Wild Crop Relatives: Genomic and Breeding Resources: Forest Trees

Wild Crop Relatives: Genomic and Breeding Resources

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Chittaranjan Kole Editor

Wild Crop Relatives: Genomic and Breeding Resources Forest Trees

Editor Prof. Chittaranjan Kole Director of Research Institute of Nutraceutical Research Clemson University 109 Jordan Hall Clemson, SC 29634 [email protected]

ISBN 978-3-642-21249-9 e-ISBN 978-3-642-21250-5 DOI 10.1007/978-3-642-21250-5 Springer Heidelberg Dordrecht London New York Library of Congress Control Number: 2011922649 # Springer-Verlag Berlin Heidelberg 2011 This work is subject to copyright. All rights are reserved, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilm or in any other way, and storage in data banks. Duplication of this publication or parts thereof is permitted only under the provisions of the German Copyright Law of September 9, 1965, in its current version, and permission for use must always be obtained from Springer. Violations are liable to prosecution under the German Copyright Law. The use of general descriptive names, registered names, trademarks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. Cover design: deblik, Berlin Printed on acid-free paper Springer is part of Springer Science+Business Media (www.springer.com)

Dedication

Dr. Norman Ernest Borlaug,1 the Father of Green Revolution, is well respected for his contributions to science and society. There was or is not and never will be a single person on this Earth whose single-handed service to science could save millions of people from death due to starvation over a period of over four decades like Dr. Borlaug’s. Even the Nobel Peace Prize he received in 1970 does not do such a great and noble person as Dr. Borlaug justice. His life and contributions are well known and will remain in the pages of history of science. I wish to share some facets of this elegant and ideal personality I had been blessed to observe during my personal interactions with him. It was early 2007 while I was at the Clemson University as a visiting scientist one of my lab colleagues told me that “somebody wants to talk to you; he appears to be an old man.” I took the telephone receiver casually and said hello. The response from the other side was – “I am Norman Borlaug; am I talking to Chitta?” Even a million words would be insufficient to define and depict the exact feelings and thrills I experienced at that moment!

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The photo of Dr. Borlaug was kindly provided by Julie Borlaug (Norman Borlaug Institute for International Agriculture, Texas A&M Agriculture) the granddaughter of Dr. Borlaug.

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I had seen Dr. Borlaug only once way back in 1983 when he came to New Delhi, India to deliver the Coromandal Lecture organized by Prof. M.S. Swaminathan on the occasion of the 15th International Genetic Congress. However, my real interaction with him began in 2004 when I had been formulating a seven-volume book series entitled Genome Mapping and Molecular Breeding in Plants. Initially, I was neither confident of my ability as a series/book editor nor of the quality of the contents of the book volumes. I sent an email to Dr. Borlaug attaching the table of contents and the tentative outline of the chapters along with manuscripts of only a few sample chapters, including one authored by me and others, to learn about his views as a source of inspiration (or caution!) I was almost sure that a person of his stature would have no time and purpose to get back to a small science worker like me. To my utter (and pleasant) surprise I received an email from him that read: “May all Ph.D.’s, future scientists, and students that are devoted to agriculture get an inspiration as it refers to your work or future work from the pages of this important book. My wholehearted wishes for a success on your important job.” I got a shot in my arm (and in mind for sure)! Rest is a pleasant experience – the seven volumes were published by Springer in 2006 and 2007, and were welcome and liked by students, scientists, and their societies, libraries, and industries. As a token of my humble regards and gratitude, I sent Dr. Borlaug the volumes. And here started my discovery of the simplest person on Earth who solved the most complex and critical problem of people on it – hunger and death. Just one month after receiving the volumes, Dr. Borlaug called me one day and said, “Chitta, you know I cannot read a lot now-a-days, but I have gone through only on the chapters on wheat, maize and rice. Please excuse me. Other chapters of these volumes will be equally excellent, I believe.” He was highly excited to know that many other Nobel Laureates including Profs. Arthur Kornberg, Werner Arber, Phillip Sharp, G€unter Blobel, and Lee Hartwell also expressed generous comments regarding the utility and impact of the book series on science and the academic society. While we were discussing many other textbooks and review book series that I was editing at that time, again in my night hours for the benefit of students, scientists, and industries, he became emotional and told me to forget about my original contributions and that I deserved at least the World Food Prize, if not Nobel Prize for peace like him. I felt honored but really very ashamed as I am aware of my almost insignificant contribution in comparison to his work, and was unable to utter any words for a couple of minutes! In another occasion he wanted some documents from me. I told him that I will send them as attachments in emails. Immediately he shouted and told me: “You know, Julie (his granddaughter) is not at home now and I cannot check email myself. Julie does this for me. I can type myself in type writer but I am not good in computer. You know what, I have a Xerox machine and it receives fax also. Send me the documents by fax.” Here was the ever-present child in him. Another occasion is when I was talking with him in a low voice, and he immediately chided me: “You know that I cannot hear well now-a-days; I don’t know where Julie has kept the hearing apparatus, can’t you speak louder?” Here was the fatherly figure who was eager to hear each of my words! I still shed tears when I remember during one of our telephone conversations he asked: “You know I have never seen you, are you coming to Texas in the near future by chance?” I remember we were going through a financial paucity at that time and I could not make a visit to Texas to see him, though it would have been a great honor.

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In late 2007, whenever I tried to talk to Dr. Borlaug, he used to beckon Julie to bring the telephone to him, and in course of time Julie used to keep alive communications between us when he slowly succumbed to his health problems. The remaining volumes of the Genome Mapping and Molecular Breeding in Plants were published in 2007, and I sent him the volumes. I wished to learn about his views. During this period he could hardly speak and write. Julie prepared a letter on his behalf that read: “Dear Chitta, I have reviewed the seven volumes of the series on Genome Mapping and Molecular Breeding in Plants, which you have authored. You have brought together genetic linkage maps based on molecular markers for the most important crop species that will be a valuable guide and tool to further molecular crop improvements. Congratulations for a job well done.” During one of our conversations in mid-2007, he asked me what other book projects I was planning for Ph.D. students and scientists. I told him that the wealth of wild species already utilized and to be utilized for genetic analysis and improvement of domesticated crop species have not been deliberated in any book project. He was very excited and told me to take up the book project as soon as possible. By that time I had a huge commitment to editing book volumes and could not start the series he was so interested about. His sudden demise in September 2009 kept me so morose for a number of months that I did not even communicate my personal loss to Julie. But in the meantime, I formulated a ten-volume series on Wild Crop Relatives: Genomic and Breeding Resources for Springer. And whom else to dedicate this series to other than Dr. Borlaug! I wrote to Julie for her formal permission and she immediately wrote me: “Chitta, Thank you for contacting me and yes I think my grandfather would be honored with the dedication of the series. I remember him talking of you and this undertaking quite often. Congratulations on all that you have accomplished!” This helped me a lot as I could at least feel consoled that I could do a job he wanted me to do and I will always remain grateful to Julie for this help and also for taking care of Dr. Borlaug, not only as his granddaughter but also as the representative of millions of poor people from around the glove and hundreds of plant and agricultural scientists who tries to follow his philosophy and worship him as a father figure. It is another sad experience of growing older in life that we walk alone and miss the affectionate shadows, inspirations, encouragements, and blessings from the fatherly figures in our professional and personal lives. How I wish I could treat our next generations in the same way as personalities like Dr. Norman Borlaug did to me and many other science workers from around the world! During most of our conversations he used to emphasize the immediate impact of research on the society. A couple of times he even told me that my works on molecular genetics and biotechnology, particularly of 1980s and 1990s, have high fundamental importance, but I should also do some works that will benefit people. This advice elicited a change in my approach to science and since then I have been devotedly endeavored to develop crop varieties enriched with phytomedicines and nutraceuticals. Inspiration, advices, and blessings of Dr. Borlaug have influenced both my personal and professional life, particularly my approach to science, and I dedicate this series to him as a token of my regards and gratitude, and in remembrance of his great contribution to science and society and above all his personal affection for me.

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Dedication

I emailed the above draft of the dedication page to Julie for her views and I wish to complete my humble dedication with great satisfaction with the words of Julie who served as the living ladder for me to reach and stay closer to such as great human being as Dr. Borlaug and expressing my deep regards and gratitude to her. Julie’s email read: “Chitta, Thank you for sending me the draft dedication page. I really enjoyed reading it and I think you captured my grandfather’s spirit wonderfully. . .. So thank you very much for your beautiful words. I know he would be and is honored.” Clemson, USA

Chittaranjan Kole

Preface

Wild crop relatives have been playing enormously important roles both in the depiction of plant genomes and the genetic improvement of their cultivated counterparts. They have contributed immensely to resolving several fundamental questions, particularly those related to the origin, evolution, phylogenetic relationship, cytological status and inheritance of genes of an array of crop plants; provided several desirable donor genes for the genetic improvement of their domesticated counterparts; and facilitated the innovation of many novel concepts and technologies while working on them directly or while using their resources. More recently, they have even been used for the verification of their potential threats of gene flow from genetically modified plants and invasive habits. Above all, some of them are contributing enormously as model plant species to the elucidation and amelioration of the genomes of crop plant species. As a matter of fact, as a student, a teacher, and a humble science worker I was, still am and surely will remain fascinated by the wild allies of crop plants for their invaluable wealth for genetics, genomics and breeding in crop plants and as such share a deep concern for their conservation and comprehensive characterization for future utilization. It is by now a well established fact that wild crop relatives deserve serious attention for domestication, especially for the utilization of their phytomedicines and nutraceuticals, bioenergy production, soil reclamation, and the phytoremediation of our ecology and environment. While these vastly positive impacts of wild crop relatives on the development and deployment of new varieties for various purposes in the major crop plants of the world agriculture, along with a few negative potential concerns, are presented, the need for reference books with comprehensive examinations of the wild relatives of all the major field and plantation crops and fruit and forest trees is indeed imperative. This was the driving force behind the inception and publication of this series. Unlike the previous six book projects I have edited alone or with co-editors, this time it was very difficult to formulate uniform outlines for the chapters of this book series for several obvious reasons. Firstly, the status of the crop relatives is highly diverse. Some of them are completely wild, some are sporadically cultivated and some are at the initial stage of domestication for specific breeding objectives recently deemed essential. Secondly, the status of their conservation varies widely: some have been conserved, characterized and utilized; some have been eroded completely except for their presence in their center(s) of origin; some are at-risk or endangered due to genetic erosion, and some of them have yet to be explored. The third constraint is the variation in their relative worth, e.g. as academic model, breeding resource, etc. and/or potential as “new crops”. The most perplexing problem for me was to assign them to different volumes dedicated to crop relatives of diverse crops grouped based on their utility. ix

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This can be exemplified with Arabidopsis, which has primarily benefited the Brassicaceae crops but also facilitated genetic analyses and improvement in crop plants in other distant families; or with many wild relatives of forage crops that paved the way for the genetic analyses and breeding of some major cereal and millet crops. The same is true for wild crop relatives such as Medicago trunculata, which has paved the way for in-depth research on two crop groups of diverse use: oilseed and pulse crops belonging to the Fabaceae family. The list is too long to enumerate. I had no other choice but to compromise and assign the crop relatives in a volume on the crop group to which they are taxonomically closest and to which they can make the greatest contributions. For example, I placed the chapter on Arabidopsis in the volume on oilseeds, which deals with the wild relatives of Brassicaceae crops. However, we have tried to include deliberations pertinent to the individual genera or species of the wild crop relatives to which the chapters are devoted. Descriptions of the geographical locations of origin and genetic diversity, geographical distribution, karyotype and genome size, morphology, etc. have been included for most of them. Their current utility status – whether recognized as model species, weeds, invasive species or potentially cultivable taxa – is also delineated. The academic, agricultural, medicinal, ecological, environmental and industrial potential of both the cultivated and/or wild allied taxa are discussed. The conservation of wild crop relatives is a much discussed yet equally neglected issue albeit the in situ and ex situ conservation of some luckier species were initiated earlier or are being initiated now. We have included discussions on what has happened and what is happening with regard to the conservation of the crop relatives, thanks to national and international endeavors, in most of the chapters and also included what should happen for the wild relatives of the so-called new, minor, orphan or future crops. The botanical origin, evolutionary pathway and phylogenetic relationship of crop plants have always attracted the attention of plant scientists. For these studies morphological attributes, cytological features and biochemical parameters were used individually or in combinations at different periods based on the availability of the required tools and techniques. Access to different molecular markers based on nuclear and especially cytoplasmic DNAs that emerged after 1980 refined the strategies required for precise and unequivocal conclusions regarding these aspects. Illustrations of these classical and recent tools have been included in the chapters. Positioning genes and defining gene functions required in many cases different cytogenetic stocks, including substitution lines, addition lines, haploids, monoploids and aneuploids, particularly in polyploid crops. These aspects have been dealt in the relevant chapters. Employment of colchiploidy, fluorescent or genomic in situ hybridization and Southern hybridization have reinforced the theoretical and applied studies on these stocks. Chapters on relevant genera/species include details on these cytogenetic stocks. Wild crop relatives, particularly wild allied species and subspecies, have been used since the birth of genetics in the twentieth century in several instances such as studies of inheritance, linkage, function, transmission and evolution of genes. They have been frequently used in genetic studies since the advent of molecular markers. Their involvement in molecular mapping has facilitated the development of mapping populations with optimum polymorphism to construct saturated maps and also illuminating the organization, reorganization and functional aspects of genes and genomes. Many phenomena such as genomic duplication, genome reorganization,

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self-incompatibility, segregation distortion, transgressive segregation and defining genes and their phenotypes have in many cases been made possible due to the utilization of wild species or subspecies. Most of the chapters contain detailed elucidations on these aspects. The richness of crop relatives with biotic and abiotic stress resistance genes was well recognized and documented with the transfer of several alien genes into their cultivated counterparts through wide or distant hybridization with or without employing embryo-rescue and mutagenesis. However, the amazing revelation that the wild relatives are also a source of yield-related genes is a development of the molecular era. Apomictic genes are another asset of many crop relatives that deserve mention. All of these past and the present factors have led to the realization that the so-called inferior species are highly superior in conserving desirable genes and can serve as a goldmine for breeding elite plant varieties. This is particularly true at a point when natural genetic variability has been depleted or exhausted in most of the major crop species, particularly due to growing and promoting only a handful of so-called high-yielding varieties while disregarding the traditional cultivars and landraces. In the era of molecular breeding, we can map desirable genes and polygenes, identify their donors and utilize tightly linked markers for gene introgression, mitigating the constraint of linkage drag, and even create pyramid genes from multiple sources, cultivated or wild taxa. The evaluation of primary, secondary and tertiary gene pools and utilization of their novel genes is one of the leading strategies in present-day plant breeding. It is obvious that many wide hybridizations will never be easy and involve near-impossible constraints such as complete or partial sterility. In such cases gene cloning and gene discovery, complemented by intransgenic breeding, will hopefully pave the way for success. The utilization of wild relatives through traditional and molecular breeding has been thoroughly enumerated over the chapters throughout this series. Enormous genomic resources have been developed in the model crop relatives, for example Arabidopsis and Medicago. BAC, cDNA and EST libraries have also been developed in some other crop relatives. Transcriptomes and metabolomes have also been dissected in some of them. However, similar genomic resources are yet to be constructed in many crop relatives. Hence this section has been included only in chapters on the relevant genera or species. In this book series, we have included a section on recommendations for future steps to create awareness about the wealth of wild crop relatives in society at large and also for concerns for their alarmingly rapid decrease due to genetic erosion. The authors of the chapters have also emphasized the imperative requirement of their conservation, envisaging the importance of biodiversity. The importance of intellectual property rights and also farmers’ rights as owners of local landraces, botanical varieties, wild species and subspecies has also been dealt in many of the chapters. I feel satisfied that the authors of the chapters in this series have deliberated on all crucial aspects relevant to a particular wild genus or species in their chapters. I am also very pleased to present many chapters in this series authored by a large number of globally reputed leading scientists, many of whom have contributed to the development of novel concepts, strategies and tools of genetics, genomics and breeding and/or pioneered the elucidation and improvement of particular plant genomes using both traditional and molecular tools. Many of them have already retired or will be retiring soon, leaving behind their legacies and philosophies for us to follow and practice. I am saddened that a few of them have passed away during

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preparation of the manuscripts for this series. At the same time, I feel blessed that all of these stalwarts shared equally with me the wealth of crop relatives and contributed to their recognition and promotion through this endeavor. I would also like to be candid with regard to my own limitations. Initially I planned for roughly 150 chapters devoted to essential genera or species of wild crop relatives. However, I had to exclude a few of them either due to insignificant progress made on them during the preparation of this series, my failure to identify interested authors willing to produce acceptable manuscripts in time or authors’ backing out in the last minute, leaving no time to find replacements. I console myself for this lapse with the rationale that it is simply too large a series to achieve complete satisfaction on the contents. Still I was able to arrange 126 chapters in the ten volumes, contributed by 380 authors from 39 countries of the world. I extend my heartfelt thanks to all of these scientists, who have cooperated with me since the inception of this series not only with their contributions, but also in some cases by suggesting suitable authors for chapters on other genera/species. As happens with a mega-series, a few authors had delays for personal or professional reasons, and in a few cases, for no reason at all. This caused delays in the publication of some of the volumes and forced the remaining authors to update their manuscripts and wait too long to see their manuscripts in published form. I do shoulder all the responsibilities for this myself and offer my sincere apologies. Another unique feature of this series is that the authors of chapters dedicated to some genera/species have dedicated their chapters to scientists who pioneered the exploration, description and utilization of those wild genera/species. We have duly honored their sincere decision with equal respect for the scientists they rightly reminded us to commemorate. Editing this series was, to be honest, very taxing and painstaking, as my own expertise is limited to a few cereal, oilseed, pulse, vegetable, and fruit crops, and some medicinal and aromatic plants. I spent innumerable nights studying to attain the minimum eligibility to edit the manuscripts authored by experts with even life-time contributions on the concerned genera or species. However, this indirectly awakened the “student-for-life” within me and enriched my arsenal with so many new concepts, strategies, tools, techniques and even new terminologies! Above all, this helped me to realize that individually we know almost nothing about the plants on this planet! And this realization strikingly reminded me of the affectionate and sincere advice of Dr. Norman Borlaug to keep abreast with what is happening in the crop sciences, which he used to do himself even when he had been advised to strictly limit himself to bed rest. He was always enthusiastic about this series and inspired me to take up this huge task. This is one of the personal and professional reasons I dedicated this book series to him with a hope that the present and future generations of plant scientists will share the similar feelings of love and respect for all plants around us for the sake of meeting our never-ending needs for food, shelter, clothing, medicines, and all other items used for our basic requirements and comfort. I am also grateful to his granddaughter, Julie Borlaug, for kindly extending her permission to dedicate this series to him. I started editing books with the seven-volume series on Genome Mapping and Molecular Breeding in Plants with Springer way back in 2005, and I have since edited many other book series with Springer. I always feel proud and satisfied to be a member of the Springer family, particularly because of my warm and enriching working relationship with Dr. Sabine Schwarz and Dr. Jutta Lindenborn, with whom

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I have been working all along. My special thanks go out to them for publishing this “dream series” in an elegant form and also for appreciating my difficulties and accommodating many of my last-minute changes and updates. I would be remiss in my duties if I failed to mention the contributions of Phullara – my wife, friend, philosopher and guide – who has always shared with me a love of the collection, conservation, evaluation, and utilization of wild crop relatives and has enormously supported me in the translation of these priorities in my own research endeavors – for her assistance in formulating the contents of this series, for monitoring its progress and above all for taking care of all the domestic and personal responsibilities I am supposed to shoulder. I feel myself alien to the digital world that is the sine qua non today for maintaining constant communication and ensuring the preparation of manuscripts in a desirable format. Our son Sourav and daughter Devleena made my life easier by balancing out my limitations and also by graciously tolerating my sparing some time rightly deserved by them and constantly supporting me in the publication of this series. I take the responsibility for any lapses in content, format and approach of the series and individual volumes and also for any other errors, either scientific or linguistic, and will look forward to receiving readers’ corrections or suggestions for improvement. As I mentioned earlier this series consists of ten volumes. These volumes are dedicated to wild relatives of Cereals, Millets and Forage Grasses, Oilseeds, Legume Crops and Forages, Vegetables, Temperate Fruits, Tropical and Subtropical Fruits, Industrial Crops, Plantation and Ornamental Crops, and Forest Trees. This volume “Wild Crop Relatives: Genomic and Breeding Resources – Forest Trees” includes 8 chapters dedicated to Alnus, Corylus, Cryptomeria, Eucalyptus, Juglans, Quercus, Santalum, and Trigonobalanus. The chapters of this volume were authored by 16 scientists from 4 countries of the world, namely Australia, India, Japan, and the USA. It is my sincere hope that this volume and the series as a whole will serve the requirements of students, scientists and industries involved in studies, teaching, research and the extension of forest trees with an intention of serving science and society. Clemson, USA

Chittaranjan Kole

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Contents

1

Alnus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 Brian D. Vanden Heuvel

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Corylus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Thomas J. Molnar

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Cryptomeria . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Yoshihiko Tsumura

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Eucalyptus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Robert J. Henry

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Juglans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Keith Woeste and Charles Michler

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Quercus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Preston R. Aldrich and Jeannine Cavender-Bares

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Santalum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 131 Madhugiri Nageswara Rao, Jaya R. Soneji, and Padmini Sudarshana

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Trigonobalanus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145 Weibang Sun, Yuan Zhou, Chunyan Han, Gao Chen, and Yanling Zheng Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161

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Abbreviations

ABA AFLP AMOVA ARS ATSC BGCI BTS CAPS CDF cpDNA ECM ECP/GR EFB EST ETS EUCAGEN EUFORGEN FFI FFPRI FISH FTGRCF FTSGCS GIS HAD HJG HTIRC HTS IAA IBPGR IEA INRA ISSR ITS LD MAS MDF MDS

Abscisic acid Amplified fragment length polymorphism Analysis of molecular variance Agriculture Research Service (USDA) Australian Tree Seed Centre Botanic Gardens Concervation International Big-tree structure Cleaved amplified polymorphic sequence Co-dominant forest Chloroplast DNA Ectomycorrhizal European Cooperative Program on Plant Genetic Resources Eastern filbert blight Expressed sequence tag External transcribed spacer Eucalyptus Genome Network European Forest Genetics Resources Programme Forest Frontiers Initiative Forestry and Forest Products Research Institute Fluorescence in situ hybridization Forest Tree Genetic Resources Conservation Forest Forest Tree Superior Gene Conservation Stand Geographical information systems Heavily disturbed by human activities Hydrojugloneglucoside Hardwood Tree Improvement and Regeneration Center Huge-tree structure Indole-3-acetic acid International Board of Plant Genetic Resources International Energy Agency French National Institute for Agricultural Research Inter-simple sequence repeat Internal transcribed spacer Linkage disequilibrium Marker-assisted selection Mono-dominant Forest Mono-dominant structure xvii

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Abbreviations

MDSD ML MOE MP Mya Mybp NALB NCGR NJ nptII OSU PCR PICME PR PRP QTL RAPD rbcL RBS rDNA RFLP RNAi rRNA RT-PCR RT-PCR rubisCO SCAR SMP SNP SSCP SSR STS SW TPS UCD uidA UPGMA USD USDA USDA VAM ybp

Mono-dominant structure in developing Maximum likelihood Module of elasticity Maximum parsimony Million years ago Million years before present North Atlantic Land Bridges National Clonal Germplasm Repository Neighbor joining Neomycin phosphotransferase II gene Oregon State University Polymerase chain reaction Platform for integrated clone management Population in recovery Proline rich protein Quantitative trait loci Random amplified polymorphic DNA Ribulose-bisphosphate carboxylase-L (gene) Relatively balanced structure Ribisomal DNA Restriction fragment length polymorphism RNA interference Ribosomal RNA Real-time PCR Reverse transcription PCR Ribulose-1,5-bisphosphate carboxylase oxygenase Sequence characterized amplified region Supplemental mass pollination Single nucleotide polymorphism Single-stranded DNA conformation polymorphism Simple sequence repeat Sequence tagged site Sprouting woods Terpene synthases University of California, Davis b-glucoronidase A gene Unweighted pair group method of arithmatic average Unstable structure in developing United States Department of Agriculture United States Department of Agriculture Vesicular arbuscular Years before present

Contributors

Preston R. Aldrich Department of Biological Sciences, Benedictine University, Birck Hall 341, 5700 College Road, Lisle, IL 60532–0900, USA, [email protected] Jeannine Cavender-Bares Department of Ecology, Evolution and Behavior, University of Minnesota, 1987 Upper Buford Circle, St. Paul, MN 55108, USA, [email protected] Gao Chen Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Botanic Garden, Kunming 650204, Yunnan, China Chunyan Han Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Botanic Garden, Kunming 650204, Yunnan, China Robert J. Henry Queensland Alliance for Agriculture and Food Innovation, The University of Queensland, St Lucia 4072, Australia, [email protected] Charles Michler USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, Pfendler Hall, 715 W. State St., West Lafayette, IN 47907, USA, [email protected] Thomas J. Molnar Department of Plant Biology and Pathology, Rutgers University, Foran Hall, 59 Dudley Road, New Brunswick, NJ 08901, USA, [email protected] Madhugiri Nageswara Rao IFAS, University of Florida (University of Florida, IFAS) Citrus Research & Education Center, University of Florida, IFAS, 700 Experiment Station Road, Lake Alfred, FL 33850, USA, [email protected] Jaya R. Soneji University of Florida, IFAS, Citrus Research & Education Center, 700 Experiment Station Road, Lake Alfred, FL 33850, USA, [email protected] Padmini Sudarshana Monsanto Research Center, #44/2A, Vasant’s Business Park, Bellary Road, NH-7, Hebbal, Bangalore 560092, India, padmini. [email protected] Weibang Sun Kunming Botanic Garden, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, China, [email protected] Yoshihiko Tsumura Department of Forest Genetics, Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan, [email protected]

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Brian D. Vanden Heuvel Department of Biology, Colorado State UniversityPueblo, Pueblo, CO 81001, USA, [email protected] Keith Woeste USDA Forest Service, Northern Research Station, Hardwood Tree Improvement and Regeneration Center, Purdue University, Pfendler Hall, 715 W. State St., West Lafayette, IN 47907, USA, Yanling Zheng Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Botanic Garden, Kunming 650204, Yunnan, China Yuan Zhou Kunming Institute of Botany, Chinese Academy of Sciences, Kunming Botanic Garden, Kunming 650204, Yunnan, China

Contributors

Chapter 1

Alnus Brian D. Vanden Heuvel

1.1 Introduction Alnus Mill. (Betulaceae), often referred to by its common name alder, is a genus composed of monoecious trees and shrubs distributed throughout the northern Hemisphere and limited in the southern Hemisphere along the Andes in South America. Species of Alnus were long thought of as forest weeds; however, since the emergence of an industry for alder wood products in the 1980s, considerable attention has been paid to the ecology, management, and genetic improvement of Alnus (Xie et al. 2002; Xie 2008). In addition to their commercial value, alder species are important because they have the ability to associate with Frankia, a nitrogen-fixing soil actinomycete. Frankia strains provide their hosts with a source of fixed nitrogen, a nutrient that limits plant growth. The host plant, in turn, provides fixed carbon to the Frankia strain (Baker and Schwintzer 1990). Plants that associate with Frankia have a distinct advantage over other plants because they are not as limited by nitrogen and can inhabit nitrogen-poor soils. Therefore, beyond wood products and biomass uses, species of Alnus play an important role in ecosystem development by securing unstable surfaces and participate in the first stage of plant succession on both wet and dry land soils in forests (Mejnartowicz 2001, 2007) and promote growth of other plants by adding nitrogen into the soil (Klemmedson 1979). It is important to point out initially that, unlike other more traditional crop plant/wild relative systems, Alnus has just recently become a target of research. Therefore,

Brian D. Vanden Heuvel Department of Biology, Colorado State University-Pueblo, Pueblo, CO 81001, USA e-mail: [email protected]

we have very limited knowledge of the baseline genetic variation within and among species and populations, the genetic architecture for desired traits, the geographic pattern of genetic variation, the extent of genotype by environment interaction, and heritability (Xie et al. 2002). This chapter focuses on what we currently know about the diversity of the genus Alnus, both within the genus and within selected species, current research on genetic resource management of some alder species, and broad patterns of Frankia strain distribution and diversity as they relate to alder distribution across a geographical mosaic of environments.

1.2 Taxonomy, Morphology, Reproductive Biology, Ecology, and Geographic Distribution of the Genus Alnus 1.2.1 Basic Taxonomy Alnus Mill. (Betulaceae) lacks a consensus classification. Since it was first described, Alnus has undergone many revisions (Furlow 1979), each varying widely in the ranks assigned to taxa and the number of species. Numbers of species within Alnus have ranged from 20 to 35 (Furlow 1979; Hall and Maynard 1979; Bond 1983). Confusion about the number and circumscription of species within Alnus arises primarily from the lack of clear morphological delimitations between taxa (Hall and Burgess 1990), specifically leaf morphology. Variations in leaf morphology show a continuum within and between taxa, making it difficult to define boundaries among species (Steele 1961; Parnell 1994). Nonetheless, the genus Alnus is a well-defined

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_1, # Springer-Verlag Berlin Heidelberg 2011

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group, easily clustered by morphological similarities and separated from the genus Betula L. (Betulaceae), its closest ally, by several discontinuities. Most notably, Alnus is recognized by its woody infructescences with persistent scales, bud structure, and the number of stamens (Furlow 1979). Within the genus Alnus, three distinct evolutionary lines have been identified and are treated as subgenera. The three subgenera are distinguishable by the bud structure, whether the pistillate catkin is exposed over the winter months, and when pollination occurs. The three subgenera of Alnus are subgenus Alnus, subgenus Clethropsis, and subgenus Alnobetula. Alnus subgenus Alnus contains the majority of the species. It is distinguished by having stalked shoot buds, pistillate catkins closed over winter, and is pollinated in late winter or early spring (Furlow 1979). Members of Alnus subgenus Alnus are found across the range of the genus, throughout North America, Europe, and along the Andes southward to Argentina. Many of the species of Alnus currently used and also those species under evaluation within agriculture and lumber industries are in this subgenus, including Alnus rubra Bongard (Red Alder) A. cordata Desf. (Italian Alder), A glutinosa (L.) Gaertner (Black Alder), and A. acuminata Kunth (Andean Alder). Alnus subgenus Clethropsis is recognized by stalked shoot buds, pistillate catkins open and pollinated in fall (Furlow 1979). Only three species are found within this subgenus, including Alnus formosana Makino (Formosan Alder) found on Taiwan, A. maritima (Marsh.) Muhl. Ex Nutt. (Seaside Alder) found on the Delmarva Peninsula on the east coast of North America, and disjunct populations in Oklahoma and Georgia, and A. nitida Endl. (Himalayan Alder). Because of their aesthetic appeal and drought tolerance, these three species, specifically A. maritima, have been gaining heightened interest as horticultural crops (Schrader and Graves 2004). Alnus subgenus Alnobetula is characterized by shoot buds without stalks and pistillate catkins produced and pollinated in late spring. Currently there is only one species, Alnus viridis (Chaix)DC. (Green Alder), in this subgenus, yet authors often raise the subspecies currently circumscribed under viridis to specific level. Although the habit of A. viridis is small and is not used for a lumber source, it has attracted interest as a candidate for reforestation in temperate forest ecosystems in both North America and Europe (Roy et al. 2007).

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1.2.2 Basic Morphology Members of the genus Alnus are woody, ranging in size from large trees with a single trunk to small shrubs with many trunks. Most of the species found in warmer climates take on a “tree-like” habit while those species found in cooler climates are “shrubby”. Even those species, which attain sizes considered arboreal, often have multiple trunks. It has been postulated that the large, arborescent habit is ancestral and the shrubby habit is an adaptation in derived lineages to the cooler climate (Furlow 1979). In most species of Alnus, the bark is smooth, but in some of the larger species, it can take on the form of large plates. The presence of smooth bark linked with a shrubby habit has been postulated by Hall (1952) as a set of neotenic characters. Stem diameters range in size from 1 cm to as large a 2 m, and twigs have a characteristic triangle shaped pith (Furlow 1979). The leaves of species of Alnus are arranged alternately on the stems and the veins are conspicuously pinnate. Leaf margins vary from entire to deeply serrate to cuneate. Overall, species of Alnus vary considerably in leaf structure, both inter and intraspecifically (Furlow 1979; Lecerf and Chauvet 2008). The wood of Alnus has long interested plant anatomists due to the interesting evolutionary series displayed by the species with respect to ray morphology. Within Alnus, Bailey (1911, 1912) discusses the evolution of multiseriate rays from uniseriate rays and the apparent reversal of the trend in a few species. The evolution of ray morphology within Alnus has further been discussed by a host of other authors (see Furlow 1979). Of all of the economic uses of Alnus, wood production is by far the most important. Alnus wood is primarily made up of vessels and fiber tracheids. Alder wood has high machining properties (Malkoc¸og˘lu and ¨ zdemir 2006) and wood quality (Bekhta et al. 2009), O making it a highly sought after wood product. Species of Alnus are monecious; the unisexual flowers are borne in staminate and pistillate catkins. Staminate catkins are pendant, while pistillate catkins are erect on the stems. In most species, the pistillate catkins are clustered and inserted just below a solitary or small group of axillary staminate catkins. At maturity, the pistillate catkin becomes woody and conelike. The cones of Alnus are useful for identification of

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species and are also one of the best ways to differentiate Alnus from its sister group, Betula.

1.2.3 Reproductive Biology Alnus is pre-dominately wind pollinated, although there are a few reports that insects are sometimes attracted to staminate catkins (Furlow 1979). The age at which flowers are produced in Alnus is not known for all species. There are reports that Alnus in Alaska (A. viridis ssp. sinuata) flowered at the age of seven years or earlier (Lawrence 1958). Furlow (1979) reports that in a common garden in east Lansing, Michigan, A. maritima flowered in 3 years, A. viridis ssp. sinuata, A. incana ssp. rugosa, and A. serrulata all flowered in 5 years. The larger species of A. rubra, A. rhombifolia, and A. acuminata did not flower until they were relatively large. It might be expected that in northern or subalpine climates early reproduction, coupled with a small, shrubby habit, would be advantageous, while in more temperate climates a more tree-like habit and delayed reproduction would be favored (Hall 1952). It is hypothesized that alders are primarily outcrossing, although some studies of genetic diversity within populations indicate high coancestry of alleles within individuals as the result of consanguineous matings (Gibson et al. 2008). Alder seeds do not have wings and can only be dispersed by wind 30–60 m from the mother tree (Mejnartowicz 2007). Although the seeds have no wings, they do possess air bladders and can be dispersed by waterways great distances. If these waterways have periodic flooding, dispersal is increased even more. Given that most of Alnus species are associated with wet habitats, this seed dispersal strategy allows alders to colonize new territories and migrate into other alder populations (Mejnartowicz 2007).

1.2.4 Geographic Distribution The genus Alnus has seven major distribution centers according to Furlow (1979): (1) western North America from southern Alaska to northern Mexico; (2) coastal eastern North America from Nova Scotia to the Gulf of Mexico (absent from the Caribbean); (3) high elevation centers of Mexico, central, and South America; (4)

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coastal eastern Asia; (5) Himalayas; (6) the mountains of Iran, Asia Minor, and southern Europe; and (7) circumpolar Europe, Asia, and North America. The oldest Alnus fossil, a catkin, dates to the midEocene (33–55 Mybp), but Alnus-like pollen has been reported from the late Cretaceous (83–85 Mybp), earlier than any fossils for the other genera in the family Betulaceae (Miki 1977). Given the distribution of known fossils and the recent molecular phylogeny (see below), it appears the Betulaceae first originated in a Mediterranean climate in Laurasia during the late Cretaceous (89–65 Mya) (Laurasia was the northern supercontinent formed after Pangaea broke up during the Jurrasic and included what are now North America, Europe, Asia, Greenland, and Iceland). Fossil evidence suggests that all six genera within the Betulaceae, including Alnus, were recognizably differentiated by the early Eocene (55 mybp) (Chen et al. 1999). By the early Tertiary (65 mybp), movement between Eurasia and North America was possible, and the range of Alnus probably increased. The distribution of Alnus to Africa and to Taiwan probably occurred later, during the Pleistocene (1.8 mybp–11,000 ypb) when sea levels were lower (Chen et al. 1999).

1.2.5 Ecology Most of Alnus species are associated with wet habitats (Furlow 1979). These include standing water, stream banks, bogs, and wet montane environments. Also, Alnus species grow in full sunlight, with the exception of A. viridis ssp. crispa, which can be an understory component of some conifer woods. Alnus is unique in the Betulaceae as it is the only genus within the family to associate with Frankia, a filamentous bacteria (actinomycetes) that fixes N2 and induces N2-fixing root nodules on a broad range of “actinorhizal plants”. Actinorhizal plants, in turn, are defined by their ability to form root nodules when in symbiosis with Frankia. Within the root nodule, Frankia fixes nitrogen that is transported to the host plant in amounts sufficient to supply most of plants’ nitrogen requirements. This symbiosis allows actinorhizal plants to invade and proliferate in soils that are low in combined nitrogen. To date, all species of Alnus examined have been shown to nodulate (Benson et al. 2004).

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Given the ability to form root nodules with Frankia, species of Alnus can inhabit nitrogen-poor soils. Therefore, Alnus plays an important role in ecosystem development by securing unstable surfaces participating in the first forest stage of plant succession (Mejnartowicz 2001, 2007), and promoting growth of other plants by adding nitrogen into the soil (Roy et al. 2007).

1.3 Genetic Variation in Alnus

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2n ¼ 28 progenitor had two 5S rDNA regions (Oginuma et al. 2000). Meiosis appears irregular and pollen quality poor in the Alnus individuals with 2n ¼ 42 (Gram et al. 1942; Jaretzky 1930). These include A. cordata, A. subcordata, and A. orientalis. In comparison, those plants with 2n ¼ 56 and 2n ¼ 28, meiosis appears normal and almost all pollen formed is high quality (Gram et al. 1942). The only other reported problems with meiosis in Alnus are the putative hybrids between A. incana ssp. rugosa and A. serrulata (Woodworth 1929, 1930, 1931).

1.3.1 Chromosome Numbers Given the relatively small size of the genus Alnus, the genus demonstrates high polyploidy and an extensive chromosome number sequence. Chromosome counts from species of Alnus have yielded 2n ¼ 14, 28, 42, 56, 70, 84, and 112 (Furlow 1979; Oginuma et al. 2000). The diploid chromosome number of 2n ¼ 14 has been found in Alnus hirsute var. microphylla, A. pendula, and A. serrulatoides, suggesting the base number in Alnus is x ¼ 7. Although this is an extensive series, most of species of Alnus are 2n ¼ 28 and are considered tetraploids (Furlow 1979; Oginuma et al. 2000) In 2000, Oginuma et al. tested whether the dramatic ploidy series seen in Alnus was produced through successive allopolyploid events. It was suggested based on previous data that the amount of 5S rDNA does not change by allopolyploid condition, allowing one to compare amounts of 5S rDNA to chromosome number for correlations and additive patterns. When the Alnus chromosomal series was investigated using in situ hybridization of 5S rDNA, some Alnus taxa with 2n ¼ 28 (Alnus hirsute var. microphylla and A. pendula) were found to have two 5S rDNA signals, while A. serrulatoides (2n ¼ 28) was found to have four 5S rDNA signals (Oginuma et al. 2000). These results suggest that although 2n ¼ 28 is by far the most common chromosome number in Alnus, 2n ¼ 28 may have been arrived at by multiple pathways and involved complex genome evolutionary histories. Therefore, two species of Alnus may have the same chromosome number but arrived at that number in very different ways. Investigations of the species of Alnus with high chromosome numbers (A. japonica 2n ¼ 56, A. sieboldiana 2n ¼ 84, and A. firma 2n ¼ 112) all showed predicted additive polyploidization of 5S rDNA signal if the base

1.3.2 Hybridization The existence of interspecific hybrids in Alnus has been well documented. Hybrids in natural populations have been recorded and studied in North America, Europe, the Russian Far east, and Japan. Documented hybrids include Alnus glutinosa  incana (Hylander 1957; Parnell 1994; Banaev and Bazˇant 2007), Alnus serrulata  rugosa (Steele 1961; Furlow 1979); Alnus sinuate  crispa (Bousguet et al. 1989, 1990), and Alnus glutinosa  rubra (Hall and Burgess 1990). Natural hybrids, specifically between Alnus incana and A. glutinosa, have been reported to possess many economically valuable properties (Banaev and Bazˇant 2007). Hybrids of A. incana  glutinosa have been found to have greater drought resistance when compared to each of the parent species, less demand for fertility (Kobendza 1956; Kundzinsh 1957), higher wood quality (Pirags 1962), and better resistance to some forms of rot (Fer and Sˇedivy 1963). Hall and Burgess (1990) reported that fast growing, early flowering hybrids of Alnus incana  glutinosa and A. glutinosa  rubra bred true through the F2 generation. Further, A. glutinosa  rubra showed hybrid vigor when grown under greenhouse conditions and showed levels of resistance to the European alder leafminer, Fenusa dohrnii. Given that interspecific hybrids form between many species pairs in Alnus, hybridization may be an important strategy for species improvement. Hall and Burgess (1990) reports that Alnus as a genus has relatively poor tolerance to moister stress, especially the species (A. rubra, A. incana, and A. glutinosa), which are primarily used for biomass and wood production.

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This trait has hampered field establishment and largescale biomass production. Instead of identifying and selecting for drought-tolerant individuals within these targeted species, a better strategy may be to bring desired traits, such as tolerance to moister stress, from other species of Alnus, like A. cordata or A. maritima, through hybridization (Hall and Burgess 1990; Schrader et al. 2005).

1.3.3 Studies of Genetic Variation Within Alnus Species As a pioneer tree species, alders are included in the first forest stage of plant succession on wet, riparian sites (Mejnartowicz 2001). Yet, alders are also a component of climax forest communities on many soil types (Mejnartowicz 2007). These two life histories predict very different genetic structures. It has been theorized and found that pioneer tree species reveal much higher genetic diversity than climax tree species (Wehenkel et al. 2006). Further, it is predicted that in pioneer species, allelic variation is distributed between individuals, while in climax species, allelic variation is distributed in individuals as heterozygous loci (Mejnartowicz 2007). Given that species of Alnus can in some circumstances be pioneer species and in others be a member of a climax community, studies of Alnus population genetic structure are very interesting (Mejnartowicz 2007). Complicating the life histories of populations of Alnus, species of Alnus, like other woody species with large geographic ranges, outcrossing breeding systems, and seed dispersal using wind and water, have a relatively higher genetic diversity within species and populations, but lower genetic diversity among populations than woody plants with other traits (Hamrick et al. 1992). Studies of genetic variation within species and populations of Alnus have been studied in Alnus rubra (Hamann et al. 1999; Xie et al. 2002), A. maritima (Schrader and Graves 2002, 2004; Gibson et al. 2008), A. serrulata (Gibson et al. 2008), A. glutinosa (Prat et al. 1992; King and Ferris 1998; Mejnartowicz 2007), A. rugosa (Bousquet et al. 1988; Huh 1999), A. japonica (Huh 1999), and A. crispa (Bousquet et al. 1987, 1988).

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In a study of Alnus glutinosa, Mejnartowicz (2007) examined the genetic structure of 12 populations in Poland using isozymes. The study was designed to address the inter- and intrapopulation variation, gene flow between populations, and correlations between geography and genetic similarity. He found that 74% of the loci examined were polymorphic, a high number for plants and in agreement with the prediction that climax species have individuals with a high frequency of heterozygous loci. Also, different populations did not differ significantly in the proportion of polymorphic loci, suggesting that there is no correlation between the level of genetic polymorphism and site conditions. Although the number of polymorphic loci is high (74%), the measurements of heterozygosity appeared low. The average observed heterozygosity was 0.2 and a positive fixation index of F ¼ 0.305, indicating a 30% deficiency in heterozygotes. These results suggest either inbreeding, vegetative reproduction from root suckers, or low efficiency of both pollen and seed dispersal has led to lower than expected heterozygotes. Steiner and Gregorius (1999) observed large degrees of self-pollination in an A. glutinosa population, but did not see a drop in seed production, implying that inbreeding is possible in these populations and may be responsible for the low heterozygosity numbers. Lastly, Mejnartowicz (2007) found no significant correlation between genetic and geographic distance between populations. Only 8.9% of the total variation was due to interpopulation differences, and gene flow estimates showed that reproductive barriers do not separate populations of A. glutinosa. Very similar results were found in a study of A. japonica in East Asia by Huh (1999). He found a high proportion of polymorphic loci (76%), low heterozygosity, and a high fixation index (F ¼ 0.502), and only 9.5% of the total variation was due to interpopulation differences. These results indicate that populations of A. japonica, like populations of A. glutinosa in Poland, are highly polymorphic, yet the individuals have low heterozygosity, and there appears to be no significant correlation between genetic and geographic distance between populations. Both of the above studies are in alignment with the prediction by Hamrick et al. (1992) that species with large geographic ranges and outcrossing breeding systems will have relatively higher genetic diversity within species and populations, but lower genetic diversity among populations.

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In 2008, Gibson et al. compared the population genetic diversity using allozymes between a common, widespread species of Alnus, A. serrulata, and a rare species A. maritima in order to evaluate the influence of small population size and extreme isolation on genetic diversity. A. maritima exists in only three locations: the Delmarva Peninsula of Delaware and Maryland, South Central Oklahoma, and Northwest Georgia (Schrader and Graves 2004). They found, as might be expected, that genetic diversity was lower in A. maritima than the common, widespread cogener A. serrulata (A. maritima He ¼ 0.217, A. serrulata He ¼ 0.268) and inbreeding higher in A. maritime (A. maritima f ¼ 0.483, A. serrulata f ¼ 0.269). Further, the partitioning of the genetic variation was higher in A. maritima (Y ¼ 0.278) than A. serrulata (Y ¼ 0.197). All the three results are generally consistent with expected values for rare and widespread species of similar life history traits (Cole 2003; Gibson et al. 2008). Although the estimation of genetic diversity was low in A. maritima, the overall pattern of population structure and genetic diversity is not strikingly dissimilar from other species of Alnus in North America (Gibson et al. 2008). Estimations of genetic diversity in A. crispa (Bousquet et al. 1987) and A. rugosa (Bousquet et al. 1988) were similar to A. maritime. What is striking about the A. maritima data, when compared to other Alnus species, is the high genetic differentiation between populations. Most species of Alnus have high gene flow among networks of populations along a watercourse and therefore considerably less genetic differentiation among populations. The high genetic differentiation seen in A. maritima undoubtedly is due to the extreme isolation of the three populations, and gene flow is rare to non-existent. The three populations of A. maritima were studied by Schrader and Graves (2004) using inter-simple sequence repeat (ISSR) markers, and they concluded that morphological and ISSR variation was sufficient enough to warrant subspecies designations (subspecies maritima, oklahomensis, and georgiensis). They also concluded from their data that the Oklahoma population diverged first, and the Georgian and Delmarva populations were more closely related and diverged later. Interestingly, the later allozyme data (Gibson et al. 2008) suggest that the Oklahoma and Georgia populations are more similar to the Delmarva population. Both results, though, support the idea that the

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highly disjunct population structure of A. maritima is a result of natural range reduction and not humanmediated establishment in Oklahoma and Georgia (Gibson et al. 2008). The genetic structure of 19 populations of Alnus rubra in British Columbia was examined by Xie et al. (2002). A. rubra (red alder) is the most common hardwood tree in the Pacific northwest of North America. Although it makes up only a small proportion of the forest resources of the Pacific northwest, it has been gaining attention due to the increased demand for red alder wood products. The goal of this study was to understand the baseline genetic variation and population structure for use in understanding and harnessing the adaptive variation of economically important traits (Xie et al. 2002). They found that the number of alleles per locus were 1.72, 52% of the loci were polymorphic, and total expected genetic diversity (0.133) were all below what has been reported for long-lived woody species. Further, they found low among-population differentiation (8%), compared to other species that outcross and disperse seed by wind. The limited among-population differentiation was almost entirely attributed to island populations versus mainland populations. The authors conclude that, if saving genetic resources is the goal, selecting at least one mainland and one island population will contain the species local genetic variation (Xie et al. 2002). Overall, the species of Alnus show extensive gene flow, most likely due to their pollination strategy (wind) and seed dispersal (water), making populations show little correlation between genetic and geographic distance, with the exception of A. rubra, which displays strong genotype x environment interactions. Further, individuals show low heterozygosity, but loci in populations are highly polymorphic.

1.4 Phylogeny of Alnus The Betulaceae is composed of six genera and about 130 species (Mabberely 1988). The family is mostly distributed throughout the temperate regions of the northern Hemisphere, with the exception of species of Alnus, which are found throughout Central America south to Argentina. The genus Alnus is the only actinorhizal genus within the Betulaceae. The angiosperm rbcL phylogeny of Chase et al. (1993) strongly

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supported the classification Betulaceae within Fagales (a relationship long recognized based on morphology). More recent studies of this group (Manos and Steele 1997) have placed the family in a subclade with Casuarinaceae and Ticodendraceae. Recent molecular phylogenies for the Betulaceae suggest two clades or lineages (Chen et al. 1999). One lineage contains the four genera - Corylus, Ostryopsis, Carpinus, and Ostrya – and the other includes Alnus and Betula. These results suggest that Betula is the closest relative to Alnus, and Alnus diverged early in the evolution of the Betulaceae. The early divergence of Alnus agrees with dated fossils for Alnus (Miki 1977; Chen et al. 1999). Fossil evidence also suggests that all six genera in the Betulaceae, including Alnus, were differentiated by the early Eocene (55 mybp) (Chen et al. 1999). This observation suggests that if the ability to nodulate with Frankia was ancestral in the Betulaceae, loss of that ability occurred very early on in the evolution of the family (Benson et al. 2004). Two separate authors conducted independent phylogenies of the genus Alnus based on the internal transcribed spacer (ITS) of the nuclear ribosomal repeat. Navarro et al. (2003) examined 18 species and constructed phylogenetic trees using neighbor joining (NJ), maximum parsimony (MP), and maximum likelihood (ML) search strategies. Overall, they detected three major clades corresponding to the three subgenera Alnus, Clethropsis, and Alnobetula (see Sect. 1.2.1). They also discovered that A. nepalensis, often positioned within subgenus Clethropsis with A. nitida, A. maritima, and A. formosana, fell within subgenus Alnus sister to A. japonica. Subgenus Clethropsis also displayed an increased substitution rate in comparison with the other clades corresponding to the other two subgenera. Based on their trees, Northeast Asia was inferred as the origin of the genus, based on the number of species from that region that were present in the basal, deep lineages identified. Further, fewer trans-continental migrations would have to be inferred based on the tree topology if Northeast Asia was the center of origin. Chen and Li (2004) examined 34 species using the ITS region. As expected, the phylogenies produced by Chen and Li (2004) are very close in topology to the previously published trees in Navarro et al. (2003), with the exception of having many more taxa sampled. Chen and Li (2004) also identify three major clades corresponding to the three subgenera. They report that subgenus Clethropsis is sister to subgenus Alnus. In a

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basal position is subgenus Alnobetula. Chen and Li (2004) also report that the central and South American Alnus taxa appear to have been derived from Asian ancestral taxa and migrated to central and South America via the Bering land bridge. Hall and Burgess (1990) postulated that in may be possible to introduce desired characteristics into economically important Alnus species through hybridization (see Sect. 1.3.2). In fact, interspecific hybrids have been reported between Alnus glutinosa  incana, Alnus serrulata  rugosa, Alnus sinuate  crispa, and Alnus glutinosa  rubra (see Sect. 1.3.2). When the parent taxa of these hybrids are examined within the topology of the trees produced by Navarro et al. (2003) and Chen and Li (2004), all are either in a sister and very near sister relationships, indicating hybrids are only formed by very closely related species. This may limit the possibility of Hall and Burgess’s (1990) recommendation of using hybridization as a tool for acquiring desired traits in Alnus species (see Sect. 1.5)

1.5 Evaluation of Alnus for Biomass Production Hall and Burgess (1990) published a summary of a workshop on alder improvement sponsored by the International Energy Agency (IEA) Forestry Energy Agreement. Alnus has attracted the interest of energy plantation systems because alders can symbiotically fix nitrogen and therefore lessen the drain on the soil nutrients after frequent harvests for biomass. Within this summary, Hall and Burgess identify three problems/objectives: (1) a limited number of Alnus species have been evaluated; (2) the availability of seed sources, especially for hybrids, is limited; and (3) there is a need for better exchange of information among groups of people working with Alnus to promote advantages and identify problems with selected Alnus species. The report attempts to summarize what we know about the first two problems ca. 1990. Hall and Burgess (1990) report on an evaluation trial, which examined 39 seed lots representing five species of Alnus. The seedlings were placed in nursery beds in four countries (Belgium, Canada, UK, and the USA) in identical planting designs, although the authors only report on the trials from Canada and USA. They first report that some seed lots had much

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lower germination rates than expected, yielding very few seedlings for study. A. acuminata was particularly hard to grow from seed because the seed lot was not cold-hardy and was quickly lost at most locations. They continue to report that fall dormancy for the seedlings was highly variable. The percentage of seedlings that were dormant for each test entry ranged from 91% to 0% and only 20 of the 39 entries had more than 50% of their seedlings dormant by the first of October. This is important because in Ames, Iowa, where the USA planting occurred, killing frosts typically occur by October 10. The delay of dormancy was unexpected and many of the seedlings in this study, from multiple seed lots and species, were lost. As a general pattern, A. glutinosa and A. rubra entries had the highest dormancy rates and therefore survival rates, followed by A. incana, A. cordata, and A. acuminata entries in that order. The A. acuminata entries showed no sign of dormancy and were dead by the end of October (Hall and Burgess 1990). Growth performance of the trees in the study by Hall and Burgess (1990) showed that A. glutinosa and A. incana had the highest growth rates and survival in the Canadian plots after a particularly hot, dry growing season. A. rubra had decent survival rates (68%), yet almost no growth, and the A. cordata entries showed the lowest growth and survival. During the same summer in Iowa, the USA plots also experienced a hot, dry growing season. In the USA plots, A. cordata entries actually had the highest survival, followed by A. glutinosa, A. incana, and A. rubra. Given the extreme water stress the seedling encountered that summer, very few individuals showed growth, independent of species. The authors comment that in the face of global warming, A. cordata and A. glutinosa show promise as biomass producers with their relatively good survival under extreme water stress (Hall and Burgess 1990). The study report by Hall and Burgess highlights the challenges and potentials of Alnus in the biomass industry. It appears that seed lots are quite variable in their germination rates. Also, the relatively poor tolerance Alnus shows to water stress will be a limitation to its inclusion in biomass plantations. Drought tolerance may be improved by selecting on drought tolerance variation within species such as A. rubra or A. glutinosa, or attempting to transfer drought tolerance from A. cordata by hybridizing it with species of Alnus better suited to biomass production.

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1.6 Development and Improvement of Genetic Stocks in Alnus rubra Alnus rubra (red alder), once regarded as a forest weed, is now gaining interest as a commercial tree and more concentration has been focused on its maintenance, growth, and genetics (Xie 2008). Given the new demand for A. rubra wood, natural stands are disappearing, and producers are turning to plantations of red alder (Xie 2008). Tanaka et al. (1997) reports that 2.5 million red alder seedlings are planted each year by a single corporation. Xie (2008) reports that over one million seedlings of red alder are expected to be planted in British Colombia alone. The increased demand for red alder wood suggests that a genetic improvement program would be worthwhile. Red alder contains sufficient variation in traits of interest to producers (i.e. height, stem volume, biomass, ecophysiological traits, etc.) (DeBell and Wilson 1978; Ager 1987; Hook et al. 1990; Dang et al. 1994; Xie and Ying 1994; Hamann et al. 1999) and using seed sources or selected genotypes ideal for local conditions can enhance productivity (Hamann et al. 2000). In order to identify ideal seed sources and appropriate genetic material of A. rubra, long-term provenance tests at multiple sites is needed, which can accurately identify areas for seed collection best for particular plantation environments (Hamann et al. 2000). Hamann et al. (2000) reports on early results from a large-scale, long-term provenance test in British Columbia. They tested the assumption that local sources of seed are optimal and used geographical information systems (GIS) to identify seed transfer zones and guidelines. They found significant genotype x environmental interactions, and seedlings grown near the site of seed collection showed superior performance, suggesting local adaptation of A. rubra populations. They generated general seed transfer guidelines and found, using GIS, that a transfer of seed 100 km in a northern or southern direction was associated with a decline of 2.5% in survival and about 5 cm in height after 2 years of growth. Xie (2008) reports on a 10-year provenance-progeny testing program for red alder. Results of the program indicate that trees planted near their site of origin performed better, and two regions (northern and southern) were identified with the boundary of 52 N. Interestingly, individual seedlings had about 5% decrease

1 Alnus

in stem volume and 6% decrease in mortality for every 1 latitude north or south the seedling was transferred from its seed source. These results suggest strong local adaptation (Xie 2008). Further, the levels of additive genetic variation showed that between 23% and 29% gains in stem volume could be gained in a rotation age of 40 years selecting the top 20 individuals, and selection of individuals could occur at age 6 (Xie 2008). The studies by Hamann et al. (2000) and Xie (2008) indicate that there is significant genotype x environmental interactions and strong local adaptation in A. rubra. Increasing yield, either height or biomass, is possible in A. rubra, but linking seed source to the local plantation environment appears to be essential.

1.7 Frankia and Alnus The actinobacterial genus Frankia contains filamentous bacteria (actinomycetes) that fix N2 and are defined by their ability to induce N2-fixing root nodules on a broad range of “actinorhizal plants”. Actinorhizal plants, in turn, are defined by their ability to form root nodules when in symbiosis with Frankia (Benson et al. 2004). Frankia fixes nitrogen in the root nodule, and the resulting ammonia is transported to the host plant in amounts sufficient to supply most of the plant’s nitrogen requirements. The host plant, in turn, provides Frankia within the nodule fixed sugar for growth (Benson et al. 2004). This symbiosis allows actinorhizal plants to colonize substrates low in nitrogen (Roy et al. 2007). The phylogeny of the genus Frankia has been elucidated using the 16S rRNA gene, the genes for nitrogen fixation (nif genes) and by other genes (Benson and Clawson 2000). All analyses done to date agree that the genus is comprised of three major clades, often referred to as groups (Groups 1, 2 and 3). Specificity of Frankia strains to host species and vice versa is not demonstrated at the “group” level; each group has different and sometimes overlapping plant specificity, physiological properties, and symbiotic interactions (Benson et al. 2004). Within each group are definable subgroups that constitute “genospecies,” as defined by DNA–DNA homology studies (An et al. 1985; Dobritsa and Stupar 1989; Fernandez et al. 1989; Normand et al. 1996; Benson and Clawson 2000). In general, Group 1 Frankia strains form nodules on members the order Fagales, including the three plant

9

families Betulaceae, Myricaceae, and Casuarinaceae (Benson et al. 2004). “Alder strains” within group 1 generally infect most species of alder tested in greenhouse experiments, with some variability in effectiveness depending on the plant/symbiont combination. To date, all alder species examined have been shown to nodulate. Alder strains are also generally able to infect members of the Myricaceae (Benson et al. 2004). A few strains of Frankia from Group 3 have also been shown to nodulate alders, but do so only rarely and are poorly effective (Bosco et al. 1992; Lumini and Bosco 1996). Estimations of nitrogen fixation rates in actinorhizal alders are comparable to those in legumes. Alnus rubra, A. glutinosa, and A. viridis have been found to fix nitrogen in the range of 40–300 kg N/ha/year as compared to Alfalfa and clover that can fix 57–300 kg N/ha/year and 104–160 kg N/ha/year, respectively (Hibbs and Cromack 1990; Zuberer 1998; Pepper 1999; Hurd et al. 2001). Nitrogen fixation rates have been found to vary with stand age, density, and alder–Frankia combinations (HussDanell 1990; Myrold and Huss-Danell 2003). Alnus spp. are often used as a “trapping plant” for Frankia strains in order to study Frankia strain distribution and diversity, largely because alder seeds are readily available and easily germinated and are infected by a wide variety of Group 1 and 3 Frankia strains (Benson et al. 2004). Except for a few environments, such as at the foot of retreating glaciers in Alaska (Kohls et al. 1994), Frankia strains infective on Alnus spp. are cosmopolitan and seem to persist independently of host plants as saprophytes. When trapping studies are done, estimates of Frankia strains infective on Alnus vary from a few per gram of soil to several thousands per gram in soils both near and removed from actinorhizal plants (Van Dijk 1979; Smolander 1990; Smolander and Sarsa 1990; Myrold et al. 1994; Markham and Chanway 1996; Maunuksela et al. 1999). Frankia strains specific to Alnus hosts seem to persist in soil long after the hosts have been removed and are also found outside of the normal geographic ranges of Alnus species, most likely due to wind action spreading Frankia spores (Wollum et al. 1968; HussDanell and Frej 1986; Smolander and Sundman 1987; Arveby and Huss-Danell 1988; Paschke and Dawson 1992; Maunuksela et al. 1999). For example, Alnus species nodulated at every site in New Zealand tested, even though Alnus is a very recent arrival to New Zealand (Benecke 1969; Benson et al. 2004).

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The nodulation capacity of soils for Alnus is affected by fertility (Kohls and Baker 1989; Sanginga et al. 1989; Thomas and Berry 1989; Myrold and Huss-Danell 1994; Myrold et al. 1994; Yang 1995), season (Myrold and Huss-Danell 1994), water availability (Schwintzer 1985; Dawson et al. 1986; Nickel et al. 1999), physiological status of Frankia strains (Myrold and Huss-Danell 1994), acidity (Griffiths and McCormick 1984; Smolander and Sundman 1987; Zitzer and Dawson 1992; Crannell et al. 1994), and by the type of plant cover (Huss-Danell and Frej 1986; Smolander and Sundman 1987; Smolander et al. 1988; Smolander 1990; Smolander and Sarsa 1990; Myrold and Huss-Danell 1994; Markham and Chanway 1996; Zimpfer et al. 1999). There is some evidence that Frankia strains infective on Alnus spp. sort by soil type. Within Frankia, it is possible to characterize strains as either sp(+) (containing sporangia) or sp() (devoid of sporangia) nodules (Schwintzer 1990). In British Columbia, sp() nodules of A. rubra dominate in near the coast, with no sp(+) nodules observed. As sampling moved inland, the proportion of sp(+) nodules increased up to 53% of the total (Markham and Chanway 1996). Also, as soil acidity rises, Frankia strains with the sp(+) phenotype become more prominent (Weber 1986; Holman and Schwintzer 1987; Kashanski and Schwintzer 1987). Overall, Frankia strains infective on Alnus are extremely cosmopolitan and have been found in soils lacking actinorhizal plants, suggesting alder Frankia strains can live independently of the association, not requiring continuous symbiotic interaction. It is likely that their wide distribution is related to the ranges of their hosts (Alnus extends throughout the northern hemisphere and into South America). Their abundance in New Zealand testifies to their ability to grow as saprophytes in the absence of Alnus, as well as their ability to spread.

1.8 Summary and Recommendations for Future Actions Alders are used primarily for their wood, either in the form of wood products or biomass. The value of alder logs has increased rapidly since the 1980s because of an emerging industry for alder wood products, which has attracted the interest of land managers, wood

B.D. Vanden Heuvel

producers, and researchers. Beyond their use in wood products, Alnus also play an important role in ecosystem development. Alnus has always been considered an outcrossing species given its life history strategy of wind pollination. Estimations of heterozygosity and numbers of heterozygotes within natural populations suggest inbreeding may be possible and common. Self-pollination has been documented (Steiner and Gregorius 1999) but did not result in a drop in seed production, implying that inbreeding is possible in these populations and may be responsible for the low heterozygosity. We know little about the possibility of using selfing as a tool for reproduction in Alnus or its possible effects in natural populations. Alnus displays a fantastic chromosome series for a genus of such small size. Most of the species are 2n ¼ 28 and are considered tetraploids. There have been reports of meiotic problems in some of the hexaploid individuals (2n ¼ 42), but other individuals with 2n ¼ 56 (octoploids) appear normal. Although many of the species of Alnus are (2n ¼ 28), species may not have arrived at this number in exactly the same way, suggesting complex chromosomal evolution. These past events may make hybridization and breeding using multiple species difficult. When Alnus sp. are targeted for genetic improvement to increase growth, stem volume, and growth rate, one strategy for improvement is to select on variation for the traits of interest present in the natural populations. Using this strategy depends on the existence of genetic variation. All estimations of genetic diversity within Alnus species are high, relative to other plant species, and have shown highly polymorphic loci. Further, species of Alnus show extensive gene flow, most likely due to their pollination strategy (wind) and seed dispersal (water), making populations show little correlation between genetic and geographic distance, with the exception of A. rubra. Xie (2008) estimated that genetic variation could be selected on and a gain in stem volume between 23 and 29% could be achieved. It is important to note that, with respect to A. rubra, there was significant genotype  environmental interactions and evidence of strong local adaptation for specific genotypes. This suggests that seed sources used in plantations should be selected from local environments and nearby natural populations and not from distant populations. Hall and Burgess (1990) suggested that hybridization may be a different

1 Alnus

strategy for genetic improvement of Alnus for commercial interests, specifically with respect to drought and water stress tolerance. Alnus, as a genus, has relatively poor tolerance to water stress, and this trait is an inhibitor for the use of Alnus in large-scale biomass plantations. Hall and Burgess (1990) argues that drought tolerance may be improved in Alnus species by selecting drought tolerance variation within species such as A. rubra or A. glutinosa, or attempting to transfer drought tolerance from A. cordata by hybridizing it with species of Alnus better suited to biomass production. Yet, when the parent taxa of published hybrids are examined within the topology of the phylogenies for Alnus, hybrids appear to be formed from parent taxa that are closely related. This may limit the possibility of Hall and Burgess’s (1990) recommendation of using hybridization as a tool for acquiring desired traits in Alnus species. The possibility of using hybridization in the improvement of Alnus still needs investigation. Alnus species are actinorhizal, meaning that they can enter into a symbiosis with Frankia, a N2-fixing filamentous bacteria. Frankia fixes nitrogen in the root nodule and the resulting ammonia is transported to the host plant in amounts sufficient to supply most of plants’ nitrogen requirements. This ability makes Alnus an attractive plant commercially because it can be grown in substandard soil conditions and does not require additional nutrients for growth. To date, all alder species examined have been shown to nodulate with Frankia. Although there are reports that Frankia infection on Alnus may sort by soil type, and nodulation capacity of soils varies, Alders appear to nodulate wherever they find themselves, precluding needing to “seed” Frankia within an alder plantation. We still know very little about alder host preference of Frankia strain, the signaling pathway(s) between Frankia and alder host for nodule formation, or if specific plant and Frankia genotype combinations yield high nitrogen fixation rates.

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13 Pirags DM (1962) Process of growth and structure of wood of hybrid alder (Alnus hybridus A.B.) in the Latvian SSR. Latvian Agricultural Academy, Elgava, p 19 Prat D, Leger C, Bojovic S (1992) Genetic diversity among Alnus glutinosa (L.) Gaertn. populations. Acta Oecologia 13:469–477 Roy S, Khasa DP, Greer CW (2007) Combining alders, frankiae, and mycorrhizae for the revegetation and remediation of contaminated ecosystems. Can J Bot 85:237–251 Sanginga N, Danso SKA, Bowen GD (1989) Nodulation and growth response of Allocasuarina and Casuarina species to phosphorus fertilization. Plant Soil 118:125–132 Schrader JA, Gardner SJ, Graves WR (2005) Resistance to water stress of Alnus maritima: intraspecific variation and comparisons to other alders. Environ Exp Bot 53:281–298 Schrader JA, Graves WR (2002) Intraspecific systematic of Alnus maritima (Betulaceae) from three widely disjunct provenances. Castanea 67:380–401 Schrader JA, Graves WR (2004) Systematics of Alnus maritima (seaside alder) resolved by ISSR polymorphisms and morphological characters. J Am Soc Hortic Sci 129:231–236 Schwintzer CR (1985) Effect of spring flooding on endophyte differentiation, nitrogenase activity, root growth and shoot growth in Myrica gale. Plant Soil 87:109–124 Schwintzer CR (1990) Spore-positive and spore-negative nodules. In: Gillings M, Holmes A (eds) Plant Microbiology. Garland Science/BIOS Scientific, London/New York, pp 177–193 Smolander A (1990) Frankia populations in soils under different tree species with special emphasis on soils under Betula pendula. Plant Soil 121:1–10 Smolander A, Sarsa ML (1990) Frankia strains in soil under Betula pendula: behavior in soil and in pure culture. Plant Soil 122:129–136 Smolander A, Sundman V (1987) Frankia in acid soils of forests devoid of actinorhizal plants. Physiol Plant 70:297–303 Smolander A, Van Dijk C, Sundman V (1988) Survival of Frankia strains introduced into soil. Plant Soil 106:65–72 Steele FL (1961) Introgression of Alnus serrulata and Alnus rugosa. Rhodora 63:297–304 Steiner W, Gregorius HR (1999) Incompatibility and pollen competition in Alnus glutinosa: evidence from pollination experiments. Genetica 105:259–271 Tanaka Y, Brotherton P, Hostetter S, Chapman D, Dyce S, Belanger J, Johnson J, Duke D (1997) The operational planting stock quality testing program at Weyerhaeuser. New Forest 13:423–437 Thomas KA, Berry AM (1989) Effects of continuous nitrogen application and nitrogen preconditioning on nodulation and growth of Ceanothus griseus var. horizontalis. Plant Soil 118:181–187 Van Dijk C (1979) Endophyte distribution in the soil. In: Gordon JC, Wheeler CT, Perry DA (eds) Symbiotic nitrogen fixation in the management of temperate forests. Oregon State University Press, Corvallis, OR, USA, pp 84–94 Weber A (1986) Distribution of spore-positive and spore-negative nodules in stands of Alnus glutinosa and Alnus incana in Finland. Plant Soil 96:205–213 Wehenkel C, Bergmann F, Gregorius HR (2006) Is there a tradeoff between species diversity and genetic diversity in forest tree communities? Plant Ecol 185:151–161

14 Wollum AG II, Youngberg CT, Chichester FW (1968) Relation of previous timber stand age to nodulation of Ceanothus velutinus. J Forest Sci 14:114–118 Woodworth RH (1929) Cytological studies in the Betulaceae. II. Corylus and Alnus. Bot Gaz 88:383–399 Woodworth RH (1930) Cytological studies in the Betulaceae. III. Parthenogenesis and polyembryology in Alnus rugosa. Bot Gaz 89:402–409 Woodworth RH (1931) Polyploidy in the Betulaceae. J Arnold Arbor 12:206–217 Xie C, Ying CC (1994) Genetic variability and performance of red alder (Alnus rubra) in British Columbia. In: Proceedings of the ecological management of BC Hardwoods, Richmond, pp 110–113 Xie CY (2008) Ten-year results from a red alder (Alnus rubra Bong.) provenance-progeny testing and their implications for genetic improvement. New Forest 36:273–284

B.D. Vanden Heuvel Xie CY, El-Kassaby YA, Ying CC (2002) Genetics of red alder (Alnus Rubra Bong.) populations in British Columbia and its implications for gene resources management. New Forest 24:97–112 Yang Y (1995) The effect of phosphorus on nodule formation and function in the Casuarina-Frankia symbiosis. Plant Soil 176:161–169 Zimpfer JF, Kennedy GJ, Smyth CA, Hamelin J, Navarro E, Dawson JO (1999) Localization of Casuarina-infective Frankia near Casuarina cunninghamiana trees in Jamaica. Can J Bot 77:1248–1256 Zitzer SF, Dawson JO (1992) Soil properties and actinorhizal vegetation influence nodulation of Alnus glutinosa and Elaeagnus angustifolia by Frankia. Plant Soil 140:197–204 Zuberer DA (1998) Biological dinitrogen fixation: introduction and non-symbiotic. In: Sylvia DM, Fuhrmann JJ, Hartel PG, Zuberer DA (eds) Principles and applications of soil microbiology. Prentice Hall, Upper Saddle River, NJ, pp 295–321

Chapter 2

Corylus Thomas J. Molnar

2.1 Introduction The Corylus L. genus contains a wide diversity of deciduous shrub and tree species that are important components of many temperate forests across the Northern Hemisphere, all bearing edible nuts. Its most widely known and well-studied member, the European hazelnut (Corylus avellana L.), is also an economically valuable commercial tree nut crop, ranking fifth in world production behind cashews (Anacardium occidentale L.), almonds [Prunus dulcis (Miller) D.A. Webb], walnuts (Juglans regia L.), and chestnuts (Castanea spp.) (FAOSTAT 2010). The top hazelnut producing country in the world is Turkey, which typically produces more than 70% of the world’s crop, which was 1,052,001 tons in 2008. Turkey is followed by Italy, which produces around 15–20% of the total, and the US, which produces 160 species) and secondarily in the southeastern United States. The genus Quercus includes several major monophyletic lineages (Fig. 6.2) after Manos et al. (1999) and Manos and Stanford (2001), recognized as distinct taxonomic sections. The white oaks (section Quercus), live oaks (series Virentes), golden cup or intermediate oaks (section Protobalanus), and red oaks (section Lobatae) are present in, if not restricted to, the Americas. The white oaks extend into Eurasia along with the Cerris or black oaks (section Cerris), while the cycle cup oaks (subgenus Cyclobalanopsis) are entirely Asian. The oaks are evergreen or winter-deciduous trees or shrubs with simple, alternate leaves. Leaf blades may be lobed or unlobed, pinnately veined, margins toothed (red oaks, section Lobatae, Fig. 6.3) or not toothed (white oaks, section Quercus, Fig. 6.3). Flowers are unisexual and wind pollinated. Fruit is an acorn with subtending cupule, maturing in the first year (all North American white oaks) or second year (most North American red oaks).

6.2.3 Hybridization and the Oak Species Oaks rank among the most recognizable trees at the genus level but among the most problematic for

Fig. 6.3 Leaf and acorn morphologies typical of section Quercus (white oaks) and section Lobatae (red oaks) [Images from USDA-NRCS (2009) and Britton and Brown (1913)]

categorizing at the species level. Since gene pool management is influenced by species delineations, we will attempt to clarify some issues in this area before proceeding. The genus Quercus is ubiquitous in many regions of the globe. It has a distinctive morphology, and historically has played an important role in human culture and industry (Ciesla 2002; Logan 2005), all contributing to its recognizability. Moreover, Quercus species do tend to possess a phenotypic cohesion that allows one to readily distinguish “good” members of a species without much difficulty. Yet close inspection of groups of individuals reveals gradations and fuzzy rather than sharp boundaries between species.

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This phenomenon has been recognized for some time and widely attributed to the propensity for oak species to form hybrids in nature (Palmer 1948; Muller 1952; Burger 1975; Van Valen 1976). Controlled crosses verified that Quercus species readily form hybrids with other members of the same section, though intersectional crosses are not formed (Cottam et al. 1982). With the further aid of molecular genetic markers it is now clear that natural hybrids are common in several groups in the Fagaceae, including Quercus as well as Castanea and Lithocarpus (Nixon 1997a). In Fig. 6.4, we show the hybridization network for North American Quercus species. Each node represents a species of oak, and a link has been formed between all pairs of species for which there is a hybrid described in the United States Department of Agriculture (USDA) Silvics Manual (Burns and Honkala 1990). Note the high frequency of hybrid formation within a section (white
white oak, red
red oak), except for species with disjunct distributions such as the west coast oaks versus those common east oaks of the Rocky Mountains. Note also the absence of described hybrids between red and white oaks. The most highly connected white oak is Q. stellata (11 described hybrids) and for the red oaks Q. velutina (12 described hybrids). Two other factors, in addition to recurrent hybridization, likely contribute to overlap in oak species phenotypes, namely incomplete lineage sorting and phenotypic plasticity. Species that no longer cross with one another may nevertheless share most polymorphisms if there has been insufficient time for differences to accrue since the speciation event. Muir and Schlotterer (2005) have proposed that much of the genetic overlap between the European white oaks Q. robur and Q. petraea is due to this shared ancestral polymorphism. In either event, both hybridization and incomplete lineage sorting yield overlapping gene pools. On the other hand, phenotypic plasticity could contribute to phenotypic overlap between oaks even in the face of genetic differences. Phenotypic plasticity has been well described in Quercus, both physiological and morphological (e.g., Bostad et al. 2003; Quero et al. 2006), and it is thought that some of this variation serves an adaptive function (Abrams 1994). Oaks are notoriously variable in their leaf characters, across seasons and even within the same canopy (Blue and Jensen 1988; Bruschi et al. 2003), even though leaves are frequently used for identifications.

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Regardless of causation, the data show that the gene pools of oak species overlap considerably in their content (see next two sections), differing mainly by allele frequencies but not diagnostic alleles. Sectional gene pools are more readily resolved, where red and white oaks form distinct clusters based on nuclear markers (Guttman and Weigt 1989) and monophyletic clades according to chloroplast and ITS markers (Manos et al. 1999). But within a section the oak species tend to overlap in allelic composition within mixed stands (e.g., Aldrich et al. 2003b) and across regions (e.g., Bodenes et al. 1997b). Thus, at both the genotypic and phenotypic levels, oak species generally do not possess traits that are useful as both necessary and sufficient conditions to distinguish them from all other oak species. Nevertheless, molecular approaches that integrate across the entire genome demonstrate some promise for distinguishing species and resolving phylogenetic relationships (Pearse and Hipp 2009). All this begs the question, what species concept should be used to classify oaks? If hybridization is widespread then the biological species concept is not suitable since it defines species on the basis of reproductive isolation (Mayr 1942). Of the many other species concepts in use, actually none are fully suited to the oak problem since they each assume some form of essentialism, that members of a species should all share some basic element that distinguishes them from others, which do not belong in that group. This emphasis on shared, derived character states as diagnostic markers of a lineage, a central tenet of the cladistic approach to phylogenetics (Hennig 1979), often does not apply well at or below the species level. Instead, many researchers at this level have gravitated toward the phenetic clustering approaches developed by the early numerical taxonomists (e.g., Sneath and Sokal 1973) that emphasize shared (instead of shared-derived) characters – since traits are not fully “derived” at this stage of evolution. Taken a step further, the contemporary inclination is to fully embrace the reticulate nature of species-level gene pool dynamics and focus on network representations rather than bifurcating trees (e.g., Posada and Crandall 2001; Huson and Bryant 2006; see also Fig. 6.4). We adopt the view that oaks can be described adequately using a flexible terminology based on a network of shared traits, like the views held by the philosopher Ludwig Wittgenstein. Pigliucci (2003)

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Fig. 6.4 Hybridization network of North American Quercus species. Data from Burns and Honkala (1990)

recognized that much of the confusion surrounding the species problem, in general, was not empirical in its nature, but philosophical and linguistic. He noted the correspondence between the cluster concepts of the

pheneticists and the “family resemblance” concept developed by Wittgenstein (1958) which maintained that most categories in human language are assemblages of referents to items that share a family

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resemblance – without all of them actually sharing some defining element. Wittgentstein used the simple concept of a “game,” defying anyone to pull out a single feature that runs through all possible activities that we might label as a game (would it be a ball, bat, dice, cards, jump rope, dart. . .?). The actual uses of a word, like “game,” he claimed, form “a complicated network of similarities overlapping and criss-crossing: sometimes overall similarities, sometimes similarities of detail” (Wittgenstein 1958, }66), not unlike the oak hybridization network that we present here (Fig. 6.4). And indeed this is how oak gene pools are managed, through identifications grounded in clusters or networks of character states. Botanical keys can provide a definitive (albeit at times arbitrary) call on species identifications, provided the requisite plant characters are available for inspection, and the person has time to scrutinize that one specimen. In stand management, however, identifications are usually made in the field where foresters, scientists, and laypersons are inclined to form flash judgments of oak identities based on collections of readily observed, cooccurring traits. This field-Gestalt method is at times referred to as the whole-tree silvics approach to identification (Tomlinson et al. 2000; Aldrich et al. 2003b). When the number of key traits rises above some critical (and often unspoken) level, the individual is typed as X, if another set of traits co-occur then Y. Intermediate specimens go un-noted or may be cut down, but are certainly excluded from breeding programs – thereby reinforcing what counts as being a member of a given species. In this regard, the oak species concept, in practice, works just fine. As Pigliucci (2003) remarked, Wittgenstein conveys the proper sense in the following: But this is not ignorance. We do not know the boundaries because none have been drawn . . . We can draw a boundary – for a special purpose. Does it take that to make the concept usable? Not at all! (Wittgenstein 1958, }69).

6.2.3.1 Quercus Section Quercus Roughly 200 species of Quercus section Quercus comprise the white oak group (Nixon and Muller 1997), distributed throughout much of the Northern Hemisphere. Section Quercus includes evergreen or deciduous trees or shrubs, with leaf blades lobed or unlobed, margins usually entire but if toothed then

never bristle-tipped (Fig. 6.3). Acorns mature within a single season. The most heavily studied oaks are the European white oaks, principally Q. robur (Pedunculate Oak or English Oak) and Q. petraea (Sessile Oak or Durmast Oak). They tend to occupy different microhabitats in a stand, Q. robur preferring richer, wetter, more alkaline soils compared to Q. petraea (Becker and Levy 1990; see Saintagne et al. 2004). There exists an impressive array of studies regarding molecular genetic variation in Q. robur and/or Q. petraea, including but not limited to the following: isozymes (Zanetto et al. 1994; Gomory 2000), ribosomal DNA (rDNA) (Petit and Kremer 1993; Muir et al. 2001), chloroplast DNA (Petit and Kremer 1993; Cottrell et al. 2002; and see Sect. 6.4.4), proteins (Barreneche et al. 1996; Jorge et al. 2005; see also Sect. 6.7.4.2), and anonymous DNA markers (Moreau et al. 1994; Bodenes et al. 1996; Cervera et al. 2000; Zoldos et al. 2001; Mariette et al. 2002; and see below), along with a variety of QTL studies (see Sect. 6.6.3.4). Q. robur and Q. petraea exhibit genetic differences in allele frequencies only, not in diagnostic alleles. This conclusion is reached after numerous genotyping projects using several marker types, some covering a large fraction of the genomes. For example, Bodenes et al. (1997a) queried 2,800 random amplified polymorphic DNA (RAPD) fragments and found that only 2% displayed allele frequency differentiation between the species, and no fragments were exclusive to one species. Scotti-Saintagne et al. (2004b) compiled variation for 389 molecular markers in Q. robur and Q. petraea, collected from several different sites, and found that only 12% of the loci displayed significant species-level differentiation. Coding regions showed more differentiation than did non-coding regions. They were able to locate roughly half of the total markers on a Q. robur map (see Sect. 6.6.3.3), and the loci associated with species differences were spread across nine linkage groups. These findings show that the Q. robur and Q. petraea genomes overlap extensively in composition, and the differences are distributed in clusters across the genome. Notwithstanding these genetic results, there is ample evidence that the designations Q. robur and Q. petraea represent biological entities. In a largescale study of leaf morphology, Kremer et al. (2002) showed that Q. robur and Q. petraea maintain a stable bimodal distribution in mixed stands, despite

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overlapping distributions. Curtu et al. (2007) studied morphological and genetic markers in a natural stand from Romania containing Q. robur, Q. petraea, Q. pubescens, and Q. frainetto. Using isozymes, microsatellites, and a Bayesian method to infer genetic clusters, they found evidence that the oak gene pool was partitioned into four populations, each corresponding in a reasonable fashion with the species identifications based on morphology. Approximately 3.4% of the population appeared to be of first-generation hybrid origin. Several reports on QTL variation are available for Q. robur and Q. petraea. Since white oak species are distinguished largely based on their leaf morphologies (Gailing 2008), we will consider leaf QTL variation here (see Sect. 6.6.3.4 for other oak QTLs). Saintagne et al. (2004) studied 15 leaf traits that are typically used to classify Q. robur and Q. petraea. Species explained little of the variation in size-related traits, but species explained much of the variation observed in several other leaf characters: petiole length and ratio (80% of variation), pubescence (77%), venation (67–70%), and sinus width (59%). Overall they detected significant QTLs for 13 of the 15 leaf traits, with on average 1–3 genes controlling each. The five traits most associated with species differentiation were localized on 6–9 linkage groups. Leaf pubescence is noteworthy since Nixon (1997b) specifies that foliar trichomes are diagnostic in North American white oaks given the often-times wide variation in leaf shapes. Saintagne et al. (2004) showed that leaf pubescence was an important difference between Q. robur and Q. petraea even though 16% of the typically glabrous Q. robur leaves exhibited trichomes. More importantly, they detected two QTLs for leaf pubescence, each explaining 12.9–17.2% of the phenotypic variance, and one of these QTLs co-localized with another QTL for another species-associated trait (petiole length). Thus, the characters distinguishing Q. robur and Q. petraea are under polygenic control and distributed broadly throughout the genome in clusters. Interestingly, Gailing (2008) examined the same set of leaf traits but using a distinct cross and found that some QTLs were preserved while several were not. Gailing (2008) performed a cross between Q. robur from Germany and Croatia, whereas Saintagne et al. (2004) had used a French cross. The two independent approaches converged on the same co-localized QTLs for number and percentage of intercalary veins

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(linkage group 3) and for number of veins (linkage group 5). However, the other QTLs exhibited poor correspondence between the studies (though see Gailing et al. 2005 for successful transfer of oak flushing date QTLs across families). Gailing (2008) suggests that this outcome is likely due to dissimilar genetic backgrounds and environments. The conclusion remains that species-level traits are under polygenic control, but the fact that only two QTLs were stable across genetic backgrounds and environments attests to the complexities of species differences in the oaks. In North American white oaks, Craft and Ashley (2006) used microsatellite DNA analysis to examine population differentiation among three species, Quercus alba, Q. bicolor, and Q. macrocarpa, occurring in both pure and mixed stands in northeastern Illinois. They detected no strong genetic groupings using individual-based Bayesian clustering or principal components analyses. Using classical F statistics, they found significant but low genetic differentiation. They also found that some intraspecific comparisons were as genetically differentiated as interspecific comparisons, with the two populations of Q. alba exhibiting the highest level of genetic differentiation. Their work indicates that the three species do not represent distinct and differentiated genetic entities. In contrast, Cavender-Bares and Pahlich (2009) found clear and significant differentiation between two sympatric sister live oak species, Q. geminata and Q. virginiana, in Florida using nuclear microsatellites. These species show a 2-week difference in flowering time, which likely provides a reproductive isolating mechanism. The genetic differentiation was matched by clear physiological and ecological differentiation (CavenderBares and Pahlich 2009). Flowering time separation has been hypothesized in earlier work (Nixon 1985) to provide a leaky reproductive barrier that is sufficient to maintain distinct identities between these species.

6.2.3.2 Quercus Section Lobatae There are approximately 195 species of red oak (Quercus section Lobatae; Jensen 1997), a group indigenous to the Americas. These are trees or shrubs, evergreen or deciduous, with lobed or unlobed leaves, margins typically toothed and bristle-tipped though at times entire (Fig. 6.3). Acorns mature in 2 years, rarely one.

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Fig. 6.5 Leaf and acorn morphologies of several red oak species (Quercus section Lobatae; images from USDA-NRCS 2009; Britton and Brown 1913)

Phenotypic variation has been described and quantified in several red oak studies. Richard Jensen’s contributions are notable in this area and one should consult his work for a systematic and thorough appreciation of naturally occurring variation in the group (e.g., Jensen 1977, 1995; Jensen and Eshbaugh 1976; Jensen et al. 1984, 1993). Leaf forms take quite a range in shapes across red oak species (Fig. 6.5), and can vary considerably by position within a canopy (Blue and Jensen 1988), as in the white oaks (Bruschi et al. 2003). Hybrids with intermediate morphologies are described between many of the red oak species as noted in Jensen (1995) and depicted in Fig. 6.4. Much less information is available on the genetic basis of species differences in the red oaks compared to white oaks, but the evidence to date suggests that red oak gene pools are poorly differentiated. Guttman and Weigt (1989) examined ten red oak and eight white oak species using isozyme markers and found that the red and white oaks were readily distinguished from one another, but the red oaks were less resolved from one another compared to the white oaks. Moreover, within-species phylogeographic signal is very

weak within the broadly distributed Northern Red Oak, Q. rubra, across its native range in North America (Magni et al. 2005), compared to the strong signal resolved for European white oaks. These factors could indicate a more recent evolutionary origin for red oak species, leaving their gene pools more articulated. Trelease (1924) suggested this based on biogeography though more recent phylogenetic and paleobotanical evidence suggests otherwise (see Manos et al. 1999). Nevertheless, it is probably safe to say that species boundaries in the red oaks are at least as ragged as in the well-studied white oak section. Jensen’s group has conducted several detailed studies of red oaks of northern Wisconsin that illustrate patterns of variation in the section. Jensen et al. (1993) described clinal variation in leaf traits (17 landmarks) for Q. rubra (Northern Red Oak), Q. ellipsoidalis (Northern Pin Oak), and putative hybrids in natural populations of the Apostle Islands. Hokanson et al. (1993) appraised isozyme variation in the same system and found little differentiation among populations. Tomlinson et al. (2000) characterized leaf and isozyme variation in 30 mother trees and their progeny,

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showing that Q. ellipsoidalis could be distinguished from Q. rubra and hybrids based on overall phenotypic characters (whole-tree silvics), though Q. rubra was not separable from hybrids. Aldrich et al. (2003b) described another ramified red oak gene pool in an old-growth stand in Indiana. They quantified microsatellite variation (15 loci) in a mixed community containing three red oak species, Q. rubra (Northern Red Oak), Q. shumardii (Shumard Oak), and Q. palustris (Pin Oak). These taxa tend to retain phenotypic cohesion and partition the habitat, with Q. palustris occupying wet, poorly drained sites, Q. rubra preferring well-drained, xeric sites, and Q. shumardii occupying intermediate areas. Professional foresters and academic dendrologists typed the species using whole-tree silvic methods. Aldrich et al. (2003b) found high genetic variation within species but moderate differences among species. A Bayesian clustering approach suggested the existence of three populations comprised of (a) pure Q. rubra, (b) Q. rubra, Q. shumardii, and their hybrids, and (c) Q. rubra, Q. shumardii, Q. palustris, and their hybrids. The procedures, conditions, and results were similar to those of Curtu et al. (2007) who resolved four genetic clusters in a mixed stand of four European white oak species, though the white oaks were better resolved genetically. In Mexico, Gonzalez-Rodriguez et al. (2004a) described the extensive hybrid zone between Q. laurina and Q. affinis and found within this zone that molecular variation did not correspond with morphological variation.

6.3 Distribution and Ecology Generic diversity in the Fagaceae is centered in Southeast Asia, whereas species diversity is more uniformly distributed. Species of Quercus are especially prominent in the Northern Hemisphere where they can come to dominate a stand, such as in temperate seasonally dry forests. They are most diverse in Mexico (Valencia 2004). On a local scale, oaks occupy a variety of habitats though they tend toward well-drained upland areas. Oaks have beneficial associations with seed dispersal agents such as squirrels and with mycorrhizal fungi. Numerous herbivores feed on the oaks and a variety of gall wasps use oaks for habitation.

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6.3.1 Global Distribution Oaks are distributed on five continents including Europe, Asia, North Africa, North America, and Central and South America. There are some 255 species in the New World (Manos et al. 1993; Nixon 2006), with 162 species in Mexico (Valencia 2004). Fossil evidence indicates that oaks originated in China during the Eocene. Black oaks (section Cerris) and white oaks (section Quercus sensu latu) are thought to have diverged in Asia by the Eocene (56–35 Mya). Black oaks are further divided into two groups: a semideciduous Cerris group and an evergreen Ilex group (Manos and Stanford 2001; Manos et al. 2001). Section Quercus dispersed to the New World during the Oligocene and is hypothesized to have undergone a rapid radiation subsequently, giving rise to three major New World clades: the red oaks, white oaks, and intermediate oaks (Daghlian and Crepet 1983; Manos et al. 1999) (Fig. 6.2). Phylogenetic and paleobotanical evidence suggests that section Quercus s. s. evolved at middle latitudes in the Americas and subsequently migrated to the Old World prior to the break up of land bridges linking the northern continents (Manos et al. 1999). Migration could have thus occurred in the general time frame between the Late Eocene (ca. 40 Mya) and the Middle Miocene (ca. 15 Mya) (Tiffney 1985). White oak (section Quercus) fossils appeared in Asia in the Miocene and Pliocene (Zhou 1993). Fossil data indicate that oaks were evergreen in their ancestral state and subsequently evolved deciduousness (Manos and Stanford 2001). Phylogenetic hypotheses in the New World oaks suggest convergent evolution in various traits associated with climate and habitat specialization, including leaf lifespan, growth rates, and vulnerability to drought (Tucker 1974; Cavender-Bares et al. 2004a), whereas little to no differentiation has occurred in the morphology of flowers and fruit (Manos et al. 1999, 2001; Oh and Manos 2008).

6.3.2 Ecological Distribution Oaks tend to be distributed in well-drained upland areas and often in montane areas. There are widespread lowland oaks, however, including the live

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oaks (series Virentes), and there are oaks associated with rivers and wetlands that are able to tolerate some degree of flooding, such as Q. lyrata and Q. laurifolia. In northern central Florida, which lies at the confluence of the northern temperate flora and the southern subtropical flora, oaks are hyperdiverse. Due to the potential for close relatives to competitively exclude one another, the coexistence of multiple congeners in this region presents a challenge to explain (CavenderBares et al. 2004b), particularly given the lack of major elevational gradients that occur in other regions of high oak diversity (Whittaker 1956; Platt and Schwartz 1990). Associations of suites of ecophysiological traits and species ecological distributions indicate that oaks specialize into different niches associated with soil moisture and fire regime, thus allowing them to partition the landscape and maintain their diversity (Cavender-Bares and Holbrook 2001; Cavender-Bares et al. 2004a, b). Furthermore, they show a pattern of phylogenetic overdispersion (Fig. 6.6) in which

Fig. 6.6 Schematic of phylogenetic overdispersion (co-occurring species are less related to each other than expected by chance) in the three major oak dominated communities in Florida (adapted from Cavender-Bares et al. 2004a, b). Oaks within each of the major phylogenetic lineages occur in each

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distantly related species (those from distinct clades) co-occur together in communities rather than species from the same lineage (Cavender-Bares et al. 2004a). This phenomenon has been observed in other oak dominated systems (Whittaker 1969; Mohler 1990). The pattern may be caused by convergent evolution in ecological traits important for habitat specialization. Density-dependent mechanisms that operate at the clade level, such as resistance to disease (see Sect. 6.4.3) or to herbivores (see Sect. 6.3.7), may also prevent closely related oaks from co-occurring.

6.3.3 Acorn Properties and Dispersal The acorn fruit is comprised mostly of cotyledon that contains high levels of energy-rich lipid, making them an important food source for many mammals and birds, including squirrels, jays, woodpeckers,

community, and respective physiological traits match each environment, indicating convergent evolution. Vulnerability to different diseases or acorn properties that promote contrasting predator behavior may promote the coexistence of oaks from different lineages

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grackles, wild turkey, mice, chipmunks, deer, black bears, and more than 140 other species of vertebrates in North America (Van Dersal 1940). Tree squirrels play a notorious and well-documented roll in acorn dispersal (Steele and Koprowski 2001). Squirrels cache the seeds in the ground and then fail to recover some of these stored reserves (Van Dersal 1940). They also often only partially consume acorns, leaving the embryo intact, permitting subsequent germination and survival (Steele et al. 1993). By transporting and scatter hoarding acorns to individual sites just below the leaf litter, squirrels reduce the probability of seed predation and desiccation and increase the chances of germination, root establishment, and winter survival (Steele and Koprowski 2001). Red and white oaks have contrasting tannin and lipid levels, which influence feeding decisions at different times of the year (Smallwood and Peters 1986). Red and white oaks also differ in their dormancy and germination patterns in the temperate zone, with white oak acorns germinating immediately upon falling in autumn and red oak acorns undergoing dormancy throughout the winter period and germinating in the spring. Gray squirrels apparently distinguish between the dormant red oak acorns and rapidly germinating white oak acorns, selectively dispersing and caching the former and eating the latter. Differential acorn dispersal and caching preferences due to contrasting fat and tannin content have given rise to the differential dispersal hypothesis (Steele and Koprowski 2001) in which red oaks are thought to disperse more rapidly than white oaks. These contrasting dispersal and caching preferences are likely to contribute to forest structure and may help explain the observation that red and white oak species tend to co-occur more often than expected by chance (Fig. 6.6; Cavender-Bares et al. 2004a). Higher dispersal rates of red oak acorns also might help explain the lower genetic differentiation observed among some red oak species (see Sect. 6.2.3.2).

6.3.4 Mast Seeding Mast seeding, the synchronous production of large crops of seeds, has been frequently documented in oak species (Liebhold et al. 2004). Sork (1993) found evidence supporting the hypothesis that mast seeding has evolved as an anti-predator adaptation by which

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large seed crops during mast years satiate the seed predators and allow survival of some of the seeds. This work indicates that there should be stronger selective forces for evolution of masting in tropical oaks compared to temperate oaks. Cues for masting are likely to be linked to climatic factors. Asynchrony in masting between species, particularly those of different lineages (Mohler 1990) could help explain phylogenetic overdispersion and coexistence of distantly related oaks. Asynchrony in masting between species has been attributed to differences in seed production caused by the varying numbers of years (1 or 2) required to mature seeds in white versus red oaks, whereas intraspecific variation in the synchrony of seed production has been related to variation in habitat conditions (Liebhold et al. 2004). The timing of masting in oaks has been associated with population cycles of insects, including gypsy moths (Lymantria dispar). One hypothesis for this association is that the tissue quality of leaves changes during mast years to contain lower secondary metabolites. The higher nutritive quality of leaves increases larval survival and leads to moth outbreaks (Sela˚s 2004).

6.3.5 Physiology While oaks, in general, are characterized as drought adapted (Abrams 1990), they inhabit a wide range of environments with respect to soil moisture, hydroperiod, and fire regime. Oaks tend to have deeppenetrating root systems (Stone and Kalisz 1991), enabling them to maintain relatively high pre-dawn water potentials during drought, and they often have thick leaves, which prevent wilting. Oaks frequently maintain a higher rate of photosynthesis at low leaf water potentials and high vapor pressure deficits than co-occurring species of other genera (Abrams 1990). In general, they tend to have high epidermal resistance, minimizing water loss when stomata are shut, although such resistances vary with species and tend to be higher in evergreen species than in deciduous species (Kerstiens 1996; Cavender-Bares et al. 2007). Deciduous oaks have been reported as having notoriously long vessels (Cochard and Tyree 1990). The xylem anatomy of temperate oaks has often been characterized as ringporous given that many temperate species produce large early wood vessels that hydraulically support the

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spring flush of leaves followed by much narrower latewood vessels. Earlywood vessels embolize partway through the season (Sperry et al. 1994), while the narrower latewood vessels, which presumably have smaller pores in their intervessel membranes, are more resistant to drought-induced cavitation and can sustain water transport for the rest of the season. However, many subtropical or tropical oak species have diffuse porous xylem anatomy in which vessels size distributions do not change throughout the year (CavenderBares and Holbrook 2001). In the southeastern United States, oak diversity is very high, maintained in part by the heterogeneity in soil moisture and fire regime. Individual species show variation in physiological and life history traits that tend to match their ecological distributions (CavenderBares and Holbrook 2001; Cavender-Bares et al. 2004a, b). For example, species that are adapted to fire regimes that are severe, predictable, and occurring on the order of 25–50 years intervals have high rhizome resprouting capacity whereas species occurring in mesic to hydric resource rich habitats where fire occurs only rarely tend to achieve tall maximum heights to compete for light at the top of the canopy (Cavender-Bares et al. 2004b). Likewise, species from xeric environments tend to show resistance to droughtinduced embolism but cannot support large hydraulic fluxes. In contrast, species from mesic environments are characterized by large hydraulic fluxes (CavenderBares and Holbrook 2001). In the seasonally dry tropical forests of Costa Rica, live oaks show unusually high resistance to leaf wilting (Brodribb and Holbrook 2006).

6.3.6 Herbivory Oak leaves support a diverse herbivore community. In one study, 138 species of leaf-chewing insects were found on leaves of Quercus alba and Q. velutina, sampled in southeastern Missouri, USA (Le Corff and Marquis 1999). Reduction in herbivore load by birds has been shown to have positive fitness consequences in oaks (Marquis and Whelan 1994). Oaks produce a diverse array of sublethal plant secondary compounds, including phenolics and hydrolyzable tannins, which are thought to provide defense by reducing insect oviposition, feeding, and biomass gain of

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herbivores (Lill and Marquis 2001). In experimental and observational studies examining impacts of these secondary compounds in leaves of Q. alba and Q. velutina on herbivores, specialist herbivores were more likely than generalists to be negatively affected by condensed tannins (Forkner et al. 2004), and defoliation levels were generally lower where concentrations of phenolics were higher (Forkner and Marquis 2004). Sclerophylly, leaf pubescence, high lignin content, and other leaf traits and architectural characteristics of oaks may also serve as important plant defenses. For example, architectural traits that minimize leaf-to-leaf contact in oaks may be defensive traits against leaf-tying caterpillars (Marquis et al. 2002). In experimental manipulations of leaf pubescence, higher leaf hair density reduced generalist caterpillar densities (Lill et al. 2006).

6.3.7 Oak Galls Of the oak herbivores, the oak gallwasps (Cynipidae: Cynipini) are known to have highly specialized associations with their oak hosts. Oak gallwasps are a species-rich lineage (ca. 1,000 species) that induces structurally complex galls on oaks and other Fagaceae (Abe et al. 2007). They are able to bypass oak chemical defenses (phenolics and condensed tannins) by inducing the development of host plant tissues that have elevated nutritive value but low concentrations of toxic secondary plant metabolites (Cornell 1983). Little is known about the process of gall induction. Gallwasps feed on and reproduce in these tissues. Despite their ability to circumvent toxic secondary compounds, host shifts are very constrained (Stone et al. 2009). Oak gallwasp genera are usually specific to a single oak section, and within section Cerris, to a single species group (Stone et al. 2009). Specificity does not extend to the level of the species, and gallwasps frequently gall multiple oaks in the same section or species group. The Western Palaearctic gallwasp fauna are best studied and include 150 species in 10 genera. While most of these species are highly specialized, at least two groups of the Western Palaearctic gallwasps (including Andricus and Calirhytis genera) show obligate alternation between oak sections during their lifecycle. In the deep split between section Cerris and section Quercus,

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diversification of the gallwasps is closely associated but post-dates diversification of the oaks. Inter- and intraspecific phylogeographies suggest that gallwasps have pursued their oak hosts for at least 20 my across much of the Palaearctic (Stone et al. 2009). These data also support the hypothesis that Western Palaearctic and Nearctic gallwasps are derived from an initial radiation in ancient Asian or Beringian oak forests, and that the divide between Palaearctic Cerris and White oak gallwasps represents the deepest divide in the Cynipini. The extreme host conservatism within some of the major clades of oak gallwasps suggests metabolically intimate aspects of the plant-gall inducer interaction. Stone et al (2009) have suggested that oaks and oak gallwasps represent an example of a coevolutionary arms race between host plant susceptibility and gall inducer virulence.

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fine roots compared to other ECM host species. However, an increase in number of studies using molecular methods has demonstrated the feasibility of these methods for oak-ectomycorrhizal associations (e.g., Cooke et al. 1999; Pinkas et al. 2000; Nechwatal et al. 2001; Giomaro et al. 2002; Avis et al. 2003; Kennedy et al. 2003; Dickie and Fitzjohn 2007; Cavender-Bares et al. 2009). Lack of ectomycorrhizal symbionts has been implicated in some forest systems in restricting oak regeneration into old fields (Dickie et al. 2007) but not in others (Klemens et al. 2010). ECM fungal colonization has also been proposed as one of several competing hypotheses to cause monodominant oak stands (Boucher 1981), although evidence for this is limited.

6.4 Conservation Biology 6.3.8 Mycorrhizal Associates In ecological settings, oaks are functionally obligately associated with a diversity of ectomycorrhizal (ECM) fungi (included in the Ascomycota and Basidiomycota) and benefit from the symbiosis in terms of growth, seedling establishment, and survival (e.g., Avis et al. 2003; Smith et al. 2007a, b; Morris et al. 2008a). It is also now known that oaks associate to some extent with vesicular arbuscular mycorrhizal (VAM) fungi (Dickie et al. 2001). While a large number of ECM taxa (~250) are specific at the family or genus level (Molina et al. 1992; Ishida et al. 2007), it is not known how commonly specificity occurs below the genus level. Individual fungi can function differently on different host plants with different degrees of penetration into and between root cells depending on the host (Taylor and Bruns 1999; Villarreal-Ruiz et al. 2004). Contrasting ECM communities were found on the roots of sympatric oaks in California, demonstrating that fungal preferences at the host plant species level can be important in ECM assemblages within the oak genus (Morris et al. 2008b). Similarly, an experimental study of two containerized oak hosts across an experimental hydrologic gradient demonstrated that ECM fungal communities differed between a white oak (Q. montana) and a red oak (Q. palustris) (Cavender-Bares et al 2009). Oak ECM fungi are difficult to work with because oaks have very small

Most oaks maintain fairly high levels of genetic variation within their populations and relatively less variation among populations (e.g., Aldrich et al. 2005a). This may be due to their propensity to outcross via wind pollination and to their longevity (Hamrick et al. 1992). There remain large standing wild populations of many of the oak species, and at present there is little effort to actively manage the gene pools, particularly in the USA. Unfortunately, this does not account for mismanagement, such as high-grading and poor nursery practices, for the possible loss of rare or endemic alleles before they are detected, or for demographic failure in parts of the range and pathogen epidemics that can wipe out entire populations in short order, or global climate change. Except for these factors, the oak gene pools appear sound.

6.4.1 Conservation Initiatives Oak gene pools have not received as much attention, in part due to the ubiquity of oaks and the high variation found in those that have been studied, though see Jacobs and Davis (2005) for considerations of the genetic implications of hardwood nursery practices in the eastern United States. On the other hand, oak demography has received considerable attention in the USA over the past 50 years due to regeneration failure noted in many areas (see Sect. 6.4.2, see also a review

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on artificial regeneration of oaks by Dey et al. 2008). The European Union has funded a network of agencies under the EVOLTREE program (http://www.evoltree. eu/), which seeks to determine the impact of climate change on forest ecosystems. Some of the focus will be on gene pool and genome structure of trees, with oaks as the model organism. Several useful reviews on the genetics and genomics of adaptation in tree species are relevant here (Gonzalez-Martinez et al. 2006; Neale 2007; Savolainen et al. 2007; Neale and Ingvarsson 2008). As for ex situ management, oaks can be propagated through grafting (e.g., Kothencz et al. 2001) although oak lines immortalized by grafting are subject to reversion wherein shoots of the mutant revert to the wild type phenotype. Oak seeds do not store terribly well, though the National Seed Laboratory offers resources for management and propagation (http://www.nsl.fs.fed.us/). In addition, the US National Arboretum maintains the Woody Landscape Plant Germplasm Repository, which includes several oak accessions (http://www.usna.usda.gov/Research/ wlpgr.html).

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census of oaks in the canopy but an appraisal of the overall regenerative potential of the stands. Of course major shifts in the local abundances of mesic vs. xeric sites due to climate change could have profound effects on forest composition and oak demography. There are several other factors that contribute to oak demographic problems. In some Californian oaks, the spatial isolation of populations appears to be an issue where Sork et al. (2002a, b) found a reduction in oak neighborhood sizes over time, attributing this to the fragmentation of the habitat and decline of pollen donor density. In many areas it is the invasive plant species that threatens oak regeneration, such as Ailanthus altissima competing with oaks for highlight environments in the eastern United States (Hu 1979; Huebner 2003; Rebbeck et al. 2005). Exotic pests and pathogens pose an immediate threat to oak forests (see next section), and although we are currently surrounded by oaks, we only need look to Castanea dentata (the American chestnut, also within the Fagaceae) to see how rapidly a pathogen (chestnut blight, Cryphonectria parasitica) can sweep through and remove a canopy dominant taxon (Fig. 6.7).

6.4.2 Oak Regeneration Failure The demographic profile of oaks in the USA appears to be changing, and several causal agents have been identified (reviewed in Abrams 1992, 2003; Abrams and Nowacki 1992; Lorimer 1980, 1993). One factor appears to be a change in the disturbance regime of some forests. Oaks are good competitors on xeric sites where canopies tend to remain open, but oaks are rather poor competitors on closed-canopy, mesic sites at least in part because they are not as shadetolerant as many other taxa, such as Acer saccharum (sugar maple). Stands whose canopies and understories have been kept open for the past century or two through logging, grazing, and fires are subject to a successional shift toward shade-tolerant taxa when these disturbances are suppressed. Consequently, many of these mesic stands still contain numerous large oaks in the canopy but little if any oak regeneration, the subcanopy dominated primarily by shade tolerants like maple (Parker et al. 1985; Aldrich et al. 2005b). Seed and seedling predation by deer and rodents can exacerbate this condition. Thus, a proper accounting of a landscape is not simply the adult

Fig. 6.7 Castanea (chestnut) adult in the wild prior to chestnut blight (image from USDA-NRCS 2009)

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6.4.3 Pests and Pathogens Oaks are susceptible to a number of pathogens and pests. Oak wilt and Sudden Oak Death are two fungal diseases of primary concern.

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grafts between individuals and removing potential spore producing host trees. Triazole fungicides have been used as a means of chemical control (Koch et al. submitted).

6.4.3.2 Sudden Oak Death 6.4.3.1 Oak Wilt Ceratocystis fagacearum (Bretz) Hunt, the oak wilt pathogen, is recognized as one of the most destructive diseases to afflict oak species in the USA. Oak wilt was first recognized in North America in 1944 and is now widespread in eastern, midwestern, and southern states (Koch et al. submitted). The distribution and development of oak wilt in eastern and midwestern United States oak forests has been closely linked to changes in forest stand composition, forest management practices, and pathogen dissemination facilitated by human and vector activity, and there is concern about its increasing spread into southern and western states, exemplified by the Texas outbreak (Wilson 2001). Oak wilt is a vascular fungal disease that blocks the xylem vessels preventing water transport (Juzwik 2000). The red oaks (section Lobatae) are generally more susceptible than white oaks (section Quercus). White oaks have narrower vessels and block the spread of the disease with tyloses. However, within the white oak clade, semi-evergreen live oaks (Quercus fusiformis Small and Q. virginiana Mill., series Virentes) are also susceptible. Oak wilt has caused massive losses of live oaks in central Texas. High susceptibility and mortality in live oaks in Texas was not anticipated given the relatively limited oak mortality caused by the disease in the deciduous forests of the north, central, midwestern, and mid-Atlantic United States. The intensity of oak wilt in Texas is attributed to a number of factors related to host characteristics and the ability of the pathogen to adapt to limiting environmental conditions (Appel 1995). Wilt symptoms begin at the crown of the tree, but are manifested differently in different lineages (Fowler 1953; Juzwik 2000). The fungus spreads to susceptible trees either via dissemination of infective spores by beetles or by underground root grafts that connect the vascular systems of individual trees, of the same species or of closely related species. Mechanical methods for controlling oak wilt have focused on severing root

A recent epidemic of Phytophthora ramorum, the nonnative invasive pathogen that causes Sudden Oak Death in coastal woodlands of California, is causing severe mortality in some oak species belonging to the red oak (Lobatae) group. P. ramorum has killed tens of thousands of native coast live oak and tanoak trees in California, and the pathogen does not seem to differentially select among genotypes or closely related species of red oaks that are susceptible (Dodd and Kashani 2003). Mortality due to the disease was first observed in 1995 in tanoak trees (Lithocarpus densiflorus), although P. ramorum was not identified as the pathogen until 2000. Subsequently, the disease was observed on coast live oak (Q. agrifolia), California black oak (Q. kelloggii), and Shreve’s oak (Q. parvula var shrevii) (McCreary 2007). In California forests, bay laurel (Laurus nobilis, Lauraceae) is the primary carrier of P. ramorum because it acquires a non-lethal but highly contagious leaf infection (Rizzo et al. 2005). The pathogen also survives and produces chlamydospores in forest soils over summer, providing a possible inoculum reservoir at the onset of the fall disease cycle (Fichtner et al. 2007). Trunk bleeding, presence of beetles, presence of the fungus Hypoxylon thouarsianum, and tree mortality through time are visible signs of P. ramorum infection (Kelly et al. 2008). P. ramorum infects a very wide diversity of plants beyond bay laurel, many of which serve as foliar hosts and sources of inoculum. As a result, there is heightened concern that the pathogen could spread to the diverse oak woodlands of the eastern United States (Hong et al. 2005; Venette and Cohen 2006; SnoverClift et al. 2007; Tooley and Kyde 2007). Reconstruction of the epidemic using molecular population genetic methods indicates historical human spread (Mascheretti et al. 2008) and highlights unintended linkages between the horticultural industry and potential impacts on forest ecosystems (Rizzo et al. 2005). Stringent efforts have been undertaken to halt the spread of the pathogen by the horticultural industry outside of California. These efforts were initiated after

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two large-scale nurseries in Southern California were implicated in having transported the pathogen via infected camellia plants to 176 locations in 21 states. Over a million infected plants were destroyed and annual inspections are now mandatory in western nurseries that ship interstate (McCreary 2007).

6.4.4 Phylogeography The literature on oak phylogeography is too extensive to do justice in the space provided here. For brevity, we focus on the intensively studied European white oaks, noting only several highlights, and refer the reader to reviews. We do not treat the growing phylogeography literature on oaks in North and Central America (e.g., Grivet et al. 2008, Craft and Ashley 2007, Gonzalez-Rodriguez et al. 2004a, b, Cavender-Bares et al. 2011), and Asia (e.g., Okaura et al. 2007, Shih et al. 2006, Kanno et al. 2004) nor on the European black oaks (e.g., Cosimo et al. 2009, de Heredia 2007, Lumaret et al. 2005). The chloroplast genome is maternally inherited in oaks (Dumolin et al. 1995), and chloroplast markers have been used effectively to resolve haplotypic variation across Europe for the major white oak species (e.g., DumolinLapegue et al. 1997). Much of this work has entailed polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using primers that span intergenic regions in the chloroplast (see Sect. 6.6.3.1). Petit et al. (2002a, b) reviewed the research by a consortium of 16 laboratories, which to date have detected 32 chloroplast DNA variants from 12,214 individual trees collected from 2,613 populations in 37 countries. The studies have revealed strong phylogeographic structure, compared with the weak structure detected in North American red oak (Magni et al. 2005; and see Sect. 6.2.3.2). White oak haplotypes found in northern Europe are also present in the south, though the reverse does not hold, suggesting that most mutations arose prior to the expansion out of southern refugia following the last glacial retreat. Even though regions can carry numerous haplotypes, it is common to encounter patches of several 100 kms dominated by a single haplotype, often cutting across white oak species boundaries. The authors suggest this is strong evidence for episodic, long-distance migrations out of the glacial refugia.

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This work has revealed some interesting natural history dynamics for the two most widespread and heavily studied of the European white oaks, Q. robur and Q. petraea. A variety of sources indicate that Q. robur has more pioneer tendencies including a greater capacity to disperse its seed (Petit et al. 2004). Thus, it is thought that Q. robur might have been among the first migrants out of the glacial refugia. The later successional Q. petraea would then have arrived and crossed with the established Q. robur populations, though there is an asymmetry in the mating success between the species. Q. petraea readily crosses to Q. robur, but the reverse does not hold. This has been found in controlled crosses (Steinhoff 1993; though see Steinhoff 1998) and in natural stands (Bacilieri et al. 1996b). Eventually the shadetolerant Q. petraea is able to replace the less tolerant Q. robur in mixed stands (Bacilieri et al. 1996b) – demographically and at the level of the genome. The overall dynamic has the effect of leaving the pioneer Q. robur chloroplast genome intact; however, the nuclear genes of Q. robur are gradually replaced or “swamped” by those of the late successional Q. petraea due to the asymmetric hybridization and introgression. This remarkable example lends some clarity as to why oak species can be problematic to demarcate.

6.5 Stocks and Lines Much of oak germplasm diversity remains in natural populations, and most managed lines are not far removed. There is some information available on how these natural lines perform through common garden, or provenance, studies. A limited amount of oak germplasm has been incorporated in breeding programs as improved lines. Many have been drawn directly from a wild tree that displayed superior form through transfer of acorns or cuttings (see Sect. 6.6.2). Some lineages have been subjected to rounds of selection, though typically not for many generations. A small and diverse set of lineages, drawing on all these sources, have been designated as cultivars. Other lines represent mutants that may be more of academic interest and include polyploids or single-gene mutants that affect the phenotype in some fashion that might illuminate a path for domestication.

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6.5.1 Provenance Studies A “provenance” is the location of a population and a “provenance study” is a common garden planting that brings together at one site a variety of germplasm from different parts of a species range. Provenance studies may prove critical in the coming years if it becomes necessary to assist oak species in their migration and adaptation to a changing climate (Ma´tya´s 1996). The US Department of Agriculture has detailed information on seed zones (or “plant hardiness zones”), which provide information on whether one could expect transplantation success (maps accessible online at http://www.usna.usda.gov/Hardzone/ushzmap.html). However, shifting climates will alter much of this hard-won information, and provenance studies will be needed to reestablish the information. Unfortunately in the USA, many hardwood provenance studies were abandoned during the transition to molecular-based breeding in the 1970s and 1980s (Woeste and McKenna 2004). Though some of the sites still exist, their lack of care has left many overgrown and of much less use for testing propositions as originally intended. Both the locations and the findings of provenance studies can be difficult to ascertain, since documents and reports often circulate through local institutions (state and local forestry, university departments) or at regional meetings (e.g., Weigel et al. 2005). Internet access is changing some of this, where for example one can find reviews of provenance work on the European white oaks Q. robur and Q. petraea (e.g., Kleinschmit and Kleinschmit 2000; see also Kleinschmit 1993) along with information on longterm oak plantings in Europe (e.g., Hubert 2005). Reports on several US oak provenance studies can now be accessed through the USDA-Forest Service website (http://www.treesearch.fs.fed.us/).

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breaks in continuity within breeding programs (Aldrich 2008). As a consequence, the cultivars cannot rival domesticates of corn or wheat. Nevertheless, there are numerous lineages that offer improved material that in some cases has been recognized among breeders for many years. McArdle and Santamour (1985a, b, 1987) summarized information on 119 valid names for cultivars of Q. robur (English oak), another 75 cultivars of white oaks (excluding Q. robur), and 59 cultivars outside of section Quercus (the non-white oaks). Included here is the “Gobbler”, a cultivar of Q. acutissima noted for its abundant acorn production and ability to satisfy wild turkeys. A more detailed and current repository of oak cultivar information is available online through the International Oak Society. Oak cultivars and groups are tracked, verified, and databased by the International Oak Society, and information can be accessed online through the Oak Name Database (Trehane 2007). Naming conventions follow the International Code of Nomenclature for Cultivated Plants (ICNCP). We retrieved information from the database on the top ten oak species based on their number of legitimate cultivars or Groups (Table 6.1). Nearly half (45.6%) of the world’s legitimate oak cultivars or groups are from either Q. robur or Q. petraea. This reflects the importance of these species in Europe and the longer time that Europeans have been breeding oaks. In North America, the white oak Q. alba is an important timber species and ranks well in number of cultivars, though the red oak (section Lobatae) cultivar pool is better developed there, at least among the top ten species. Two of the taxa in this abbreviated list are entirely of hybrid origin (Q.
hispanica and Q.
undulata). A total of 25 oak cultivars have been trademarked or have a restricted name, but 80% of these derive from species not in the top ten list, i.e., from oak taxa with fewer cultivars of any kind.

6.5.2 Cultivars

6.5.3 Polyploids and Other Mutants

Improved oak stock comes in many forms and with varied backgrounds, and includes such lines as “Argenteo-Marginata” or “Variegate English Oak.” Trees in general have a shallow domestication history because of their longevity and because of the frequent

Chromosomal mutations occur at a low rate in natural Quercus populations. Naujoks et al. (1995) found 1 in 400 Q. robur trees exhibited anomalous isozyme banding, altered leaf morphology, large stomata, and a triploid chromosome count. The authors took

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Table 6.1 Cultivar pools of the top 10 oak species ranked by number of legitimate cultivars or groups recognized by the International Oak Society Section/Species Common name Region Total Legit Hybrids Tm Cerris Q. cerris Turkey Oak E, As 37 20 0 0 Q.
hispanicaa Lucombe Oak 34 17 34 0 Lobatae Q. palustris Pin Oak NA 28 18 0 1 Q. rubra Northern Red Oak NA 31 19 0 0 Q. velutina Black Oak NA 11 9 1 1 Quercus Q. alba White Oak NA 20 14 0 1 Q. ilex Holly or Holm Oak E, Af 23 16 0 0 Q. petraea Sessile of Durmast Oak E 64 46 0 0 Q. robur Pedunculate or English Oak E, As 269 161 0 3 Q.
undulatab Wavyleaf Oak 18 11 18 0 Lesser taxa Miscellaneous species 232 123 72 20 Total 767 454 124 25 Information source: Trehane (2007) Oak name database, International Oak Society. Accessed Jan 2009 (http://www.oaknames.org/) Region, native distribution: As Asia, Af Africa, E Europe, NA North America Total: Total number of cultivars and groups listed, legitimate and illegitimate Legit: Legitimate cultivar or group name, published in literature and conforming to International Code of Nomenclature for Cultivated Plants (ICNCP) rules Hybrids, cultivars, or groups of hybrid origin noted within this taxon Tm, trademarked or restricted name a Q. cerris
Q. suber b Q. gambelii
several other species

cuttings and rooted the specimen. Zoldos et al. (1998) found two populations of Q. petraea containing several individuals with extra chromosomes. Dzialuk et al. (2007) also found ploidy variation in Q. robur and Q. petraea. To our knowledge these chromosomal variants have not been integrated into production lines. Higher chromosomal mutation rates also have been detected in “natural” Q. robur oak populations contaminated with radiation from the Chernobyl accident (Kalaev and Butorina 2006). Synthetic oak polyploids can be generated through different treatments. Bueno et al. (1997) used stress signaling, or starvation followed by heat shock, to alter the gametophytic trajectory in anther cultures, inducing doubled haploids. Alternatively, antimitotic agents such as colchicine, oryzalin, and amiprophos-methyl (APM) can be used to disrupt meiosis in anther cultures. Pintos et al. (2007) compared these chemical treatments in Q. suber (cork oak) and found superior yield with oryzalin. Sometimes ploidy instability is an unwanted by-product of the somatic embryogenesis process, such as an 8% rate of tetraploidy detected

in Q. robur clones that were cultured over 7 years (Endemann et al. 2001). Other studies of oak cultures show no evidence of ploidy instability (e.g., Q. suber, Loureiro et al. 2005). Then there are a limited number of additional described mutants in Quercus. Some have been systematically studied such as the ML mutant, a chlorophyll-deficient line of Q. petraea that exhibits enhanced resistance to powdery mildew, Erysiphe cichoracearum (Repka 2002). This mutant accumulates reactive oxygen species, enhancing the defense response.

6.6 Crop Improvement Quercus domestication is still in its infancy though great progress has been made in the past 10–15 years. Here we consider some impediments to oak domestication brought by the natural history of the organism and by some human practices. We then

6 Quercus

cover different breeding approaches and the progress to date in oak domestication. In particular, we consider traditional, molecular, and biotechnology-based breeding such as in vitro culturing. Because of considerable progress in the area of oak molecular breeding, we treat this subject in greater detail (see Sect. 6.6.3). Included here are resources for Quercus in molecular genetic markers, crosses and pollinations, and linkage and QTL mapping.

6.6.1 Impediments to Improvement Aldrich (2008) considered the domestication history of forest trees, and most of the generalizations made therein hold for the genus Quercus. Despite a long relationship between humans and oak trees (Ciesla 2002; Logan 2005), several factors have contributed to the slow progress of changing the oak phenotype, most notably generation time and breeding directly from natural populations. Oaks have a long generation time, some living to be hundreds of years (see Burns and Honkala 1990), which can work against the domestication process. Delayed onset of reproduction can require that a breeder wait 10–15 years before considering an artificial cross using an elite tree. Hand pollinations in Quercus are difficult, good seed crops can arise sporadically separated by several years of poor yield, and the acorns of the red oaks typically require 2 years to reach maturity. Moreover, it can take decades before traits of interest become evident, such as wood quality and growth form. All this contributes to slow progress as well as discontinuities in breeding programs. Most “domesticated” oak lineages are not many generations removed from wild populations (see Sect. 6.6.2). This has the benefit of repeatedly tapping natural reserves for genetic variation that should promote gains in breeding, except there have been serious problems in continuity of breeding efforts in oak programs and other hardwoods (Woeste and McKenna 2004). The proverbial wheel has been re-invented several times as breeding efforts and programs have been abandoned for various reasons, such as in the USA during the 1970s and 1980s when attention shifted away from traditional breeding and toward biotechnology.

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Perhaps the most pernicious aspect of human interactions with wild populations of trees has been that inadvertent selection pressures likely have worked against the improvement of timber traits over the millennia. Selection and harvest of the biggest and best trees for their wood removes these individuals from the gene pool. This practice of “high-grading” has the opposite effect one would like on the domestication process, though the magnitude of impact cannot be determined in most cases due to poor records. But with the possible exception of the long-standing European work with Q. robur and Q. petraea, it is safe to say that we have only recently begun to leave the hunter-gatherer stage of our relationship with the oaks.

6.6.2 Traditional Breeding Traditional silviculture forms the foundation of oak breeding programs. A valuable resource in this area is the The Ecology and Silviculture of Oaks (Johnson et al. 2002). There are other web-based resources on oak silviculture in the USA, such as through the USDA-Forest Service website (http://www.fs.fed.us/ publications/) and through state and local forestry extension services (e.g., http://web.extension.uiuc.edu/ forestry/publications/index.html). At Purdue University, the Hardwood Tree Improvement and Regeneration Center (HTIRC, Michler et al. 2005) is a collaborative enterprise involving federal, state, and private organizations involved in combining traditional breeding methods with biotechnology in the advancement of hardwood resources, including oaks (http:// www.agriculture.purdue.edu/fnr/HTIRC/index.htm). Traditional breeding methods borrow heavily from the classical and quantitative genetic approaches used in crop and livestock breeding. The common garden experiment, or provenance trial, still figures prominently as it serves to evaluate pre-existing adaptations in the gene pool (see Sect. 6.5.1). Phenotypic selection can occur any time from the point of selecting germplasm from a superior tree in the wild through the point of production and deployment of elite lines in plantations. Controlled crosses are possible with the oaks, though sometimes openpollinated canopies are utilized. In fact, much of the traditional oak breeding done today utilizes only slightly improved germplasm. Jacobs and Davis

108 Table 6.2 Narrow sense heritabilities for traits in Quercus Trait(s) h2 Height 0.64 (0.48–0.80) Diameter 0.62 (0.55–0.70) Height 0.60 (0.50–0.70) Vessel area 0.60 Sapwood rings 0.57 Bud burst 0.33 (0.15–0.51) Diameter 0.28 (0.09–0.46) Growth 0.14 (0.04–0.23) Mean 0.47

P.R. Aldrich and J. Cavender-Bares

Species Q. robur Q. pagoda Q. pagoda Q. petraea/Q. robur Q. petraea/Q. robur Q. robur Q. robur Q. robur

References Bogdan et al. (2004) Adams et al. (2007) Adams et al. (2007) Kanowski et al. (1991) Savill et al. (1993) Scotti-Saintagne et al. (2004a) Bogdan et al. (2004) Scotti-Saintagne et al. (2004a)

h 2, midpoint heritabilities (value or range reported)

(2005) reviewed hardwood tree nurseries in the eastern United States and found that only 6.8% of hardwood seedlings derived from improved stock, compared to 36% for conifers. Moreover, it is often the case that seed source is unknown since many nurseries accept anonymous collections and pool these before planting. These are areas worth improving if oak breeding is to keep a reasonable pace. Studies to date indicate that natural oak populations maintain sufficient additive genetic variation to serve as a source for breeding programs. Average heritabilities for several oak traits that we review here are around h2 ¼ 0.47 (Table 6.2). Adams et al. (2007) used 37 half-sib families representing eight provenance from the eastern United States to estimate heritabilities of h2 ¼ 0.55–0.70 for diameter and height in Q. pagodae (cherrybark oak). Heritabilities were high for height (h2 ¼ 0.62–0.78) and diameter (h2 ¼ 0.28–0.65) in Q. robur from an open-pollinated progeny test of 21 Slavonian plus trees (Bogdan et al. 2004). Kanowski et al. (1991) reported a high heritability (h2 ¼ 0.60) for crosssectional area of earlywood vessels in Q. robur and Q. petraea, a trait that may influence the development of fissures in wood. Savill et al. (1993) reported a high heritability (h2 ¼ 0.57) for the number of sapwood rings in Q. robur and Q. petraea. The genetic gain from selecting for volume in a Q. serrata seed orchard was estimated at 4.0–9.1% for 50% selection intensity (Kang et al. 2007). Mosedale et al. (1996) examined wood qualities in Q. petraea and Q. robur and found that heartwood ellagitannin content and wood density were both under strong genetic control though this was not the case for wood color (see Sect. 6.7.5.1).

6.6.3 Marker-Assisted Breeding The application of molecular markers is an active area in oak breeding as this method can be used to hasten the process of selection during a life cycle that is long and protracted. There are a variety of molecular marker types and a variety of applications that are relevant to breeding. Some markers are used to appraise variation in natural stands, whereas others to resolve the structure of variation within the individual genome. Chief among the latter applications are the molecular maps, and the attachment of phenotypic effects onto such a map in the form of QTL. All can be used as a handle to track the establishment of desirable traits in a lineage. Useful information on molecular marker systems and their applications in the management of plant genetic resources, including forest trees, is maintained through Wageningen UR (http://www.cgn.wur.nl/UK/CGN+Plant+Genetic +Resources/Research/Molecular+markers).

6.6.3.1 Molecular Genetic Markers A variety of molecular markers are available for Quercus. Commonly used phylogenetic sequences have been studied such as the ITS of the ribosomal DNA (rDNA; Muir et al. 2001; Bellarosa et al. 2005). An extensive program of research on oak phylogeography has been developed for the European white oaks Q. robur and Q. petraea using chloroplast and mitochondrial markers (e.g., Petit et al. 1997; Taberlet et al. 1998; Cottrell et al. 2002; see also Sect. 6.4.4). These primer sequences for the organellar genomes

6 Quercus Table 6.3 SNP and SSR markers developed for Quercus Marker type Genome N SNP/SSR mtDNA 4 SNP/SSR cpDNA 3 SNP/SSR cpDNA 9 SNP/SSR cpDNA 17 SNP/SSR cpDNA 14 SSR Nuclear 3 SSR Nuclear 17 SSR Nuclear 9 SSR Nuclear 32 SSR Nuclear 14 SSR Nuclear 16 SSR Nuclear 10 SSR Nuclear 11 EST-SSR Nuclear 20 EST-SSR Nuclear 1,328a EST-SSR Nuclear 931a

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Species Q. robur Q. robur Q. robur Q. petraea/robur Q. petraea Q. macrocarpa Q. petraea/robur Q. myrsinifolia Q. robur Q. rubra Q. rubra Q. mongolica Q. mongolica Q. mongolica Q. rubra Q. alba

References Demesure et al. (1995) Taberlet et al. (1991) Demesure et al. (1995) Deguilloux et al. (2003a) Sebastiani et al. (2004) Dow et al. (1995) Steinkellner et al. (1997) Isagi and Suhandono (1997) Kampfer et al. (1998) Aldrich et al. (2002) Aldrich et al. (2003a) Ueno and Tsumura (2008) Mishima et al. (2006) Ueno et al. (2008) Fagaceae Project Fagaceae Project

Many of the other sequences available at Molecular Ecology Resources database (http://tomato.bio.trinity.edu/) Untested sequences available at Fagaceae Genomics Web (http://www.fagaceae.org/web/db/index)

a

work in most oak species and are reported in Dumolin et al. (1995), Deguilloux et al. (2003a), and Sebastiani et al. (2004) (Table 6.3), and many are available online through the Molecular Ecology Resources database (http://tomato.bio.trinity.edu/). At the population level, single locus codominant markers are available for Quercus in the form of allozymes (e.g., Redkina et al. 2008), single-strand conformation polymorphisms (SSCPs) (e.g., Bodenes et al. 1996), though mainly as microsatellites (SSRs or simple sequence repeats). Such markers have been used in a variety of studies regarding the oak gene pool, characterizing diversity (Aldrich et al. 2005a), clonal structure (Ainsworth et al. 2003), mating system (Bacilieri et al. 1996a, b), paternity analysis, and pollen dispersal (Nakanishi et al. 2004), inferring the number of pollen donors in a canopy (Lexer et al. 2000), hybridization (Nason et al. 1992), detection of seed contamination (Lexer et al. 1999), seed dispersal (Dow and Ashley 1996), origin of trees in a stand (Lefort et al. 1998), origin of wood samples (Deguilloux et al. 2004), and criminal forensics (Craft et al. 2007). Primer sequences for microsatellite loci are reported for the white oaks in Dow et al. (1995), Kampfer et al. (1998), Steinkellner et al. (1997), Mishima et al. (2006), and Ueno and Tsumura (2008), for the red oaks in Aldrich et al. (2002, 2003a), and section Cyclobalanopsis in Isagi and Suhandono (1997) (see also Table 6.3 and the Molecular Ecology

Resources database as noted above). Note also that Lepais et al. (2006) provide a high-throughput protocol for multiplex amplification of a panel of 10 microsatellites in the white oaks (section Quercus). In addition to the random nuclear SSR markers, there are increasing resources for microsatellites associated with transcribed regions of the genome. Ueno et al. (2008) report a set of 20 primer pairs for SSR loci derived from inner bark ESTs in Q.mongolica var. crispula (Table 6.3). In addition, the Fagaceae Project has posted to its website (http://www.fagaceae.org/ web/db/index) sequence contigs from 454-based EST sequencing in oaks (see Sect. 6.7). Available there are 6,187 SSR locus-containing sequences from Q. rubra (section Lobatae, Northern Red Oak), 1,358 of which have suggested primer sequences. For Q. alba (section Quercus, White Oak), there are 4,350 SSR loci of which 931 have suggested primer sequences. Many of these loci display in silico evidence of polymorphism. Although codominant markers such as SSRs have desirable properties for many kinds of study, medium to high density genome screens often utilize dominant markers such as random amplified polymorphic DNAs (RAPDs) and various fingerprinting methods like amplified fragment length polymorphisms (AFLPs). These are readily applied to non-model organisms since they do not pre-suppose knowledge of a specific DNA sequence in the focal species. Several recent

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studies have applied RAPD markers to oaks (e.g., Song et al. 2002; Yakovlev and Kleinschmidt 2002; Schiller et al. 2006; Gonzalez-Rodriguez et al. 2005). Some of the larger RAPD studies utilize a fairly high marker density, such as Lee et al. (2007) who applied 144 primers to several white oak species, and Barreneche et al. (1998) who constructed a linkage map for Q. robur based largely on 271 RAPD markers. The latter study also applied a minisatellite marker as did Fladung and Ziegenhagen (1998) and Kumar and Rogstad (1998) in their studies of oaks. More recent fingerprinting work often involves intersimple sequence repeats (ISSRs) and AFLPs. Lopez-Aljorna et al. (2007) used both SSR and ISSR markers to fingerprint elite Q. suber trees, and there are now numerous studies of oaks using AFLPs (Q. robur and Q. petraea: Bakker et al. 2001; Coart et al. 2002; Mariette et al. 2002; Q. crispula and Q. dentata: Ishida and Kimura 2003; Q. ellipsoidalis: Hipp and Weber 2008). Note that Cervera et al. (2000) provide an AFLP protocol optimized for several tree species including oak. Pearse and Hipp (2009) used AFLPs to generate a reliable phylogeny resolved at the species level.

6.6.3.2 Crosses and Pollination Quercus is predominantly wind-pollinated, with various factors influencing pollen production including genetic, atmospheric, and biotic agents (RodriguezRajo et al. 2005). Schueler et al. (2005) describe considerable variability in pollen viability and sensitivity to sunlight. These factors can in turn affect acorn production (e.g., Cecich and Sullivan 1999). Acorns mature during the first year in all North American white oaks (section Quercus) but require 2 years in the North American red oaks (section Lobatae). Oaks are monecious but are predominantly outcrossers, seemingly through some combination of protandry (e.g., Q. alba, Burns and Honkala 1990) and/or self-incompatibility. Q. ilex is considered highly selfincompatible and Yacine and Bouras (1997) described slower pollen tube growth and reduced seed set in selfed flowers, and elevated rates of ovule abortion in mixed pollinations. Nevertheless, low levels of selffertilization have been reported (s < 5%), and biparental inbreeding or mating between relatives can be common (Schwarzmann and Gerhold 1991; Sork et al.

P.R. Aldrich and J. Cavender-Bares

2002a, b; Fernandez and Sork 2005; Ferna´ndezManjarre´s et al. 2006; Pakkad et al. 2008). Controlled crosses are described between Q. robur and Q. petraea (Aas 1991; Steinhoff 1998). Steinhoff (1998) reported highest success rates for outcrossing to other individuals of the same species (Q. robur, 11.3%; Q. petraea, 5.8%), lesser rates for interspecific hybrids (Q. robur
Q. petraea, 5.3%; Q. petraea
Q. robur, 0.8 %), and lowest for selfing (Q.robur, 1.3%; Q. petraea, 0%). In controlled crosses, introgression is feasible as Olrik and Kjaer (2007) report that a Q. petraea
Q. robur hybrid was able to backcross to both parent species, with only a slight bias toward the Q. robur genome. This stands in contrast to an earlier study (Steinhoff 1993) and to studies of natural populations that have suggested there exists a barrier to nuclear gene exchange in the Q. robur-to-Q. petraea direction (Bacilieri et al. 1996b; see Sect. 6.4.4). Advanced generation crosses are atypical in the oaks, mainly due to the generation time problem. To our knowledge there are no publicly available F2 or backcross lines. F1 crosses are feasible and reported, and in some instances publicly available (see Sect. 6.5.2 and below). Several mapping populations in Q. robur and Q. petraea are soon available from the EVOLTREE program. This entails the following single pedigrees: Q. robur (375 genotypes), Q. petraea (127 genotypes), and Q. robur
Q. petraea (151 genotypes). Populations for association mapping are also going public and will include: one Q. robur population (296 genotypes), three Q. petraea populations (1,251 genotypes), and one Q. robur
Q. petraea population (296 genotypes).

6.6.3.3 Linkage Mapping Barreneche et al. (1998) produced the first oak linkage map (in Q. robur), which has been used in a variety of structural and comparative genomic applications (see next section, also Sects. 6.7.3 and 6.7.4). The map includes 307 total markers of the following types and abundances: RAPDs (n ¼ 271), microsatellites (n ¼ 18), sequence characterized amplified regions (SCARs; n ¼ 10), isozymes (n ¼ 6), minisatellite (n ¼ 1), and 5S rDNA (n ¼ 1). This meiotic linkage map was constructed from a controlled cross between two French trees yielding a two-generation full-sib

6 Quercus

pedigree (94 progeny). The mapping design was based on the two-way pseudo-testcross (Grattapaglia and Sederoff 1994), yielding a male and female map that was then merged using intercross markers. Both the male and female maps resolved 12 linkage groups (the haploid number in oak), and only slightly different map lengths (maternal, 893.2 cm; paternal, 921.7 cm). Total genome coverage was 85–90%. The map can be viewed at NCBI using Mapviewer (http://www.ncbi.nlm.nih.gov/mapview). Since then other oak linkage maps have been produced through extension of the original French mapping population (e.g., Saintagne et al. 2004), and other maps have been generated based on distinct crosses. Porth et al. (2005b) describe linkage mapping of osmotic stress induced genes in two maps, the intraspecific Q. robur cross (3P*A4) and an interspecific cross of Q. robur
Q. petraea (11P*QS29). Gailing (2008) report a QTL leaf morphology study based on a linkage map constructed from another intraspecific Q. robur cross, though here between geographically distant parents, one from Germany and the other Croatia. Most recently, a linkage map has been produced for the red oaks through work initiated through the Hardwood Tree Improvement and Regeneration Center (HTIRC) at Purdue University (unpublished data). Other mapping of the Quercus genome has involved the cytogenetic localization of specific genes or gene families, comparative mapping of loci across species, and QTL mapping of phenotypic traits (see next section). As an example of cytogenetic mapping, Chokchaichamnankit et al. (2008) used fluorescence in situ hybridization (FISH) to map ribo-

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somal genes onto the karyotypes of 15 species from the Fagaceae, including several oaks. For a recent review of genes mapped to Quercus and Castanea (chestnut), see Kremer et al. (2007) where they describe genes for bud burst, hypoxia, osmotic stress, and developmental stage-dependent expression. A number of comparative studies of the oak genome have used the markers of Barreneche et al. (1998) map to query the genomes of other species (see also Sects. 6.6.3.4 and 6.7.4). For example, Saintagne et al. (2004) expanded the size of the original mapping population from 94 full-sibs to 278 in their study of leaf QTLs distinguishing Q. robur and Q. petraea (see Sect. 6.2.3.1). As for non-QTL comparative studies, Barreneche et al. (2004) used microsatellite markers to explore synteny between Q. robur and Castanea sativa (European chestnut). They were able to anchor 19 markers into two previously constructed linkage maps for Quercus and Castanea, and after some local sequencing resolved seven linkage groups and another two regions based each on a single matched marker. The Barreneche et al. (1998) map for Q. robur can be compared to other species using the CMap Comparative Map Viewer hosted by the Fagaceae Project website (http://www.fagaceae.org/cgi-bin/ cmap/). Additional linkage mapping information and resources can be found in the following. Aldrich (2008) reviewed approaches and progress in the mapping of forest tree species, Kremer et al. (2007) reviewed both genetic mapping and comparative genetic mapping in the Fagaceae, and Plomion et al. (2007) reported on the availability of mapping resources for oak through the EVOLTREE program,

Table 6.4 Quantitative trait locus (QTL) findings in Quercus robur Trait(s) QTLsa Bud burst 32.0 (32) Stomatal density and growth 18.0 (18) Water use efficiency 10.0 (10) Rooting ability 10.0 (10) Leaf morphology 10.0 (10) Leaf morphology 7.5 (6–9) Waterlogging tolerance 5.0 (5) Height growth 3.0 (2–4) Mean 11.9 a

%b 7.0 (3–11) 9.8 (3.6–15.9) 20.0 (20) 9.1 (4.4–13.8) 6.6 (3.6–9.6) – (–) 9.0 (>9%) 11.5 (4–19) 10.4

Midpoint number of significant QTLs detected (value or range reported) Midpoint percentage of the variance explained by the QTLs (value or range reported)

b

References Scotti-Saintagne et al. (2004a) Gailing et al. (2008) Brendel et al. (2008) Scotti-Saintagne et al. (2005) Gailing (2008) Saintagne et al. (2004) Parelle et al. (2007a, b) Scotti-Saintagne et al. (2004a)

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including sets of SSR and SNP markers. And as noted earlier, the Fagaceae Project has made available numerous EST sequences containing SSR loci, these from both Q. rubra and Q. alba.

6.6.3.4 Quantitative Trait Loci (QTL) Mapping There are now several QTL studies of oak involving Q. robur as at least one parent in the cross. Table 6.4 shows summary information for these. For a detailed consideration of leaf QTLs distinguishing Q. robur and Q. petraea, see Sect. 6.2.3.1. For a recent review of QTL detection in the Fagaceae, see Kremer et al. (2007). Comparisons across QTL studies can be interesting but must be made with the caveat that different traits are at times composites of several lesser traits, though these concerns are certainly diminished when the traits and protocols are identical (e.g., Saintagne et al. 2004 and Gailing 2008). Still, Gailing (2008) showed the importance of genetic background and environment in their influence on outcomes even when other aspects remained the same. Though the conditions of the studies reviewed here varied, the average number of QTLs that were identified was around 11.9 per trait, with an average of 10.6% of the phenotypic variation explained. This average number of QTLs is close to the total number of linkage groups in Quercus (n ¼ 12), though there is the tendency for the QTLs to cluster together such that any one trait is not necessarily effected by variation at all the chromosomes. The estimate also falls within the range noted by Orr (2001) who summarized work in a variety of species and noted that most QTLs fell between 1 and 20 loci.

6.6.4 Biotechnology Breeding Tree breeding through biotechnology includes in vitro methods of propagation that should hasten the rate of domestication and permit mass production of elite lines. Genetic transformations hopefully will contribute through enhancements in wood quality and growth rates along with improved tolerance to drought, pests, and pathogens. These areas are aptly reviewed in a variety of contexts including the biotechnology of

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hardwoods (Merkle and Nairn 2005; Pijut et al. 2007), wood biotechnology (Boerjan 2005), biotechnology and plantation forests (Fenning and Gershenzon 2002), and methods for accelerated tree improvement (Nehra et al. 2005). Wilhelm (2000) reviewed oak somatic embryogenesis, and we offer a brief update here. Unfortunately, Quercus is not as readily cultured and transformed as other tree species like poplar, but progress has been made. Most of the oak work has been published on section Quercus, particularly the European white oaks Q. robur, Q. petraea, and Q. suber. Reports of successful methods also exist for section Cerris (Q. accutissima, Q. cerris, Q. serrata) and section Lobatae (Q. rubra). Several in vitro reports exist for Q. robur (Juncker and Favre 1994; Sanchez et al. 1996; Chalupa 2000; Vidal et al. 2003; Toribio et al. 2004; Valladares et al. 2006) and Q. petraea (San-Jose et al. 1990; Cvikrova et al. 2003). Corredoira et al. (2006) provide a detailed morphohistological study of the development of somatic embryos of leaf cultures from a 100-year-old Q. robur tree. However, there have been several reports of chromosomal instability in Q. robur during somatic embryogenesis (Endemann et al. 2001; Wilhelm et al. 2005). Transformation systems are described for Q. suber as well (Romano et al. 1992; Hernandez et al. 2003; Sanchez et al. 2005; Alvarez et al. 2004; Alvarez and Ordas 2007). Somatic embryogenesis studies on this species include cyclin gene expression associated with adventitious rooting (Neves et al. 2006), effects of abscisic acid (ABA) and indole-3-acetic acid (IAA) on embryo maturation (Garcia-Martin et al. 2005), and small heat shock proteins (Puigderrajols et al. 2002). Reports on chromosomal stability suggest that Q. suber is stable in its ploidy during embryogenesis (Bueno et al. 2003; Loureiro et al. 2005; Lopes et al. 2006). In vitro work has been done with a few other oaks outside section Quercus. Transformations described for section Cerris include Q. cerris (Tsvetkov and Hausman 2005), Q. acutissima (Kim et al. 1997), and Q. serrata (Sasamoto and Hosoi 1992). In section Lobatae, Vengadesan and Pijut (2009a, b) review the work already done on Q. rubra (Northern Red Oak) and report successful methods for in vitro propagation by somatic embryogenesis and plant regeneration for this species.

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6.7 Genomics Resources Quercus entered the genomics age with the publication of the Barreneche et al. (1998) linkage map. Since then there have been gains in the area of oak structural genomics (described earlier), including the aforementioned publications on QTL mapping of oak traits. But much of the emphasis in oak genomics has been in the area of functional genomics. Though research in this area still lags far behind that on other model organisms such as Arabidopsis and Populus, considerable progress is anticipated through the development of major new oak genomics initiatives that we describe below before reviewing progress to date.

6.7.1 Genome Structure Several studies have described oak karyotypes and overall genome structure and content. We have already treated single-copy regions of the genome in several sections. Here we consider the basic karyotype and genome size, along with some surveys of repetitive DNAs.

6.7.1.1 Karyotype The chromosomal count of Quercus is 2n ¼ 24 (D’Emerico et al. 1995, 2000). This cytogenetic work has concentrated on the European members of section Quercus (white oaks; Q. dalechampii, Q. petraea, Q. pubescens, and Q. robur) and section Cerris (Q. cerris, Q. coccifera, Q. trojana, Q. suber), along with a few other species. Reports from other geographic regions concur with the same count (e.g., North Korea, Q. acutissima, Baranec and Murin 2003). By comparison, Populus has 2n ¼ 38 and Arabidopsis 2n ¼ 10. Cytomorphological work, cytogenetic banding methods, in situ hybridization, and other physical mapping techniques indicate a strongly conserved genome organization within Quercus (Zoldos et al. 1999, 2001; D’Emerico et al. 2000). This holds true for comparisons between Europe and North America, and across ecophysiological classes, namely evergreen or deciduous. Nevertheless, slight karyotypic variability has been detected as differences in intrachromoso-

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mal and interchromosomal asymmetry across species, noted by D’Emerico et al. (1995, 2000). For further discussion of ploidy variants in Quercus, see Sect. 6.5.3.

6.7.1.2 Genome Size and Composition Oaks have a moderate-sized genome, though large in comparison to current model genomes. Estimates of genome size in Quercus range from 1.84 to 2.00 pg/2C (Favre and Brown 1996; Zoldos et al. 1998), almost twice the size of the Populus genome (1.2 pg/2C, Bradshaw and Stettler 1993) and nearly five times the size of the Arabidopsis genome (0.41 pg/2C, Bennett and Leitch 2005). The oak estimates largely derive from the European members of sections Quercus and Cerris. The same studies reported GC content ranging from 39.9% to 42.1%. Repetitive DNA often comprises a large fraction of eukaryotic genomes, and repeats seem well represented in Quercus as well. As for tandem repeats, Zoldos et al. (1999) found 18S–5.8S–26S rRNA genes at one major and one minor locus, and the 5S rDNA at a separate single locus. This was true of all the oak species they examined, though rRNA gene copy number at each of the loci varied across species (1,300–4,000) according to a dot-blot survey. As for dispersed repeats, Zoldos et al. (2001) used representational difference analysis to subtract the Q. suber genome from the Q. robur genome, yielding a library of 400 DNA sequences representing genome differences. They examined in detail seven of these sequences and found that each had a high similarity to known retrotransposons. Hybridizations indicated that these repeats were present as 100–700 copies in the Q. robur genome.

6.7.2 Oak Genomics Initiatives Technology is advancing at such a pace that the distinction between model and non-model organism is beginning to erode. Moreover, oak is recognized as a keystone species in Northern Hemisphere ecosystems, and so is already a model organism of an ecological variety. These factors contribute to the rise of oak genomics initiatives on both sides of the Atlantic.

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6.7.2.1 European The Europeans continue to lead in the area of oak genetics, genomics, and domestication. Their focus has been on the European white oaks, mainly Q. robur, Q. petraea, and to a lesser extent Q. suber and Q. ilex. Much of the progress already achieved and described in the following sections derives from work out of INRA, the French National Institute for Agricultural Research (http://www.inra.fr/) led by or associated with Antoine Kremer’s team. He continues a leadership role in the new EVOLTREE network (Plomion et al. 2007, see also http://www.evoltree.eu/), a European Union-funded federation of research units with the goal to assess the impact of climate change on forests, using oak as a model organism. The genomics arm of the program anticipates the following research on oak: large-scale studies of adaptive genetic variation, large collections of ESTs, SSRs, and SNPs, mapping and QTL projects, BAC libraries, microarrays, and genome sequencing including full sequences for the mitochondrial and chloroplast genomes.

6.7.2.2 North American Three US-based agencies provide notable direct contributions to the oak genomics effort. Emphases here include centralized bioinformatics resources, EST libraries, applications of biotechnology to tree improvement, and integration of genomics resources within the Fagaceae with an ancillary goal for the reintroduction of Castanea (chestnut) back into the wild. The Fagaceae Project is a multi-institutional collaboration including NC State, Penn State, and Clemson Universities, the American Chestnut Foundation, the US Forest Service, and others. They are producing considerable genomics work on Castanea (chestnut) but also Fagus (beech) and Quercus (oak). They maintain the Fagaceae Genomics Web (http://www.fagaceae.org/web/db/index), which is a portal to a variety of genome-related resources for the family, particularly EST libraries. These resources are currently available to the public and we describe some of them in the following sections. The Hardwood Tree Improvement and Regeneration Center (HTIRC, http://www.agriculture.purdue. edu/fnr/HTIRC/index.htm) is another collaborative

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enterprise that includes academia (Purdue University), governmental agencies (US Forest Service and Indiana State Forestry), private foundations (American Chestnut Foundation, Walnut Council), various members of the wood products industry, and others. They are active in the development and transfer of technologies associated with tree improvement through traditional and biotechnology means for the major hardwoods of the Central Hardwoods Region of the USA, principally Juglans (both walnut and butternut), Prunus (cherry), and Quercus (oak, especially Q. rubra, Northern Red Oak). Thirdly, the Dendrome Project (http://www.fagaceae.org/web/db/index) provides a central portal for access to several databases on tree genomics and genetics, although their bias is for conifers.

6.7.3 Structural Genomics The primary structural genomic resources for oaks are the linkage maps constructed in the white oaks Q. robur and Q. petraea (Barreneche et al. 1998; Porth et al. 2005b; Gailing 2008) and the red oak Q. rubra (HTIRC, unpublished data). We describe these maps in Sect. 6.6.3.3. The white oak map can be viewed using Mapviewer at NCBI (http://www.ncbi. nlm.nih.gov/mapview) or CMap at the Fagaceae Project website (http://www.fagaceae.org/cgi-bin/cmap/). Others have probed the oak genome for content with respect to specific genes or gene families. For example, Porth et al. (2005b) located osmotic stress genes in Q. robur and in a Q. robur
Q. petraea cross. They began with 25 cDNAs derived from osmotically stressed Q. petraea callus tissue (Porth et al. 2005a) and, using SNPs, were able to position 13 of 14 genes on at least one of the two maps. Zoldos et al. (2001) used representational difference analysis to subtract the genome of Q. suber from that of Q. robur. The library of 400 clones representing differences contained a number of sequences identified as retrotransposons. They tested seven of these clones against hybridization in other oak species and found positive hybridization in Q. petraea but not in Q. cerris, C. coccifera, Q. ilex, or Q. palustris, though three clones did hybridize to Q. virginiana which is the species most closely related to Q. robur–Q. petraea.

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This pattern suggests that the retrotransposons arose in the common ancestor of this group. Comparative mapping has been done between Quercus and Castanea (chestnut) (Barreneche et al. 2004) using microsatellite markers. They tested for cross-species amplification of SSR markers and found 47% of Quercus markers would transfer as would 63% from Castanea. From this set of anchor markers, they were able to integrate 19 into linkage maps that had already been produced for the two species. Subsequent sequencing verified the homology of the markers. Casasoli et al. (2006) extended this comparative work, combining the SSR markers with sequence tagged site (STS) markers produced from oak EST information. This yielded a comparative map based on 55 orthologous markers. They compared QTL positions for three adaptively important traits, finding a shared position of control of bud burst but not for height or carbon isotope discrimination. Outside of linkage maps and related technologies, there are limited resources for Quercus in the area of structural genomics at this point. Its genome size (1.84–2.00 pg/2C, Favre and Brown 1996; Zoldos et al. 1998) is roughly twice that of Populus (1.2 pg/2C, Bradshaw and Stettler 1993). Given that the full sequence of popular is now available (Tuskan et al. 2006), there are useful opportunities for comparative genomic research on the tree habit and other topics that might improve the domestication of oaks. Much work is underway on restoration of the chestnut, also a member of the Fagaceae, including two BAC libraries that have been constructed through the Fagaceae Project for Castanea mollissima (Chinese chestnut). The EVOLTREE program plans to generate several key structural genomics resources for oak including a 10
BAC library and large-scale sequencing that includes the full oak chloroplast and mitochondrial genomes (Plomion et al. 2007).

6.7.4 Functional Genomics Much interesting work has been done on the functional genomics of trees. Large-scale EST studies of trees are commonplace now, and public resources are increasingly available. The public availability of the poplar chip has also promoted research. Tang et al. (2003)

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provide a general review of progress in the functional genomics of wood quality and properties. Most of the genomics research to date in Quercus entails studies of gene expression, mainly at the RNA level though more recently at the protein level as well. Most of this work has been done on the European white oaks, notably Q. robur, Q. petaea, Q. suber, and Q. ilex. Many studies have focused on expression of a single gene or gene family, using hybridizations, real-time polymerase chain reaction (RT-PCR), and other methods. Broader surveys query the full transcriptome using hybridizations, subtractive hybridizations, and macroarrays, and several EST libraries have been constructed for oaks, even a red oak (Q. rubra). As for protein expression, a proteome project is underway for the leaf of the Holm oak, Q. ilex.

6.7.4.1 Transcriptome Several oak studies have targeted the expression of a single gene or gene family. The stress response is a frequent topic, with recent work on small heat shock proteins in cork tissue and apical meristems (Pla et al. 1998) and in somatic embryos (Puigderrajols et al. 2002). Oxidative stress studies exist as well, including work on calmodulin genes in flooded seedlings (Folzer et al. 2006), type 2 metallothionein in cork tissue (Mir et al. 2004), and non-symbiotic hemoglobin in seedlings (Parent et al. 2008). Genes associated with fungal interactions are of interest, from the positive mycorrhizal association to the negative pathogenic interaction. One study targeted class III chitinases in root tissue during pre-mycorrhizal interactions (Frettinger et al. 2006) and another cinnamyl alcohol dehydrogenase defense in response to infection by Phytophthora cinnamomi (Coelho et al. 2006). Other work has been developmental stage specific, as in a differentially expressed gene in juvenile-like and mature shoots (Gil et al. 2003), or in micropropagated tissue during adventitious rooting wherein expression of a B-type cyclin gene was characterized (Neves et al. 2006). Larger surveys of oak transcriptomes have been conducted using hybridizations, RT-PCR, ESTs, and macroarrays. Kruger et al. (2004) used subtractive hybridization and differential expression to query Q. robur transcripts up-regulated in the pre-mycorrhizal phase in a micropropagation system. RT-PCR has been used successfully to examine seasonal variation

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in the transcripts involved in cork biosynthesis and regulation using candidate genes (Soler et al. 2008). It also has been applied to the transcriptome of Q. petraea bud burst (Derory et al. 2006). Several expressed sequence tag (EST) libraries have been published as well, including one for Q. petraea bud burst (Derory et al. 2006) and an inner bark EST library for Quercus mongolica var. crispula that Ueno et al. (2008) used to explore microsatellite marker development. EST-QTL maps have even been generated for osmotic stress-induced genes in Q. robur
Q. petraea (Porth et al. 2005a, b) and a broader comparison of adaptive traits including bud burst and height growth in oak versus chestnut (Quercus robur
Castanea sativa, Casasoli et al. 2006). Public availability of oak expression data is growing, with nearly half a million (n ¼ 489,780) Quercus EST sequences in public databases, as far as we know. There are 9,420 Quercus EST sequences presently listed with GenBank, all from the white oaks (section Quercus). Species representation is as follows: Q. robur (n ¼ 3,499), Q. mongolica subsp. crispula (n ¼ 3,385), Q. petraea (n ¼ 2,300), and Q. suber (n ¼ 236). A notable sequencing effort is underway at the Fagaceae Project, and public access to these sequences is provided through their website. They have pooled multiple above-ground tissues (including buds, cambium, flowers, fruit, phloem, and stems) to produce RNA, and then used 454-technology to sequence through the cDNAs. They have pursed sequencing in the two main timber species for North America, one for each oak section. Q. alba (White Oak, section Quercus) has a total of 203,206 EST sequences and Q. rubra (Northern Red Oak, section Lobatae) has 277,154 ESTs, totaling 480,360 sequences for these two species. Also available on the Fagaceae Project website are the contigs and unigene sets for these projects, along with similar resources for other members of the Fagaceae, namely Fagus grandifolia (American beech, 64,253 ETSs), Castanea mollissima (Chinese chestnut, 847,952 ESTs), and C. dentata (American chestnut, 398,783 ESTs). The French, in association with EVOLTREE, also have a forest tree genomics initiative (FOREST, http://www.genoscope.cns.fr/spip/Quercus-Forestfrench-initiative.html) producing Q. robur ESTs for differentiating xylem (n ¼ 9,529), leaves (n ¼ 7,097), and roots (n ¼ 19,177), along with Q. petraea ESTs for buds (n ¼ 9,990).

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The poplar genome array from Affymetrix represented a major step forward for tree functional genomics, and oak microarrays are in the planning stages. Oak macroarrays have already been used in research. For example, Frettinger et al. (2007, see also a review in Herrmann and Buscot 2007) used a cDNA array to explore transcriptional changes in pre-mycorrhizal roots and in ectomycorrhizae. Soler et al. (2007) used a cDNA array to explore suberin biosynthesis and cork differentiation in Q. suber, and Derory et al. (2006) to examine bud burst in Q. petraea. Public releases of oak microarrays are on the horizon. Following its participation in the Human Genome Project, the French Genoscope program (http://www.genoscope.cns.fr) has changed its focus to environmental genomics. In association with the EVOLTREE and FOREST programs, Genoscope states that a unigene set of 16,000 elements from Q. robur and Q. petraea ESTs is now ready to be printed on a microarray (as of January 15, 2009). On an annual basis, the EVOLTREE program plans to issue public releases of subsets of the ESTs in its possession in the form of microarrays, through the PICME (Platform for Integrated Clone Management; http://www.picme.at). This program of the Austrian Research Centers GmbH – ARC will make available to the scientific community the ESTs of a centralized database for a variety of species including Quercus but also Populus, Pinus, and Ipomoea.

6.7.4.2 Proteome There are numerous reports on protein variation in Quercus though most of these are isozyme studies that focus on a few select metabolic enzymes (e.g., Schnabel and Hamrick 1990; Berg and Hamrick 1993). Such studies can be very informative as to the gene pool structure, wherein members of the genus typically display a large amount of protein-based genetic variation within populations and little differentiation among populations, as is typical of longlived, outcrossing, woody species (Hamrick et al. 1992). Isozymes, as with other marker types, have not revealed fixed differences between oak species (see also Sect. 6.2.3). This is so even in high-throughput research as in Barreneche et al. (1996) in which they used two-dimensional gel electrophoresis to query protein variation in Q. robur and Q. petraea. In the

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scoring of 530 polypeptide spots, 101 were polymorphic, three spots displayed a frequency difference between species, but no spots were specific to one or the other species. More recently, Jorge et al. (2005) announced the Q. ilex (Holm oak) leaf proteome project. They report the first stages of a protocol optimized for assessing both analytical and biological variance in protein abundance. Methods include two-dimensional gel electrophoresis, tandem mass spectrometry, de novo sequencing, and sequence similarity searches against non-oak databases for identifications. Their initial report describes 35 identifications (out of 43 analyzed spots) mainly of proteins involved in photosynthesis and energy-based metabolism, with ribulose1,5-bisphosphate carboxylase oxygenase (RubisCO) especially well represented.

6.7.5 Metabolomics Most published biochemical research on oaks has targeted specific chemical groups or pathways and includes research in chemosystematics, acorn nutritive chemistry (e.g., Ozcan and Baycu 2005), isoprenoid emissions (e.g., Loreto et al. 1998), chemical deterrents to herbivory, stress physiology, and considerable work on secondary metabolites including tannins and other heartwood extractives. 6.7.5.1 Wood Chemistry There has been much attention given to the chemistry of wood formation in trees. Many of the approaches are integrative and include genomics research to identify genes participating in relevant biochemical pathways. As noted, most of this work has occurred in Populus, though Eucalyptus is a rising presence in this area along with the conifers, especially Pinus. For example, sequenced ESTs from the poplar secondary xylem have revealed genes active in lignin and cellulose biosynthesis (e.g., Sterky et al. 1998; Hertzberg et al. 2001). Recent reviews of various aspects of wood formation chemistry and genomics can be found in Tang et al. (2003) and Farrokhi et al. (2006). There has been much less work on the temperate hardwood metabolome in the area of wood chemistry.

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Robinia pseudoacacia (Black Locust) has provided insights into the chemistry of hardwood formation, wherein Yang et al. (2004) queried gene expression at the sapwood-heartwood transition zone using a cDNA microarray for 2,567 unigenes. They used this system to describe variation in 569 genes that displayed differential expression across seasons. As for oaks, Soler et al. (2007) recently combined subtractive hybridization and cDNA microarrays to explore the basis of suberin biosynthesis during cork formation in Q. suber. They isolated and identified sequences from cork tree bark, printed them on an array, and queried cork versus xylem tissue, resolving several genes specific to interfacing with suberin monomers. Much of the research to date on oak wood chemistry has focused on heartwood extractives, a diverse collection of secondary metabolites that includes the soluble polyphenolic compounds known as tannins (gallotannins and ellagitannins). These compounds have historically been used to cure or tan leather, though they also influence coloration (and value) of wood, and readily leach out of wooden casks or barrels thereby influencing the flavor of wine (reviewed in Puech et al. 1999; see also Doussot et al. 2002; and Sect. 6.8.3). Not surprisingly, much of this work has been done on Q. robur and Q. petraea in Europe, with a couple of reports from Q. laevis. Puech et al. (1999) summarized findings on the natural variation in tannin concentrations in oak heartwood, finding that there was considerable variation within and among trees, provenance, and species. But there seems to be a reasonable genetic basis to variation in the trait, even though the environment can have a strong influence. Mosedale et al. (1996) and Mosedale and Savill (1996) found evidence for strong genetic control of heartwood ellagitannin concentration in Q. petraea and Q. robur, though a large amount of the total variation was due to geographic origin, and heartwood color appeared to arise from environmental factors. Snakkers et al. (2000) describe extractive variation in Q. petraea across geography, silvicultural treatment, and stem. In Quercus laevis, Klaper et al. (2001) used microsatellites to show a significant correlation between genetic relatedness and leaf phenolics though there also appeared to be seasonal variation. Other studies support the notion that oak heartwood extractives can have a strong environmental determinant (e.g., Masson et al. 1995; Prida et al. 2006).

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6.8 Domestication and Commercialization The economic impact of wild oak populations is considerable through their collective contributions to the structure and function of forested ecosystems (see Sect. 6.3). Although most oak lines are not heavily domesticated, the commercial contributions of both wild and managed populations are large most notably as a source of wood and paper products. Lesser but notable contributions also exist in the areas of food and beverages.

6.8.1 Wood and Paper Products Oaks are an important source of hardwood lumber (Johnson et al. 2002; reviewed in Bowyer et al. 2007). Finer grades are used in veneer, furniture, flooring, and other building materials. Lesser grades are used in pallets, crates, and railroad ties. Oak species are usually lumped together at the mill and sold either as red or white oak. Red oaks play a greater economic role in the US market (i.e., in their native range), whereas the white oaks are economically important in many areas of the globe. We have already considered the selection and breeding of wood properties in other sections including general wood quality (see Sect. 6.6.3.4) as well as extractives that influence the color of wood (see Sect. 6.7.5.1). There is more to say regarding extractives in the context of wine and whiskey making (see below). Additional wood-related topics of commercial importance include recent proteomic research on wood decay fungi (Abbas et al. 2005) and a burst of interest in forensic identification of wood origins. Much can happen between the forest or plantation and the lumber yard, and sometimes buyers would like to know the provenance of the wood they are purchasing. Forest certification establishes that certain timber has been harvested from sustainably managed forests, and checking that the chain of custody is as advertised is of growing economic importance. Molecular markers can be used to type wood as to its geographic origin. This is especially true in places such as Europe where there is a geographically extensive and dense database on the composition of the gene pool for some agricultural species, such as the oaks. Chloroplast

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microsatellites have proven especially useful in the typing of oak wood to the stand-level (e.g., Deguilloux et al. 2002, 2003b). Dried oak wood and oak wood as old as 600 years have been used successfully in PCRbased assays of this type (Dumolin-Lape`gue et al. 2002). Among the forest industries benefiting from enhanced typing, the cooperage industry that makes oak barrels for the aging of wine and whiskey must know the geographic source of the wood used in barrels since this is critical to the flavor imparted to the end product (Deguilloux et al. 2004). Other applications of forensic techniques include court cases involving wood theft and criminal cases where the geographic source of botanical evidence is relevant (e.g., Craft et al. 2007). Since oaks are common one would anticipate their frequent appearance in such cases. For a more general consideration of wood typing, see Nielsen and Kjær (2008).

6.8.2 Food Products Oaks serve as a food source for a variety of organisms of economic importance to humans, including wildlife, a silkmoth, and a variety of mushrooms. Although indigenous cultures ate acorns and recipes can be found for acorn-based flour, the extensive preparations required to remove the tannins has caused acorns to fall off the human menu. Modern usage of oaks as a human food source is more often indirect. Clearly oaks serve as mast for a variety of wildlife, some of which humans eat, such as deer. Acorn chemistry and nutrition has been studied, and we know for instance their elemental composition (e.g., Ozcan and Baycu 2005) and their potentially therapeutic properties associated with free-radical scavenging (Jin et al. 2005). Oak leaves sustain a diverse fauna of herbivores, some of which are of economic interest such as the Japanese oak silkmoth Antheraea yamamai (Oishi et al. 2005). And oak wood is commonly used in the culture of mushrooms for human consumption. For example, oak wood chips are useful in growing Hericium (Ko et al. 2005) and Pleurotus (Suzuki and Mizuno 1997). But it is the truffle (genus Tuber) that fetches the highest price among edible fungi. This grows below ground near the base of oak trees, is notoriously hard to find, and expensive. Techniques are now available to detect and monitor the abundance of Tuber mycelia

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in the soil, exemplified by the real-time PCR study of an oak orchard by Suz et al. (2008).

6.8.3 Wine and Whiskey The wine and whiskey industries utilize oak wood in the production of barrels (cooperage), corks for bottles, and yeast indigenous to oak bark for flavoring. Casks and barrels used to age wine and whiskey are often made from oak heartwood wherein the wood serves a dual function as container but also contributor to the flavor of the wine and whiskey (Feuillat and Keller 1997; Puech et al. 1999; Doussot et al. 2002; see also Sect. 6.7.5.1). Secondary metabolites such as the tannins and other heartwood extractives leach from the wood during the aging, influencing the end product flavor. The wood chemistry is highly variable by species, geography, and wood treatment such as natural drying versus toasting. Quercus suber (cork oak, section Cerris) is a dominant source of cork, and recent genomic work is exploring the molecular genetic basis of cork development (Soler et al. 2007). The yeast Saccharomyces cerevisiae is used in wine making and researchers have begun exploring the genetic characteristics of different yeast strains collected from oak bark (Mesa et al. 2000; Aa et al. 2006; Wang and Bai 2008). The hope is that some of these naturally occurring yeast varieties would improve wine flavor during fermentation and aging. These are important areas in which Quercus research leads that done in Populus and Arabidopsis.

6.9 Conclusion Oaks and humans have interacted for many millennia, yet the character of the interactions has remained largely unchanged until only recently. We are moving from a state of near complete ignorance regarding the content of the oak genome to a place where we now know much of the protein coding sequence, through EST studies. It is likely that the whole genome will be sequenced not that far in the future. Domestication will advance at a much more rapid pace, building upon the numerous projects that have taken hold in the last several decades. A challenge for the immediate

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future is to take stock of the wild populations of oaks to ensure that naturally occurring biodiversity is retained for incorporation into the cultivars that will derive from domestication programs. Although the oak gene pools appear sound in many regards, there are numerous reasons to be cautious in our optimism and to manage the presently ubiquitous resource with care so that this critical species continues to play a dominant role in the planet’s ecosystems and our economies.

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.

Chapter 7

Santalum Madhugiri Nageswara Rao, Jaya R. Soneji, and Padmini Sudarshana

7.1 Basic Botany of the Species The genus Santalum, commonly known as sandalwood, belongs to the family Santalaceae (Fig. 7.1). The genus is composed of approximately 16 species and their variants, many of which are geographically and probably reproductively isolated (Applegate et al. 1990). Several species, most notably S. album, are highly valued as they produce extremely aromatic heartwood and oil (Uma Shaanker et al. 2000; Nageswara Rao 2004; Nageswara Rao et al. 2007a). Though S. album species, known commonly as Indian sandalwood, is indigenous to Peninsular India (Srinivasan et al. 1992), disagreement persists as to whether it is native to India or was introduced for cultivation over 2,000 years ago from the Timor Islands of Indonesia (Roxburgh 1820; Sprague and Summerhayes 1927; Fischer 1928, 1938; Tuyama 1939; St. John 1947; Thirawat 1955; Shetty 1977; Mc Kinnell 1990; Rai 1990) or Australia. S. lanceolatum is believed to have originated in Australia, S. austrocaledonicum in New Caledonia, and S. yasi in Tonga, while S. ellipticum, S. freycinetianum, S. haleakalae, and S. paniculatum are endemic to Hawaiian Islands (Merlin et al. 2006; Thomson 2006). S. album is distributed between 30
N and 40
S from Indonesia in the West to Jaun Fernandez Islands in the East and from Hawaiian Archipelago in the North to New Zealand in the South. S. austrocaledonicum and S. yasi have limited plantings, outside of its native range, in Australia for

M. Nageswara Rao (*) IFAS, University of Florida (University of Florida, IFAS) Citrus Research & Education Center, University of Florida, IFAS, 700 Experiment Station Road, Lake Alfred FL 33850, USA e-mail: [email protected]

trial purposes (Thomson 2006). S. austrocaledonicum has also been planted in Fiji and the Cook Islands. S. ellipticum, S. freycinetianum, S. haleakalae, and S. paniculatum have primarily been planted inside of their natural range for economic or preservation purposes (Merlin et al. 2006). S. acuminatum is wide spread in all Australian mainland states (George 1984). Sandal grows naturally in a variety of climates from warm desert in Australia, through seasonally dry monsoon climate in India, eastern Indonesia, and Vanuatu, to subtropical climate with almost uniform rainfall in Hawaii and New Caledonia (Applegate et al. 1990). It is adaptable to most soil conditions but grows well in light to medium, well-drained soils (Merlin et al. 2006). Most sandal species are small trees or large shrubs, attaining a height of about 5–20 m or more and girth of 1–2.5 m with slender drooping and/or erect branching (Fig. 7.2a). They are slow growing root hemiparasites, with roots tapping the root systems of surrounding trees for water, minerals, and nutrients (Stemmermann 1977). Scott (1871) for the first time reported the parasitic nature of sandal. Requirement of host for proper growth of sandal was demonstrated in a field study by Ananthpadmanabha et al. (1984). Depending on the sandal species and the location, the host trees may vary though they seem to rely on nitrogen fixing trees such as Acacia and Casuarina, along with many other legumes, shrubs, herbs, and grasses. A wide range of hosts are utilized by S. album in India and occasionally it self-parasitizes (Rai 1990), while in Timor it associates with numerous species such as Eugenia, Casurina, Cassia, Schleichera, Pterocarpus, etc. (Suriamihardja and Suriamihardja 1993). In Australia, S. spicatum and S. lanceolatum parasitizes Acacia, Eremophlia, Melaleuca, etc. (Applegate and McKinnell 1993).

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_7, # Springer-Verlag Berlin Heidelberg 2011

131

132 Fig. 7.1 Taxonomic position of Santalum

M. Nageswara Rao et al. Kingdom Plantae

Division Magnoliophyta

Subdivision Magnoliophytina

Class Magnoliopsida

Subclass Rosidae

Superorder Santalanae

Order Santalales

Suborder Santalineae

Family Santalaceae

four ploidy levels, ranging from diploidy to octoploidy, and differ significantly from one another in DNA content (Harbaugh 2008). S. album is diploid with 2n ¼ 20, while other members of the genus, particularly S. acuminatum and S. macgregorii, are believed to be tetraploid (Kulkarni et al. 1998; Byrne et al. 2003a). Most of the Australian species (S. acuminatum, S. lanceolatum, S. murrayanum, S. obtusifolium, and S. spicatum) are diploids, with the exception of S. leptocladum, which is a tetraploid. S. austrocaledonicum from Vanuatu and New Caledonia and S. yasi from Fiji are also diploids, while S. macgregorii from Papua New Guinea and S. ellipticum, S. freycinetianum, S. haleakalae, and S. paniculatum from Hawaiian Islands are tetraploids. S. insulare may be a putative hexa- or octaploid (Harbaugh 2008). Incongruence between nuclear and chloroplast trees in the positions of two taxa, S. boninense and S. macgregorii, shows that hybridization and possibly allopolyploidization may have played a role in the evolution of the genus (Harbaugh and Baldwin 2007).

Subfamily Santaloideae

Tribe Santalaceae

Subtribe Santalinae Genus Santalum

The leaves of sandal are opposite and decussate, and sometimes show whorled arrangement. The tree starts flowering at an early age of 2–4 years and flowers twice a year from March to May and September to December. Flowers (Fig. 7.2b and Table 7.1) are unscented, purplish brown, and borne in axillary or terminal cymose panicles (Srinivasan et al. 1992). Petals are tetra or pentamerous, fused to the perianth (Hewson and George 1984). The ovary is semi-inferior and unilocular yielding one single-seeded drupe (Fig. 7.2c). Seeds are naked, i.e. lack testa. However, S. spicatum has dry and fibrous fruits enclosing a hard nut (Jones and Plummer 2008). S. album and S. spicatum are outcrossing insect-pollinated trees; however, self-pollination is possible (Rugkhla 1997). Birds disperse sandal fruits. Seedling establishment has been found to be positively associated with seed size (Hegde et al. 1991). Limited work has been done on the cytological features of sandal. The genus Santalum appears to include

7.2 Taxonomy and Phylogeography Santalum has been taxonomically grouped entirely on a few morphological characters such as floral tube color, ratio of corolla length to width and placement of ovary. They were divided into five sections or genus. The first section Santalum was described as usually having reddish corollas that are longer than wide and partly superior ovaries (Skottsberg 1930a; Stemmermann 1980; Wagner et al. 1999) and covered species such as S. album, S. austrocaledonium, S. boninense, S. lanceolatum, S. macgregorii, S. obtusifolium, and S. yasi occurring in Australia, Indonesia, India, New Caledonia, Vanuatu, Bonin Islands, Papua New Guniea, Fuji, or Tonga. Solenantha was the second section covering S. freycinetianum and S. haleakalae species both of which were endemic to the Hawaiian Islands and were described based on their longer perianth tubes, smaller ovaries, and absence of hair proximal to the filaments (Tuyama 1939). S. ellipticum and S. paniculatum occurring in the Hawaiian Islands formed the third section of Hawaiiensia and described as having white, green, brown, or orange corollas that are as wide as long, and inferior ovaries (Skottsberg 1930a; Stemmermann 1980; Wagner et al. 1999). Polynesica formed the fourth

7 Santalum

133

a

b

c

Fig. 7.2 (a) Sandalwood (Santalum album L.) tree in its natural habitat, (b) Fruits, and (c) Flowers

section with species such as S. fernandezianum and S. insulare occurring in Juan Fernandez Islands, Society Islands, Marquesas Islands, Pitcairn Islands, Austral Islands, or Cook Islands. Section Polynesica has characters similar to Hawaiiensia and could be distinguished from it based on their partly superior ovaries (Skottsberg 1930a). S. acuminatum, S. murrayanum, and S. spicatum occurring in Australia formed the fifth section of Eucarya (Harbaugh and Baldwin 2007). Morphological similarities between Hawaiiensian and Polynesican sections have led to the hypothesis that they may be closely related (Skottsberg 1930a, b) or may be synonyms (Fosberg and Sachet 1985). Recently, molecular phylogenetic analyses have demonstrated that sections Hawaiiensia and Polynesica are more closely related to other taxa of Santalum than to one another (Harbaugh and Baldwin 2007). Phylogeographical analysis has been carried out in sandal. It relies on interpreting patterns of congruence or lack of congruence between the geographical distribution of chlorotypes and their genealogical relationships (Avise 2000). If clades of closely related chlorotypes are geographically restricted and occur in proximity to each other, they represent a pattern of congruence, which indicates long-standing pattern of highly restricted

gene flow (Butaud et al. 2005). Harbaugh and Baldwin (2007) reported a genus-wide phylogenetic analysis for Santalum, using a combination of 18S–26S nuclear ribosomal [internal transcribed spacer (ITS) and external transcribed spacer (ETS)] and chloroplast (30 trnK intron) DNA sequences, and provided new perspectives on relationships and biogeographic patterns among the widespread and economically important sandal. Indonesia has been reported to be the possible source of S. album in India and Australia (Roxburgh 1820; Sprague and Summerhayes 1927; Tuyama 1939; St. John 1947; Mc Kinnell 1990; Rai 1990). Several specimens of S. album from India and Australia were phylogenetically identical suggesting a recent dispersal, consistent with the movement of this species by people (Harbaugh and Baldwin 2007). Another study also reported a lack of differentiation between Timorese and Indian germplasm of S. album supporting the hypothesis that S. album was introduced to India in very recent geological time, possibly through human mediation (Jones 2008). The origin of Santalum in Australia and at least five putatively bird-mediated, long-distance dispersal events out of Australia (with two colonizations of Melanesia, two of the Hawaiian Islands, and one of the Juan

Fruit color

Fruit length Fruit shape

Leaf length Leaf width Leaf shape and characteristics

Ovary

Flower size

Flower fragrance

Tree height Bole diameter at breast height Canopy diameter Flower color 2–4 m Reddish cream

2–4 m 20 cm

1,900–2,700 m Small tree

S. haleakalae Endemic to Maui

Reddish purple to black

8–24 mm Drupes

4.0–9.0 cm 1.3–7.5 cm Acute to rounded apex, green to a bit glaucous

Produce sweet fragrance Flowers are as long as wide Ovary inferior

3–7 m Greenish to tinged brown, orange

3–10 m 1m

450–2,550 m Shrubs to small tree

S. paniculatum Endemic to Hawaii

10–12 mm Drupes Black to purple black Purple to black

8–24 mm Drupes

2.5–7.5 cm 2.5–8.0 cm 2.0–6.0 cm 2.0–4.5 cm Ovate, obovate or Ovate, elliptic or orbicular, stiff to obovate, glossy coriaceous upper and dull/ surfaces pale lower

Produce weak Produce sweet fragrance fragrance Flowers are as long Flowers are as long as wide as wide Ovary partly inferior Ovary inferior

3–10 m Red to yellow

1–13 m 80 cm

250–950 m Shrubs to small tree

560–950 m Shrubs to small tree 1–12 m 30 cm

S. freycinetianum Endemic to Hawaii

S. ellipticum Endemic to Hawaii

1–5 m Greenish to tinged brown, orange, salmon Fragrant Produce fragrance Produce sweet fragrance 3.0–5.0 mm long 4.5 mm long Flowers are as long as wide Ovary semi-inferior Ovary inferior or half Ovary inferior inferior 3.0–8.0 cm 5.0–9.0 cm 2.5–6.1 cm 3.0–5.0 cm 2.0–3.5 cm 1.7–4.0 cm Opposite, decussate, Simple, obovate, Ecliptic to orbicular, ovate, or ovate glabrous, shiny at ovate, or obovate elliptical, shiny the top, dull light in shape, leathery at the top, dull green below to succulent green below 10–12 mm 10–15 mm 9–12 mm Globose to ellipsoid, Subglobose or Glaucous fleshy drupe ellipsoid Purple to black Drupe, purplish black Purple to black

5–12 m 40–50 m

4–10 m 4–8 m Greenish to purplish Greenish white to brown cream

12–20 m 1–1.5 m

Table 7.1 Botanical description of Santalum species Santalum album S. austrocaledonicum Distribution Australia, India, New Caledonia, Indonesia, New Vanuatu Zealand Elevation 650–1,200 m 5–800 m Plant size Small tree Shrubs to small tree

Reddish purple to black

12 mm Ellipsoid drupe

8–12 m Greenish to dark red, tinged brown Produce fragrance 3.0–4.5 mm long Ovary inferior or half 6.0–7.0 cm 1.5–2.0 cm Simple, narrow to broadly lanceolate, shiny green

0–300 m Shrubs to small tree 8–10 m 40–50 m

S. yasi Fiji, Niue, Tonga

134 M. Nageswara Rao et al.

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Fernandez Islands) has been supported by congruent trees based on maximum parsimony, maximum likelihood, and Bayesian methods. The phylogenetic data also provide the best available evidence for plant dispersal out of the Hawaiian Islands to the Bonin Islands and eastern Polynesia (Harbaugh and Baldwin 2007). Phylogeographical analysis of S. album using chloroplast genes showed different haplotypes for the western Ghat population, when compared with the other two major geographic regions (eastern Ghat and Deccan plateau) from Peninsular India (Nageswara Rao 2004). The geographical difference and genetic structure in the study also appeared to be consistent with the presence of three major clusters (western Ghat, eastern Ghat, and Deccan plateau) differentiated across the sandal populations in Peninsular India (Nageswara Rao et al. 2007a; Fig. 7.3). Phylogeographical analysis of S. insulare, endemic to the islands of eastern Polynesia, using chloroplast microsatellite markers separated the populations sampled into three clusters, each cluster corresponding to one geological archipelago: Marquesas Islands, Society Islands, and Cook-Austral Archipelago (Butaud et al. 2005). The diversity and phylogeographic patterns within the chloroplast genome of S. spicatum were analyzed. The chloroplast diversity structured into two main clades that were geographically sepa-

Savandurga Siddarabetta Sakrebayalu Tavrekoppa Devarayanadurga Kasaragodu Haliyala Targodu Sonda Kalasa Mananthwadi Yellapura Kivara Yercadu Tirumala Metupalyam Chitoor Chitteri

rated, one centered in the southern (semi-arid region) and the other in the northern (arid) region in Australia suggesting a fragmentation due to climatic instability. The phylogeographic pattern in the chloroplast genome was congruent with that detected in the nuclear genome (Byrne et al. 2003b).

7.3 Conservation Initiatives Despite being a renewable plant resource, sandal populations in India, Indonesia, the South Pacific, and Australia are declining due to over-harvesting (Fig. 7.4) and illegal poaching of native stands, changes in landuse patterns, grazing, poor natural regeneration, and/or spike disease (Loneragan 1990; Rai and Sharma 1990; Srinivasan et al. 1992; Meera et al. 2000; Nageswara Rao et al. 2007a, b, 2008a). Since almost all of the extraction is from natural populations, the pressure on the existing populations has been tremendous (Radomiljac et al. 1998; Nageswara Rao et al. 2001a, b; Suma and Balasundaran 2003). This overexploitation has led to a steady decrease in the availability of S. album in India (Nageswara Rao 2004; Nageswara Rao et al. 2007a, 2008c), S. insulare in eastern Polynesia (Butaud 2004), S. spicatum in

D. Plateau

W.Ghat

E.Ghat 0

2000

4000

6000 Linkage Distance

8000

10000

Fig. 7.3 Dendrogram clustering of sandal populations in peninsular India (Nageswara Rao et al. 2007a)

12000

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M. Nageswara Rao et al. 3500

y = 2E+24e-0.0246x R2 = 0.3912

3000

Quantity (tonnes)

2500

2000

1500

1000 500

1950 1951 1952 1953 1954 1955 1956 1957 1958 1959 1960 1961 1962 1963 1964 1965 1966 1967 1968 1969 1970 1971 1972 1973 1974 1975 1976 1977 1978 1979 1980 1981 1982 1983 1984 1985 1986 1987 1988 1989 1990 1991 1992 1993 1994 1995 1996 1997 1998 1999 2000 2001 2002 2003 2004

0

Fig. 7.4 Extent of quantity of sandalwood extracted in Karnataka, India between 1950 and 2004 (Nageswara Rao et al. 2007a, 2008c)

0.4 BRT

Observed Heterozygosity

S.durga

T.koppa

0.36

Targodu Sonda Haliyala

0.32

S.betta

Kivara

S.bayalu D.durga

Yellapura K.godu Kalasa

0.28

Yercadu M .wadi M .palyam

Tirumala

Chitteri

0.24 Chitoor 0.2 74

75

76

77 78 Longitude (Degree East)

79

80

Fig. 7.5 Relationship between observed heterozygosity with longitude in sandalwood populations across peninsular India (Nageswara Rao 2004; r ¼ 0.51; p < 0.05)

Australia (Byrne et al. 2003a, b), and S. austrocaledonicum in Vanuatu (Bottin et al. 2005). To safeguard the natural populations of sandal, effective measures need to be taken. Critical information on the distribution and status of sandal resources throughout India have been collected and used to develop a comprehensive distribution map, which would directly help the conservation efforts and would also serve as a bench mark to further monitor

the changes in the species landscape over time (Nageswara Rao et al. 2007a, 2008d). This distribution map will be of great importance in identifying the hot-spots of genetic diversity and will be helpful in formulating management plans to conserve the genetic resources of this species (Nageswara Rao et al. 2001b, 2002, 2007a; Nageswara Rao and Soneji 2009). This study also highlighted a strong negative correlation with increasing longitude for the sandalwood populations

7 Santalum

in Peninsular India (Fig. 7.5). The occurrence of six biotypes of sandal in India have been reported (Kulkarni and Srimathi 1982). Efforts have been made to establish ex situ conservation gardens for S. album at different sites in India and artificial regeneration of sandal in areas where the stock of sandal growth is poor has also been suggested (Srinivasan et al. 1992; Nageswara Rao et al. 2001b, 2008c). Three clonal banks, one each at Gottipura, Bangalore Division, Karvetinagar at Chittor Division and at Kurumbapatti at Salem, have been established. Besides the clonal orchards, there have been efforts to establish a germplasm bank at Gottipura, Bangalore Division (Srinivasan et al. 1992). Eight sandal-bearing areas have been recognized as potential provenances in India (Jain et al. 1998). Using advanced Geographic Information System ecological niche modeling (DIVA-GIS), the map for possible potential occurrence of sandal genetic resources in the Peninsular India have been developed (Nageswara Rao et al. 2008d). Studies have also highlighted the possible role of protected areas in conserving the sandal resources in India (Nageswara Rao et al. 1998, 2001b, 2007b, c; Ravikanth et al. 2009). For a long-term management and conservation of sandal, “Forest Gene Bank” model has also been proposed (Nageswara Rao et al. 1999, 2008b). Conservation programs have been implemented for critically endangered S. insulare (Butaud et al. 2005). Heavy harvesting, grazing, and fires coupled with disrupted gene flow and possibly genetic drift of S. lanceolatum from Victoria and New South Wales in the mid- to late-1800s has led to its being listed as a threatened taxon under Schedule II of the Victorian Flora and Fauna Guarantee Act 1988 (Warburton et al. 2000). The restriction and isolation of the remnant S. lanceolatum populations has led to the development of a conservation strategy for the species in Victoria (Johnson 1996). Establishment of new stands of S. lanceolatum has also been proposed as a conservation objective (Warburton et al. 2000). In situ and ex situ conservation strategies have been formulated in Pacific islands for the effective conservation of S. yasi (Padolina 2007). Seed collection efforts are being made by the Forestry Department of Vanuatu to establish a gene pool collection to conserve S. austrocaledonicum (Padolina 2007). A variety of Santalum plantings have been established to ensure conservation of genetic diversity for species, such as S. album, from a range of locations in India, Timor and

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Australia, S. austrocaledonicum from New Caledonia, and S. macgregorii from Papua New Guinea (Vernes 2001). S. macgregorii seed has also been distributed for ex situ conservation in Australia (Queensland and western Australia) and the Pacific (Thomson and Bosimbi 2000).

7.4 Genetic Diversity Though a number of efforts are being made to conserve sandal, the lack of basic information on the extent of natural genetic variability of sandal populations has been a major handicap in formulating conservation plans (Nageswara Rao 2004). Studying the genetic diversity of sandal could lead to the identification of “hot-spots” of genetic variability, which can then be targeted for their efficient conservation, sustainable utilization of genetic resources, and/or genetic improvement programs. A number of studies have been carried out to understand the genetic diversity of a few species of sandal. Genetic diversity in S. album has been studied using isozymes (Nageswara Rao et al. 1998, 2007a; Angadi et al. 2003), random amplification of polymorphic DNA (RAPDs; Shashidhara et al. 2003; Suma and Balasundaran 2003), and restriction fragment length polymorphism (RFLPs; Jones 2008). Using eight populations, Angadi et al. (2003) identified eight provenances of S. album indicating that the populations used were of separate varieties or races. Suma and Balasundaran (2003) reported low degree of variability within five provenances of S. album. This might have been due to fragmentation of a previously large population, resulting in loss of genetic variation, least amount of gene flow between the provenances and differentiation of population due to random drift. RAPDs were found to be effective in distinguishing 51 genotypes of S. album (Shashidhara et al. 2003). Nageswara Rao et al. (2007a, b) used 19 populations to understand the extent of diversity remaining within the natural populations of sandal occurring in Peninsular India and have been able to identify the “hot-spots” of genetic resources of S. album. RFLP analysis of 233 S. album genotypes also revealed low levels of genetic diversity (Jones 2008). This information will be of great use in genetic improvement programs.

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RFLP analysis of S. spicatum populations collected from semi-arid and arid regions of western Australia showed differences in population structure between these regions. Populations in the arid region showed higher levels of genetic diversity and a greater number of rare alleles than those in the semi-arid region. The degree of differentiation between the populations is related to the interaction and relative influences of both drift and gene flow (Byrne et al. 2003a). These regional differences are congruent with the presence of different chloroplast lineages (Byrne et al. 2003b) and consistent with the identification of arid and semiarid ecotypes based on morphological variation (Fox and Brand 1993). Allozymes and RAPDs were used to determine the extent of clonality in remnant populations of S. lanceolatum, which is represented by only one small population of unique clones in Victoria, southeastern Australia. Both allozymes and RAPDs detected no variation within populations, suggesting that each population consisted of a single genet. The study also indicated that each of the five remnant S. lanceolatum populations existed as a single unique clone, recruiting individuals only by vegetative reproduction reflecting on the history of disturbance and fragmentation of the populations due to harvesting, clearing, grazing, and fires coupled with disrupted gene flow and possibly genetic drift. These populations also showed little or no fruit production due to pollen sterility, pollen-pistil incompatibility or pistil dysfunction (Warburton et al. 2000). Chloroplast microsatellite markers have been used to analyze 11 populations of S. insulare collected from islands of eastern Polynesia where it is an endemic. The gene flow between populations was found to be low. The gene diversity index varied among the archipelagoes but was high for the total population. Genetic structure was characterized by high levels of differentiation between archipelagoes (36% of total variation) and between islands, but differentiation between islands varied according to the archipelago (Butaud et al. 2005). Another study was carried out on the same populations of S. insulare using nuclear microsatellites. This study indicated that clonality was a frequent phenomenon in S. insulare. As a consequence of insularity, the genetic diversity within populations was also lower than the values assessed in species distributed on the mainland. This may also be due to over-exploitation of S. insulare (Lhuiller et al. 2006). S. austrocaledonicum is another insular

M. Nageswara Rao et al.

tree which is confined to the archipelago of New Caledonia. Microsatellite analysis of 17 populations of S. austrocaledonicum indicated that the genetic diversity was lowest in the most recent islands when compared with the oldest ones. At both population and island levels, island age and isolation seemed to be the main factors influencing the amount of genetic diversity suggesting a strong influence of insularity on the genetic diversity and structure of S. austrocaledonicum.

7.5 Role in Crop Improvement Through Traditional and Advanced Tools Sandal is a pre-dominantly outbreeding species though its flower structure was designed for self-pollination (Sindhu Veerendra and Ananthapadmanabha 1996). Apomixis and/or parthenocarpy have not been noticed in sandal while bees, butterflies, and beetles are the pollinating agents. Although sandal is being utilized for centuries, hardly any studies have been carried out for its genetic improvement. The only tree improvement work being carried out is the selection of superior phenotypes from naturally occurring populations. The species also has a complex ecology, which has hampered field trails. Heritability studies for desirable traits such as tree form, health, early heartwood onset, high heartwood content, and essential oil yield have not been conducted extensively (Jones 2008). Studies conducted on genetic diversity will provide the researchers with information on the level of variation within and between sandal populations and help them in assessing its potential for genetic improvement. For example, RFLP analysis in S. spicatum populations collected from semi-arid and arid regions of western Australia showed polymorphisms, which may represent genotypic variation in gene structure and function that could in turn affect important phenotypic traits such as form, growth rate, and heartwood oil production (Byrne et al. 2003a, b). Clonal reproduction of S. lanceolatum has been used to advantage, as regrowth from pulled trees appears to result in healthier root suckers (Bristow et al. 2000). Conventional breeding of sandal for introgression of new genetic information can be an expensive and difficult task because of their long generation time, sexual incompatibility, and heterozygous nature (Rugkhla 1997). Also traditional crossing and selection trials would take at least

7 Santalum

40–50 years to evaluate, provided a suitable phenotype screen, which takes into account the environmental variability, is established. However, if genetically superior seedlings could be identified at the nursery stage, or even propagated in vitro, sandal could be planted with certainty of better long-term returns (Jones and Plummer 2008). Seeds are mainly used for natural regeneration and artificial propagation, even though the seedlings of sandal are extremely heterozygous due to outcrossing (Sanjaya et al. 2006). Dibbling of seeds in bushes, sowing of seeds on the moulds, planting of container-raised seedlings (Rai and Sharma 1986), and vegetative propagation through grafting, air layering, and root suckers (Rao and Srimati 1977) are the other artificial methods of sandal propagation. However, the production of clones is time consuming (Srimati et al. 1995). Sandal is also recalcitrant to in vivo and in vitro propagation (Sanjaya et al. 2003), though tissue culture has been used to achieve high-frequency regeneration in vitro. Various explants such as hypocotyl, endosperm, nodal, and internodal segments, protoplasts, zygotic embryo, leaves, and nodal stem segments with dormant axillary buds have been used (Bapat and Rao 1978; Lakshmi Sita et al. 1980; Bapat and Rao 1984, 1985; Rao and Bapat 1992; Rugkhla and Jones 1998; Rai and McComb 2002; Mujib 2005; Sanjaya et al. 2006). Early attempts to develop somatic embryogenesis for sandal propagation focused on indirect embryogenesis (Rao and Rangaswamy 1971; Lakshmi Sita et al. 1979, 1980; Rai 2005). However, when somatic embryos were obtained through callus, the conversion or germination of these embryos into plantlets was problematic (Rao and Bapat 1995). Rai (1996) and Rai and McComb (2002) reported the regeneration of sandal plantlets from somatic embryos developed from mature zygotic embryos of sandal through direct somatic embryogenesis. Direct somatic embryogenesis from nodal and seed explants has also been reported (Rugkhla and Jones 1998). Sandal plants have also been successfully micropropagated by in vivo methods using mature plants (Lakshmi Sita et al. 1982). Somatic embryos have been used to develop synthetic seeds in sandal (Bapat and Rao 1988), which could be germinated in vitro as well as in vivo (Fernandes et al. 1992). The synthetic seeds could also be germinated after storage at 4
C for 45 days (Bapat and Rao 1988). In vitro regeneration techniques can be used to clone

139

superior lines and are needed for Agrobacteriummediated gene transfer techniques and protoplast fusion (Rugkhla and Jones 1998). However, the bottleneck is in vitro rooting, which limits the widespread application of micropropagation techniques in sandal (Sanjaya et al. 2006). As high efficiency in vitro regeneration systems are available for sandal, application of transgenic technology may offer a faster route for its genetic improvement. To date, there are only two reports of transformation of sandal. The first study reported a system for the introduction and expression of foreign genes in torpedo-cotyledonary stage embryos of sandal (Shiri and Rao 1998). These somatic embryos were inoculated by pricking and cells of disarmed Agrobacterium strains carrying b-glucuronidase (uidA) and neomycin phosphotransferase II (nptII) genes on the binary vectors pKIWI105, pBI121, and pIG121-Hm were directed at the wound sites. Transgenic plants were regenerated from embryogenic cultures derived from these transformed somatic embryos, and the transgenic nature was confirmed by gus and nptII assays as well as polymerase chain reaction (PCR) with insert-specific primers. More recently, an efficient method for the transformation of embryogenic cell suspension cultures of sandal has been described (Shekhawat et al. 2008). Embryogenic cell suspension cultures were transformed with A. tumefaciens strain EHA105 harboring the binary vector pCAMBIA 1301 containing a gusA gene interrupted by a modified castor bean catalase intron and an hptII gene conferring resistance to hygromycin. Plantlets were regenerated from the transformed embryogenic cells. Expression of b-glucuronidase in the suspension cultures was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and gus histochemical staining and stable insertion of T-DNA into the host genome was confirmed by Southern blot analysis. These reports have opened new vistas for the transformation of sandal with gene(s) resistant to various sandal diseases and for metabolic engineering to increase and/or modify the essential oil yield.

7.6 Genomics Resources Developed As yet, no attempts have been made to map or sequence the sandal genome or transcriptome.

140

However, attempts have been made to isolate and clone a proline-rich protein (PRP) cDNA from leaves of S. album (Bhattacharya and Lakshmi Sita 1998, 1999). PRPs are regarded as reactive biopolymers which are involved in maintenance of cell wall integrity and may be involved in systemic resistance to protoplasma-like pathogens. Genomic library was constructed using nuclear DNA prepared from tender leaves of S. album and screened using heterologous probes to isolate the PR1 genomic homolog. The coding region for PR1 gene was obtained by restriction mapping and hybridization (Bhattacharya and Lakshmi Sita 1999). To identify genes involved in essential oil biosynthesis, particularly terpene synthases (TPS) in S. album, degenerate TPS primers used amplified two genomic TPS fragments, one of which enabled the isolation of two TPS cDNAs, SamonoTPS1 (1,731 bp), and SasesquiTPS1 (1,680 bp) from leaf and wood tissues (Jones et al. 2008). The terpenoids produced by SamonoTPS1 and SasesquiTPS1 were not found in substantial quantities in the distilled oil of sandal, suggesting that additional TPS genes are expressed in S. album, which may contribute to the majority of heartwood essential oil. The gene structure and expression properties of TPS may be exploited through modification or selection of specific genotypes conducive to high essential oil production. The genes responsible for the production of essential oil in S. album, which is considered unique and is preferred in the preparation of various perfumes, flavors, cosmetics, etc., when identified and isolated will be of great economic importance. The gene can not only be introduced in other species of sandal but also in faster growing annual plants to provide quicker and easier route to obtaining the fragrant essential oil. Isolation and characterization of microsatellite loci in S. austrocaledonicum has been carried out (Bottin et al. 2005).

7.7 Scope for Domestication and Commercialization Heartwood and oil of sandal tree are of great commercial importance and extensively used in perfumes and medicine. The heartwood, being closely grained and amenable to carving, is one of the finest woods for making idols, boxes, and other curios of exquisite beauty. It is also used as an astringent, bitter, antipyretic, and a cooling agent. The bark when powdered is

M. Nageswara Rao et al.

an important raw material for the manufacture of incense sticks. The bark extract has been reported to behave as a chemosterilant and as an insect growth inhibitor (Shankaranarayana et al. 1979). The oil is considered to be unique and used for the preparation of top-class and sophisticated scents, perfumes, flavors, cosmetics, toiletries, beauty aids, and herbal medicine (Srinivasan et al. 1992). The seeds are used for treating diuretic, hypotensive, antitumorogenic, antiviral activities, and a number of skin diseases (Kirthikar and Basu 1987; Desai and Shankaranarayana 1990). With the dwindling of natural stands of sandal, attempts have been made to replant sandal. The last 10 years have seen an increase in the establishment of tree farms of S. album and S. spicatum in Australia. Around 1,750 ha of S. album plantations have been developed on irrigated land in Ord River Irrigation Area (ORIA) in northern western Australia by managed investment scheme (MIS) companies, while the area of tree farms of S. spicatum have steadily increased to over 10,000 ha (www.tfsltd.com.au; http://www.fpc.wa.gov.au/pdfs/ industry_plans/sandalwood_idp.pdf). It is anticipated that at the current rate of planting the tree farm estate is likely to reach 50,000 ha by the year 2020. No other country has embarked on a significant program of sandal establishment. The seemingly simple process of growing seedlings, planting them in the tree farm, and maintaining trees until harvest has been very difficult. Silviculture of sandal tree farms is complex as all species of sandal are root parasites (Radomiljac et al. 1998; Brand 2002; Woodall and Robinson 2002a, b). Both S. album and S. spicatum required a short-term pot host to be grown alongside and at the time of planting into the tree farm, a medium-term host must be well established nearby and a suitable long-term host must eventually be present. It is believed that the wood with finest odor is obtained from the driest region particularly on red or stony ground (Gunther 1952) and that yield of oil will be much higher than those grown in fertile tracts (Singh 1911; Gildemeister and Hoffman 1928; Bhatnagar 1965). It has been observed that trees extracted from open fields or edges of plantation yield more heartwood than those of comparative size extracted from adjoining forests (Wilson 1915; Mitchell 1941). The hosts also seem to influence the heartwood formation besides the growth and development (Rama Rao 1911). Srimathi and Kulkarni (1980) were of the view that heartwood formation is dependent on general factors of the individual tree and

7 Santalum

phenotypic factors play only a secondary role. Age of sandal tree and color of heartwood influences the quality and content of sandal oil (Shankaranarayana and Venkatesan 1982; Shankaranarayana and Parthasarathi 1984). Heartwood from young trees (around 10 years of age) contains 0.2–2% of oil and that from the mature trees (around 30–50 years of age) contains 2.8–5.6% of oil. Sandal oil content markedly decreases along the length of the tree (root: 3.5–6.3% to tip; stem: 3–5%; and branches: 1–3%). Generally, there is a decrease of about 45% in oil content from root to tip and about 20% from core to periphery (Shankaranarayana and Parthasarathi 1987). It would be beneficial if the plantation industry takes into consideration these observations for obtaining a better stand of sandal population.

7.8 Conclusion Natural stands of sandal are found in Australia, Hawaii, India, eastern Indonesia, New Caledonia, and Vanuatu. Phylogeographical analysis in sandal relied on interpreting the patterns of congruence or lack of congruence between the geographical distribution of chlorotypes and their genealogical relationships. Natural sandal populations are declining due to over-harvesting and illegal poaching of native stands, changes in land-use patterns, grazing, poor natural regeneration, and/or spike disease (Nageswara Rao et al. 2007a, 2008c). To safeguard the natural populations of sandal, effective measures need to be taken. Critical information on the distribution and status of sandal resources has been collected and used to develop a comprehensive distribution map. Genetic diversity of sandal has also been mapped. Such information would directly help the conservation efforts and would also serve as a bench mark to further monitor the changes in the species landscape over time (Nageswara Rao et al. 2008d). Efforts are underway for in situ and ex situ conservation of sandal. For commercial propagation of sandal, tree farms are being established. With the plantation sandalwood industry growing rapidly in Asia and Australia, there is a substantial need for well-characterized germplasm. Knowledge and utilization of the genes involved in essential oil production may advance efforts toward sandalwood tree improvement, further development of sustainable sandalwood plantations, and thereby conservation efforts to protect

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sandalwood trees in natural forests. The establishment of high efficiency tissue culture regeneration systems will facilitate the application of transgenic technology for the genetic improvement of sandal. To identify the genes of economic importance in sandal, a genetic mapping and sequencing project needs to be initiated.

References Ananthpadmanabha HS, Rangaswamy CR, Sharma CR, Nagaveni HC, Jain SH, Krishnappa HP (1984) Host requirement of sandal. Indian Forest 110:264–268 Angadi VG, Jain SH, Shankaranarayana KH (2003) Genetic diversity between sandal populations of different provenances in India. Sandalwood Res Newslett 17:4–5 Applegate GB, Chamberlain JC, Daruhi G, Feigelson JL, Hamilton L, Mc Kinnell FH, Neil PE, Rai SN, Rodehn B, Statham PC, Stemmermann L (1990) Sandalwood in the Pacific: a state-of-knowledge synthesis and summary. In: Hamilton L, Conrad CE (eds) Proceedings of the symposium on Sandalwood in the Pacific, vol 122. Honolulu, HI. USDA Forest Service Gen Tech Rep PSW, pp 1–11 Applegate GB, McKinnell FH (1993) The management and conservation status of Santalum species occurring in Australia. In: McKinnell FH (ed) Sandalwood in the Pacific region. ACIAR, Canberra, Australia, pp 5–11 Avise JC (2000) Phylogeography: The History of Formation and Species. Harvard Univ Press, Cambridge, MA, USA Bapat VA, Rao PS (1978) Vegetative propagation of sandalwood plants through tissue culture. Can J Bot 56:1153–1156 Bapat VA, Rao PS (1984) Regulatory factors for in vitro multiplication of sandalwood tree (Santalum album): 1. Shoot bud regeneration and somatic embryogenesis in hypocotyl cultures. Proc Indian Acad Sci Plant Sci 93(1):19–28 Bapat VA, Rao PS (1985) Regeneration of somatic embryos and plantlets from stem callus protoplasts of the sandalwood tree (Santalum album L.). Curr Sci 54:978–982 Bapat VA, Rao PS (1988) Sandalwood plantlets from synthetic seeds. Plant Cell Rep 7(6):434–436 Bhatnagar SP (1965) Studies in angiospermic parasites (No. 2) Santalum album – the sandalwood tree. National Botanical Gardens, Lucknow, India, Bull No 112 Bhattacharya A, Lakshmi Sita G (1998) cDNA cloning and characterization of a proline (or hydroxyproline)-rich protein from Santalum album L. Curr Sci 75:697–701 Bhattacharya A, Lakshmi Sita G (1999) Isolation and characterization of PR1 homolog from the genomic DNA of sandalwood (Santalum album L.). Curr Sci 77(7):959–963 Bottin L, Verhaegen D, Tassin J, Olivieri I, Vaillant A, Bouvet JM (2005) Genetic diversity and population structure of an insular tree, Santalum austrocaledonicum in New Caledonian archipelago. Mol Ecol 14:1979–1989 Brand JE (2002) Review of the influence of Acacia species on establishment of sandalwood (Santalum spicatum) in Western Australia. Conserv Sci W Aust 4:125–129

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143 (ed) Handbook of nature conservation: global, environmental and economic issues. Nova Publ, USA, pp 119–146 Nageswara Rao M, Uma Shaanker R, Ganeshaiah KN (2001b) Protected areas as refugias for genetic resources: are sandal genetic resources safe in our sanctuaries? In: Ganeshaiah KN, Uma Shaanker R, Bawa KS (eds) Tropical ecosystems: structure, diversity, and human welfare. Science Publ, Enfield, NH, USA, pp 121–124 Nageswara Rao M, Uma Shaanker R, Ganeshaiah KN (2002) Mapping the reek. Down to Earth 10:18 Padolina C (2007) An overview of forest genetic resource conservation and management in the Pacific. Acta Hortic 757:37–42 Radomiljac AM, Anathapadmanabha HS, Welbourn RM, Satyanarayana Rao K (1998) The effect of sandal wood availability on the craftsman community. In: Sandal and its products. ACIAR Proc (84), Publication-Australian Centre for International Agricultural Research, Canberra, Australia, p 204 Rai R (2005) Somatic embryogenesis in sandalwood. In: Jain SM, Gupta PK (eds) Protocol for somatic embryogenesis in woody plants. Springer, Dordrecht, Netherlands, pp 497–504 Rai SN (1990) Status and cultivation of sandalwood in India. In: Hamilton L, Conrad CE (eds) Proceedings of the symposium on sandalwood in the Pacific. Honolulu, Hawaii. USDA For Serv Gen Tech Rep PSW USA, pp 66–71 Rai SN, Sharma CR (1986) Relationship between height and diameter increment of sandal (Santalum album L.). Van Vigyan 24:105–138 Rai SN, Sharma CR (1990) Depleting sandalwood production and rising prices. Indian Forest 116:348–355 Rai VR (1996) Direct somatic embryogenesis from mature embryos of sandalwood. Sandalwood Res Newslett 5:4 Rai VR, McComb J (2002) Direct somatic embryogenesis from mature embryos of sandalwood. Plant Cell Tissue Organ Cult 69:65–70 Rama Rao M (1911) Host plants of the sandal tree. Indian Forest Rec 2(4):159–207 Rao PS, Bapat VA (1992) Micropropagation of Sandalwood (Santalum album L.). In: Bajaj YPS (ed) Biotechnology in agriculture and forestry, high-tech and micropropagation II, vol 18. Springer, Heidelberg, Germany, pp 193–210 Rao PS, Bapat VA (1995) Somatic embryogenesis in sandalwood Santalum album L. In: Jain S, Gupta PK, Newton RJ (eds) Somatic embryogenesis in woody plants. Klewer Academic Publ, Dordrecht, Netherlands, pp 153–170 Rao PS, Rangaswamy NS (1971) Morphogenic studies in tissue culture of parasite Santalum album L. Biol Plant 13:200–206 Rao PS, Srimati RA (1977) Vegetative propagation of Sandal (Santalum album L.). Curr Sci 46:276 Ravikanth G, Nageswara Rao M, Ganeshaiah KN, Uma Shaanker R (2009) Impacts of harvesting on genetic diversity of NTFP species: implications for conservation. In: Uma Shaanker R, Joseph GC, Hiremath AJ (eds) Management, utilization, and conservation of non-timber forest products in the south asia region. Universities Press, Bangalore, India, pp 53–63 Roxburgh W (1820) Santalum. In: Carey W (ed) Flora indica, vol I. Mission, Calcutta, India, pp 442–445 Rugkhla A (1997) Intra-specific and inter-specific hybridisation between Santalum spicatum and S. album. PhD thesis, Murdoch Univ, Perth, Western Australia

144 Rugkhla A, Jones MGK (1998) Somatic embryogenesis and plantlet formation in Santalum album and S. spicatum. J Exp Bot 49:563–571 Sanjaya BM, Anathapadmanabha HS, Rai VR (2003) In vitro and in vivo micrografting of Santalum album shoot tips. J Trop Forest Sci 15:234–236 Sanjaya MB, Rathore TS, Rai VR (2006) Micropropagation of an endangered Indian sandalwood (Santalum album L.). J Forest Res 11:203–209 Scott J (1871) Notes of horticulture in Bengal. No 2, Loranthaceae, the mistletoe order, their germination and mode of attachment. J R Hort Soc 2:287 Shankaranarayana KH, Parthasarathi K (1984) Compositional differences in sandal oils from young and mature trees and in oils undergoing color change on standing. Indian Perfum 28: 138–141 Shankaranarayana KH, Parthasarathi K (1987) On the content and composition of oil from heartwood at different levels in sandal. Indian Perfum 31:211–214 Shankaranarayana KH, Shivaramakrishnan VR, Ayyar KS, Sen PK (1979) Isolation of a compound from the bark of sandal and it’s activity against some lipidopterous and coleopterous insects. J Entomol Res 3:116–118 Shankaranarayana KH, Venkatesan KR (1982) Chemical aspects of sandalwood oil in cultivation and utilization of aromatic plants. Atakal CK, Kapoor RPL (CSIR), Jammu, India, pp 406–411 Shashidhara G, Hema MV, Koshy B, Farooqi AA (2003) Assessment of genetic diversity and identification of core collection in sandalwood germplasm using RAPDs. J Hort Sci Biotechnol 78:528–536 Shekhawat UKS, Ganapathi TR, Srinivas L, Bapat VA, Rathore TS (2008) Agrobacterium-mediated genetic transformation of embryogenic cell suspension cultures of Santalum album L. Plant Cell Tissue Organ Cult 92:261–271 Shetty RH (1977) Is sandal exotic? Indian Forest 811:804 Shiri V, Rao KS (1998) Introduction and expression of marker genes in sandal wood (Santalum album L.) following Agrobacterium-mediated transformation. Plant Sci 131:53–63 Sindhu Veerendra HC, Ananthapadmanabha HS (1996) The breeding system in sandal (Santalum album L.). Silvae Genet 45(4):188–190 Singh P (1911) Memorandum on the oil value of sandalwoods from Madras. Forest Bull No 6. FRI and Colleges, Dehra Dun, India Skottsberg C (1930a) The geographical distribution of the sandalwoods and its significance. Proc fourth Pacific Sci Congress, Java, Indonesia 3:435–440 Skottsberg C (1930b) Further notes on Pacific sandalwoods. Meddelanden fran Go¨teborgs Botaniska Tra¨dgard 5:135–145 Sprague TA, Summerhayes VS (1927) Santalum, Eucarya, and Mida. Bull Miscellaneous Information Royal Botanic Gardens, Kew 5:193–202 Srimathi RA, Kulkarni HD (1980) Preliminary findings on the heartwood formation in sandal (Santalum album L.). Report of the Sandal Research Centre, Bangalore, India, p 5 Srimathi RA, Venkateshan KR, Kulkarni HD (1995) Guidelines for selection and establishment of seed stands, seed production areas, plus trees and clonal seed orchards for sandal (Santalum album L.). In: Srimathi RA, Venkateshan KR,

M. Nageswara Rao et al. Kulkarni HD (eds) Recent advances in research and management of sandal (Santalum album L.) in India. Associated Press, New Delhi, India, pp 281–299 Srinivasan VV, Shivaramakrishnana VR, Rangaswamy CR, Anathapadmanabha HS, Shankaranarayan KH (1992) Sandal. Indian Council of Forestry Research and Education, Dehra Dun, India St. John H (1947) The history, present distribution, and abundance of sandalwood on O‘ahu, Hawaiian Islands: Hawaiian plant studies 14. Pacific Sci 1:5–20 Stemmermann RL (1977) Studies of the vegetative anatomy of the Hawaiian representative of Santalum (Santalaceae), and observations of the genus Santalum in Hawaii. Master’s Thesis, Univ of Hawaii, USA Stemmermann RL (1980) Observations on the genus Santalum (Santalaceae) in Hawai‘i. Pacific Sci 34:41–53 Suma TB, Balasundaran M (2003) Isozyme variation in five provenances of Santalum album in India. Aust J Bot 51: 243–249 Suriamihardja H, Suriamihardja S (1993) Sandalwood in Nusa Tenggara Timur. In: McKinnell FH (ed) Sandalwood in the Pacific Region. ACIAR, Honolulu, HI, USA, pp 39–43 Thirawat S (1955) Spike disease of sandal. Indian Forest 81:804 Thomson L, Bosimbi D (2000) Santalum macgregorii – PNG sandalwood. CSIRO/PNG Forest Research Institute/ACIAR project; Domestication of Papua New Guinea’s Indigenous Forest Species. Australian Tree Seed Centre, CSIRO Forestry and Forest Products, Yarralumla, ACT, Australia Thomson LAJ (2006) Santalum austrocaledonicum and S. yasi (sandalwood), ver. 2.1. In: Elevitch CR (ed) Specific profiles for Pacific Island agroforestry (www.traditionaltree.org). Permanent Agriculture Resources, Hawaii, USA, pp 1–21 Tuyama T (1939) On Santalum boninense, and the distribution of the species of Santalum. J Jap Bot 15:697–712 Uma Shaanker R, Ganeshaiah KN, Nageswara Rao M (2000) Conservation of sandal genetic resources in India: problems and prospects. In: International conference on science and technology for managing plant genetic diversity in the 21st Century, Kuala Lumpur, Malaysia, p 73 Vernes T (2001) Preliminary results from Santalum macgregorii ex situ conservation planting. Sandalwood Res Newslett 13: 6–7 Wagner WL, Herbst DR, Sohmer SH (1999) Santalum. In: The manual of the flowering plants of Hawaii, vol 2. Univ of Hawaii Press, Honolulu, Hawaii, USA, pp 1218–1222 Warburton CL, James EA, Fripp YJ, Trueman SJ, Wallace HM (2000) Clonality and sexual reproductive failure in remnant populations of Santalum lanceolatum (Santalaceae). Biol Conserv 96:45–54 Wilson CC (1915) Sandalwood (a) a parasite, (b) susceptibility to fire, (c) damage by borers, (d) spike disease. Science 188:1018–1021 Woodall GS, Robinson CJ (2002a) Direct seedling Acacias of different form and function as hosts for sandalwood (Santalum spicatum). Conserv Sci W Aust 4:130–134 Woodall GS, Robinson CJ (2002b) Same day plantation establishment of the root hemiparasite sandalwood [Santalum spicatum (R.Br.) A D.C.:Santalaceae] and its hosts. J Roy Soc W Aust 85:37–42

Chapter 8

Trigonobalanus Weibang Sun, Yuan Zhou, Chunyan Han, Gao Chen, and Yanling Zheng

8.1 Introduction The broadly circumscribed genus Trigonobalanus includes three species: T. verticillata from Sulawesi, Borneo, and the Malaya Peninsula, T. excelsa from the tropical forests of Colombia, South America, and T. doichangensis distributed in southern China and northern Thailand (Hsu et al. 1981; Nixon and Crepet 1989). Alternatively, on the basis of the unique pollen (peroblate shape and presence of endoaperature) of T. doichangensis, whorled phyllotaxy with interpetiolar stipules in T. verticillata and lack of bud scales in T. excelsa, Nixon and Crepet (1989) proposed dividing the genus into three monotypic genera: Formanodendron, Trigonobalanus, and Colombobalanus, respectively. At the time of their study, chromosome numbers (2n ¼ 42, 40, and 44) were known for T. verticillata (Hou 1971). Chen et al. (2007) determined that T. doichangensis has a chromosome number of 2n ¼ 14 and concluded that the base chromosome number of Trigonobalanus is x ¼ 7. Although recognizing the variability within the genus, Chinese experts prefer the broader systematic delineation of Trigonobalanus (Sun et al. 2004, 2006, 2007). Based on morphology and biogeography, Wu et al. (2003) proposed Fagaceae to constitute four subfamilies – Castaneoideae, Trigonobalanoideae, Fagoideae, and Quercoideae (Jones 1986; Takhtajan 1997; Li 1999). Trigonobalanus has been considered a key to an understanding of the phylogeny and biogeography of Fagaceae. The genus shares with both Fagus and

Quercus morphologies that are considered ancestral (Forman 1964; Crepet and Nixon 1989; Nixon and Crepet 1989) and has an extensive fossil record (Manos and Stanford 2001). Molecular studies have placed Trigonobalanus basal in a clade that also includes Quercus, Chrysolepis, and Castanea and have resolved this clade as sister to Fagus (Manos and Steele 1997). Biogeographical studies indicate that intercontinental variance of Trigonobalanus occurred earlier than in both Quercus and Fagus and involved dispersal across the North Atlantic Land Bridges (Manos and Steele 1997). Among the three extant species, T. doichangensis was widely recognized that it is the only one distributed in China (Hsu et al. 1981). However, recent investigations have confirmed that T. verticillata is distributed in Hainan Province (Ng and Lin 2008). Thus, China hosts two of the extant species of the genus Trigonobalanus. T. doichangensis was placed on the national Rare and Endangered Species List of China in 1984 because of its limited distribution and the destruction of its habitat within China (Fu 1992; Sun et al. 2004) and it is also China’s second-ranked taxon for priority of national protection (Anon 1999) because of its endangered status and its scientific value in providing evidence on the phylogeny and phytogeography of the Fagaceae and the biogeography of the Chinese flora. For conserving T. doichangensis, a scientifically and ecologically important plant species in Fagaceae, we have carried out comprehensive studies since 2000, and this chapter is mainly a summary of our work on T. doichangensis.

W. Sun (*) Kunming Botanic Garden, Kunming Institute of Botany, Chinese Academy of Sciences, Kunming 650204, Yunnan, China e-mail: [email protected]

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5_8, # Springer-Verlag Berlin Heidelberg 2011

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8.2 Geographical Distribution and Population Ecology of T. doichangensis T. doichangensis is a national rare and endangered plant of China (Fu 1992). It is restricted to four sites in southwest Yunnan, China and one site in ChiangRai, northern Thailand (Sun et al. 2006; Fig. 8.1). Our investigations revealed that four community types are currently extant in Yunnan: isolated individuals, sprouting woods, mono-dominant forest (MDF), and co-dominant forest (CDF) (Sun et al. 2004). The habitats have been severely damaged and the populations there are facing a high risk of extinction according to sun’s research (Sun et al. 2006). The adult phase of T. doichangensis is reached when a tree attains a height of about 4 m. The flowering and fruiting time varies slightly among populations and/or across the micro-habitats (Sun et al. 2004). In comparison with other Fagaceous plants in the community, T. doichangensis has an inverse flowering and fruiting period from October to May (Li 1994). At present, the vegetation destruction caused by agricultural land expansion and cuttings of the species for fuelwood are forcing endangering its habitats. The alien species Ageratina adenophra, Chromolaena odorata, and

Fig. 8.1 Geographical distribution of the five extant populations of T. doichangensis (after Sun et al. 2007). CR Chiang Rai population, ML Menglian population, LC Lancang population, XM Ximeng population, CY Cangyuan population

W. Sun et al.

Tithonia diversifolia have already invaded into communities in shrubbery layer, and they are inhibiting the species regeneration (Sun et al. 2006). Except this, according to the result of wild survey on the T. doichangensis community, Li (1994) indicated the floristic elements of the community are tropical and subtropical floristic region (90.25% of the total species). Sun et al. (2006) further indicated the population of T. doichangensis showed different characteristics in their tree-age structure and floristic composition. Four community types were recognized during their investigation (Table 8.1): (1) Type IsI (isolated individuals): Individuals of T. doichangensis were often found in secondary woods, by roadsides, in mixed woods or farmland, and occasionally within the evergreen broadleaf forests. The formation of Type IsI is due to heavy cutting and vegetation destruction. (2) Type SW (sprouting woods): Type SW is the result of fuelwood cutting by indigenous people. Investigations show that the indigenous ethnic groups of Dai, Wa, and Laku are familiar with T. doichangensis and have realized that the tree can sprout easily after top-cutting and thinning and thus they have adopted the methods of “alternate cutting or thinning cutting” for the primitive sustainable use of the tree as fuelwood. As a result of these practices, plants of T. doichangensis in these woods showed some unique characteristics in tree

8 Trigonobalanus

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Table 8.1 Population characteristics of T. doichangensis in different communities (cited from Sun et al. 2006) Communities SW MDF CDF Localities

Menglian

Lancang

Plots

1,020 N, 30 >90 800 195 24 5.1 13.5/0.2

1,450 W, 40 >90 400 140 35 5.6 9.5/0.3

59 15 26

73 21 6

45 ‚ƒ HAD

30 ‚ƒ PR

Altitude (m) Slope Coverage (%) Size (m2) TD no. in the plot TD no. per 100 m2 TD Avg. height (m) Hst/Shst of TD (m) % of TD  20 m % of TD  4–20 m % of TD 2–3 m % of TD  1.0 m % of dead TD Avg. height of DP (m) Accompanier no. Main companions (TP) Evaluation

Cangyuan BTS 1,550 SE, 40 >95 3,000 41 2 17.9 35/0.4 51 44 5

25 „… RSS

RBS 1,590 S, 30 >95 600 101 17 6.8 32/0.28 6 55 23 16 17 1.5–10 27 „… USD

MDS 1,730 S, 30 >95 400 118 30 14.4 20/0.2 0.8 92 5 3 15 3.5–18 25 „… MDSD

SW Sprouting wood, MDF Mono-dominant forest, CDF Co-dominant forest, HTS Huge-tree structure, RBS Relatively balance structure, MDS Mono-dominant structure, TD Trigonobalanus doichangensis, Hst Highest, Shst Shortest, Avg. Average, DP dead plants, TP top layer,  Vaccinium bracteatum, ‚ Castanopsis echinocarpa, ƒ Castanopsis hytrix, „ Castanopsis calathiformis, … Schima wallichii, HAD heavily disturbed by human activities, PR Population in recovery, RSS relatively stable structure, USD unstable structure in developing, MDSD mono-dominant structure in developing

shape, tree height structure, and associated floristic composition. In this community, the tallest T. doichangensis was about 13 m and the average height was around 5 m. Some 60% of T. doichangensis reached the reproductive phase and about 25.6% of the individuals were below 1 m in height. T. doichangensis was the dominant species in woods, and 50 species of accompanying higher plants were present. (3) Type MDF: Plants of T. doichangensis in type MDF are found as small mono-dominated patches scattered in the secondary evergreen broadleaf forest. The tallest plant of T. doichangensis was about 10 m and the average height was around 5–6 m. Approximately 70% of the trees in the plots were mature with a height of 4 m. Around 20% of the plants were 2–3 m in height, while seedlings and young trees represented only 6% of the total population. Accompanying higher plants were represented by some 30 species and most of the woody species were the same as in Type SW, but there were far fewer herbaceous plants and epiphytes. (4) Type CDF: Plants of T. doichangensis in Type CDF often formed a mosaic of mono-dominant patches in the primitive evergreen broadleaf forest. In this type of community, T. doichangensis grows

naturally without destructive disturbance from human activities. Based on the tree, height-grade Type CDF can be divided into three ranks. These are big tree structure (BTS), relatively balanced structure (RBS), and mono-dominant structure (MDS) of mature trees (Table 8.1).

8.3 Cytology and Karyotype 8.3.1 Base Chromosome Number of Genus Trigonobalanus Of the three species of Trigonobalanus, the base chromosome number of T. verticillata is x ¼ ca. 21 (2n ¼ 40, 42, and 44) (Hou 1971), and the five extant populations of T. doichangensis (Table 8.2) indicated that the base chromosome number of the species is x ¼ 7 (2n ¼ 14) (Chen et al. 2007; Sun et al. 2007). After carefully studying Hou’s original article and the photographs therein (his chromosome numbers are doubted due to unclear illustrations) and comparing

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all records and references of T. verticillata stored in the herbarium of the Royal Botanic Gardens, Kew, we propose that the mitotic number of 2n ¼ 42 for T. verticillata is probably the correct one. We may therefore conclude that T. verticillata is a hexaploid (2n ¼ 6x ¼ 42, x ¼ 7) derived from the ancestral T. doichagnensis. In fact, our recent cytological observations on T. verticillata populations from Fraser’s Hill in Malaysia (N 3 430 1500 , E 101 440 4000 , and altitude of 970 m) and Hainan (N 19 020 1900 , E 109 310 1500 , and altitude of 1,460 m) in China (Chen and Sun 2010) have already further confirmed the base chromosome number of x ¼ 7 for genus Trigonobalnus.

Table 8.2 Localities, geographical positions, altitudes, and voucher numbers of the five investigated T. doichangensis populations (cited from Chen et al. 2007) Population Locality Altitude Voucher (KUN) code (m) ML Menglian county, 1,100 SWB02T01-20 Yunnan, China LC Lancang county, 1,500 SWB02T21-40 Yunnan, China XM Ximeng county, 1,040 SWB02T41-60 Yunnan, China CY Cangyuan county, 1,730 SWB02T61-80 Yunnan, China 1,237 SWB02T081-100 CR Chiang Rai Province, Thailand

Fig. 8.2 Cytological features of the five T. doichangensis populations (cited from Chen et al. 2007). (a) Interphase nuclei of simple chromocenter type. (b) The prophase chromosomes of all populations in this study were of the proximal interstitial type. (c) Two B chromosomes were observed at prophase and

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8.3.2 Karyotype of T. doichangensis The interphase nuclei of the five T. doichangensis populations in China and Thailand (Table 8.2) had the same distribution pattern of chromatin, and according to Tanaka (1971, 1977), the pattern could be categorized as the simple chromocenter type (Fig. 8.2) (Chen et al. 2007). Heterochromatin and euchromatin segments were clearly observed at mitotic prophase in individuals of all the T. doichangensis populations (Chen et al. 2007). The heterochromatin segments were located in the proximal regions that were deeply stained, which indicated early condensation. While the euchromatin segments in the distal regions of chromosomes were lightly stained and extended, indicating late condensation (Fig. 8.2; Chen et al. 2007). Two B-chromosomes were commonly observed at prophase and prophase-metaphase, but rarely observed at metaphase (Fig. 8.2; Chen et al. 2007). The prophase chromosomes of all the populations belonged to the proximal interstitial type. The karyomorphological characteristics of the five populations (Fig. 8.2) are described as follows (Chen et al. 2007). The karyotype formula of five populations are 2n ¼ 14 ¼ 12m (2SAT) + 2sm + 2Bs (CR), 2n ¼ 14 ¼ 14m (2SAT) + 2Bs (LC), 2n ¼ 14 ¼ 12m (2SAT) + 2sm + 2Bs (XM), 2n ¼ 1 ¼ 14m (2SAT) + 2Bs (ML), and 2n ¼ 14 ¼ 12m (2SAT) + 2sm + 2Bs (CY) (Fig. 8.2). The ratio of the longest to the shortest

prophase-metaphase, but less at metaphase (arrows). (d–h) Mitotic metaphases. (d) CR population, 2n ¼ 14. (e) LC population, 2n ¼ 14. (f) XM population, 2n ¼ 14. (g) ML population, 2n ¼ 14. (h) CY population, 2n ¼ 14. Scale bars ¼ 10 mm

8 Trigonobalanus

chromosome was from 1.48 to 2.06 (Fig. 8.2). The karyotype asymmetries of five populations are classified as 1A (CR), 1A (LC), 2A (XM), 1A (ML), and 2B (CY), respectively (Fig. 8.2).

8.3.3 Implications of the Cytological Data Stebbins (1971) and Stace (2000) considered that almost all polyploids ultimately come from diploids. As all the five T. doichangensis populations are invariably diploid (2n ¼ 14) and T. verticllata is polyploid (2n ¼ 42), we suspect that T. doichangensis might represent the basal lineage of the genus Trigonobalanus, which may have originated from southeastern Asia (Zhou 1999; Manos and Stanford 2001). Trigonobalanus was probably broadly distributed (perhaps over the whole northern Hemisphere) during the Tertiary period or even earlier (Zhou 1992). However, extreme climate changes in the late Tertiary period brought on an extinction and habitat disappearance of a large number of species. Certainly, such global changes could cause species of Trigonobalanus to be diminished or even extinguished in their distribution ranges. In this way, T. doichangensis became a relict, currently restricted to some scattered populations in South Yunnan of China and North Thailand (Sun et al. 2006). As a relic species, the B-chromosome existed in T. doichangensis might be related to the strong climate changes in late Tertiary period (Zhou 1999). And the B-chromosome may contribute to the low production of the fertile nuts of the species (less that 10%) (Sun et al. 2006).

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8.4 Population Genetics of T. doichangensis 8.4.1 Genetic Diversity Five populations (ML, LC, XM, CY, and CR) were sampled that represent each of the known sites of occurrence. Within each population, 19 or 20 individuals at least 20 m apart were selected at random. Young, fully expanded leaves were harvested in January 2002 when the trees were in flower and fruit (T. doichangensis can have both flowers and fruits at the same time). Total DNA was extracted from 1–2 g of silica dried leaves using the modified CTAB method. The amplified fragments were scored for the presence (1) and absence (0) of homologous bands, the matrix of random amplified polymorphic DNA (RAPD) markers was inputted POPGENE software for further statistical analyses. UPGMA (unweighted pair group method arithmetic average) clustering was conducted according to Nei’s genetic distance using PAUP4.0b10 software. Eighty-three (52.8%) of the fragments were polymorphic across all populations (Table 8.2; Sun et al. 2007). For population XM, 34.9% of fragments were polymorphic, followed by CY, ML, and CR with 33.3, 32.8, and 20.7%, respectively (Table 8.2). Population LC had the fewest polymorphic (10.9%) and six private fragments. None of the other populations had private fragments but instead shared fragments with one or more other populations (Sun et al. 2007). Some fragments, although not unique to a population, were relatively rare and were shared between only two of the five populations (Table 8.3). Each population

Table 8.3 The percentage of polymorphic fragments (% Polym), estimated population diversity (HS), total genetic diversity (HT), number of invariant (In), polymorphic (Pl), and total fragments for each population of T. doichangensis (cited from Sun et al. 2007) Population code % Polym HT HS In Fragment (Pl) Total LC 10.9 0.033 131 16 (6.2) 147 CR 20.7 0.054 115 30 (2) 145 CY 33.3 0.104 100 50 (1) 150 ML 32.8 0.096 94 46 (1) 140 XM 34.9 0.108 97 52 (2) 149 Mean 52.8 0.160 0.079 107 39 146 The numbers of fragments that are private (bold) and rare (shared by two populations) are given in parentheses beside the number of polymorphic fragments

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possessed at least one of the eight rare fragments that were amplified (Sun et al. 2007). The estimated genetic diversity within populations (HS) also indicated that the LC population was the least diverse (HS ¼ 0.033) followed by CR (HS ¼ 0.054) (Table 8.2). The remaining three populations had similar HS values (0.108 for XM, 0.104 for CY, and 0.096 for ML). The estimated species genetic diversity of T. doichangensis was HT ¼ 0.160, the genetic diversity within populations was 0.079 (Table 8.4; Sun et al. 2007). It is apparent that genetic variation of among population was greater than that of within population.

8.4.2 Genetic Differentiation The estimated genetic differentiation among populations (FST) and that among regions (FSC) were 0.530 and 0.579, respectively (Table 8.3; Sun et al. 2007). And the genetic differentiation within populations (FCT) was 0.116 (Table 8.4). The results meant that the genetic variation among population accounted for 53.0% of the total variation and strong gene differentiation was present among populations (Sun et al. 2007). Pair-wise fixation indices indicated that LC was the most distinct population (Table 8.4). The lowest pair-wise (FST) values occurred when ML was compared with CY (0.288) or XM (0.338) (Table 8.4; Sun et al. 2007). The estimated pair-wise gene flow among populations ranged between NM ¼ 0.085 (LC and CR) and 0.618 (ML and CY)

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and averaged 0.276 across all pairs of populations (Table 8.5)

8.4.3 Genetic Distance and Cluster Analysis On the basis of individual pair-wise comparisons, the genetic identity among the five populations ranged from 0.792 to 0.952 (Table 8.6; Sun et al. 2007), with a mean of 0.888. The genetic pair-wise distances varied among populations from 0.049 to 0.234 (Table 8.5), with a mean of 0.121 (Sun et al. 2007). Gene flow (NM) was only 0.276 among populations, and gene flow among populations was very low. In agreement with the low value for the estimated gene flow between populations, all individuals from each population formed a single cluster (Fig. 8.3; Sun et al. 2007). Estimated distances between individuals within population LC (Yunnan, China) indicated that the trees sampled were quite similar in their RAPD fragment profiles (Fig. 8.3). The ML, CY, and XM populations (Yunnan, China) had relatively more divergence between individuals. The Thailand population (CR) had individuals that were intermediate in their overall similarity on the basis of RAPD fragments and genetic diversity (Table 8.3). In comparison with fagaceous species, the genetic variation of T. doichangensis was relatively lower, and over half genetic variation existed among populations (Hs ¼ 0.079). Approximately 53% of all fragments

Table 8.4 Analysis of molecular variation (AMOVA) among the two regions, northern Thailand and Yunnan Province, China, among populations and within populations for T. doichangensis (Sun et al. 2007) Source of variation Fixation value Among regions (FSC) 0.579 Among populations (FST) 0.530 0.116 Within populations (FCT)

Table 8.5 Fixation index (FST) and estimated number of migrants (NM) (below diagonal) among T. doichangensis populations (cited from Sun et al. 2007) LC CR CY ML XM LC – 0.747 0.698 0.710 0.730 CR 0.185 – 0.491 0.415 0.562 CY 0.108 0.259 – 0.288 0.354 ML 0.102 0.352 0.618 – 0.338 XM 0.092 0.195 0.456 0.491 –

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LC CR CY ML XM

LC – 0.146 0.190 0.188 0.234

CR 0.864 – 0.088 0.061 0.122

CY 0.827 0.916 – 0.049 0.070

ML 0.829 0.941 0.952 – 0.062

XM 0.792 0.886 0.932 0.940 –

XM

CY

ML

CR

LC

Table 8.6 Nei’s genetic identity (I) and genetic distance (D) (below diagonal) among T. doichangensis populations (cited from Sun et al. 2007)

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Fig. 8.3 UPGMA phenogram based on Nei’s genetic distance and 157 RAPD fragments from 99 T. doichangensis trees representing each of the five known extant populations (CY Can-

gyuan, China; XM Ximeng, China; LC Lancang, China; ML Menglian, China; CR Chiangrai, Thailand) (cited from Sun et al. 2007)

resolved in T. doichangensis were polymorphic. This level of polymorphism was low compared with T. verticillata where 79% of AFLP fragments

were polymorphic (Kamiya et al. 2002). Similar to the levels of polymorphic fragments, the estimated species genetic diversity of T. doichangensis

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(HT ¼ 0.160) was within the range of genetic diversity found in other Fagaceae species but lower than HT ¼ 0.198 for T. verticillata (Kamiya et al. 2002).

8.5 Reproductive Biology of T. doichangensis 8.5.1 Blooming and Fruit Habits, Microspore Genesis, and Development of Male Gametes T. doichangensis may flower when its height is about 4 m, and the starting date of blooming is slightly different among populations or population’s microclimatic habitants (Sun et al. 2004). In general, the plants in the higher altitude and further north latitude may have an earlier starting date of blooming. But the duration of blooming and fruiting is mostly from October until next May, and this phenological character is obviously different in comparison to that of other fagaceous species within the same populations. Observations on the microspore genesis and the development of male gametes in T. doichangensis revealed that the anther is 4-sporangiate and its anther wall formation conforms to the dicotyledonous type (Zeng and Sun 2004; Fig. 8.4). And also the tapetum is of the glandular type and its most cells are two-nucleate, and cytokinesis at meiosis of microspore mother cells is simultaneous and tetrads are tetrahedral, occasionally decussate (Zeng and Sun 2004; Fig. 8.6). Besides, mature pollen grains are two-celled. Therefore, it can be concluded that microspore genesis and development of male gametes in T. doichangensis are normal, and they are not the factors caused the lower rate of fructification (Zeng and Sun 2004; Figs. 8.5 and 8.6).

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Fig. 8.5 (a) An young anther in transverse section (T.S.) with four sporangia; (b) Secondary sporogenous cells; (c) Microspore mother cells when the middle layers begin to degenerate; (d) Microsporocyte at the meiosis and full-developed tapetum; (e) Microspore tetrads when tapetum begins to disintegrate; (f) Tapetum at the stage of uninucleate microspore; (g) Tapetum at the stage of vacuolated period of uninucleate microspore; (h) Anther wall of the time of the pollen spread; (i) Binucleate cells of tapetum; (j) Multinucleolate characters in tapetal cells. a 200; b, e 500; c, h 400; d, f, g, i, j 1,000 (cited from Zeng and Sun 2004)

c

Fig. 8.4 Inflorescences and infructescence of T. doichangensis; (a) Staminate inflorescences, (b) Pistillate inflorescences, and (c) Fructescence

8 Trigonobalanus Fig. 8.6 (a) Microsporocyte at the telophase I of meiosis; (b) Microsporocyte at the telophase II of meiosis; (c) Formation of microspore tetrads surrounded by callose; (d) Tetrahedral miscrospore tetrads; (e) Decussate microspore tetrads; (f) Young microspores just released from tetrads; (g) Microspore grows and becomes rounded, its wall begins to thicken; (h) vacuolated period of uninucleate microspore; (i) Uninucleate microspores; (j) Bicellular pollen; (k) The mature bicellular pollen grain. a–d, g–k  1,200; e, f  1,000 (cited from Zeng and Sun 2004)

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8.5.2 Ovule Abortion and Embryo Development According to our recent research, in T. doichangensis, the ovary has three locules with two ovules per locule. Observation on the ovary in transverse section showed that six ovules in each ovary were generated at an early stage, and at the developmental anaphase one of two ovules per chamber were well developed, while another one was aborted. Meanwhile, both axile and parietal placentas were observed, and thus T. doichangensis may show a transition trend from axile to parietal placentation. The ovule is anatropous and is enclosed by outer and inner integuments. The following three phenomena were also observed in the young fruits: (1) only one of the six ovules per ovary developed into an embryo and the others were aborted. The percentage of the one-ovule developed nuts was less than 10%; (2) in some fruits all the six ovules in the ovary were aborted and the ovary was lignified; and (3) the ovules in some fruits seem to be developed but the embryos

e

d

h

k

were membranous. It can be concluded that considerable embryo abortion occurs in T. doichangensis.

8.6 Propagation of T. doichangensis Producing a large number of plants through seeds could allow for conservation of the widest range of genetic diversity and would negatively affect the least its natural regeneration and population expansion. Vegetative propagation and in vitro techniques for rapid and mass propagation could offer possibilities to conserve germplasm and multiply desirable genotypes. For re-introduction planning and ex situ conservation, a holistic approach including conventional (vegetative propagation and seed germination) and in vitro techniques should be used for the propagation of T. doichangensis. Fruits (nuts) of T. doichangensis are both of the dispersal unit and germination unit. T. doichangensis

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a

b

Fig. 8.7 The anatomic feature of T. doichangensis nuts; (a) Nuts of seeds well developed (b) Nuts of seeds aborted

bears female flowers with one ovary. Each ovary has six ovules but only one has the potential to develop into a seed with an embryo. Some ovaries, however, have only aborted ovules and the fruits are either empty or lignified (Sun et al. 2006; Fig. 8.7) reported that the germination percentage of T. doichangensis was low and considerable seed abortion occurred in the species. It was observed that there was no apparent correlation between percentage of fruit fill (i.e., fruits containing seeds) and fruit morphological traits (Zheng and Sun 2008). Therefore, it is not possible to sort out fruits filled with seeds according to the fruit morphological characters. According to our observations of consecutive years, fully formed nuts collected at the same time differ obviously in color (dark and light), and dark-colored fruits have significantly higher rate of well-developed seeds (W. Sun, unpublished report). The variation in seed abortion between the two kinds of fruits may result from phenology. It could be concluded that sampling strategy must be taken into consideration when determining sample size of fruit collection for conservation or reforestation. Dark-colored fruits began to germinate after 3 days of incubation on moistened filter papers, with a peak after 5–6 days, followed by occasional germination thereafter. Percentage of fruit germination varied from 28.4  7.42 (2006) to 39.0  13.9 (2008) among years, from 0 to 59.6  3.42 among individuals (Zheng and Sun 2008). Germination percentage, germination index, and vigor index presented significant variation among populations and among individual trees within populations. The three germination-related indices had weak correlations with fruit morphological traits. Therefore, it could be concluded that the fruit size of T. doichangensis does not relate to its fruit germination capacity. Selecting fruits on the

basis of mass is not an appropriate way to enhance germination for reforestation projects. Moreover, collecting fruits from various individuals at each of the populations will be the preferred strategy to conserve the most genetic diversity of the species. So far, some 2,000 saplings propagated from seeds (botanically called nuts) (Fig. 8.8) have been conserved ex situ at the Kunming Botanic Garden, Chinese Academy of Sciences. Vegetative propagation is widely employed for multiplication of genetically superior genotypes. Although the factors affecting rooting capacity of stem cuttings have not been entirely elucidated (Dick and Leakey 2006), it has often been reported that plant age (Black 1972), pretreatment with auxin, substrates, leaf areas could affect rooting efficiency (Desrochers and Thomas 2003; Copes and Mandel 2000). According to our preliminary study, vegetative propagation of T. doichangensis with lignified cuttings is rather difficult. The leaves on cuttings will turn brown and shed within one month of culture and no rooting was occurred. Semi-lignified cuttings, with or without apical shoot, showed some rooting capacity (Fig. 8.9), however, the obtained rooting percentage was low. Further study should be conducted to maximize rooting success through selection of appropriate combination of pre-planting treatments. Micropropagation with shoots originated from 4-year-old seedlings failed. Brown substance exuded at the base of shoots and the shoots would die within 1 month. Cotyledonary nodes as one kind of explants have been used by some authors (Pradhan et al. 1998; Vengadesan et al. 2002; Jha et al. 2004). With explants of cotyledonary nodes originated from juvenile seedlings, 20–25 shoots/explants can be induced within 4 months (Fig. 8.10). And over 90% of the plantlets

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b

a

e

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f

d

Fig. 8.8 Seeding tests of T. doichangensis; (a) Seeds (nuts); (b), (c), and (d) Tests of the mixed-medium seeding; (e) Test of perlite seeding; and six cotyledons and the first two leaves

Rooting

Fig. 8.9 The rooted semi-lignified cuttings of T. doichangensis

Rooted cuttings

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Rooted shoots

Induced shoots

Fig. 8.10 Micropropagation of T. doichnagensis, with explants of cotyledonary nodes originated from juvenile seedlings

were successfully acclimatized and established in perlite media (Zheng et al. unpublished report).

8.7 Conservation Considerations and Population Re-enforcement Program

2.

8.7.1 Conservation Consideration (Cited from Sun et al. 2006) Protection and restoration of natural habitats is the best and cheapest method of preserving the biological diversity and stability of global ecosystem. Undoubtedly, the long-term survival of T. doichangensis is dependent on habitat conservation. However, only one of the four extant populations of T. doichangensis in China has been legally preserved by government ownership and others are facing a high risk of habitat disappearance. Perhaps more than most endangered plant species in China, the unique characteristics of T. doichangensis’ population isolation and the complex community interaction exemplify the importance of habitat preservation and in situ conservation. Nevertheless, both in situ and ex situ measures are needed for preserving T. doichangensis and its genetic diversity, and following aspects should be particularly considered. 1. Preservation of the habitat and population of T. doichangensis in Canyuan must be reinforced. The Canyuan population is the only one of the four populations in China, which has been well

3.

4.

5.

protected inside a national natural reserve. Therefore, the population is a vital resource for further research on the origin and evolution, eco-biological characteristics and genetic realities of T. doichangensis. Furthermore, the population also has a great value in population restoration research. As T. doichangensis populations in Menglian, Lancang, and Ximeng are still exposed to high habitat destruction and cutting for fuelwood, it is essential that new in situ conservation sites in these area should be urgently planned. These new sites will play an important role in habitat recovery and population restoration. Although cross-planting is often controversial in plant conservation planning, it is proposed that seedling cross-planting between different populations of T. doichangensis should be considered. At least, the potential impacts of this measure should be studied to determine if low gene flow would be enhanced and if overall diversity would be increased. As young T. doichangensis plants propagated from seeds can tolerate temperatures below 2 C at Kunming (Sun et al. 2004), it may be cultivated for fuelwood and as a landscaping plant in northern parts of the Tropic of Cancer. However, the mixed planting of trees propagated from various populations is essential. Ex situ efforts need to be undertaken to preserve genetic diversity and multiply specimens. This applies, in particular, to those populations outside of the well-protected national nature reserve. Ideally, ex situ sites will be close to natural reserves or to botanical gardens. Propagation from seeds is preferred, since it would be the least detrimental

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to the extant populations and would include the widest range of genetic diversity. Optimally, seeds should be taken from many individuals from each of the populations.

8.7.2 Population Re-enforcement T. doichangensis Based on the large number of saplings ex situ conserved at the Kunming Botanic Garden, the population re-enforcement action of T. doichangensis supported by the Botanic Gardens Conservation International (BGCI), together with the governmental program of “Returning Farmland to Forest”, has been launched since 2007. Some 247 selected saplings of T. doichangensis have been planted in Banli village of Lancang, where the species occurring. With observations on growing, ecological and biological characteristics, the saplings are growing well. It can be expected that re-enforced individuals can be developed as an ecologically functional population in its natural habitat (Fig. 8.11).

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8.8 Perspective of the Genus Trigonobalanus in Future Genus Trigonobalanus has long generated considerable interest because of its hemispheric disjunction and its morphological characters, some of which are unique in Fagaceae. Because of difficulty to attain the materials of T. verticillata and T. excelsa, most of our research concentrated on T. doichangensis. Following works still need to be done.

8.8.1 Conservation Biology Protection and restoration of natural habitats is the best and cheapest method of preserving the biological diversity and stability of global ecosystem (Lande 1988). Undoubtedly, the long-term survival of T. doichangensis is dependent on habitat conservation. However, only one of the four extant populations of T. doichangensis in China has been legally preserved by government ownership and others are facing a high risk of habitat disappearance. Both in situ and ex situ

a

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Fig. 8.11 Propagated T. doichnagensis were re-enforced in the natural habitat; (a) Saplings from seeds, (b) Re-enforcing site, (c and d) Planting with the local people

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measures are needed for preserving T. doichangensis and its genetic diversity, fortunately, our team is doing the work through cooperation with some international organization, such as Forest Frontiers Initiative (FFI) and BGCI.

8.8.2 Pollination Ecology T. doichangensis is an endangered species in China. The genus Trigonobalanus is a transitional phase in Fagaceae. Research on polymorphism in pollination syndrome (wind vs. generalist insect) also contributes to understand the reproductive trait evolution in the family.

8.8.3 Ecological Physiology The physiological function of leaf should be measured, the seasonal modification of wax deposition, and the impact of epicuticular wax on gas-exchange as well as photoinhibition in T. doichangensis, with wax-covered leaf surfaces and the stomata also partially occluded by wax, is an interesting study. Epicuticular waxes decrease cuticular water loss? The temperature of leaves without wax is lower than that of wax-covered leaves? The wax coverage at the entrance of stomata in T. doichangensis increase resistance to gas diffusion and as a consequence decrease stomatal conductance, transpiration, and photosynthesis? These question needs to be answered in the future research.

8.8.4 Recommendation of Other Immediate Measures The following aspects are needed to be considered immediately. 1. Systematic relationships within Trigonobalanus need to be clarified. Manos and Stanford (2001) indicated that T. verticillata was sister to T. doichangensis in Asia. But, Oh and Manos (2008) indicated that T. verticillata was sister to T. excelsa.

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More molecular data are needed to support the precise relationship among the three species. 2. Chromosome numbers of T. excelsa need to be counted since of the three species of Trigonobalanus, the chromosome numbers are known for two, T. verticillata and T. doichangensis. The basic chromosome number of these taxa is x ¼ 7, which is unique in Fagaceae, even in Fagales. 3. Systematic position of Trigonobalanus in Fagaceae should be studied. The transitional characteristics of Trigonobalanus have caused a series of taxonomic problems in Fagaceae. It has been divided into different taxa in different treatments. Based on morphological observations, Jones (1986) suggested that there are four subfamilies in Fagaceae. Nixon and Crepet (1989) divided Fagaceae into two subfamilies. However, combined analysis of all sequences yielded parsimony trees identifying three basic lineages in Fagaceae: Fagus, Trigonobalanus, and the remaining genera (Li et al. 2004; Chen et al. 2008; Oh and Manos 2008). 4. Historical biogeography of Trigonobalanus should be revisited. The evergreen genus Trigonobalanus shows a disjunct continental distribution, which provides a single, yet important data point in evaluating the biogeographic history of Fagaceae. Trigonobalanus are subtropical members of the Fagaceae, and both its modern and extensive fossil distributions suggest an old biogeographical history (Manos and Stanford 2001). A 2.21% sequence divergence between the Asian species and the New World relict Trigonobalanus excelsa led Manos and Stanford (2001) to estimate a divergence time of ca. 37 million years (Manos and Stanford 2001). The combination of fossil and modern distributions with molecular dating suggests that the continuous distribution of Trigonobalanus was most likely achieved via the North Atlantic Land Bridges (NALB) during the Paleocene before the complete formation of the Turgai Strait (Manos and Stanford 2001; Tiffney and Manchester 2001). Using the cpDNA molecular clock, Kamiya et al. (2002) provided a clue about the migration routes and divergence time of T. verticillata, the divergence of the Fraser’s Hill population from the others was estimated to be 16.7 Mya. The 95% upper confidence limit indicates that T. verticillata reached the Malay peninsula not earlier than the Oligocene.

8 Trigonobalanus

Paleobotanical evidence has shown that the Oligocene and early Miocene were periods of relatively dry and cool climates in Southeast Asia (Morley 1998; Whitmore 1998). The cooler climates could have allowed the montane species to expand to lower altitudes and migrate to lower latitudes. This suggests that T. verticillata could have expanded to the south and subsequently migrated as far as Borneo during this period. Early Miocene paleogeographical reconstruction in the Sunda–Sahul region suggests that one possible migration route would have been the corridor across the Malay peninsula and Borneo (Morley and Flenley 1987). Morley (1998) also suggested that warm and moist climatic conditions prevailed during the initial parts of the middle Miocene (ca. 15 Mya) throughout large part of East and Southeast Asia. Thus, a large area of the regions was covered by tropical lowland evergreen forest species. Species of Trigonobalanus, which prefer a higher altitude in the montane zone, would have become isolated. A new distribution record for T. verticillata from Hainan Island, South China greatly expands the distribution range of this species (Ng and Lin 2008) from 7 N to 19 N. Stebbins (1971) and Stace (2000) considered that almost all polyploids ultimately come from diploids. As the population of T. verticillata in Hainan Island, China, is diploid, we suspect that T. verticillata might represent the basal lineage of the genus Trigonobalanus, which may have originated in southeastern Asia. From there, it possibly spread southwards to Celebes, Borneo, and Malaya, generating the hexaploid T. verticillata populations. To compare the genetic divergence between the Malay population and the Chinese population and to measure the origin and disjunction time of this ancient relict species, further field observations and molecular clock data are certainly required.

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Index

5S rDNA, 4, 110 5S rRNA, 22 16S rRNA, 9 454-Technology, 116 A Abiotic stress, 72 Acorn, 94, 95, 110 chemistry, 118 properties, 98–99 Actinorhizal, 11 Adaptation, 34 Afforestation, 49 Affymetrix, 116 AFLPs. See Amplified fragment length polymorphism Agriculture Research Service (ARS), 21 Agrobacterium, 139 Agroforestry, 79 Allergens, 53 Allergic reaction, 57 Allogamous, 59 Allopolyploid, 4 Allozyme, 53 Alnus, 1–11 A. acuminata, 3 A. cordata, 4 A. firma, 4 A. glutinosa, 4 A. incana ssp. rugosa, 3, 4 A. japonica, 4 A. maritima, 2 A. nitida, 2 A. orientalis, 4 A. pendula, 4 A. rhombifolia, 3 A. rubra, 3 A. serrulata, 3, 4 A. serrulatoides, 4 A. subcordata, 4 A. viridis, 2 American hazelnut, 17 Amplified fragment length polymorphism (AFLP), 23, 57, 109 Andean Alder, 2 Angiosperm, 6 Angophora, 66 Antioxidant, 42

Antipyretic, 140 Antisense, 84 Antiseptic, 70 Antitumorogenic, 140 Antiviral, 140 Arabidopsis, 113 Arboretum, 21, 52 Aromatic heartwood, 131 ARS. See Agriculture Research Service Artificial forests, 53 Association genetics, 71 Astringent, 140 B BAC. See Bacterial artificial chromosome Backcross, 83 hybrids, 38 Bacterial artificial chromosome (BAC) library, 23 Bay laurel, 103 B-chromosome, 148 Beaked hazel, 18 Betula, 2 Bifurcating tree, 92 Biodiversity, 119 Biogeographical studies, 145 Biogeography, 96, 145 Biomass, 5, 42, 72 gain, 100 industry, 8 plantation, 8 production, 7–8 Biosynthesis, 71 Biotechnology, 84–85 Black Alder, 2 Black oak, 91 Black walnut, 77 Blooming, 152 Bridge species, 83 Butternut, 80–81 C California black oak, 103 Candidate, 2, 39 gene, 56, 115–116 tree, 53

C. Kole (ed.), Wild Crop Relatives: Genomic and Breeding Resources, Forest Trees, DOI 10.1007/978-3-642-21250-5, # Springer-Verlag Berlin Heidelberg 2011

161

162 CAPS. See Cleaved amplified polymorphic sequence Carpinus, 7 Castanea, 20, 90 CDF. See Co-dominant forest Cerris, 91 Charcoal, 67 Chemosterilant, 140 Chemotaxonomy, 70–71 Chemotype, 70 Chestnuts, 90 Chinese tree hazel, 19 Chlorophyll deficiency, 22 Chloroplast genome, 66, 135 Chloroplast DNA (cpDNA), 52, 72, 140 array, 116 library, 56, 84 microarrays, 117 Chromosomal stability, 112 Cleaved amplified polymorphic sequence (CAPS), 54 Clonal, 57, 82 propagation, 21 reproduction, 138 structure, 109 Clonality, 138 Coast live oak, 103 Co-dominant forest (CDF), 146 Colchicine, 84 Cold hardiness, 38 Cold-hardy, 28, 36, 83 Common walnut, 77 Comparative mapping, 115 Consensus linkage map, 57 Conservation, 20–21 biology, 101–104 efforts, 82 initiatives, 82, 101–102 Cork biosynthesis, 115–116 colurna, 37 Corylus, 7, 15–44 C. americana, 16, 17 C. avellana, 15 C. californica, 16, 36 C. chinensis, 16, 19, 39 C. colurna, 19, 37 C. colurna L., 16 C. cornuta, 18 C. cornuta even, 36 C. fargesii, 40 C. ferox, 16, 40 C. ferox var. tibetica, 40 C. heterophylla, 16–18 C. jacqemontii, 16 C. jacquemontii, 39 C. kweichowensis, 17 C. mandshurica, 18 C. papyraceae, 16 C. sieboldiana, 16, 18, 37 seed and plants, 36 Corymbia, 66, 71 Cosmetics, 140 cpDNA. See Chloroplast DNA

Index Crop vulnerability, 82 Cross-planting, 156 Cryopreservation, 22 Cryptomeria, 49–60 C. japonica, 49 C. japonica var. radicans, 49–50 C. japonica var. sinensis, 51 Cultivar, 105 Cyanogenic glycosides, 71 Cycle cup oak, 91 Cytogenetic stocks, 71 D Deciduous tree, 77 Deforestation, 20 Deformity, 53 Distorted segregation, 56–57 Diuretic, 140 Domestication, 72, 106 Dormancy, 99 Doubled haploid, 106 Drought, 37 tolerance, 8 Dwarfism, 49 E Eastern filbert blight (EFB), 22 resistance, 32, 42 Ecological, 49, 89 distribution, 97–98 physiology, 158 Ecology, 3–4 Ecosystem development, 4 ECP/GR. See European Cooperative Program on Plant Genetic Resources Ectomycorrhizal (ECM) fungi, 101 EEC. See European Economic Community Embryo development, 153 Endangered, 16, 69 plant, 146 species, 68 Essential oil, 140 ESTs. See Expressed sequence tags Eucalypts, 65 Eucalyptus, 65–73 E. benthamii, 70 E. consideniana, 68 E. drummondii, 68 E. grandis, 68, 72 E. graniticola, 68 E. rudis, 68 Eucalyptus Genome Network (EUCAGEN), 72 Euchromatin, 148 EUFORGEN. See European Forest Genetics Resources Programme European Cooperative Program on Plant Genetic Resources (ECP/GR), 78–79 European Economic Community (EEC), 78–79 European Forest Genetics Resources Programme (EUFORGEN), 78 European hazelnut, 15

Index Evergreen, 94 EVOLTREE, 102 Expressed sequence tag (EST), 56, 72, 84, 90 library, 116 sequence, 116 Ex situ collection, 78 Ex situ conservation, 60, 70, 137, 153 Ex situ propagation, 70 External transcribed spacer (ETS), 133 F Fagus, 145 Fatty acid, 41 Female map, 111 FFPRI. See Forestry and Forest Products Research Institute Firewood, 77 First-generation hybrid, 95 FISH. See Fluorescence in situ hybridization Fixation index, 5 Flavonoid, 84 Flavor, 140 Floristic composition, 146 Flowering phenology, 55 Fluorescence in situ hybridization (FISH), 111 Forest ecosystem, 102 Forestry and Forest Products Research Institute (FFPRI), 52 Forest tree genetic resources conservation forest (FTGRCF), 52 Forest Tree Superior Gene Conservation Stand (FTSGCS), 52 Fossil evidence, 77 Frankia, 1 strain, 10 Fructification, 152 FTGRCF. See Forest tree genetic resources conservation forest FTSGCS. See Forest Tree Superior Gene Conservation Stand Full-sib progenies, 83 Functional genomics, 115–117 G GenBank, 22, 84 Gene, 5, 9, 10, 22 expression, 115 flow, 5, 137 pool, 90, 91 Generic diversity, 97 Genetic, 1, 5–11 conservation, 52 differentiation, 6, 54, 59, 99, 150 distance, 150 diversity, 5, 21, 59, 65, 137–138 erosion, 68 identity, 21, 150 overlap, 92 stock, 8–9, 71 structure, 5, 59, 71 transformation, 72 Genome, 16, 23, 52 sequence, 66 size, 49, 66, 113 structure, 113

163 Genome-wide association, 59 Genomic, 56, 59 library, 140 selection, 59 Genotyping, 57 Geographical information systems (GIS), 8 Geographic structure, 70 Germplasm, 78 diversity, 104 exchange, 27 preservation, 21 Gigantism, 49 GIS. See Geographical information systems Glacial refugia, 104 Global distribution, 97 ecosystem, 156 warming, 8 Golden cup, 91 Grafting, 21, 84 Growth performance, 8 H Habitat, 3, 17, 36, 70, 89, 97 disappearance, 149 recovery, 156 specialization, 98 Hardwood, 105 Hardwood Tree Improvement and Regeneration Center (HTIRC), 107 Hazelnut, 15 kernel, 41 oil, 41 Heartwood, 140 Herbivores, 97 Herbivory, 100 Heritabilities, 108, 138 Heterochromatin, 148 Heterosis breeding, 71–72 Heterozygosity, 5 High-throughput sequencing, 84 Himalayan tree hazel, 20 HJG. See Hydrojuglone glucosides HTIRC. See Hardwood Tree Improvement and Regeneration Center Hybrid hazelnut, 24 Hybridization, 4–5, 92 Hybrid vigor, 85 Hydrojuglone glucosides (HJG), 84 Hypotensive, 140 I IBPGR. See International Board of Plant Genetic Resources ICNCP. See International Code of Nomenclature for Cultivated Plants IEA. See International Energy Agency Inbreeding, 5 depression, 56 Indian tree hazel, 19 In situ conservation, 52, 68–70 Institut de Recerca I Technologia Agroalimenta`ries (IRTA), 20

164 Institut National de la Recherche Agronomique (INRA), 79, 114 Intellectual property rights (IPR), 73 Intermediate oak, 91 Internal transcribed spacer (ITS), 7, 22, 81, 90, 133 International Board of Plant Genetic Resources (IBPGR), 78 International Code of Nomenclature for Cultivated Plants (ICNCP), 105 International Energy Agency (IEA), 7 Intersectional hybridization, 83 Inter-simple sequence repeat (ISSR), 6, 110 Interspecific, 4, 15, 22–24 breeding, 24 cross, 31 hybridization, 15 hybrid, 4, 24, 35, 71–72 Intraspecific comparisons, 95 Introgression, 72, 83 Invasive species, 68 In vitro preservation, 21 IPR. See Intellectual property rights IRTA. See Institut de Recerca I Technologia Agroalimenta`ries ISSR. See Inter-simple sequence repeat Italian Alder, 2 ITS. See Internal transcribed spacer J Juglans, 77–85 J. ailantifolia, 83 J. cinerea, 83 J.  intermedia, 79 J. nigra, 81 J. regia, 77 K Karyotype, 66, 81, 113 L Landscape plants, 42 LD. See Linkage disequilibrium Lignin content, 71 Lineage sorting, 92 Linkage, 49, 84 disequilibrium (LD), 59 group, 57 map, 56 mapping, 110–112 Lipid, 99 Live oak, 91 Local conservation, 73 Lumber, 77 M Macroarrays, 115 Male gametes, 152 Male map, 111 Male-sterile, 53 Male strobili, 56 Managed investment scheme (MIS) companies, 140 Mapping, 57, 71, 90, 107 design, 111 population, 111

Index Marker-assisted breeding, 108–112 Marker-assisted selection (MAS), 23 Mass propagation, 153 Mast seeding, 99 Maximum likelihood (ML), 7 Maximum parsimony (MP), 7 MDF. See Mono-dominant forest Melaleuca alternifolia, 70 Metabolome, 117 Metabolomics, 117 Microfibral angle, 71 Micropropagation, 21, 84, 139, 154 Microsatellites, 55, 71, 109 Microsatellite sequence repeat (SSR), 22 Microspore genesis, 152 ML. See Maximum likelihood Model forest tree, 68 plant, 68 Molecular, 7, 15, 16, 66, 68 breeding, 57 genetic map, 71 phylogeny, 3 Mono-dominant forest (MDF), 146 Mono-dominant structure (MDS), 147 Monophyletic, 92 origin, 51 Morphotaxonomy, 70 MP. See Maximum parsimony Mutant, 53, 105–106 Mutation, 104–106 breeding, 84 Mycorrhizal associates, 101 N NALB. See North Atlantic Land Bridges Naphthoquinone metabolism, 84 National Center for Biotechnology Information (NCBI), 84, 111 National Clonal Germplasm Repository (NCGR), 20, 78 Neighbor joining (NJ), 7 Nitrogen fixation, 9 North Atlantic Land Bridges (NALB), 158 Nucleotide diversity, 59 O Oak, 89 gall, 100 gallwasps, 100 microarray, 116 wilt, 103 Omote-sugi, 49–50 Open-pollination, 28 Ord River Irrigation Area (ORIA), 140 Ornamental, 30, 34, 37 hybrids, 72 traits, 42 Osmotic stress, 114 Ostrya, 7 Ostryopsis, 7 Outcrossing, 5, 65

Index Ovule abortion, 153 Oxidative stress, 115 P Paleobotanical evidence, 159 Paper products, 118 Paperbark tree hazel, 19 Perfumes, 140 Persian walnut, 77 Phenolics, 100 Phenotypic, 57, 78, 107, 111 plasticity, 92 variation, 96 Photoinhibition, 158 Phylogenetic, 7, 52, 81 analyses, 133 overdispersion, 98 relationships, 92 tree, 7 Phylogeny, 6–7, 9, 145 Phylogeographic signal, 96 Phylogeography, 80–81, 104, 132–135 Physical map, 52 Phytochemicals, 42 Phytogeography, 145 Phytophthora ramorum, 103 P. ramorum, 103 PICME. See Platform for Integrated Clone Management Plantation systems, 7 Platform for Integrated Clone Management (PICME), 116 Pollen, 23, 28, 31–39 allergy, 57 fecundity, 57 flow, 68 Pollination ecology, 158 Pollinosis, 57 Polymerase chain reaction (PCR), 139 Polyploid, 49, 105–106 POPGENE, 149 Population genetics, 149 Populus, 89, 113 Progeny testing, 59 Proline-rich protein (PRP), 140 Propagation, 84, 153 Proteome, 115–117 Provenances, 137 studies, 105 trial, 80 Pseudo-testcross, 111 Pulp, 67, 72 Q Quantitative trait loci (QTL), 57, 71, 89, 112 Quercus, 89–119, 145 Q. affinis, 97 Q. laurina, 97 Q. petraea, 89 Q. robur, 89 Q. stellata, 92 Q. velutina, 92

165 R Random amplified polymorphic DNA (RAPD), 23, 54, 57, 78, 94, 109, 137, 149 Red alder, 2, 8 Red oak, 91 Reforestation, 2, 55, 154 Regeneration, 102 Repetitive DNA, 113 Reproductive biology, 152 Restriction fragment length polymorphism (RFLP), 49, 104, 137 RNA interference (RNAi), 57 Rootstock, 21, 83 S S-allele, 23 Sandal, 131 Sandalwood, 131 Santalum, 131–141 S. acuminatum, 131 S. album, 131 S. austrocaledonicum, 131 S. boninense, 132 S. ellipticum, 131 S. freycinetianum, 131 S. haleakalae, 131 S. insulare, 132 S. lanceolatum, 131 S. leptocladum, 132 S. macgregorii, 132 S. murrayanum, 132 S. obtusifolium, 132 S. paniculatum, 131 S. spicatum, 131 Scents, 140 Secondary metabolites, 99 Seed abortion, 154 Selection, 54 Self-compatibility, 22 Self-fertilization, 55–56 Self-pollination, 10 Semi-domesticated, 77 Sequence tagged site (STS), 52, 115 Sexual incompatibility, 138 Shade tolerance, 55 Shreve’s oak, 103 Siberian hazel, 17, 34 Silviculture, 107, 140 Simple sequence repeat (SSR), 22, 54, 72 Single-nucleotide polymorphism (SNP), 59, 71 Single-stranded DNA conformation polymorphism (SSCP), 56 SMP. See Supplemental mass pollination Snow, 55 fall, 59 pressure, 59 SNP. See Single-nucleotide polymorphism Soil reclamation, 36 Somatic, 53, 84, 115 embryogenesis, 112, 139 embryo, 112, 139 Spiny chestnut, 20

166

Index

Spring flush, 99–100 SSCP. See Single-stranded DNA conformation polymorphism SSR. See Simple sequence repeat Staminate, 33 Structural, 90, 110 genomic resource, 114 genomics, 114–115 STS. See Sequence tagged site Subtractive hybridization, 115 Sudden oak death, 103–104 Sugi, 49 Supplemental mass pollination (SMP), 56 Symbiosis, 3

T. excelsa, 145 T. verticillata, 145 Triploid, 49 Turkish tree hazel, 18

T Tannin, 99, 100, 117 Tanoak tree, 103 T-DNA, 139 Terpene, 140 Terpene synthase (TPS), 140 Terpenoid, 140 Tetraploid, 49, 84 Tetraploidy, 106 Threatened, 20, 82 population, 70 taxon, 137 Tibetan hazel, 20 Timber, 57 Toiletries, 140 Transcriptome, 115–116 Transcript, 115–116 Transformation, 112 Transgenic eucalypts, 72–73 Tree-age structure, 146 Tree nut, 15 Tribal use, 68 Trigonobalanus, 145–159 T. doichangensis, 145

V Vegetation, 73 destruction, 146 Vegetative propagation, 73, 154 Vesicular arbuscular mycorrhizal (VAM) fungi, 101

U UNESCO, 52 United States Department of Agriculture (USDA), 20, 30, 82, 92, 107 Unweighted pair group method arithmetic average (UPGMA), 149 Ura-sugi, 49–50 USDA. See United States Department of Agriculture

W Weediness, 85 Weeds, 68 Whiskey, 119 White oak, 89 Wild survey, 146 Wine, 119 Winter injury, 34 Wood, 1–6 chemistry, 117 density, 57, 108 products, 118 quality, 57 strength, 57 X Xylem, 103, 116, 117 anatomy, 99