cM total). Severson et al. showed that cDNA markers are colinear in Ae. aegypti and Ae. albopictus.85 These studies using molecular markers suggest a large (57 cM) increase in the recombinational size of the Ae. albopictus genome. Furthermore, most of this appears to be due to increased recombination on chromosome III. It is uncertain whether these differences are due to variation in DNA amount or differences in the distribution and frequency of chiasmata on chromosome III of the two species. Aedes species are known to vary widely in chiasmata distribution and frequency.86,87 The lengths of the three linkage maps involving morphological and enzyme loci calculated from observed chiasmata frequencies were 62, 86 and 80 cM, respectively (total 228 cM in Ae. aegypti).88 However, large stretches of all three linkage maps were devoid of any markers particularly on linkage group III on which the 17 observed markers were clustered in a 44-unit map while the chiasmata based model predicts an 80-unit map. A linkage map of Armigeres subalbatus constructed using 26 RFLP markers involving cDNA clones from Ae. aegyti resulted in overall lengths of linkage group I, II and III as 51, 72, 58, respectively (181 cM total) and except for one marker, the linear order was the same as in Ae. aegypti.89 A similar RFLP linkage map has been constructed for Culex pipiens using a relatively small sample of 21 cDNA clones from Ae. aegypti. The comparative linkage maps for chromosomes II and III in Cx. pipiens and Ae. aegypti reflect whole arm translocations.27


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Zheng et al. (1996) mapped 131 microsatellite markers in Anopheles gambiae. Chromosomes I, II and II were respectively 49 cM, 72 cM and 94 cM in length (215 cm total).90 Integration of RAPD markers into this map increased the overall density of markers without affecting the overall length.91 It is instructive to consider linkage map size, an indication of the amount of recombination on individual chromosomes, relative to the genome sizes discussed earlier in this review. The observed total linkage map sizes are: 165 cM in Ae. aegypti, 225 cM in Ae. albopictus, 166 in Cx. pipiens, 181 in Ar. subalbatus, and 215 cM in An. gambiae. These do not correspond in any way to the respective genome sizes of 0.83, 0.86–1.32, 0.54–1.02, 1.12 and 0.27 pg/haploid genome respectively in these species. The relationship of physical to recombination distance is approximately 3–6 Mbp DNA/cM in Ae. aegypti, Ae. albopictus and the two other culicine species studied, and 1.2 Mbp DNA/cM in Anopheles gambiae.5 Thus there appears to be little relationship between genome size and recombination frequency. The frequency of recombination remains high in An. gambiae despite having a genome size 1/3 to 1/5 the size of the Aedes and other culicine species genomes. DNA reassociation kinetic analysis has shown that the amount of repetitive DNA sequences in culicine species is generally much higher than that in Anopheles species.69-71 Since recombination is considerably restricted in chromosomal regions rich in repeated DNA sequences,92 overall, Anopheles would be expected to show higher recombination rates. Also, this predicts a closer relationship between physical and linkage maps in Anopheles. Furthermore, the fact that the size of the linkage maps do not vary by more than 60 cM in these three species suggests that the number of chiasmata have remained relatively constant despite increases in genome size and chromosome length in the evolution of Culicidae.

Ethological differentiation, reproductive isolation and speciation Analyses of these inter-related areas involved several Aedes species with particular emphasis on those that comprise the Aedes (Stegomyia) scutellaris group. This group is divided into two subgroups: the scutellaris subgroup consisting of some 34 species which are largely allopatric in distribution on the islands of the South Pacific, generally endemic to single island or groups of island; and the albopictus subgroup with 11 described species having a predominantly sympatric distribution in Southeast Asia.3 This distribution suggests that speciation in the former subgroup occurred after gene flow was interrupted by the development of oceanic barrier. A comparison of allozyme and morphological relationships based on numerical taxonomic methods showed that adult morphological traits and allozymes are congruent with geographical distributions and classical taxonomic assignment in the Ae. scutellaris subgroup and certain other (Stegomyia) species. Furthermore

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allozyme relationships at the group and subgroup levels were consistent with previous taxonomic treatments and all species fell into independent and cohesive clusters.93 Genetic comparisons were also based on interspecific hybridization compatibilities and the levels of fertility and viability of the hybrids experimently produced in the laboratory among the Ae. scutellaris group and certain other Aedes species.3,94 The development of reproductive isolation in the island-dwelling species has been largely a random event such that no association was found between Nei’s unbiased genetic distances and compatability.95 The relative tempo of speciation was estimated by comparing patterns of allozyme relationships with morphology, hybridization, and geological history in allopatric island-dwelling mosquitoes.95 Assuming that molecular evolution occurs at a relatively constant rate, and that Nei’s genetic distance roughly estimates time since divergence, calibration of a known geologic event, e.g. the separation of two islands in the South Pacific, was used to correlate zoogeography with allozyme and ethological differentiation and to obtain reasonable estimates of geological time frames of the onset and tempo of speciation events in the group. A molecular clock, based on the ratio between experimentally determined genetic distances between two given species and divergence time (one unit of Nei’s distance = 11 million years) inferred from a known geologic event allowed estimates of geologic time frames ranging from 3 mya to approx 7 mya for the observed speciation events in the group following their geographic isolation.95 In addition, ethological divergence and reproductive isolation among eight species in the South Pacific Ae. sutellaris subgroup was investigated by giving females a simultaneous choice between males of their own species and males of another species thereby determining isolation indices. The degree of ethological isolation between species pairs thus measured was associated with the timing of their geographic isolation following the fragmentation of the Outer Melanesian Arc 2–10 mya and on other published studies of allozyme variation and percent egg hatch from interspecific hybridization.96 To answer the critical question whether sympatry between closely related but non-interfertile species can promote the evolution of prezygotic (ethological) reproductive isolation, three sibling species of the Ae. albopictus subgroup and six strains of Ae. albopictus with known dates of laboratory colonization were employed. The data showed that selection for reinforcement of ethological isolation can occur in sympatric populations where strong postmating reproductive isolation already exists.97 In the case of Ae. scutellaris subgroup species the production of fairly fertile hybrids in the laboratory among several species pairs and no hybrid breakdown in two filial generations suggest that allopatry preempts imposition of any selection pressure for the development of reproductive isolating mechanisms.


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In conclusion, the data from Ae. scutellaris group species allowed important insights concerning the onset, tempo, and intensity of reproductive isolation and genetic differentiation, in relation to their contrasting distribution patterns, with geological time frames.

Variation in and genetics of vector competence Extensive variation exists within and among species in their ability or inability to transmit important arboviral and other diseases such as malaria and filariasis. Hardy et al.98 presented an exhaustive review of intrinsic factors that affect vector competence of mosquitoes for arboviruses. Although the underlying molecular pathway of this fundamental vector characteristic is not yet clearly delineated, extensive data from laboratories around the world, including those from Notre Dame, have unequivocally established that differential ability to transmit a particular pathogen can be a characteristic of individuals or populations as well as of species. Furthermore, a particular geographic strain is often associated with different profiles of oral susceptibility and horizontal transmission rates. For example, Boromisa et al.99 examined vector competence of eight geographic strains of Aedes albopictus from N. America and Asia and one North American strain of Ae. aegypti to Fiji strain of Dengue-1 serotype virus. The Asian strains were derived from known recent dengue non-endemic, endemic, and epidemic history. All strains tested except the Houston strain, showed high susceptibility to midgut infection. However, there were marked differences in percent disseminated infection and transmission rates among the various strains tested. It is ironic that whereas single genes for aphid and leafhopper transmission of plant viruses have been known for more than half a century, the goal of establishing genetic basis of susceptibility of any of the mosquito transmitted arboviruses has remained illusive. This likely is due to complexity of viral transmission by mosquito species, involving several seemingly independent steps and barriers such as ingestion, replication, and dissemination of the virus from the hemocoel, the salivary glands and finally the ability to transmit. In all probability, these interrelated steps are controlled by different genetic factors and their interaction with the intrinsic environment of the specific vector population. Furthermore, numerous extrinsic environmental factors doubtlessly also come into play. In a presumptive genetic study, strain differences in basal lamina thickness, assessed by measurement of transmission electron micrographs, and disseminated infection rates of DEN-1 virus were compared among three laboratory strains of Ae. albopictus.100 Mean basal lamina thickness for the NEW ORLEANS and the HOUSTON strains were significantly greater than those for the OAHU strain, which exhibited a higher disseminated infection rate than the former two. Although the basal lamina thickness among the F1 progeny of reciprocal crosses of the

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OAHU and HOUSTON strains were intermediate between the parental strains, they were too variable to be useful in genetic studies. Measurements of basal laminae among individuals of the NEW ORLEANS strain, with disseminated or nondisseminated infections, failed to demonstrate a role of basal lamina thickness as a modulator of DEN-1 virus dissemination across the midgut epithelium of the Ae. albopictus, in contrast to the demonstration of this phenomenon, apparently of considerable importance in the Ae. triseriatus – La Crosse virus system.101 In contrast to the above paradox in delineating genetic basis of arbovirus vector competence, the genetics of mosquito – Plasmdium interaction has been well characterized. The susceptibility of Anopheles mosquitoes to Plasmodium infection may range from 100 percent where all mosquitoes transmit malaria to complete refractoriness. Furthermore, the infection success of Pl. falciparum has been shown to be variable depending upon the geographic origin of both the parasite and the mosquito.102 The molecular interactions of malarial parasite with their host tissues within the mosquito have been resolved and the molecular mechanisms responsible for pathogen destruction, such as melanotic encapsulation and immune peptide production have been characterized.103 Three genes responsible for the encapsulation of the malarial parasite in An. gambiae have been physically localized to discreet regions of polytene chromosome complement.104 Acknowledgements: I thank Prof. V.P. Sharma to invite me, on behalf of the National Academy of Sciences of India, to contribute an overview of the patterns of evolutionay differentiation and the mechanics of speciation in mosquitoes in connection with the Academy’s commemoration of the bicentennial of Sir Charles Darwin’s birth on February 12, 1809.

References 1.

Darwin C (1859) The Origin of Species by Means of Natural Selection. Murray, London

2.

Kitzmiller JB (1953) Mosquito Genetics and Cytogenetics. Separata da Rev Bras de Malariol e D Trop 5:285–359

3.

Rai KS, Pashley DP, Munstermann LE (1982) Genetics of speciation in aedine mosquitoes. In: Recent Advances in Genetics of Insect Disease Vectors. Steiner WMW, Tabachnick WJ, Rai KS, Narang SK (Eds.), Stipes, Champaign, IL

4.

Rai KS (1999) Genetics of mosquitoes. J Genetics 78:163–169

5.

Rai KS, Black IV WC (1999) Mosquito Genomes: Structure, Organization and Evolution. Adv Genetics 41:1–33

6.

Knight KL, Stone AA (1977) Catalog of the Mosquitoes of the World (Diptera Culicidae), 2nd edition, College Park, MD. Entomol Soc Am

7.

Knight KL (1978) Supplement to a Catalog of the Mosquitoes of the World. College Park, MD. Entomol Soc Am

8.

Ward RA (1984) Second supplement to ‘A catalog of the mosquitoes of the world’ (Diptera: Culicidae). Mosq Syst 16:227–270


213

9.

Ward RA (1992) Third supplement to A catalog of the mosquitoes of the world (Diptera: Culicidae). Mosq Syst 24:177–230

10.

Service MW (1993) Mosquitoes (Culicidae). In: Medical Insects and Arachnids. Lane RP, Crosskey RW (Eds.), Chapman and Hall, New York

11.

Edwards FW (1932) Genera Insectorum. Diptera, family Culicidae. Fasc. 194. Wytsman PAG (Ed.), Verteneuill, Brussels, Desmet

12.

Wilson JT (1963) Continental Drift. Sci Am 208:86–100

13.

McClelland GAH (1967) Speciation and Evolution in Aedes. In: Genetics of Insect Vectors of Disease. Wright JW, Pal R (Eds.), Elsevier, New York1

14.

Ross HH (1951) Conflict with Culex. Mosq News 11:128–132

15.

Belkin JN (1968) Mosquito studies (Diptera: Culicidae). VII. The Culicidae of New Zealand. Contr. Am Ent Inst 3:1–182

16.

Rohdendorf B (1974) The historical development of diptera. Alberta Press, Edmonton

17.

Rick EF (1970) Fossil history. In: Insects of Australia. Melbourne Univ. Press, Victoria

18.

Munstermann LE, Conn JE (1997) Systematics of mosquito disease vectors (Diptera: Culicidae): Impact of molecular biology and cladistic analysis. Ann Rev Entomol 42: 351–369

19.

Oosterbroek P, Courtney G (1995) Phylogeny of the nematocerous families of Diptera (Insecta). Zool J Linn Soc 115:267–231

20.

Miller BR, Crabtree MB, Savage HM (1997) Phylogenetic relationships of the Culicomorpha inferred from the 18S and 5.8S ribosomal DNA sequences (Diptera: Nematocera). Ins Mol Biol 6:105–114

21.

Pawlowski, J, Szadziewski R, Kmieciak D, Fahmi J, Bitta G (1996) Phylogeny of the infraorder Culicomorpha (Diptera: Nematocera) based on 28S RNA gene sequences. Syst Ent 21:167–178

22.

Rai KS (1963) Comparative study of mosquito karyotypes. Ann Entomol Soc Am 56: 160–170

23.

White GB (1980) Academic and applied aspects of mosquito cytogenetics. In: Insect Cytogenetics. Blackman RL, Hewitt GM, Ashburner M (Eds.), Blackwell Scientific Publications, London

24.

Rao PN, Rai KS (1987a) Comparative karyotypes and chromosomal evolution in some genera of Nematocerous (Diptera: Nematocera) families. Ann Entomol Soc Am 80:321–332

25.

Rao PN, Rai KS (1990) Genome evolution in the mosquitoes and other closely related members of superfamily Culicoidea. Hereditas 113:139–144

26.

Matthews TC, Munstermann LE (1994) Chromosomal repatterning and linkage group conservation in mosquito karyotype evolution. Evolution 48:146–154

27.

Mori A, Severson DW, Christensen BM (1999) Comparative Linkage Maps for the Mosquitoes, Culex pipiens and Aedes aegypti, based on Common RFLP Loci. J Heredity 90:160–164

28.

Kreutzer RD (1978) A mosquito with eight chromosomes: Chagasia bathana Dyar. Mosq News 38:554–558

29.

Baimai V, Rattanarithikul R, Kajchalao U (1993a) Metaphase Karyotypes of Anopheles of Thailand and Southeast Asia. I. The Hyrcanus Group. Am Mosq Control Assoc 9:59–67

30.

Baimai V, Rattanarithikul R, Kajchalao U (1993b) Metaphase Karyotypes of Anopheles of

214


Thailand and Southeast Asia. II. The Maculatus Group, Neocellia Series, subgenus Cellia. Mosq Syst 25:116–123 31.

BaimaiV R, Rattanarithikul R, Kajchalao U (1995) Metaphase Karyotypes of Anopheles of Thailand and Southeast Asia: IV. The Barbirostris and Umbrosus Species Groups, Subgenus Anopheles (Diptera: Culicidae). J Am Mosq Control Assoc 11:323–328

32.

McDonald PT, Rai KS (1970) Correlation of linkage groups with chromosomes in the mosquito, Aedes aegypti. Genetics 66:475–485

33.

Gilcrist BM, Haldane JBS: Sex linkage and sex determination in a mosquito, Culex molestus. Hereditas 33:175

34.

McClelland GAH (1947) Sex-linkage in Aedes aegypti. Trans Roy Soc Trop Med Hyg 56:4 (Abst.) (1962)

35.

Baker RH, Sakai RK, Mian A (1971) Linkage group-chromosome correlation in a mosquito: translocations in Culex tritaeniorhynchus. J Hered 62:90–100

36.

Dennhöfer L (1972) Die Zuordnung der Koppelungsgruppen zu den Chromosomen bei der Stechmücke Culex pipiens L. Chromosoma 37:43–52

37.

Rai KS (1980) Evolutionary cytogenetics of Aedine mosquitoes. Genetica 52/53: 281–290

38.

Severson DW, Mori A, Kassner VA, Christensen BM (1995) Comparative linkage maps for the mosquitoes Aedes albopictus and Aedes aegypti based on common RFLP loci. Insect Mol Biol 4:41–45

39.

White MJD (1973) In: Animal Cytology and Evolution. 3rd edition, Cambridge Univ. Press, UK

40.

Mauldin I (1970) Preliminary Studies on Karyotypes of five species of Glossina. Parasitology 61:71–74

41.

Boyes JW (1967) The Cytology of Muscoid Flies. In: Genetics of Insect Vectors of Diseases. Wright J, Pal R (Eds.), Elsevier Publishing Co., New York

42.

Rothfels KH (1979) Cytotaxonomy of Black Flies (Simuliidae). Ann Rev Entomol 24:507– 39

43.

Charlesworth B (1978) A model for the evolution of Y chromosomes and dosage compensation. Proc Natl Acad Sci (USA) 75:5618

44.

Steinemann M, Steinemann S, Lottspeich F (1993) How Y chromosomes become genetically inert. Proc Natl Acad Sci (USA) 90:5737–5741

45.

Morell V (1994) Rise and Fall of the Y Chromosome. Science 263:171–172

46.

Rice WR (1994) Degeneration of a Nonrecombining Chromosome. Science 263:230–232

47.

Rice WR (1996) Evolution of the Y chromosome in animals. Bioscience 46:331–343

48.

Motara MA, Rai KS (1977) Chromosomal differentiation in two species of Aedes and their hybrids revealed by giemsa C-banding. Chromosoma 64:125–132

49.

Motara MA, Rai KS (1978) Giemsa C-banding patterns in Stegomyia mosquitoes. Chromosoma 70:51–58

50.

Motara MA (1982) Giemsa C-banding in four species of mosquitoes. Chromosoma 86:319–323

51.

Gatti M, Santini G, Pimpinelli S, Coluzzi M (1977) Fluorescence banding techniques in the identification of sibling species of the Anopheles gambiae complex. Heredity 38: 105–108


215

52.

Baimai V (1988) Constitutive heterochromatin differentiation and evolutionary divergence of karyotype in Oriental Anopheles (Cellia). Pac Sci 42:13–27

53.

Marchi A, Mezzanotte R (1990) Inter- and intraspecific heterochromatin variation detected by restriction endonuclease digestion in two sibling species of the Anopheles maculipennis complex. Heredity 65:135–142

54.

Mezzanotte R, Marchi A, Ferrucci L (1979) Idenfication of sex chromosomes and characterization of the heterochromatin in Culiseta longiareolata (Macquart, 1838). Genetica 50:135–139

55.

Fraccaro M, Laudani U, Marchi A, Tiepolo L (1976) Karyotype, DNA replication and origin of sex chromosomes in Anopheles atroparvus. Chromosoma 55:27–36

56.

Marchi A, Rai KS (1986) Cell cycle and DNA synthesis in Aedes aegypti. Canad J Gent Cyt 20:243–247

57.

Green CA, Baimai V, Harrison BA, Andre RG (1985) Cytogenetic evidence for a complex of species within the taxon Anopheles maculatus (Diptera: Culicidae). Biol J Linn Soc 24:321–328

58.

Baimai V (1988) Constitutive heterochromatin differentiation and evolutionary divergence of karyotype in Oriental Anopheles (Cellia). Pac Sci 42:13–27

59.

Redfern CPF (1981) Satellite DNA of Anopheles stephensi Liston (Diptera: Culicidae). Chromosomal Location and Under-Replication in Polytene Nuclei. Chromosoma 82: 561–581

60.

MacLain DK, Rai KS, Fraser MJ (1987) Intraspecific and interspecific variation in the sequence and abundance of highly repeated DNA among mosquitoes of the Aedes albopictus subgroup. Heredity 58:373–381

61.

Kumar A, Rai KS (1991a) Organization of a cloned repetitive DNA fragment in mosquito genomes (Diptera: Culicidae). Genome 34:998–1106

62.

Kumar A, Rai KS (1991b) Chromosomal localization and genomic organization of cloned repetitive DNA fragments in mosquitoes (Diptera: Culicidae). J Genet 70:189–202

63.

Kuma A, Rai KS (1992) Conservation of a highly repeated DNA family of Aedes albopictus among mosquito genomes (Diptera: Culicidae). Theor Appl Genet 83:557–564

64.

Munstermann LE, Marchi A, Sabatini A, Coluzzi M (1985) Polytene chromosomes of orthopodomyia pulcripalpis (Diptera, Culicidae). Parassitologia 27:267–277

65.

Munstermann LE, Marchi A (1986) Cytogenetic and isozyme profile of Sabethes cyaneus. J Heredity 77:241–248

66.

Jost E, Mameli M (1972) DNA content of nine species of Nematocera with special reference to the sibling species of the Anopheles maculipennis group and the Culex pipiens group. Chromosoma 37:201–208

67.

Rao PN, Rai KS (1987b) Inter- and intraspecific variation in nuclear DNA content in Aedes mosquitoes. Heredity 59:253–258

68.

Rao PN, Rai KS (1990) Genome evolution in the mosquitoes and other closely related members of superfamily Culicoidea. Hereditas 113:139–144

69.

Black WC IV, Rai KS (1988) Genome evolution in mosquitoes; intraspecific and interspecific variation in repetitive DNA amounts and organization. Genet Res 51:185–196

70.

Warren AM, Crampton JM (1991) The Aedes aegypti genome: Complexity and organization. Genet Res Camb 58:225–232

71.

Besansky NJ, Powell JR (1992) Reassociation kinetics of Anopheles gambiae (Diptera: Culicidae). DNA J Med Entomol 29:125–128

216


72.

Davidson EH, Galua GA, Angerer RC, Britten RJ (1975) Comparative aspects of DNA organization in metazoa. Chromosoma 51:253–259

73.

Wells R, Royer H, Hollenberger CP (1976) Non-Xenopus like DNA organization in the Chironomus tentans genome. Mol Gen Genetics 147:45–51

74.

Palmer MJ, Black WC IV (1997) The importance of DNA reassociation kinetics in insect molecular biology. In: The Molecular Biology of Insect Disease Vectors: A Methods Manual. Crampton J, Beard CB, Louis C (Eds.), Chapman and Hall Publishers, New York

75.

KumarA, Rai KS (1990) Intraspecific variation in nuclear DNA content among world populations of a mosquito, Aedes albopictus (Skuse). Theor Appl Genet 79:748–752

76.

MacLain DK, Rai KS, Fraser MJ (1987) Intraspecific and interspecific variation in the sequence and abundance of highly repeated DNA among mosquitoes of the Aedes albopictus subgroup. Heredity 58:373–381

77.

Palmer M (1996) Petitpierre E. Relationship of Genome Size to Body Size in Phylan semicostatus (Coleoptera: Tenebrionidae). Ann Entomol Soc Am 89:221–225

78.

Walker LI, Spotorno AE, Sans J (1991) Genome size variation and its phenotypic consequences in Phyllotis rodents. Hereditas 115:99–107

79.

Cavalier-Smith T (1985) Introduction: The evolutionary significance of genome size. In: The Evolution of Genome Size. Cavalier-Smith T (Ed.), Wiley, New York

80.

Ferrari J, Rai KS (1989) Phenotypic correlates of genome size variation in Aedes albopictus. Evolution 43:895–899

81.

Birago CA: Bucci ED, Frontali C, Zenobi P (1982) Mosquito infectivity is directly related to the proportion of repetitive DNA in Plasmodium berghei. Mol Biochem Parasit 6:1–12

82.

Severson DW, Mori A, Zhang Y, Christensen BM (1993) Linkage map for Aedes aegypti using restriction fragment polymorphism. J Hered 84:241–247

83.

Antolin MF, Bosio CF, Cotton J, Sweeney WP, Black IV WC (1996) Rapid and dense linkage mapping in a wasp (Bracon hebetor) and a mosquito (Aedes aegypti) with Single Strand Conformation Polymorphisms Analysis of Random Amplified Polymorphic DNA markers. Genetics 143:1727–1738

84.

Mutebi JP, Black IV WC, Bosio CF, Sweeney Jr WP, Craig Jr GB (1997) Linkage map for the Asian tiger Mosquito Aedes (Stegomyia) albopictus, based on SSCP analysis of RAPD markers. J Hered 88:489–494

85.

Severson DW, Mori A, Kassner VA, Christensen BM (1995) Comparative linkage maps for the mosquitoes, Aedes albopictus and Aedes aegypti, based on common RFLP Loci. Insect Mol Biol 4:41–45

86.

Dev V, Rai KS (1984) Genetics of speciation in the Aedes (Stegomyia) scutellaris group (Diptera: Culicidae). V. Chromosomal relationships among five species. Genetica 64:83–92

87.

Sherron DA, Rai KS (1984) Genetics of speciation in the Aedes (Stegomyia) scutellaris group (Diptera: Culicidae). 4. Chromosomal relationships of Aedes cooki with four sibling species. Can J Genetic Cytol 26:237–248

88.

Munstermann LE, Craig GB Jr (1979) Genetics of Aedes aegypti. Updating the linkage map. J Hered 70:291–296

89.

Ferdig MT, Taft AS, Severson DW, Christensen BM (1998) Development of a comparative genetic linkage map for Armigeres subalbatus using Aedes aegypti RFLP markers. Genome Research 8:41–47


217

90.

Zheng L, Benedict MQ, Cornel AJ, Collins FH, Kafatos FC (1996) An integrated genetic map of the African human malaria vector mosquito, Anopheles gambiae. Genetics 143: 941–952

91.

Dimopoulos GL, Zheng L, Kumar V, Torre A, Kafatos FC, Louis C (1996) Integrated genetic map of Anopheles gambiae: Use of RAPD polymorphisms for genetic, cytogenetic and STS landmarks. Genetics 143:953–960

92.

Charlesworth B, Langley CH, Stephan W (1986) The evolution of restricted recombination and the accumulation of repeated DNA sequences. Genetics 112:947–962

93.

Pashley DP, Rai KS (1983) Comparison of allozyme and morphological relationships in some Aedes (Stegomyia) mosquitoes (Diptera: Culicidae). Ann Entomol Soc Am 76:388–394

94.

Dev V, Rai KS (1985) Genetic relationships among certain species of the Aedes (Stegomyia) sutellaris group (Diptera: Culicidae). Ann Trop Med Parasitol 79:325–331

95.

Pashley DP, Rai KS, Pashley DN (1985) Patterns of allozyme relationships compared with morphology, hybridization, and geologic history in allopatric island-dwelling mosquitoes. Evolution 39:985–997

96.

Mclain DK, Rai KS, Rao PN (1985) Ethological divergence in allopatry and asymmetrical isolation in the south pacific Aedes scutellaris subgroup. Evolution 39:998–1108

97.

Mclain DK, Rai KS (1986) Reinforcement for ethological isolation in the southeast asian Aedes albopictus subgroup (Diptera: Culicidae). Evolution 40:1346–1350

98.

Hardy JL, Houk EJ, Kramer LD, Reeves WC (1983) Intrinsic factors affecting vector competence of mosquitoes to arboviruses. Ann Rev Entomol 28:229–262

99.

Boromisa RD, Rai KS, Grimstad PR (1987) Variation in the vector competence of geographic strains of Aedes albopictus for dengue-1 virus. J Am Mosq Cont Assoc 3:378–386

100.

Thomas RE, Wu WK, Verleye D, Rai KS (1993) Midgut basal lamina thickness an. dengue-1 virus dissemination rates in laboratory strains of Aedes triseriatus (Diptera Culicidae). J Med Entomol 30:326–331

101.

Grimstad PR, Walker ED (1991) Aedes triseriatus (Diptera: Culicidae) and La Cross virus. Nutritional deprivation of larvae affects the adult barriers to infection and transmission. J Med Entomol 28:378–386

102.

Hume JCC, Tunnicliff M, Ranford C, Day KP (2007) Susceptibility of Anopheles gambiae and Anopheles stephensi to tropical isolates of Plasmodium falciparum. Malaria J 6:139

103.

Beernsten BT, James AA, Christensen BM (2000) Genetics of mosquito vector competence. Microbiol Molecular Biol Rev 64:115–137

104.

Collins HH, Zheng L, Paskewitz SM, Kafatos (1997) Progress in the map based cloning of the Anopheles gambiae genes responsible for the encapsulation of malarial parasites. Ann Trop Med Parasit 91:517–522

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14 The saga of pollination biology Rajesh Tandon* and H.Y. Mohan Ram** *Department of Botany, University of Delhi, Delhi - 110 007, India [email protected] **INSA Honorary Senior Scientist, 194, SFS DDA Flats, Mukherji Nagar, Delhi - 110 009, India [email protected]

Abstract: Developmental events leading to seed set in plants are broadly categorized into three phases of pollination biology: (1) formation of male and female gametes (2) dispersal and deposition of pollen grains on the pistils of individual of the same species and (3) pollen – pistil interaction leading to double-fertilization. Whereas the first and the third are under genotypic control, the second is largely influenced by the ecological conditions. Charles Darwin was among the first to recognize the functional components of pollen transfer or pollination. In accordance with the theme, we highlight the various aspects of pollination biology pertaining to the second event, which are fascinating and require careful observations. Pollination is a fundamental and vital process in the perpetuation of angiosperms. The process of transfer of pollen to a receptive stigma of the same plant species is accomplished with utmost accuracy amidst various challenges such as immobility of plants and the vagaries of environment that carry the pollen. Flowering plants have accommodated both biotic and abiotic means of pollen transfer. A vast array of floral adaptations such as showiness, colouration, nectar guide-marks on petals, nectaries and floral rewards ensure the targeted delivery of pollen to a conspecific stigma. The combination of floral adaptations – structure, aggregation, scent and display–result in an interplay of selection pressures that maximizes pollination efficiency. Consequently, every forager that visits a flower to seek rewards may not be a pollinator but a robber. Besides the structural organization of the flower, its functional morphology, blooming time, longevity and the innate genetic mechanisms further contribute to screening for fidelity in pollination. As pollination is a mutualistic interaction, the ecological domain has a crucial role in the timing of its occurrence. There are genetic consequences of the pattern of pollen-mediated gene flow in a community. On the other hand, a pollinator is only a forager which opportunistically seeks for a reward and relies on flowers of the other species, when a particular species ceases to bloom. Thus, studies on plant–pollinator interaction are now pursued at different levels of organization with growing emphasis on conserving and maintaining the networks. This paradigm shift arises because there is perceptive apprehension that habitat loss, fragmentation of ecosystems, notably V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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forests, dislocation of overlapping of flowering time and the life cycles of pollinators due to changing climatic conditions and anthropogenic activities are likely to threaten mutualistic interactions. Keywords: Pollination, Pollination syndrome, Generalization and specialization, Floral cues, Biotic Pollination, Abiotic pollination, Mutualism

Introduction Sexual reproduction in flowering plants essentially follows a sequence of events involving transfer of pollen grains (carrying two male gametes) onto a conspecific stigma, growth of the pollen tube through the style and ovary and subsequent release of the two male gametes into the embryo sac and eventually the fusion of one male gamete with the egg (female gamete) to form a zygote and the other male gamete with the polar nuclei to trigger endosperm (double fertilization). The development of male (pollen) and female (egg) gametes and their interaction (pollen–pistil interaction) are under the control of mating genotypes with the exception of self-pollination. The transfer of pollen (pollination) from dehisced anthers to pistil largely involves biotic and abiotic components of the ecosystem. Pollination is a crucial and complex ecological process. Being immobile, plants themselves are not capable of carrying the pollen. Nevertheless, the inability of plants to move has not restricted them to exchange genes among different individuals, as they have successfully accommodated a variety of organisms and abiotic means as carriers (modes of pollination) to transfer pollen from anthers to stigma of the same species. Among these modes of pollination, biotic pollination (zoophily) occurs in nearly three-fourths of the flowering plants which rely on around 2,00,000 animal pollinator species.1 Although abiotic pollination does not play a predominant mode, it occurs in grasses, bamboos, sedges and palms growing in a wide range of habitats. That the dynamics of pollination is complex can be experienced by conducting a single study on pollination biology in any flowering plant. It is not merely an event of transfer of pollen to the stigma of the same species; rather an infallible display of an intricate mechanism that brings connectivity, accuracy and coherence of biological events to ensure multiplication, variation, perpetuation and adaptation of plants. Modularity of plants is another feature that accentuates the complexity in pollination mechanism, as pollen may arrive from the flowers borne on different braches (ramets) of the same plant (geitonogamy) or from other plants (xenogamy). Consequently, the breeding system and eventually the genetic structure of a species population is influenced by pollination pattern; types, availability and behavior of the foragers; arrangement and opening of flowers in an inflorescence; functional

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morphology of the flowers including the cues and rewards (pollen, nectar and wax) for attracting the floral foragers and the time of maturation of anthers and pistil (dichogamy). Schmid has brought out an excellent review of the work on pollination biology done for over two centuries and has traced the evolution of approaches providing invaluable information.2 Even the summary of the enormous information and insights contained in Schmids’s account is difficult to accommodate in this paper. The contribution of the Indian biologists to the fundamental and applied aspects of pollination biology has been discussed in detail by Shivanna and Mohan Ram.3 This essay outlines the aspects of plant-pollinator interaction, highlights the current trends of pollination ecology and exemplifies some of the recent research carried out in India. This is a small but sincere effort to honour the legendary biologist Charles Darwin, whose bicentenary is being observed throughout the world. His work has stimulated biologists around the world and has dramatically changed our understanding and outlook on organic evolution and has brought respectability to biology as an autonomous science.3a

Resurgence of interest in pollination ecology The present day interdisciplinary field of pollination ecology is largely a legacy of the fascinating records on pollination gifted to posterity by Sprengel and Charles Darwin.4–7 Kölreuter was the first to establish the notion that insects are crucial for seed set in certain plants.8 He even carried out artificial hybridization between two plants. Later, Darwin (1859, 1862) used pollination as an example for establishing his theory on adaptations and co-evolution.5 His books on floral biology and pollination—The various contrivances by which orchids are fertilized by insects5; The effect of cross and self-fertilization in the vegetable kingdom6 and The different forms of flowers on plants of the same species,7 attest to his sharp powers of observation connecting the meaning of form and function and his deep understanding of the significance of pollination in flowering plants. One of the most astounding observations made by Darwin was the prediction of a possible pollinator that would pollinate a foot and half-long spurred orchid, Angraecum sesquipedale, in Madagascar.5 Although he could not locate the pollinator, what foretold was later successfully confirmed in 1903 with the identification of a moth, Xanthopan morgani predicta which had a 22-cm-long tongue to sip out nectar seated deep in the spur of the orchid and effect pollination in the process. These studies elaborated the functional significance of the morphology of the flower and have triggered unending studies on exploring the pollinators and their ingenious contrivances. The saga still continues and the secrets appear never ending. By the middle of the twentieth century the unification of approaches used in studying the functional floral morphology and the evolutionary aspects

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gave fresh impetus to pollination biology.9 Several books such as Flower pollination in the Phlox family,10 The Principles of Pollination Ecology11 and the edited volumes – Handbook of experimental pollination biology;12 and Pollination biology13 – have been instrumental in the resurgence to investigate and validate various aspects of pollination biology with novel approaches. Recently, the techniques to pursue pollination biology have been updated in compilations—Pollen biology: a laboratory manual14; Pollination ecology: a practical approach15; Techniques for pollination biologists16 and Field methods in pollination ecology.17 In the past one decade, there is renewed drive to protect mutualism in the tropics so that loss of diversity in the fragmented areas could be arrested.18 This is particularly so because tropical areas abound with outbreeders (including self-incompatible and dioecious or subdioecious species, particularly trees), with obligatory reliance on pollinators.19 Pollination biology in India has been largely pursued as a component of reproductive biological studies. Considering the extent of diversity and the potential of pollination in the maintenance of agroecosystems,20 the information on pollination from India is abysmally little.21 In recent years there has been increased realization to conserve bioresources in the hotspot regions of India. Several economically important species including those which provide non wood forest products (NWFPs) and timber have been investigated.22–27 Certain oleo-gum-resin yielding trees such as Acacia senegal, Boswellia serrata, Butea monosperma, Commiphora wightii and Sterculia urens from the dry deciduous ecosystems have shown interesting pollination syndromes, sexual systems and breeding strategies. In a community-wide survey of pollination modes in the trees of Kakachi forest in Western Ghats, Devy and Davidar28 have shown that ~75% of the trees are specialized to a single pollinator group and the remaining are polylectic; the diversity in pollination modes is comparable to other lowland tropical forests of the world. At present, there are no studies from India, which have covered the entire diversity of life-forms to establish the community level pollination-systems and the pollinator networking.

Biotic pollination A great majority of plant species are pollinated by animals (zoophily) rather than water (hydrophily) or wind (anemophily). This is obvious due to the co-occurrence of enormous plant and animal diversity in the tropical and subtropical regions where resources are abundant. Pollinators in general seek for rewards among which pollen and the nectar are the primary resources. Besides these, some foragers collect wax, oils, resins as floral rewards;29–31 others find a brooding place for their own reproduction and survival.


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Entomophily is most widespread and among these, bees (tribe Aipdae) being the most important and efficient pollinators.18,32 More than 66% of the 1500 crop plant species of the world are pollinated by bees, which essentially account for 15–30% of food production.33 It is believed that the social behavior of maintaining a huge colony and the need to provide pollen and honey to developing larvae have contributed towards the evolutionary specialization of the body parts in the bees. Among the honeybees, the giant Asian honeybee, Apis dorsata (Fig. 1C), is the predominant pollinator and is also capable of foraging in full moon light conditions in the night.34,35 Other Apidae members – Apis mellifera, Apis cerana, Apis florea and Trigona spp. are the other important pollinators of plantation crops in the tropics.36,37 Strobilanthes kunthianus is a semelparous species that comes to flower once every 12 years in the southern part of Western Ghats (Kerala/Tamil Nadu border). Mass flowering in the species attracts a variety of insects comprising bees and butterflies. However, only Apis cerana indica and certain ants are the frequent foragers and are the efficient pollinators ensuring 100% pollination and high seed set.38,39 Flowering behavior in bamboos has always been a botanical curiosity as it affects several dynamic features of the ecosystem. Bees are believed to be the major pollinators in the species so far studied,40 although the role of wind can not be ruled out in many of them. For example, Jijeesh et al. have reported wind pollination in Pseudoxytenanthera monadelpha.41 Butterflies frequently visit a number of herbaceous and woody flowering plants. However, only a few plant species are effectively pollinated. In Western Ghats, it has been shown that four out of the 86 tree species are primarily pollinated by the butterflies.28 Anitha and Prasad observed ~16 species of butterflies in gregariously flowering S. kunthianus, but none appeared to be involved in pollination.38 In India, ornithophily was reported in a few species belonging to families Bignoniaceae, Fabaceae, Loranthaceae, Malvaceae, Myrtaceae and Verbenaceae.42 Subramanya and Radhamani have reviewed the reports on plants that are visited by birds and bats to forage their flowers.43 Nearly 60 species of birds visit around 100 species and in the majority (~80%), more than one species of birds visit the flowers. However, only a few are believed to aid in pollination44 and others may act as nectar robbers.45 Thus, merely foraging or visiting of birds is not an indication of successful ornithophily in a plant. For example in B. monosperma, out of seven species of visiting birds, only the purple sunbird (Nectarinia asiatica) is effective in bringing about pollination.25 Most of the birds are reported to be nectar robbers.46 Thus, to establish the obligate necessity of birds as pollinators, it is essential to analyze their foraging behavior, pollen load and importantly, whether or not their visit to unpollinated flowers results in fruit-set. Among the variety of animal pollinators, there are unbelievable pollinators such as cockroaches,47 mosquitoes48 and lizards.49 Even giraffes were suggested to

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be pollinators of the knob thorn Acacia (A. nigrescencs).50 However, a subsequent study has shown that foraging by giraffes leads to low fecundity and only bees are effective.51 A frequently cited mode of pollination in the text-books is by snails and slugs. Malacophily is among the rarest instances of pollination.52 Snails are largely implicated as pests of crops and other plants as they eat away the fleshy floral organs. However, in Volvulopsis nummularium (Convolvulaceae), a rainy season prostrate herb that occurs in Delhi region, the pollination mechanism is present as a strange guild involving a terrestrial garden snail (Lamellaxis gracile) (Fig. 1D) and honeybees (Apis cerana indica), both of which contribute to successful cross-pollination and fruit set.52 On cloudy days the bee visits were lacking and on the bright sunny days the snails come first followed by the bees. Although the plants are self-compatible, obligate autogamy is prevented by ensuring crossing, mediated by snails and bees. This is the first report on malacophily in India. There is need to explore the possibility of more cases of malacophily in the wet and humid areas where snails are abundantly found. Thrips were mainly considered phytophagous insect pests. While demonstrating the significance of change in the colour of flower in an inflorescence of a particular variety of Lantana camara, Mathur and Mohan Ram53 established the role of thrips in pollination (thripophily). Several instances of thripophily have been reported from the members of Asteraceae, Solanaceae and Fabaceae54, reaching to the conclusion that thripophily is a common phenomenon in nectarproducing flowers and tropical forest trees of the genus Shorea in Malaysia.55 Although mediation of pollinators is mandatory in obligate outbreeders, their role is essential in certain self-compatible plants as well. For example, certain self-compatible leguminous crops exhibit autosterility because of the presence of a thick cuticle over the stigma. In these plants, visit by insects (tripping) causes rupturing of the cuticle over the stigma to release the stigmatic exudates. The latter facilitates germination of self-pollen.56 Conversely, insect visitation may induce dehiscence of anthers. In Incarvillea emodi (Bignoniaceae), nectar foraging by Apis mellifera, pressurizes the appendages of anthers to release the pollen grains and carries them while moving out of the flower.57 Pollen shedding is negligible in the absence of the pollinator.

The question of legitimacy Faegri and Pijl proposed a beautiful correlation between the flower types that animals prefer to visit.11 This relationship is termed “pollination syndrome” and essentially refers to suites of flower characteristics (morphology, colour, nectar and/or pollen as food) that attract a particular pollinator to visit them and discourage others from accessing the rewards or visit them. This means that bright


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Figure 1. (A) Flower colour signals and pollination. Top view of a variety of Lantana camara showing unopened pink-tipped flower buds in the centre, freshly opened yellow flowers ready to be pollinated, surrounded by post-pollinated flowers that have changed colour to prevent the foragers Figure 1. (B–E) Diversity in pollination modes. (B) Bat pollination in Oroxylum indicum, photographed late 9 in the evening using a tele-lens and a flash). A short-nosed bat, Cynopterus sphinx, approaching a flower bud in the inflorescence. Assured of the source of nectar, the bat develops constancy with the flowers and forcibly opens the corolla to sip the nectar: (C) The giant Asian honey bee, Apis dorsata, is the most frequent pollinator of many wild and domesticated plants. This picture shows a honeybee foraging the male flowers of kusum, Schleichera oleosa, a source of lac resin: (D) Enlarged view of a terrestrial garden snail (Lamellaxis gracile) with a conspicuous shell foraging the flower of a garden weed (Volvulopsis nummularis). The snail consumes the anthers and pollen but does not damage the pistil. Pollination occurs when the snail transfers the pollen deposited on the shell to the stigma of the same or of another flower resulting in seed-set. (E) Pre-anthesis cliestogamy in Polypleurum stylosum (Podostemaceae). Disposition of stamens and the pistil in the flower of the aquatic plant (spathe has been removed). The anthers are entangled with the bilobed stigma to facilitate autogamy. See text for details

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coloured flowers with copious nectar that lack fragrance and bloom during the day time are likely to be visited by birds to result in pollination, and such plants are called ornithophilous. Similarly plants pollinated by ants (myrmecophily), beetles (cantherophily), flies (myophily), dung and carrion flies (sapromyophily) honeybees (melittophily), butterflies (psychophily), moths (phalaenophily), snails and slugs (malacophily), reptiles, birds (ornithophily) rodents (therophily), marsupials and bats (cheiropterophily) are characterized by their respective pollination syndromes. Among these, the bats, moths, geckos and owls are associated with nocturnal pollination.58 Some experimental studies have shown that the syndrome concept holds true in certain cases and it usually helps in designing the field-based experiments59,60. Most of the flowers with restricted floral phenologies and complex structure may follow such a specialized relationship. For example, in Oroxylum indicum occurring in North India, there is a specialized relationship and the interaction follows the concept of pollination syndrome. The flowers are bat-pollinated (cheiropterophilous), dull-coloured, emit a fetid smell and bloom only for a short period in the night. As the floral phenology is confined to night time, bats gain access to the long erect racemes above the canopy (Fig. 1B). The structural organization of the flower, the placement and angle of the flower on the inflorescence axis allow only a suitable sized bat – Cynopterus sphinx to legitimately forage the flower and prevent over-sized bats to do so.61 In Malaysia and Thailand, a different species of bat, Eonycteris spelaea, with similar size and foraging behavior has been reported earlier.62, 63 This study establishes that O. indicum has strong affiliation of the plant for bat pollination and the principal pollinator of specialized flowers may vary in time and space. Similarly, Ravenala madagascariensis (Strelitziaceae), popularly known as the traveller’s tree, is shown to be pollinated by lemurs in Madagascar,64 while elsewhere its flowers are either explosively bird pollinated65 or foraged by large squirrels in Brazil (personal communication). In India, this plant produces seeds in Wayanad, (Kerala) (personal observation). The pollinator is yet to be established. The extent of extreme one-to-one relation, known as nursery pollination syndrome, is exhibited by a few species such as Ficus and their agaonid wasp pollinators66 and Yucca and Tegiticula,67 the moth pollinator. In nursery pollination syndromes, the flowers become the brooding site for pollinators. These extreme specializations are fragile as both the counterparts may perish if one were to disappear. However, recent studies have shown that the syndrome concept is imperfect in many cases, as several pollinators of distant taxonomic groups may visit the same flower with equal efficiency. Additionally, the syndromes are defined as we perceive the flowers and their colours and not as the pollinators see them.68 In Butea monosperma, popularly known as the “flame of the forest” for usually possessing bright India-orange coloured flowers, is “conceptually” meant


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for bird pollination. Detailed pollination ecology of the species has established that successful pollination requires legitimate foraging (pollination in return of rewards without damaging the flower) because the nectar is deep seated in the keel petals and the margin of the keel is the only opening to access it.25 Among a variety of birds, only the purple sunbirds (Nectarinia asiatica) are successful to legitimately do so. Besides the sunbirds, the common Three-striped squirrel, Funambulatus tristiatus, is equally effective in pollination. Instances of co-pollination in the otherwise specialized flower-bearing plants are increasingly mounting, indicating that a generalized relation involving one plant and many pollinators is believed to be of more common occurrence.69–71 Finally, the mismatching in the components of the syndrome concept is exemplified by the instances of ambophily. In Plantago lanceolata, 72 Hormathophylla spinosa,73 Buxus balearica74 exhibit ambophily, in which flowers are pollinated by insects as well as wind.75 A recent assessment of syndrome concept based on six communities from three continents has shown that pollination syndrome does not describe the diversity of floral phenotype or predict the pollinators of plant species.60

Floral display and cues Flowers advertise in a number of ways to establish a relation with the pollinators and optimize pollination efficiency. Besides the size and arrangement of flowers in an inflorescence, scent and pigmentation of petals are the crucial attractants.11,76–78 A small solitary flower may not have the same effect on a forager than a group of small flowers with compacted arrangement in an inflorescence. In such plants, the inflorescence serves as pollination unit.79 In acacias, individual flowers are very small; their compact arrangement into a long spike or globose inflorescence not only imparts a mass effect but also results in intense fragrance around the flowers and rapid pollination.80 Repeated visits by the foragers make them learn the cues81 to develop floral constancy.6, 82 Constancy with flowers by the foragers is acquired by learning experience and leads to targeted pollination of flowers of the conspecific plants while minimizing the loss of pollen. Floral constancy is more pronounced in mature foragers suggesting that they learn flower handling initially by visiting a number of flower forms and memorizing before narrowing on to a particular one or visit similar forms in different species.83 By using mutant lines in snapdragon (Antirrhinum spp.) that lacked only the conical cells on the petals, Whitney et al. showed that the conical cells assist in the landing of the bumblebees.84 The foraging bumblebees could use colour cues to discriminate between the petals with flat surface from those having conical cells.

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Besides the major floral contrivances that result in various forms, pigmentation on the petals has a significant effect on pollination efficiency. This has been aptly demonstrated by developing near isogenic lines (NIL) in two sister species of monkeyflowers (Mimulus spp.).85 The wild-type M. lewsii has pink flowered flowers and is pollinated by bumblebees, whereas M. cardinalis is red-flowered and is pollinated by hummingbirds. The Yellow Upper (YUP) allele at a single locus controls the yellow carotenoid pigments in the petals of pink flowered M. lewsii. NIL Substitution to alternative alleles in these species showed that M. cardinalis NILs with M. lewsii YUP allele had dark pink flowers which attracted 78-fold more bee visits than by hummingbirds. The M. lewsii NILs with yup allele had yellow-orange flowers which attracted 68-times more visits by the hummingbirds than on the wild type. Flowers often convey information about the presence or absence of rewards by means of changing their petal colour. In Lantana (Fig. 1A), the freshly opened flowers are yellow and attract thrips, butterflies or even birds (such as red-vented bulbul) to nectar rich yellow flowers whereas the pink flowers lack nectar and the change in the colour is caused by pollination and production of delphinidine glucoside.86 Change in colour is not always unidirectional. In Desmodium setigerum, tripping initiates change in colour from lilac to white. If the flower remains unpollinated even after tripping, it reverses its colour back to deep turquoise or lilac for a second time to receive pollen.87 Preference to yellow colour by other insects such as bumblebees was demonstrated in an ingenious experiment conducted by Chitka and Walker.88 They employed untrained bumblebees bred in dark for generations and exposed them to large prints of famous paintings – “Sunflowers” by Vincent van Gogh, “A vase of flowers” by Paul Gauguin, “Pottery” by Patrick Caulfield and “Still life with a beer mug” by Fernand Léger. van Gogh’s sunflowers with predominant yellow colour proved most attractive to the bees. Olfactory cues (fragrance) are an important attractant to pollinators, which intensify at the peak time of blooming. The floral scent emanates from the secretory tissue termed osmophores,89 which may contain 1–200 volatile compounds.90 A selective role of scent in attracting the pollinators and repelling the non-pollinators has been suggested by many workers.91,92 Attraction may be particular type of species or rewards91 and even to the unpollinated flowers for a second chance of pollination.93 Floral scents in many orchids (~30,000) that offer no reward94,95, mimic the pheromones of female insects to sexually deceive the male insect pollinators.96 In an Australian orchid, Chiloglottis trapeziformi,97 a novel compound, chiloglottone mimics the pheromone of the female wasp (Neozelboria cryptoides) to sexually deceive the males. In Ophyris sphegodes, by using gas chromatography-electromagnetic detection methods, Schiestl et al. have shown that there are 15 compounds which elicit electroantennographic


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responses in male antennae and copulation attempts in the male bees (Andrena nigroaenea).98 The experiment demonstrated that these compounds were similar to those present in the cuticular wax for protection. Generally the bird-pollinated flowers lack fragrance.11 However, flowers of several hummingbird pollinated species contain volatiles in their floral headspace, which are common to vegetative parts.99 These studies indicate that during the course of evolution the compounds present in the vegetative parts assumed the function of attracting pollinators in one way or the other.

Nocturnal pollination studies in India Research on nocturnal pollination is beginning to be taken up in India. In earlier studies,100 only cursory observations were made on visits made by bats to flowers. Whether or not their visits lead to fruit-set, was not reported. This is understandable because of challenges posed by nocturnal observations, poor technology for photographing/filming in darkness and above all by assuming that bats are probably more efficient diaspore dispersers than pollinators.28 Out of presumably 28 bat visited plants43 (Subramanya & Radhamani 1993), only two – Ceiba pentandra (Bombacaceae),101,102 and recently Oroxylum indicum (Bignoniaceae) have been investigated from India.61 Most bees are diurnal pollinators. However, recent work103 has demonstrated that carpenter bee, Xylocopa tenuiscapa, is capable of effecting pollination in dark in the self-incompatible Heterophragma quadriloculare. Somanathan and co-workers have shown that a nocturnal bee Xylocopa tranquebarica from the seasonal cloud forest in India can fly under starlight conditions and is even endowed with colour vision under extremely dim light.104

Pollination constraints in the introduced crops Many plants suffer from serious losses in fruit and seed production due to inadequate pollination. In hermaphroditic species, the combined effect of the distribution pattern of individuals in the population and the extent of index of self-incompatibility limits fecundity. In dioecious plants, seed set may vary due to skewed distribution of sexes in the population and if the species is wind-pollinated, inclement weather conditions may also affect the yield. Absence of legitimate pollinators of plants in the wild may also adversely affect fruit-set.57 Yield can be sustained in crop plants through artificial pollination or by increasing the pollinator visitation rates. In the East African oil palm (Elaeis guineensis) presently grown on a commercial scale, fruit-set is accomplished by wind as well as by insect-pollination. In Malaysia, fruit yield was initially low because the specific weevil pollinator, Eledobious kamaroonicus, had not been

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introduced along with the crop. Introduction of imported weevils drastically improved the fruit-set in the plantations. Oil palm exhibits temporal dioecism at the population level, as it bears male and female inflorescences in alternate years. The yield depends upon the extent of balanced sex expression in a season. In bad years, when there are few male plants in the plantation or under inclement weather conditions, the yield is low. Work done on oil palm in India has shown that assisted pollination sustains the regular yield.105 Oil palm pollen grains respond to long term storage for 8–10 years under cryo (–198°C) conditions.106 The other approach to improve insect visitation rates include enhancement of native pollinator populations. Significant improvement in the yield was noticed in clover (Trifolium pratense) after the nesting sites of its pollinator, Bombus spp. were increased around the crop field.107,108 Pollination may become a major constraint due to competition between native wild bees with the introduced colonies of domesticated honeybees, rendering poor yield at places where agricultural fields are managed in the vicinity of natural populations. Intensification of agricultural fields and increased reliance on domestic bees may adversely affect the native pollinator diversity in a particular area. For example in the Western Ghats, lesser cardamom (Elettaria cardamomum) is extensively planted with areca nut as a cash crop. On the basis of observation made at three sites, Sinu and Shivanna showed that the wild stingless bee, T. iridipennis serves as the main pollinator of cardamom at sites where the competition with domesticated honey bees is low.37 Urgency to conserve the native pollinators is being realized as insurance because of serious decline in the populations of domestic bees such as Apis mellifera, owing to diseases and heavy use of insecticides.109,110

Abiotic pollination In this category, wind pollination (anemophily) is predominant and water pollination (hydrophily) occurs in a few genera.111,112 Wind pollination Wind pollination is found in nearly 10% of the families in flowering plants.113 Based on phylogenetic analyses, it is believed that anemophilous plants are derived from their entomophilous ancestors114,115 and probably at 65 independent times.116 In general, wind-pollinated plants produce unisexual flowers, copious amount of dry pollen and occupy more open and wind dominated habitats.75 However, in tropical environments airborne pollen have also been recorded from entomophilous, especially the buzz pollinated species117 or plants with ambophilous floral traits.118 In these plants foraging by insects facilitates dispersal of pollen grains in the air but the may not actually pollinate the flowers.119 However, in certain Indian grasses


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pollen transfer to stigma has been observed.120 In closed tropical conditions wind pollination may not always correlate completely with anemophilous syndrome. In wind-pollinated Cicca acida and Emblica officinalis floral traits are conducive enough for successful release and trap airborne pollen but in the deciduous Madhuca indica and evergreen Mimusops elengi, they do not.121 As natural conditions vary in the distribution range of these species, it is important to assess their breeding systems, particularly the extent of autogamy, from different areas to establish the prevailing adaptive value of their floral traits. Unlike animal pollinated trees, wind-pollinated species suffer from acute pollination limitation in natural populations especially if the plants are sparsely distributed and are self-incompatible.122 In ambophilous oil palm, the airborne pollen density varies according to the number of male plants in the plantation. During the male dominated phase pollen density is limited up to 30 m from the source.105 The dispersal distance may vary according to the source height of the plants123 and habitat conditions. There is paucity of information on the pollination dynamics of wind-pollinated species form India. Individual contribution to total fecundity from wind and biotic pollination is important but difficult to gather from tall trees, as they can not be contained for establishing the exclusivity of modes or limitation of pollination as in the herbaceous species. Friedman and Barrett recorded that at least in nine out of ten herbaceous wind-pollinated plants, pollen was not a limitation.122 Water pollination Hydrophily occurs in nearly 30 genera18 and is believed to be a rare phenomenon. Many aquatic plants such as Cabomba, Nymphoides, Hottonia, and Potamageton exhibit entomophily, rather than hydrophily. Depending upon whether the flowers are exposed to air before pollination or remain submerged, hydrophily is either ephydrophily or hyphydrophily, respectively. Hyphydrophily is usually shown by marine angiosperms such as Amphibolis antartica, Zostera marina and Najas. Ephydrophily is more common and reported in plants such as Lemna trisulca;124 Vallisneria;125 Ruppia, Callitriche, Elodea and Hydrilla.126 In Ceratophyllum, plants are completely submerged and pollen grains are not released until the abscised anthers float up to the surface of water. The germinating pollen grains are released after the dehiscence of anthers and the elongated pollen tubes sink to effect pollination (Mohan Ram and Sehgal).126a In Callitriche palustris and C. heterophylla,127 the unisexual flowers are borne on the same plant, but in separate leaf axils. When the plants are completely submerged, the pollen grains have been noticed in a few cases to germinate inside the anthers of the partially developed male flowers and their tubes traverse through the filament and reach the ovule of the pistillate flower to effect fertilization; this mechanism is known as internal geitonogamy.

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Podostemaceae, inhabiting fast-flowing steams, rivers and cataracts attached firmly to the rocks, is the largest family of aquatic angiosperms and its members occur in the tropical and the sub-tropical regions of the world. Pollination system in Podostemaceae has been studied in a few species and variously interpreted. Since the majority of species have small, naked flowers with closely placed stamen and pistil, spontaneous autogamy seems to be the most probable mechanism.128–130 However, there have been reports of insect pollination in Mourera and Tulasneantha131 and speculations of wind pollination in Marathrum rubrum132 and some new world Podostemaceae.133 Cleistogamy and pre-anthesis cleistogamy (Fig. 1E) have also been reported from Podostemaceae134-136 which further corroborate the general occurrence of self-pollination in the family. Unfortunately, the family has not received the much warranted attention and still remains a neglected group of plants for further studies. Mutualism at peril The success of pollination event in a species is believed to be an evolutionarily stabilized mutualistic episode and in a community, pollination systems of different species contribute to a pollination network. As the collection of rewards from the flowers is intricate in the ecological network of animal-food web,137 even minor changes in the environment may disproportionately affect the dynamics of pollination systems. Effect of climate change, particularly global warming, is being best witnessed in the shifts in the flowering period of many plant species.138,139 Although detailed long-term phenological observations in the tropics are deficient,138 recent changes in the tropical cloudiness140 and monsoon dynamics141 may seriously affect the reproductive biology of many plant species in the tropical belt. Several tree species in northern India such as, Rhododendron and Reinwardtia are reported to have advanced their blooming period by nearly a month. In many tropical deciduous trees of India, in which the reproductive strategies have developed in response to monsoonic bioclimate,142 early flowering (Madhuca latifolia, Mangifera indica) to prolonged flowering (as in Cassia fistula) has been observed (personal observation). Such changes and shifts are likely to create gaps in the flowering episodes at the community level that may allow invasive plants to occupy the flowering niche. As the invasive species are known to proliferate intensely, the divergence in mutualism is likely to affect the native plant species. Many invasive plants such as Ageratum conyzoides, Chromolaena odorata, Eichhornia crassipes, Eupatorium adenophorum, Ipomoea carnea, Lantana camara, Mikania micrantha, Mimosa invisa, Parthenium hysterophorus and Prosopis juliflora have naturalized in several parts of India and are threatening the natural communities. Chromolaena odorata has invaded in the entire buffer area of silent valley (personal observation). The impacts of such plants on the


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pollinator diversity are yet to be analyzed and strategies are to be developed to eradicate their dominance in the species rich areas. Conclusion Evolution and the subsequent radiation of flowering plants is a reflection of their ability to adapt to a variety of habitats and their diversity and often flexibility, in the modes of reproduction. The enormous diversity in flowering plants amazed Charles Darwin and he discussed many of his careful observations based on evolution of co-adaptive traits in plants and their pollinators. The fascinating observation made by him on pollination mechanism in orchids is still an inspiration to pollination biologists. The dependence of flowering plants on pollinators, including a variety of animals and abiotic modes for pollen mediated gene flow has motivated biologists to use interdisciplinary approaches to explore the fascinating area of pollination biology. Amidst fresh challenges such as - habitat modification, climate change declining biodiversity, assessment of genetic resources in related gene pools, management of agroecosystems while sustaining future food security,143 gene flow from the genetically modified organisms and threats from the invasive plants, there is need to recombine conventional approaches with powerful molecular tools to design future course of investigation in pollination biology. It is ironical that Indian efforts on pollination biology mismatch the magnitude of the country’s biodiversity and its deep concern on the conservation of the flora and enhanced crop production. Acknowledgment: We thank Ms. Priyanka Khanduri for her able assistance in the preparation of this chapter.

References 1.

Anonymous (2007) Status of pollinators in North America, Committee on the Status of Pollinators in North America, National Research Council. The National Academies Press, Washington, DC

2.

Schmid R (1975) Two hundred years of pollination biology: An overview. Biologist 57:26–35

3.

Shivanna KR, Mohan Ram HY (1993) Pollination biology: Contributions to the fundamental and applied aspects. Curr Sci 65:226–233

3a.

Mayr E (2004) What Makes Biology Unique? Considerations on the Autonomy of a Scientific Discipline. Cambridge University Press, UK

4.

Sprengel CK (1793) Das entdeckte Geheimniss der Natur im Bau und in der Befruchtung der Blumen. Friedrich Vieweg der Ältere, Berlin

5.

Darwin CR (1862) The various contrivances by which orchids are fertilized by insects. John Murray, London

234


6.

Darwin C (1876) The effects of cross and self fertilization in the vegetable kingdom. John Murray, London

7.

Darwin CR (1877) The different forms of flowers on plants of the same species. John Murray, London

8.

Kölreuter JG (1761) Vorläufige Nachricht von einigen das Geschlecht der Pflanzen betreffenden Versuchen und Beobachtungen. Leipzig

9.

Mitchell RJ, Irwin RE, Flanagan RJ, Karron JD (2009) Ecology and evolution of plant– pollinator interactions. Ann Botany 103:1355–1363

10.

Grant V, Grant KA (1965) Flower pollination in the phlox family. Columbia University Press, New York

11.

Faegri K, van der Pijl L (1979) The principles of pollination ecology. Pergamon Press, Oxford

12.

Jones CE, Little RJ (1983) Handbook of experimental pollination biology. Van Nostrand Reinhold, New York

13.

Real L (1983) Pollination biology. Academic Press, New York

14.

Shivanna KR, Rangaswamy NS (1992) Pollen biology: A laboratory manual. Springer Verlag, Berlin

15.

Dafni A (1992) Pollination Ecology: A practical approach. Oxford University Press, Oxford

16.

Kearns CA, Inouye DW (1993) Techniques for pollination biologists. Colorado Press

17.

Dafni, A, Kevan PG (2003) Field methods in pollination ecology. Ecoquest, Cambridge, Canada

18.

Corlett RT (2004) Flower visitors and pollination in the Oriental (Indomalayan) region. Biol Rev 79:497–532

19.

Bond WJ (1994) Do mutualisms matter? Assessing the impact of pollinator and disperser disruption on plant extinction. Philosophical Transactions of the Royal Society of London Series B. Biol Sci 344:83–90

20.

Allen-Wardell G, Bernhardt P, Bitner R, Burquez A, Buchmann S, Cane J, Cox P, Dalton V, Feinsinger P, Ingram M, Inouye D, Jones CE, Kennedy K, Kevan P, Koopowitz H, Medellin R, Medellin-Morales S, Nabhan GP (1998) The potential consequences of pollinator declines on the conservation of biodiversity and stability of food crop yields. Conser Biol 12:8–17

21.

Vasudeva R, Lokesha R (1993) Plant-pollinator interactions. Curr Sci 65:198–201

22.

Menon S, Shivanna KR, Mohan Ram HY (1993) Pollination ecology of Dalbergia sissoo - an important Indian tree legume. In: International Union of the Study of Social Insects (IUSSI). Veeresh GR, Shaanker U, Bangalore KN (Eds.), Indian Chapter

23.

Gupta P, Shivanna KR, Mohan Ram HY (1999) Apomixis and polyembryony in the guggul plant, Commiphora wightii. Ann Botany 78:67–72

24.

Tandon R, Shivanna KR, Mohan Ram HY (2001) Pollination biology and the breeding system of Acacia senegal. Bota J Linnaean Soc 135:251–262

25.

Tandon R, Shivanna KR, Mohan Ram HY (2003) Reproductive biology of Butea monosperma (Fabaceae). Ann Botany 92:715723

26.

Sunnichan VG, Mohan Ram HY, Shivanna KR (2005) Reproductive biology of Boswellia serrata, the source of salai guggul, an important gum-resin. Botan J Linnaean Soc 147: 73–82


235

27.

Sunnichan VG, Mohan Ram HY, Shivanna KR (2004) Floral sexuality and breeding system in gum karaya tree, Sterculia urens. Plant Systematics and Evolution 244:201–218

28.

Devy MS, Davidar P (2003) Pollination systems of trees in Kakachi, a mid-elevation wet evergreen forest in Western Ghats, India. Am J Botany 90:650–657

29.

Buchmann SL (1987) The ecology of oil flowers and their bees. Ann Rev Ecol Systemat 18:343–369

30.

Dafni A, Kevan PG, Husband B (2005) Practical pollination biology. Enviroquest Ltd., Cambridge, Canada

31.

Roulston TH, Cane JH (2000) Pollen nutritional content and digestibility for animals. Plant Systemat Evolut 222:187–209

32.

Keven PG, Baker HG (1983) Insects as Flower Visitors and Pollinators. Ann Rev Entomol 28:407–453

33.

McGregor SE (1976) Pollinating agents and their comparative value. In insect pollination of cultivated crop plants, agriculture handbook. USDA 496:19–23

34.

Dyer FC (1985) Nocturnal orientation by the Asian honey bee Apis dorsata. Animal Behavior 33:769–74

35.

Rao SP, Atluri JB, Reddi CS (2001) Intermittent mass blooming, midnight anthesis and rockbee pollination in Pterocarpus santalinus (Fabaceae). Nordic J Botany 21:271–276

36.

Slaa EJ, Chaves LAS, Malagodi-braga KS, Hofstede FE (2006) Stingless bees in applied pollination: practice and perspectives. Apidologie 37:293–315

37.

Sinu PA, Shivanna KR (2007) Pollination Ecology of cardamom (Elettaria cardamomum) in the Western Ghats, India. J Trop Ecol 23:493–496

38.

Anitha K, Prasad SN (2007) Mass flowering and pollinators of Strobilanthes consanguinea in the Western Ghats, South India. Curr Sci 92:1680–1681

39.

Sharma M, Kuriakose G, Shivanna KR (2008) Reproductive strategies of Strobilanthes kunthianus, an endemic, semelparous species in southern Western Ghats, India. Botan J Linnaean Soc 157:155–163

40.

Koshy KC, Harikumar D, Narendran TC (2001) Insect visits to some bamboos of Western Ghats, India. Curr Sci 81:833–838

41.

Jijeesh CM, Seethlakshmi KK, Beena VB, Raveendran VP (2009) Recent flowering of endemic bamboo – Pseudoxytenanthera monadelpha (Thw.) Soderstrom and Ellis, in Munnar, India. Phytomorphology 59:35–39

42.

Ali SA (1932) Flower birds and bird flowers in India. J Bombay Natu History Soc 35: 573–605

43.

Subramanya S, Radhamani TR (1993) Pollination by birds and bats’. Curr Sci 65(3): 201–209

44.

Davidar P (1983) Similarity between flowers and fruits in some flowerpecker pollinated mistletoes. Biotropica 15:32–37

45.

Kannan P (1978) Nectar-feeding adaptations of flower-birds. J Bombay Natu History Soc 75:1036–1050

46.

Maloof JE, Inouye DW (2000) Are nectar robbers cheaters or mutualists? Ecology 81: 2651–2661

47.

Nagamitsu T, Inoue T (1997) Cockroach pollination and breeding system of Uvaria elmeri (Annonaceae) in a lowland mixed-dipterocarp forest in Sarawak. Am J Botany 84: 208–213

236


48.

Thien LB (1969) Mosquito pollination of Habenaria obtusata (Orchidaceae). Am J Botany 56:232–237

49.

Olesen JM, Valido A (2003) Lizards as pollinators and seed dispersers: An island phenomenon. Trends Ecol Evolu 18:177–181

50.

Du Toit JT (1992) Winning by a neck. Natu History 8:29–32

51.

Fleming PA, Hofmeyr SD, Nicolson SW, Du Toit JT (2006) Are giraffes pollinators or flower predators of Acacia nigrescens in Kruger National Park, South Africa? J Trop Ecol

52.

Sarma K, Tandon R, Shivanna KR, Mohan Ram HY (2007) Snail-pollination in Volvulopsis nummularium. Curr Sci 93(6):826–831

53.

Mathur G, Mohan Ram HY (1978) Significance of petal colour in thrips-pollianted Lantana camara. Ann Botany 42:1473–1476

54.

Ananthakrishanan T (1993) Role of thrips in pollination. Curr Sci 65:262–264

55.

Appanah S, Chan HT (1981) Thrips: The pollinators of some dipterocarps. Malaysian Forester 44:234–252

56.

Shivanna KR, Owens SJ (1989) Pollen-ovule interactions (Papilionoideae). In: Advances in Legume Biology. Stirton CH, Zarucchi JL (Eds.), Monographs in Systematic Biology, Missouri Botanical Garden

57.

Verma S, Kaul V, Magotra R, Koul, AK (2008) Pollinator-induced anther dehiscence in Incarvillea emodi (Bignoniaceae). Curr Sci 94:1372–1274

58.

Autumn K, Jindrich D, DeNardo D, Mueller R (1999) Locomotor performance at low temperature and the evolution of nocturnality in geckos. Evolution 53:580–599

59.

Fenster CB, Armbruster WS, Wilson P, Thomson JD, Dudash MR (2004) Pollination syndromes and floral specialization. Annu Rev Ecol Evolut System 35:375–403

60.

Ollerton1 J, Alarco J, Waser NM, Price MV, Watts S, Cranmer L, Hingston A, Peter CI, Rotenberry J (2009) A global test of the pollination syndrome hypothesis. Ann Botany 103:1471–1480 Vikas, Gautam M, Tandon R, Mohan Ram HY (2009) Pollination biology and breeding system of Oroxylum indicum in Western Himalaya. J Trop Ecol 25:93–96 Srithongchuay T, Bumrungsri, S, Sripao-Raya E (2008) Pollination ecology of the late-successional tree, Oroxylum indicum (Bignoniaceae) in Thailand. J Trop Ecol 24: 477–484 Gould E (1978) Foraging behavior of Malaysian nectar-feeding bats. Biotropica 10: 184–193 Kress J (1993) Coevolution of plants and animals: Pollination of flowers by primates in Madagascar. Curr Sci 65:253–257 Mabberley DJ (1997) In: The Plant-book: A Portable Dictionary of the Vascular Plants. 2nd edition, Cambridge University Press, UK Machado CA., Robbins N, Gilbert MTP, Herre EA (2005) Critical review of host specificity and its coevolutionary implications in the fig fig-wasp mutualism. Proc Nat Acad Sci USA 102:6558–6565 Pellmyr O (2003) Yuccas, yucca moths, and coevolution: A review. Ann Missouri Botan Garden 90:35–55 Howe HF, Westley LC (1988) Ecological relationships of plants and animals. Oxford University Press Johnson SD, Steiner KE (2000) Generalization versus specialization in plant pollination systems. Tren Ecol Evol 15:140–143

61. 62.

63. 64. 65. 66.

67. 68. 69.


237

70.

Kandori I (2002) Diverse visitors with various pollinator importance and temporal change in the important pollinators of Geranium thunbergii (Geraniaceae). Ecol Res 17:283–294

71.

Waser NM (2006) Plant–pollinator interactions: From specialization to generalization. University of Chicago Press, Chicago

72.

Stelleman P (1984) Reflections on the transition from wind pollination to ambophily. Acta Botanica Neerlandica 33:497–508

73.

Gómez JM, Zamora R (1996) Wind pollination in high mountain populations of Hormathophylla spinosa (Cruciferae). Am J Botany 83:580–585

74.

De la Bandera MC, Traveset A (2006) Breeding system and spatial variation in the pollination biology of the heterocarpic Thymelaea velutina (Thymelaeaceae). Plant System Evol 257:9–23

75.

Culley TM, Weller SG, Sakai AK (2002) The evolution of wind pollination in angiosperms. Tren Ecol Evol 17:361–369

76.

Kevan PG (1997) Pollination biology and plant breeding system. In: Pollen biotechnology for crop production and improvement. Shivanna KR, Sawhney VK (Eds.), Cambridge University Press, UK

77.

Chitka L, Shonida A, Troje N, Menzel R (1994) Ulravoilet as component of flower reflection and colour perception of Hymenoptera. Vision Res 34:1489–1508

78.

Dafni A, Lehrer M, Kevan PG (1997) Spatial flower parameters and insect spatial vision. Biol Rev 72:239–282

79.

Arroyo MTK (1981) Breeding system and pollination biology in Leguminosae. In: Advances in Legume Systematics. Part I. Polhill RM, Raven PG (Eds.), Proceedings of International Legume Conference, Royal Botanic Gardens, Kew

80.

Bernhardt P (1989) Floral ecology of Australian acacias. In: Advances in Legume Biology. Stirton CH, Zarucchi JL (Eds.), Monographs in Systematic Biology, Missouri Botanical Garden

81.

Yoshioka Y, Ohashi K, Konuma A, Iwata H, Ohsawa R, Ninomiy S (2007) Ability of bumblebees to discriminate differences in the shape of artificial flowers of Primula sieboldii (Primulaceae). Ann Botany 99:1175–1182

82.

Heinrich B (1976) The foraging specializations of individual bumblebees. Ecological Monographs 46:105–128

83.

Wilson P, Stine M (1996) Floral constancy in bumble bees: handling efficiency or perceptual conditioning? Oecologia 106:493–499

84.

Whitney HM, Chittka L, Bruce TJA, Glover1 BJ (2009) Conical Epidermal Cells Allow Bees to Grip Flowers and Increase Foraging Efficiency. Curr Biol 19:948–953

85.

Bradshaw HD, Schemske DW (2003) Allele substitution at a flower color locus produces a pollinator shift in two monkeyflower species (Mimulus). Nature 426:176–178

86.

Mohan Ram HY, Mathur G (1984) Flower colour changes in Lantana camara. J Experim Botany 35:1656–1662

87.

Willmer P, Stanley D, Steijven K, Matthews I, Nuttman C (2009) Bidirectional Flower Color and Shape Changes Allow a Second Opportunity for Pollination. Curr Biol 19:919–923

88.

Chittka L, Walker J (2006) Do bees like Van Gogh’s Sunflowers? Optics Laser Technol 38:323–328

89.

Vogel S (1990) Radiación adaptiva del síndrome floral en las familias Neotropicales. Boletín de la Académia Nacional de Ciéncias, Córdoba, Argentina 59:5–30

238


90.

Dobson HEM, Raguso RA, Knudsen JT, Ayasse M (1995) Scent as an attractant. In: Practical Pollination Biology. Dafni A, Kevan PG, Husband BC (Eds.), Enviroquest Ltd., Canada

91.

Dobson HEM, Bergström G (2000) The ecology of pollen odors. Plant Systemat Evolut 222:63–87

92.

Kessler D, Baldwin IT (2006) Making sense of nectar scents: the effects of nectar secondary metabolites on floral visitors of Nicotiana attenuate. Plant J 49:840–854

93.

Raguso RA, Willis MA (2005) Synergy between visual and olfactory cues in nectar feeding by wild hawkmoths, Manduca sexta. Animal Behav 69:407–418

94.

Kullenberg B (1961) Studies in Ophrys pollination. Zool Bidrag Uppsala 34:1–340

95.

Brodmann J, Twele R, Francke W, Yi-bo L, Xi-qiang S, Ayasse1 M (2009) Report Orchid Mimics Honey Bee Alarm Pheromone in Order to Attract Hornets for Pollination. Curr Biol 19:1368–1372

96.

Ledford H (2007) Plant biology: The flower of seduction. Nature 445:816–817

97.

Schiestl FP, Peakall R, Mant J, Schulz C, Ibarra F, Franke F (2003) The chemistry of sexual deception in an orchid-wasp pollination system. Science 302:437–438

98.

Schiestl FP, Ayasse M, Paulus HF, Lo¨fstedt C, Hansson BS, Ibarra F, Francke W (1999) Orchid pollination by sexual swindle. Nature 399:421–422

99.

Knudsen JT, Tollsten L, Groth I, Bergstro¨m G, Raguso RA (2004) Trends in floral scent chemistry in pollination syndromes: floral scent composition in hummingbird-pollinated taxa. Botan J Linnaean Soc 146:191–199

100.

McCann C (1939) Notes on the fulvous fruit-bat (Rousettus leschenaultia Desm.). J Bombay Natu Hist Soc 41:805–816

101.

Singaravelan N, Marimuthu G (2004) Nectar feeding and pollen carrying from Ceiba pentandra by Pteropodid bats. J Mammal 85(1):1–7

102.

Nathan PT, Raghuram H, Elangovan V, Karuppudurai T, Marimuthu G (2005) Bat pollination of Kapok tree, Ceiba pentandra. Curr Sci 88:1679–1681

103.

Somanathan H, Borges RM, Warrant EJ, Kelber A (2008) Visual ecology of Indian carpenter bees I: Light intensities and flight activity. J Comparat Physiol 194:97–107

104.

Somanathan H, Borges RM (2001) Nocturnal Pollination by the Carpenter Bee Xylocopa enuiscapa (Apidae) and the Effect of Floral Display on Fruit Set of Heterophragma quadriloculare (Bignoniaceae) in India. Biotropica 33:78–89

105.

Tandon R, Manohara TN, Nijalingappa BHM, Shivanna KR (2001) Pollination and pollenpistil interaction in Oil Palm, Elaeis guineensis. Ann Botany 87(6):831–838

106.

Tandon R, Chodhury R, Shivanna KR (2007) Cryopreservation of oil palm pollen. Curr Sci 92:182–183

107.

Donovan J, Weirs S (1978) Development of hives for field population increase, and studies on the life cycles of the four species of introduced bumble bees in New Zealand. N Z J Agricul Res 21:733–756

108.

Macfarlane RP, Griffin RP (1985) Assessment of bumble bees and honey bees as pollinators of red clover. Proceedings of the 4th Australasian Conference on Grassland Invertebrate. Eco1ogy 11:4–120

109.

Nabhan GP, Buchmann SL (1996) Pollination services: biodiversity’s direct link to world food stability. In: Ecosystem Services. Daly G (Ed.), Island Press, Washington

110.

Kreman C, Williams M, Thorp RW (2002) Crop pollination from native bees at risk from agricultural intensification. Proc Nat Acad Sci USA 99:16812–16816


239

111.

Richards AJ (1986) In: Plant Breeding Systems. George Allen and Unwin, London

112.

Kearns C, Inouye D, Waser N (1998) Endangered mutualisms: the conservation of plantpollinator interactions. Ann Rev Ecol Systemat 29:83–112

113.

Ackerman JD (2000) Abiotic pollen and pollination: ecological, functional, and evolutionary perspectives. Plant Systemat Evol 222:167–185

114.

Li J, Bogle AL, Donoghue MJ (1999) Phylogenetic relationships in the Hamamelidoideae inferred from sequences of TRN non-coding regions of chloroplast DNA. Harvard Papers Botany 4:343–356

115.

Hu S, Dilcher DL, Jarzen, DM, Taylor DW (2008) Early steps of angiosperm–pollinator coevolution. Pro Nat Acad Sci USA 105:240–245

116.

Linder PH (1998) Morphology and the evolution of wind pollination. In: Reproductive biology in systematics, conservation and economic botany. Owens SJ, Rudall PJ (Eds.), Royal Botanic Gardens, Kew

117.

Bush MB, Rivera R (2001) Reproductive ecology and pollen representation among neotropical trees. Global Ecol Bio 10:359–367

118.

Peeters L, Totland O (1999) Wind to insect pollination ratios and floral traits in five alpine Salix species. Canad J Botany 77:556–563

119.

Reddi EUB, Reddi CS (1984) Pollination ecology of Euphorbia geniculata (Euphorbiaceae). J Bombay Nat His Soc 81:571–582

120.

Pant DD, Nautiyal DD, Chaturvedi SK (1982) Insect pollination in some Indian glumiflorae. Beitraege zur Biologie der Pflanzen 57:229–236

121.

Reddi C, Reddi EU (1984) Wind pollination in two tropical tree species of Euphorbiaceae. Proc Indian Nat Sci Acad 850:66–80

122.

Friedman J, Barrett SCH (2009) Wind of change: new insights on the ecology and evolution of pollination and mating in wind-pollinated plants. Ann Botany 103:1515–1527

123.

Atluri JB, Venkata Ramana SP, Subba Reddi C (2004) Explosive pollen release, windpollination and mixed mating in the tropical tree Shorea robusta Gaertn (Dipterocarpaceae). Curr Sci 86:1416–1419

124.

den Hartog Segal (1964) A new classification of water-plant communities. Acta Botanica Neerlandica 13:367–393

125.

Kausik SB (1939) Pollination and its influences on the behavior of the pistillate flower in. Am J Botany 26:207–211

126.

Ernst-Schwarzenbach M (1945) Kreuzungsversuche an Hydro-charitaceaen. Arch JuliusKlaus Stiftung 20:22–41

126a. Sehgal A, Mohan Ram HY. Comparative developmental morphology of two populations of Ceratophyllum L. (Ceratophyllaceae) and their taxonomy. Botanical J Linnaean Soc 82:343–356 127.

Philbrick CT (1984) Pollen tube growth within vegetative tissue of Callitriche (Callitrichaceae). Am J Botany 71:882–886

128.

Khosla C, Mohan Ram HY (1993) Morphology of flower, fruit and seed in Polypleurum stylosum. Aquatic Botan 46:125–128

129.

Khosla C, Shivanna KR, Mohan Ram HY (2000) Reproductive biology of Polypleurum stylosum (Podostemaceae). Aquatic Botany 67:143–154

130.

Khosla C, Sehgal A (2009) Pollination biology of Indotristicha ramosissima (Podostemaceae: Tristichoideae). Aquatic Botany 91:51–56

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131.

Cook CDK (1988) Wind pollination in aquatic angiosperms. Ann Missouri Botan Garden 75:768–777

132.

Philbrick CT, Novelo RA (1998) Flowering phenology, pollen flow, and seed production in Marathrum rubrum (Podostemaceae). Aquatic Botany 62:199–206

133.

Rutishauser R (1997) Structural and developmental diversity in Podostemaceae (riverweeds). Aquatic Botany 57:29–70

134.

Khosla C, Shivanna KR, Mohan Ram HY (2001) Cleistogamy in Griffithella hookeriana (Podostemaceae). S Afr J Botany 67:24–328

135.

Philbrick CT, Vomela M, Novelo RA (2006) Preanthesis cleistogamy in the genus Podostemum (Podostemaceae). Rhodora 108(935):195–202

136.

Sehgal A, Sethi M, Mohan Ram HY (2009) Development of the floral shoot and pre-anthesis cleistogamy in ss(Podostemaceae). Botan J Linnaean Soc 59:229–236

137.

Bascompte J, Jordano P, Melian CJ, Olesen J (2003) The nested assembly of plant-animal mutualistic networks. Proc Nat Acad Sci USA 100:9383–9387

138.

Abu-Asab M, Peterson PM, Shetler SG, Orli SS (2001) Earlier plant flowering in spring as a response to global warming in Washington, DC, area. Biodive Conservat 10:597–612

139.

Cleland EE, Chuine I, Menzel A, Mooney HA, Schwartz MD (2007) Shifting plant phenology in response to global change. Trend Ecol Evolut 22:358–365

140.

Wielicki BA, Wong T, Allan RP, Slingo A, Kiehl JT, Soden BJ, Gordon CT, Miller AJ, Yang S, Randall DA, Robertson F, Susskind J, Jacobowitz H (2002) Evidence for large decadal variability in the tropical mean radiative energy budget. Science 295:841–844

141.

Kumar KK, Rajgopalan B, Cane MK (1999) On the weakening relationship between the Indian monsoon and ENSO. Science 284:2156–2159

142.

Singh KP, Kushwah (2006) Diversity of flowering and fruiting phenology of trees in a tropical deciduous forest in India. Ann Botany 97:265–276

143.

Lonsdorf E, Kremen C, Ricketts T, Winfree R, Williams N, Greenleaf S (2009) Modelling pollination services across agricultural landscapes. Ann Botany 103:1589–1600

15 Darwinian evolution and post developments in genomics Arun Kumar Sharma Center of Advanced Study, Department of Botany, University of Calcutta, Kolkata - 700019, India

Abstract: The status of Darwin’s theory of Natural Selection in ‘Origin of Species’ and the impact of recent developments in genetics on evolution form the theme of the paper. Despite the conflicting views of the role of mega and micro evolution as visualized by Darwin, his theory, followed by theory of inheritance and chromosome theory of heredity form the very foundation of our knowledge of evolution and continuity of life from generation to generation. In the post Darwinian phase, the importance of the regulatory sequence in the complex expression of genes controlling evolution has been emphasized. The role of high amount of non coding repeat sequences as well as mobile sequences in evolution has been discussed. Finally, the emergence of RNA as the primitive molecule of life and the present role of small RNAs in defence, growth, differentiation and indirectly evolution of species have been indicated. Keywords: Evolution, Natural selection, Post-Darwinian genetics

Basic concept The discourse on evolution has all along been a topic of special interest to any biologist dealing with the origin of life itself. Since the origin of living organisms, and gradually the diversification to eukaryotic systems, Darwin’s theory of Natural Selection and Origin of Species1 and later the Descent of Man stood the test of time and almost universally accepted. The origin of species arising through random minute variations, and selection of the fittest through struggle for existence were visualized. Darwin fully believed that evolution follows a tree like growth with different branches of tree representing separate forms of life.2 However, even after the publication of the ‘Origin of Species’, 150 years ago, there have been nine different species concept, and according to some even more. Each concept has its merits and demerits but the most universally accepted is that ‘complete reproductive isolation’ is the crucial parameter for future of V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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speciation.3,4 Mayr’s concept of species5 involves ‘potentially inbreeding natural populations which are reproductively isolated from other such groups’6 concurs with the genic view of speciation on principle, that is, the evolution of reproductive isolation barrier with divergence as the key factor. However the importance of allopatry in reproductive isolation is an important factor as well. Fortunately, within a few years of Darwin’s publication of the paper in Origin of Species, Gregor Mendel’s theory of inheritance was published which formed the base of our knowledge of heredity. Just as Darwin’s theory envisaged the continuity of life through preexisting ones, Mendel’s ideas provided a base for their inheritance from generation to generation.

Darwin and Mendel There has, however, been efforts at post-Darwinian enthusiasm to bring together Mendel’s laws of heredity7 and Darwin’s on evolution. Several authors were in favour of Mendel but some biometricians accepted Darwinian selection but rejected Mendelism. Views were also expressed that natural selection as ineffective and evolution as occurring through macro mutation or rather gene change with major effect. Hybrids and polyploidy too in speciation were known to be widespread in plants. Even Ernst Mayr and Theodosius Dobzhansky suggested major changes rather than a continuum. In any case, (i) the laws of inheritance of Mendel,7 (ii) chromosome theory of heredity,8 and (iii) the establishment of gene concept,9 along with natural selection, without underestimating the importance of genetic drift and isolation, form the very foundation of our understanding of evolution – the origin and development of life forms in this planet. The later discovery of the double helical nature of the gene by Watson and Crick10 in 1953 and genetic code showing that the code is same in lilies, mosquito and man, brought distinct evidence of life – all united in a common descent. Evidence of extra planetary life beyond earth, however, cannot be ruled out. In later years, the chemical nature of the genes, and gene action, and their role in the expression of characters were lucidly manifested in the Central Dogma of DNA → RNA → Protein. The genetic code, and the simplicity embodied in it, provided the very chemical basis of evolution enshrined in Darwin-Mendelian concept. Undoubtedly the Laws of inheritance, Chromosome Theory of Heredity and Gene concept provided a solid foundation of genetic and chemical basis of both mega and microevolution of Darwin. However, the modality of bringing alterations both major and minor, has been greatly influenced by the gene structure of eukaryota on the one hand and novel gene sequences, forming major components of chromosome structure on the other. Of these, Repeated DNA sequences11–13 and Transposons14–16 need special mention because of their role in evolution.

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Recent developments on genetics with impact on evolution Regulatory sequences The coding regions of genes are no doubt the materials for evolution, but simultaneously evidences came up indicating profound role of regulatory DNA — the ‘cis element’ as a crucial factor in evolution. As genes control all characters, the expression of a character is the result of a series of chemical reactions set up and triggered at the gene level. Moreover, as demonstrated, each character is controlled by many genes, which are not necessarily located at the adjacent site and even at the same chromosome. The expression is thus coordinated by regulatory sequences – the cis elements – lying often far away from the target genes. The mutations in regulatory elements, the ‘cis elements’17 underlie many alterations and may provide the organism to have a long life and such DNA may not necessarily be located very near the genes. It is evident that mutations in coding regions and cis-regulatory changes both play a role in evolution. This is true also for several mammals which are similar in genomic content and proteins, but the difference is more due to the product of changes emanating out of – when, where and to what degree these genes function and express. Arguments have been put forward that (Sean Carrol, University of Wisconsin, vide Pennisi17 that genes involved in body patterns and plans are so wide ranging, affecting variety of tissues, at different stages of development, that a mutation in their coding region may create catastrophe. But rather changes in ‘cis’ elements which often bring about coordination and control have specific limited effect. Microevolutionary changes can be traced to cis-regulatory sequences too. Each element may serve as the base for a peculiar ‘transcription factor’, stimulating or multiplying gene expression. Because of this effect of modulation, there can be innumerable cis element combinations which allow genes to express in specific time, space and degree. There are several cases in plants where cis elements have proved to be very important. Teosinte the ancestor of modern maize had several stalks as compared to single stalk of corn. This change has been shown to be linked to a difference in DNA sequences17 – several thousand bases away for the gene ‘teosintebranched’. This indicates the role of non-coding ‘cis’ elements in corn evolution. As each character is the result of multiple effects – a mutation in cis-regulatory sequence much removed from the original site is crucial for evolution. This fact has been taken to explain why chimpanzee and man are so similar in genomic content but difference in general, is due to the function and expression of the genes at different time, stage and extent, that is, when, where and to what degree, these genes should express.

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In the light of the evidences obtained so far, the role of regulatory sequences such as transcription factors in species adaptation and evolution can hardly be overestimated. These ideas are still in a nascent state but the importance of coding and regulatory sequences have been clearly demonstrated in the evolution of species.

Gene structure The single gene – single protein concept of Beadle and Tatum18 needs to be looked at with the establishment of split nature of the gene made up of intron (non essential) and exon (essential) sequences.19 Despite the fact, that normal messenger processing following transcription is the rule, alternate splicing resulting in different combinations of exons as recorded to occur under certain conditions, result in more than one protein from a single gene locus. Latest studies, indicate that exons from two different genes may also undergo fusion to yield a fused transcript.20 Further complexity is added by RNA editing whereby the Guide RNA incorporates different nucleotides in the messenger transcript, before processing after transcription. As a result, the resulting protein is quite different from the protein, coded in the primary transcript from the master template of DNA. All such variations result in alterations in polypeptide structure, ultimately generating from a single gene locus. In addition, there may be ‘Post Translational Modifications’ as well which make Proteome more complex, deviating from the total message coded in the primary transcript. The variations in the structural make up of gene product significantly influences the expression of new characters involved in origin of species in Darwinian concept.

Repeat sequences of DNA Along with the further development in the understanding of structure and expression of genes, the chromosomes of eukaryotes in general, harbor huge mass of repeated or amplified DNA sequences11 occupying the major segments of chromosomes and are often termed as ‘Selfish’ or Junk21 with not much appreciable effect,13 as well as Dynamic need mention.22,23 It is presumed that these sequences multiply and exist as the cell environment is congenial for their survival. Moreover, the intergenic repeat sequences, structural obesity at the chromosome level as well as the non coding sequences and transcripts, call for a deeper analysis of their role in chromosome evolution. Conserved noncoding sequences in human system are associated with a large number of genes and their very survival for a long period of evolution may indicate a selective value. The location of some of these sequences has also been such as to indicate their possible role in brain functioning, may be even wiring of brain cells, cell signalling and growth.24 In the plant system, the repeated sequences occupy the major portion of the genome.25


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Their role in the control of non specific functions have been established.23,26 They also confer adaptation to extremely stress environment recorded in different plant genera.27 The importance of adaptation is one of the key factors in the evolution of species is evident.

Mobile Sequences Further to non-coding, repeats performing some non specific functions the mobile genes – transposons, initially studied by Barbara McClintock, capable of jumping from one segment to another has led to complexity in the concept of genome structure – its functioning and even evolution.14–16 In the plant system, in maize, transposon activity has been responsible for increasing genome size from 1 to 2.4 billion bases. In the chromosome structure, the transposons have been shown to be present in the flanking region of centromeres and telomeres and may serve as epigenetic regulators of the genome. Almost half of the human genome consists of mobile elements (LINE) which can jump around the genome. Some of these mobile elements add to diversity and are vital for regulation and human evolution.28 Mobile elements present in 50% of Primate genome indicate strong diversity and influence on evolution of genome vis-à-vis speciation.29

Gene Order: Synteny Lately new ideas have come up which have a profound influence on concepts and mechanics of species evolution. The comparative analysis of cereal genomes reveals that they are composed of a few similar genomic building blocks with conserved sequences. By simple rearrangement of these blocks, and amplification of some of the repetitive sequences contained within them, it is possible to reconstitute the chromosome complements of rice, wheat, maize, sorghum, millet and sugarcane, from the cleavage of a single chromosome.30,31 This similarity in the genetic maps of several plant genomes and the order of DNA sequences is termed ‘synteny’.30 There is striking collinear conservation in the gene order, between wheat, maize, sorghum, sugarcane, millet and rice, even though they differ in size, chromosome number and taxonomic distance. The existence of an ancestral order of all the DNA sequences possibly in one chromosome in the grasses, if not in the entire plant kingdom has been tentatively presumed. Similar syntenic arrangements have been recorded in animal system as well. The role of synteny in evolution of species can hardly be overestimated.

RNA world The most significant development in Post Darwinian Mendelian phase is the understanding of the RNA world.32–34 It is visualized that the first molecule to

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appear on earth in the primitive, dark anaerobic inhospitable world about 4 billion years back was a RNA molecule. The DNA came much later about, 3.6 billion years back where the role of RNA molecule became principally as the messenger. Lately, new classes of RNA have been discovered namely, micro RNA (miRNA) and Short interfering RNA (siRNA).35–37 Micro RNAs These RNAs, 20 to 25 bp long, are coded by the genome and couple with mRNA for silencing. Its role in transcription, silencing, viral resistance and metazoan evolution is now well recognized.38–40 The role of short RNAs in checking transposon activity is also demonstrated. In fact, later findings may show that41 there is a direct correlation of increasing morphological complexity with micro RNA number viz., sponges (8), sea anemone (50), Drosophila (147) and human (677). The antiquity of micro RNA can hardly be questioned as their origin is rather evident in metazoan evolution before multicellularity emerged.

Conclusion All these developments at the post-Darwinian era in the recent past, ranging from single nucleotide polymorphism to split genes, repeat DNA and mobile sequences, have direct bearing on the minute variations which Darwin witnessed as well as major changes. But the discovery of the RNA world – the initiator RNA molecule of evolution and later the tiny world of RNA as mentioned above surpass all others in their impact on evolution at the post-Darwinian phase. It is visualized now that non coding RNAs may represent a rich substrate for innovations in evolution in eukaryota.42 More startling is the fact, that despite their established antiquity almost prior to metazoan evolution these molecules are exerting at present a profound influence on growth, differentiation and indirectly the evolution which Darwin visualized, through their involvement in chromatin dynamics and gene regulation.

References 1.

Darwin C (1859) On the Origin of Species by Means of Natural Selection or the Preservation of Favoured Races in the Struggle for Life. John Murray

2.

Pagel M (2008) Natural Selection 150 years on. Nature 457:808–811

3.

Coyne A (1996) Speciation in action. Science 1972:700–701

4.

Coyne JA, Orr HA. Speciation. Sinauer Associates, Inc.

5.

Mayr E (1994) Reasons for the failure of theories. Phil Sci 61:529–533

6.

Reznick DN, Ricklefs RE (2008) Darwin’s bridge between microevolution and macroevolution. Nature 457:837–842


247

7.

Mendel G (1986) Plant Hybridization. British Nat Hist Soc, Oliver and Boyd, Edinburgh

8.

Sutton NS (1903) The Chromosomes in heredity. Biol Bull 4:213–251

9.

Bateson W, Punnett RC (1905) Reports on the Evolution Committee of the Royal Society. Harrison and Sons, London

10.

Watson JD, Crick FHC (1953) Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature 171:737–738

11.

Britten RJ, d Kohne DE (1968) Repeated sequences in DNA. Science 161:529–540

12.

Banerji M, Sharma AK (1979) Variation in DNA content. Experientia 35:42–43

13.

Schmidt T, Heslof Harrison JS (1998) Genomes, Genes, Junk: The large scale organization of plant chromosomes. Trends Plant Sci 3:195–199

14.

Kunze R, Sandler H, Lonnig WE (1997) Plant Transposable Elements. In: Advances in Botanical Research. Gallow JA (Ed.), Academic Press, New York

15.

Craig R, Geileot M, Lambowitz AM (2002) In: Mobile DNA II. AMG Press

16.

Walker T, Sabot F, Hunvan A, Bennetzen JL, Capy P, Chalhoab B, Flovell A, Leroy P, Morgante M, Panaud O, Panx E, Sunnignel P, Schulman AH (2009) A unified classification of transposable elements. Nat Rev Genetics 8:875–982

17.

Pennisi E (2008) Deciphering genetics of evolution. Science 321:760–763

18.

Beadle GW, Tatum EZ (1941) Genetic control of biochemical reactions in neurospora. Proc Natl Acad Sci USA 27:499–506

19.

Gilbert W (1979) Split genes. Science 204:1979

20.

Parra G, et al. (2006). Genome Res 16:37

21.

Orgel L, Crick FHC (1980) Selfish DNA – the ultimate parasite. Nature 204:604–7

22.

Sharma AK (1983) In: Additional Genetic Materials in Chromosomes. George Allen & Unwin, Oxford Chromosomes Conference, London

23.

Sharma AK (1986) A critical review of some terminologies used for additional DNAs in plant chromosomes: DNA Systematics. Plants II. CRC Press, Florida

24.

Hayden EC (2009) The other strand. Nature 457:776–779

25.

Cavalier-Smith T (2005) Economy, speed and size matter: evolutionary forces driving nuclear genome miniaturization and expansion. Ann Bot 5(5):147–175

26.

Sharma AK (1984) Dynamic DNA and chromosome structure. In Genetics – New frontiers. Proc Inter Congr Genetics 1:205–212

27.

Mukherjee S and Sharma AK: In situ nuclear DNA content in perennial fast and slow growing Acacias from arid zones. Cytobios75:33–36 (1993)

28.

Kyudong Han, et al. (2007) Mobile DNA in old world monkeys. Science 316:238–240

29.

Ostertag EM, Kazazian HH (2005) Genetics: Lines in mind. Nature 435:890–891

30.

Moore G (1995) Cereal genome evolution: pastoral pursuits with ‘Lego’ genomes. Curr Opin Genet Develop 5:717–724

31.

Moore G, Foote T, Helentjaris T, Deros K, Kurata N, Gate M (1995) Was there a single ancestral cereal chromosome? Trends Genet 11:81–82

32.

Gilbert W (1986) The RNA World. Nature 319:618

33.

Grobhans H, Filipowicz (2008) The expanding world of small RNAs. Nature 451: 414–416

34.

Curninci P (2009) The long and short of RNAs. Nature 457:974–975

248


35.

Moazed D (2009) Small RNAs in transitional gene silencing and genome defence. Nature 457:413–420

36.

Cullen BK (2009) Viral and cellular messenger targets of viral micro RNAs. Nature 457:421–425

37.

Hayden EC (2008) Thousands of proteins affected by miRNAs. Nature 454:562

38.

Makeyev EG and Maniatis T: Multilevel regulation of gene expression by micro RNAs. Science 319:1789–90

39.

Grimson A, Srivastava M, Fahey B, Woodcrift BJ, Rosaria-Chiang H, King N, Degnan BM, Rokhsar DS, Bartel DPL (2008) Early origin and evolution of micro RNAs and interacting RNAs in animals. Nature 455:1193–1197

40.

Chen K, Rajewsky N (2007) The evolution of gene regulation by transcription factors and micro RNAs. Nat Rev Genetics 8:93–103

41.

Technan K (2008) Small regulatory RNAs pitchin. Nature 455:1184–1185

42.

Amarel PP, Inger ME, Mercer TR, Mattick JS (2008) The eukaryotic genome as an RNA machine. Science 319:1287–88

16 Pathogen pressure and molecular evolutionary genetics of innate immunity genes in humans Partha P. Majumder Indian Statistical Institute and TCG-ISI Centre for Population Genomics, 203, B. T. Road, Kolkata - 700108, India [email protected]

Abstract: We report results of an extensive and comprehensive study of genetic diversity in 12 genes of the innate immune system in a population of eastern India. Almost half of the 548 DNA variants discovered was novel. DNA sequence comparisons with human and chimpanzee reference sequences revealed evolutionary features indicative of natural selection operating among individuals, who are residents of an area with a high load of microbial and other pathogens. The haplotype structures in India are significantly different from those of European-American and African-American populations, indicating local adaptation to pathogens. Most of the human haplotypes are many mutational steps away from the ancestral (chimpanzee) haplotypes, indicating that humans may have had to adapt to new pathogens. We have tested the opposing views concerning evolution of genes of the innate immune system that (a) being evolutionary ancient, the system may have been highly optimized by natural selection and therefore should be under purifying selection and (b) the system may be plastic and continuing to evolve under balancing selection. We have found that in these genes, there is (a) generally an excess of rare variants (b) high, but variable, degrees of extended haplotype homozygosity, (c) low tolerance to nonsynonymous changes and (d) essentially one or a few high-frequency haplotypes, with star-like phylogenies of other infrequent haplotypes radiating from the modal haplotypes. Purifying selection is the most parsimonious explanation operating on these innate immunity genes. This genetic surveillance system recognizes motifs in pathogens that are perhaps conserved across a broad range of pathogens. Hence, functional constraints are imposed on mutations that diminish the ability of these proteins to detect pathogens. Keywords: DNA resequencing, Single nucleotide polymorphism, Haplotype, TLR, Defensin, CAMP, MBL2, Neutrality tests, Haplotype networks, Extended haplotype homozygosity, Purifying selection

Introduction The September-October 1997 issue of American Scientist carried a review of the book titled “Darwin’s Black Box: The Biochemical Challenge to Evolution.” V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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The book is authored by Michael Behe, and the review was written by Robert L. Dorit. I reproduce, verbatim, some sentences (italicized for convenience) from this review: “Will you honestly tell me (and I should be really much obliged) whether you believe that the shape of my nose was ordained and ‘guided by an intelligent cause’?” In exasperation, but not without humor, Charles Darwin posed this question to Charles Lyell in 1860, the year Darwin dealt with the maelstrom unleashed by the publication of The Origin of Species. In both the popular and the scientific press, Darwin had to contend with the wrath of those for whom the notion of a living world based on accident, time and natural selection was simply too disquieting. Look around, Darwin’s critics argued, and see the evidence of design. And where there is design, there must be a designer. Today, when we are able to look around at the molecular level, we find patterns; patterns that are consistent with the theory of evolution propounded by Darwin; not the handiwork of a designer. Some of our recent findings on patterns of variation at the molecular level in genes of the innate immune system are reported in this article, and we examine whether and how these patterns can be explained in the context of the Darwinian theory. Vertebrates defend themselves against pathogens using the immune system. The vertebrate immune system has two distinct arms – the innate and the adaptive. Invertebrates only possess the innate immune system. They lack the mechanisms for generating diverse antigen receptors by recombination. Therefore, invertebrates do not possess B and T cells. The innate immune system responds very quickly (usually within minutes) to an infection. The proteins involved in this system detect broad cues from invading pathogens and responds. Diverse and evolutionarily conserved families of Pattern Recognition Receptors (PRRs) of the innate immune system detect broad and conserved patterns that differ between pathogenic organisms.1 The innate immune system provides signals to the adaptive immune system to initiate response. The adaptive immune response usually becomes effective after several days or weeks after infection, but provides antigenic specificity required for complete elimination of the pathogen and generation of immunologic memory. Quick recognition of broad signatures of pathogens by the innate immune system leads to the rapid mobilization of immune effector and regulatory mechanisms that provide the host with three critical advantages2: (i) initiating the immune response (both innate and adaptive) and providing the inflammatory and co-stimulatory context for antigen recognition; (ii) mounting a first-line of defense, thereby holding the pathogen in check during the maturation of the adaptive response; and (iii) steering the adaptive immune system towards the cellular or humoral responses most effective against the particular infectious agent. Important genes of the innate immune system include cathelicidin antimicrobial peptide (CAMP), defensins, mannose binding lectin (MBL2) and toll-like receptors (TLRs). CAMP is one of the major antimicrobial peptides of

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the human innate immune system in the intestinal tract.3 Cathelicidins are small (12–100 amino acids) cationic peptides that possess broad-spectrum antimicrobial activity, and share features with defensins.3 While humans and mice each possess a single cathelicidin, other mammalian species, such as cattle and pigs, express many different cathelicidins.4 The defensins code for small cationic, cysteinerich peptides. These cationic peptides exhibit a broad-spectrum of activity against gram-positive and gram-negative bacteria, fungal species and viruses,5 by interacting with negatively charged molecules on the surface of pathogens and permeating their membranes. On the basis of the position and bonding of six conserved cysteine residues, defensins in vertebrates are divided into two categories, designated as - and -defensins.6 While -defensins are produced primarily by intestinal Paneth cells and neutrophils, the -defensins are primarily produced by epithelial cells. MBL is a calcium dependent serum protein that plays a role in the innate immune response by binding to carbohydrates on the surface of a wide range of pathogens.7 It is also an important component of the complementactivation pathway for activation of macrophages by forming membrane-attack complexes (MACs). The TLRs are a class of single membrane-spanning, noncatalytic receptors that recognize structurally conserved molecules derived from microbes once they have breached physical barriers such as skin or intestinal tract mucosa.8 They are characterized by an extracellular leucine-rich repeat (LRR) domain for ligand recognition and an intracellular tail that bears a homology to the conserved interleukin-1 receptor (TIR) for signal transduction. Ten TLRs (TLR1–TLR10), each with its own ligand specificity, have been identified. Diversification of the genes of the innate immune system have taken place during evolution possibly in response to the diversification of microbes, especially pathogenic microbes. Gene families, such as TLRs, evolved by gene duplication and individual members of these families have evolved different but related functions, possibly to protect the host against a larger set of diverse pathogens. Individual members of these gene families also exhibit high levels of polymorphism, which is consistent with Haldane’s (1949)9 prediction that maintenance of polymorphisms in genes governing host-pathogen interaction is driven by rapid rates of microbial evolution. Indeed, evidence for overdominant selection (heterozygote advantage) has been documented at the major histocompatibility complex class I loci, in which the rate of non-synonymous (amino acid altering) nucleotide substitutions have been found to be significantly greater than synonymous substitutions in the antigen recognition site.10 With respect to the genes of the innate immune system, however, there is debate whether these genes are continuing to evolve or whether being evolutionarily ancient they have been highly optimized by natural selection.11 Specific polymorphic variants in innate immunity genes have been found to be associated with human diseases (12–15) and some investigations have been carried out on the mode and tempo of evolution of some genes of the innate immune system.12–15

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Nature and extent of variation in innate immunity genes in India We have assayed inter-individual variation at the DNA level in 12 genes of the innate immune system. These genes were: CAMP, defensins (DEFA4, DEFA5, DEFA6 and DEFB1), MBL2 and TLRs (TLR1, TLR2, TLR4, TLR5, TLR6 and TLR9). DNA resequencing of these genes was carried out on samples collected, with written informed consent, from 171 individuals belonging to Indo-European speaking groups resident in eastern India. On each sample, ~75 kb of DNA was resequenced. A total of 548 variations were detected, of which 229 were polymorphic. No polymorphic variant was observed in CAMP. Most variants (528; 96%) were single nucleotide changes. However, 20 (4%) changes were single- or multi-nucleotide insertions/deletions (INDELs). One tri-allelic variation (alleles: T, A and G) was also detected in TLR1 (rs4540055). A large number (259 of 548; 47%) of the detected variants were novel, i.e. unreported in dbSNP (build 126) prior to our submission to dbSNP. Of these novel variations, ~10% were polymorphic. From these data it is clear that the observed frequencies of DNA variants and polymorphisms are, respectively, ~7.4 and ~3.1 per kb (further details are provided in Bairagya et al.16). Among the 548 variations observed, 92 were in coding regions, of which 50 were non-synonymous. Of these 92 variants in coding regions, 21 (23%) were polymorphic; 11 (52%) were non-synonymous and 10 (48%) were synonymous. Since population genetic theory suggests that the most frequent allele is the oldest17 and therefore likely to be the ancestral allele, we compared our data at the variable positions with the corresponding data from the chimpanzee reference sequence (UCSC Genome Browser; March 2006 assembly). While the major allele at the majority (412 of 544; 76%) of variable sites in our data coincided with the ancestral (chimpanzee reference sequence) allele, at the remaining 132 (24%) sites the major allele is not the ancestral allele. Such observations have been made earlier.18 It is not unlikely to observe non-ancestral alleles to be major alleles in humans, when these pertain to loci that are influenced by natural selection. Indirectly, our observation lends support to natural selection acting on the innate immunity genes.

Extensive global variation in haplotype structures Haplotypes were reconstructed using genotype data at the polymorphic loci and their frequencies were estimated. For most genes, three or four haplotypes were in high frequencies. For most genes, the Human Reference Sequence Haplotype (build 36) was not the most common haplotype in the Indian study population and for some genes (e.g. DEFA4) the reference sequence haplotype was not even

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observed. The haplotype diversity values were very high (from 62% for TLR9 to 88% for DEFA6) for most genes. The ratio of SNPs to haplotypes varied from 0.41 (DEFA6) to 2.08 (TLR6). Haplotypes based on some specific loci in MBL2 are known to be associated with MBL serum concentration variability.7 MBL serum concentration influences susceptibility to and clinical course of many infectious and chronic diseases.19 The relevant details about these haplotypes and their frequencies observed in our Indian study population are presented in Table 1. Recently,14 it has been shown that there is a strong continental structuring of haplotype frequencies at two nonsynonymous sites (rs4986790, Asp > Gly and rs4986791, Thr > Ile) in TLR4. This structuring was inferred to be due to the interaction between our innate immune system and the infectious pressures in particular environments during the outof-Africa migration of modern humans. The observed percent frequencies of the Asp/Thr, Asp/Ile, Gly/Thr and Gly/Ile haplotypes in our data were, respectively, 86.52, 2.33, 1.16 and 9.94. For the variants in these innate immunity genes that were also assayed in the HapMap project (10 genes), we compared our data with the HapMap data (HapMap Data Release 21a/phaseII 07 January, on NCBI B35 assembly, dbSNP build 125). We found that there are three nucleotide positions (1 in DEFB1, rs2978873; 1 in TLR1, rs1873196; and 1 in TLR2, rs4696480) that are monomorphic in all four HapMap populations (Chinese [CHB], Japanese [JPT], African [YRI], Caucasian [CEU]), but are polymorphic in our study population. None of these variants is in a coding region, however. On the other hand, there are 26 positions that are polymorphic in at least one of the four HapMap populations, but are monomorphic in our study population. There is also a tremendous variation in allele frequencies at highly polymorphic loci (i.e. allele frequencies ranging between 0.2 and 0.8 in at least one population) among the HapMap populations and our study population from India. There is also considerable variation in haplotype frequencies (Fig. 1), calculated on the basis of loci that were common to HapMap and our study populations. (Because of lack of data in HapMap database for TLR5, haplotype frequencies could not be compared for this gene.)

Search for natural selection We have used these data to to assess the nature and extent of selective forces in shaping the contemporary genetic diversity of these genes. In particular, we sought to test whether overdominant selection operates on the TLR genes, because TLR proteins contain LRR domains (characterized by a segment LxxLxLxxNxL, in which “L” is Leu, Ile, Val, or Phe and “N” is Asn, Thr, Ser, or Cys and “x” is any amino acid) that are responsible for molecular recognition.20 Similar to the antigen recognition site of the MHC locus, individuals who are heterozygous at the variant

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Figure 1. Frequencies of haplotypes >10% in the study population (IND) and the four HapMap populations. (Haplotype identification and estimation of haplotype frequencies were based on SNPs that are common among these populations. Because of lack of data in HapMap database for TLR5, haplotype frequencies could not be compared for this gene. Haplotypes with frequencies 3 SNPs per kb) and variants (>7 variants per kb) in the innate immunity genes in the India population were significantly higher (p < 0.02). Even compared to the more recent estimate28 of one SNP per 0.88 kb, the SNP density observed in this study is higher. There is, of course, variation in SNP density across autosomal chromosomes. The range of variation (see Table 1, Ref. 26) is one SNP per 1.19 kb (chromosome 22) to one SNP per 2.18 kb (chromosome 19); thus, the SNP density in the innate immunity genes observed in the Indian population exceeds the highest density observed for any autosomal chromosome in the human genome. In fact, at the genome-wide level, there are very few genomic regions where SNP density exceeds 2.5 SNPs per kb (see Fig. 1 of The International HapMap Consortium, 2007). Thus, compared with all existing data, the extent of genetic variation in the human innate immunity genes observed in India is very high. We have found that most of the innate immunity genes have been influenced by purifying selection, even though the strengths of the signatures of purifying selection are variable. For most genes, only one or two haplotypes were present in high frequencies; evolutionary variants that have arisen to create new haplotypes were either eliminated or were present at very low frequencies, being perhaps transient to being eliminated. Non-synonymous variations in these genes are not tolerated, as indicated by the ratios of rates of non-synonymous to synonymous changes being 45 °C due to higher proportion of alkanes) as well as with lower Tm (≤ 25 °C due to alkenes).62 Therefore, there is lack of correlation between Tm of cuticular lipids and cuticular water loss in several insect taxa from diverse habitats. Thus, there is general lack of supporting evidence for the role of cuticular lipids in changing cuticular permeability. If reduction in cuticular water loss can not be explained on the basis of epicuticular lipids, it may be accomplished by changes in other portions of the cuticle. A single study has shown a possible link between body melanisation and desiccation resistance e.g. darker mutant strain (ebony) of

Figure 2. Schematic representation of three different modes of water conservation in ectothermic insects

Testing the melanism-desiccation hypothesis: A case study in Darwinian evolution

287

Table 1 A comparison of the outcome of laboratory vs. natural selection for the mechanistic basis of desiccation resistance in Drosophilids Laboratory selection

Natural Selection

Body water

Accumulation of body water in haemolymph

No variation in water contents

Dehydration tolerance

Lack of differences

Show variation

Cuticular water loss

Negative correlation

Negative correlation

Cuticular lipids

Lack of differences

In some taxa, trait variation impacts desiccation resistance while not in others.

Body size

Lack of differences

Lack of differences (within population)

Body melanisation

Not analyzed

Positive correlation with desiccation

D. melanogaster is desiccation resistant as compared with non-melanic mutant strain (yellow).50 In this brief review, we raised a hypothesis that flies differing significantly in melanisation level (darker vs. lighter) might evidence correlated differences in desiccation resistance and its mechanistic basis i.e. reduction in cuticular water loss.

Melanisation - desiccation hypothesis Melanism seems to have evolved independently through diverse mechanisms in various taxa and different ecological factors could be responsible for selective responses.17 Increased body melanisation at higher altitudes as well as latitudes is generally considered to be adaptive for thermoregulation. Physiological traits such as body melanisation and desiccation resistance have been investigated independently in diverse insect taxa at three levels: within population, between populations and among species.5,9 A substantial number of Drosophila studies have reported clinal variations in both these traits along latitude.5,41 Correlations between these traits remained unexplored in wild and laboratory populations of ectothermic insect taxa including Drosophilids. In insects, there are several investigations on the rate of water loss in laboratory selected strains for desiccation resistance but such analyses have not been considered in context of body melanisation.45,49 Association between pigmentation and desiccation resistance was initially proposed by Kalmus.50 He compared desiccation resistance in wild-type flies to that of yellow (light body color) and ebony (dark body color) mutants in D. melanogaster. Similar desiccation assays were also performed for wild type as well as yellow mutant flies in D. simulans, D. pseudoobscura, and D. subobscura.

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The ebony mutants survived longer as compared with yellow mutants. Fraenkel and Rudall51 and Pryor52 concluded that the darkening and hardening traits of the cuticle are due to the same biochemical processes which may involve cross linking of proteins with melanin. Brisson et al.37 investigated desiccation resistance in D. polymorpha and found that dark flies from open dry environments survived longer in a desiccating environment than lighter flies inhabiting dark humid forest environments. A possible link between melanisation and desiccation can be shown if laboratory selected strains for higher melanisation evidence increase in desiccation resistance; and if within population differences in assorted groups (darker and lighter phenotypes) of body melanisation evidence significant parallel changes in desiccation resistance and its mechanistic basis i.e. rate of water loss/hr. Accordingly, we analyzed darker and lighter phenotypes of body melanisation in six wild Drosophila melanogaster populations from closely located highland and lowland sites at three different latitudinal locations (Table 2). In large population samples, within population variability made it possible to assort non-overlapping phenotypes of body coloration [i.e. lighter ( 45%)] for all the populations which were investigated for desiccation resistance and rate of water loss/hr (Table 2). Wild populations of Drosophila melanogaster from diverse geographical locations showed significant associations between

Figure 3. Relationship between % body melanisation and desiccation resistance in wild caught flies assorted into two groups of darker (> 45% melanisation) and lighter phenotypes (< 30% melanisation) for each of the five highland populations of D. melanogaster

11.15

11.21

24.12

24.36

30.44

31.06

--

--

--

Coonoor

Deesa

Mount Abu

Chandigarh

Shimla

mean ± s.e.

s.d.

r values

Lat. (ºN)

Calicut

Pop.

--

--

--

2202

347

1195

136

1747

10

Alt. (m)

--

5.64

21.33±2.30

30.40±2.74

24.00±2.16

22.40±1.97

17.00±1.68

20.50±1.74

14.00±1.14

Light

--

9.69

54.34±3.85

69.02±4.99

60.00±4.02

55.10±4.12

48.00±3.40

52.12±3.02

42.00±2.56

Dark

% Melanisation

0.99±0.08

3.45

12.50±1.41

18.00±1.96

13.00±1.59

14.00±1.76

10.00±1.34

12.00±1.36

8.00±1.24

Light

0.99±0.07

6.18

23.16±2.52

33.00±4.01

26.00±3.60

24.00±3.72

19.00±3.26

22.00±3.14

15.00±2.80

Dark

Desiccation hr.

0.98±0.10

0.42

0.036±0.0017

0.030±0.0005

0.033±0.0007

0.035±0.0004

0.038±0.0005

0.037±0.0002

0.043±0.0003

Light

0.98±0.09

0.79

0.028±0.0030

0.016±0.0006

0.023±0.0007

0.026±0.0007

0.032±0.0008

0.033±0.0005

0.038±0.0006

Dark

CWL (mghr.-1)

Table 2 Data on mean ± s.d. of percent body melanisation, desiccation hours and rate of cuticular water loss/ hr (CWL) in wild caught assorted groups of darker and lighter phenotypes of six populations of Drosophila melanogaster

Testing the melanism-desiccation hypothesis: A case study in Darwinian evolution 289

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darker phenotypes and higher desiccation resistance while lighter phenotypes showed lesser desiccation resistance (Fig. 3). Significant reduction in rate of water loss/hr in darker phenotypes could be indirect evidence in favor of cuticular impermeability due to higher body melanisation.7 In conclusion, analysis of populations of Drosophila melanogaster demonstrates that body melanisation is phenotypically variable and subject to natural selection pressure for increased melanisation under colder and drier conditions. Our data are consistent with the increased desiccation being a consequence of selection on melanisation. Body melanisation may result in cuticular impermeability for reducing water loss under increasing dehydrating conditions.7,42

Evidences for melanisation - desiccation hypothesis High vs. low potential of body melanisation and desiccation stress resistance across species Several investigations have shown rapid response to laboratory selection for desiccation resistance in D. melanogaster.45,53,54 Thus, genetic variation for desiccation resistance appears to be directly or indirectly under selection. However, various studies on the mechanistic basis of desiccation resistance in various Drosophila species have shown that rate of cuticular water loss are negatively correlated with desiccation resistance.7,55,56 For ectothermic insects, more than 90% loss of body water involves cuticular transpiration under desiccation stress.57 If desiccation resistance evolves through changes in cuticular permeability, the target of selection might be cuticular components either cuticular lipids or cuticular melanisation. However, such a possibility has not been investigated in natural populations as well as through laboratory selection experiments. For desiccation resistance, water proofing role of cuticular lipids has been reported for terebrionid beetles, scorpions and grasshoppers. 58,59,47 Cuticular impermeability results due to changes in the amount and/or composition of cuticular lipids.46,60,61 According to Gibbs,62 there is lack of correlation between Tm of cuticular lipids and cuticular water loss in several insect taxa. Thus, the water proofing role of cuticular lipids may not be a universal mechanism for regulating water loss. However, it is not clear whether species specific differences in body melanisation can impact their stress resistance levels. For generalist species, it is generally assumed that populations have abundant genetic variations in quantitative traits for adaptation.63 By contrast, there are limited data on the evolutionary responses of stress related traits in tropical Drosophila species from humid habitats.64 A comparison of geographical variations for body melanisation, desiccation resistance and cuticular water loss per fly in D. melanogaster as well as in


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D. ananassae suggested non-overlapping species specific clinical variation (Fig. 4). The most resistant northern populations of D. ananassae are comparable to the least resistant southern populations of D. melanogaster for all the desiccation related traits as well as body melanisation. Thus, the baseline resistance levels are significantly different across these two species. For sympatric populations of these species, selection pressures due to climatic conditions might differ if they vary in their behavioral evasion of desiccation stress under wild conditions. This is possible because both the species are linked with domestic habitats which may offer protection from desiccation stress. In this respect, these species may encounter differential exposure to climatic stress conditions resulting in different slope values for desiccation resistance across species. Alternatively, if there are direct selection pressures on body melanisation which differ significantly across species, we may expect similar response to selection (similar slope values) but with significant differences in intercept or baseline resistance level which correspond to equatorial regions (Fig. 4 A–C). Ectothermic insect taxa may be thermal generalists, or high- or low temperature specialists. Sensitivity to temperature and humidity has the potential to influence the ecology, behavior and adaptive fitness of different insect species. Thus, the extent to which ectotherms can tolerate changes in their ambient thermal environments can be critical in determining their distribution and abundance. On the Indian subcontinent, southern tropical habitats and northern subtropical regions have impacted distribution patterns of D. melanogaster and D. ananassae. However, it is not known which ecophysiological traits are influenced by varying climatic conditions. The contrasting levels of body melanisation correspond with species specific desiccation resistance i.e. body melanisation could be the target of climatic selection. For D. ananassae, inability to colonize high latitudes as well as altitudes may be limited by its quite lower levels of body melanisation. Thus, for these two Drosophila species, variability for body melanisation may be a limiting factor for ecological success under variable climatic conditions. This is supported by distribution of D. ananassae along latitude which has shown a steeper negative slope (b = 2.20 ± 0.21) as compared with D. melanogaster (b= 0.94 ± 0.11; Fig. 4D). Such data suggest that significant reduction in % distribution of D. ananassae along latitude matches with its desiccation sensitivity. However, for D. melanogaster, the changes in % distribution are significantly lower which correspond with its high desiccation resistance level.65 Evidences from seasonal adaptations In montane localities of subtropical regions, winter is the dry season and ectothermic Drosophilids are expected to evolve desiccation resistance to cope with drier climatic conditions. Seasonal variations in quantitative traits are generally assessed by differences in thermal conditions66,67 but evolutionary

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Figure 4. Geographical variation in % body melanisation (A); desiccation resistance (B); and cuticular water loss hr-1 (C) in twelve sympatric populations of D. melanogaster and D. ananassae. The slope values for each trait are quite similar across species but intercept values differ significantly. Regression analysis of changes in % species distribution (D); as a function of RHcv of site of origin of populations

responses due to other environmental factors have received lesser attention.68–71 Seasonal variations have been reported for body size (low heritability) in different Drosophila species72,73; and for desiccation resistance (which show high heritability) in a single population of D. simulans.74 Under seasonally varying environments, wing color patterns in different lepidopterans confer adaptive responses such as crypsis and thermoregulation.75,76 Thus, seasonally varying environments impose strong natural selection and cause rapid phenotypic changes in quantitative traits.70,77,78 However, there are no data on the impact of seasonal climatic variations on desiccation related traits in different Drosophila species from the subtropical regions. Body melanisation shows seasonal plasticity but its role in conferring adaptations to desiccation stress is not clear. Melanic flies are expected to have tighter cross-linkage of the cuticular proteins which would make it less permeable.79,80 If body melanisation is linked with desiccation resistance, seasonal changes in body color due to phenotypic plasticity may impact desiccation related


293

traits. Thus, we addressed the following questions: (a) Do quantitative traits (body size, body melanisation and desiccation related traits) vary seasonally in wild populations? (b) Do plastic changes in body size and cuticular lipids correlate with desiccation resistance? (c) Whether plasticity for body melanisation impacts desiccation resistance and cuticular water loss? We examined seasonal changes in body size, body melanisation, cuticular lipids, desiccation resistance and cuticular water loss in six altitudinal populations of D. melanogaster. Wild caught as well as laboratory reared individuals were analyzed to find genetic and plastic effects for these traits. Further, we examined whether seasonal plastic changes in body melanisation are linked with desiccation resistance and cuticular water loss in D. melanogaster. Finally, we considered spatial as well as temporal changes in body melanisation and desiccation related traits which might be in agreement with the co-gradient hypothesis. Our data have evidenced considerable changes in body melanisation and desiccation related traits across two seasons (autumn and winter). However, seasonal plastic effects for body size are not correlated with desiccation resistance. Further, there is lack of changes in cuticular lipids which do not account for variations in desiccation resistance across seasons. To the best of our knowledge, adaptive significance of body color plasticity to cope with seasonally varying desiccation stress has not been previously analyzed. Following are the main findings of our study: Assessment of plastic vs. genetic changes

Insects can cope with seasonal environments through phenotypic plasticity which allows a single genotype to produce different phenotypes. In temperate regions, some studies have compared clines for body size in wild-caught individuals with their laboratory progeny based on common garden experiments.81,82 However, changes in clinal patterns under seasonally varying conditions have not been reported so far. Thus, in order to test genetic as well as plastic effects, we compared slope values for clinal variation in wild vs. laboratory populations for desiccation related traits. Changes in slope values for quantitative traits across seasons are expected due to plastic responses. By contrast, lack of differences in slope values would suggest the absence of plastic effects for quantitative traits. The slope values for desiccation related traits were found to be significantly higher for winter as compared with autumn under wild conditions i.e. plastic effects were 1.64 fold higher for body melanisation as well as desiccation resistance across seasons (Table 3). By contrast, laboratory analysis of seasonal populations found no differences in slope values. Interestingly, the ratios of slope values based on wild data across seasons were identical for body melanisation, desiccation resistance and cuticular water loss. Thus, in montane localities, phenotypic plasticity for body melanisation in D. melanogaster leads to darker flies in winter and lighter

-4.5 ± 0.37 -2.2 ± 0.30 2.04

2.8 ± 0.34 1.43

Desi. h. 4.0 ± 0.39

CWL

5.8 ± 0.37 1.45

8.4 ± 0.48

1.07

% Mel

0.16 ± 0.015

4.84***

2.32**

4.29***

0.48 ns

Wild /Lab. t- test(p)

0.15 ± 0.014

Lab. (L)

WL

Wild (W)

Slope values *10-3 ( Autumn)

-3.0 ± 0.39

7.0 ± 0.74

13.6 ± 1.0

0.20 ± 0.02

Wild

2.50

2.34

1.25

2.01 **

4.88***

7.31 ***

1.50 ns

Wild/Lab. t-test (p)

-2.0 ± 0.31 1.50

2.8 ± 0.45

5.8 ± 0.38

0.16 ± 0.015

Lab.

Slope values*10-3 (Winter )

1.50

1.75

1.61

1.36

1.01

1.01

1.01

1.00

Wild/ Wild Lab/ Lab.

Plasticity across seasons

Table 3. Comparison of elevational slope values *10-3 (b ± S.E. = regression coefficients) for wild and their laboratory progeny; and plastic effects based on their ratios for four quantitative traits in seasonal populations of D. melanogaster. Plastic effects across seasons are also shown. The slope values for different traits in wild vs. laboratory progeny were compared statistically on the basis ‘t’ - test. ** p < 0.01; ***p < 0.001; ns = non-significant

294 Nature at work: Ongoing saga of evolution


295

Figure 5. Histograms showing changes in % body melanisation of (A) 2nd +3th+4th abdominal segments; (B) 5th +6th +7th segments; and (C) total melanisation per fly in seasonally varying wild - caught individuals from a highland population (Chail - 2226 m a.s.l.) of D. melanogaster

flies during autumn season; and such plastic effects correspond with changes in desiccation resistance across seasons. Plasticity of body melanisation

In D. melanogaster, the developmental specification of the three anterior (2nd, 3rd and 4th) and the three posterior abdominal segments (5th, 6th and 7th) are respectively controlled by the homeotic genes: Abdominal-A and Abdominal-B.83 There is a lack of phenotypic correlations between these two groups of abdominal

296


segments i.e. (2nd + 3rd + 4th) vs. 5th +6th + 7th) for all the populations suggesting their independent genetic control (Fig. 5 A–C). For 2nd to 4th abdominal segments, we obtained non-significant differences in slope values for two different seasons. By contrast, the 5th, 6th and 7th segments showed significant differences in slope values for the two seasons. Plastic effects for three posterior abdominal segments (5th +6th +7th) were two fold higher. An interesting observation is that there were no plastic changes for the three anterior abdominal segments (2nd+3rd+4th). However, the magnitude of the genetic effect (ca. 40%) was similar for (2nd+3rd+4th) and (5th + 6th + 7th) segments. Therefore, due to plastic effects, darker flies in winter are more desiccation resistant and show reduced cuticular water loss as compared with the lighter flies of the autumn season. We may infer that plastic changes in (5th +6th + 7th) segments are correlated with plastic changes in desiccation resistance across seasons. In D.melanogaster, plastic effects for body melanisation and desiccation resistance exceed the magnitude of genetic differences along a cline. There are 40% higher trait values (% melanisation, desiccation hours and cuticular water loss) along the altitudinal cline based on either autumn or winter data. By contrast, for each population, trait divergence across seasons was 50% higher for desiccation related traits. Thus, D. melanogaster has evolved developmental plasticity for body melanisation as an adaptive response to seasonally varying environments. In D. melanogaster, spatial and temporal variations for quantitative traits result from genotype-environment effects. We observed variations in desiccation related traits associated with environmental gradient along altitude. Our data indicate that body melanisation is influenced by environmental conditions during different seasons which impact desiccation resistance. We found significant genetic and plastic effects by comparing slope values along a cline for wild vs. laboratory reared progeny for autumn and winter seasons. The adaptive role of plastic changes described here has not been reported previously. The major conclusion is that plasticity for body melanisation confers desiccation resistance under seasonally varying climatic conditions in the montane localities.84 Evolution of behavioral traits through natural selection Behavioral differences between closely related species are common e.g. different species of Drosophila sing different courtship songs. By contrast, when behavioral variation within a species corresponds to variation in environmental conditions, it is an evidence of past evolution. For example, sitter and rover represent foraging behavioral phenotypes in D. melanogaster which are controlled by diallelic variation at a locus i.e. fors and forR. Laboratory studies of Drosophila populations raised in high- and low-density conditions showed clear divergence in behavior linked to foraging gene (for). Significant increase in the frequency of fors in low density populations; and of forR under high-density conditions support the contention that


297

foraging divergence represents experimental evidence for behavioral evolution in Drosophila.85 Because of the influence of genes on behavior, natural selection can result in the evolution of behavioral traits in populations. Evidences for such evolution are behavioral divergence between as well as within species. In species of Ladybird beetles that harbor melanic polymorphism, dark- colored individuals benefit from increased mating success via thermoregulation (Brakefield, 1984). 32 However, fitness benefits of body color polymorphisms in diverse insect taxa have received lesser attention so far. For body melanisation, evolutionary response of thermal effects has been tested experimentally in different insect taxa from temperate regions10,27 but lesser attention has been paid to other abiotic factor i.e. humidity. We tested this hypothesis in Drosophila jambulina which exhibits color dimorphism controlled by a single locus but its ecological significance is not clear. Dark and light morphs differ significantly in body melanisation, desiccation resistance, rate of water loss, mating activity and fecundity. Interestingly, this species lacks clinal variation for body size, desiccation resistance and life history traits. For body melanisation, lack of geographical variation as well as plastic effects is not in agreement with a thermal melanism hypothesis. However, based on field data, there are seasonal changes in phenotypic frequencies of dark and light body color morphs which correlate significantly with variation in humidity levels. In order to test which of the climatic variables could be a selective agent in the tropics, we raised the following hypotheses: (a) changes in phenotypic frequencies of dark and light morphs may occur through thermal effects and/or humidity changes; (b) humidity changes (dry vs. wet) may impact mating propensity (assortative matings) for different body color morphs; (c) if body melanisation confers desiccation resistance, dark morph is expected to prevail under dry seasons while the reverse may occur for light morph under humid conditions. Under short-term (8 hours) desiccation stress, we observed higher number of assortative matings, longer copulation period and increased fecundity for dark strains as compared with light strains. By contrast, both the morphs when exposed to high humid conditions exhibited higher assortative matings and fecundity for light strains as compared with dark strains. In tropical populations of D. jambulina, body color polymorphism seems to be maintained through humidity changes as opposed to thermal melanism. Thus, seasonal changes in the frequency of body color morphs in this tropical species supports melanism–desiccation hypothesis. Variation in phenotypic frequencies through humidity changes

On the Indian subcontinent, Tave does not vary along latitude as well as longitude. There is lack of correlation between Tave and latitude (r = 0.19 ± 0.44; ns) and also with longitude (r = –0.11 ± 0.70; ns). By contrast, humidity changes are significant along latitude (r = – 0.92 ± 0.10) as well as longitude (r = 0.96 ± 0.20).

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Figure 6. Field data on seasonal changes in the % frequency of dark and light morphs in (A & B) northern vs. southern population; and (C & D) in western vs. eastern population of D. jambulina. For southern and eastern localities, changes in humidity levels are quite low as compared with northern and western localities. The number of wild caught individuals was 200–240 for each season as well as locality

Field data have shown significant seasonal variation in the % frequency of dark and light morphs i.e. greater frequency of melanic morph in autumn as compared to rainy season (Fig. 6 A–C). Longitudinal localities are characterized by similar thermal conditions but higher humidity levels correspond with increased frequency of lighter morph in the eastern locality (Guwahati, 91.35°E; Fig. 6D). Data on varying humidity conditions under laboratory set up has also shown changes in the frequencies of dark and light morphs. Thus, seasonally varying humidity seems to be the principle selective agent for adaptive changes in the frequencies of dark and light body color morphs.


299

Figure 7. Comparison of mating propensity (mated pairs and copulation period) in control vs. flies exposed either to desiccation stress or to high humidity condition. (A) Bar diagrams for mated pairs; (B–D) Changes in copulation period are shown as a function of mating speed for dark and light morphs of D. jambulina. Data are based on analyses of twenty isofemale lines. For all correlation values, p < 0.001

Mating propensity under dry and wet conditions

In order to test whether mating propensity of dark and light morphs in D. jambulina varied with seasons, we attempted no choice mating experiments and the data on observed matings under control and stressful conditions are given in Table 4 and Figure 7A. Mating propensity were estimated on the basis of two components of mating process (mating speed and copulation period) and finally in terms of fecundity differences in four types of observed matings (homospecific and heterospecific reciprocal matings; Table 4). For control experiments, homospecific and heterospecific matings did not differ significantly i.e. under laboratory conditions, matings with no choice method are random between dark and light

300


Table 4 Data on the basis of no choice method for frequency of mated pairs (MP), mean ± SD for mating speed (MS), copulation period (CP) and fecundity under control and stressful (dry and wet) conditions for D. jambulina. For each experiment (A–C), there were twenty replicates. *** p < 0.001, ns = nonsignificant. Humidity levels were 68 ± 2 % for control; 5 ± 2 % for desiccation stress; and 85 ± 2 % for high humidity. Experiment

Type of Mated pairs

(A) Control

MP (%)

MS (min) m ± SD

CP (min) m ± SD

Fecundity m ± SD

1.

Light ♀ * Light ♂

26.27

9.38 ± 1.38

6.24 ± 1.31

24.4 ± 2.18

2.

Dark ♀ * Dark ♂

28.20

7.15 ± 1.46

7.50 ± 1.29

28.0 ± 2.07

3.

Light ♀ * Dark ♂

23.03

9.23 ± 1.15

6.00 ± 1.35

24.9 ± 2.61

4.

Dark ♀ * Light ♂

22.50

9.32 ± 1.07

6.19 ± 1.16

21.6 ± 2.44

ns

ns

Contingency χ

ns

ns

(B)

1.

----Light ♀ * Light ♂

20.53 14.0 ± 1.41

4.21 ± 1.43

20.1 ± 2.90

Desiccation

2.

Dark ♀ * Dark ♂

64.47

5.15 ± 1.12

11.4 ± 1.14

44.2 ± 3.45

stress

3.


8.70

10.0 ± 1.20

5.08 ± 1.23

23.7 ± 2.84

(8 hours)

4.


6.30

10.2 ± 1.33

5.32 ± 1.02

24.5 ± 3.80

2

Contingency χ

-----

2

(C)

1.

Light ♀ * Light ♂

*** 64.44

*** 6.49 ± 1.39

*** 10.0 ± 1.31

*** 35.9 ± 3.25

High humidity

2.

Dark ♀ * Dark ♂

22.22 11.0 ± 1.27

6.13 ± 1.06

22.9 ± 3.11

(120 hours)

3.


6.11

10.1 ± 1.34

6.29 ± 1.25

25.4 ± 3.61

4.


7.23

8.12 ± 1.28

7.47 ± 1.19

24.1 ± 2.66

Contingency χ

2

-----

***

***

***

***

morphs. However, when dark and light morphs were subjected to short-term desiccation stress, mating propensity between dark ♀ x dark ♂ increased about two fold as compared with control experiments (Table 4). The homospecific matings between light ♀ x light ♂ and reciprocal heterospecific matings decreased significantly (p < 0.001). By contrast, flies exposed to high humidity, under no choice mating conditions, exhibited two fold higher mating propensity for light ♀ x light ♂. However, all other mating combinations (dark ♀ x dark ♂) and reciprocal heterospecific matings showed significant reduction (p < 0.001; Table 4). These significant changes in homospecific mating propensity under desiccation or high humidity conditions as compared with control are illustrated in Figure 7A. Thus, the observed differences in mating propensity are a reflection of the type of humidity conditions experienced by different body color morphs. Regression analyses of data on two important components of mating propensity (mating speed and copulation period) under control and stressful conditions are shown in Figure 7B–D. Population means for mating speed (MS) and copulation period (CP) for all types of mating under no choice method are given in Table 4. For all matings under stressful conditions, mating speed and copulation period were negatively correlated i.e. if the MS was longer, CP was shorter and vice versa.


301

Figures 7B to D clearly demonstrate that under desiccation stress, copulation period is longer and mating speed is short for dark morph as compared with light morph (Fig. 7C). Under high humidity conditions, we observed a reverse trend i.e. light morph showed significantly longer copulation period as compared with dark morph (Fig. 7D). Interestingly, under control conditions, there is a significant overlap in mating propensity for dark and light morphs (Fig. 7B). For each experiment, the slope values for both the morphs were quite similar. Thus, regression analysis of copulation period as a function of mating speed helped in discriminating mating propensity under control vs. stressful conditions. In tropical regions, seasonal changes in precipitation cause desiccation stress in autumn. D. jambulina has adapted to wet (rainy season) and dry (autumn) seasons by modifying the frequencies of color morphs through assortative matings. Therefore, humidity rather than temperature is the primary selection agent for body color polymorphism. Field and laboratory data on D. jambulina show evidences in favor of melanism–desiccation hypothesis. Like D. jambulina, other species of montium species subgroup are expected to provide similar evidences. Thus, seasonally varying humidity conditions in the tropics can maintain body color polymorphism and desiccation resistance in D. jambulina.

Conclusions Possible role of body melanisation in conferring desiccation resistance was initially demonstrated for ebony mutant strains of D. melanogaster.50 Subsequently, darker and lighter laboratory strains of D. polymorpha were shown to differ in desiccation resistance.37 For altitudinal populations of D. melanogaster, assorted dark and light flies exhibited significant differences in desiccation resistance and cuticular water loss.24 Furthermore, seasonal data on wild living flies have shown frequency changes of dark and light morphs in D. jambulina which correspond with varying humidity levels. We also observed the impact of short-term desiccation stress or high humidity on mating propensity under no choice experiments. We found a consensus between wild vs. laboratory data on the role of body melanisation in desiccation resistance in this species. Thus, change in humidity level is the principle agent of natural selection which modifies the frequencies of dark and light morphs in D. jambulina. The melanism–desiccation hypothesis finds support from studies on seasonal variations,84 body color mutant strains,50 use of assorted dark and light individuals from a given population43; and on the basis of assortative matings either for dark morph under desiccation stress or light morph under high humidity conditions.8 Finally, laboratory selection experiments on body melanisation and their possible impact on desiccation resistance can form evidence in favor of melanism–desiccation hypothesis but there are no data to support such contentions. However, future investigations are needed to support melanism–desiccation hypothesis across diverse insect taxa.

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Acknowledgement: The help and support of Prof. Dr. K.S. Rai and Prof. Dr. V.P. Sharma is gratefully acknowledged. This work was supported by a research project of UGC, New Delhi.

References 1.

Mortan RA (1993) Evolution of Drosophila insecticide resistance. Genome 36:1–7

2.

Ford EB (1975) In: Ecological Genetics. 4th edition, Chapman & Hall, UK

3.

Endler JA (1986) In: Natural Selection in the Wild. Princeton University Press, Princeton

4.

Powell JR (1997) In: Progress and Prospects in Evolutionary Biology - The Drosophila Model. Oxford University Press, Oxford

5.

Parkash R, Munjal AK (2000) Evidence of independent climatic selection for desiccation and starvation tolerance in Indian tropical populations of Drosophila melanogaster. Evol Ecol Res 2:685–699

6.

Parkash R, Sharma V, Kalra B (2009 ) Correlated changes in thermotolerance traits and body color phenotypes in montane populations of Drosophila melanogaster: Analysis of within and between population variations. J Zool (In Press)

7.

Parkash R, Rajpurohit S, Ramniwas S (2008) Parallel changes in body melanisation and desiccation resistance in highland vs. lowland populations of D. melanogaster. J Insect Physiol 54:1050–1056

8.

Parkash R, Singh S, Ramniwas S (2009) Seasonal changes in humidity level in the tropics impact body color polymorphism and desiccation resistance in Drosophila jambulina – Evidence for mealnism-desiccation hypothesis. J Insect Physiol 55:358–368

9.

Majerus MEN (1998) Melanism: Evolution in Action. Oxford University Press, Oxford

10.

Brakefield PM, Willmer PG (1985) The basis of thermal melanism in the ladybird, Adalia bipunctata: Differences in reflectance and thermal properties between morphs. Heredity 54:9–14

11.

Ellers J, Boggs CL (2002) The evolution of wing color in Colias butterflies: Heritability, sex-linkage, and population divergence. Evolution 56:836–840 Kennington WJ, Killeen JR, Goldstein DB, Partridge L (2003) Rapid laboratory evolution of adult wing area in Drosophila melanogaster in response to humidity. Evolution 57(4): 932–936 Roskam JC, Brakefield PM (1999) Seasonal polyphenism in Bicyclus (Lepidoptera: Satyridae) butterflies: Different climates need different clues. Biol J Linnean Soc 66: 345–356 Hollocher H, Hatcher JL, Dyreson EG (2000) Evolution of abdominal pigmentation differences across species in the Drosophila dunni subgroup. Evolution 54:2046–2056 Hollocher H, Hatcher JL, Dyreson EG (2000) Genetic and developmental analysis of abdominal pigmentation differences across species in the Drosophila dunni subgroup. Evolution 54:2057–2071 Llopart A, Elwyn S, Lachaise D, Coyne JA (2002) Genetics of a difference in pigmentation between Drosophila yakuba and D. santomea. Evolution 56:2262–2277 True JR (2003) Insect melanism: The molecules matter. Trend Ecol Evol 18:640–647

12.

13.

14. 15.

16. 17. 18.

De Jong PW, Gussekloo SWS, Brakefield PM (1996) Differences in thermal balance, body temperature and activity between non-melanic and melanic two-spot ladybird beetles (Adalia bipunctata) under controlled conditions. J Exper Biol 199:2655–2666


303

19.

Dombeck I, Jaenike J (2004) Ecological genetics of abdominal pigmentation in Drosophila falleni: a pleiotropic link to nematode parasitism. Evolution 58:587–596

20.

Ellers J, Boggs CL (2004) Functional ecological implications of intra-specific differences in wing melanism in Colias butterflies. Biol J Linnean Soc 82:79–87

21.

Mani MS (1968) Ecology and Biogeography of high altitude Insects. In: The Hague. Junk W (Ed.)

22.

Rapoport EH (1969) Gloger’s rule and pigmentation of Collembola. Evolution 23: 622–626

23.

Pool JE, Aquadro CF (2007) The genetic basis of adaptive pigmentation variation in Drosophila melanogaster. Molecular Ecology 16(4):2844–2851

24.

Parkash R, Sharma V, Kalra B (2008) Climatic adaptations of body melanisation in Drosophila melanogaster from Western Himalayas. Fly 2:111–117

25.

Ohnishi S, Watanabe TK (1985) Genetic analysis of color dimorphism in the Drosophila montium subgroup. Jap J Genetics 60:355–358

26.

Watt WB (1968) Adaptive significance of pigment polymorphism in Colias butterflies, I: Variation in melanin in relation to thermoregulation. Evolution 22:437–458

27.

Berry RJ, Willmer PG: Temperature and the color polymorphism of Philaenus spumarius (Homoptera: Aphrophoridae). Ecol Entomol 11:251–260 (1986)

28.

Kettlewell B (1973) The Evolution of Melanism. The study of a recurring necessity; with special reference to industrial melanism in Lepidoptera. Clarendon Press, London

29.

Roland J (1982) Melanism and diel activity of alpine Colias (Lepidoptera; Pieridae). Oecologia 53:214–221

30.

Guppy CS (1986) The adaptive significance of alpine melanism in the butterfly Parnassus phoebus (Lepidoptera, Papilionidae). Oecologia 70:205–213

31.

Kingsolver JG, Wiernasz DC (1991) Seasonal polyphenism in wing-melanin pattern and thermoregulatory adaptations in Pieris butterflies. Am Natu 137:816–830

32.

Brakefield PM (1984) Selection along clines in the ladybird Adalia bipunctata in the Netherlands. A general mating advantage to melanics and its consequences. Heredity 1984 53:37–49

33.

Verhoog MD, Breuker CJ, Brakefield PM (1998) The influence of genes for melanism in the activity of the flour moth, Ephestia kuehniella. Anim Behav 56:683–688

34.

Rettenmeyer CW (1970) Insect mimicry. Annu Rev Entomol 15:43–74

35.

Brakefield PM (1985) Polymorphic Muellerian mimicry and interaction with thermal melanism in ladybirds and a soldier beetle: a hypothesis. Biol J Linnean Soc 26:243–268

36.

Wittkopp PJ, Caroll SB, Kopp A (2003) Evolution in black and white: Genetic control of pigment patterns in Drosophila. Trend Genet 19:495–504

37.

Brisson JA, Toni DCD, Duncan I, Templeton AR (2005) Abdominal pigmentation variation in Drosophila polymorpha: Geographic variation in the trait and underlying phylogeography. Evolution 59:1046–1059

38.

Komai T (1956) Genetics of ladybeetles. Adv Genetics 8:155–188

39.

Lusis JJ (1973) Taxonomic relations and geographical distribution of forms in beetles of the genus Adalia Mulsant. Prob Genet Evol 1:5–12

40.

Rhamhalinghan M (1988) Seasonal variations in the color patterns of Coccinella septempunctata L. (Coleoptera: Coccinellidae) in Nilgiri Hills, India. J Bombay Natu Hist Soc 85:551–558

304


41.

Rajpurohit S, Parkash R, Ramniwas S (2008) Body melanisation and its adaptive role in thermoregulation and tolerance against desiccating conditions in Drosophilids. Entomol Res 38:49–60

42.

Parkash R, Ramniwas S, Rajpurohit S, Sharma V (2008) Variations in body melanization impact desiccation resistance in Drosophila immigrans from Western Himalayas. J Zool 276:219–227

43.

Parkash R, Rajpurohit S, Ramniwas S (2009) Impact of darker, intermediate and lighter phenotypes of body melanisation on desiccation resistance in Drosophila melanogaster. J Insect Sci 9:97–103

44.

Markow TA, O’Grady PM (2006) Drosophila: A guide to species identification and use. Academic Press, UK

45.

Gibbs AG, Chippendale AK, Rose MR (1997) Physiological mechanisms of evolved desiccation resistance in Drosophila melanogaster. J Exp Biol 200:1821–1832

46.

Hadley NF (1994) Water relations of Terrrestrial Arthropods. Academic Press, San Diego, California

47.

Rourke BC (2000) Geographic and altitudinal variation in water balance and metabolic rate in a California grasshopper, Melanoplus sanguinipes. J Exp Biol 203:2699–2712

48.

Parkash R, Kalra B, Sharma V (2008) Changes in cuticular lipids, water loss and desiccation resistance in a tropical Drosophilid: Analysis of within population variation. Fly 2: 189–197

49.

Hoffmann AA, Parsons PA (1993) Direct and correlated responses to selection for desiccation resistance – a comparison of Drosophila melanogaster and Drosophila simulans. J Evolut Biol 6:643–657

50.

Kalmus H (1941) The resistance to desiccation of Drosophila mutants affecting body colour. Proc R Soc Lond B 130:185–201

51.

Fraenkel G, Rudall KM (1940) A study of the physical and chemical properties of the insect cuticle. Proc R Soc Lond Ser B 129:1–35

52.

Pryor MGM (1940) On the hardening of the cuticle of insects. Proc R Soc Lond B 128: 393–407

53.

Hoffmann AA, Parsons PA (1989) An integrated approach to environmental stress tolerance and life history variation: desiccation tolerance in Drosophila. Biol J Linnean Soc 37:117– 136

54.

Telonis-Scott M, Guthgridge KM, Hoffmann AA (2006) A new set of laboratory-selected Drosophila melanogaster lines for the analysis of desiccation resistance: response to selection, physiology and correlated responses. J Exp Biol 209:1837–1847

55.

Kimura K, Shimozawa T, Tanimura T (1985) Water loss through the integument in the desiccation–sensitive mutant, parched, of Drosophila melanogaster. J Insect Physiol 31: 573–580

56.

Gibbs AG, Matzkin LM (2001) Evolution of water balance in the genus Drosophila. J Exp Biol 204:2331–2338

57.

Gibbs AG, Fukuzato F, Matzkin LM (2003) Evolution of water conservation mechanism in Drosophila. J Exp Biol 206:1183–1192

58.

Edney EB (1977) Water Balance in Land Arthropods. Springer-Verlag, Berlin

59.

Zachariassen KE (1988) Water loss in insects from arid and humid habitats in Eastern Africa. Acta Entmologica Bohemoslov 85:81–93


60.

305

Toolson EC (1984) Interindividual variation in epicuticular hydrocarbon composition and water loss rates of the Cicada Tibicen dealbatus (Homoptera: Cicadidae). Physiol Zool 57:550–556

61.

Gibbs AG (1998) Water-proofing properties of cuticular lipids. Am Zool 38:471–482

62.

Gibbs AG (2002)Lipid melting and cuticular permeability: New insights into an old problem. J Insect Physiol 48:391–400

63.

Hoffmann AA, Weeks AR (2007) Climatic selection on genes and traits after a 100 year-old invasion: a critical look at the temperate-tropical clines in Drosophila melanogaster from eastern Australia. Genetica 129:133–147

64.

Hoffmann AA, Hallas RJ, Dean A, Schiffer M (2003) Low potential for climatic stress adaptation in a rainforest Drosophila species. Science 301:100–102

65.

Parkash R, Kalra B, Sharma V (2009) Impact of body melanisation on contrasting levels of desiccation resistance in a circumtropical and a generalist Drosophila species. Evolutionary Ecology

66.

Kingsolver JG (1995) Fitness consequences of seasonal polyphenism in western white butterflies. Evolution 49:942–954

67.

Holloway GJ, Marriott CG, Crocker HJ (1997) Phenotypic plasticity in hoverflies: The relationship between color pattern and season in Episyrphus balteatus and other Syrphidae. Ecol Entomol 22:425–432

68.

Tauber MJ, Tauber CA, Nyrop JP, Villani MG (1998) Moisture, a vital but neglected factor in the seasonal ecology of insects: Hypothesis and tests of mechanisms. Environ Entomol 27:523–530

69.

Chown SL, Nicolson SW (2004) Insect Physiological Ecology: Mechanisms and Patterns. Oxford University Press, Oxford

70.

Danks HV (2007) The elements of seasonal adaptations in insects. Can Entomol 139:1–44

71.

Brakefield PM, Pijpe J, Zwaan BJ (2007) Developmental plasticity and acclimation both contribute to adaptive responses to alternating seasons of plenty and of stress in Bicyclus butterflies. J Biosci 32;465–475

72.

Tantawy AO (1965) Studies on natural populations of Drosophila. III Morphological and genetic differences of wing length in D. melanogaster and D. simulans in relation to season. Evolution 18:560–570

73.

Kari JS, Huey RB (2000) Size and seasonal temperature in free-ranging Drosophila subobscura. J Thermal Biol 25:267–272

74.

Mckenzie JA, Parsons PA (1974) The genetic architecture of resistance to desiccation in populations of D. melanogaster and D. simulans. Aust J Biol Sci 27:441–456

75.

Gotthard K, Nylin S (1995) Adaptive plasticity as an adaptation: A selective review of plasticity in animal morphology and life history. Oikos 74:3–17

76.

Stoehr AM, Goux H (2008) Seasonal phenotypic plasticity of wing melanisation in the cabbage white butterfly, Pieris rapae L. (Lepidoptera: Pieridae). Ecol Entomol 33: 137–143

77.

Shapiro AM (1976) Seasonal polyphenism. Evol Biol 9:259–333

78.

Tauber CA, Tauber MJ: Insect seasonal cycles (1981) Genetics and evolution. Ann Rev Ecol System 12:281–308

306


79.

Walter MF, Black BC, Afshar G, Dermabon AY, Wright TRF, Biessmann H (1991) Temporal and spatial expression of the yellow gene in correlation with cuticle formation and dopa decarboxylase activity in Drosophila development. Develop Biol 147:32–45

80.

Wittkopp PJ, Vaccaro K, Carroll SB (2002) Evolution of yellow gene regulation and pigmentation in Drosophila. Curr Biol 12: 1547–1556

81.

Coyne JA, Beecham E (1987) Heritability of two morphological characters within and among natural populations of Drosophila melanogaster. Genetics 117:727–737

82.

Imasheva AG, Bubli OA, Lazebny OE (1994) Variation in wing length in Eurasian natural populations of Drosophila melanogaster. Heredity 72:508–514

83.

Carroll SB (1995) Homeotic genes and the evolution of arthropods and chordates. Nature 376:479–485

84.

Parkash R, Sharma V, Kalra B (2009) Impact of body melanisation on desiccation resistance in montane populations of D. melanogaster: Analysis of seasonal variation. J Insect Physiology 55:898–908

85.

Sokolowski MB, Bauer SJ, Wai-Ping V, Rodriguez L, Wong JL, Kent C (1986) Ecological genetics and behaviour of Drosophila melanogaster larvae in nature. Animal Behav 32:403–408

19 Origin and evolution of human malaria parasite, P. falciparum and P. vivax Nidhi Datta* and Virender Singh Chauhan** International Center for Genetic, Engineering and Biotechnology, Aruna Asaf Ali Marg, PO Box 10504, New Delhi - 110067, India *[email protected], **[email protected]

Abstract: The global occurrences of malaria make it the major killer among all diseases. Many initiatives are being taken worldwide to control the disease. But, despite all these initiatives, its incidences are increasing constantly. Antimalarial drug resistance and lack of method to assess efficacy of any control initiatives have also worsen the situation. In this scenario phylogenetic study of malaria pathogen, can be effective in helping to develop new drugs and vaccines for the disease as well as to understand host parasite interactions and evolution of drug resistance in the pathogen. Malaria is likely originated in tropical Africa and later spread to other parts of the world due to human movement. Presence of plastid in Plasmodium sp. indicates its affinity with cynobacteria and green algae. Recent phylogenetic studies have indicated that all the four human malaria sp. are remotely related to each other. Close relation between P. falciparum and chimpanzee malaria parasite (P. reichenowi) and P. vivax and malaria parasite species of Asian monkeys has been observed. P.vivax shows higher genomic diversity than P. falciparum which indicates that it is older than the latter. Evolutionary studies suggest origin of P. falciparum and P. vivax in Africa and Asia respectively, and support ancient population expansions for both Plasmodium sp. Resistance in Plasmodium sp. for widely used antimalarial drugs was also developed along with spread of the disease It is Darwin’s fundamental contribution that fired the imagination of scientist’s interest in evolutionary studies, and these studies have already proved of much value in understanding the origin, spread and control measures in malaria. Keywords: Darwin, Malaria, P. falciparum, P. vivax, Phylogeny

Introduction The evolution of organisms is well-established scientific knowledge and has important applications in agriculture, medicine, and other areas. Charles Robert Darwin, known as the originator of the modern theory of evolution, was born V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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on February 12, 1809. In 1859, he published his most important book “On the Origin of Species by Means of Natural Selection.1 In Origin, he gathered the best available evidences for evolution and explained the process of natural selection, the role of hereditary variation, and considered possible objections to his theory. As for most path breaking theories that challenge conventional wisdom, it took sometime for its acceptance, but by the middle of 19th century, naturalists accepted the evolution of organisms. The theory of natural selection is now considered as the central organizing principle of biology. In an address to the American Association of Biology Teachers in 1973, the evolutionist Theodosius Dobzhansky emphasized on the importance of evolution by saying that “Nothing in biology makes sense except in the light of evolution”. Human malaria is an endemic parasitic disease in most tropical and subtropical ecosystems worldwide. Malaria infects more than 600 million people, causing up to two to three million deaths, mostly in sub-Saharan Africa every year.2 However, malaria exhibits few forms of genetic resistance too. Patients of sickle cell anemia show some kind of protection; specifically, a child with the genetic disorder has high chances to survive the acute illness of malaria. Presence of heterozygous HbS provides survival advantages, over people with both the genes for normal hemoglobin.3–4 These genes are able to transmit to next generation and hence spread resistance in a population. People of Africa and their progenies also show resistance against P. vivax. The reason behind this could be the absence of Duffy antigen which is the receptor through which merozoites of P. vivax enter into red blood cell.5 Lack of this receptor does not allow the pathogen to invade the host blood cells and provides him resistance against the disease. Thalassemic patients also show some level of resistance against the disease. However, despite this genetic resistance, cases of malaria infection have increased drastically during the last few years and there is no direct method to check affectivity of malaria control measures. Efficacy of any malaria control measure is generally assessed by its impact on the number of infected individuals, clinical cases registered, treatment failure etc. However, most of the times this process does not give a clear picture of the situation and is also not able to show small changes in pathogen population. In this scenario, study of parasite genetic diversity could be the best way to detect the effect of intervention and any slight changes in the population. Knowledge of evolutionary history of parasite population does not only help to check the affectivity of any disease control measure but also assists to understand the development of genetic resistance in a species which is extremely useful to avoid any disastrous situation to human kind. Study of the genetic basis and evolutionary pattern of resistance is helpful in direct monitoring6 and epidemiology surveillance studies.7 It also encourages to test and use different parasitic antigens as vaccines candidate and can contribute to development of new drugs and vaccine.

Origin and evolution of human malaria parasite, P. falciparum and P. vivax

309

Phylogeny of malaria Phylogenetic analyses provide information on the origins, evolutionary history and relationships between organisms. Serious research work on molecular phylogenetics of malaria parasites began in 1991.8,9 Most early conclusions drawn from such studies were based on Small Subunit (SSU) rRNA, the Circumsporozoite Protein (CSP),8-11 mitochondrial gene for cytochrome13-14 and the gene for the housekeeping enzyme adenylosuccinate lyase.15-16 Plasmodium belongs to the phylum Apicomplexa. Phylogenetic investigators suggested its origin with kingdom of plants, fungi and animals around one billion years ago.17 Presence of plastid in Plasmodium and its genetic similarity with cynobacteria and green alga also indicates close affinity between them.18 Four Plasmodium species viz. P. falciparum, P. malariae, P. ovale and P. vivax are parasitic to humans. Earlier it was thought that all human malaria parasites are closely related, but recent works have shown that all the four human malaria sp. are only remotely related to each other.14 It was conjunctured that Plasmodidae were first appeared in the blood stream and have been transmitted by blood sucking anthropods.19 Some studies suggested that the origin of Plasmodium occurred through host switching from a non-human parasite to human parasite. Close relation of some species of Plasmodium to parasitic species of birds than to species parasitic to human or primates supports this hypothesis.8-9 Through several genetic studies, avian origin of malaria parasite proposed earlier8,9,11,15 was later refuted14 and P. falciparum was placed within the clade of mammalian parasites. The single gene hypothesis determined that ‘Plasmodium’ is paraphyletic relative to genus ‘Haemoproteus’ and ‘Hepatocystis’. Genus Haemoproteus belongs to malaria parasite of amphibian, reptiles and birds while Hepatocystis represents the evolutionary stem of malaria parasite of mammals.19 However, connectivities between mammals, bird and lizard Plasmodium parasites were also found. On the other hand, the four gene hypothesis did not show any paraphyletic relation between Plasmodium and avain Haemoproteus. In the case of malaria parasite of primates, the genus Hepatocystis and quartan group is considered as most ancestral, followed by the tertian group. Subgroup Laverania (P. falciparum and P. reichenowi) is probably the most recent.20 Avian and reptilian malaria parasites also show host shift and the lateral transmission of Plasmodium parasite from monkey host to humans is a common occurrence.21,22 Transmission from humans to monkey has been achieved through experiments22 but it may also occur naturally. The evolution of parasite may or may not always be in sync with the evolution of the host. The pace of evolution of parasite and evolution of the hosts sometimes does not match with each other. Some members of a species of host adapt themselves with the changing environment more easily than the others. Even the effect of environment is also not equal on the host and internal parasites present in the host.23 The difference of time between the change in environment

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for the host and the parasite results in the difference in rate of evolution and ultimately evolution of parasite is slower than the host. One notable example of this inconsistency is of malaria species from a southeast asian bird that groups with lizard malaria parasite.24 It seems that malaria originated in tropical Africa and remained localized in the Paleolithic and Mesolithic periods. In the Neolithic period, human population increased and likewise the malaria infection and its movement spread to Mesopotamia, India, South China, and from Nile Valley to Mediterranean shores and ultimately to other parts of the world.20 Prehistoric man must have been a victim of malaria in ancient time and ultimately developed some resistance to it.25 It seems that the resistance to P. vivax developed earlier than to other human plasmodia. Some recent studies support these observations.26–30 Introduction of new and resistant parasite genotype could be due to population movement. During the course of time, Plasmodium also developed resistance against commonly available drugs. Cloroquine resistance (CR) in P. falciparum was first reported in 1978 in non-immune travelers from Kenya and Tanzania.31–32 Similar kinds of reports of CR were also come from Madagascar,33 Tanzania34 and Kenya.35 It is hypothesized that the CRPF spread to Africa from Asia due to the mobility of man. The genetic similarity between parasites of Africa and Asian origin and difference with South America and Papua New Guinea parasite support this hypothesis.36 Thailand, Kwa Zulun Natal,37 Malawi, Kenya and S. Africa are some of the countries which have replaced CQ as a first line drug. P. vivax resistance against CQ has also been reported from Guyana (S. America),38 Sumatra (Indonesia),39 Myanmar and Mumbai (India)40 and Brazilian Amazon region, and particularly for reappearance of parasite after treatment with CQ from Vietnam.41

Evolutionary history of P. falciparum A study based on CSP and rRNA revealed close relation between P. falciparum and P. reichenowi (a rare parasite of chimpanzees).14,15, 42–44 There are different views about their divergence. Some believed that the two species likely diverged from each other between 5–8 million years ago.10,11 This hypothesis is consistent with the time of divergence between human and Chimpanzee. A very recent study favors hypothesis of evolution of P. falciparum from P. reichenowi, most likely by a single host transfer from Chimpanzee to human between 10,000 to 2–3 MYA. This transfer may have happened due to two genetic mutations in Cytidine monophosphate-N-acetylneuraminic acid hydroxylase (CMAH) gene and in EBA -175, respectively.45 CSP and cyt-b tree both shows close relation of P. falciparum with P. reichenowi.46 Close association between both sp. has also been revealed by the analysis of mitochondrial DNA sequence polymorphism. A study of mitochondrial haplotypes isolates of P. falciparum from several endemic areas showed a wave of migration from Africa to southeast Asia and


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South America suggesting origin of P. falciparum in Africa and colonization in Southeast Asia and South America.47 Variation in the mitochondrial DNA sequences showed a sudden increase in the African malaria parasite population that was prominent about 10,000 years ago, and after that it migrated to other regions.48 Study on Microsatellite DNA has suggested, African P. falciparum species as the most ancient. It seems that P. falciparum was derived from one single parasite, a cenancestor, within the last 5,000–50,000 years.49 There is a consistency between the antigenic polymorphisms and the recent origin of the world populations of P. falciparum. Strong natural selection leads to rapid allele substitutions or polymorphisms. Extent of polymorphism at neutral evolving loci indicated towards a common ancestor for P. falciparum sp. which was not recent than 1,50,000–200,000 years ago.50 Balanced polymorphisms at certain antigen encoding loci ruled out the hypothesis that this species underwent a recent extreme bottleneck but occurrence of less extreme bottlenecks could be possible. Such bottlenecks could have occurred at the time of origin of modern humans and again at the origin of West African agriculture.51–52 However, it shows that these bottlenecks were not so severe as to eradicate all of the polymorphism present in P. falciparum, some of which is clearly ancient and selectively maintained. Future phylogenetic analyses may help to identify close relatives of P. falciparum and better understand the evolutionary history of these parasites.

Evolutionary history of P. vivax Till mid-1900s, P. vivax was the most widespread and prevalent of the human malaria parasite. It’s ability to complete its sporogonic cycle at a comparatively minimum lower temperature (16°C) helps it to establish stable foci of transmission in the temperate zones. P. vivax shows extraordinary phenotypic diversity and is found in a broad range of ecotypes. It can survive long in liver through its dormant stages, which ultimately leads to relapses of the infection, weeks after the initial infection.53 The relatively stable GC-rich genome of P. vivax (which has a GC content double to that of P. falciparum) has few di-nucleotide microsatellite markers, and the (TA) and (CA) motifs are rare in P. vivax.54 It also possesses highly polymorphic genome which may present some challenges for malaria control initiatives.55 Most recent studies on origin of P. vivax are based on the mitochondrial genome, nuclear genome and the microsatellite loci. Phylogenetic studies showed origin of P. vivax from a malarial parasite of non-human primates as a result of a host switch.13 Genetic affinity of P. vivax with Asian primates malaria has also been reported14,56 Phylogenetic analysis based on mitochondrial cyt b gene showed close proximity of P. vivax with human and monkey malaria parasite from south east asia.13 There are some phylogenetic evidences that P. vivax derived from Plasmodium found in Southeast Asian macaques.57 There are abundant

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evidences available which shows association of P. vivax to a monophyletic group of Plasmodium species parasitizing old world monkeys (Cercopithecoidea) and suggested spreading of the sp. along with its hosts.10,13,14 It was considered that P. vivax co-evoluted with Africans human population58 probably between 45,000– 81,000 years,56 but a study of sequences of complete mitochondrial genome of isolates of P. vivax, P. malariae, P.ovale, P. knowlesi, P. simium, P. frzagile revealed sequence divergence between P. vivax and P. knowlesi and placed it’s mitochondrial ancestors at around 2–3 million years ago.59 It gives a possibility of transfer of P. vivax from a monkey host to H. erectus in Southeast Asia sometime before 1 million years ago and subsequently spread across southern Asia into Africa through the H. erectus population. It supports findings of a previously given hypothesis on the Asian origin and close relation with Asian monkey’s of P. vivax based on the nuclear-encoded -tubulin and CDC-2 genes of 10 species of Plasmodium.56 Asian populations of P. vivax are considered the oldest. Diversity between Plasmodium sp. Two principal human malaria parasites, P. falciparum and P. vivax, seem to be very different in origin and in phylogenetic resemblance to other species of Plasmodium.

Evolution of Plasmodium parasite as suggested by Richard Carter and Kamini N. Mendis.61


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Further, they both differ not only in the mortality and morbidity they cause but also in the percentage of nucleotide composition in their respective genomes. The African populations of P. falciparum are highly diverse at the DNA level among other populations and in P. vivax the Asian population shows maximum diversity. These studies support African origin of P. falciparum and Asian origin of P. vivax. P. vivax shows higher genomic diversity than P. falciparum which suggests that P. vivax is older than P. falciparum. P. vivax has close genetic affinity with malaria parasite species of Asian monkeys, P. falciparum showed genetic similarity with P. reichenowi and chimpanzee.3 Like other Plasmodium sp., P. falciparum and P. vivax both have 14 haploid chromosome in their genome. The P. vivax genome contains a much lower AT (~55%) content in comparison to the genome of P. falciparum (~80%).60,62 On genetic variation, some reports have suggested a low level of genetic variation in P. falciparum and thus a relatively recent expansion, while other studies revealed a high level of genetic variation, indicating a large population size that has been maintained over hundreds of thousands of years.63 Similar level of diversity between P. falciparum and P. vivax confirmed that both the parasites underwent ancient population expansions. This strengthens the previous hypothesis that these two species were parasites of the hominid lineage before the origin of modern H. sapiens and expanded with population expansion of their host.3 Studies on population structure have provided important indications as to the origin, dispersal and stability of multilocus genotypes and quantify how alleles are spatially dispersed and how the overall genetic diversity is organized. Both P. falciparum and P. vivax use their genetic diversity to fight against anti-malarial drugs and the host immunity. However, the mechanisms of maintenance of such diversities are not known. Thus, study of genetic variations and evolutionary history of Plasmodium parasite genes and populations is critical in understanding several mechanisms in malaria, especially in identifying drug and vaccine targets, understanding virulence pattern, host–parasite interactions for determining source of parasite for imported malaria and for determining genes for drug resistance.7,64,65 On February 12, 2009, world has celebrated 200th birthday of Charles Darwin and 150 years of his magnificent intellectual achievement ‘On the Origin of Species’. He died in 1882, just 2 years after Alphonse Laveran identified Plasmodium, the parasite that caused malaria. Since then, a great deal of development has been achieved in malaria treatment procedures, as well as in issues like drug resistance by malaria parasites, effect of changing human behavior like agriculture and urbanization on the malaria parasite, adaptation by mosquitoes to changing environment and pesticides. Role of natural selection and development of resistant strains of malaria parasite,66 and in insects exposed to pesticides undergo strong natural selection through adaptive mechanisms to survive.67 These

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issues can be resolved by evolutionary studies of the parasite which can help to develop potential remedial measures. Today only because of Darwin’s work and guidance, we have some understanding about the complex evolution of Malaria. On the occasion of two centuries of Darwin, the biologists should acknowledge our gratitude to him and remember his outstanding contributions, which are still so relevant and useful ammunition in the continuing battle to save human lives from infectious diseases.

References 1.

Darwin C (1859) In: On the Origin of Species by Means of Natural Selection. John Murray, London

2.

Snow RW, Guerra CA, Noor AM, Myint HY, Hay SI (2005) The global distribution of clinical episodes of Plasmodium falciparum malaria. Nature 434:214–217

3.

Allison AC (1954) Protection afforded by sickle, cell trait against subtertian malaria infection. Br Med J I:290–294

4.

Allison AC (1964) Polymorphism and natural selection in human populations. Cold spring Harbor Symp Quant Biol 29:137–149

5.

Allison AC (2009) Genetic control of resistance to human malaria. Curr Opin Immunol 21:1–7

6.

Das A, Bajaj R, Mohanty S, Swain V (2007) Genetic diversity and evolutionary history of Plasmodium falciparum and P. vivax. Curr Sci 92(11):1516

7.

Rodríguez-Morales AJ, Benitez JA, Franco-Paredes C (2006) Impact of imported malaria on the burden of disease in northeastern Venezuela. J Travel Med 13:15–20

8.

Waters AP, Higgins DG, McCutchan TF (1991) Plasmodium falciparum appears to have arisen as a result of a lateral transfer between avian and human hosts. Proc Natl Acad Sci USA 88:3140–3144

9.

Waters AP, Higgins DG, McCutchan TF (1993) Evolutionary relatedness of some primate models of Plasmodium. Mol Biol Evol 10(4):914–923

10.

Escalante AA, Ayala FJ (1994) Phylogeny of the malarial genus Plasmodium, derived from rRNA gene sequences. Proc Natl Acad Sci USA 91:11373–11377

11.

Escalante AA, Barrio E, Ayala FJ (1995) Evolutionary origin of human and primate malarias: evidence from the circumsporozoite protein. Mol Biol Evol 12:616–626

12.

McCutchan TF, Kissinger JC, Touray MG, Rogers MJ, et al. (1996) Comparison of circumsporozoite proteins from avain and mammalian malaria. Proc Natl Acad Sci USA 93:11889–11894

13.

Escalante AA, Freeland DE, Collins WE, Lal AA (1998) The evolution of primate malaria parasites based on the gene encoding cytochrome b from the linear mitochondrial genome. Proc Natl Acad Sci USA 95:8124–8129

14.

Perkins SL, Schall JJ (2002) A molecular phylogeny of malarial parasites recovered from cytochrome b gene sequences. J Parasitol 88(5):972–978

15.

Escalante AA, Goldman IF De RP De WR Collins WE Qari SH Lal AA (1997) Phylogenetic study of the genus Plasmodium based on the secondary structure-based alignment of the small subunit ribosomal RNA. Mol Biochem Parasitol 90:317–321


315

16.

Kedzierski L, Escalante AA, Isea R, Black CG, Barnwell JW, Coppel RL (2002) Phylogenetic analysis of the genus Plasmodium based on the gene encoding adenylosuccinate lyase. Infect Genet Evol 1(4):297–301

17.

Ayala FJ, Escalante A, Lal A, Rich S (1998) Evolutionary relationships of human malarias. In: Malaria: Parasite Biology, Pathogenesis, and Protection. Sherman IW (Ed.), American Society of Microbiology, Washington, DC

18.

Kohler S, Delwiche CF, Denny PW, Tilney LG, Webster P, Wilson RJM, Palmer JD, Roos DS (1997) A plastid of probable green algal origin in apicomplexan parasites. Science 275:1485–1489

19.

Bray RS (1957) Studies on the exo-erythrocystic cycle of the genus Plasmodium. Lewis, London

20.

Bruce-Chwatt LJ (1965) Paleogenesis and paleo-epidemiology of primate malaria. Bull Wld Hlth Org 32:363–387

21.

Contacos PG, Coatney GR (1963). J Parasit 49:912

22.

Chin W, Contacos PG, Coatney GR, Kimball HR (1965) A natuarally acquired quotidian type malaria in man transferable to monkeys. Science 149:865

23.

Moskovskij SD (1948). Tr Akad Med Nauk SSSR 1:155

24.

Martinsen ES, Perkins SL, Schall JJ (2008) A three-genome phylogeny of malaria parasites (Plasmodium andclosely related genera): Evolution of life-history traits and host switches. Mol Phylogenet Evol 47:261–273

25.

Russell PF (1955) Man’s mastery of malaria. Oxford University Press, London

26.

Coatney GR (1963). J Parasit 49:919

27.

Hirsch A. Handbook of geographical and historical pathology. New Sydenham Society (English translation) 1883–1886, London

28.

Koch R (1898). Arb K Gesundh Amt 14:292

29.

Stephans, JW, Cristophers SR (1900). Rep Malar Com Roy Soc Series 3:4

30.

Garnham PCC (1963) An introduction to protozoal immunity. In: Immunity to Protozoa. Garnham PCC, Pierce AE, Roitt I (Eds.), Blackwell, Oxford

31.

Campbell CC, Chin W, Collins WE, Teutsch SM, Moss DM (1979) Chloroquine-resistant Plasmodium falciparum from East Africa: cultivation and drug sensitivity of the Tanzanian I/CDC strain from an American tourist. Lancet 35(II):1151–1154

32.

Fogh S, Jepsen S, Effersoe P (1979) Chloroquine-resistant Plasmodium falciparum malaria in Kenya. Trans R Soc Trop Med Hyg 73:228–229

33.

Aronsson B, Bengtsson E, Bjorkman A, Pehrson PO, Rombo L, Wahlgren M (1981) Chloroquine-resistant falciparum malaria in Madagascar and Kenya. Ann Trop Med Parasitol 75:367–373

34.

Kihamia CM, Gill HS (1982) Chloroquine-resistant falciparum malaria in semi-immune African Tanzania. Lancet 2:43

35.

Markwalder KA, Meyer HE (1982) Possible sulfadoxine-pyrimethamine resistance in Plasmodium falciparum malaria from Kenya. Trans R Soc Trop Med Hyg 76:281

36.

Wellems TE Pantons LJ Gluzman IY Do RV, Gwadz RW, Walker-Jonah A, Krogstad DJ (1990) Chloroquine-resistant malaria. J Infect Dis 184:770–776

37.

Bredenkamp BL, Sharp BL, Mthembu SD, Durrheim DN, Barnes KL (2001) Failure of sulphadoxine-pyrimethamine in treating Plasmodium falciparum malaria in KwaZulu-Natal. S Afr Med J 91:970–972

316


38.

Phillips EJ Keystone JS Kain KC (1996) Failure of combined chloroquine and high-dose primaquine therapy for Plasmodium vivax malaria acquired in Guyana, South America. Clin Infect Dis 23:1171–1173

39.

Baird JK, Wiady I, Fryauff DJ, Sutanihardja MA, Leksana B, Widjaya H, Kysdarmanto Subianto B (1997) In vivo resistance to chloroquine by Plasmodium vivax and Plasmodium falciparum at Nabire, Irian Jaya, Indonesia. Am J Trop Med Hyg 56:627–631

40.

Kshirsagar NA, Gogaty NJ, Rajgor D, Dalvi SS, Wakde M (2000) An unusual case of multidrug- resistant Plasmodium vivax malaria in Mumbai (Bombay). India Ann Trop Med Parasitol 94:189–190

41.

Phan GT, de Vries PJ, Tran BQ, Le HQ, Nguyen NV, Nguyen TV, Heisterkamp SH, Kager PA (2002) Artemisinin or chloroquine for blood stage Plasmodium vivax malaria in Vietnam. Trop Med Int Health 7:858–864

42.

Qari SH, Shi YP, Pieniazek NJ, Collins WE, Lal AA (1996) Phylogenetic relationship among the malaria parasites based on small subunit rRNA Gene sequences: monophyletic nature of the human malaria parasite, Plasmodium falciparum. Mol Phylo Evol 6(1):157–165

43.

Leclerc MC, Hugot JP, Durand P, Renaud F (2004) Evolutionary relationships between 15 Plasmodium species from new and old world primates (including humans): an 18S rDNA cladistic analysis. Parasitology 129(6):677–684

44.

Martinsen ES, Perkins SL, Schall JJ (2008) A three-genome phylogeny of malaria parasites (Plasmodium and closely related genera): Evolution of life-history traits and host switches. Mol Phylogenet Evol 47(1):261–273

45.

Rich SM, Leendertz FH, Guang Xu G, LeBreton M, et al. (2009) The origin of malignant malaria. http://www.pnas.org/content/early/2009/07/31/0907740106 Gysin J (1998) Animal models: Primates. In: Malaria: Parasite Biology, Pathogenesis, and Protection. Sherman IW (Ed.), American Society of Microbiology, Washington, DC Conway DJ, Fanello C, Lloyd JM, Al-Joubori BM, Baloch AH, et al. (2000) Plasmodium falciparum malaria is traced by mitochondrial DNA 1. Mol Biochem Parasitol 111(1): 163–171 Joy DA, Feng X, Mu J, Furuya T, Chotivanich K, et al. (2003) Early origin and recent expansion of Plasmodium falciparum. Science 300:318–321 Rich SM, Ayala FJ (2000) Population structure and recent evolution of Plasmodium falciparum. Proc Natl Acad Sci USA 97(13):6994–7001 Hughes AL, Verra F (2002) Extensive polymorphism and ancient origin of Plasmodium falciparum. Trends Parasitol 18:348–351 Coluzzi M (1999) The clay feet of the malaria giant and its African roots: hypotheses and inferences about origin, spread and control of Plasmodium falciparum. Parassitologia 41:277–283 Hughes AL, Verra F (2001) Very large long-term effective population size in the virulent human malaria parasite Plasmodium falciparum. Proc R Soc Lond B Biol Sci 268:1855–1856 Mendis K, Sina BJ, Marchesini P, Carter R (2001) The neglected burden of Plasmodium vivax malaria. Am J Trop Med Hyg 64:97–106 Carlton JM (2001) Profiling the malaria genome: a gene survey of three species of malaria parasite with comparison to other apicomplexan species. Mol Biochem Parasitol 118:201–210 Feng X, Carlton JM, Joy DA, Mu J, et al. (2003) Single-nucleotide polymorphisms and genome diversity in Plasmodium vivax. Proc Natl Acad Sci USA 100:8502–8507

46. 47.

48. 49. 50. 51.

52.

53. 54.

55.


317

56.

Escalante AA, Cornejo OE, Freeland DE, Poe AC, Durrego E, Collins WE, Lal AA (2005) A monkey’s tale: the origin of Plasmodium vivax as a human malaria parasite. Proc Natl Acad Sci USA 102:1980–1985

57.

Cornejo OE, Escalante AA (2006) The origin and age of Plasmodium vivax. Trends Parasitol 22(12):558–563

58.

Ayala FJ, Escalante AA, Rich SM (1999) Evolution of Plasmodium and the recent origin of the world populations of Plasmodium falciparum. Parassitologia 41:55–68

59.

Jongwutiwes S, et al. (2005) Mitochondrial genome sequences support ancient population expansion in Plasmodium vivax. Mol Biol Evol 22:1733–1739

60.

Carlton JM, et al. (1999) Karyotype and synteny among the chromosomes of all four species of human malaria parasite. Mol Bio chem Parasitol 118:201–210

61.

Carter R, Mendis KN (2002) Evolutionary and historic aspects of the burden of malaria. Clin Microbiol Rev 564–594

62.

Gardner MJ, Shallom SJ, Carton JM, et al. (2002) Genome sequences of the human malaria parasite Plasmodium falciparum. Nature 19:531–534

63.

Hey J (1999) Parasite populations: The puzzle of Plasmodium. Curr Biol 9: R5652–R5657

64.

Severini C, et al. (2004) Risk of Plasmodium vivax malaria reintroduction in Uzbekistan: genetic characterization of parasites and status of potential malaria vectors in the Surkhandarya region. Trans R Soc Trop Med Hyg 98:585–592

65.

Centers for Disease Control and Prevention (2004) Multifocal autochthonous transmission of malaria – Florida. MMWR Morb Mortal Wkly Rep 53:412–414

66.

Bloland PB (2001) Drug resistance in malaria. WHO/CDS/CSR/DRS/2001.4

67.

Müller P, Warr E, Stevenson BJ, Pignatelli PM, Morgan JC, Steven A, Yawson AE, Mitchell SN, Ranson H, Hemingway J, Paine MJ, Donnelly MJ (2008) Field-caught permethrinresistant Anopheles gambiae overexpress CYP6P3, a P450 that metabolises pyrethroids 1. PLoS Genet 4(11):e1000286

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20 Evolutionary trends in soil-inhabiting alaimid nematodes Mahlaqa Choudhary* and M. Shamim Jairajpuri** Section of Nematology, Department of Zoology, Aligarh Muslim University, Aligarh - 202002, UP, India *[email protected], **[email protected]

Abstract: In the present article, morphological characteristics with reference to evolutionary patterns of the nematodes inhabiting soil and belonging to the Order Alaimida on the basis of their sensory structures – amphids have been discussed. The alaims are simple nematodes, having a thin slender body and unarmed stoma, more or less gradually widening esophagus single or paired female gonads, single testis, simple and short spicules, few ventromedian supplements and no caudal glands or spinneret. Depending on the type of amphids two different routes of evolution i.e ‘Alaimus group’ and ‘Amphidelus group’ have been traced. A third line that is, ‘Cristamphidelus group’ has diverged mainly on the basis of numerous prominent longitudinal ridges. Possible evolutionary pattern of thirteen genera of alaims has also been discussed. ‘Alaimus group’ having amphidial apertures which are small, pore-like or indistinct and posteriorly placed, diverges into the genus Alaimus and Cosalaimus respectively. In ‘Amphidelus group’ the amphidial apertures are very large, transverse slit – like or oval or even longitudinally oval, placed generally close to the head. Ten different genera coming under this group i.e Amphidelus, Caviputa, Etamphidelus, Laxamphidelus, Megamphidelus, Metamphidelus, Paramphidelus, Postamphidelus, Scleralaimus, Scleramphidelus, have been discussed. These seem to have evolved from one single Amphidelus-like ancestor. Scleramphidelus have large amphids but instead of having a cup-shaped appearance transform to a stirrup-shaped form and also the margins are sclerotized. Probably, later three lines arose such as Paramphidelus slit-like, crescentshaped amphids, Etamphidelus, oval amphids with margins sclerotized and Metamphidelus having small, irregularly oval ones also with the same feature of the amphid margins. The genus Postamphidelus, as the name means posteriorly directed amphids, derive the character of these sensory structures from Paramphidelus. On the other hand, Etamphidelus gave rise to Megamphidelus with very large amphids and Caviputa where only the males have large, oval, kidney-shaped amphids. Lastly Caviputa gave rise to those alaims that have amphids which are oval and bilobed-Scleralaimus and Laxamphidelus with amphids that are kidney-shaped. ‘Cristamphidelus group’ has become a little more advanced in comparison to other alaims in the sense that it has evolved dorylaim characters such as presence of long slender spicules and also a small gubernaculum. Keywords: Alaimida, Nematodes, Evolutionary trends, Amphids and Soil V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Introduction The soil-inhabiting nematodes are, by and large rather, simple and primitive in their body structure and functions. They were discovered only in the late eighteenth century. The nematodes occur in very large numbers and are estimated to consist of about 5,00,000 species of which 30,000 have been described so far. It is said that the nematodes comprise of more than 90% of all metazoan of the world. They have successfully adapted to live, nearly in every ecological niche from marine to freshwater, polar regions to the tropics, as well as the highest to the lowest of elevations. These ubiquitous and in many respects bizarre animals live in marine, freshwaters and terrestrial environments, where they outnumber all other animals, both as individuals and in species count, can also be parasitic, both on plants and animals. The nematodes are placed at a very low level of hierarchial classification of invertebrates, somewhere close to Rotifera, Gastrotricha, Kinorhyncha, Nematomorpha, etc. They are good biological models for various kinds of studies as in genetics, ageing studies, molecular biology, mainly because of their features such as small size, transparent bodies, constant number of cells, shorter life cycles, etc. Their structural, biological and habitat diversities have made them extremely important for a broad spectrum of studies. Their numbers like that of Arthropoda is very large but unlike the later their bodies are not protected by a chitinous exoskeleton and hence cannot conserve water specially when on land. As soon as nematodes encounter atmospheric air, their bodies undergo dehydration. It has to overcome this condition by undergoing cryptobiosis, if it cannot, it may eventually die. This way, these organisms counteract their inability of having a chitinous coat, by hiding themselves. They are always enveloped in a thin film of water as long as they are hiding within the soil or may flourish in aquatic habitats. They represent a wide range of biodiversity. Nematodes also represent an important transitional stage of evolution of animals on earth. Being pseudocoelomates, they form an important link between acoelomates and the true coelomates. Being a variable group, new forms are being added to the number and hence no generalizations can be made regarding their morphology which could satisfactorily apply to all species. Because of the fact that the body is soft and due to the lack of solid shell or skeleton, fossilization is not possible, and consequently there are only a few known but poor fossil records. The oldest impression from Eocene of a Mermithida species, Heydonius antiquus1 which showed that the worm that was wriggling from the abdomen of a beetle was a nematode. In the absence of fossils and also that a huge ecological diversity is displayed by nematodes, it is difficult to correctly trace the phylogeny or geohistory of the group. Their evolution and phylogeny may only be reconstructed from the structure of recent living forms, relationships within and without the group

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and perhaps from their ontogeny. No reliable data is available as far as the phase of their phylogeny is considered, that is whether it is progressive or regressive. Their ancestors also seem to have disappeared entirely without much trace. The evolution which has produced diversity in the form and function and also the interrelationships, can, however, be inferred while studying the contemporary species of nematodes. The richness of species and the variety of their form and habitat suggests that they are comparatively of recent origin and are a dynamically developed group of animals. The alaims or alaimid nematodes that belong to the Order Alaimida2 can be easily recognized in a mixed nematode suspension by having a very thin and slender body and a simple appearance that is without cuticular appendages, absence of setae on head or body, non-annulated cuticle (under light microscope), narrow lip region, pore or slit-like amphids, small unarmed stoma, more or less gradually widening esophagus, single or paired female gonads, single testis, simple and predominantly very short spicules, few ventromedian supplements, no caudal glands or spinneret. The size of the body can range from being small (0.4 mm) to large (4 mm). In this article, stress has been laid on the morphological characteristics of alaim nematodes with reference to their evolution.

Morphological characteristics of alaim nematodes with reference to evolution Due to the lack of fossil remains, there is no concrete proof of when, where and in what possible shape nematodes in general appeared for the first time. Their ancestors, the primitive nematodes, the kind of evolutionary line followed by them, factors that influenced them to follow various lines of evolution. Their unparallel adaptations and advance stage of speciation and also that most ancient nematodes that survive even today are the ones which are supposedly the most advanced representatives of the group, are some of the questions that can be answered through indirect means and that too may be done by profound study of recent forms. The above aspects are very important as far as the evolution is concerned. We can try to put the present forms into an evolutionary series or we can connect them by their relationships. Therefore, in order to do this we can in short discuss the morphological characteristics and thereby evaluate the features phylogenetically as ones that are primary, primitive and also secondary features which are indicative of an advance development of nematodes (Fig. 1). Alaimid nematodes usually have a cuticle that is smooth, whether the smooth cuticle is more primitive than the annulated is difficult to decide. Lip region is somewhat rounded continuous with the body contour, sometimes offset, truncated and conoid in shape. Most of the species have six lips of equal size having a papilla

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on inner and outer margins. Stoma is simple unarmed and triradiate. Cephalic setae are absent, generally these are found in aquatic forms, whether absence of setae is primitive or not is still to be understood (dorylaims are regarded to be more advanced as they are without setae). Amphids and their apertures play an important role in the taxonomy of alaims. They are a pair present, one on each side of the proximal end of body. They may be pore-like, a little posteriorly placed from the anterior end, amphidial ducts between fovea and sensillar pouches is rather short, it is distinctive of Alaimus group. They may also be very prominent, their apertures may be large to very large, transverse, slit-like or oval, close to the head or a little below, amphidial duct between fovea and sensilla is very long. These amphids are not superficial organs, but lie below the cuticle and are said to be modified forms of missing labial papillae.3 If we say that it is a primitive character (originating from papillae) then in case of soil-inhabiting species which are regarded to be more advanced, this character persists and the different genera of alaims are differentiated largely on the basis of this feature. Consequently, it is regarded to be an advanced feature. Pharynx is clearly marked as an anterior part that is narrow, tubular while the posterior region is the swollen part often referred to as a glandularium, enlargement can be about one-fourth to one-sixth of esophageal length. The glandularium also contains three or five uninucleate glands, the glands are not very distinct but their nuclei are very clear, the orifice of the glands open into the lumen of pharynx. The five gland composition may possibly have evolved from the enlargement of dorsal nucleus and to counterbalance this, the subventral nuclei became double. Excretory system is simple and excretory pore occupies a variable position in the oesophageal region. It is also important taxonomically. Alaimids have either a single female reproductive system (monodelphic) or a set of double gonads (didelphic or amphidelphic). If monodelphic, then the reproductive branch can be anteriorly (prodelphic) or posteriorly (opisthodelphic) directed. In monoprodelphic condition vulva is situated post-equatorial. While in mono-opisthodelphic condition, the vulva is pre-equatorial. Amphidelphic condition has both the functional branches. Female reproductive branch is represented by an ovary, oviduct, sometimes spermatheca, uterus and vagina which in turn opens to the outside by vulva. According to Andrássy,3 single branched, unpaired gonad is a primitive character while the paired gonad condition is said to have developed later due to the gradual shifting of the vulva. Male reproductive system usually consists of a testis, vas deferens, ejaculatory duct, cloaca, additional sensory structures, spicules, guiding apparatus and ventromedian supplements. In alaim nematodes, male reproductive system is represented by the presence of a single testis (monorchic). It may also be outstretched and its tip is directed toward the anterior end of body. Spicules in this group are paired, usually equal in size, cuticularized and


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ventrally arcuate. They are guided by a set of protractor and retractor muscles. A variety of types of spicules are seen. Spicules can be straight, robust, cylindroid and provided with rounded ends. Ventromedian supplements vary in number from species to species and it is a good taxonomic character. Gubernaculum, which supports and guides the spicules, is absent in the alaims except in the genus Cristamphidelus where it is present as a small piece (Fig. 2). Length of tail is usually dependent on anal body diameter. Tail is an important feature in the group of alaims. Various sizes and shapes such as elongated, regularly tapering or flagelliform, mucronated, conoid, lanceolate, etc. are found and this character helps in the classification of these nematodes. Sometimes the two sexes may have the same type of tail but there can be differences also.

Figure 1. Alaimus teres (A) Entire female, (B) Entire male, (C) Anterior region (D) Basal region of esophagus, (E) Female gonad, (F) Female tail, (G) Male posterior region

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Scleralaimus (amphids large transversely oval, bilobed)

Megamphidelus (amphids large, longitudinal, oval)

Laxamphidelus (amphid apertures kidney shaped)

Caviputa (males with large, oval, kidney shaped amphids)

Postamphidelus (slit-like amphids, placed far from anterior end) Paramphidelus (slit-like, crescent-shaped amphids)

Cosalaimus (Indistinct amphids)

Alaimus (pore like amphids)

Etamphidelus Metamphidelus (Oval amphids (amphids irregularly with sclerotized margins) oval, small with sclerotized margins)

Scleramphidelus (large, stirrup-shaped amphids, opening slit-like to oval with sclerotized margins)

Amphidelus Cristamphidelus (large amphids, (transverse amphidial aperture, gubernaculum absent) gubernaculum present)

Alaims with pore-like amphids

Alaims with large, prominent amphids

Alaim ancestors Figure2. Evolutionary tree of Alaimida


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Evolutionary aspect De Man4 for the first time proposed the genus Alaimus for those nematodes having long and slender bodies, minute pore-like amphid apertures and a dorylaimoid type of esophagus. Micoletzky5 classified alaims in the family Alaimidae as nematodes with simple appearance, without cuticular appendages, cephalic or somatic setae, having non-annulated cuticle under light microscope, simple rounded head, pore or slit-like amphids, very small unarmed stoma, more or less gradually widening esophagus, simple or paired female gonads, mostly few ventromedian supplements without caudal glands and spinneret. Chitwood6 considered the families Tripylidae, Mononchidae and Alaimidae into an assemblage of unrelated groups and of independent evolutionary lines. Clark7 revised the diagnosis of Alaimina and Dorylaimina placing both Suborders under Enoplida. Gerlash8 and Andrássy5 placed the alaims in the superfamily Oxysomatoidea Filipjev9 of the Order Enoplida, basically on the characters such as absence of cephalic setae and caudal glands. Amphids are extremely small, pore-like or conspicuous, one or two ovaries, gubernaculum lacking, pre-anal supplements, papilliform, etc. The later because of characters such as absence of stomatal armature, distinct ducts connecting the fovea and sensillar pouches, cephalic sensillae in two separate circlets, spermatheca found in females (in some only) and a single outstretched testis, Siddiqi2 suggested that alaims should be placed under a separate Order Alaimida within subclass Enoplia. He rejected the view presented by Maggenti10 which was that Enoplia developed primarily along two lines one marine and the other terrestrial, the later line included dorylaims. Therefore, he was of the opinion that nematodes representing different evolutionary lines independently invaded the niche “land” and successfully occupied it. He, therefore placed only one suborder Alaimina under Alaimida (found on land) and Oxystomina that are sea-dwelling were placed separately. Bongers11 recognized Alaimidae but assigned it to suborder Tripylina under Enoplida. Among alaims, again depending on the type of amphids, two different routes of evolution can be traced, the “Alaimus group” that has amphid apertures which are small, pore-like or indistinct very posteriorly placed (about 1/3–1/2 of esophagus length) on amphidial duct short, includes the genus Alaimus de Man.4 Cosalaimus Siddiqi12, which is also in this group has evolved further by acquiring longitudinal ridges, and at the same time, the amphids have become indistinct. The other, “Amphidelus group” has amphid apertures that are large to very large, transverse, slit-like or oval, or longitudinally oval, generally close to head, about (one-fifth labial widths) from anterior end. Amphidial duct between fovea and sensilla are long to very long. Within this group, 10 different genera have been reported, that is Amphidelus, Caviputa, Etamphidelus, Laxamphidelus, Megamphidelus, Metamphidelus, Paramphidelus, Postamphidelus, Scleralaimus,

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Figure 3. Different types of alaimid amphids. (A) Alaimus, (B) Amphidelus, (C) Cristamphidelus, (D) Cosalaimus, (E) Scleramphidelus, (F) Paramphidelus, (G) Etamphidelus, (H) Metamphidelus, (I) Postamphidelus, (J) Megamphidelus, (K) Caviputa, (L) Scleralaimus, (M) Laxamphidelus


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and Scleramphidelus (Fig. 3). Amphids, being a particular characteristic of alaims is always a pair on each side of the proximal end of the body. The shape and structure varies in all the 10 genera. Very close to the Amphidelus group, a third line that is,Cristamphidelus group has diverged mainly on the basis of numerous (30–60) fine longitudinal ridges, slit-like amphids, characteristic S-shaped vagina, long and slender spicules and the presence of a small gubernaculum. In the earlier two groups, represented by Alaimus and Amphidelus, there is an absence of gubernaculum, however, in the Cristamphidelus group, the presence of gubernaculum shows that this may be close to dorylaims . In Amphidelus group, the amphids are large but the gubernaculum is absent. Scleramphidelus is the genus that has large amphids like Amphidelus but a slight diversion has taken place, where amphids have become stirrup-shaped, the opening is slit-like to oval and their margins are sclerotized. From this, three lines very close to one another, that is, Paramphidelus having slit-like, crescent-shaped amphids, Etamphidelus with oval amphids (sclerotized margins) and Metamphidelus with irregularly oval, small amphids and also with sclerotized margins have diverged out. The slit-like crescent-shaped amphids of Paramphidelus later evolved into those that were pushed behind, far from the anterior end, which is a characteristic feature of Postamphidelus. Etamphidelus later on, gave rise to two types that is, Megamphidelus having amphids that are large and longitudinally oval, while Caviputa in which females have no amphids but the males have large oval, kidneyshaped amphids, though according to Siddiqi, 1993)12 Caviputa comes close to Alaimus de Man (1880)4 but differs from it in having prominent outer labial sensillae and didelphic gonads. Two genera i.e., Scleralaimus and Laxamphidelus have amphids that are large transversely oval, bilobed and amphids that are kidneyshaped respectively having possibly evolved from Caviputa. Acknowledgement: One of us, Prof. M. Shamim Jairajpuri is thankful to INSA, New Delhi for providing financial assistance.

References 1.

Taylor AL (1935) A review of the fossil nematodes. Procf Helmintholog Society, Washington 2:47–49

2.

Siddiqi MR (1983) Phylogenetic relationships of the soil nematode orders Dorylaimida, Mononchida, Triplonchida and Alaimida, with a revised classification of the subclass Enoplia. Pak J Nematol 1(2):79–110

3.

Andrássy I (1976) Evolution as a basis of the systematization of nematodes. London

4.

De Man JG (1880) Die einheimischen, frei in der Erde und im süssen Wasser lebenden Nematoden. Vorläufiger Bericht und descriptiv-systematischer Theil. Tijdschrift der Nederlandsche Dierkundige Vereenigin 5:1–104

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5.

Micoletzky H (1922) Die freilebenden Erd-Nematoden mit besonderer Berücksichtigung der Steiermark und der Bukowina, zugleich mit einer Revision sämtlicher nicht mariner, freilebender Nematoden in Form von Genus-Beschreibungen und Bestimmungsschlüsseln. Archiv für Naturgeschichte, Abteilung A 87:1–650

6.

Chitwood BG (1937) A revised classification of the Nematoda. Papers Helminth Moscow 67–79

7.

Clark WC (1961) A revised classification of the Order Enoplida (Nematoda). N Z J Sci 4:123–50

8.

Gerlach SA (1966) Bemerkungen zur Phylogenie der Nematoden, Mit zwei Anhangenm: 1. Klasification der Oxystominidae, 2. Das Mannche von Onchulus longicaudatus Cobb, 1920. Mitteilungen aus der biologischen Bundesanstatt fur land und Forstwirtschaft, Berlin-Dahlem, 118:35–39

9.

Filipjev IN (1918) Nématodes libres marins des environs des Sebastopol. Part I (22 parts). Travaux Lab Zool et Stat Biol Sebastopol Ac Sci Russie 2:1–350

10.

Maggenti AR (1983) Nematode higher classification as influenced by species and family concepts. In: Concepts in Nematode Systematics. Stone AR, Platt HM, Khalil LF (Eds.), Academic Press, London

11.

Bongers T (1988) De Nematoden van Nederland Utrecht

12.

Siddiqi MR (1993) Nematodes of tropical rainforests. 1. Six new genera and eighteen new species of alaims. Afro-Asian J Nematol 3:60–80

21 Evolution of the cerebral cortex in amniotes: Anatomical consideration of neuronal types U.C. Srivastava* and R.C. Maurya** Department of Zoology, University of Allahabad, Allahabad - 211002, India *[email protected], **[email protected]

Abstract: The cerebral cortex is a thin sheet of nervous tissue in the roof of the cerebrum of amniotes. The cerebral cortex of reptiles is divided into four areas viz. medial, dorsomedial, dorsal and lateral cortices having different neuronal components. The areas of the hippocampal complex of birds are subdivided into a dorsal parahippocampal region and a ventral hippocampus. The different neuronal types divide the hippocampus of birds into five fields viz. medial and lateral hippocampus, parahippocampal area, central field of parahippocampal area and crescent field. The cerebral cortex of mammals is a complex structure. The neocortex of mammals shows the typical six layered structure in the frontal, parietal, temporal and occipital lobes having typical pyramidal and nonpyramidal neurons which show phenotypic variations. Neuronal types in cerebral cortex of reptiles, birds and mammals have been compared to determine homologous structures. The possible homologous structures have been mentioned in final comments. Keywords: Cerebral cortex, Hippocampus, Amniotes, Pyramidal, Nonpyramidal neurons

Introduction Cerebral cortex is a structure that constitutes the outermost layer of the cerebrum. The term pallium is used for cerebral cortex coating the surface of the cerebrum (cerebral hemisphere). In the anatomical nomenclature, the cerebral cortex is divided into neocortex (isocortex) and allocortex. The neocortex constitutes 95.6% of the cerebral cortex and is six layered structure. The allocortex is divided into paleocortex and archicortex. The latter is synonym of hippocampus1. The periallocortex or the mesocortex are the terms used to differentiate the transitional zone between the neocortex and allocortex. V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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From the beginning of 19th century, different investigators have differentiated the hippocampus into different regions and used varied terminology/nomenclatures. Cajal2 divided the hippocampus into region superior and region inferior while Rose3 divided the hippocampus into five regions naming H1–H5. Lorente de Nó4 identified four regions using the term CA1–CA4. In fact, he was the first to use the term cornu ammonis for the hippocampus. Of late, Federative committee on anatomical terminology (1998) gave the newest anatomical nomenclature and divided the hippocampus in four regions naming CA1–CA4 which may be written as I–IV. In addition, the term gyrus dentate has been used for fascia dentate. How the cerebral cortex of mammals should be compared to that of reptiles and aves is one of the oldest and most intensely debated questions in the field of comparative neurobiology. In this article the neuronal classes of the cerebral cortex of reptiles, aves and mammals have been compared to show homologous structures for evolutionary interpretations.

Cerebral cortex of reptiles The entire cerebral hemisphere of reptiles from rostral to caudal region is divisible into pallium (includes cortical layers) and subpallium (include septal complex, dorsal ventricular ridge, striatal complex and amygdaloid complex). The cerebral cortex of reptiles is differentiated into four areas having distinct cellular composition. These are named according to their relative mediolateral position as medial cortex (MCx), dorsomedial cortex (DMCx), dorsal cortex (DCx) and lateral cortex (LCx). The cerebral cortex shows three-layered structure consisting of a cell layer (cl) with highly packed neuronal cell bodies sandwiched between cell-sparse outer (opl) and inner plexiform layer (ipl).5-12 The outermost layer-І has only few neuronal somata and also the dendrites ascending from subjacent layers. There are only few somata in layer-I of different reptilian species viz. lizard8,11 and snakes6 whereas in tegu lizard5 neurons are absent in layer-I. Layer-II of the cerebral cortex of reptiles is characterized by the presence of densely packed neuronal somata in P. hispanica,9 M. carinata,11,13 and snakes.6–7 It also contains dendrites descending from outer layer-І and ascending from inner layer-ІІІ. The sheet of somata in the cell layer-ІІ is interrupted by a discontinuity in the different regions of cerebral hemisphere. The cortex of all the reptiles except crocodilian14-15 is characterized by a distinct discontinuity in layer-II.16 Layer-II of the dorsal cortex is placed closer to the ventricle than that of the medial and lateral cortices. In many species layer-II of the dorsal cortex is overlapped by the adjacent cortical layers forming medial and lateral superpositions, whereas in snakes such as Eryx or in turtles15,17 little overlapping is observed. The significance of this discontinuity and overlapping is not clear but the basic trilaminar pattern is replaced by

Evolution of the cerebral cortex in amniotes: Anatomical consideration of neuronal types

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five-layered annulus of cortex with dendrites being exchanged between the densely packed somata of overlapping layers.11 The somata are loosely packed in layer-III of the cerebral cortex of reptiles9,11-13,18 and snakes.6–7 It also contains dendrites descending from layer-І & ІІ and ascending processes from ependymal layer. Below the inner plexiform layer an ependymal layer is also observed just above the ventricle (V). Ependymal layer has been observed in all the cortical areas except the lateral cortex.

Neuronal types in reptiles By using classical Golgi-impregnation method19 different investigators described the different number and types of the neuronal classes in all the cortical areas in ophidian and lacertilian groups. In the medial cortex, only one type of neuron in snake genera Natrix and Boa,7 five types in the lizard L. pityusensis,20 P. hispanica9 and M. carinata11, seven types in H. flaviviridis10 have been reported. In case of dorsomedial cortex one type of neuron in the lizard A. agama21 and three types in each layer of snake’s dorsomedial cortex22 have been reported. In the dorsal cortex, four types of neurons in the lizard P. algirus23 and five types in M. carinata11 have been reported. In lateral cortex, four types of neurons are present in all the three layers of snakes,24 whereas three types were observed in the M. carinata11 and four types were present in the H. flaviviridis.25 Of late, different neuronal types described in lacertilian species are pyramidal (may be inverted and bipyramidal), multipolar, monotufted, bitufted, stellate, candelabra like monotufted, monotufted monopolar and monotufted bipolar neurons. Further, they have been classified on the basis of the presence or absence of spines. Different investigators have used different methodology to reveal the morphology of the neurons and neuropil in the cerebral cortex of reptiles.6,7,9,11,13,18,21,26–30 These investigations are important. Various neuronal types described by different investigators in medial, dorsomedial, dorsal and lateral cortex of reptiles have been given in Tables 1 to 4.

Cerebral cortex of birds The boundaries of the avian hippocampus have not been well established39-41, which is very important for understanding the functions of the avian hippocampus. The hippocampal complex (dorsomedial forebrain) of birds, separated from the rest of the cerebral hemisphere by lateral ventricle, is a narrow curved strip of tissue present on the dorsomedial surface of telencephalic hemisphere. Anatomically, the hippocampal complex is subdivided into two main structures, a dorsal parahippocampal area and a ventral hippocampus. The hippocampus is widest dorsally at the junction with the parahippocampal area, and it tapers ventrally

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Table 1 Neuronal types of the medial cortex of various species reported by different investigators S. No. Author (year) 1. Ramón, 189631; Ramón y Cajal, 190432 2. Minelli, 196633 3. 4. 5. 6. 7. 8.

Northcutt, 196734 Regidor et al., 197435 Ulinski, 19777 Wouterlood, 198121 Shen and Kriegstein, 198636 Berbel et al., 198720

Animal Types of neurons C. chamaeleon Pyramidal cells L. viridis, L. muralis I. iguana L. galloti

Very large pyramidal neurons; Vertical bipolar neurons Double pyramidal cells; Small projecting cells m1B, 1C, 1D, 1E

Snakes (Natrix Six types of candelabra cells and Boa) Six types of candelabra cells A. Agama Turtle

Stelllate cells; Pyramidal cells

L. Pityusensis

Sparsely spinous horizontal neurons; Spinous pyramidal neurons; Spinous bitufted neurons; Small, sparsely spinous pyramidal neurons; Spinous multipolar neurons Heavily spiny granular monotufted, medium sized neurons; Heavily spiny bitufted neurons; Spiny bitufted neurons; Sparsely spiny bitufted neurons; Superficial multipolar neurons Monopolar neurons; Monotufted bipolar neurons; Candelabra neurons; Bitufted neurons Aspinous bipolar neurons; Aspinous monotufted monopolar neurons; Aspinous monotufted bipolar neurons; Spinous monotufted monopolar neurons; Spinous monotufted bipolar neurons; Spinous bitufted bipolar neurons; a. Heavily spinous bitufted bipolar neurons; b. Spinous bitufted bipolar neurons; c. Sparsely spinous bitufted bipolar neurons; Spinous multipolar neurons

9.

Luis de la Iglesia P. hispanica and Lopez Garcia, 19979

10.

Srivastava et al., M. carinata 200711

11.

Maurya and Srivastava, 200610; Maurya, 200925

H. flaviviridis


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Table 2 Neuronal types of the dorsomedial cortex of various species reported by different investigators S. No.

Author (year)

Animal

Types of neurons

1.

Wouterlood, 198121

A. Agama

Large pyramidal cells of one types

2.

Srivastava et al., 200711

M. carinata

Pyramidal neurons; Inverted pyramidal neurons; Bipyramidal neurons; Multipolar neurons; Candelabra neurons

3.


H. flaviviridis

Bitufted neurons; Pyramidal neurons; Inverted pyramidal neurons; Bipyramidal neurons; Multipolar neurons; Candelabra like monotufted neurons

Table 3 Neuronal types of the dorsal cortex of various species reported by different investigators S. No. Author (year)

Animal

Types of neurons

1.

Garcia Verdugo et al., 198337 L. galloti

2.

Guirado et al., 198723

P. algirus

Bitufted neurons; Multipolar neurons; Pyramidal neurons

3.


M. carinata

Monotufted neurons; Bitufted neurons; Pyramidal neurons; Bipyramidal neurons; Candelabra neurons

4.

Maurya, 200925

H. flaviviridis Monotufted neurons; Bitufted neurons; Multipolar neurons; Pyramidal neurons

Six types of neuronal soma

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Table 4 Neuronal types of the lateral cortex of various species reported by different investigators S. No.

Author (year)

Animal

Types of neurons

1.

Ulinski and Rainey, 198024

Snakes (C. constrictor, T. sirtalis, N. sipendon)

Bipyramidal neurons; Pyramidal neurons; Polymorphic neurons

2.

Garcia Verdugo et al., 198438 L. galloti

3.


M. carinata

Bipyramidal neurons; Pyramidal neurons; Polymorphic neurons

4.

Maurya, 200925

H. flaviviridis

Monotufted neurons; Bitufted neurons; Pyramidal neurons; Stellate neurons

Four basic types of neuronal soma

with the septum.39,41–45 On the basis of the presence of different neuronal types,39,45 the hippocampal complex have been divided into five different fields viz. medial (HCm) and lateral (HCl) hippocampus, parahippocampal area (APH), central field of parahippocampus (PHc) and crescent field (CF). The adjacent region, corticoid complex (CC) occupies the dorsolateral surface of the telencephalic pallium in zebra finch, Taeniopygia guttata and Strawberry finch, Estrilda amandava.39,45 The corticoid complex is divided into two subfields viz an intermediate corticoid area (CI) and a dorsolateral corticoid area (CDL). The extent of these two areas varies considerably in different species of birds.39,45-46 The intermediate corticoid region and various regions of hippocampal complex progressively disappear in the caudal region at the level of the cerebellum. The avian dorsomedial forebrain or hippocampal complex is involved in memory processes associated with spatial behaviour, food storing.40-41,47-52 The hippocampal volume of the food storing passerine species is larger than non food storing species.49,53-55

Neuronal types in birds On the basis of Golgi study, the neurons of hippocampus of birds have been classified into two main groups. The predominant cell types were projection neurons with spinous dendrites and local circuit neurons having sparsely spinous and aspinous dendrites. In HCm area pyramidal, multipolar, bitufted, monotufted; in PHc and CF region multipolar neurons have been observed. The neurons of


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the corticoid complex are classified into three main cell groups: predominant projection neurons, local circuit neurons and stellate neurons. These neurons are sub classified into pyramidal neurons (located only in CI) and multipolar neurons (located both in CI and CDL). The stellate neurons of the CI have a small round or ovoid cell body that extends 4–6 long thin dendrites. The spines on the dendrites are moderately distributed. The axon of these neurons originates either from cell body or from a dendrite, and ramifies locally. The above mentioned neuronal types and their projections have been completely described in strawberry finch, Estrilda amandava by Srivastava et al.56 The comparative account of different neuronal types of hippocampal complex, corticoid complex and visual wulst in different bird species has been shown in Table 5 to 7. Table 5 Neuronal types of the hippocampal complex of various species reported by different investigators S. No. Author (year)

Animal

Types of neurons

Gallus gallus

Pyramidal neurons/ Bipyramidal; Long axon multipolar neurons; Short axon multipolar neurons; Horizontal neurons

1.

Mollá et al., 1986

2.

Montagnese et al., 199639

Taenopygea guttata

Bitufted neurons (Pyramidal); Multipolar; Local circuit neurons (dense pericellular, basket, radial, netlike)

3.

Tömböl et al., 200041

Gallus domesticus and Columba livia

Pyramidal neurons; Pyramidal-like neurons; Multipolar; Local circuit neurons (small/ medium, large/ medium); Stellate neurons (Columba livia)

4.


Estrilda amandava

Pyramidal neurons; Pyramidal-like neurons; Multipolar; Bitufted neurons; Monotufted neurons; Local circuit neurons

57

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Table 6 Neuronal types of the corticoid complex of various species reported by different investigators S. No. Author (year)

Animal

1.

Montagnese et al., Taenopygea guttata 199639

2.


Estrilda amandava

Types of neurons Bitufted neurons (Pyramidal); Multipolar; Local circuit neurons (radial, horizontal, bipolar/multipolar); Stellate neurons Pyramidal neurons; Multipolar; Horizontal neurons; Local circuit neurons (small, medium sized multipolar); Stellate neurons

Table 7 Neuronal types of the visual wulst of various species reported by different investigators S. No. Author (year)

Animal

Types of neurons

1.

Watanabe et al., 198358.

Coturnix coturnix japonica

Type I; Type II; Type III; Type IV

2.

Tömböl and Maglóczky, 199059.

Gallus domesticus

Type 1d; Type 1b; Type 1a, Type 1c; Local circuit neurons (small, medium, large sized, GABA positive neurons); Stellate

3.

Montagnese et al., 199639.

Taenopygea guttata Multipolar; Local circuit neurons (small and medium sized with few spines); Stellate

4.

Chand P, 200960.

Estrilda amandava

Pyramidal neurons (Moderately spinous, Sparsely spinous); Multipolar (Highly spinous, Moderately spinous, Sparsely spinous) Local circuit neurons (small and medium sized), Aspinous; Stellate, Sparsely spinous


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Cerebral cortex of mammals The cerebral cortex of mammals: a sheet of nervous tissue in the telencephalic roof is commonly referred to as gray matter due to the predominance of cells which appears grayish brown. These neurons are connected to other neurons within the brain by axons located beneath the cortex and within the cortex. The neocortex of mammals shows the typical six layered structure in the frontal, parietal, temporal and occipital lobes, corresponding to the names of skull plates that protect them. The characteristics of six different layers of different fields are different. Among eutherians, Golgi study of the specific sixth layer of cerebral cortex in brains of insectivore, chiroptera, rodentia, lagomorpha, artiodactyla, carnivore and primate revealed differences in cortical thickness, soma types, soma size, dendritic extents and orientation of neurons.61–62 Neuronal morphology and distribution of calcium binding proteins on cortical neurons have been found to be similar in cetacea and artiodactyla but differ considerably from that in primates, carnivores and rodents. This shows several species and order specific patterns which can be used to assess taxonomic affinities among species.63 Pyramidal cells in prefrontal cortex of new world monkey are relatively simple in structure while inferotemporal cells are highly branched and spinous64, although it is generally agreed that the cortical areas have the same complements of neuronal cell types.65 In some cases there are distinct types of neurons which are only found in particular cortical areas or species, such as Meynert and Betz giant pyramidal neurons, spindle neurons and double bouquet cells.66-68 The enlargement and differentiation of the giant pyramidal Betz and Meynert subtype cells can be viewed as a correlate of specialization of functionally distinct regions of the neocortex in the primates.69 In addition, the pyramidal cells sampled from different areas of different primate species, show differences in dendritic arbor size and spine density.64,67,70–74 In addition to this, the pyramidal neurons exhibit a differential distribution among cortical layers and regions, and some of them are differentially represented among species.75 Dendritic spines, a characteristic feature of typical mammalian pyramidal neurons represent important structural specialization of eutherian isocortical neurons and provide most of the postsynaptic sites of axon terminating upon pyramidal neurons.76–77 Differences in pyramidal neuron’s spine density could reflect important functional differences in the isocortex78 and in spine density of pyramidal neurons of visual areas in marmoset monkey.70 These phenotypic differences in pyramidal neurons may reflect some evolutionary trends.

Neuronal types in mammalian cerebral cortex In general, two main types of neurons have been described in mammalian cerebral cortex: pyramidal and nonpyramidal types. The specialization in these neurons

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shows homology and differences not only among the different mammalian species but also in sauropsids (reptiles and birds). The different types of neurons described by different investigators in mammalian species in the cerebral cortex are given in Table 8.

Table 8 Neuronal types of the mammalian cerebral cortex of various species reported by different investigators S.N. Author (year)

Animal/region

Types of neurons

1.

Economo, 192779 Human cortex

2.

Mitra, 195580

Visual, somatosensory and motor cortex of cat

Fusiform neurons

3.

Adrianov and Mering, 195981

Layer II of Broadmann’s area

Small, medium and large pyramidal neurons

4.

Tunturi, 197182

Ectosylvian auditory cortex of Dog

Small, medium and large pyramidal; star; special; double bouquet and fusiform cells

5.

Gilbert and Kelly, Visual cortex of Cat 197583

6.

Lund et al., 1977, Visual cortex of Macaque Pyramidal and stellate cells Monkey 197984-85

7.

Garey and Saini, 198186

Monkey

Multipolar, triangular, bipolar, capsular and neurons with fine axon-like dendrites

8.

Garey et al., 198587

Visual cortex of bottle nose Dolphin

Pyramidal, multipolar/stellate and spindle shaped cells

9.

Ferrer et al., 198661–62

Rat, mouse, hamster, vole, rabbit, dwarf bat, hedgehog, cat, dog, cow, sheep and man; sixth layer of cerebral cortex

Pyramidal neurons (large, medium, small or flattened and atypical or triangular); Multipolar pyramidal; Inverted pyramidal; Fusiform neurons; Horizontal neurons; Fan-shaped neurons; Martinotti cells; Multipolar neurons; Bitufted neurons

Small, medium and large pyramidal neurons

Pyramidal and stellate cells

Cont’d on next page...


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...Cont’d from previous page

S.N. Author (year)

Animal/region

Types of neurons

10.

Ferrer, 1986

Mole (Talpa europaea)

Presumptive projection neurons and Local circuit neurons (bipolar, multipolar, double bouquet cells, spiny stellate and star pyramidal cells)

11.

Fitzpatick and Henson, 199489

Auditory cortex, mustached bat

Spiny cells (pyramidal, inverted pyramidal and extraverted pyramidal cells, spiny stellate, multiform cells). Non-spiny cells (multipolar, bitufted and bipolar cells)

12.

Tyler et al., 199890

Dunnart, quakka (both marsupials) and Macaca

13.

Hassiotis and Ashwell, 200378

Australian echidna

14.

Hof and Sherwood, 200591

50 species of 12 mammalian orders –

Pyramidal neurons, spiny stellate neurons, wide arbor basket neurons, medium arbor basket neurons, robust columnar axon cells, double bouquet cells, chandelier neurons, neurons with simple beaded axon arbors, layer 4 type beta 3 neurons. Pyramidal, spinous bipolar, aspinous bipolar, spinous bitufted, aspinous bitufted, spinous multipolar, aspinous multipolar and neurogliaform. Pyramidal neurons, multipolar neurons

88

Primary motor cortex of primates and primary visual cortex of primates Cingulated cortex and insula of hominids

Meynert cells, Betz cells

Spindle cells with vertical, fusiform morphology

Comparison of the neuronal types of the cerebral cortex in reptiles, birds and mammals The medial cortex of reptiles and the hippocampal fascia dentata of mammals have been considered homologous centers on the grounds of their common cytoand chemo-architectonical patterns, their position in cortical circuitry, and their

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late ontogenesis.9,10, 92–93 The neurons of the cell layer-II of the medial cerebral cortex of reptilian species show resemblance with the granule cells present in the hippocampal formation of different mammalian species such as rat,4,94–97 rabbit,98,99 monkey,100 primates101 and human.102 The dentate granule cell layer of mammals is occupied by homogeneous cell population which is very similar in different reptiles,2,4,98 where most morphological differences are of the soma position in the granule layer. Basal dendrites are transitory in immature dentate granule cells of rodents,94,97 frequent in ectopic granule cells,96 and are considered as aberrant characteristic in granule cells under experimental conditions.103–104 Moreover, basal dendrites are rare in mature granule cells of rabbits,2,98 but they are regularly seen in granule cells of other mammalian species105 including primates and humans.2,98,101–102,106 The morphology of dentate granule cells with basal dendrites is very much similar to different types of spiny bitufted bipolar neurons in the cell layer-II of medial cortex of the reptiles.9,10,12 The neurons of the hippocampal complex of the birds share some common features with the neurons present in the homologous structures of the reptilian and mammalian telencephalon. In the hippocampus of birds such as chick and homing pigeon, the dominant projection neurons are the multipolar neurons39, 41,45 unlike the situation in the mammalian hippocampus, where the only projection neurons are pyramidal. In the mammalian hippocampus, the pyramidal neurons are located only in the pyramidal layer while in the hippocampus of the birds the dominant multipolar projection neurons were found in all layers, but the pyramidal and pyramidal-like neurons were located only in the pyramidal layer-II.39,41,45 The spinous and highly branched monotufted neurons present in the lateral hippocampus of birds39,45 and extending their projection towards the pia seems to be similar with those of the candelabra cells,107 granular neurons9 or spiny monotufted neurons10 present in the cell layer-II of the medial cortex in different lizard species. The bitufted neurons present in the lateral and medial hippocampus of the bird39,41–42, 45 also show resemblance with those found in the bitufted and bipyramidal neurons of medial and dorsomedial cortex of the lizards.9–10,21,26 According to Casini et al.108 the hippocampus would correspond to a part of Ammon’s horn, APH to the subiculum, and hyperpallium densocellulare (HD) to the entorhinal cortex. Using Golgi method, Montagnese et al.39 suggested that the medial arm of the V-shaped layer corresponds to Ammon’s horn, the central field of the parahippocampus to the subiculum, and the intermediate corticoid to the entorhinal cortex. They proposed an absence of a dentate gyrus and mossy fibers because of the absence of granule cells in the hippocampus, as did Tömböl et al.41 for the chicken and pigeon. In zinc staining, the hippocampus is stained entirely homogeneously in the chicken and zebra finch,39,109–110 although the mossy fiber bundle from the dentate gyrus to CA3 is very apparent in mammals.


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Similarities between reptilian medial and dorsomedial cortex with avian hippocampus and parahippocampus include the position medial and dorsal to the ventricle, a more or less pronounced three layered organization,26,111–112 neuronal types,9–12, 21, 26, 34 and efferent and afferent projections.26,108,113–114 The dorsomedial cortex of reptiles shows resemblance with the parahippocampal area of birds due to its position dorsal to the ventricle and medial hippocampus, and the presence of multipolar neurons in the parahippocampal area of birds with medium/large cell body and four to six spinous dendritic branches.39,45 The pattern of extra cortical afferences and efferences of the dorsomedial cortex neurons,112,115 and the intra cortical scheme of connections of the lizard cerebral cortex have a clear resemblance to that of the mammalian hippocampus and the entorhinal olfactory cortex.116 The dendritic tree pattern of pyramidal and bipyramidal neurons with their dendritic spines of dorsomedial cerebral cortex of reptiles27 are comparable with the corresponding elements on pyramidal neurons of the CA3 (Cornu ammonis 3) area of the mammalian hippocampus because it emits a prominent commissural-contralateral projection117 and also because it is the main recipient of the zinc-positive ‘lizard mossy fibers’ coming from the medial cortex.27,11 8 Anatomically, reptilian dorsal cortex resembles with the isocortex of mammals in many respect. The three neurons of reptilian dorsal cortex namely bitufted neurons, multipolar neurons and pyramidal neurons have formed the basis for comparing the medial aspect of dorsal cortex being homologous to the mammalian hippocampal formation,119–120 whereas the lateral aspect of the dorsal cortex has been compared to the mammalian isocortex, or at least to part of it.107, 121 Anatomically the dorsal cortex of reptiles differ from the isocortex in being three layered instead of six layered and in lacking columnar organization.122 The dorsal cortex is a structure unique to reptiles, and its relationship to structures in mammalian brain is of great theoretical interest.122–126 The pyramidal neurons of the hyperpallium apicale (HA) of birds39,41,45,58–60, 127 are comparable with the layer V-VI of the mammalian neocortex.128–131 Sparsely spinous pyramidal neurons of the HD are slightly similar with the pyramidal neurons found in the layer-III of the echidna somatosensory cortex and layer-V of bottlenose dolphin visual cortex.78,87 The multipolar neurons of the laminae: interstitial nucleus of the HA, hyperpallium intercalatum and HD of wulst of the birds39,45,58–60,127 show similarities with the sparsely spinous multipolar cells of the layer II-III of the mammalian visual cortex132 and also with the spinous multipolar neurons of the layer III and IV of echidna somatosensory cortex,78 spiny cells without apical dendrites of the rat Sm1 cortex133 and type 7 cells in primate somatosensory cortex.134 The local circuit neurons observed in the HA, HI and HD of birds39,41,59,127 are comparable with the chandelier cells of the somatosensory134–135 and visual cortex of the different mammalian groups.85,136–145 The sparsely spinous

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stellate neurons of the wulst of birds, strawberry finch,60 zebra finch,39 Japanese quail58 show resemblance with the spiny stellate neurons of the layer III, IV and VI of the visual cortex of bottlenose dolphin87 and other mammals.78,146–147 The stellate cells are also referred to as local plexus neurons because of the limited spread of axonal arborization.148 The wulst of birds is comparable to dorsal isocortex comprising occipital, parietal and sensorimotor parts of the frontal cortex.122,125–126,149–150 The visual and somatosensory areas of the wulst resemble the primary visual and somatosensory regions of the occipital and parietal cortex in mammals.150–151 The wulst lamination, however, is inverted as compared to the mammalian isocortex.152 It is inferred that the superficial wulst layer is comparable to deep isocortical layers (V-VI) in that it projects to both striatum and brainstem. The middle layers of wulst are comparable to the middle isocortical layer (IV) in that they receive primary sensory thalamic inputs. The deepest layer of the wulst is comparable to the superficialmost isocortical layers (II-III) in that it projects to the striatum and other telencephalic targets but not to the brainstem.130–131 Wulst of birds includes a somatomotor area,153–154 which could neither be found in lizards112,155–156 nor in turtles157 and shows somatosensory thalamic projections to the cortex. Like the avian wulst,128 the dorsal cortex (DC) of turtles displays a visual area that includes the pallial thickening, which receives a projection from the dorsal lateral geniculate nucleus of the thalamus.157 In lizards, this projection reaches just the pallial thickening.155,158 The reptilian pallium has a three-layered cortex, consisting of a medial, a dorsomedial part (both similar to the mammalian hippocampal formation), a lateral (olfactory) cortex,107 and finally a dorsal cortex (comparable to the wulst of birds) which receives visual projections from the dorsal lateral geniculate nucleus, as well as some somatosensory input.131 The dorsal cortex of reptiles and its avian equivalent, the wulst, are considered to be homologous to both the striate or primary visual cortex and the somatosensory cortex of mammals, as all these structures receive similar sensory projections.131,149,159–160 More precisely, the visual projections from the retina to the thalamic dorsal lateral geniculate nucleus terminate in the posterior dorsal cortex/wulst of sauropsids and in the striate cortex of mammals, respectively. In addition, the somatosensory spinothalamic and the dorsal column-medial lemniscus pathways project to the anterior dorsal cortex/ wulst of sauropsids and to the somatosensory cortex of mammals.131,161 In the corticoid complex (CI) of birds, dominant pyramidal neurons possess spinous apical dendrites that give rise to oblique side branches and several basal dendrites. The axon collaterals of these neurons show connections with their own dendritic spines and other neuronal dendritic spines. Some of these collaterals run laterally towards the CDL and APH regions.39,41,45 The pyramidal cells of mammals have one dominant large apical dendrite that often branches close to


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the soma, whereas the basal dendrites extend extensively in all directions within the deep layers. The apical dendrites and the axon collaterals extend towards the superficial layers, sometimes even reaching the pial surface.162–164 A major afferent pathway to the mammalian hippocampus comes from the entorhinal cortex.165 The projection from the corticoid complex to the HCC in birds108 might originate from CI pyramidal neurons, which can be compared with the projections of the entorhinal cortex to the various parts of hippocampus in mammals.162–164,166–170 The spinous multipolar neurons of the birds are uniformly distributed in the CI and CDL regions and their axons run towards the ventricular wall, whereas the axon collaterals of CDL region run parallel to the entire width of CDL. The CI receives some axon collaterals from the adjacent regions, such as the CDL and APH and vice versa.39,56,127,171–173 The polymorphic multipolar neurons of the entorhinal cortex of mammals have medium-sized somata with spherical to slightly pyramidal shape. These neurons have multipolar spiny dendritic arborizations that extend for a long distance in all directions, instead of having dominant apical dendrites.162–164,169 The horizontal cells of the CDL region of birds39–56 are comparable with the horizontal neurons of entorhinal cortex of mammals characterized by sparsely spiny apical dendrites extending to the pial surface and slightly spiny basal dendritic plexus that extend horizontally, whereas their axon travels up to the angular bundle.162–163,170,174–175 In the entorhinal cortex of mammals, the somata of stellate cells extend dendritic arbor comprising multiple, roughly equal-sized, primary dendrites that branch widely in the mediolateral entorhinal cortex.175–178 The axons of stellate neurons run towards the angular bundle from a primary dendrite or the base of the soma.175 The stellate neurons of the birds39,127, 171–172,179–180 correspond to the stellate neurons of the entorhinal cortex of mammal, having an equal-sized primary spiny dendritic tree. The lateral cortex of lizards may be considered homologous to the mammalian olfactory cortex as it receives the bulk projection from the principal olfactory bulb.181–182 It shows a highly laminated efferent to ipsilateral medial cortex, a projection that can be considered as a ‘lizard perforant path’ in comparison with that of the mammalian perforant path arising from the entorhinal cortex and ending in the hippocampus and outer molecular layer of the dentate gyrus.182 In addition to the pattern of the extracortical afferences and efferences,115 the intracortical scheme of connections of the lizard cerebral cortex also has a clear resemblance to that of the mammalian hippocampus and the entorhinal olfactory cortex.116 The lateral cortex of the lizard forms an intermediate region by which olfactory information is distributed to the other cortical areas,24 showing a similarity with birds in which the CI and CDL regions act as intermediate platforms from which efferent and afferent connections originate and extend to the adjacent areas. The pigeon hippocampus has reciprocal ipsilateral connections with the hippocampus

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and the CDL.183 Thus it can be concluded that the lateral cortex in reptiles serves as an interface between the olfactory bulb and other cortical areas and that the CI and CDL in birds carry out the same work between adjacent regions of hippocampus, whereas the same function is carried out in the mammals by the entorhinal cortex between neocortex and hippocampal formation.184 Thus, all these regions in distant animal groups work as a gateway through which bidirectional information passes.

Final comments The comparison of the morphology and connections of the neuronal classes of the cerebral cortex of reptilian, aves and mammals shows following important outcomes: 1. The medial cortex of reptiles shows homology with the hippocampus of birds and fascia dentata of mammals. 2. The dorsomedial cortex of reptiles shows homology with the parahippocampal area of birds and CA3 region of mammalian hippocampus. 3. The dorsal cortex of reptiles shows homology with visual wulst of birds and mammalian isocortex. 4. The lateral cortex of reptiles shows homology to corticoid complex of birds and to entorhinal olfactory cortex of mammals. These findings may provide a further basis of investigations of homology to establish a more detailed correspondence between subdivisions of hippocampal region of cerebral cortex in amniotes.

References 1.

Schwerdtfeger WK (1984) Structure and fiber connections of the hippocampus: A comparative study. Adv Anat Embryol Cell Biol 83:1–74

2.

Ramón y Cajal S (1911) Histologie du Systeme Nerveux de l’Homme et des Vertebres. A Maloine, Paris

3.

Rose M (1927) Die sog. Riechrinde beim Menschen und beim Affen. II. Teil des Allocortex bei Teir und Mensch. J Psychol Neurol 34:261–401

4.

Lorente de Nó R (1934) Studies on the structure of the cerbral cortex. II. Continuation of the study of the ammonic system. J Psychol Neurol 46:113–177

5.

Ebbesson SOE, Voneida TJ (1969) The cytoarchitecture of pallium in the tegu lizard. Brain Behav Evol 2:431–466

6.

Ulinski PS (1974) Cytoarchitecture of cerebral cortex in snakes. J Comp Neurol 158:243– 266

7.

Ulinski PS (1977) Intrinsic organization of snake medial cortex: An electron microscopic and Golgi study. J Morphol 152(2):247–279

8.

Smeets WJAJ, Hoogland PV, Lohman AHM (1986) A forebrain atlas of the lizard Gekko gecko. J Comp Neurol 254:1–19


345

9.

Luis de la Iglesia JA, Lopez-Garcia C (1997) A Golgi study of the principal projection neurons of the medial cortex of the lizard Podarcis hispanica. J Comp Neurol 385:528–564

10.

Maurya RC, Srivastava UC (2006) Morphological diversity of the medial cortex neurons in the common Indian wall lizard, Hemidactylus flaviviridis. Natl Acad Sci Lett 29:375–383

11.

Srivastava UC, Maurya RC, Shishodiya U (2007b) Cytoarchitecture and morphology of the different neuronal types of the cerebral cortex of an Indian lizard (Mabouia carinata). Proc Nat Acad Sci India 77:331–347

12.

Srivastava UC, Maurya RC, Chand P (2009) Cyto-architecture and neuronal types of the dorsomedial cerebral cortex of the common Indian wall lizard (Hemidactylus flaviviridis). Arch Ital Biol 147:21–35

13.

Abhinav, Srivastava UC (2006) Neuroanatomy of cerebral cortex of an Indian lizard Mabouia carinata. J Appl Biosci 32(2):157–160

14.

Crosby EC (1917) Forebrain of Alligator mississippiensis. J Comp Neurol 271:325–402

15.

Platel R, Beckers HJA, Nieuwenhuys R (1973) Les champs corticaux chez Testudo hermanii (Reptile chelnien) et chez Caiman crocodylus (Reptile crocodilian). Acta Morph Neerl Scand11:121–150

16.

Beckers HJA, Platel R, Nieuwenhuys R (1971) Les aires corticales de quelques reptiles squamates (Lacerta viridian, Chamaeleo lateralis, Monopeltis guentheri). Acta Morphol Neerl Scand 9:337–364

17.

Northcutt RG (1970) The telencephalon of the western painted turtle (Chrysemys picta belli). Illinois Biological Monographs Urbana: University of Illinois Press

18.

Lopez-Garcia C, Molowny A, Nacher J, Ponsoda X, Sancho-Bielsa F, Alonso-Llosa G (2002) The lizard cerebral cortex as a model to study neuronal regeneration. An Acad Bras Cienc 74(1):85–104

19.

Colonnier M (1964) The tangential organization of the visual cortex. J Comp Neurol 98:327–344

20.

Berbel PJ, Martinez-Guijarro FJ, Lopez-Garcia C (1987) Intrinsic organization of the medial cerebral cortex of the lizard Lacerta pityusensis. A Golgi study. J Morphol 194:276–286

21.

Wouterlood FG (1981) The structure of the mediodorsal cerebral cortex in the lizard Agama agama: A Golgi study. J Comp Neurol 196(3):443–458

22.

Ulinski PS (1979) Intrinsic organization of snake dorsomedial cortex: An electron microscopic and Golgi study. J Morphol 161(2):185–210

23.

Guirado S, Davila JC, De la Calle A, Marin-Giron F (1987) A Golgi study of the dorsal cortex in the lizard Psammodromus algirus. J Morphol 194:265–274

24.

Ulinski PS, Rainey WT (1980) Intrinsic organization of snake lateral cortex. J Morphol 165:85–116

25.

Maurya RC (2009) Identification and classification of different types of neurons of the cerebral Cortex of the common Indian wall lizard, Hemidactylus flaviviridis. D. Phil Thesis, University of Allahabad, India

26.

Ulinski PS (1976) Structure of anterior dorsal ventricular ridge in snakes. J Morphol 148:1–21

27.

Martinez Guijarro FJ, Berbel PJ, Molowny A, Lopez Garcia C (1984) Apical dendritic spines and axonic terminals in the bipyramidal neurons of the dorsomedial cortex of lizards (Lacerta). Anat Embryol (Berl.) 170:321–326

28.

Berbel PJ (1988) Cytology of medial and dorsomedial cortices in lizards: A Golgi study.

346


In: The Forebrain of Reptile. Current Concept on Structure and Function. Schwerdtfeger WK, Smeets W (Eds.), Karger, Basel 29.

Luis de la Iglesia JA, Lopez-Garcia C (1997) A Golgi study of the short axon interneurons of the cell layer and inner plexiform layer of the medial cortex of the lizard Podarcis hispanica. J Comp Neurol 385:565–598

30.

Molnar Z, Metin C, Stoykova A, Tarabykin V, Price DJ, Francis F, Meyer G, Dehay C, Kennedy H (2006) Comparative aspects of cerebral cortical development. Eur J Neurosci 23:921–934

31.

Ramón y Cajal P (1896) Estructura del encefalo del camaleon. Rev Trimest Micrograf 1:46–82

32.

Ramón y Cajal S (1904) Estructura comparada de la corteza cerebral. In: Textura del Sistema Nervioso del Hombre y de los Vertebrados. Vol. II, Imprenta y libreria de Nicolas Moya, Madrid

33.

Minelli G (1966) Architettura delle corteccie di alcuni Rettili (Lacerta muralis, Lacerta viridis, Testudo graeca, Crocodilus acutus). Arch Zool Ital 51:543–573

34.

Northcutt RG (1967) Architectonic studies of the telencephalon of Iguana iguana. J Comp Neurol 130:109–147

35.

Regidor J, Martin-Trujillo JM., Lopez-Garcia C, Martin-Giron F (1974) Estudio citoarquitectonico de la carteza cerebral de reptiles. II. Tipologia dendritica y destribucion neuronal en areas corticales de Lacerta galloti. Trab Inst Cajal Invest Biol 66:1–32

36.

Shen JM, Kriegstein AR (1986) Turtle hippocampal cortex contains distinct cell types, burst-firing neurons, and an epileptogenic subfield. J Neurophysiol 56:1626–1649

37.

Garcia Verdugo JM, Lopez Garcia C, Berbel Navarro P, Soriano Garcia E (1983) Ultrastructure of neuronal cell bodies in dorso-medial cortex of Lacerta galloti. J Hirnforsch 24(3):307–314

38.

Garcia-Verdugo JM, Berbel Navarro P, Regidor Garcia J, Lopez Garcia C (1984) Ultrastructure of neuronal cell bodies in the medial cortex of Lacerta galloti. J Hirnforsch 25:187–196

39.

Montagnese CM, Krebs JR, Meyer G (1996) The dorsomedial and dorsolateral forebrain of the zebra finch, Taeniopygia guttata: A Golgi study. Cell Tissue Res 283:263–282

40.

Casini G, Fontanesi G, Bingman VP, Jones TJ, Garliardo A, Ioale P, Bagnoli P (1997) The neuroethology of cognitive maps: contributions from research on the hippocampus and homing pigeon navigation. Arch Ital Biol 135:73–92

41.

Tömböl T, Davies DC, Németh A, Sebestény T, Alpár A (2000) A comparative Golgi study of chicken (Gallus domesticus) and homing pigeon (Columba livia) hippocampus. Anat Embryol 201:85–101

42.

Tömböl T, Davies DC, Németh A, Alpár A, Sebestény T (2000) A Golgi and a combined Golgi/GABA immunogold study of local circuit neurons in the homing pigeon hippocampus. Anat Embryol 201:181–196

43.

Atoji Y, Yamamoto Y, Suzuki Y (2001) Distribution of NADPH diaphorase-containing neurons in pigeon central nervous system. J Chem Neuroanat 21:1–22

44.

Falougy H, Benuska J (2006) History, anatomical nomenclature, comparative anatomy and functions of the hippocampal formation. Bratisl Lek Listy 107:93–108

45.

Srivastava UC, Chand P, Maurya RC (2007) Cytoarchitectonic organization and morphology of the cells of hippocampal complex in strawberry finch (Estrilda amandava). Cell Mol Biol 53:103–120


347

46.

Atoji Y, Wild JM (2005) Afferent and efferent connections of the dorsolateral corticoid area and a comparison with connections of the temporo-parieto-occipital area in the pigeon (Columba livia). J Comp Neurol 485:165–182

47.

Bingman VP, Ioale P, Casini G, Bagnoli P (1990) The avian hippocampus: Evidence for a role in the development of homing pigeon navigational map. Behav Neurosci 104:906–911

48.

Sherry DF, Jacobs LF, Daulin SJC (1992) Spatial memory and adaptive specialization of the hippocampus. Trends Neurosci 15:298–303

49.

Hampton RR, Shettleworth SJ (1996) Hippocampal lesions impair memory for location but not for color in passerine birds. Behav Neurosci 110:831–835

50.

Patel SN, Clayton NS, Krebs JR (1997) Spatial learning induces neurogenesis in the avian brain. Behav Brain Res 89:115–128

51.

Strasser R, Bingman VP (1997) Goal recognition and hippocampal formation in the homing pigeon (Columba livia). Behav Neurosci 111:1245–1256

52.

Volman SF, Grubb TC, Jr Schuett KC (1997) Relative hippocampal volume in relation to food-storing behaviour in four species of woodpeckers. Brain Behav Evol 49:110–120

53.

Krebs JR, Sherry DF, Healy SD, Perry VH, Vaccarino AL (1989) Hippocampal specialization of food-storing birds. Proc Natl Acad Sci USA 86:1388–1392

54.

Sherry DF, Vaccarino AL, Buckenham K, Herz RS (1989) The hippocampal complex of food-storing birds. Brain Behav Evol 34:308–317

55.

Sherry DF, Vaccarino AL (1989) Hippocampus and memory for food caches in black-capped chickadees. Behav Neurosci 103:308–318

56.

Srivastava UC, Chand P, Maurya RC (2009) Neuronal classes in the corticoid complex of the telencephalon of the strawberry finch, Estrilda amandava. Cell Tissue Res 336:393–409

57.

Mollá R, Rodriguez J, Calvet S, Garcia-Verdugo JM (1986) Neuronal types of the cerebral cortex of the adult chicken, Gallus gallus. A Golgi study. J Hirnforsch 27:381–390

58.

Watanabe M, Ito H, Masi H (1983) Cytoarchitecture and visual receptive neurons in the wulst of the Japanese quail (Coturnix coturnix japonica). J Comp Neurol 213:188–198

59.

Tömböl T, Maglόczky Z (1990) Cytoarchitecture of chicken wulst: A Golgi study in cell types and their maturation after hatching. Acta Morphol Hung 38:35–53

60.

Chand P (2009) Neuronal classes in the Central Nervous System of a bird, Estrilda amandava. D. Phil Thesis, University of Allahabad, India

61.

Ferrer I, Fabrigues I, Condom E (1986) A Golgi study of the sixth layer of the cerebral cortex. I. The lissencephalic brain of rodentia, lagomorpha, insectivora and chiroptera. J Anat 145:217–234

62.

Ferrer I, Fabrigues I, Condom E (1986) A Golgi study of the sixth layer of the cerebral cortex. II. The gyrencephalic brain of carnivora, artiodactyla and primates. J Anat 146:87–104

63.

Hof PR, Glezer II, Condé F, Flagg R, Rubin MB, Nimchinsky EA, Vogt Weisenhorn DM (1999) Cellular distribution of calcium-binding proteins paravalbumin, calbindin, and calretinin in the neocortex of mammals: Phylogenetic and developmental patterns. J Chem Neuroanat 16:77–116

64.

Elston GN, Benavides-Piccione R, DeFelipe J (2001) The pyramidal cell in cognition: A comparative study in human and monkey. J Neurosci 21:1–5

65.

Rockel AJ, Hiorns RW, Powell TPS (1980) The basic uniformity in structure of the neocortex. Brain 103:221–224

66.

Nimchinsky EA, Gilissen E, Allman JM, Perl DP, Erwin JM, Hof PR (1999) A neuronal

348


morphologic type unique to humans and great apes. Proc Natl Acad Sci USA 96: 5268–5273 67.

DeFelipe J, Alonso-Nanclares L, Jon I, Arellano JI (2002) Microstructure of the neocortex: Comparative aspects. J Neurocytol 31:299–316

68.

Ballesteros-Yáñez I, Muñoz A, Contreras J, Gonzalez J, Rodriguez-Viega E, Defelipe J (2005) The double bouquet cell in the human cerebral cortex and a comparison with other mammals. J Comp Neurol 486:344–360

69.

Sherwood CC, Lee PWH, Rivara CB, Holloway RL, Gilissen EPE, Simmons, RMT, Hakeem A, Allman JM, Erwin JM, Hof PR (2003) Evolution of specialized pyramidal neurons in primate visual and motor cortex. Brain Behav Evol 61:28–44

70.

Elston GN, Tweedale R, Rosa MGP (1999) Cellular heterogeneity in cerebral cortex, A study of the morphology of pyramidal neurones in visual areas of marmoset monkey. J Comp Neurol 415:33–51

71.

Elston GN (2005) A study of pyramidal cell structure in the cingulated cortex of the macaque monkey with comparative notes on inferotemporal and primary visual cortex. Cereb Cortex 15:64–73

72.

Elston GN, Rosa MGP (1997) The occipitoparietal pathway of the macaque monkey: Comparison of pyramidal cell morphology in layer III of functionally related cortical visual areas. Cereb Cortex 7:432–452

73.

Benavides-Piccione R, Ballesteros-Yáñez I, DeFelipe J, Yuste R (2002) Cortical area and species differences in dendritic spine morphology. J Neurocytol 31:337–346

74.

Elston GN, DeFelipe J (2002) Spine distribution in cortical pyramidal cells: A common organizational principle across species. Prog Brain Res 136:109–133

75.

Hof PR, Glezer II, Nimchinsky EA, Erwin JM (2000) Neurochemical and cellular specializations in mammalian neocortex reflect phylogenetic relationships: Evidence from primates, cetaceans and artiodactyls. Brain Behav Evol 55:300–310

76.

Feldman ML (1984) Morphology of the neocortical pyramidal neuron. In: Cerebral Cortex, Vol. I. Cellular Components of the Cerebral Cortex. Peters A, Jones EG (Eds.), Plenum Press, New York

77.

Nieuwenhuys R (1994) The Neocortex, an overview of its evolutionary development, structural organization and synaptology. Anat Embryol 190:307–337

78.

Hassiotis M, Ashwell KW (2003) Neuronal classes in the isocortex of a monotreme, the Australian echidna (Tachyglossus aculeatus). Brain Behav Evol 61:6–27

79.

Economo Von C (1927) Zellaufbau der Gross hirnrinde des Menschen. Verlag Von Juliur Springer, Berlin

80.

Mitra NL (1955) Quantitative analysis of cell types in mammalian neocortex. J Anat 89:467–483

81.

Adrianov OS, Mering TA (1959) In: Atlas of the Canine Brain. Domino EF (Ed.), Government Publication of Medical Literature, Moscow

82.

Tunturi AR (1971) Classification of neurons in the ectosylvian auditory cortex of the Dog. J Comp Neurol 142:153–166

83.

Gilbert CD, Kelly JP (1975) The projections of cells in different layers of the cat’s visual cortex. J Comp Neurol 163:81–106

84.

Lund JS, Booth RG, Lund RD (1977) Development of neurons in the visual cortex (area 17) of the monkey (Macaca nemestrina): A Golgi study from fetal day 127 to postnatal maturity. J Comp Neurol 164:187–304


349

85.

Lund JS, Henry GH, MacQueen CL, Harvey AR (1979) Anatomical organization of the primary visual cortex (area 17) of the cat: A comparison with area 17 of macaque monkey. J Comp Neurol 184:599–618

86.

Garey LJ, Saini KD (1981) Golgi studies of the normal development of neurons in the lateral geniculate nucleus of the monkey. Exp Brain Res 44:117–128

87.

Garey LJ, Winkelmann E, Brauer K (1985) Golgi and Nissl studies of the visual cortex of the Bottlenose Dolphin. J Comp Neurol 240:305–321

88.

Ferrer I (1986) Golgi study of the isocortex in an insectivore: The common European mole (Talpa europea). Brain Behav Evol 29:105–114

89.

Fitzpatrick DC, Henson OW (1994) Cell types in mustached bat auditory cortex. Brain Behav Evol 43:79–91

90.

Tyler CJ, Dunlop SA, Lund RD, Harman AM, Dann JF, Beazley LD, Lund JS (1998) Anatomical comparison of the macaque and marsupial visual cortex: Common features that may reflect retention of essential cortical elements. J Comp Neurol 400:449-468

91.

Hof PR, Sherwood CC (2005) Morphological neuronal phenotypes in the neocortex reflect phylogenetic relationships among certain mammalian orders. Anat Rec 287A:1153–1163

92.

Molowny A, Lopez-Garcia C (1978) Estudio citoarquitectonico de la corteza cerebral de reptiles. III. Localizacion histoquimica de metales pesados y definicion de subregiones Timm-positive en la corteza de Lacerta galloti, Chalcides sp. y Tarentola mauritanica. Trab Inst Cajal Invest 70:55–74

93.

Lopez-Garcia C, Molowny A, Martinez-Guijarro FJ, Blasco-Ibanez JM, Luis de la Iglesia JA, Bernabeu A, Garcia-Verdugo JM (1992) Lesion and regeneration in the medial cerebral cortex of lizards. Histol histopath 7:725–746

94.

Seress L, Pokorny J (1981) Structure of the granular layer of the rat dentate gyrus: A light microscopic and Golgi study. J Anat 133:181–195

95.

Wiliams RS, Matthysse S (1983) Morphometric analysis of granule cell dendrites in the mouse dentate gyrus. J Comp Neurol 215:154–164

96.

Marti-Subirana A, Soriano E, Garcia-Verdugo JM (1986) Morphological aspects of the ectopic granule-like cellular population in the albino rat hippocampal formation: A Golgi study. J Anat 144:31–47

97.

Lubbers K, Frotscher M (1987) Fine structures and synaptic connections of identified neurons in the rat fascia dentate. Anat Embryol 177:1–14

98.

Ramón y Cajal S (1904b) Focus olfativos cuaternarios o asta de Ammon y fascia dentata. In: Textura del Sistema Nervioso del Hombre y de los Vertebrados. Vol. II, Imprenta y libreria de Nicolas Moya, Madrid

99.

Stensaas LJ (1968) The development of hippocampal and dorsolateral pallial regions of the cerebral hemisphere in fetal rabbits. VI. Ninety millimeter stage, cortical differentiation. J Comp Neurol 132:93–108

100.

Duffy CJ, Rakic P (1983) Differentiation of granule cell dendrites in the dentate gyrus of the rhesus monkey: A quantitative Golgi study. J Comp Neurol 214:224–237

101.

Seress L, Frotscher M (1990) Morphological variability is a characteristic feature of granule cells in the primate fascia dentate: A combined Golgi/electron microscope study. J Comp Neurol 293:253

102.

Al-Hussein S, Al-Ali S (1995) A Golgi study of cell types in the dentate gyrus of the adult human brain. Cell Mol Neurobiol 15:207–220

350


103.

Hartmann D, Frotscher M, Sievers J, Pehlemann FW (1989) Altered development of the dentate gyrus after neonatal destruction of meningeal cells: Analysis of fiber connections and cell morphology. In: Dynamics and Plasticity in Neuronal Systems. Elsner N, Singer W (Eds.), Thieme Verlag, New York

104.

Heimrich B, Frotscher M (1991) Differentiation of dentate granule cells in slice cultures of rat hippocampus: A Golgi/electron microscopic study. Brain Res 538:263–268

105.

Buhl EH, Dann JF (1990) Basal dendrites are a regular feature of hippocampal granule cells in flying fox hippocampus. Neurosci Lett 116:263–268

106.

Seress L, Mrzljak L (1987) Basal dendrites of granule cells are normal features of the fetal and adult dentate gyrus of both monkey and human hippocampal formations. Brain Res 405:169–174

107.

Ulinski PS (1990) The cerebral cortex of reptiles. In: Cerebral Cortex. Vol. 8A. Comparative Structure and Evolution of Cerebral Cortex. Jones EG, Peters A (Eds.), Part I. Plenum Press, New York

108.

Casini G, Bingman VP, Bagnoli P (1986) Connections of the pigeon dorsomedial forebrain studied with WGA-HRP and 3H-Proline. J Comp Neurol 245:454–470

109.

Faber H, Braun K, Zuschratter W, Scheich H (1989) System-specific distribution of zinc in the chick brain. A light and electron-microscopic study using the Timm method. Cell Tissue Res 258:247–257

110.

Montagnese CM, Geneser FA, Krebs JR (1993) Histochemical distribution of zinc in the brain of zebra finch (Taeniopygia guttata). Anat Embryol 188:173–187

111.

Lohman AHM, Mentink GM (1972) Some cortical connections of the tegu lizard (Tupinambia teguixin). Brain Res 45:325–344

112.

Guirado S, De La Calle A, Gutierrez A, Davila JC (1989) Serotonin innervation of the cerebral cortex in lizards. Brain Res 488:213–220

113.

Bruce LL, Butler AB (1984) Telencephalic connections in lizards. I. Projections to cortex. J Comp Neurol 229:585–601

114.

Hoogland PV, Vermeulen-Van Der Zee E (1989) Efferent connections of the dorsal cortex of the lizard Gekko gecko studied with Phaseolus vulgaris-leucoagglutinin. J Comp Neurol 285:289–303

115.

Bruce LL, Butler AB (1984b) Telencephalic connections in lizards. II. Projections to anterior dorsal ventricular ridge. J Comp Neurol 229:602–615

116.

Olucha FE, Martinez-Garcia F, Poch L, Schwerdtfeger WK, Lopez-Garcia C (1988) Projections from the medial cortex in the brain of lizards: Correlation of anterograde and retrograde transport of horseradish peroxidase with Timm staining. J Comp Neurol 276:469–480

117.

Martinez-Guijarro FJ, Desfilis E, Lopez-Garcia C (1990) Organization of the dorsomedial cortex in the lizard Podarcis hispanica. In: The forebrain in non-mammals. New aspects of structure and development. Schwerdtfeger WK, Germroth P (Eds.), Springer-Verlag, Berlin

118.

Lopez-Garcia C, Martinez-Guijarro FJ (1988) Neurons in the medial cortex gives rise to Timm-positive boutons in the cerebral cortex of lizards. Brain Res 463:207–217

119.

Lacey DJ (1978) The organization of the hippocampus of the Fence lizard: A light microscopic study. J Comp Neurol 182:247–264

120.

Guirado S, Real MA, Padial J, Andreu MJ, Davila JC (1998) Cholicystokinin innervation of the cerebral cortex in a reptile, the lizard Psammodromus algirus. Brain Behav Evol 51:100–112


351

121.

Ten Donkelaar HJ (1998) Reptiles. In: The central nervous system of vertebrates. Vol. 2. Nieuwenhuys R, Ten Donkelaar HJ, Nicholson C (Eds.), Springer-Verlag, Berlin

122.

Reiner A (1993) Neurotransmitter organization and connections of turtle cortex: Implications for the evolution of mammalian brain. Development 125:3719–3729

123.

Powers AS (1990) Brain mechanisms of learning in reptiles. In: Neurobiology of comparative cognition. Kesner RP, Olton DS (Eds.), Erlbaum: Hillsdale NJ

124.

Reiner A (1991) A comparison of neurotransmitter-specific and neuropeptide-specific neuronal cell types present in the dorsal cortex of reptiles with those present in the isocortex of mammals. Brain Behav Evol 38:53–91

125.

Butler AB (1994) The evolution of the dorsal thalamus of jawed vertebrates, including mammals: Cladistic analysis and a new hypothesis. Brain Res Rev 19:29–65

126.

Butler AB (1994) The evolution of the dorsal pallium in the telencephalon of amniotes: Cladistic analysis and a new hypothesis. Brain Res Rev 19:66–101

127.

Tömböl T (1995) Golgi structure of telencephalon of the chicken. Abaevo, Budapest

128.

Karten HJ, Hodos W, Nauta WJH, Revzin AM (1973) Neural connections of the ‘visual wulst’ of the avian telencephalon. Experimental studies in the pigeon (Columba livia) and owl (Speotyto cunicularia). J Comp Neurol 150:253–277

129.

Shimizu T, Cox K, Karten HJ (1995) Intratelencephalic projections of the visual Wulst in pigeons (Columba livia). J Comp Neurol 359:551–572

130.

Veenman CL, Wild JM, Reiner A (1995) Organization of the avian ‘corticostriatal’ projection system: A retrograde and anterograde pathway tracing study in pigeons. J Comp Neurol 354:87–126

131.

Medina L, Reiner A (2000) Do birds possess homologues of mammalian primary visual, somatosensory and motor cortices? Trends Neurosci 23:1–12

132.

Peters A, Fairen A (1978) Smooth and sparsely spined stellate cells in the visual cortex of the rat: A study using a combined Golgi-electron microscope technique. J Comp Neurol 181:129–172

133.

Simons DJ, Woolsey TA (1984) Morphology of Golgi-Cox impregnated barrel neurons in rat SmI cortex. J Comp Neurol 230:119–132

134.

Jones EG (1975) Varieties and distribution of non-pyramidal cells in the somatic sensory cortex of the squirrel monkey. J Comp Neurol 160:205–267

135.

Szentágothai J (1975) The ‘module-concept’ in cerebral cortex architecture. Brain Res 95:475–496

136.

Somogyi P (1977) A specific ‘axo-axonal’ interneuron in the visual cortex of the rat. Brain Res 136:345–350

137.

Somogyi P (1979) An interneuron making synapses specifically on the axon initial segment of pyramidal cells in the cerebral cortex of the cat. J Physiol (London) 296:18–19

138.

Fairén A, Valverde F (1979) Specific thalmo-cortical afferents and their presumptive targets in the visual cortex: A golgi study. In: Development and Chemical specificity of Neurons. Vol 51. Cuénod M, Kreutzberg GW, Bloom FE (Eds.), Progress in Brain Research

139.

Fairén A, Valverde F (1980) A specialized type of neuron in the visual cortex of cat: A Golgi and electron microscope study of chandelier cells. J Comp Neurol 194:761–779

140.

Tömböl T (1976) Golgi analysis of the internal layers (V-VI) of the cat visual cortex. Exp Brain Res Suppl 1:292–295

141.

Tömböl T (1978) Some Golgi data on visual cortex of the rhesus monkey. Acta Morphol Acad Sci Hung 26:115–138

352


142.

Tömböl T (1978) Comparative data on the Golgi architecture of interneurons of different cortical areas in cat and rabbit, In: Architectonics of the cerebral cortex. Brazier MAB, Petsche H (Eds.), Raven Press, New Yark

143.

Peters A, Regidor J (1981) A reassessment of the forms of nonpyramidal neurons in area 17 of cat visual cortex. J Comp Neurol 203:685–716

144.

Somogyi P, Freund TF, Cowey A (1982) The axo-axonic interneuron in the cerebral cortex of the rat, cat and monkey. Neurosci 7:2577–2607

145.

Valverde F (1983) A comparative approach to neocortical organization based on the study of the brain of the hedgehog (Erinaceus europaeus). In: Ramón y Cajal’s Contribution to the Neurosciences. Grisolía S, Guerri C, Samson F, Norton S, Reinoso-Suárez F (Eds.), Elsevier, Amsterdam

146.

Fairén A, De Felipe A J, Regidor J (1984) Non-pyramidal neurons. General account. In: Cerebral Cortex, Cellular components of the Cerebral Cortex. Vol. 1. Peters A, Jones EG (Eds.), Planum Press, New York

147.

Lorente de Nó R (1938) Analysis of the activity of the chains of internuncial neurons. J Neurophysiol 1:207–244

148.

Peters A, Saint Marie RL (1984) Smooth and sparsely spinous nonpyramidal cells forming local axonal plexuses. In: Cerebral Cortex. Vol. 1, Peters A, Jones EG (Eds.), Planum Press, New York

149.

Nauta WHJ, Karten HJ (1970) A general profile of the vertebrate brain, with sidelights on the ancestry of the cerebral cortex. In: The Neurosciences, Second Study Program. Heimer L, van Hoesen GW, Trimble M, Zahm SD (Eds.), Schmitt Rockefeller: University Press

150.

Karten HJ (1991) Homology and evolutionary origins of the ‘neocortex’. Brain Behav Evol 38:264–272

151.

Karten HJ, Shimizu T (1989) The origins of neocortex: Connections and lamination as distinct events in evolution. J Cogn Neurosci 1:291–301

152.

Reiner A, Karten HJ (1983) The laminar source of efferent projections from the avian Wulst. Brain Res 275:349–354

153.

Wild JM (1992) Direct and indirect ‘cortico’-rubral and rubro-cerebellar cortical projections in the pigeon. J Comp Neurol 326:623–36

154.

Wild JM, Williams MN (2000) Rostral wulst in passerine birds. I. Origin, course, and terminations of an avian pyramidal tract. J Comp Neurol 416:429–450

155.

Lohman AHM, Van Woerden-Verkley I (1978) Ascending connections to the forebrain in the tegu lizard. J Comp Neurol 182:555–594

156.

Neary TJ, Wilczynski W (1977) Ascending thalamic projections from the obex region in ranid frogs. Brain Res 138:529–533

157.

Hall WC, Ebner FF (1970) Thalamotelencephalic projections in the turtle (Pseudemys scripta). J Comp Neurol 140:101–122

158.

Kenigfest N, Martinez-Marcos A, Belekhova M, Font C, Lanuza, E, Desfilis E, MartinezGarcia F (1997) A lacertilian dorsal retinorecipient thalamus: A re-investigation in the old-world lizard Podarcis hispanica. Brain Behav Evol 50:313–334

159.

Karten HJ (1997) Evolutionary developmental biology meets the brain: The origins of mammalian neocortex. Proc Natl Acad Sci (USA) 94:2800–2804

160.

Shimizu T, Karten HJ (1993) Multiple origins of neocortex: Contributions of the dorsal ventricular ridge. In: Brain and Behavior in Birds. Zeigler HP, Bischof JH (Eds.), MIT Press


353

161.

Wild JM (1997) The avian somatosensory system: The pathway from wing to Wulst in a passerine (Chloris chloris). Brain Res 759:122–34

162.

Hamam BN, Kennedy TE, Alonso A, Amaral DG (2000) Morphological and electrophysiological characteristics of layer V neurons of the rat medial entorhinal cortex. J Comp Neurol 418:457–472

163.

Hamam BN, Amaral DG, Alonso AA (2002) Morphological and electrophysiological characteristics of layer V neurons of the rat lateral entorhinal cortex. J Comp Neurol 451:45–61

164.

Canto CB, Wouterlood FG, Witter MP (2008) What does the anatomical organization of the entorhinal cortex tell us? Neural Plast Article ID 381243, doi:10.1155/2008/381243

165.

Rosene DL, Van Hoesen GW (1987) The hippocampal formation of the primate brain. A review of some comparative aspects of cytoarchitecture and connections. In: Cerebral cortex Further aspects of cortical function, including hippocampus. Vol. 6. Jones EG, Peters A (Eds.), Plenum, London

166.

Alonso A, Klink R (1993) Differential electroresponsiveness of stellate and pyramidal-like cells of medial entorhinal cortex layer II. J Neurophysiol 70:128–143

167.

Klink R, Alonso A (1997) Muscarinic modulation of the oscillatory and repetitive firing properties of entorhinal cortex layer II neurons. J Neurophysiol 77:1813–1828

168.

Gloveli T, Schmitz D, Empson RM, Dugladze T, Heinemann U (1997) Morphological and electrophysiological characterization of layer III cells of the medial entorhinal cortex of the rat. Neuroscience 77:629–648

169.

Gloveli T, Dugladze T, Schmitz D, Heinemann U (2001) Properties of entorhinal cortex deep layer neurons projecting to the rat dentate gyrus. Eur J Neurosci 13:413–420

170.

Canto CB, Ganter P, Moser EI, Moser MB, Witter MP (2006) Neuron diversity in the medial entorhinal cortex of the rat. Soc Neurosci Abst 32:68

171.

Tömböl T, Csillag A, Stewart MG (1988) Cell types of the hyperstriatum ventrale of the domestic chicken (Gallus domesticus): A Golgi study. J Hirnforsch 29:319–334

172.

Tömböl T, Csillag A, Stewart MG (1988) Cell types of the paleostriatal complex of the domestic chicken (Gallus domesticus): A Golgi study. J Hirnforsch 29:493–507

173.

Kröner S, Gottmann K, Hatt H, Güntürkün O (2002) Electrophysiological and morphological properties of cell types in the chick neostriatum caudolaterale. Neuroscience 110:459– 473

174.

Lorente de Nó R (1933) Studies on the structure of the cerebral cortex. J Psychol Neurol 45:381–438

175.

Lingenhohl K, Finch DM (1991) Morphological characterization of rat entorhinal neurons in vivo: Soma-dendritic structure and axonal domains. Exp Brain Res 84:57–74

176.

Schwartz SP, Coleman PD (1981) Neurons of origin of the perforant path. Exp Neurol 74:305–312

177.

Klink R, Alonso A (1993) Ionic mechanisms for the subthreshold oscillations and differential electroresponsiveness of medial entorhinal cortex layer II neurons. J Neurophysiol 70:144–157

178.

Klink R, Alonso A (1997) Morphological characteristics of layer II projection neurons in the rat medial entorhinal cortex. Hippocampus 7:571–583

179.

Erichsen JT, Bingman VP, Krebs JR (1991) The distribution of neuropeptides in the dorsomedial forebrain of the pigeon (Columba livia): A basis for regional subdivisions. J Comp Neurol 314:478–492

354


180.

Krebs JR, Erichsen JT, Bingman VP (1991) The distribution of neurotransmitter-related enzymes in the dorsomedial telencephalon of the pigeon (Columba livia). J Comp Neurol 314:467–477

181.

Hoogland PV, Vermeulen-Van Der Zee E (1995) Efferent connections of the lateral cortex of the lizard Gekko gecko: Evidence for separate origins of medial and lateral pathways from the lateral cortex to the hypothalamus. J Comp Neurol 352:469–480

182.

Martinez-Garcia F, Amiguet M, Olucha F, Lopez-Garcia C (1986) Connections of the lateral cortex in the lizard Podarcis hispanica. Neurosci Lett 63:39–44

183.

Atoji Y, Wild JM, Yamamoto Y, Suzuki Y (2002) Intratelencephalic connections of the hippocampus in pigeons (Columba livia). J Comp Neurol 447:177–199

184.

Kerr KM, Agster KL, Furtak SC, Burwell RD (2007) Functional neuroanatomy of the parahippocampal region: The lateral and medial entorhinal areas. Hippocampus 17: 697–708

22 Medicinal and aromatic plants: A case example of evolving secondary metabolome and biochemical pathway diversity Suman P.S. Khanuja*, Tripta Jhang** and Ajit Kumar Shasany*** *Former Director, CIMAP (CSIR), C41-42, Double Storey, Ramesh Nagar, New Delhi - 110015, India [email protected], [email protected] **Genetic Resources & Biotechnology Division, Central Institute of Medicinal and Aromatic Plants (CSIR), Lucknow - 226015, India [email protected], ***Senior Scientist, Genetic Resources & Biotechnology Division, Central Institute of Medicinal and Aromatic Plants (CSIR), Lucknow 226015 [email protected]

Abstract: The unique capability of plants, in spite of being immobile in strict sense, to defend and respond precisely to environmental stresses whether biotic or abiotic is relatable to their ability to synthesize an array of phytochemicals as metabolites. We find these plants compounds useful for health care as nutraceuticals, drugs and medicines and even most attractive fragrances and flavors. This huge diversity of low molecular weight compounds is represented by secondary metabolites that confer the power of responding to stimuli in plants. Hence, the network of metabolic pathways in plant species represents the pool of functions and chemical diversity leading to biomolecules such as alkaloids, flavanoids, terpenoids, glycosides, etc. Although the structures of approximately 50,000 such metabolites have already been elucidated, there are probably hundreds of thousands of such compounds which we are not able to detect or decipher within the existing limitation of detection. Only a few of these are part of ‘primary’ metabolic pathways (those common to all organisms). The rest are termed ‘secondary’ metabolites; this term is historical and was initially associated with inessentiality but we know today their necessity in defence to signals and stimuli. In the last decade, the research on plant secondary metabolism has been aiming to understand genetic basis from synthesis to regulation of plant compounds at molecular level. With these gene and genome studies, fascinating insights into the creation of genetic diversity of secondary metabolism have become evident. This has been leading to rising inquisition about the mechanisms of gene recruitment and diversification for novel functions. After more than 100 years of ignorance, biologists have reached a stage to recognize the causal connection between gene diversity and plasticity of secondary metabolism in its indispensable ecological role in the dynamic interactions of the plant V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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kingdom with its continuously changing environment. Nevertheless, the complexities are many folds. The multitude of metabolites found in living organisms and the calculated, unexpected small number of genes identified during genome sequencing projects pose more questions to the biologists. Several processes on the posttranscriptional and posttranslational level lead to the formation of enzyme diversity through structural and functional variant forms explaining partially this surprising situation. Further, lower enzyme specificity may also contribute to metabolome diversity. The bottom-line is the evolution of these genes which may provide multiple forms and thereby leading to metabolic diversity even for any cause including adaptation. The situation of pathway diversity across species and genera breaks the conventional taxonomic barriers and brings in chemo-taxonomic basis as new dimension to explain the ever evolving metabolome and hence vertical to horizontal genomic flow for the natural combinatorial biological chemistry working in plant kingdom as no surprise. Keywords: Medicinal plants, Aromatic plants, Biochemical pathways, Secondary metabolism, Metabolome, Diversity, Evolution, Pathway genes, Metabolic enzymes

Introduction Plants like other living forms in nature tend to conserve the energy in the metabolic activities but are known to produce and accumulate a large number of crucial and structurally very diverse molecules/metabolites. These metabolites directly may not serve as essential molecules of life for not being involved in the primary processes of growth and development. But such molecules are present in almost all plant forms from fungi to higher plants and are termed as secondary metabolites.1 Secondary metabolites, however, are known to have crucial role in plant defence (against herbivores, microbes, viruses or competing plants) and signaling or to attract pollinating or seed dispersing animals. Thus, secondary metabolites represent plant’s adaptive functions essential for its own survival and reproductive fitness aiding in its natural selection during evolution. The huge phytochemicals diversity found in plant kingdom is the result of different selection pressure conditions that plants have been successfully coping with through evolution. Medicinal and aromatic plants have become most attractive category of plants both for researchers as well as industry mainly because of the abundant presence of these prized secondary metabolites which find diverse uses from drugs to industrial and agricultural or environmental applications. Structures of these secondary metabolites appear to have evolved during evolution in such a way that they can mimic the structures of endogenous substrates, hormones, neurotransmitters or other ligands. The distribution pattern of secondary metabolite in a given plant is complex but very dynamic as it precisely links with different tissues/developmental stages. Plant metabolites of this nature can exist as proactive/pro-drug molecules that become activated upon wounding, infection or in

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the body of a herbivore. The biosynthesis of some secondary metabolites is induced upon wounding or infection and is made de novo, for example, phytoalexins.2 This quality of plants represents their capability too of producing enormous pool of molecules dynamically through the metabolic pathways diversity. Perhaps this diversity also is in an ever-evolving state through the continuum of genetic pool recombination, reshuffling and mutations happening and getting accumulated across the Darwinian forces and selection pressures. This state has been taken up here as the case study of evolving genomes in plants and discussed at length.

Evolving plant genomes: Emergence of duplicate genes, their clustering and divergence Understanding the evolutionary process leading to the emergence, distribution, diversification and selection of genes involved in plants’ metabolite biosyntheses is possible by visualising the ancestral forms vis-à-vis present phenotypes whether in primary or secondary metabolism. It is believed that in primary metabolism the new genes/alleles mainly arise by gene duplication followed by divergence.3,4 This equips the organism with one gene that maintains the original function and a second copy that is not restricted by natural selection. This second copy can then accumulate mutations until, rarely, it has acquired a new function and might then become fixed in the population. So gene duplication is assumed to be the major driving force for diversification and gene recruitment.1,5 If a gene that directs an essential function is duplicated, the duplicate is released from the stringent function of the mother gene; it may either be eliminated by inactivation (e.g. pseudogenization) or recruited for modified or new functions. There are at least two routes for duplicate genes to be recruited and functionalized. (1) Continuous modification of its function during plant speciation, as consequence of which large gene families with rich functional diversity arise and (2) the duplicate is recruited for a new stringent function in a novel biochemical environment, as consequence, a new single copy gene originates.6 Further elaborating this rationale are the following examples and cases. Amyrin synthases in oats are entirely distinct from other plants. The gene AsbAS1 seems to have arisen by duplication and divergence of a cycloartenol synthase-like gene, and later clustering with other genes required for distinct steps in avenacin biosynthesis in a region of the genome that is not conserved in other cereals. The components of this gene cluster are required for at least four clearly distinct enzymatic processes (2,3-oxidosqualene cyclization, amyrin oxidation, glycosylation and acylation), it is unlikely that the cluster has evolved as a consequence of duplication of a common ancestor. Although clusters of paralogous genes are common in plants (e.g. gene clusters for rRNA and specific disease resistance), reports of clusters of genes that do not share sequence relatedness and whose products contribute to a single selectable function are rare.7 With a

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series of mutants and their functional characterization, it has been shown that AsbAS1 might have evolved from an ancestral cycloartenol synthase-like gene cycloartenol by duplication and rapid sequence divergence. The close relatedness of AsbAS1 to cycoartenol synthases is interesting because, although cycloartenol synthase and amyrin synthase both use, 2,3-oxidosqualene as a substrate, but the structures of the cyclization products generated are quite different. Although gene clusters for secondary metabolites are not well documented in plants, they are common in fungal biosynthetic components, specific pathway regulators and for auto-resistance to the end-product.8 Transmission of these self-contained ‘gene cassettes’ by horizontal gene transfer has also been suggested as an explanation for the persistence of clustering in fungi although recent phylogenetic analyses underestimate the significance of vertical transmission.9 Clustering facilitates the inheritance of the genes for selective advantage as a single functional unit. Disruption of the gene cluster may lead to failure in producing desired pool of protective chemicals and further could result in the accumulation of deleterious intermediates.7 Synthesis of avenacins, like many other plant secondary metabolites, is highly tissue specific and under strict developmental control. Such situations may also confer undefined selective advantages associated with physical proximity and position effects. Intimate coadaptation of all enzymes of a single pathway is likely to be important as an additional mechanism for strict control and containment of secondary metabolites and their pathway intermediates during synthesis. This coadaptation may extend to physical interactions among pathway components, which would aid the channelling of metabolic intermediates within multienzyme complexes.10 Genes for secondary metabolism may in turn be derived from genes for primary metabolism by gene duplication and divergence or possibly also by allelic polymorphism. Similarly, another group of the plant enzymes, terpene synthases (which collectively mediate production of a diverse class of natural products) are predicted to be derived from genes for primary metabolism by duplication and consequent divergence in structural and functional specialization. The example of the clustering of polyketide synthases (PKSs) genes from different plant species shows them to be into two categories. The two clusters represent chalcone synthases (CHSs) and nonchalcone-synthases of angiosperms. The latter apparently originated from ancient duplication of an ancestral CHS gene. During angiosperm speciation, it seems, one gene retained the essential CHS function while the duplicate underwent functional diversification. Under environmental selection pressure new polyketide synthases might have evolved, producing a rich diversity of polyketides that can be distinguished by their biogenetic starter units, the hierarchy through number of added C2-units and the mechanism involved in cyclization.6


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The other example for the gene duplication worth mentioning is the evolution of benzoxazinone (DIMBOA) and indole.7 Molecular characterization of DIMBOA pathway indicates that non-homologous genes are organized in a gene cluster. The first gene in the sequence (BX1) is believed to have originated from a duplication of the gene encoding the unknown subunit of ubiquitous tryptophan synthase followed by recruitment of the second gene duplicate for the emission of volatile indole. Thus, both gene duplicates acquire completely new functions. The first being involved in the biosynthesis of defence compounds under developmental control of young seedlings, the second, encoding indole-3-glycerol phosphate lyase (IGL) is induced in mature leaves in response to herbivore damage. IGL catalyzes the formation of indole as one of the volatiles in maize emitted as signals in tritrophic defence (the volatile signals allure parasitoids to their prey, i.e. the maize herbivores). Another example relates to homospermidine synthase (HSS), which has already been mentioned as the first pathway-specific enzyme in the biosynthesis of pyrrolizidine alkaloids. HSS evolved by duplication of the gene encoding desoxyhypusine synthase (DHS).11 DHS takes part in the posttranslational activation of the eukaryotic initiation factor 5A (eIF5A). It is evident that HSS retained all kinetic and molecular properties of DHS except the ability to bind the eIF5A precursor protein.12 The ability of synthesizing homospermidine from putrescine and spermidine, a side activity already existent in DHS, ultimately became the core-activity of HSS. Again the duplicate of an essential gene of primary metabolism here was recruited for an entirely divergent function in secondary metabolism. The third example has also been described by Hartmann6 that concerns acyltransferases operating with 1-O-ß-acetalesters (1-O-glucose esters) as acyl donors instead of coenzymeA thio-esters. These enzymes, which play important role in plant phenylpropanoid metabolism, have most likely evolved from serine carboxy peptidases by gene duplication and new functionalization.13,14 Gene duplications are relatively frequent events within genomes and have a high impact on the evolution of new biological functions.15,16 The most likely event after gene duplication is the production of pseudogenes from one of the gene copies by knockoff mutations. However, in rare cases, one copy may acquire a completely new function as a result of beneficial mutations within its regulatory and structural components. According to a model proposed by Hughes, 17 the evolution of functionally distinct daughter genes is preceded by a period in which the ancestral gene is bifunctional. This bifunctionality is accomplished by the deoxypussine synthase (DHS) protein that originally possesses homospermidine synthesizing activity in many separate angiosperm lineages, later becoming the exclusive activity of the other gene copy. The HSS-coding gene copy presumably lost the protein-modifying activity of DHS and escaped the strong selection pressure on this essential function of primary metabolism. Nevertheless, its remaining ability

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to synthesize homospermidine became the object of selection pressure from herbivores enabling some plants to recruit the gene copy to establish the first step in the biosynthesis of defence compounds. There are several examples known in which genes have been independently recruited to a single function within a gene family. For instance, the resistance of insects to specific toxins seems to have been acquired through the independent recruitment of paralogous genes belonging to the cytochrome P450 superfamily.18,19 Plant terpene synthases, such as limonene synthase, have been shown to be repeatedly recruited within their gene family from other terpene synthases20 enabling some plants to recruit the gene copy to establish the first step in the biosynthesis of defence compounds. In case of Solanum habrochaites (formerly Lycopersicon hirsutum) f. typicum LA1777 and cultivated tomato Solanum lycopersicum (formerly Lycopersicon esculentum), it has been observed that the glandular type VI trichomes present on the leaves and stems accumulate monoterpenes in S. lycopersicum21 but sesquiterpenes, as insecticidal carboxylic acid derivatives, are accumulated in LA1777.22 Enzymes related to germacrene C synthase mediate the accumulation of a group of structurally similar compounds termed class I sesquiterpenes (cI-Ss) in LA1777 and S. lycopersicum.23 This represents the existence of a mechanism of secondary metabolites controlling the production of two distinct groups of sesquiterpenes from different precursor pools in the wild species. Such a partition in sesquiterpene biosynthesis could be the result of metabolite channeling, through two distinct farnesyldiphosphate (FPP) synthases in the cytoplasm for the secondary metabolites that are associated either with cI-S synthase or with plastidial transporters and the cII-S synthase.

Case examples of pathway genes diversity for secondary metabolism in plants Secondary metabolites of high structural as well as functional similarities are well known to occur simultaneously across even unrelated families of the plant kingdom. For example, the anti-tumor alkaloid camptothecin (inhibitors DNAtopoisomerase) has been found in the following unrelated orders and families Nothapodytes foetida (Celastrales), Pyrenacantha klaineana (Icacinaceae), Camptotheca acuminate (Cornales: Nyssaceae), Ophiorrhiza mungos, O. pumila, O. filistipula (Rubiales: Rubiaceae), Ervatamia heyneana (Apocynaceae) and Mostuea brunonis (Loganiaceae). 2 Consequently, the co-occurrence of a structural class in two taxa could, but not necessarily, be an indication of a monophyletic relationship. This could be due to convergent evolution or differential gene expression wherein, it is likely that in some cases the genes that encode the enzymes for the production of a given structure or structural skeleton might have evolved early during evolution. These genes were not lost during phylogeny


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but got switched off and on again at some later point.24 An example of lupaine pathway shows that genes evolved early during evolution were turned on in some plants using the alkaloids as chemical defence substances but remained turned off in most instances.25 Wink and Mohamed26 to study the phylogenetics of the Leguminosae by selecting representative taxa covering a broad range of tribes established a large rbcL data set and using secondary metabolites as chemical defence traits, reducing the entire Leguminosae to 95 taxa. Quinolizidine alkaloids are frequently present in all taxa of the subfamily Papilionoideae except the tribe Crotalarieae.27 Crotalaria species sequester pyrrolizidine alkaloids and/or nonprotein amino acids but not Quinolizidine alkaloids. In the genus Lotononis, some taxa produce quinolizidine alkaloids and others produce pyrrolizidine alkaloids. Since Crotalaria and Lotononis have derived from same ancestors, producing quinolizidine alkaloids but not pyrrolizidine alkaloids, the genes encoding biosynthetic enzymes of quinolizidine alkaloid formation must still be present. More likely the quinolizidine alkaloid genes have been turned off in Crotalaria and partially in Lotononis. The formation of pyrrolizidine alkaloids (which are typical secondary metabolite of the Boraginaceae and some Asteraceae) instead appears to be a new acquisition for chemical defence, which probably evolved independently. The protease inhibitors (i.e. trypsin and chymotrypsin inhibitors) distribution pattern is also similar to quinolizidine alkaloids.28 The members of the Caesalpinoideae and many Mimosoideae accumulate protease inhibitors in their seeds, where they serve concomitantly as chemical defence and nitrogen storage compounds. Within the Papilionoideae, protease inhibitors are prominent in the tribes Vicieae, Trifolieae, Cicereae, Abreae, Galegeae, Loteae, Phaseoleae, and Tephrosieae, but are not described in the Mirbelieae. Withanolides represent a group of steroidal lactones with strong insecticidal properties which appear to be restricted to the family Solanaceae.29,30 Withanolide producing genera are typical for the tribe Physaleae, but isolated occurrences have been reported for Brugmansia (Datureae), Hyoscyamus (Hyoscyameae), Lycium (Lycieae), Jaborosa (Jaboroseae), Nicandra (Nicandreae) and Browallia (Browallieae) serving as chemical defence compounds in the plants producing them. Therefore, these compounds constitute important fitness traits and represent adaptive characters with some, but usually have limited value as a taxonomic marker.26 All members of a monophyletic group share a chemical characteristic; favouring their use as a taxonomic marker. In other instances, a particular secondary metabolite may occur in several unrelated clades and/or plant families.26,28 The erratic secondary metabolite distribution can be due to simple convergence, where genes encoding a particular biosynthetic pathway evolved independently in several parts of a phylogeny. There is evidence however for an alternative explanation: In several cases, it is apparent that ancestral members of a group evolved the

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biosynthetic capacity to produce a specific secondary metabolite. The absence of such a trait in phylogenetically derived groups is probably due to differential gene expression, where the corresponding genes are not lost but switched off. Since secondary metabolites play a vital role as defence and signal compounds, their occurrence apparently reflects adaptations and particular life strategies embedded in a particular phylogenetic framework.2 Khanuja et al.31 while assessing the interspecific as well as intraspecific relationships in Mentha at molecular (DNA) level, observed that M. gracilis Sole Cardiaca showed a much higher similarity with M. spicata as well as M. arvensis, which amongst themselves showed rather a greater distance. This indicated that the species might have evolved as a natural hybrid between M. arvensis and M. spicata. The GC and GC/MS of the oil quality for metabolite components of 20 accessions of M. piperita32 showed close relationship of mutant ‘Kukrail’ with Japanese oil, whereas other two accessions were similar to Chinese oil as analysed in the component plot. These genotypes were released as varieties by the names of ‘CIM -Indus’ (high pulegone and menthofuran content) and’ CIMMadhuras’ (peppermint plant having aroma with high acceptability respectively by CIMAP. Similarly, the aromatic grass species Cymbopogon, in the analysis for essential oil biosynthetic components indicated remarkable variation among various species.33 The major essential oil components citral ‘a’ and ‘b’, were detected in Cymbopogon pendulus, C. flexuosus and C. citratus with highest in C. Citratus where as it was not detected in C.winteianus (Jowii) The hierarchical cluster analysis based on essential oil composition placed C. winterianus distantly from all other taxa followed by the hybrid Jamrosa. This hybrid contained high geraniol (68%), low citral (less than 2%) and trace of citronellal (0.5%) in the essential oil composition. The genomic synteny in essential oil compositions along with the differences indicated gene duplication followed by variations to create the metabolite diversity during speciation.

Domain swapping and neo-funtionalization: Another level of functional divergence Domain swapping represents an independent mechanism for the generation of new composite genes with or without prior gene duplications.34 It is theoretically possible for a new allele in one of the plant’s genetic loci to be selected for if it encodes the ability to make a new defence compound, whereas the older alleles still specify the synthesis of another defence compound that however may be no longer effective. Thus, in secondary metabolism, there is a potential for new genes to keep evolving without a prior gene duplication event. In such cases, these orthologous genes in related species might encode proteins with different functions1. It is widely believed that enzymes with more active and specialized


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function evolve divergently from enzymes with promiscuous function. This process is thought to be closely associated with the evolution of metabolic pathways.35–37 It has been observed that that the recruitment of single enzymes from other metabolic pathways might significantly drive the evolution of both enzymes and metabolic pathways.35 It is believed that enzymes with promiscuous functions can be initially shared by two distinct metabolic pathways. Enzymes with promiscuous function might give organism novel metabolic capabilities and, thus, render them adaptable to different environments. When the gene is duplicated, one enzyme is free to abandon the role it had in the previous pathway and thus can specialize its function for the new pathway, and vice versa. This results in divergent molecular evolution of enzymes and a mosaic or patchwork evolution of metabolic pathways. If multiple steps in a metabolic network are catalyzed by a series of promiscuous enzymes, although inefficient, this network would be able to produce a large library of natural products. If any of these products were to be captured by positive selection, the metabolic network could then converge on the pathway through divergent evolution of each component enzyme.38 In a research study, a combination of domain swapping and reciprocal sitedirected mutagenesis was carried out in grand fir between (–)-(4S)–limonene synthase (LS) and (–)-(4S)-limonene/ (–)-1S, 5S–α-pinene synthase (LPS) by Katoh et al.39 Exchange of the predicted helix D through F region in LS gave rise to an LPS-like product outcome. Whereas reciprocal substitutions of four amino acids in LPS (two in the predicted helix D and two in the predicted helix F) altered the product distribution to that intermediate between LS and LPS. This resulted in a 5-fold increase in relative activity. Based on the results of these chimeric studies, reciprocal point mutations were made in each parent monoterpene synthase to provide five single and 33 multiple site mutants of LS, and five single and 12 multiple site mutants of LPS, and were successfully expressed and evaluated. The most effective in altering monoterpene olefin distribution was V384L, a position predicted by modeling to reside in helix D. In this mutant, the proportions of generated α- and β-pinene were reversed and the level of β-phellandrene was found to be nearly doubled. These experiments were conducted, in conjunction with modeling of the two enzymes to know the critical amino acids for product determination. But indirectly this denotes the change in domains among highly homologous genes can lead to change the product distribution itself. In another study, domain swapping experiments between Cl(-)  PINS ( -pinene synthase) and Cl TS ( -terpinene synthase) and between Cl(+)LIMS2 (limonene synthase) and Cl  TS were conducted40 to identify domains within the monoterpene synthase enzymes determining the product specificity. Similarly, a sesquiterpenes cyclase (CASC2) showing 77% amino acid identity with the previously cloned sesquiterpene cyclase CASC1 of Capsicum annuum failed to express in Escherichia coli. However, the chimeric construct of CASC2 in which

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the amino terminal 164 amino acid was substituted by the equivalent portion of either CASC1 or tobacco sesquiterpene, the cyclase was capable of expressing the functional sesquiterpene cyclase activities.41 Relatively closer similarity of GES (geraniol Synthase) and LIS ((R)-linalool synthase protein pairs in Ocimum basilicum indicates that further terpene biosynthetic diversity is continuing to be generated in the basil lineage by gene duplication and divergence.42 This two genes have been found to produce only a single product, either linalool or geraniol, but not both. But the results of domain-swapping experiments by the same group also demonstrated that it is possible to generate a monoterpene synthase that can synthesize both geraniol and linalool. Here, multiple amino acids contribute to such a dual selectivity but such an enzyme is not found in nature till date. Another phenomenon of importance observed relates to the occult biosynthetic capacities of plant either constitutive or induced. This confers them an unlimited potential to produce a large array of different compounds that can be activated when novel substrates become available.43 Through biotechnological route, overexpression of Clarkia breweri floral gene linalool synthase (LIS), an enzyme that catalyzes the formation of (S)-linalool from the monoterpene precursor geranyl diphosphate was attempted in tomato fruit.44 Interestingly, accumulation of (S)-linalool (expected) and 8-hydroxylinalool (unexpected) was observed. In this case, the availability of novel substrate due to expression of a foreign gene enabled endogenous ‘occult’ hydroxylase activity to act on (S)-linalool. When the Clarkia LIS gene was overexpressed in carnation flowers, linalool in the transgenic flowers further metabolized to linalool oxides. Thus, the overexpression of an identical gene in different target tissues and organisms could gave rise to distinct phenotypes, according to the metabolism present or induced in the target organism that might interact with the novel products generated. Similarly, from lemon basil (O. basilicum L. cv. Sweet Dani) geraniol synthase (GES) gene was overexpressed in tomato fruits to modify the aroma and flavor.45 Besides high levels of geraniol, eleven novel additional metabolites sharing a common chemical backbone accumulated. These were derived from geraniol i.e the monoterpene alcohols nerol and citronellol. This way, monoterpene aldehydes geranial, neral and citronellal, the monoterpenol esters geranyl, neryl and citronellyl acetate, geranic and neric acid and rose oxide accumulated in such transgenic tomatoes. In another study,46 the sweet basil type that possesses OMT activities converting methylate chavicol to estragole, was compared with a distinct basil type that accumulates only methyl eugenol (a 3 methoxylated estragole derivative). When chavicol was provided as a substrate it was methylated to estragole in cell-free extracts, although the plant itself does not contain estragole. The “lemon-scented” basil line 197 is also known to contain citral (a mixture of the monoterpene aldehydes geranial and neral) but lacks volatile phenylpropenes in its essential oil. And yet cell-free extracts derived from this lemon-scented line


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could readily convert O-methylate chavicol to estragole, eugenol to methyl eugenol or isoeugenol to methyl-isoeugenol although none of these compounds are present in the plant. This further indicates the presence of ‘silent’ O-methyltransferase activities in basil lines that may readily accept novel substrates if the ability to produce those substrates is acquired by breeding or mutation, easily yielding to novel chemotypes. Biosynthesis of tryptophan, a precursor for indole alkaloids, is well known for the feedback inhibition mechanism to regulate its own production. Hence, its availability can be well understood as a limiting factor in biosynthesis of indole alkaloids. However, the introduction of Arabidopsis thaliana feedback-resistant anthranilate synthase (AtAS) and induction of tryptophan decarboxylase (TDC) in Catharanthus roseus hairy roots did not significantly improve downstream alkaloids even though the levels of early alkaloid precursors tryptamine and tryptophan increased.47 This suggests that the availability of the early amino acid precursor is not limiting for TIA (Terpenoid indole alkaloids) biosynthesis, which confirms the finding that the availability of secologanin was the important ratelimiting step in TIA biosynthesis.48 A superfamily is supposed to be a group of enzymes related by divergent evolution.49 Among these, the plant terpene synthase superfamily is interesting and the most cited example to explain divergent molecular evolution. These enzymes share a strikingly similar active site scaffold comprising several -helices and catalyze the formation of diverse terpenes from several different classes of prenyl diphosphates through wide varieties of carbocation rearrangements. Terpene synthase subfamilies within angiosperms are more closely related to each other than are members in the same subfamily from gymnosperms. In each subfamily, terpene synthases from the same or related species are more closely related to each other than are ones from different species with similar catalytic mechanism based secondary metabolites.20 These observations indicate that divergence in terpene synthase subfamilies evolved after angiosperms and gymnosperms separated and that of terpene synthases within each subfamily arose after a series of subsequent speciation events.38 All terpene synthases so far described show promiscuous function. Among those, -humulene and dselinene synthases are very promiscuous sesquiterpene synthases that are constitutively expressed in Abies grandis, each catalyzing the formation of at least 52 and 36 sesquiterpenes, respectively. In addition, these enzymes can use geranyl diphosphate as a substrate and catalyze the formation of monoterpenes. Although the specific roles of these enzymes have not been identified, it is thought that they might create chemical libraries that are important in general defence against microbial invasion. By contrast, many other terpene synthases have highly specialized functions and are often found to have very specific roles in the formation of bioactive metabolites. For example, (+)-D-cadinene, vetispiradiene and 5-epi-aristolochene synthases catalyze the

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first reaction step of phytoalexin (anti-fungal agents) production in various plant species and yield their respective sesquiterpenes with more than 98, 90 and 70% selectivity, respectively. Thus, it has been suggested that terpene synthases with specific functions have evolved from ones with promiscuous function after having been captured by positive selection.38 Acknowledgments: Prof. Sushil Kumar, former Director, CIMAP who happens to be our teacher showing the path through the pathways in plants and life inspiring this writing attempt.

References 1.

Pichersky E, Gang DR (2000) Genetics and biochemistry of secondary metabolites in plants: An evolutionary perspective. Trend Plant Sci 5:439–445

2.

Wink M (2003) Evolution of secondary metabolites from an ecological and molecular phylogenetic perspective. Phytochem 64:3–19

3.

Ohno S (1990) In: Evolution by Gene Duplication. Springer, Verlag

4.

Pichersky E (1990) Nomad DNA – a model formovement and duplication of DNA sequences implant genomes. Plant Mol Biol 15:437–448

5.

Ober D (2005) Seeing double-gene duplication and diversification inplant secondary metabolism. Trends Plant Sci 10:444–449

6.

Hartmann T (2007) From waste products to ecochemicals: Fifty years research of plant secondary metabolism. Phytochemistry 68:2831–2846

7.

Gierl A, Frey M (2001) Evolution of benzoxazinone biosynthesis andindole production in maize. Planta 213:493–498

8.

Ward TJ, Bielawski JP, Kistler HC, Sullivan E, O’Donnell K (2002) Ancestral polymorphism and adaptive evolution in the trichothecene mycotoxin gene cluster of phytopathogenic Fusarium. Proc Natl Acad Sci USA 99:9278–9283

9.

Kroken S, Glass NL, Taylor JW, Yoder OC, Turgeon BG (2003) Phylogenomic analysis of type I polyketide synthase genes in pathogenic and saprobic ascomycetes. Proc Natl Acad Sci USA 100:15670–15675

10.

Burbulis IE, Winkel-Shirley B (1999) Interactions among enzymes of the Arabidopsis flavonoid biosynthetic pathway. Proc Natl Acad Sci USA 96:12929–12934

11.

Ober D, Hartmann T (1999) Homospermidine synthase, the firstpathway-specific enzyme of pyrrolizidine alkaloid biosynthesis,evolved from deoxyhypusine synthase. Proc Natl Acad Sci USA 96:14777–14782

12.

Ober D, Harms R, Witte L, Hartmann T (2002) Molecular evolution by change of function: alkaloid-specific homospermidine synthase retained all properties of deoxyhypusine synthase except binding theeIF5A precursor protein. J Biol Chem 278:12805–12815

13.

Milkowski C, Strack D (2004) Serine carboxypeptidase-like a cyltransferases. Phytochemistry 65:517–524

14.

Stehle F, Brandt W, Milkowski C, Strac D (2006) Structure determinants and substrate recognition of serine carboxypeptidase-like acyltransferases from plant secondary metabolism. FEBS Lett 580:6366–6374


367

15.

Wagner A (1998) The fate of duplicated genes: Loss or new function? Bioessays 20: 785–788

16.

Kondrashov FA, Rogozin IB, Wolf YI, Koonin EV (2002) Selection in the evolution of gene duplications. Genome Biol 3:1–9

17.

Hughes AL (1994) The evolution of functionally novel proteins after gene duplication. Proc R Soc Lond B Biol Sci 256:119–124

18.

Scott JG, Wen Z (2001) Cytochromes P450 of insects: The tip of the iceberg. Pest Manag Sci 57:958–967

19.

Wilson TG (2001) Resistance of Drosophila to toxins. Annu Rev Entomol 46:545–571

20.

Bohlmann J, Meyer-Gauen G, Croteau R (1998) Plant terpenoid synthases: Molecular biology and phylogenetic analysis. Proc Natl Acad Sci USA 95:4126–4133

21.

Besser K, Harper A, Welsby N, Schauvinhold I, Slocombe S, Li Y, Dixon RA, Broun P (2009) Divergent Regulation of Terpenoid Metabolism in the Trichomes of Wild and Cultivated Tomato Species. Plant Physiol 149:499–514

22.

Li L, Zhao Y, McCaig BC, Wingerd BA, Wang J, Whalon ME, Pichersky E, Howe GA (2004) The tomato homolog of Coronatine-Insensitive1 is required for the maternal control of seed maturation, jasmonate-signaleddefense responses, and glandular trichome development. Plant Cell 16:126–143

23.

van der Hoeven RS, Monforte AJ, Breeden D, Tanksley SD, Steffens JC (2000) Genetic control and evolution of sesquiterpene biosynthesis in Lycopersicon esculentum and L. hirsutum. Plant Cell 12:2283–2294

24.

Wink M, Witte L (1983) Evidence for a wide spread occurrence of the genes of quinolizidine alkaloid biosynthesis. Induction of alkaloid accumulation in cell suspension cultures of alkaloid-free species. FEBS Letters 159:196–200

25.

Wink M (1992) The role of quinolizidine alkaloids in plant insect interactions. In: Insect– Plant Interactions. Bernays EA (Ed.), CRC Press, Boca Rato

26.

Wink M, Mohamed GIA (2003) Evolution of chemical defence traits in the Leguminosae: Mapping of distribution patterns ofsecondary metabolites on a molecular phylogeny inferred fromnucleotide sequences of the rbcL gene. Biochem. Syst Ecol 31:897–917

27.

Kinghorn AD, Balandrin MF (1984) Quinolizidine alkaloids of the Leguminosae: Structural types, analysis, chemotaxonomy, and biological activities. In: Alkaloids, Chemical and Biological Perspectives. Pelletier WS (Ed.), Wiley, New York

28.

Wink M, Waterman P (1999) Chemotaxonomy in relation to molecular phylogeny of plants. In: Biochemistry of Plant Secondary Metabolism, Annual Plant Reviews. Wink M (Ed.), Sheffield Academic Press and CRC Press

29.

Hegnauer R (1973) Chemotaxonomie der Pflanzen. Birkha user Verlag, Basel

30.

Harborne, JB, Baxter H (1993) Phytochemical Dictionary. In: A Handbook of Bioactive Compounds from Plants. Taylor & Francis, London

31.

Khanuja SPS, Shasany AK, Srivastav A, Kumar S (2000) Assessment of genetic relationships in Mentha species. Euphytica 111:121–125

32.

Shasany AK, Gupta S, Gupta MK, Singh AK, Naqvi AA, Khanuja SPS (2007) Chemotypic comparison of AFLP analyzed Indian Peppermint germplasm to selected peppermint oils of other countries. J Essent Oil Res 19:138–145

33.

Khanuja SPS, Shasany AK, Pawar A, Lal RK, Darokar MP, Naqvi AA, Rajkumar S, Sundaresan V, Lal N, Kumar S (2005) Essential oil constituents and RAPD markers to

368


establish species relationship in Cymbopogon Spreng (Poaceae). Biochem Syst Ecol 33:171–186 34.

Doolittle RF (1995) The multiplicity of domains in proteins. Ann Rev Biochem 64: 287–314

35.

Schmidt S, Sunyaev S, Bork P, Dandekar T (2003) Metabolites: A helping hand for pathway evolution? Trends Biochem Sci 28:336–341

36.

O’Brien PJ, Herschlag D (1999) Catalytic promiscuity and the evolution of new enzymatic activities. Chem Biol 6:R91–R105

37.

Jensen RA (1976) Enzyme recruitment in evolution of new function. Ann Rev Microbiol 30:409–425

38.

Yoshikuni Y, Keasling JD (2007) Pathway engineering by designed divergent evolution. Curr Opin Chem Biol 11:233–239

39.

Katoh S, Hyatt D, Croteau R (2004) Altering product outcome in Abies grandis (−)-limonene synthase and (−)-limonene/(−)-α-pinene synthase by domain swapping and directed mutagenesis. Arch Biochem Biophys 425:65–76

40.

Tamer MKE, Lucker J, Bosch D, Verhoeven HA, Francel WAV, Schwab W, Tunen AJV, Voragen AGJ, Maagd RA, Bouwmeester H (2003) Domain swapping of Citrus limon monoterpene synthases: Impact on enzymatic activity and product specificity. Arch Biochem Biophys 411:196–203

41.

Back KNJ, Lee SB, Song JH, Shin DH , Kim HY (2000) Cloning of a sesquiterpene cyclase and its functional expression by domain swapping strategy. Mol Cells 10:220–225

42.

Iijima Y, Davidovich-Rikanati R, Fridman E, Gang DR, Bar E, Lewinsohn E, Pichersky E (2004) The biochemical and molecular basis for the divergent patterns in the biosynthesis of terpenes and phenylpropenes in the peltate glands of three cultivars of basil. Plant Physiol 136:3724–3736

43.

Lewinsohn EG (2009) Phytochemical diversity: The sounds of silent metabolism. Plant Sci 176:161–169

44.

Lucker J, Bouwmeester HJ, Schwab W, Blaas J, van der Plas LH, Verhoeven LH (2001) Expression of Clarkia S-linalool synthase in transgenic petunia plants results in the accumulation of S-linalyl-beta-d-glucopyranoside. Plant J 27:315–324

45.

Davidovich-Rikanati R, Tadmor S, Iijima Y, Bilenko N, Bar E, Carmona B, Fallik E, Dudai N, Simon JE, Pichersky E, Lewinsohn E (2007) Enrichment of tomato flavor by diversion of the early plastidial terpenoid pathway. Nat Biotechnol 25:899–901

46.

Xie ZJ, Kapteyn DR, Gang A (2008) Systems biology investigation of the MEP/terpenoid and shikimate/phenylpropanoid pathways points to multiple levels of metabolic control in sweet basil glandular trichomes. Plant J 54:349–361

47.

Leonard E, Weerawat R, O’Connor S, Prather KJ (2006) Opportunities in metabolic engineering to facilitate scalable alkaloid production. Nat Chem Biol 5:292–272

48.

Shukla AK, Shasany AK, Gupta MM, Khanuja SPS (2006) Transcriptome analysis in Catharanthus roseus leaves and roots for comparative terpenoid indole alkaloid profiles. J Experimen Botany 57:3921–3932

49.

Glasner ME, Gerlt JA, Babbitt PC (2006) Evolution of enzyme superfamilies. Curr Opin Chem Biol 10:492–497

23 Conservation of Himalayan bioresources: An ecological, economical and evolutionary perspective Lok Man S. Palni* and Ranbeer S. Rawal** *G. B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora 263 643 (Uttarakhand), India *[email protected], **[email protected]

Abstract: The Himalayan ranges are the youngest and loftiest among the mountain systems of the world. They represent a highly complex and diversified system both in terms of biological and physical attributes. Their vulnerability toward natural and human-induced disturbances is well recognized. On account of richness and uniqueness of biodiversity elements, the region has been recognized as one of the 34 global biodiversity hotspots. It represents 3 sub-centers (west Himalaya, east Himalaya and north east region) of plant origin, which, respectively, contribute 125, 82 and 132 species of wild relatives. The eastern Himalaya and north eastern sub-centers are known for contribution to Musa and Citrus diversity. The prevailing primitive agricultural systems in the region and conscious and unconscious selections by indigenous farming communities have contributed toward enormous enrichment of genetic diversity in the form of land races. Diversity of representative natural ecosystems (grasslands and woodlands) and richness of endemic bioresources have added to the ecological significance of the Himalaya. Particularly, the alpine grasslands and the forests of the region exhibit unique features. Furthermore, medicinal and wild edible plants add substantially to the ecological and economic value of the region. However, Himalayan ecosystems and their components are highly vulnerable both due to geological reasons and on account of stress caused by increased pressure of population. Also, there are growing indications that the ill effects of these factors may be exacerbated on account of the impact of climate change. This would affect the very sustenance of the indigenous communities living in the uplands as well as downstream areas. Therefore, there is an urgent need for making conscious efforts for conserving all representative systems. In this context, the existing conservation area network in the region, which appears to be stronger than the country average, is one welcome initiative. This network, however, requires strengthening to provide adequate coverage to all representative ecosystems, particularly in north east. Need for a major shift in the conservation approach through community support, and by way of promotion of sustainable use concept, has been suggested to ensure conservation of Himalayan bioresources. This is pivotal for maintaining vital stocks of unique, often endemic elements, for the natural evolutionary processes to continue. V. P. Sharma (ed.), Nature at Work: Ongoing Saga of Evolution © The National Academy of Sciences, India 2010

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Keywords: Indian Himalayan region, Biodiversity hotspot, Center of origin, Endemics, Community participation, Protected area network

Introduction Charles Robert Darwin, who derived pleasure in inventing a whole new science from the small, commonplace features of the Earth that most of us mortals often ignore; he was able to transform the then prevailing contemporary thinking across diverse spheres of human society. The Origin of Species,1 which established theory of Natural Selection, subsequently popularized as ‘Survival of the Fittest’, marks a watershed in the history of scientific, philosophical, theological ideas. In spite of considerable changes in evolutionary theory over time, the content, arguments and reasoning provided in his great revolutionary work remain relevant even to this date. Among others, the complexity and interdependence of natures’ elements, the entangled bank as he describes in the concluding paragraph, holds the truth of the life’s diversity which manifests itself in its myriad of forms. ‘It is interesting to contemplate an entangled bank, clothed with many plants of many kinds, with birds singing on the bushes, with various insects flitting about, and with worms crawling through the damp earth, and to reflect that these elaborately constructed forms, so different from each other, and dependent on each other in so complex a manner, have all been produced by laws acting around us……………………There is grandeur in this view of life, with its several powers, having been originally breathed into a few forms or into one; and that, whilst this planet has gone cycling on according to the fixed law of gravity, from so simple a beginning endless forms most beautiful and most wonderful have been, and are being, evolved’.1 Any attempt, small or big, to describe such complexity of nature would be a tribute in itself to the great scientific-visionary. Recognizing this, in subsequent pages, we have tried to explore complexities and interdependence of various elements of the Himalayan Ecosystem. Our attempt is to find out a few workable solutions under changing ecological and economic scenarios in the region which could help in maintaining vital stocks of unique Himalayan biodiversity elements and help in the natural evolutionary processes to continue. The mountains, which cover nearly one quarter of Earths’ land area, host over 12% of global human population. More importantly, >50% of global human population draws benefits directly or indirectly from resources and services emanating from the mountains. These regions, although well recognized for their evolutionary significance and ecological value manifested by ecosystem integrity, adaptability and services, have largely remained marginalized from economic development perspectives. However, in the aftermath of Rio Earth

Conservation of Himalayan bioresources: An ecological, economical and evolutionary perspective

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Summit and the implementation of its Agenda 21, awareness about the mountains has increased manifold, and the region is being institutionalized on mainstream developmental agenda. In this context, on account of richness (diversity), representativeness and uniqueness, the mountain biodiversity elements have gained considerable attention. The Himalaya: A global biodiversity hotspot While considering the mountains as a special focus, the Himalaya, which represents youngest but complex mountain system on the surface of earth, which is still evolving, has been recognized among the 34 global biodiversity hotspots.2 On account of its being in evolving state, the ecosystem components in the region exhibit great dynamism.This majestic mountain system covers nearly 7.5 lakh km2 area (spanning over 3000 km in length, and rising from < 300 to > 8000 m asl) and stretches from northern Pakistan on the west to northeastern region of India through northwestern states of India, Nepal and Bhutan. The region has a discrete geographic and ecological entity. It produces a distinctive climate of its own and influences the climate of much of Asia.3 However, the variations in topographical features along three-dimensional frame work (i.e. latitudinal: SouthNorth; longitudinal: East-West; altitudinal: Low-High) cause diversity in climate and habitat conditions within the region.4 This leads to overwhelming richness of biodiversity elements and to their uniqueness. The region serves as a rich repository of plant and animal wealth in diverse ecological systems. These ecosystems reflect a mosaic of biotic communities at various spatial and organizational levels. Diversity and uniqueness features with respect to biodiversity of Himalayan hotspot are included (Table 1). The Indian Himalayan Region (IHR), with geographical coverage of over 5.3 lakh km2, constitutes a large proportion of the hotspot and, therefore, contributes greatly to richness and representativeness of its biodiversity components at all levels (i.e. genes, species and ecosystems). Administratively IHR (Fig. 1) covers 10 Table 1 Diversity and uniqueness of biodiversity in the Himalayan biodiversity hotspot [Source: Ref. 2] Taxonomic group Species Percent Endemism Endemic species Plants Mammals Birds Reptiles Amphibians Freshwater fishes

10,000 300 977 176 105 269

3,160 12 15 48 42 33

31.6 4.0 1.5 27.3 40.0 12.3

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states entirely (i.e. Jammu and Kashmir, Himachal Pradesh, Uttarakhand, Sikkim, Arunachal Pradesh, Nagaland, Manipur, Mizoram, Tripura and Meghalaya), and two states partially (i.e. hilly districts of Assam and West Bengal). The region represents nearly 3.8% of total human population of the country and exhibits diversity of ethnic groups (171 of a total 573 scheduled tribes in India) which inhabit remote inhospitable terrains.5 On account of its contribution (biophysical values) to the Himalayan biodiversity hotspot, IHR holds a significant position.6 While considering maintenance or conservation of its representative biodiversity values (Table 2), the region needs to be examined critically for its evolutionary, ecological and economic contributions.

Himalayan bioresources: Evolutionary and biogeographic contributions The Himalaya, which owes its origin to the collision between two land masses (the Peninsular Indian Plate and the Eurasian Plate) about 60–70 million years

Figure 1. Indian Himalayan region with three centers of plant origin. [Modified after Refs. 6, 22]


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Table 2 Representativeness of Himalayan biodiversity Representation Levels

References

Total number

% of India

Eco-regions Communities

16

-

[60]

Vegetation types Forest types Formation types Species

21 10 11

26 -

[32] [61] [7]

Angiosperms Gymnosperms Pteridophytes Bryophytes Lichens Fungi Mammals Birds Reptiles Amphibians Fishes Specific groups

8000 44 600 1737 1159 6900 241 528 149 74 218

47 81 59 61 59 53 65 43 35 36 17

[62] -do-do-do-do-do[63] -do-do-do-do-

Medicinal Wild edible Trees

1748 675 723

23 67 28

[38] [44] [64]

ago, is the youngest and the tallest mountain system in the world, and is most susceptible to landslips and erosion owing to the presence of residual stresses and highly compressed and tectonized rocks.7 However, the Himalayan orogeny does not relate to a single event, the western part being more recent.8 The temporal and spatial variations caused by diversity in geological orogeny has resulted in to a marked difference in climate and physiography, and consequently in distribution pattern of biotic elements. Also, the spatial position (at the juncture of five major biogeographic regions- Palaearctic, Mediterranean, Indo-Chinese, Indo-Malayan and Peninsular India) and heterogeneous dispersion of biodiversity elements has caused complexity in biogeographical patterns of the region. The erosion of the young Himalaya resulted in production of alluvia that led to filling and drying up of the Tethys Sea in northwest India and also led to the formation of the Indus and Gangetic alluvial plains.9 Considering the wide ranging affinities of the biota, the IHR (Table 3) consists of three biogeographic zones and seven provinces.10,11

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Table 3 Biogeographic divisions of IHR [Source: Refs. 10,11] Biogeographic Biogeographic Geographical Major Biome Zones Provinces area of India Representation (%) Trans Himalaya 1A: Ladakh Mountains 3.3 Tundra 1B: Tibetan Plateau 2.3 Alpine The Himalaya 2A: North west 2.1 Alpine, temperate, Himalaya Subtropical 2B: West Himalaya 1.6 -do2C: Centaral Himalaya 0.2 -do2D: East Himalaya 2.5 -doTropical evergreen forest, very moist Northeast India 9A: Brahamputra Valley 2.0 sal forest, tropical Grasslands Tropical evergreen, tropical moist 9B: Northeast Hills 3.2 deciduous, subtropical, Temperate forests, wetlands

Cradle of flowering plants Considering the evolutionary contribution, the east Himalayan biogeographic province along with the North East Indian biogeographic zone has great significance. This region is rich in biodiversity and harbors largest number of endemics and Schedule I species as compared to any other part of India.12 This region represents a confluence of the Indo-Malayan and Indo-Chinese biogeographical realms. Also, it exhibits intermixing of the Himalayan and Peninsular Indian elements. The region was considered as cradle of flowering plants,13 which represents some of the primitive angiosperm families including Magnoliaceae, Degeneriaceae, Himantandraceae, Eupomatiaceae, Winteraceae, Trochodendraceae, Tetracentraceae and Lardizabaleaceae. The specific primitive genera include Alnus, Aspidocarya, Betula, Decaisnea, Euptelea, Exbucklandia, Haematocarpus, Holboellia, Houttuynia, Magnolia, Magnelietia, Pycnarrhena and Tetracentron.9,14 The region is known for high evolutionary activities which are clearly evident from the cytogeographic studies on selected genera like Rhododendron, Camellia, Magnolia, Budleia, etc.15–20


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Table 4 Distribution of diversity of wild relatives in the Himalaya. [Source 22] Category Distribution in Himalayan Sub-centers West Himalaya Eastern Himalaya North Eastern Region Cereals and millets 29 07 16 Legumes Fruits Vegetables Oilseeds Fibers Spices and condiments Miscellaneous Total spp. Diversity

09 37 25 06 04 10

05 32 12 03 04 09

06 51 27 01 05 13

05 125

10 82

13 132

Centers of Diversity Further, the IHR contributes considerably in the form of wild relatives of several crop plants and domesticated animals. Of the total 8 sub-centers of plant origin, the region represents 3 sub-centers (e.g. Western Himalaya, Eastern Himalaya and North Eastern Region). These sub-centers, respectively, contribute 125, 82, and 132 species of wild relatives (Table 4).21–22 Based on extensive explorations across the world, Vavilov considered the north eastern region of India as ‘Hindusthan Centre of Origin of Cultivated Plants’, which is very important for tropical and subtropical fruits, cereals, etc. This region forms the richest reservoir of genetic variability of many groups of crop plants. The taxonomical and cytogenetic studies have revealed AssamBurma-Siam-Indo-China region as the center of origin of Musa.23 Greatest diversity, with some being endemic, of this genus is known form this region. Banana in north east India grows wild along the hill slopes of Arunachal Pradesh, Meghalaya and Assam. The region is also rich in Citrus wealth with nearly 64 taxa of Citrus growing wild. Also, it is regarded as the center of origin for five species of palms of commerce – coconut, areca nut, palmyra palm, sugar palm and wild date palm.24 In addition, it contributes a whole range of medicinal and aromatic plants with their origin in the region and wild progenitors of a number of ornamentals like Primula, Rhododendron and huge diversity of Orchids. Among wild and domesticated faunal elements, region harbors wild chicken, zebu, mithun, yak, etc.9

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The prevailing primitive agricultural system of raising crops under stress conditions in the region have resulted in much variability, particularly in adaptive traits. Most of the hilly terrain in the eastern Himalaya is under shifting cultivation and several promising, agronomically and physiologically well adapted types/ land races belonging to diverse crop species occur in this region. The specialized habitats in the western part of Himalaya (e.g. cold arid climate) are also responsible for promoting variation in specific traits. In addition, in isolated pockets, various ethnic groups grow their own preferred locally selected cultivars. All these factors have contributed to enormous enrichment of genetic diversity in land races through conscious and unconscious selections by indigenous farming communities in the Himalaya.25 Emerging issues Realizing the above uniqueness, there is a need to place special attention to maintain the evolutionary significance of the region. Apart from inventorization/ documentation of such genetic resources, their evaluation for use and maintenance both as in situ and ex situ gene banks assumes priority. Ecogeographical surveys in areas of diversity will be required to mark out precise pockets for in situ conservation, and integration of such efforts with monitoring of diversity in bioresources is called for. Apart from seed storage in genebanks, in vitro conservation measures assume greater importance, with overall emphasis on complementary conservation strategies.22, 26 This can, however, be achieved only through a holistic understanding of existing ‘man and nature’ relationships. Among others, the practicable solutions for achieving conservation goals with respect to these genetic resources may be drawn from a diverse set of best practices performed by the indigenous communities.

Himalayan bioresources: Ecological contributions The biogeographical diversity (Table 3) has endowed the region with rich and representative biodiversity elements (Table 2), and it serves as a rich repository of plant and animal wealth in diverse ecological systems. These ecosystems reflect a mosaic of biotic communities at various spatial and organizational levels along three spatial transitions: (1) East to West transition: over 2500 km in length, this transition represents cool and moist conditions of east Himalaya (i.e. Arunachal Pradesh) to cold dry conditions of west (i.e. Ladakh). (2) South to North transition: representing tropical/subtropical conditions of southern most Himalaya (i.e. adjoining the Indus-Gangetic plains) to cold desert (i.e. Tibetan Plateau) in the north. This transition results into a width ranging from 240 to 350 km.


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(3) Low to High altitude transition: ranging from tropical and subtropical conditions of low altitudes ( 8000 m asl). While considering the conservation of bioresources of such a complex mosaic, among various important ecological considerations, representativeness of ecosystems and uniqueness (endemism) of species deserve priority mention.

Ecosystem representation Considering representation of wide structural and functional diversity, the region is broadly grouped in to two major natural terrestrial ecosystems: (1) the Grasslands, and (2) the Woodlands. The alpine meadows represent natural grassland ecosystems in the Himalaya which are widely distributed between two ecotones (tree line and snow line) from extremely dry trans/north west Himalaya to humid east Himalaya. In view of the variations in structure/composition of biodiversity elements in response to climatic conditions two types of alpine grassland ecosystems are discernible in the Himalaya: (1) Alpine Arid Pastures (AAP) – largely represented in cold desert areas of the trans and north west Himalaya these pastures show specialized characters for both plant and animal forms and occur in relatively low densities, often in geographically isolated populations, which are vulnerable to local extinction.27 While these ecosystems are characterized by dominance of xerophytic plant elements they also provide special habitats for certain localized animals such as Tibetan wild ass (Equus kiang kiang), Tibetan wolf (Canis lupes chanco), ibex (Capra ibex sibirica), Ladakh urial (Ovis orientalis), Markhor (Capra falconeri), Tibetan gazelle (Procarpa picticaudata) and widely distributed high-altitude fauna, for example, bharal (Pseudois nayaur), snow leopard (Uncia uncia), Indian wolf (Canis lupus), brown bear (Ursus arctos isabellinus); and (2) Alpine Moist Pastures (AMP) – they represent typical alpine meadows usually extending across the Himalaya occupying the southern slopes, and are known to be the rich habitats with high productivity. Besides, the unique representative assemblages of floral and faunal elements, value of these ecosystems in sustenance of local pastoral communities is well recognized. These ecosystems, on account of higher productivity, support large populations of wild and domestic ungulates. Species such as bharal, Himalayan thar (Hemitragus jemlahicus), Himalayan musk deer (Moschus chrysogaster), brown bear, red fox (Vulpes vulpes) and the Himalayan monal (Lophorus impejanus) depend substantially on these ecosystems.27 For the representative alpine grasslands of the region, annual live shoot production (382–409 g/m2) has been reported high (example central Himalaya) as compared to reported values (13–348g/m2) for alpine grasslands elsewhere.28 These grasslands, despite intense grazing pressure, maintain good vegetation cover, high production and high species diversity, indicating that these meadows

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are grazophil communities, adapted to withstand grazing.29 Also, these alpine meadows have greater productivity and stocking capacity (7.13 sheep/ha) than the values reported for other cold climatic zones (0.4–2.0 sheep/ha).30 Interestingly, variation in height, manner of leaf deployment, such as vertical vs horizontal spread of shoots and periodicity of leafing enables a number of species to occupy same site, by reducing competition for vital resources in these ecosystems.31 Similarly the woodland ecosystems in the region can be grouped into two categories: (1) the forests and (2) the scrubs. Under both the categories various sub categories, in different parts of the Himalaya, are recognizable. While attempting the zonation of vegetation types many researchers have considered the Himalaya as a transition between tropical and temperate regions of Northern Hemisphere, and between dry west and humid east of the Himalaya.32–35 Major shifts in vegetation types are thus discernible across two simultaneously operating environmental gradients (i.e. warm to cool: temperature; and dry to humid: moisture conditions). Considering the close proximity of vegetation boundaries with climatic zones, the Himalayan woodlands can be grouped into following five broad zones (terminology largely follows the one used for northern temperate types35): (1) tropical/sub tropical (represented by three dominant/physiognomic types – Moist evergreen broadleaf forest Tropical Dry Deciduous and Semi evergreen Forests; (2) warm temperate (characterized by two dominant types – dry deciduous forests and needle leaf forests); (3) temperate (represents maximum diversity of dominant types which mainly include broadleaf evergreen forests, broadleaf deciduous forests temperate needleleaf evergreen forests); (4) sub alpine/cool temperate (dominated by Sub alpine conifer forests, Sub alpine broadleaf forests and (5) alpine (represented by alpine scrublands which include two distinct types (Alpine dry scrub and Alpine moist scrub). In view of the spatial geographical position of the Himalaya and uniqueness of its ecosystems, it is pertinent to assess the extent of representativeness in biological components of different ecosystems. Such studies assume significance while native species populations, with their natural interactions in naturally structured communities, are considered as best indicators of biological integrity. Unfortunately, such information is lacking in the region. However, while considering the ecological characteristics of Himalayan forests it is suggested that Himalayan forests differ significantly in the following attributes: (1) tree phenology, chemistry and form; (2) physiological responses; (3) ecosystem processes and (4) vegetation types.3 Uniqueness of biodiversity: Endemism Endemism is yet another important attribute which needs consideration while developing conservation strategies for the Himalayan biodiversity hotspot. Among


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floristic elements, besides nearly 32% of species being endemic, the region represents 71 endemic genera and five endemic families (i.e. Tetracentraceae, Hamamelidaceae, Circaeasteraceae, Butomaceae, and Stachyuraceae). A few families, for example Berberidaceae and Saxifragaceae, represent >90% species endemic to the Himalaya. A large number of orchids, many representing neo endemic taxa, have been recently reported from Sikkim and Arunachal Pradesh. Likewise, of the nearly 300 recorded mammal species across the region, 12 are endemic to the Himalaya. The endemics include the golden langur (Trachypithecus geei) – very restricted range in the Eastern Himalaya; the Himalayan tahr (Hemitragus jemlahicus); and the pygmy hog (Sus salvanius) – species restricted to grasslands in the Terai-Duar savannah and grasslands in the Manas National Park. The Namdapha flying squirrel (Biswamoyopterus biswasi) also represents the only endemic genus in the Himalaya, but is a poorly known species described on the basis of a single specimen taken from Namdapha National Park. Of the 979 bird species recorded from the region, 15 are endemics, including one species, the Himalayan quail (Ophrysia superciliosa) representing an endemic genus. Four Endemic Bird Areas (EBAs) overlap entirely or partly with the Himalaya Hotspot. Among other groups, reptiles (177 spp.) represent 49; amphibians (124 spp.) 41; and fishes (269 spp.) 33 species as endemic to this hot spot.2 The studies in IHR have revealed that the high altitude Himalaya (alpine and sub-alpine zone) is rich in plant endemic diversity. This feature is particularly prominent in Trans/Northwest and West Himalaya, suggesting thereby that the high altitude zone of these provinces can be considered as one of the endemic centers. Among endemics, prevalence of shizoendemics is reported from the western part (Kashmir) of the region which points toward their progressiveness.36

Conservation imperatives Realizing that the Himalayan ecosystem is highly vulnerable both due to geological reasons and on account of the stress caused by increased pressure of population, exploitation of natural resources and other related challenges need special attention. These vulnerability aspects may be exacerbated due to the impact of climate change. It is possible that climate change may adversely impact the Himalayan ecosystem through increased temperature, altered precipitation patterns, episodes of drought and biotic influences. This would not only impact the very sustenance of the indigenous communities in uplands but also the life of downstream dwellers across the country and beyond. Therefore, there is an urgent need for giving special attention to sustain the Himalayan Ecosystem.6 This would require conscious efforts for conserving all the representative systems. Further, it needs to be emphasized that the endemics with restricted distribution, and most often with specialized habitat requirements, are among

380


the most vulnerable elements. In this respect the Himalayan biodiversity hotspot, with rich endemic diversity, is vulnerable to climate change. The threats include possible loss of genetic resources and species, habitats and concomitantly a decrease in ecosystem services.37 Therefore, conservation of endemic elements in representative ecosystems/habitats assumes a great significance while drawing conservation plans for the region. Toward achieving the above, we will have to shift toward contemporary conservation approaches, which include a paradigm of landscape-level interconnectivity between protected area systems. The concept advocates a shift from the species-habitat focus to an inclusive focus on expanding the biogeographic range so that natural adjustments to climate change can proceed without being restrictive.37

Himalayan bioresources: Economic contributions Traditionally, indigenous communities in the Himalaya have been dependent on bioresources to meet basic sustenance needs, notably food, fodder, fuel, fertilizer, fiber, shelter, health care, etc. More than 80% of the population in the region is involved in agriculture, animal husbandry, forestry and other biodiversity dependent vocations. Among other bioresources with direct economic value, the Himalayan region is well recognized for diversity of medicinal plants, wild edibles and other nontimber forest produce. Medicinal plants The rich plant diversity of the Himalaya has been a source of medicine for millions of people in the country and elsewhere in the world. IHR supports over 1748 (23.4% of India) plant species of known medicinal value.38 The unique diversity of medicinal plants in the region is manifested by the presence of number of native (31%), endemic (15.5%) and threatened elements (14% of total Red Data Book plant species of IHR). The economic potential of Himalayan medicinal plants and their contribution in providing novel biomolecules is well recognized.39–41 While setting priorities for medicinal plants of IHR, it is reported that41: (1) of the nearly 280 medicinal plants being used in industry 175 are from IHR; (2) more than two third (122: 69.7%) of identified species from IHR are exposed to destructive harvesting, thereby adversely affecting the resource base; (3) industry extensively uses nonnatives particularly in robust life forms (shrubs 96.8%; trees 90.9%); (4) native percentage among exploited herbs is relatively high (20.2%) and most of them are restricted to specialized alpine habitats and (5) contribution of wild-cultivated forms of medicinal plants is poor (20%).


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Wild edible plants Among the economically important bioresources of IHR, the wild edibles have emerged as potential resources for addressing issues of rural development and biodiversity conservation, particularly on account of their nutritional and pharmaceutical potential.42–43 Over 675 species (angiosperms 647, gymnosperms 7; pteridophytes 12; fungi 7 and lichens 2 species) of wild edible species belonging to 384 genera and 149 families are known from IHR.44–45 Across biogeographic provinces, richness of known wild edibles is maximum in West Himalaya (344 spp.; 50.9%); followed by East Himalaya (221 spp.; 32.7%), Central Himalaya (173 spp.; 25.6%) and Trans/North West Himalaya (169 spp.; 25.0%). Of the total reported wild edibles, 39 (5.8%) species were restricted range endemics and 93 (13.8%) near endemics.45 Consumption of wild edibles meets the protein, carbohydrate, fat, vitamin and mineral requirement of poor rural populace in the region.46 Other reports suggest wild edibles can also generate substantial income. A study in Almora district, Indian west Himalaya, reveals that the sale of Myrica esculenta fruits (Kaiphalknown for edible fruits and other by products in sub-Himalayan region) brings each household Rs. 913–3713 (US$ 26–106) per season, which was significant considering low annual per-capita income in the region.43 Likewise, other wild edibles have been identified with income generating potential.42–47 Changing perspectives With respect to medicinal plants in the region, extensive use of wild forms and destructive harvesting by the industry is a serious threat. At the same time, dependence on relatively small proportion of available stock and that too on nonnative elements suggests that currently the pharmaceutical industry in the country is utilizing only a few true Himalayan medicinal plants.41 Further, recognizing the importance of the medicinal plant resources of the region and considering the emerging trends of global market, particularly issues of commercial patenting, there is a need to build the capacity of indigenous people to harness the opportunities. Further, need to integrate traditional knowledge system of health care into the national planning process so as to ensure participation of communities in preserving the resources and perpetuating knowledge for renewed research is being emphasized at different levels. The studies have proved that wild edibles of the region are promising source of nutrients and minerals. This potential coupled with proven medicinal value of some of these species, can be harnessed for promoting their use as health food supplements. The nutritional attributes varied considerably among species implying that potential of different species needs to be harnessed for specific

382


attribute. These results have significant bearing on bio-prospecting of wild edibles for rural development in the region wherein some of these can be projected as an important resource for income generation through value addition. Realizing the potential of mainstreaming medicinal and wild plants, there is a need to bring in the much-needed coordination among different players for development of these sectors. Effective measures to support marketing efforts with appropriate fiscal and policy support are urgently needed. However, as elsewhere in the world, there is a need to bring in a paradigm shift in realizing the economic contribution of Himalayan biodiversity. The attention needs to be focused on the value of ecosystem services. Particularly, the globally referred/ recognized concept of ‘Payments for Ecosystem Services’ requires serious efforts for implementation in the region under upcoming international climate frameworks.

Bioresource management: Existing scenario Conservation area network As advocated in the Article 8 of Convention of Biological Diversity promotion of in situ conservation is being considered important for the maintenance of biodiversity. This has led to emerging international commitment for strengthening Protected Area (PA) systems. Responding positively to global commitments to bring representative coverage of biological diversity under in situ conservation through strengthening of Protected Area Network, India has created a network of PAs and other conservation areas which include a total of 700 units (i.e. 99 National Parks, 513 Wildlife Sanctuaries, 43 conservation Reserves, 4 Community Reserves, 25 Ramsar sites and 16 Biosphere Reserves). The area covered under PAs and other conservation sites (Biosphere Reserves, Ramsar sites, etc.) accounts for around 9% of the total geographical area of the country. The Himalaya, a global conservation priority, has increasingly received attention of Government of India under its PA programme (Table 5).The progression of PA network expansion (both numbers and area coverage) in the IHR has been presented (Fig. 2). However, due to poor coordination and sufficient transparency in implementation, there are incidents which suggest that the inhabitants in and around PAs are frequently not aware of PA proclamation, and issues of their rights remain unsettled. This has caused an atmosphere of resentment against PAs in the region. All these issues are deleterious to the overall objectives of preserving PA values.48 The PA coverage in the IHR (7.6%) is higher than the national average (4.75%). The existing figures of PAs is apparently indicative of the satisfactory state of network system for conservation of representative ecosystems in the region. However, a more objective analysis of facts reveals the following:

Grand Total

09 North East

27

12

Total

12

Total

07

2

East

NE Hills

2

Central

05

4

Brahmaputra valley

4

West

3

Total

Northwest

1

Tibetan Plateau

02 Himalaya

2

Ladakh Mtn

01 Trans Himalaya

No. of NPs

Province Name

Zone Name

2.7

1.54

0.63

2.98

3.41

2.74

3.05

6.50

1.92

3.14

2.38

3.67

(% of) Biozone Area

98

36

18

18

58

11

08

14

25

4

3

1

No. of WLS

Table 5 Summary of Existing Protected Area Network in the IHR [Source: Ref. 65]

4.84

2.00

1.65

2.98

6.89

9.08

7.24

5.52

5.26

5.65

7.25

4.56

(% of) Biozone Area

125

48

25

23

70

13

10

18

29

7

4

3

No. NPs + WLS

7.6

3.71

2.29

5.96

10.30

11.82

10.29

12.02

7.18

8.79

9.63

8.22

(% of) Biozone Area

Conservation of Himalayan bioresources: An ecological, economical and evolutionary perspective 383

384


Figure 2. Progression of PA Network in IHR [(A)Numbers and (B) Area]


385

● Broadly the random distribution of PAs and representative coverage well above the national average (4.75%) in each province, barring NE Hills [Ladakh Mtn: 3 units (8.22% of area); Tibetan Plateau: 4 (9.63%); North west: 29 (7.18%); West: 18 (12.02%); Central: 10 (10.29%), East: 13 (11.82%); Brahmaputra Valley: 23(5.96%) and NE Hills: 25 (2.29%)], supposedly takes care of representative habitats, ecosystems and biota along longitudinal-east to west gradient of the region. The representativeness of these elements along horizontal (latitudinal-South to North) and vertical (low to high elevation) gradients needs to be looked into for individual provinces. In this regard bigger PAs with adequate vertical and horizontal expanse emerge as better candidates. The analysis of size of existing PAs in IHR, however, reveals that: (1) PAs in Trans Himalaya are relatively large (average size 2321.1 km2, n = 7); (2) in Himalayan zone the average size (310.0 km2; n = 70) is small. Within this zone, East Himalayan province (average size 761.3 km2, n = 13) has relatively large PAs, followed by west (347.77 km, n = 18), North west (171.8 km2, n = 29) and Central Himalayan province (56.1 km2, n = 10); (3) PAs in NE biogeographic zone are invariably smaller in size–average size 132.3 km2 (Brahmaputra Valley – 171.8 km2, n = 23; NE Hills – 96.0 km2, n = 25) and (4) over 50% of PAs in Himalayan biogeographic zone are below 100 km2. These figures need to be viewed for wider conservation implications, particularly considering the fact that small PAs are inadequate to (1) preserve large contiguous tracts of representative ecosystems/habitats, (2) viable populations of large key stone species and (3) safeguard overall ecological value of ecosystems. ● As reflected, the PA coverage in NE biogeographic zone, particularly in NE hill province, is poor. Whereas, from the biodiversity point of view, considering in particular the evolutionary importance as one of the centers of plant origin, this region holds great conservation significance. Therefore, it would require immediate attention for bringing in adequate and representative areas under PA network to ensure natural evolutionary processes to continue through strict in situ protection of habitats and ecosystems of the region. Recently, recognizing the need for holistic management of biodiversity at the landscape level ‘Biosphere Reserve’ concept is being promoted in the region. The concept considers conservation of landscapes having immense biodiversity value by maintaining cultural heritage and fostering economic and human development which is culturally and ecologically sustainable. Between 1988 till date a total of seven Biosphere Reserves, which represent 43.8% of total units in India, have been designated in different states of IHR [Cold desert – Himachal Pradesh (Trans Himalaya); Nanda Devi – West Himalaya (Uttarakhand); Kangechnjunga – Sikkim (Central Himalaya); Manas and Dibru-Saikhowa – Assam (Bahmaputra Valley); Nokrek-Meghalaya (NE Hills); and Dehang Debang – Arunachal Pradesh (East Himalaya)]. Considering the ecological, economic and evolutionary significance

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of such landscapes, there is a strong need to further strengthen Biosphere Reserve network in the Himalaya.6 In spite of all these initiatives with respect to conservation area network in the Himalaya, the indications are such that community involvement in Government sponsored or otherwise implemented management programmes in the region is decreasing. This calls for reorientation of strategies and action plans.

Bioresource management: Toward contemporary thinking Finding solutions through integration of traditional and cultural values The concept of ‘natural cultural landscape’ which sees cultural and biological diversity as mutually supportive49 appears most appropriate to ensure community support for maintaining and conserving the uniqueness of biodiversity (both wild and domesticated). This calls for proper integration of cultural values and Traditional Ecological Knowledge (TEK) system with modern approaches of management. In this context, one needs to find out tangible dimensions of TEK derived from ‘intangibles’ (Box 1). It would, however, also require putting in place adequate reward systems.6 Management: Synchronizing with sustainable use As indicated in the beginning, the Himalaya has emerged as a mega biodiversity center of the world with its biodiversity components (i.e. genetic, species and ecosystems) having global importance. However, the region is always referred to as economically marginal and as a home of the worlds’ poorest whose livelihoods depend heavily on natural resources, and any change in the status of these resources affects them severely.50 In this context, the Himalayan mountains being sensitive

Box 1: Tangible dimensions of TEK derived from intangibles [Source: Ref. 49] Economic: traditional ethnobiology (lesser-known plants and animals harvested from the wild and used for food and medicine) Socioecological: the ways in which traditional societies conserve and use biodiversity to manage soil fertility, nutrient cycling and soil moisture regimes Sociocultural: the cultural, spiritual and religious belief systems of mountain people which are centered around ‘sacred species’, ‘sacred groves’ and ‘sacred landscapes’


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ecosystems for both human induced and natural perturbations have exhibited rapid changes in its resource base and thereby affecting the sustenance of its people. As a result, the indigenous communities in the region have, by and large, begun to show disinterest toward management of natural resources including the bioresources. Specifically, they feel alienated on account of current policies and programmes of conservation which consider nonuse and existence value of biodiversity as points of prime focus. In the context of above, while initiatives for strengthening of conservation areas is essentially required, there is a need for reorienting existing norms and management practices by harnessing the income generating potential of such areas for local communities so as to promote their interest and participation in conservation.6 Biodiversity conservation: Linking with livelihoods There is a strong need to promote sustainable use concept which attempts to establish linkages between conservation and economic growth, and recognizes that the bioresources are a source for sustainable income. Experiences from the region itself have revealed that people become interested in biodiversity conservation when they realize that it has immediate utility value (i.e. subsistence or income generating opportunity) attached and they can harness benefits from it. A few examples include: (1) Developing biodiversity based enterprise while conserving the resources (e.g. Oak-silk in Garhwal (Uttarakhand); traditional local paper from loktak – Daphne spp. in Nepal and Sikkim, high value caterpillar plant ‘yarsha gumbu – Cordyceps sinensis and matsutake mushroom – Tricholoma matustake in Bhutan); (2) Niche product marketing from shifting cultivation (e.g. ‘kholari’ bean from the shifting cultivation areas of Nagaland); (3) Nature valuation for accrued benefits (e.g. valuation of Kangechnjunga National Park and Khecheopalri Sacred lake in Sikkim) and (4) Ecotourism and biodiversity conservation related enterprises (e.g. conservation of snow-leopard through community based tourism in Ladakh, biodiversity ecotourism initiatives in Sikkim, expansion of Chitwan National Park through community forestry and ecotourism development in Nepal).50 Yet another strategy, which has emerged as promising, is to maximize economic benefits by adding value to biodiversity components available in immediate vicinity of indigenous communities. An example can be cited from Uttarakhand, wherein Institute of Himalayan Environment and Education (INHERE), an NGO, focuses on promotion of agrodiversity of the mountain villages by converting this diversity into high-value products for niche markets. Basic to this effort has been the conversion of, largely by default, the organic agriculture of the mountains into certified organic products by adding value.51

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Efforts made by the G.B. Pant Institute of Himalayan Environment and Development (GBPIHED) in Uttarakhand, Himachal, Sikkim and Arunachal Pradesh for capacity building of inhabitants for effective use of bioresource based rural technologies (e.g. bio-briquettes, protected cultivation, poly-pit, nursery development, medicinal plant cultivation, mushroom cultivation, etc.) have received considerable attention across IHR.52 This has led people to reorient their mindset in a way so as to view bioresources or its products as effective source of income for rural inhabitants. Ecosystem approach: Appropriate option With the growing realization that elements of biodiversity are intricately linked and focusing on management of any one, ignoring the others, cannot be sustainable, there is a felt need for considering holistic or ecosystem based approach of management. It recognizes that humans, with their cultural diversity, are an integral component of ecosystems. The above implies that management of biodiversity components in any ecosystem would require integration of research outputs and human dimensions. One example can be drawn from the role of bees as pollinators to enhance agricultural productivity. The extensive researches have proved that the decline in production of apple in Himachal Pradesh is linked with disturbances in pollination process – a reason largely unknown to many of the growers and even agricultural extension workers.53 With this realization the rental of bee hives is getting popular for pollination among apple growers of Himachal Pradesh. This realization has also led India to participate in the formulation of a global pollinator project, being coordinated by GBPIHED, with targets of management of pollinators for sustainable agriculture through ecosystem approach in the Himalaya. Yet another example of holistic view of conservation can be cited as implementation of Village Environment Action Plan (VEAP) developed by GBPIHED. This includes approach of preparing developmental plan for village ecosystem using integration of its resources (physical, biological and human) and environmental hazard management through participatory approach, of village resource mapping, monitoring and evaluation.54 Likewise the Catchment Area Protection (CAP) and Assisted Natural Regeneration (ANR) initiatives of the Institute focus on ecosystem approach for participatory management of natural water and bioresources.55

Effective valuation: Ensuring transfer of payments While considering ecosystem approach, there is a growing concern to effectively attach monitory value to biodiversity, specifically the ecosystem services and make provisions for transfer of payments (compensations) to their protectors. An


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emerging issue is attaching value to forests. Under the efforts to implement the United Nations Framework Convention on Climate Change (UNFCCC), with a growing global realization that fuller valuation of forests is necessary if forests are to be conserved by local people, progress has been made to develop carbon markets. The trends on initiatives across the world are indicative of the fact that forestry efforts could play an increasingly important role in achieving the emission reduction targets agreed by signatories to the Kyoto Protocol. Experiences from the Uttarakhand (west Himalaya), which represents one of the oldest and largest system of Van Panchayat (introduced in 1930; over 12,000 villages; nearly 30% of total forest area of the State), under ‘Kyoto- Think Global, Act Local’ project has revealed that communities can be trained to use technology to measure carbon sequestration rate in their community forests. Estimates suggest that if trained communities are able to submit proposal through proper mechanisms one Van Panchayat alone could receive up to 1.08 Lakhs per annum for carbon sequestered by their forest. The funds generated through the sale of carbon credits can be used to meet their needs and can encourage people to save their forests as ‘Carbon Sinks’.56 The recently released guidelines by the Ministry of Environment and Forests [6] also emphasize upon the need to pay proper attention for harnessing benefits from the programmes like ‘Reduced Emission from Deforestation and Degradation’ (REDD) under the United Nations Framework Convention on Climate Change (UNFCC). This would require integration and involvement of diverse stakeholder groups for developing effective and feasible mechanisms to conserve carbon within existing forests and through slowing down the rates of deforestation and degradation of forests. Watershed Management: Ultimate solution Having understood the fact that linking with livelihoods, transfer of payments for services and ecosystem approach are among essential components of effective management strategy for Himalayan Biodiversity, the extent of scale of operation remains a major concern. The forested mountain watersheds are now viewed as being vital for the ecological health across the world. In addition to their important protective role, they shelter immense biodiversity, provide food and fodder, and are an important source of fresh water. These benefits are realized not only by the upland watershed communities but also by those who live much beyond its physical boundaries in downstream areas. Considering this value, integrated watershed management approach for mountains has emerged as an effective solution. It is an on-going process which actively involves local people and decision makers – in both upstream and downstream areas– in analyzing the problems and developing appropriate solutions to ensure that the activities undertaken are well adapted to the local situations.57

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In the context of Himalaya, Environmental and Social Management Framework; Guidelines and Implementation and Monitoring manual developed for Watershed Management Directorate (Uttarakhand) sets an example which largely follows the above approach of Integrated Watershed Management.58 Conservation Awareness: A key element Notwithstanding that the above mentioned approaches deserve immediate consideration for the adequate management of biodiversity in Himalayan ecosystem, there is an urgent need for designing and implementing programmes for creating mass awareness among different stakeholders. The approaches need to take note of location specific issues and specificities in the region. While recognizing this requirement, various organizations have initiated activities in this direction. However, a critical assessment of such initiatives most often results in realization that the approach followed lacks a holistic vision. As a result their long term effectiveness remains invisible. We, therefore, feel that it is a high time for introspection by all concerned. We would, however, like to cite two examples from the region. These approaches, in our opinion, possess unique strengths and have potential to serve long term management goals for the Himalayan Bioresources. The growing concern over Environmental Education (EE) programmes calls for developing perspective guidelines for implementation in both formal and informal sectors in order to bring in a perceptible shift in the mindset of communities toward integrating conservation science with societal needs. With regard to IHR, mountain specificities need to be accordingly included in such guidelines and programmes. Approaches developed through successful initiatives in the region; both in formal [e.g. initiative by an environmental NGO – Uttarakhand Seva Nidhi (UKSN), Almora (Uttarakhand)] and informal education [e.g. approach developed and tested by G.B. Pant Institute of Himalayan Environment and Development, Almora and Gauhati based CEE- North East] need to be promoted and considered for replication across the Himalayan states with area/location specific modifications. The concerned State Governments may initiate developing programmes in this direction and ensure implementation in consultation with suitable organizations.6

Conclusion Realizing that evolution is much more than a simple change within individual species, it builds up intricate, intertwined relationships between the earth and its inhabitants, we believe there is a vast scope and need of untangling the complexities of59 Himalayan ecosystem. The representative ecosystems and their unique (endemic) elements are different from the remaining world in many ways.


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The prevailing TEK systems, and potential economic values attached with goods and services emanating from the region, provide enough space to draw plans for long term maintenance of its resources. Such plans, while addressing the issues of livelihood needs of indigenous communities, would also accommodate the emerging needs under the changing climate and economic scenario. In particular, with perceived greater vulnerability of the Himalayan ecosystem to changing climate, it is certain that the ‘struggle for existence’ is going to intensify in the region. Therefore, the adaptation process, which largely follows course of natural selection, under changing climate will determine fate of its bioresources. However, understanding the process of change and its impacts is not an easy task. It would require deep wonderings, like Darwin did, for resolving many of nature’s mysteries. Integrated and participatory efforts, with deeper understanding of intricate linkages, will prove beneficial for conserving unique Himalayan ecosystem. In the above context it is pertinent to end this essay with the following quote from the Origin of Species by Charles Darwin.1 ‘Climate plays an important part in determining the average number of species, and periodical seasons of extreme cold or drought, I believe to be the most effective of all checks……………..The action of climate seems at first sight to be quite independent of the struggle for existence; but in so far as climate chiefly acts in reducing food, it brings on the most severe struggle between the individuals, whether of the same or of distinct species, which subsist on the same kind of food…..’ ‘When a species, owing to highly favourable circumstances, increases inordinately in numbers in a small tract, epidemics……often ensue: and here we have a limiting check independent of the struggle for life’. Acknowledgements: Authors thank various individuals and organizations from where the information has been drawn for building this paper.

References 1.

Darwin C (Ed.) (2006) In: The Origin of Species. Atlantic Publishers, New Delhi, India

2.

Conservation International [http://www.biodiversityhotspots.org]

3.

Zobel DB, Singh SP (1997) Himalayan forests and ecological generalizations. Bio Science 47:735–745

4.

Khoshoo TN (1992) Plant diversity in the Himalaya: Conservation and utilization. 2nd GB Pant Memorial Lecture. Kosi-Katarmal, GBPIHED, Almora

5.

Samal PK, Palni LMS, Agrawal DK (2003) Ecology, ecological poverty and sustainable development in central Himalayan region of India. Int J Sustain Dev World Ecol 10: 157–168

6.

Governance for Sustaining Himalayan Ecosystem: Guidelines and Best Practices (2009). Ministry of Environment and Forests, New Delhi

7.

Singh JS, Singh SP (1987) Forest vegetation of the Himalaya. Bot Rev 53:80–192

392


8.

Dhar U (1996) Overview of Himalayan biodiversity. In: Changing Perspectives of Biodiversity Status in the Himalaya. Gujral GS, Sharma V (Eds.), British Council Division, New Delhi

9.

Khoshoo TN (1996) Biodiversity in Indian Himalayas: Conservation and utilization. In: Banking on Biodiversity. Shengji P (Ed.), ICIMOD, Nepal

10.

Rodger WA, Panwar HS (Eds.) (1988) In: Planning Wildlife Protected Area Network in India. Vol. 1 and 2. Wildlife Institute of India, Dehradun

11.

Rodgers WA, Panwar HS, Mathur VB (Eds.) (2000) In: Wildlife Protected Areas Network in India: A Review (Executive Eummary). Wildlife Institute of India, Dehradun

12.

MacKinnon J, MacKinnon K (Eds.) (1986) In: Review of the Protected Areas System in the Indo-Malayan Realm. Gland, IUCN

13.

Takhtajan A (Ed.) (1969) In: Flowering Plants: Origin and Dispersal. Oliver and Boyd, Edinburgh

14.

Malhotra CL, Hajra PK (1977) Status of floristic studies in Arunachal Pradesh. Bull Bot Surv India 19:61–63

15.

Janaki Ammal EK (1950) Polyploidy in the genus Rhododendron. Rhododendron Year Book 92–96

16.

Janaki Ammal EK (1952) The race history of Magnolias. Indian J Genet Pl Breed 12:82– 92

17.

Janaki Ammal EK (1952) Chromosome relationships in cultivated species of Camellia. Am Camelia Year Book 106–114

18.

Janaki Ammal EK (1953) Chromosomes and species problem in the genus Viburnum. 22:4–6

19.

Janaki Ammal EK (1954) Cytogeography of the genus Buddleia in Asia. Sci Cult 19:578– 581

20.

Janaki Ammal EK, Saunders B (1952) Chromosome numbers in species of Lonicera. Kew Bull 539

21.

Arora RK, Nayar ER : Wild relatives of crop plants in India. New Delhi: National Bureau of Plant Genetic Resources, Monogr. No. 7 (1984)

22.

Arora RK (1997) Diversity and distribution of wild relatives of crop plants in the Himalaya. In: Himalayan Biodiversity: Action Plan. Dhar U (Ed.), Gyanodaya Prakashan, Nainital

23.

Chakravorty AK (1951) Origin of cultivated banana of south-east Asia. Ind J Genet 11:34–46

24.

Rao RR (1993) Floristic diversity of eastern Himalaya – a national heritage for conservatuion. In: Himalayan Biodiversity. Dhar U (Ed.), Gyanodaya Prakashan, Nainital

25.

Arora, RK (1993) Himalayan plant resources: diversity and conservation. In: Himalayan Biodiversity. Dhar U (Ed.), Gyanodaya Prakashan, Nainital

26.

Palni LMS (1993) Conservation of plant genetic resources: the role of biotechnology. In: Himalayan Biodiversity. Dhar U (Ed.), Gyanodaya Prakashan, Nainital

27.

Johnsingh AJT, Rawat GS, Sathyakumar S, Karunakaran PV, Kuar J (2000) Prioritization of areas for biodiversity conservation of alpine zone in the trans and greater Himalaya in India. In: Setting Biodiversity Conservation Priorities for India. Singh S, et al. (Eds.), WWF–India, New Delhi

28.

Jeet Ram, Singh SP, Singh JS (1989) Plant biomass, species diversity and net primary production in a central Himalayan high altitude grassland. J Ecology 77:456–468


393

29.

Rawat GS, Rodgers WA (1987) The alpine meadows of Utter Pradesh: An ecological review. In: Proc. of National Rangeland Symposium. IGFRI, Jhansi

30.

Jeet Ram, Singh SP (1994) Ecology and conservation of alpine meadows in central Himalaya, India. In: High Altitudes of the Himalaya. Pangty YPS, Rawal RS (Eds.), Gyanodaya Prakashan, Nainital

31.

Negi GCS, Rikhari HC, Singh SP (1992) Phenology features in relation to growth forms and biomass accumulation in an alpine meadow of the central Himalaya. Vegetatio 101:161–170

32.

Schweinfurth U (1957) Die horizontale und vertkale verbreituny der vegetation in Himalaya. Bonner Geogr Abh 29:1–372

33.

Meusel H (1971) Mediterranean elements in the flora and vegetation of the west Himalaya. In: Plant Life of South west Asia. Davis PH, Harper PC, Hedge IC (Eds.), Botanical Society, Edinburghs

34.

Ohsawa M, Shakya PR, Numata M (1986) Distribution and succession of west Himalayan forest types in the eastern part of the Nepal Himalaya. Mount Res Dev 6:143–157

35.

Ohsawa M (1987) Vegetation zones in the Bhutan Himalaya. In: Life Zone Ecology of the Bhutan Himalaya. Ohsawa M (Ed.), Chiba University, Japan

36.

Dhar U (2002) Conservation implications of plant endemism in high altitude Himalaya. Curr Sci 82:141–148

37.

Sharma E, Tsering K (2009) Climate change in Himalayas: The vulnerability of biodiversity. In: Biodiversity and Climate Change. International Centre for Integrated Mountain Devlopment, Kathmandu, Nepal

38.

Samant SS, Dhar U, Palni LMS (Eds.) (1998) In: Medicinal Plants of Indian Himalaya. Himvikas Publ 13. Kosi-Katarmal, GBPIHED, Almora

39.

Nautiyal AR, Nautiyal MC, Purohit AN (1997) In: Harvesting Herbs – 2000. Singh B, Singh M (Eds.), Dehradun

40.

Dhawan BN (1997) Biodiversity: A valuable resource for new molecules. In: Himalayan Biodiversity: Action Plan. Dhar U (Ed.), Gyanodaya Prakashan, Nainital

41.

Dhar U, Rawal RS, Upreti J (2000) Setting priorities for conservation of medicinal plants – a case study in the Indian Himalaya. Biol Cons 95:57–65

42.

Maikhuri RK, Rao KS, Saxena KG (2004) Bioprospecting of Wild edibles for Rural Development in the Central Himalayan Mountains of India. Mount Res Dev 24(2): 110–113

43.

Bhatt ID, Rawal RS, Dhar U (2000) The availability, fruit yield and harvest of Myrica esculenta Buch-Ham ex D. Don in Kumaun (West Himalaya), India. Mount Res Dev 20:146–153

44.

Samant SS, Dhar U (1997) Diversity, endemism and economic potential of wild edible plants of Indian Himalaya. Int J Sustain Dev World Ecol 4:179–191

45.

Samant SS, Dhar U, Rawal RS (2001) Diversity and Distribution of Wild Edible Plants of Indian Himalaya. In: Plant Diversity of the Himalaya. Pande PC, Samant SS (Eds.), Gyanodaya Prakashan, Nainital

46.

Sundriyal M, Sundriyal RC (2001) Wild edible plants of the Sikkim Himalaya: Nutritive value of selected species. Econ Bot 55: 377–390

47.

Dhyani PP, Khali MP (1993) Fruit yield and Economics of Jelly and Jam production from fruits of some promising Ficus (fig) tree crops. Ecol Food Nutr 30:169–178

394


48.

Rawal RS, Dhar U (2001) Protected Area network in Indian Himalayan region: Need for recognizing values of low profile protected areas. Curr Sci 81:175–184

49.

Ramakrishnan PS (2009) Linking traditional ecological knowledge systems with modern approaches. In: Biodiversity and Climate Change. ICIMOD, Kathmandu, Nepal

50.

Sharma E, Chettri N, Kerkhoff E (2005) Opportunities for income generation through biodiversity conservation. ICIMOD Newsletter 48:9–11

51.

Bisht BS (2005) Enhancing life and livelihoods in the villages of Uttarakhand. ICIMOD Newsletter 48:35

52.

Anonymous: Over the years – stakeholder driven Research and Development (2007) G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora

53.

Pratap U, Ahmed F (2005) A new and more rewarding vocation for beekeepers in Himachal Pradesh, India. ICIMOD Newsletter 48:15–17

54.

Kumar K, Rawat DS, Negi GCS, Agrawal DK, Satyal GS, Palni LMS (Eds.) (2000) In: Village Environment Action Plan – Community Manual. G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora

55.

Kumar K (Ed.) (2005) Catchment Area Protection and Assisted Natural Regeneration (FTR submitted to PMU, SWAJAL, Uttarakhand). G.B. Pant Institute of Himalayan Environment and Development, Kosi-Katarmal, Almora

56.

Tewari A, Phartiyal P (2006) The carbon market as efficient livelihoods opportunity for communities of the Himalaya. ICIMOD Newsletter 49:26–27

57.

Price MF (2004) Conservation and sustainable development in mountain areas. IUCN, Gland, Switzerland

58.

Anonymous (2003) Environmental and social management framework. Guidelines prepared for Uttaranchal Decentralized Watershed Project, Govt. of Uttaranchal. Kosi-Katarmal, GBPIHED Almora

59.

Milner R (1999) Charles Darwin – Evolution of a Naturalist. University Press (India) Limited, Hyderabad

60.

Anonymous (1998) Ecoregional cooperation for biodiversity conservation in the Himalaya. Proceeding of a regional meeting organized by UNDP in cooperation with WWF and ICIMOD

61.

Champion HG, Seth SK (1968) A revised survey of the forest types of India. Manager of Publications, Delhi

62.

Singh DK, Hajra PK (1996) Floristic diversity. In: Changing Perspectives of Biodiversity Status in the Himalaya. Gujral GS, Sharma V (Eds.), British Council Division, New Delhi

63.

Ghosh AK (1997) Himalayan fauna with special reference to endangered and endemic species. In: Himalayan Biodiversity: Action Plan. Dhar U (Ed.), Gyanodaya Prakashan, Nainital

64.

Purohit AN, Dhar U (1997) Himalayan tree diversity-an update. Proc Indian Natn Sci Acad B63:187–209

65.

Anonymous (2009) National Wildlife Database. Wildlife Institute of India, Dehradun

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(a) P. Dayanandan: Figure 5, Page 5

(a) V.P. Sharma: Figure 1, Page 23

(b) V.P. Sharma: Figure 2, Page 24

(c) V.P. Sharma: Figure 3, Page 25

(d) V.P. Sharma: Figure 6, Page 28

(e) V.P. Sharma: Figure 7, Page 29

(a) Veena Tandon and Gaurangi Maitra: Figure 1, Page 37

(b) Veena Tandon and Gaurangi Maitra: Figure 2, Page 37

(c) Veena Tandon and Gaurangi Maitra: Figure 4, Page 41

(d) Veena Tandon and Gaurangi Maitra: Figure 5, Page 42

(e) Veena Tandon and Gaurangi Maitra: Figure 6, Page 42

(f) Veena Tandon and Gaurangi Maitra: Figure 8, Page 45

(g) Veena Tandon and Gaurangi Maitra: Figure 9, Page 46

~4.5 billion years

Cyanobacteria

Earth birth

Eukaryotes

Single celled

Scale

Multicellular

Mammals Vertebrate

Time

500 million years 50 million years 5 million years

= represents time required for continuous re-incarnation of a 100 year old human more than 50,000 times

(a) Amit Sharma: Figure 1, Page 61

(b) Amit Sharma: Figure 2, Page 65

(c) Amit Sharma: Figure 4, Page 67

(a) Anupam Varma and Shelly Praveen: Figure 1, Page 77

(d) Amit Sharma: Figure 5, Page 69 (b) Anupam Varma and Shelly Praveen: Figure 2, Page 78

(c) Anupam Varma and Shelly Praveen: Figure 3, Page 79

(e) Anupam Varma and Shelly Praveen: Figure 5, Page 82

(d) Anupam Varma and Shelly Praveen: Figure 4, Page 82

(f) Anupam Varma and Shelly Praveen: Figure 6, Page 84

(g) Anupam Varma and Shelly Praveen: Figure 7, Page 86

(h) Anupam Varma and Shelly Praveen: Figure 8, Page 87

(i) Anupam Varma and Shelly Praveen: Figure 9, Page 88 (j) Anupam Varma and Shelly Praveen: Figure 10, Page 89

(a) Radha Prasanna and B.D. Kaushik: Figure 1, Page 125

(a) V.V. Ramamurthy and Asha Gaur: Figure 4 (a,b,c,d,e,f), Page 186

(a)Rajesh Tandon and H.Y. Mohan Ram: Figure 1(a,b,c,d,e), Page 225

(a) Lok Man S. Palni and Ranbeer S. Rawal: Figure 1, Page 372

(b) Lok Man S. Palni and Ranbeer S. Rawal: Figure 2, Page 384

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