Life of Marsupials

LIFE OF MARSUPIALS HUGH TYNDALE-BISCOE © Hugh Tyndale-Biscoe 2005 All rights reserved. Except under the conditions d...

Author: Hugh Tyndale-Biscoe

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LIFE OF MARSUPIALS

HUGH TYNDALE-BISCOE

© Hugh Tyndale-Biscoe 2005 All rights reserved. Except under the conditions described in the Australian Copyright Act 1968 and subsequent amendments, no part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, duplicating or otherwise, without the prior permission of the copyright owner. Contact CSIRO PUBLISHING for all permission requests. National Library of Australia Cataloguing-in-Publication entry Tyndale-Biscoe, Hugh. Life of marsupials. [New ed.]. Bibliography. Includes index. ISBN 0 643 06257 2 (Hardback). ISBN 0 643 09199 8 (Paperback). ISBN 0 643 09220 X (netLibrary eBook). 1. Marsupials. I. CSIRO Publishing. II. Title. 599.2 Available from CSIRO PUBLISHING

PO Box 1139 (150 Oxford St) Collingwood VIC 3066 Australia Telephone: +61 3 9662 7666 Local call: 1300 788 000 (Australia only) Fax: +61 3 9662 7555 Email: [email protected] Web site: www.publish.csiro.au Front cover (clockwise from top left): Gray four-eyed opposum (Hugh Tyndale-Biscoe); Stages in tammar wallaby development: the unattached vesicle (Ivan Fox) and a newborn tammar (LA Hinds); Chromosome painting (JAM Graves); Feathertail glider (Ederic Slater); Julia Creek dunnart with 60-day-old litter (PA Woolley and D Walsh). Spine: Yellow-footed rock wallaby (Esther Beaton). Back cover: Male honey possum on Banksia inflorescence (PA Woolley and D Walsh).

Set in Minion 10/12 Cover and text design by James Kelly Typeset by J&M Typesetting Printed in Australia by Ligare

Contents Preface

v

1

What is a marsupial?

1

2

Reproduction and development

3

Opossums of the Americas: cousins from a distant time

103

4

Predatory marsupials of Australasia: bright-eyed killers of the night

139

5

Bandicoots: fast-living opportunists

165

6

Pygmy possums and sugar gliders: pollen eaters and sap suckers

183

7

Life in the trees: koala, greater glider and possum

219

8

Wombats: vegetarians of the underworld

267

9

Consummate kangaroos

287

Marsupials and people: past and present

365

References

385

Index

421

10

37

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Preface When the first edition of this book was written in 1970, the old debate about the inferior status of marsupials, compared to other mammals, was still active. The work reviewed then on a few species of marsupial in Australia and North America was beginning to dispel this idea but it still prevailed in other countries, particularly in the Northern Hemisphere. Thirty-five years later it is no longer an issue of importance. Now, much more is known about the past history and the present relationships of marsupials in Australia, New Guinea and South and Central America, so that the long evolution of this separate line of mammals is becoming much clearer. As well as this, there are now detailed studies on the physiology, reproduction, ecology and behaviour of representatives of all the main families of marsupials, so that comparisons and generalisations can be made with much more confidence. In the first edition of this book it was also still possible to cover the whole literature on marsupials. That is no longer possible and a small book now must be selective in its coverage and its acknowledgement of sources. However, for most topics and for most groups of marsupials there are now excellent monographs or reviews that enable the interested reader to follow any topic further. Several themes about marsupials have developed among the people who have studied them during the past 35 years and these resonate through all the work that is described here. The oldest of these themes is the remarkable convergence of adaptations seen in Australasian marsupials and mammals on other continents. When first seen by European explorers these similarities were thought to indicate close relationship but deeper understanding soon showed that these were two independent evolutionary lines responding to similar external imperatives. As well as these large convergences we can now recognise convergences between marsupials from Australasia and those from South America. Within the Australasian marsupials there are also convergences to similar food sources, such as the leaf eating koala, possums and ringtails, or the nectar-eating marsupials from four separate families. However, the most interesting outcome of the new work on marsupials has been a much greater appreciation of how marsupials have adapted to the special conditions of the Australian environment, its unpredictable climate, low fertility soils and unpalatable plants. It is an important and interesting aspect of the adaptive radiation of marsupials in Australia and raises the question how marsupials came to prevail in Australia but shared South America with other kinds of mammals: it also tells us how we must adapt to the land if we wish to live here in the long term, and what we must do to let these long time residents continue to live here also. Because it is not possible for one person to command a knowledge of so many fields as this book covers, I have depended on the expert advice of colleagues in several fields: while taking full responsibility for what is written, I am deeply grateful for the generous help of Ken Aplin, Bill Foley, Jennifer Graves, Brian Green, Stephen Ho, Peter Janssens, John Kirsch, the late Richard Mark, Lauren Marotte, Jim Merchant, David Ride, Phil Waite, Mike Westerman and Patricia Woolley. For each reading several chapters as a non-expert and thereby helping me to express things more clearly than I otherwise would have, I sincerely thank Meredith McKinney and Nicola Tyndale-Biscoe. I am also very pleased to acknowledge CSIRO: this great organisation has supported research on Australasian marsupials since 1950 and my own research for more than 40 years, so that much that is discussed in this book stems directly from that support. Then, when I began this book Brian Walker, Chief of CSIRO Wildlife and Ecology, offered me generous and stimulating hospitality to prepare it, and his successors in CSIRO Sustainable Ecosystems have graciously continued to do so, to its completion. At CSIRO Sustainable Ecosystems I have been wonderfully

well supported by many people and I especially thank Alice Kenney for preparing the figures; Margaret Hindley, Trish Kelly, Megan Edwards and Inge Newman for tracking down difficult or unusual references with speed and efficiency; and Andrew Bishop, Brian Davis and Yechiam Marks for leading me courteously through the complexities of information technology. At CSIRO Publishing I thank Paul Reekie for great patience as deadlines passed and Nick Alexander and Briana Elwood for producing the finished work with diligence and despatch. I also thank Alexa Cloud for superb copy editing. Finally, I thank Marina, who read and commented on every chapter in draft and then read the proofs, and has sustained me throughout the whole saga as one year passed into another and the end remained a mirage too far away: thank you for everything. Hugh Tyndale-Biscoe January 2005

Chapter 1


South American opossum; steel engraving from Buffon (1749).


T

his is the story of a group of mammals that were isolated from the rest of the world for many millions of years. It is set on the great southern continent of Gondwana that stretched from the Caribbean to the islands of New Guinea and included the three present-day continents of South America, Antarctica and Australasia. The characters are the marsupial mammals and the plot is how they came to be there and how they adapted to the special conditions of their vast homeland. Today marsupials only occur in Australasia and the Americas, although fossil marsupials have been discovered on every continent of the world. If they occurred on all the continents in the past, why are they not more widely distributed today? We first need to ask whether marsupials really share a common relationship closer than that to any other group of mammals. If they do, where did marsupials originate and how did they come to be where they are today? The features that were described first by the Europeans were the pouch of the female and the extraordinarily small size of the young at birth. These are their two most distinctive features and reproduction is what sets marsupials apart from other mammals and permeates the life history of every species. But what confused the early European explorers was that many marsupials closely resembled mammals more familiar to them that follow similar life styles. First European encounter with American marsupials The first marsupial brought to Europe from America was a common opossum collected by Vincente Yañez Pinzón on his first voyage to the New World in 1500. He collected a female, which had young in its pouch, and later he presented it to Queen Isabella and King Ferdinand II in Grenada. By the end of the voyage to Spain the young were gone and the mother opossum dead but the Queen inserted her fingers into the ‘second belly’ of this strange creature from the New World. This extraordinary organ and the young it enclosed caused astonishment in scientific circles throughout Europe and led to speculation about how the tiny young reached the pouch – did they grow from the teats as buds, or were they blown into the pouch from the mother’s nostrils? Both ideas had a long currency but the equally astonishing fact that they crawl to the pouch unaided by their mother was not discovered for another 420 years. The strange appearance of this New World animal, with a fox-like head and a monkey’s hands, also puzzled European scientists, who described it as the monkey-fox or ‘simivulpa’. First European contact with Australasian marsupials In 1493 Pope Alexander IV divided the world between the two major European powers – Spain to the west and Portugal to the east – so it was Spanish explorers who discovered marsupials in the Americas, while Portuguese traders made the first observations on Australasian marsupials. The earliest description was by Antonio Galvao, Station Captain of the Portuguese settlement on Ternate in the Moluccas from 1536 to 1540. He brought back to Lisbon extensive notes from which he intended to write a treatise on the Moluccas. He wrote (Jacobs 1971): Some animals resemble ferrets, only a little bigger. They are called Kusus. They have a long tail with which they hang from the trees in which they live continuously, winding it once or twice around a branch. On their belly they have a pocket like an intermediate balcony; as soon as they give birth to a young one they grow it inside there at a nipple until it does not need nursing any more. As soon as she has borne and nourished it, the mother becomes pregnant again. This is a good description of the common cuscus, Phalanger orientalis, which still lives on Ternate. Galvao’s manuscript lay forgotten in the Jesuit Library at Seville for 400 years, until discovered and published by Father Hubert Jacobs in 1971. However, the manuscript may well

3

4

Life of Marsupials

have circulated in Europe because there are other references in the 17th century to animals similar to the opossum being found in the Moluccas (eg Piso 1648). As the rivalry of Portugal and Holland for the rich takings of the Spice Islands increased, travellers also reported on the strange animals of New Holland and New Guinea. In 1606 Captain Don Diego de Prado y Tovar landed at San Millaus Bay, on the southern coast of New Guinea and wrote (quoted by Stevens 1930): Here we killed an animal which is in the shape of a dog, smaller than a greyhound, with a bare scaly tail like that of a snake, and his testicles hang from a nerve like a thin cord; they say it was the castor, we ate it and it was like venison, its stomach full of ginger leaves and for that reason we ate it. The species that most nearly fits this description (Calaby 1965) is the pademelon Thylogale brunii, which makes this the earliest European record of a member of the kangaroo family. The Dutch Captain Pelsaert, wrecked on the inhospitable Abrolhos Islands off the west coast of Australia in 1625, described another animal with the same remarkable pouch and tiny offspring within it. This was the tammar wallaby, Macropus eugenii, the second member of the kangaroo family to be described by Europeans. Then on Dirk Hartog Island, the buccaneer, William Dampier in 1699, described the banded hare wallaby, Lagostrophus fasciatus: A sort of raccoons, different from those of the West Indies chiefly as to their legs; for these have very short fore-legs; but go jumping upon them as the others do, and like them are very good meat. Other navigators were also encountering similarly bizarre animals when they made landfalls on the coast of Western Australia and, from their reports, scientists in Europe recognised the similarities to the American animals. Pallas (1766) named the cuscus from the Moluccas Didelphis orientalis but Storr (1780) 14 years later noticed that the 2nd and 3rd toes or phalanges of their hind feet are partly fused and changed it to Phalanger orientalis, the name it has today. It has also given its name to the family of Australasian possums and cuscuses, the Phalangeridae. Adaptive radiations on different continents While all the early European observers were much struck by the pouch of female marsupials, they were also struck by the astonishing similarities of Australian marsupials to familiar mammals from Europe. In the scientific names they gave them they often used Greek prefixes that meant pouch or pocket and the scientific name of the familiar mammal that the marsupial resembled. So we find Pera-meles the pouched badger, Pera-dorcas the pouched antelope, Phascol-arctos, the pouched bear, Thylo-gale, the pouched hare and Thyla-cinus, the pouched dog. As the Australian continent was explored yet more remarkable similarities were discovered, the most extraordinary being the marsupial mole, Notoryctes typhlops, which looks and behaves like the golden mole of Namibia, Eremitalpa granti, especially in that both species ‘swim’ through dry sand, which collapses behind them leaving no burrow; and the exquisite numbat, Myrmecobius fasciatus, adapted to living on termites and ants. The tree-living marsupials of Australia and New Guinea show remarkable yet superficial likenesses to the various species of lemur of Madagascar (ie Daubentonia madagascarensis) and the tree sloths (ie Bradypus tridactylus) of South America. More remarkable yet are the many physiological similarities between marsupials and other mammals in such functions as fermentation of grass in the forestomach of kangaroos and ruminants. Apart from a pouch, what do all these species that are called


marsupials have in common that tells us that they are uniquely related to one another and separate from all the other mammals that they variously resemble?

Distinctive features of marsupials The person who first looked beneath the superficial similarities to find the fundamental criteria for determining relationships between mammals was de Blainville in 1816. He took as the defining character the anatomy of the female reproductive tract (Fig. 1.1). In marsupials there are two vaginae, two uteri and two oviducts, whereas in other mammals there is a single vagina, cervix and uterus and only the oviducts are paired. He named the marsupials the Di-delphia from the Greek words for two uteri and other mammals he called the Mono-delphia. He later realised that the platypus and echidna did not fit in either group, having a reproductive tract like that of birds and reptiles with a single opening for discharging products from the gut, the bladder and the gonads, and he termed them the Ornitho-delphia, or bird-uterus. His division of mammals has stood to this day, although his terms have not been retained. Instead, the Ornithodelphia are known as the Monotremata (Greek for one hole), the Didelphia are generally called the Marsupialia (Latin for pocket or pouch) and the Monodelphia are called the Placentalia, because of the great development of the placenta as an organ of exchange during pregnancy. These terms are also unsatisfactory because they are not exclusive. Thus, the marsupials share with the monotremes a single opening, the cloaca, for the discharge of all products. Second, not all female marsupials possess a well-developed pouch, whereas the female echidna, which is a monotreme, develops a pouch during lactation. And third, all marsupials have a placenta during intra-uterine development and in some species it is a complex structure with an intimate connection to the uterus. To avoid this confusion of terms some biologists favour Huxley’s (1880) terms for the living mammals. He saw the three groups of mammals as evolutionary stages on the way to ‘true’ mammals, by which he meant the group of mammals to which we human beings belong. So he coined the terms Proto-theria, or first mammals (the monotremes), Meta-theria or halfway mammals (the marsupials) and Eu-theria or true mammals (the placentals). These terms themselves imply progress, and so are also unsatisfactory. It is probably best now to ignore their original meanings and accept the terms as neutral descriptors of the groups they have been assigned to: in this book I will refer to monotremes, marsupials and placentals. Anatomy of the reproductive organs of marsupials De Blainville was right to choose the anatomy of the reproductive organs as the central criterion of his classification because they are unequivocally distinct between the three groups of living mammals. To appreciate this we must consider the development of the kidney and the ducts that convey urine, and the genital ducts that convey gametes and embryos to and from the body. If we compare a fetus from a tammar wallaby four days before birth and a human embryo at five weeks of gestation, for example, both have the same arrangement of kidney ducts, genital ducts and gonads (Fig. 1.1). Both the genital ducts and the kidney ducts (ureters) enter a common tube, the urogenital sinus, on its dorsal side and the future bladder is on the opposite, ventral side. In later development of both groups the ureters migrate to the ventral side to enter the bladder, while the genital ducts remain dorsal. This migration of the ureters only occurs in placentals and marsupials and probably arose as an adaptation for the more effective storage of urine in the bladder. In the monotremes, the ureters still discharge into the top of the sinus and the urine must pass across the sinus to enter the bladder, by a process still not understood.

5

6

Life of Marsupials

Figure 1.1: The primary difference between marsupial and placental mammals. The relative position of the ureters and genital ducts and how it is derived from a common pattern of kidney and genital ducts at the indifferent stage in the embryo.

In the human, and all placental mammals, the ureters migrate outside and below the genital ducts, while in the tammar, as in all other marsupials, the ureters migrate inside and above the genital ducts. While the initial adaptation may have had more to do with excretion in the ancestral mammals, the route that the ureters take to reach the bladder unequivocally distinguishes living marsupials from living placentals. And it has had profound consequences for reproduction. Its most obvious effect is seen in the female reproductive tract. Each oviduct transforms into the specialised regions of Fallopian tube, uterus, cervix and vagina (Fig. 1.1). In placental mammals the left and right oviducts join in the midline to form a single vagina and, in most species, also join to form a single cervix and a single uterus. The only portions that remain separate are the two Fallopian tubes, which each receive an egg from their respective ovary.


In marsupials this joining of the oviducts cannot occur because the ureters pass between them, so there are two lateral vaginae, each arising from the common urogenital sinus posteriorly. Above the ureters the lateral vaginae loop back to the midline and become partially fused (Fig. 1.1). Then there are two cervices, two uteri and two separate Fallopian tubes. At copulation the semen is deposited in the lateral vaginae and the sperm pass through the two cervices and uteri to the Fallopian tubes, where fertilisation occurs. In the males of many species the glans of the penis is also divided and it is supposed that the semen is directed separately to each lateral vagina, but this has not been established. The same relationship of the ureters and the genital ducts in males means that the vas deferens in placental mammals loops over the ureter, whereas in marsupial males the arrangement is simpler (Fig. 1.1). At birth the young marsupial passes through a new-formed canal in the tissues between the ureters, direct from the lateral vaginae to the urogenital sinus. In almost all marsupials this extraordinary arrangement, the pseudo-vaginal or birth canal, is re-formed at each birth: only in some kangaroos, Macropus, and the honey possum, Tarsipes rostratus, does it remain open after the first birth and in these species it is called the median vagina. So, female kangaroos have three vaginas, two for sperm and one for the young at birth. It has long been held that the small size of the divided uteri and the inadequacy of the birth canal are the reasons that marsupial young at birth are so very small. Whether or not this was the cause, the young of all marsupials at birth are much smaller than the most immature of placental young.

Figure 1.2: Relationship between maternal body weight and weight of the newborn marsupial and its stage of development in different species.

7

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Life of Marsupials

Small size at birth Females of the largest living marsupials, the eastern grey kangaroo, Macropus giganteus, and the red kangaroo, Macropus rufus, weigh 28 kg and deliver one young that weighs 830 mg, half the size of a newborn mouse. The newborn kangaroo is 0.003% of its mother’s weight, compared to a newborn mouse or human baby that are about 5%: almost a two thousand-fold difference. Most marsupials at birth are between 200 mg and 400 mg (Fig. 1.2) but those of small dasyurid marsupials are under 10 mg and the very smallest newborn marsupial is that of the honey possum at 4 mg. It is little wonder that the first European explorers to see these tiny creatures were unable to believe that they were born in the normal manner or could travel unaided to the pouch and there find and attach to a teat. It seemed impossible that the young marsupials could be active and possess sensory organs or have sufficient neuromuscular coordination to move independently. But they do and, even more remarkably, they control the onset of their own birth like the much more advanced placental young do (see Chapter 2). One reason often cited for the small size of marsupials at birth is that pregnancy is very short, compared to placental counterparts. Although it is true that in some species pregnancy is very short (less than two weeks), in other species it is longer than in a comparable sized placental species. The real point is not the duration of pregnancy but the mass of young produced at the end of pregnancy. All placental mammals bring forth very much larger young, at a more advanced stage of development, because the major growth phase of the young occurs during pregnancy, via a well-developed placenta. By contrast, in marsupials almost all growth and development occurs after birth during the long and complex period of lactation, when large changes in the volume and composition of the milk occur that support the changing needs of the young. Thus, the placental female makes her major investment in reproduction during pregnancy, the female marsupial makes hers during lactation. This difference has important consequences in the ecology of the species. Control of sexual differentiation in marsupials Superficially the external genitalia of marsupials and placentals are similar but the control of development to the adult form is different in the two groups of mammals. In placental mammals sexual differentiation of the fetus takes place during gestation when the external appearance of both sexes is the same, the so-called indifferent stage. Later under the influence of testosterone secreted by the developing testes of male fetuses the genital tubercle develops into a penis and the scrotum forms behind it; in the absence of testes the same structures develop in female fetuses into the clitoris and the outer lips of the vulva, respectively; nipples and mammary glands are formed in both sexes and retained through life. The whole cascade of change from the indifferent stage to the adult form is controlled by the expression of one gene on the tiny Y chromosome, called the ‘sex determining region’ of the Y chromosome, or SRY gene. Possession of this gene directs the gonads to differentiate into testes, which then secrete testosterone and transform the other genital structures to the male pattern: in the absence of the SRY gene the fetus becomes female. The normal complement of sex chromosomes in placentals and in marsupials is two X chromosomes in females and one X and one Y chromosome in males, but an individual with one X and no Y chromosome (XO) is female and an individual with two X and one Y chromosome (XXY) is male. By contrast in marsupials the scrotum forms as two bulges in front of the genital tubercle, there is nothing equivalent to the outer lips of the vulva in female marsupials, and the pouch and mammary glands only differentiate in females. Male marsupials have a Y chromosome and the SRY gene, which directs the differentiation of the gonads to become testes and secrete testosterone leading to the differentiation of the internal genitalia and the genital tubercle to the male form: absence of the SRY gene results in the female form of the internal genitalia and genital tubercle.


However, the developing testes do not control the differentiation of mammary glands, pouch or scrotum. Indeed, scrotal bulges develop in genetic males and mammary glands and pouch in genetic females many days before the gonads can be distinguished as ovary or testis, and these organs are not affected in their later development by sex hormones. The present thinking is that these external organs in marsupials (mammary glands, pouch or scrotum) are controlled directly by the sex chromosome constitution of the tissues themselves, particularly the X chromosomes (Cooper 1993). Thus, possession of one X chromosome, as in a normal male marsupial, leads to differentiation of scrotal bulges; and possession of two X chromosomes, as in a normal female marsupial, leads to differentiation of mammary glands and a pouch (Renfree et al 1996a). In genetically abnormal tammars, XO individuals have female organs internally, as in placental species, but externally they do not have mammary glands or a pouch but do have a well-developed, empty scrotum. Conversely, XXY tammars have male organs internally and a well-developed penis, in accordance with their possession of a Y chromosome, but instead of a scrotum, they have a small pouch and mammary glands, in accordance with their possession of two X chromosomes (Sharman et al 1990). Thus, the control of sexual differentiation in marsupials has followed a different path from that followed by placentals, although the end result is deceptively similar. Physiological differences between marsupials and other mammals Marsupials, like other mammals and birds, maintain their body temperature at a fairly constant level. However, the normal body temperature of marsupials is about 35.5°C, which is 2.5°C lower than that of most placentals, which in turn are lower than birds (Table 1.1). It is not clear why this should be so but it does appear to be something that is genetically determined and affects the lives of marsupials as profoundly as their mode of reproduction. To appreciate this we need to understand the underlying physiological process of which body temperature is an outward manifestation. Since the rate of chemical reactions doubles for every rise of 10°C, the rate in marsupials must be about 25% lower than in placentals, which in turn must be about 25% lower than passerine (song) birds. This is clearly seen in the cost of maintaining a constant body temperature, which rises with increasing basal body temperature (BBT) (Table 1.1). Table 1.1: A comparison of the standard metabolic rate of terrestrial vertebrates Standard metabolic rate (SMR; kJ/kg0.75 per day); basal body temperature (BBT; °C) (after Dawson and Hulbert 1970). Reptiles Lizards

Mammals

Birds

Monotremes

Marsupials

Placentals

Non-passerine

Passerine

BBT

30

30

35.5

38.0

39.5

40.5

SMR

31

142

204

289

347

598

SMR at 38°C

82

260

260

289

301

477

To maintain their body temperature mammals and birds expend the least amount of energy when the surrounding temperature is nearly the same as their body temperature. When the surrounding, or ambient, temperature is lower more energy is required to generate heat and when it is higher than the body temperature more energy is expended in cooling devices, such as panting and sweating. The ambient temperature where minimum energy is used by the non-feeding mammal at rest is called its thermo-neutral zone. This minimum value is termed the standard metabolic rate (SMR) and it represents the energy required to maintain essential

9

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Life of Marsupials

functions of the living body at a constant body temperature. It is usually determined as the oxygen (O2) consumed or carbon dioxide (CO2) produced under these conditions in a given period of time (Table 1.2). Table 1.2: Resting body temperature (TBody), oxygen consumption and standard metabolic rate (SMR) of seven marsupials, arranged according to body mass (Hume 1999). Species

Mass

TBody

Oxygen consumption per hour

SMR

(kg)

(°C)

(mL O2/g)

(kJ/kg0.75 per day)

Planigale ingrami

0.007

34.8

2.130

310

Monodelphis brevicaudata

0.076

0.800

211

Perameles nasuta

0.667

0.479

209

Didelphis virginiana

2.403

0.380

238

Macropus eugenii

4.878

0.283

212

0.202

210

0.178

210

A

28.000

Macropus robustus

30.000

Vombatus ursinus

36.1

36.4

36.0

A

Gowland (1973).

The SMR values can be converted to energy used (Joules) if the composition of the food being eaten is known. For carbohydrate one molecule of sugar and 6 molecules of O2 are converted into 6 molecules of water and 6 molecules of CO2. This is a respiratory quotient (RQ) of 1 and the water formed is called metabolic water. For fat the RQ is 0.7 and more metabolic water is formed, while for protein the RQ is 0.8 and less metabolic water is produced. If it is assumed that the animal’s food is a mixture of carbohydrate, fat and protein, with an average RQ of 0.8, then 1 mL of O2 consumed is equivalent to 21 Joules of energy and 1 mL of CO2 produced is equivalent to 26 Joules. The values for O2 consumed by a range of marsupials of increasing body size are given in Table 1.2. Although the body temperature of all the species is much the same, the smallest species consumed 12 times more O2 per gram of body tissue than the largest species. Why is the cost of living for the small species greater than for the large species? Body size in relation to metabolic processes For all animals there is an important relationship between body mass and standard metabolic rate, which is much more pronounced for birds and mammals than for other animals. In its simplest terms the mass of the body increases by the cube power, whereas the surface area increases by the square power, so the small species has a relatively larger surface area than the larger species. Since all metabolic functions occur at surfaces the smaller species has a relatively higher metabolic rate than the larger one. This affects all sorts of physiological functions. For instance, the heart rate of the smallest mammals are about 1000 beats per minute, compared to about 70 beats per minute for humans and fewer than 10 beats per minute for large whales. Again, the strength of muscle and bone depends on the cross-sectional area, so the strengths of these tissues increase by the square power also, so that a comparison between a small and a large mammal shows the small one to be proportionately much stronger than the larger one. For instance, a female antechinus, weighing 30 g, can carry a litter of young that weighs more than she does, whereas the female kangaroo ejects her single young from the pouch when it weighs about one-tenth of her own weight.


Conversely, because the small mammal has a proportionately greater surface area than the larger species, it loses heat and water across its skin and lungs more readily. This greater energy and water loss must be made up from the food and water ingested, so that less is available for synthesis into stored material. Hence, small animals cannot survive starvation for as long as large ones. For example, a small marsupial, such as a dunnart, Sminthopsis, consumes the equivalent of its body weight each day, whereas a person consumes about 1%, so a dunnart cannot survive more than a few days without food, whereas a person can survive for several weeks. For the same reason, one large mammal takes much longer to exhaust its food supply than an equivalent mass of many small mammals. Hence the argument: 10 rabbits eat as much as one sheep. There is, thus, in body mass a nice balance of advantages and disadvantages. Under favourable conditions the small mammal converts food more rapidly and the population proliferates faster than the large species. In adverse times, however, the population of a small species will decline rapidly, as its members succumb to the lack of food or the adverse environment, whereas the members of a large species can withstand adversity for much longer. In the later chapters of this book the importance of body size in the economy of different marsupials will recur often. Comparing mammals of different size In order to compare the performances of animals of different diets, life styles and ancestry it is necessary to agree on mathematical functions that reduce the variability due to body mass. The formula that has generally been adopted is: y = bxk where y is the size-dependent variable (eg O2 consumption, heart rate or food consumption), x is body mass in kilograms, b is the intercept constant and k is the slope (Fig. 1.3). For metabolic rate the exponent 0.75 is still the best approximation for k, despite considerable variability among species. Thus, to compare the SMR of a range of mammals of differing size, the formula used is kJ/kg0.75 per day. Using this formula the average SMR of 56 marsupials from small 7 g dasyurids to large 29 kg kangaroos is 204 kJ/kg0.75per day (Table 1.1). The SMR values for individual species vary from 140 for some desert dasyurids (eg Dasycercus cristicauda, 160 for

Figure 1.3: The relationship between body mass (on a log scale) and daily energy consumption for marsupials and placentals. Closed lines show standard metabolic rate and dashed lines show field metabolic rate. Note that the slope of the field metabolic rate for marsupials is not parallel to that for placentals, the smallest species having a much higher metabolic scope.

11

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Life of Marsupials

the koala, Phascolarctos cinereus, to 310 for the tiny planigale, Planigale ingrami. This compares with the average SMR for a group of 272 placental mammals, 289 kJ/kg0.75 per day, ranging from the house mouse to the elephant. Thus, the average value for marsupials is about 70% of the average value for placental mammals. In Table 1.1 these values are compared to other terrestrial vertebrates, measured at their thermo-neutral zone and also at 38°C, the body temperature of placental mammals. What is evident from this is that the reptiles, which do not control their body temperature, have a much lower metabolic rate than the mammals and birds under both conditions. More interestingly, each group of mammals and birds has a characteristic level and each is positively correlated with body temperature. The monotremes have the lowest body temperature and the lowest metabolism, while the passerine birds have the highest. The high values of the latter are probably associated with the special requirements of flight. During the past 30 years there has been much discussion about the significance of the apparent difference between marsupials and placentals, some people arguing that life style and diet may be more significant factors in determining SMR than ancestry (see especially Lee and Cockburn 1985, McNab 1986, 1988). Thus, some placentals, such as sloths, Bradypus, have a SMR below the marsupial average and some marsupials, such as planigale and the honey possum have a SMR of, respectively, 106 and 158% of the placental average. Nevertheless, the mean value for marsupials is 30% below the mean for placental mammals, which suggests that there is a basic underlying difference in SMR, although food habits and activity are sometimes strong enough to mask phylogeny. It is not clear why the set point should vary between different kinds of animals, nor what controls it. Hulbert and Else (1999) have observed that the plasma membranes of the mitochondria and cells of vertebrates with high metabolic activity have a relatively high proportion of polyunsaturated lipids, whereas those with lower metabolic activity have membranes that are relatively mono-unsaturated. They have proposed that these differences may be the basis of the pacemaker for metabolism. This suggests that there may be a fundamental difference between the cell membranes of marsupials and placentals, which affects all metabolic processes, including respiration, excretion, water balance, nerve conduction and growth rate. The lower setting of the rate in marsupials is reflected in several other physiological functions: their heart rates, adjusted for body mass, are lower than for placentals, as are nitrogen requirements in some species. The lower SMR of marsupials also means that they have lower food requirements and water turnover rates, which may confer special advantages in adverse conditions or arid environments. Conversely, the advantage for placentals of a higher body temperature and SMR is faster nerve conduction and smooth muscle contraction, faster growth rates and faster reproduction, with the trade off in higher food requirements. In those environments where soils are rich, the climate is equable and food predictable the higher metabolic and reproductive rate of placentals is advantageous. However, in less benign environments, where the soils are infertile and climate unpredictable, the lower metabolism of marsupials may confer an advantage. The spectacular radiations of marsupials in South America and Australia alongside placentals may in part have been due to environmental constraints in these continents that favoured species that conserved limited resources. Field metabolic rates A major development in the last 20 years has been the measurement of field metabolic rate (FMR) in 28 species of marsupial, using isotope dilution techniques (see Box 1.1). This is a much more informative measure of a species’ actual metabolic needs than SMR and the difference between SMR and a species’ maximum metabolic rate provides a measure of its metabolic scope. While most marsupials have a low SMR, compared to most placentals, the FMRs of species under 100 g


Box 1.1: Measuring field metabolism To compare the metabolic strategies that different species have evolved we need to understand the various components of metabolism. These are metabolic rate, water turnover, and the nutrients (eg nitrogen) required for an animal to maintain itself in its natural environment and reproduce. Each of these three components can be measured in captive animals in feeding trials, and by the use of respirometers. The use of water labelled with radioactive or other identifiable isotopes of hydrogen and oxygen give more realistic measures from free-living animals. Doubly labelled water (3H2O, H218O), composed of tritium (3H2) or deuterium (2H2), and a non-radioactive isotope of oxygen (18O), can provide measures of respiration (CO2 production), water turnover, fat deposition and food consumption. If the sodium content of the food is known, food intake can also be estimated in the same way, using an isotope of sodium (22Na). The technique is to capture the animal, inject it with a known quantity of the particular isotope and then let the isotope equilibrate with the animal’s body tissues, a process that usually takes 2 to 6 hours. Then a blood sample is taken to measure the concentration of the isotope distributed in the body. This initial ratio of labelled to unlabelled isotope at equilibration gives a measure of total body content, or pool size, of that element: Pool size = concentration of isotope injected × sample volume concentration of isotope in sample The animal is then released and, after some days, when it is recaptured, a second sample of blood is taken and the concentration of the isotope measured again. The difference between the initial and subsequent concentrations is a measure of the dilution that has taken place in the elapsed time by respiration, and by the ingestion and excretion of water and food by the animal. These measures can be converted into field metabolic rate, water turnover rate and food consumption, respectively. For instance, oxygen turnover is measured by the dilution of the 18O isotope compared to the common isotope 16O, using a mass spectrometer. Because each of these rates is substantially affected by the body mass, different species can be compared only if the values are expressed by an allometric exponent. For metabolic rate and food intake the exponent is usually taken to be the three-quarter power of body mass (kg0.75), for water turnover it is kg0.8. However, both of these are approximations based on placental mammals and standard conditions. Green (1997) has shown that there are wide differences between species of marsupial and within a species at different times of the year, and that other exponents may more accurately reflect reality. However, we will follow convention here when comparing species in each Chapter. In order to estimate the field metabolic rate of an animal we need to measure the volume of O2 consumed or CO2 produced, and to know the available energy content of the food. The mean energy content of the food varies according to the relative composition of carbohydrate, fat and protein. For example, it is 3–4 kJ/g for insects and 6 kJ/g for mammalian flesh. However, the net energy that can be used by the animal is less than this because about 10% is lost in faeces and 8% in urinary excretion. The balance is the net metabolically available energy of the food and, with this information and the SMR (calculated as kJ/kg0.75 per day), the daily food requirements of the animal can be estimated. By comparing these several measures in different species of marsupial in later Chapters we can understand how each species is using the available resources to meet its basic requirements at different periods of its life cycle or in different environments.

13

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Life of Marsupials

body mass are much the same, which means that their metabolic scopes are greater (Fig.1.3). For instance, the fat-tailed dunnart, Sminthopsis crassicaudata, has a FMR up to seven times higher than its SMR (Nagy et al 1988). It is unusual for placental mammals to have FMR more than three times SMR, which means that the dunnart’s metabolic scope is considerably greater than equivalent placental species. This may be a definite advantage in extreme environments, since the low SMR conserves resources of the animal at rest, without limiting its metabolism when active. These several attributes of marsupial metabolism will be discussed when considering the adaptations of particular species in later chapters.

Relationships within marsupials If we accept that marsupials are a distinct group of mammals, what are the relationships within the group, and how have they evolved? For 100 years marsupials have been classified on the basis of the number and kinds of teeth and on the number of digits on the feet. These characters are undoubtedly affected by the mode of life of the possessors but have the advantage of being available in fossil remains and can be accurately dated and used to calibrate rates of change in other criteria, based on living animals. Most information comes from the structure of the molar teeth, whereas the structures of the simpler canines and incisors have only been used to determine total dentition. There may also be subtle differences in structure of the enamel and other features of these teeth that have not been disclosed so far. In classifying living marsupials the anatomy of the soft tissues can also be used to distinguish between related species or groups, as can the form and number of chromosomes. In the past 30 years new techniques in biochemistry and molecular biology have greatly extended our understanding of the relationships of modern day marsupials: amino acid analyses and immunological techniques opened up new ways to compare relationships of living species by comparing proteins, and analyses of base sequences in nuclear and mitochondrial DNA has provided an even more powerful means to determine relationships of living marsupials, and to provide increasingly precise measures of the time since related groups diverged in the past. We will begin by considering anatomical characters and then see how relationships based on the newer techniques corroborate or refute the classification of marsupials based on anatomy. Relationships based on anatomy Teeth Teeth develop in the jaws of young mammals as a coalescence of cells derived from the base of a deep groove of surface epithelium and the underlying tissues in the jaw. The initial tooth bud develops as a central core and an overlying cap of epithelium in which dentine and enamel is later laid down. The tooth bud rises to the surface of the jaw as it develops, eventually piercing the groove and erupting; other tooth buds follow successively behind it in the jaw. In the front teeth the structure remains simple with a single cusp and a single root, but the back teeth develop more cusps and roots. These secondary cusps develop, like the primary one, as subsidiary buds and their position relative to the primary cusp is characteristic of the particular tooth and for the particular species. This pattern of development has been understood for a long time but in the last 10 years the way in which the pattern is controlled genetically has become clearer through the expression of developmental genes in the tooth bud (for review see Thesleff and Sharpe 1997). One group of genes determine that the tooth bud will develop into a complex molar instead of a simple front


tooth, while other genes determine the position of the secondary buds relative to the primary bud. What is interesting is that the expression of the genes precedes by one or two days the first morphological changes in the cells of the jaw, indicating that the information about pattern is set up before the buds begin to form and not as a result of interaction between adjacent tooth buds in the jaw. Thus, gene expression predicts future cusp pattern and affects the very early stages of tooth development. In evolutionary terms small changes in gene expression could have profound effects on tooth morphology. As an example of what may become possible, Jernvall et al (2000) compared the expression of specific genes in the formation of molar teeth in mouse, Mus musculus, and vole, Microtis rossiameridionalis, which evolved from a common ancestor in the early Miocene epoch 20 million years ago. They showed that the big differences in the molar pattern between mice and voles is the result of small changes in the expression of the genes that determine the relative position of the secondary cusp, whether it is parallel to the primary cusp (in mice) or diagonal to the primary cusp (in voles). What is the relevance of this to marsupials? Marsupial teeth develop in the same manner as the teeth of placental mammals, reflecting their common ancestry more than 120 million years ago. It is, therefore, reasonable to assume that the same or similar genes control the pattern of molar cusps in marsupials. More than this, the discovery that the cusp pattern of molar teeth is controlled by the expression of particular genes brings closer the day when the evidence of palaeontology, based largely on the morphology of molar teeth, and the evidence of relationships based on molecular genetics, can be integrated more precisely. Teeth in mammals have two primary functions: the front teeth are used to bring food into the mouth, while the back teeth are used to process it for digestion, either by cutting or grinding the food into fine pieces. Teeth may also be used in aggressive or defensive displays, for grooming, or for grasping the young. The front teeth are single rooted and comprise a variable number of chisel-shaped incisors and a single set of pointed canines. Behind the canines are up to four larger teeth, each with two roots, called premolars and behind the premolars are up to four sets of molars, each with three or four roots. The structure of teeth reflects the uses to which they are put in different species and they can therefore tell us a considerable amount about the life of the possessor. This is especially useful when examining fossil specimens, which often consist only of a few teeth, because these are the most durable part of the body. The size and shape of the front teeth can tell us whether the mammal catches moving prey and what sort of prey, or whether its diet is largely of plant material. Carnivorous species have many sharp incisors, prominent canines and many sharp points on the premolars and molars. Their molars intersect in such a way as to provide many shear surfaces, like scissor blades, and less emphasis on flat, opposed surfaces where food can be crushed or ground small. In herbivorous species, by contrast, the canines are small or absent, the incisors form two opposing rows of chisel-like teeth with which herbage can be cut, the molars and premolars have fewer sharp points and shear surfaces but have a much larger area for grinding. Sanson (1985) has suggested that the different types of dentition reflect the forces required to penetrate the bodies of prey species. For instance, the impact strength of bone is about 2 kg/cm2 while that of insect cuticle is 23 kg/cm2, so puncturing the cuticle with sharp points is an easier option than crushing it. The impact strength of plant cell walls, however, is about 76 kg/cm2, which explains why the teeth of herbivores have such highly developed crushing surfaces. It also accounts for the different anatomy of the jaws of carnivores and herbivores: carnivores have the jaw muscles grouped at the back of the jaw, which allows for a wide gape but delivers less force than the forward disposition of the jaw muscles of herbivores, such as kangaroos (see Chapter 9).

15

16

Life of Marsupials

Since the earliest mammals and all the ancestral marsupials and placentals were small insectivores, the dentition of small carnivores resembles more closely the ancestral dentition of mammals from which the several types of herbivorous dentition have evolved. In the study of teeth of mammals the molars provide the greatest amount of information about diet, about relationships between species, and about the evolution of the main lines of descent from the Cretaceous period ancestors. Because of their importance in the subsequent discussion on the evolution of marsupials, some knowledge of the anatomy of molars is necessary. Diprododont Marsupial

Polyprododont Marsupial stE

Phascolarctos

stA

pa me

prd

me

Perameles

mcl

mcl

med

med

Tertiary

pad

Didelphis, Dasyurus

Placental

Alphadon

Didelphodus

Upper Cretaceous

stylar shelf

Pappotherium

Clemensia

Lower Cretaceous (Albian)

prd hyd pad

end med

Eupantothere

me

Upper Jurassic

pr

Pantothere Upper Triassic (Rhaetic)

pa

outer anterior

posterior inner

Figure 1.4: Probable evolution of molar teeth in marsupials and placentals from pantothere ancestors 250 million years ago. The lower left molar is shown in occlusal view, stippled, and the upper molar is superimposed in outline. In the upper molar the primary cusps are the (pr) protocone, (pa) paracone and (me) metacone, to which were later added posteriorly the (mcl) metaconule and laterally the (st) stylar shelf. In the lower molars the primary cusps are the (prd) protoconid, (pad) paraconid and (med) metaconid, to which were later added posteriorly the (end) entoconid and (hyd) hypoconid. Data from Romer (1966) and Archer (1976).


The structure of molar teeth can be traced from the earliest mammals, which had triangular, three-cusped teeth in the upper and lower jaws arranged so that, seen in surface view, the apex of each upper tooth (protocone) pointed inwards while the apex of each lower molar (protoconid) pointed outwards (Fig. 1.4). When the mouth closed the upper and lower molars fitted closely between each other providing a zigzag shear surface. This can still be seen in the skulls of small carnivorous marsupials (see Fig. 4.2) and placentals. At an early stage of mammalian evolution, before the separation of marsupials and placentals, the simple arrangement of three cusps was extended. In the lower jaw two additional cusps developed on the posterior face of the molars, the entoconid and hypoconid, thereby providing a basin into which the apex, or protocone, of the upper molar fitted. This provides a grinding surface in addition to the shearing component of the molar teeth. In the upper molars additional cusps developed, both between the primary three cusps and also on the outside of them. The outer series of five small cusps, called the stylar shelf, do not meet complementary parts of the lower molars so that their function in mastication is unclear. Although the earliest placentals had two stylar cusps, later placentals do not, whereas all early marsupials had the full complement of five cusps, most of which are retained in the present day American opossums (Fig. 1.4) and dasyurids (see Fig. 4.2). The presence of the stylar shelf is a diagnostic tool in differentiating between fossil placentals and fossil marsupials. Ridges may form between these several cusps and the pattern of the ridges and the relative sizes of the cusps are used to determine phylogenetic relationships between extinct and living mammals. In addition the fine surface structure of the teeth and surface scratch marks can provide further information about relationships and the type of food that was processed by the living animal. All the living American marsupials and the Australian carnivorous species have long snouts bearing a battery of simple, sharp-pointed teeth. In each jaw there are four molars, three premolars and one prominent canine, as well as four or five incisors in the upper jaw and three in the lower jaw (Fig. 1.5). On this criterion they are grouped together as the Polyprotodontia

Figure 1.5: Marsupial relationships based on teeth and feet. Upper panel to show representatives of didactyl and syndactyl feet, the latter differentiated by the small, paired digits 2 and 3 on the pes; also note the large digit 4 in kangaroos; lower panel shows representative skulls of polyprotodont and diprotodont species, differentiated by the presence of 4 or 5 incisor teeth (i) in each jaw in front of the canines (c) in all polyprotodonts and only one in each lower jaw of diprotodonts; all have four molars (m) and up to three premolars (pm). After Jones (1924) and Tyndale-Biscoe (1973).

17

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Life of Marsupials

(meaning many front teeth). The bandicoots are included in this group on this criterion but they are more omnivorous than the dasyurids and their molar teeth are squared up with an extra cusp at the back, called the metaconule (Fig. 1.4 and 5.2). The herbivorous species of Australasia have fewer premolars, the canines are small or absent and there are only one to three incisors in the upper jaw and a single large pair of incisors in the lower jaw. With fewer teeth, there is often a gap between the front teeth and the cheek teeth, called the diastema, which enables the herbage to be presented by the tongue to the battery of grinding molars. The single pair of incisors in each lower jaw is the defining character for this diverse group, which is called the Diprotodontia (meaning two front teeth). Again, there is a superficial link to one group of South American marsupials, the Caenolestidae, in which the first incisors are large and procumbent, but in them the other incisors are present. Furthermore, in the embryological development of the teeth of the Diprotodontia, the large incisor is the second, not the first, incisor so this is another case of convergence rather than indicating a close relationship. Within the Diprotodontia further distinctions can be made on the basis of teeth. As in bandicoots, they have the metaconule in the upper molars, but have lost the stylar shelf, and their lower molars are also squared up by the loss of the paraconid at the front (Fig. 1.4). Also the grinding surface of the molars is more rounded than the sharp pointed cusps of the Polyprotodontia and are variously shaped into crescents or ridges (see Fig. 7.7). This development of a grinding battery of molars reaches its greatest development in two groups of grass eaters: in the wombats the incisors in the upper jaw are reduced to one pair and all the teeth grow continuously through life in a similar manner to the teeth of rodents and rabbits; in the large kangaroos the molar teeth are high crowned, like those of sheep, and the whole battery moves forward in the jaw, the molars being used successively as the more anterior ones are worn down and shed (see Fig. 9.3). Elephants do this too, but on a grander scale. Foot structure When foot structure is used as the criterion for grouping marsupials, all the American marsupials and the dasyurid marsupials of Australia share a common feature of hind feet with five separate, subequal digits (Fig. 1.5). This is termed didactyly, or separated digits. The remaining Australian marsupials have digit 1 of the hind foot reduced to a nubbin and digits 2 and 3 partly fused and together equal in size to digit 5, and this is termed syndactyly, or fused digits. In some species, especially the large kangaroos, digit 4 is much larger and longer than the other digits (Fig. 1.5) and takes the main thrust during jumping. The semi-fused digits 2 and 3 are used in grooming by some species (see Fig. 9.2e), but whether they evolved primarily for this function or represent a progressive reduction for speedier locomotion, as in the evolution of horses and ruminants, is not clear. A long-standing paradox in understanding marsupial relationships is that bandicoots (Peramelomorphia) have a dentition like the American and Australian carnivorous marsupials but have a foot structure that is apparently identical with the Diprotodontia. This paradox is slowly being resolved in favour of bandicoots and the Diprotodontia having acquired fused toes independently. Although this is a very remarkable convergence, it is under a simple genetic control and so could have arisen more than once. Ankle bones Szalay (1982) proposed that the anatomy of the ankle bones could differentiate between American and Australasian marsupials: in all the Australian species that he examined there was a single articular facet where the ends of the long bones attach to the bones of the ankle joint, whereas in the two main families of American marsupials he found two facets. The only American species


that had undivided facets like the Australian species was a species that lives in Chile, the monito del monte, Dromiciops gliroides; this alerted biologists to look for closer links between American and Australian marsupials, which has gained strong support from the molecular studies (see Marsupial relationships based on protein analysis). While subsequent work has confirmed a closer relationship of Dromiciops with Australasian marsupials than with American species, the criterion itself has not proved to be as consistent as Szalay thought. Hershkovitz (1992) examined a much larger series of species than Szalay and found that both types of ankle joint occur among the American marsupials and among the Australasian species, so it is not an exclusive character. Brain anatomy Links between the two halves of the forebrain provide another way to distinguish relationships between seemingly similar marsupial groups. Two nerve tracts, or commissures, link the cerebral hemispheres of the forebrain: the large anterior commissure, which also links the two olfactory lobes of the forebrain, and the smaller hippocampal commissure (Johnson 1977). In placentals a third commissure, the corpus callosum, links the cerebral cortex of each side, but it is absent from monotremes and marsupials. Within the marsupials a clear distinction can be made between the Diprotodontia and the Polyprotodontia: the former group has an additional tract of fibres, called the fasciculus aberrans, that extends the links of the anterior commissure between the two sides of the cerebral cortex. The absence of this tract in the Caenolestidae supports the conclusion that their diprotodont dentition was independently acquired, and the absence of this tract in the bandicoots also supports the conclusion that syndactyly was independently evolved in them. The brain of Dromiciops has not been described but it would be pertinent to the argument about its relationship to Australian marsupials to know whether it has the fasciculus aberrans. Sperm morphology and other anatomy The morphology and fine structure of the spermatozoa of marsupials can also disclose relationships (Fig.1.6). In the two main families of American marsupials, the Didelphidae and the Caenolestidae, the sperm occur as conjoined pairs. The head of each sperm is intimately adpressed to the head of the other and they remain like this from their passage through the epididymis of the male until they reach the vicinity of the egg in the oviducts of the female. Conjugation of sperm is unknown in any placental mammal, or in any species of marsupial from Australia. Until 1982 this character was thought to separate the American and Australian marsupials from each other and represent a very old divergence. However, Dromiciops has unconjugated sperm, like Australian marsupials. This was further support for closer links between the two geographical groups. Within each family the sperm head has a characteristic shape, which distinguishes the three families of American marsupials from each other, and within the Australian marsupials, distinguishes between the Macropodidae, the Dasyuridae and the Peramelidae (Fig. 1.6). The wombats (Vombatidae) and the koala have similarly hook-shaped sperm heads distinct from any other marsupial, which supports their close relationship to each other and differences from possums. And the minute marsupial, Tarsipes, has the largest sperm of any mammal so far described. It is 360 µm in length or 4.5 times longer than a human sperm. As mentioned earlier, the head of the penis is more or less divided into two lobes in all species of South American marsupials and most Australian species. It is only partly divided in the koala and wombats, while in the Macropodidae it is a single structure and in Tarsipes there is no glans at all. Among some dasyurids the anatomy of the penis is a useful character for classification (Woolley 1982).

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Life of Marsupials

Figure 1.6: Marsupial relationships based on head and midpiece of spermatozoa. Note three forms of pairing at the head in (a) didelphid, (b) caenolestid, (c) caluromid marsupials of America and (d) unpaired sperm of the American Dromiciops and Australian phalangerid, petaurid, burramyid and macropodid species. Other family specific forms of the sperm head are shown for (e) peramelids, (f) dasyurids, (g) honey possum, Tarsipes and (h) koala, Phascolarctos cinereus, and wombats, Lasiorhinus. From Temple-Smith (1994) Reproduction, Fertility and Development 6, 423, with permission of CSIRO Publishing.

On the anatomical characteristics of the teeth, feet, brain, sperm and penis shape the 16 families of living marsupials separate into five groups, one exclusively in South America, three exclusively in Australasia and one with representatives in both regions (Table 1.3). These groups will now be used as the basis for comparing the other characters of cytology, proteins and genes. Relationships based on chromosomes The double-stranded molecules of DNA, which carry the genetic information of the individual animal in all its cells, is packaged in a variable number of pairs of chromosomes, characteristic for each species. At cell division each chromosome divides along its length by a separation of the two strands of the DNA molecule and one strand goes to each daughter cell where it synthesises the complementary strand to reconstitute the double-stranded molecule. The separation of the strands begins from a body called the centromere, which first divides and then draws the dividing DNA to opposite poles of the cell. The centromere may be at one end of the chromosome, in which case the chromosome has one arm and is called acrocentric, or it is at some point along the length of the chromosome, in which case the chromosome has two arms and is called metacentric. In placental mammals the commonest number of chromosomes is 48, that is, 23 pairs of autosomes and two sex-determining chromosomes, but between species the number ranges from 6 to 92. Marsupials have about the same amount of DNA as placentals but it is packaged in fewer, larger chromosomes: the number of chromosomes ranges from 10 in the swamp wallaby,


Table 1.3: Distribution of anatomical characters among American and Australasian marsupial families

A

Chapter Family

RegionA

Sperm

Digits 2 and 3 on hind feet

Incisor teeth

Brain Glans commissure penis

3 3 3 4 4 4 4 5 6 6 6 7 7 7 8 9

Am S Am S Am ANG ANG Aust Aust ANG Aust ANG ANG ANG ANG Aust Aust ANG

paired

separate

10/8

single

6/2

double

Didelphidae Caenolestidae Microbiotheriidae Dasyuridae Thylacinidae Myrmecobiidae Notoryctidae Peramelidae Tarsipedidae Petauridae Burramyidae Phalangeridae Pseudocheiridae Phascolarctidae Vombatidae Macropodidae

divided

single

united

single

Am, North and South America; S Am, South America; ANG, Australia and New Guinea; Aust, Australia.

Wallabia bicolor, to 32 in the rufous bettong, Aepyprymnus rufescens, with 111 species having 14 chromosomes and 37 species having 22 (Table 1.4). In both America and in Australasia there are marsupials with 14 and with 22 chromosomes, so it was for long uncertain which was the ancestral number. However, with techniques that can identify parts of the chromosomes, it is now clear that the chromosomes of all marsupials can be derived from an ancestral number of 14, composed of six pairs of autosomes and the two sex chromosomes. Using specific Giemsa dyes that disclose a pattern along the chromosome, called G-banding, rather like a bar code, it is possible to recognise portions of chromosomes of different species that have the same pattern (Rofe 1978). However, a much more precise technique, called chromosome painting (Fig. 1.7, Plate 1), was developed in 1995, which is transforming understanding of the relationships between parts of chromosomes in different species. In this technique individual chromosomes of one species are isolated and the DNA amplified by a process called polymerase chain reaction and then labelled with a fluorescent dye of a particular colour. This labelled DNA is then mixed with chromosomes from the other species to be compared. It attaches to the complementary DNA in them, and can be visualised by fluorescence microscopy. Only those parts of the chromosomes that are the same in both species will be coloured. By labelling different chromosomes with different coloured dyes it is possible to show that the chromosomes in one species are composed of parts of several chromosomes from the other. This beautiful technique now makes it possible to resolve the paradoxes of closely related species having very different numbers of chromosomes. For marsupials (De Leo et al 1999, Rens et al 2003) it is also enabling the relationships of the different orders and families to be better understood (Fig. 1.8, Plate 1), even those between Australasian and American marsupials (Rens et al 2001).

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Table 1.4: Distribution by family of chromosome numbers (karyotype) among 211 species of living marsupials Hayman (1990), with 31 additions since 1990. Family 10/11 12/13 14 15/16 Didelphidae 22 Microbiotheriidae 1 Caenolestidae 5 Thylacinidae 1 Dasyuridae 55 Myrmecobiidae 1 Notoryctidae Peramelidae 8 Thylacomyidae Vombatidae 2 Phascolarctidae Burramyidae 5 Acrobatidae 2 Phalangeridae 5 Petauridae 1 1 Tarsipedidae Hypsiprymnodontidae Potoroinae 2 Macropodinae 1 2 4 1 Sthenurinae Total 2 5 111 1

16

18 18/19 6

20

22 10

24

32

1 1 1

4

3

3 8

5

7

1 4 17

19

37

1

12

5

17

14

1

1

1

1 3

1

Total 38 1 5 1 55 1 1 8 1 2 1 5 2 8 22 1 1 8 49 1 211

By G-banding and chromosome painting the 14 two-arm chromosomes of South American species are seen to be very similar to the 14 two-arm chromosomes of Australian species, with differences being due to inversions and translocations of bits within individual chromosomes. In species that have more than 14 chromosomes the individual chromosomes can be identified as equivalent to separate parts of these 14. Thus in the koala, with 16 chromosomes, one two-arm chromosome has divided to produce two one-arm chromosomes. Similarly, the 18 chromosomes of the South American species of Monodelphis can be derived by the division of four of the two-arm chromosomes of the related Marmosa species group. In the Australasian family, Phalangeridae, the cuscuses have 14, while the related Australian brushtail possums (Trichosurus) have 20 chromosomes, the latter being derived by division of eight two-arm chromosomes followed by fusion of 4 one-armed chromosomes (Rens et al 2003). The Petauridae and Macropodidae show the greatest variation in chromosome number within each family. For the latter family, this variation has been interpreted as due to several fissions of the ancestral 14 chromosomes to give 22, and then later fusions of some one-armed chromosomes, in several different arrangements, to give the range of numbers from 10 to 32 (Glas et al 1999, Rens et al 2003). This variability reaches its most extreme expression among the rock wallabies (see Chapter 9). It is not clear what the selective advantage of arranging the DNA in different numbers of chromosomes is, although there is some evidence that the upper size limit for a single chromosome is half the length of the spindle formed at each mitotic cell division (Schubert and Oud 1997). Nor is there any clear reason for the bimodal distribution of chromosome number in marsupials from what is now agreed to be the ancestral number of 14, to 22, a shift that has


occurred in at least four families of marsupials independently. This illustrates the point, made earlier in regard to teeth and feet, that genetic changes may occur independently and yet appear to be superficially similar (Rens et al 2003). It may be that there is some fundamental advantage in arranging the DNA into 14 or 22 chromosomes, rather than into any other combination. Sex chromosomes The genes that determine the sexual differentiation of an individual mammal are usually carried on one pair of chromosomes, known as the sex chromosomes. The X chromosome is usually of normal size and contains many genes that are not directly concerned with sexual differentiation, whereas the Y chromosome is usually very small and contains few other genes than those concerned with sex determination. Most marsupials have small X chromosomes, representing less than 3% of the total DNA, and minute Y chromosomes, which do not even pair up with the corresponding X. By contrast in placental mammals the X chromosome represents more than 5% of the total DNA and the Y chromosome has some common genes with the X and does pair with it at meiosis. Jennifer Graves (1996) thinks the marsupial pattern is the original or primitive one and that at some early stage of evolution an additional part of an autosome became attached to the original placental X and Y chromosome. A similar fusion of an autosomal chromosome to the original X chromosome has occurred independently in four species of marsupials, which have large X chromosomes. For instance in the long-nosed potoroo, Potorous tridactylus, females have 10 autosomes and two large X-chromosomes, while males have 10 autosomes, one large X chromosome, one large Y and one small Y chromosome. The large Y chromosome is actually the other half of the autosome that has fused with the original X and at meiosis it is paired with one arm of the large X and the small Y with the other arm. A similar thing must have occurred independently in the swamp wallaby, which has 8 autosomes, a large X chromosome and one large and one small Y chromosome. Chromosome painting with antibodies to tammar chromosomes has shown how this came about. The long arm of the X and the whole of the large Y chromosome are homologous with chromosomes 2 and 7 of the tammar and only the short arm of the X is homologous with the tammar X chromosome (Fig. 1.7, Plate 1) (Toder et al 1997). These unusual arrangements appear to have no significance in the life of the species concerned, since closely related species have the normal XX/XY sex chromosome arrangement. Dosage compensation in female mammals Because female mammals have two X chromosomes, one from their father and one from their mother, they have a double dose of genes that reside on the X chromosome, compared to males, which only have one X chromosome from their mother. In both placentals and marsupials one of the X chromosomes in females is inactive and replicates later than the other one. In placentals the X chromosome that is inactive in any cell may be the one that came from the father or from the mother, so it is called random X inactivation. In marsupials, by contrast, it is almost invariably the X chromosome that came from the father that is inactivated, and this is termed paternal X inactivation. It has been most thoroughly studied in kangaroos but there is evidence for similar processes in other families. In the bandicoots most tissues of the body possess only one X chromosome, the second X in females and the Y chromosome in males being lost during development. Only the tissues of the gonads retain the full sex chromosome complement (see Hayman 1990), a condition termed sex chromosome mosaicism. It is clear from the above that chromosome number and arrangement reflects the relationships between marsupials derived from other criteria, but there have been many changes in the order of the chromosomes and their number that make it hard to interpret. As a primary criterion for understanding the relationships of marsupials, chromosome number is not satisfactory,

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although it can add secondary support to more comprehensive criteria, such as protein and DNA sequences. Marsupial relationships based on protein analysis With the development in the 1960s of amino acid analysers that could determine the structure of large protein molecules, it became possible to use this as a criterion for examining relationships between mammals. The first proteins to be examined were haemoglobins, albumins and some enzymes. These proteins were used to estimate the time of separation of placentals and marsupials on the basis of the number of differences in the amino acids in the comparable molecules. It was assumed that changes in the sequences and composition had changed over time by random mutation and that the rate had been constant. Neither of these assumptions could be sustained adequately: any change in the amino acid composition of the protein would be likely to affect its function and hence not be random, and the rate was determined by the known time of separation determined by fossils and so was a circular argument. The approach with albumins and enzymes of intermediary metabolism were more successful at determining relationships within closely related marsupials and between families but suffered from the same limitation that change was unlikely to be random. The most important development was John Kirsch’s (1977) comparisons between different marsupials of whole serum. With its great number of individual proteins, serology redefined the classification of marsupials for 20 years. Kirsch exploited the then new techniques of immunology to raise antibodies to the serum of one species in a rabbit and challenge other related species with the antiserum from the rabbit. If the challenged species were closely related to the original species, they would react to many antibodies in the rabbit serum; if more distantly related, they would react to fewer. By making numerous such challenges, using many representative species, Kirsch was able to build up a picture of relationships of all the main families of marsupials. His results led to a revision of the earlier classification in several ways. First, the Polyprotodontia are not a single group, the South American didelphids being distinct from the caenolestids and from the Australasian dasyurids and peramelids. Second, the two groups with fused toes, the peramelids and Diprotodontia, were not closely related. But the most surprising result was that Dromiciops of southern Chile appeared to be more closely related to the Australian dasyurid and diprotodont marsupials than to the other South American families. In subsequent years the spermatozoa of Dromiciops were found to be unconjugated, unlike those of all other South American marsupials, and the cytology (Sharman 1982) and anatomy of the ankle joints (Szalay 1982) all supported a closer relationship of this species to Australian marsupials than to South American species. Serology gave no support to a close relationship between ringtail possums (Pseudocheiridae) and the koala, despite their similar type of molar teeth, but rather confirmed a close relationship between koala and wombats, previously suggested from penis and sperm morphology. Why is serology a more powerful criterion for determining relationships than anatomy? One reason is because it deals with interactions between many different proteins, each of which may act as a foreign antigen, so that many different genes are involved. Conversely, using individual anatomical characters, fewer genes are involved and it is difficult to distinguish characters held in common from characters acquired independently by parallel evolution. For instance, the failure of adjacent toes to separate (syndactyly) may be controlled by a single gene. The same holds for comparisons between the amino acid sequences of individual proteins, such as albumin, haemoglobin or myoglobin, which have all been used as criteria for determining relationships.


Marsupial relationships based on DNA The most powerful technique available for determining relationships is the comparison of whole genomes of related species. This can be achieved in two ways. The sequence of base pairs along part of the genome can be examined by comparing the sequence with that from other species. Most of the analyses have been done using four mitochondrial genes (12S rRNA, valine tRNA, 16S rRNA and cytochrome b DNA) and two nuclear genes (exon and intron of the protamine P1 gene and the exon of inter photo receptor retinoid binding protein or IRBP). These provide information about short lengths of the total genome but, unless many sequences are used together, they suffer from the same shortcomings as amino acid sequences in proteins. The other technique, called DNA/DNA hybridisation compares differences in the entire genome, including the so-called ‘nonsense,’ or non-coding DNA, which makes up 95% of the genome. Both sequence analyses and DNA/DNA hybridisation are based on the assumption that small changes in the DNA occur at a fairly constant rate over time, so the greater the difference in the DNA between two species the longer the time since they had a common ancestor. By analysing several different types of DNA, including mitochondrial DNA, with both techniques, the separate values provide internal checks on the accuracy of the estimates. In addition, independent checks can be made when the time of separation of two related species is known from the fossil record. However, a much more cogent argument for the accuracy of this technique is that most of the DNA in the genome of all mammals does not encode for specific proteins and its function is unknown. There are three kinds of DNA in the nucleus: unique or single copy DNA, which codes for proteins and makes up 10–20% of the genome; medium repeat DNA, comprising one thousand to one hundred thousand copies, making up another 20%; highly repeated DNA with more than one million copies, which until recently was called nonsense, redundant or junk DNA because it does not code for proteins. One view is that the latter DNA may be the residue of viruses that infected the species in the distant past, were inactivated in the nucleus but were then trapped in the genome. If highly repeated DNA does not code for protein or have an important function in the life of the mammal, it is unlikely that natural selection will act against changes that occur in it. Hence, mutations in repeat DNA can be assumed to occur at random and to increase with time, so that differences between compared species are more likely to represent real differences in the time since they shared a common ancestor: such differences are time dependent. Time dependency has been tested by comparing the distances between closely related species to another that is much more distantly related. For instance, species representing several different families of marsupial can be compared with a placental species. If rates of mutation since the separation of placentals and marsupials have been constant, two things follow: the genetic distances between each of the marsupials and the placental should be approximately the same; and in every case the distance between any two marsupials should be less than the distance between them and the placental. In several independent trials using DNA/DNA hybridisation this has been found to hold. As Sibley and Ahlquist (1986) said: DNA and the morphological characters traditionally used to reconstruct phylogeny serve to provide different kinds of information. Morphology shows how natural selection has modified structure to adapt organisms to the environments, whereas DNA comparisons give a direct indication of the branching pattern and the approximate branching dates among living lineages. Morphology is functional; the DNA clock keeps time.

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Box 1.2: DNA/DNA hybridisation Each strand of the deoxyribonucleic acid or DNA molecule is made up of a sequence of four kinds of nucleotides, each composed of a five-carbon sugar (ribose) and phosphate group and one of four nucleic acids. These nucleic acids are adenine (A), thymine (T), cytosine (C) and guanine (G), which form two complementary pairs A–T and G–C, held together by hydrogen bonds. There are about three billion nucleotides in the nucleus of a mammal and a triplet of nucleotides codes for an amino acid, so the order of the nucleotides on the strand determines the order in which amino acids line up to form protein chains. Because the nucleotides are complementary, each strand of the DNA is the complement of the other and, when the cell divides, each divided strand synthesises the complementary strand in the new cell. The sugar–phosphate bonds that maintain each strand are very strong but the hydrogen bonds between each nucleotide pair, which keep the two strands together, are weak. When DNA is isolated from a cell and heated to boiling point the weak hydrogen bonds are broken and the two strands separate, without affecting the sugar–phosphate bonds of the separated strands themselves. As the strands cool they may collide with other single strands and, if the nucleotides match each other, the strands will combine. If the separate strands come from the same species, the recombination will be strong and will occur at a high temperature. However, if separated strands from two related species are mixed, not all the nucleotides will encounter a match and the combination will be weaker (Fig.1.9, Plate 2). Being weaker they will only stay together at lower temperatures. This technique of introducing single strands of DNA from different species and testing their ability to combine is called DNA/ DNA hybridisation and allows the closeness of a relationship to be measured directly by the temperature at which the strands hold together. An important assumption is that changes in the nucleotide sequence along the DNA strands occur at a fairly uniform rate. Hence, the longer two species have been separated in time, the greater the number of differences between their nucleotide sequences and the lower the temperature at which they will associate. This technique was first developed in the 1970s and has been used with increasing precision to explore the relationships of living marsupials and their origins. It does two things: it provides values for the closeness of relationships between living species when compared one with another and, by iteration of many individual comparisons, it can allow family trees to be constructed; it also indicates how long ago the separation between each group occurred. These two functions have transformed our understanding of marsupial systematics and phylogeny.

All the families of living marsupials, represented by more than 100 species, have been analysed by DNA/DNA hybridisation and the results compared to other analyses using DNA sequencing data from less complete series (Kirsch et al 1997, Springer et al 1997). These results enlarge and to a substantial degree confirm the earlier results from serology. The major division is not between South American and Australasian marsupials, as previously supposed, but between the Didelphidae, Caenolestidae and Peramelidae on the one hand and on the other the Dasyuridae, with Notoryctidae, the Microbiotheriidae (Dromiciops) and the large multifamily Diprotodontia, comprising most of the Australasian species (as summarised in Fig. 1.10). Using this outline as the best representation of marsupial relationships, it is apparent that many of the anatomical features that have previously been used to classify marsupials have arisen more than once and, therefore, do not represent special affinities (Fig. 1.11). Thus, species with 14 chromosomes are found in every major group, procumbent lower incisors are found in the caenolestids of South America and in all the diprotontids of Australia; syndactyly was almost certainly acquired independently in the peramelids and the diprotodontids, a conclusion supported by brain


commissures and the markedly different mode of reproduction in the two groups; conjugated sperm occur in only two families of South American marsupials. Other features can now be seen to have been independently acquired: the burrowing habit has arisen independently in the bilby (Macrotis lagotis), the marsupial mole (Notoryctes typhlops), the wombats, the rufous hare wallaby (Lagorchestes hirsutus), and the burrowing bettong (Bettongia lesueur); arboreal leaf eating has also evolved independently in the koala, in ringtail possums and the greater glider (Petauroides volans), and in brushtail possums and cuscuses (Phalanger); gliding membranes have evolved independently at least three times; and the pouch may have arisen independently several times also. We will return to the details of this classification as we deal with each group of families in the later chapters.

Marsupial distribution in space and time The earliest mammals appeared about 220 million years ago, before the age of dinosaurs, but they remained a small and insignificant part of the world fauna for the next 100 million years. During the early part of this long span of time the continents of the world were contiguous, so that the different kinds of mammals could spread to all of them. In South America fossils of two extinct groups of early mammals, the dryolestids and symmetrodonts, have been found, and in Australia two monotremes, Steropodon and Kollikodon, have been described from the opal fields of Lightning Ridge, NSW, dated at 110 million years ago (Archer et al 1985, Flannery et al 1995) and another mammal, Ausktribosphenus has been described from Flat Rocks, Victoria, dated to 115 million years ago (Rich et al 1997). The latter was first thought to be a placental mammal but is now considered by Kielan-Jaworowska et al (1998) to be either a symmetrodont or a multituberculate, both of which groups had a long separate evolution from the placentals and marsupials. Ausktribosphenus has left no living representatives in Australia or elsewhere but the monotremes have persisted to the present day and at least one species was present in South America in the early Tertiary period, 60 million years ago. Beginning in the Jurassic period (200–140 million years ago) the landmass of the world split into two super continents: Laurasia comprised Europe, Asia and North America; Gondwana comprised South America, Africa and Madagascar, India, Australia and Antarctica. Since the common ancestor of marsupials and placentals arose during the early Cretaceous in the northern hemisphere, after South America, Antarctica and Australia were isolated from Africa and the northern continents, the first half of their evolutionary history occurred in Asia, Europe and North America. The precise time when the two groups of mammals separated is still conjectural because the critical anatomical features that distinguish them leave no direct evidence in the hard parts that become fossils. Now that protein and DNA criteria can be used with increasing precision to determine the time that living mammals have had a separate history, the time of separation is being narrowed down to between 120 and 100 million years ago. Luo et al (2003) described the whole skeleton and skull and even impressions of fur of a small mammal that lived in China 120 million years ago. They consider that this is the earliest known marsupial on the basis of several features of its dentition, the cusp pattern of its molar teeth, the structure of its limbs and feet, and the possession of epipubic bones, found only in marsupials and monotremes. Equally interesting is the discovery in the same fossil beds of another small mammal that they consider to be a placental mammal. The next oldest fossils of both kinds of mammal occur in Asia and in North America at about 110 million years ago and then with increasing abundance up to the end of the Cretaceous at 65 million years ago.

27

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Figure 1.10: A family tree of all living marsupial families, based on DNA/DNA hybridisation and DNA sequence data. The column on the left refers to the genera of marsupials used to construct the tree and that on the right the families to which they belong. The length of the horizontal connecting lines represent the degree of relatedness and probable time since a common ancestor. Time scale at bottom is only approximate. After Kirsch et al (1997) and Springer et al (1997).


Figure 1.11: How the molecular family tree can be used to test other criteria, such as chromosome number, diprotodont incisors, syndactyl toes and sperm morphology between American and Australian marsupials. After Springer et al (1997).

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In Asia the fossils are predominantly of placental mammals and in North America, predominantly marsupial (Cifelli and Davis 2003, Kielan-Jaworowska et al 2004): there is no evidence of fossil marsupials or placental mammals in the three southern continents before the beginning of the Tertiary. Since North and South America were separated in the Cretaceous, placentals and marsupials must have reached South America across an island chain but being a minor element of the fauna left few fossils. Then the world changed. Marsupial history through the Tertiary Sixty-five million years ago an asteroid of about 10 km diameter struck the earth at a point that is today the Yucatan Peninsula of Mexico (Alvarez 1998). This resulted in the extinction of 70% of all living things in the sea and on the land. It included the demise of the large dinosaurs that had been the dominant vertebrates for 100 million years. In addition, the impact area must have closed off any connection between North and South America, so that the surviving southern fauna became isolated from the rest of the world except Antarctica and Australia. In the aftermath of the asteroid, some small mammals survived and prospered: in North America small marsupials diversified into several kinds during the early Tertiary but then died out and there was no later connection to the marsupials that evolved in South America. Thus, the great southern continent, extending from the equator to the pole and round the other side, was swept clear of most of its fauna from the Cretaceous and isolated from further input until the end of the Tertiary (Fig.1.12). The main marsupial story now takes place on the three southern continents and is intimately associated with the movements of the continents through this time.

Figure 1.12: Disposition of South America, Antarctica and Australasia at the close of the Cretaceous, showing the site of the asteroid impact in the Yucutan peninsula that isolated the continents from North America. Sites V in all three continents where fossil marsupials of the early Tertiary period (65–25 million years ago) have been found. The lower figure shows the disposition of the continents at about 25 million years ago, after their separation and when the circumpolar current had begun to flow. After Archer et al (1993) and Woodburne and Case (1996).

Continental movements through the Tertiary During the past 30 years the precise positions of all the continents and larger islands have been plotted for the whole of the Tertiary, so that their relative movements and times of separation are now understood in detail. The crucial evidence was palaeomagnetism: igneous rocks that


contain iron particles retain the magnetic field that prevailed on the earth when the rock was molten. Because the polarity of the Earth has switched from one direction to the other on many occasions through geological time, it is possible to estimate when a particular rock was molten. This technique disclosed that the youngest rocks occur in the mid-ocean ridges with progressively older rocks of complementary ages outwards on either side of the ridge. This observation can be explained only by spreading of the sea floor with new rock emerging in the mid-ocean ridge and the continents on each side moving apart. The exciting thing is how the findings of palaeontology and the position of the continents through the Tertiary complement the conclusions, based on the findings from DNA analyses, for the times of divergence of the various groups of marsupials in South America and Australasia. At the close of the Cretaceous, 65 million years ago, Gondwana had already begun to break up. Africa and India had separated from South America and Australia, respectively, but South America and Antarctica were still united at what is now the Antarctic (formerly Palmer) Peninsula, and the southern coast of Australia was contiguous with eastern Antarctica, so that the three continents still formed one super continent (Fig. 1.12). No icesheet covered Antarctica and this vast continent in high latitudes was probably rather like Siberia is today, except that it was much warmer and was heavily forested. For the next 20 to 25 million years mammals could have moved each way between South America and Australia via Antarctica. However, since its position over the South Pole has not changed substantially, any species of mammal or bird would have had to survive long polar nights on that continent. As the Australian plate moved north, shallow seas in the west gradually separated it from eastern Antarctica, the last connection being south of Tasmania. When this submerged, Australia became wholly separated from Antarctica between 45 and 38 million years ago, closing off further opportunity for migration of land animals from South America and Antarctica. The land connection between South America and the Antarctic Peninsula of Western Antarctica remained for another 15 million years, to the end of the Oligocene epoch (23 million years ago). After the final separation and the opening of Drake Passage the circumpolar current began to run as it does to this day (Fig. 1.12), bringing profound changes to the climate of Australia and Antarctica. Antarctica became progressively colder and developed a thick ice sheet that obliterated almost all life, and Australia became drier. Since the start of the Tertiary the Australian plate has been moving northwards at a fairly constant rate of 1 degree of latitude every 2.1 million years, or 50 mm per year, and this also contributed to profound changes in its climate and vegetation. While the southern half of New Guinea was a part of the Australian plate and moved north into the tropics ahead of it, for all of the early and mid Tertiary it was either submerged or separated by shallow seas from Australia. It is only in the last 5 million years that it has become a prominent landmass with high mountains and an intermittent connection with Australia (see Fig. 10.2). The only fossil marsupials from New Guinea are of this time: nothing earlier has been discovered (Flannery 1995). Climate changes through the Tertiary Estimates of the sea temperature through the Tertiary suggest that the climate of the early super continent in the south was initially very warm (20oC) but cooled to 1oC during the Oligocene, after Australia had separated from Antarctica (Kemp 1981, Galloway and Kemp 1981). A polar ice cap began to form in the late Eocene epoch (Fig. 1.13) and increased through the Oligocene, when sea ice first occurred. Sea temperatures rose again to 10oC in the early Miocene, 20 million years ago, which was associated with warm, moist climates in Australia. Then, through the second half of the Miocene the sea temperatures were again low, a thick ice sheet covered Antarctica, and much of Australia was dry. There was a return to warm moist climates in the Pliocene, 5–2 million years ago, followed by cold, dry conditions in the Pleistocene, which have persisted to the present time.

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South America in the Early Tertiary era After its isolation there followed a large radiation of placental mammals in South America, which included the ancestors of today’s sloths and armadillos (Edentata) and a wealth of other groups now extinct, such as horse-like litopterns, tapir- and camel-like forms and rhinoceroslike notoungulates (Patterson and Pascual 1972). Most of these placental mammals died out when connection with North America was resumed in the late Pliocene, 3 million years ago, being displaced by the more familiar mammals of the northern continents, such as llamas, bears and large cats. The only surviving descendants of the old Tertiary radiation are the sloths and armadillos. The marsupials in South America fared little better, with the large marsupial carnivores becoming extinct at the same time as the old placental mammals. The three surviving lines can be traced to the Early Tertiary (Fig. 1.13): the omnivorous Didelphidae, which gave rise to the large borhyaenid top carnivores (eg Thylacosmilus); the Caenolestidae, now represented by a few small insectivorous species in the high Andes and southern Chile; and the Microbiotheriidae, represented today by a single species, Dromiciops gliroides, in southern Chile (see Chapter 3). Australasia in the Early Tertiary Vegetation change At the beginning of the Tertiary, the vegetation across the great southern continent was composed of the ancient conifer genera Araucaria, Podocarpus, Phyllocladus and Dacridium, as well as the cool temperate species of flowering plants, such as Nothofagus, Casuarina and the families Myrtaceae, Proteaceae and Banksiae (Martin 1981). All of these persist to the present day in Chile, New Zealand, Tasmania, eastern Australia and New Guinea. Other early elements of the Australian flora that first appear in the Eocene were members of the families Loranthaceae (mistletoe), Cupressaceae and Graminae (grasses). Closed forests covered most of Australia through this immense span of time, while the climate was warm and moist. With the onset of the great arid period through the Oligocene, the evolution of hard-leafed (xerophilous) species began, possibly in response to the leached soils and arid conditions. This is when the genera Eucalyptus, Acacia, Melaleuca and Eremophila first appear. In the Miocene these species largely replaced the Nothofagus and other closed forests. The eucalypts were astonishingly successful in the Miocene, especially in the western, drier half of the continent. Grasses and species of Compositae also become predominant in mid Miocene and remained so thereafter. Mammal history of Australasia Until the 1990s, the mammal history of the first half of the Tertiary of Australia was unknown, the earliest fossil mammals being from the Miocene (about 25 million years ago) and recognisably modern (Fig. 1.13). What had occurred in the first 40 million years of the Tertiary? Now, at a site in southern Queensland, near Murgon, the fossil record reaches back to 55 million years ago (Godthelp et al 1992). There is some dispute about the precise age of the Murgon site, but volcanic rock overlaying it is 40 million years old, so the site must be older than this. At Murgon there are fossil marsupials, representing Australian bandicoots and dasyurids, as well as other fossils that resemble contemporaneous fossil didelphids from Peru (Djiarthia murgonensis, Godthelp et al 1999; Thylacotinga bartholomaii, Archer et al 1993) and a microbiotheriid (Chulpasia, M. Archer pers. comm. 2001). The latter discovery is of particular interest because the living microbiotheriid, Dromiciops in Chile, is more closely related to Australian possums and kangaroos than to either of the other South American families (Springer et al 1994, 1997). Conversely, the Australian bandicoots appear to have diverged from other Australian marsupials 60 million years ago (Fig. 1.10) and are more closely related to the living caenolestids of South America than to any Australian family (Palma and Spotorno 1999). These astonishing results


Figure 1.13: A summary of marsupial history in Asia, Africa, Europe and North America in the Cretaceous and Tertiary periods, and in South America, Antarctica and Australia in the Tertiary and Quarternary. The dotted lines indicate approximate times of migration between continents. The living families are listed in the same order as in Figure 1.10. After Archer et al (1999) and Marshall et al (1990).

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have led to the idea that there must have been considerable exchange both ways between South America and Australia while the connection through Antarctica persisted (Kirsch et al 1991). If there was such an exchange during the first half of the Tertiary, why did none of the placental mammals of South America reach Australia at the same time as the marsupials and undergo adaptive radiation in Australia, as they did in South America? Maybe some did reach Australia but failed to survive: two lower molar teeth and a petrosal (the bone in the skull that encloses the inner ear) discovered at Murgon have been provisionally identified as placental (Tingamara porterorum, Godthelp et al 1992), and palaeontologists from South America consider the teeth to resemble those of the early Tertiary condylarths, which gave rise to the large herbivores in South America. If these claims of relationship are sustained and the age of 55 million years for the Murgon site is confirmed, this is the best evidence yet for a direct link between the earliest South American fauna and the earliest Australian marsupial fauna. It allows for the possibility that for the first 15 million years of the Tertiary there was interchange of mammals across the super continent of South America–Antarctica–Australasia, with a number of separate lineages of marsupials evolving on one or the other part. This could account for the closer relationships of Dromiciops to the Australasian families than to the other living South American families, and for the apparently closer relationship of the Peramelidae to South American Caenolestidae than to any Australian family. Miocene fossil history in Australasia After Murgon nothing is known of mammals in Australia for another 30 million years. What took place in that long span of time remains a mystery but we can infer that much did because, when the Miocene opened about 25 million years ago, an incredible variety of marsupials appear in several sites across Australia. By far the richest of these sites is at Riversleigh, northern Queensland. Here are found representatives of almost all the present day families of marsupials, some very similar to existing species, as well as a host of other kinds that have no living descendants. In the earliest deposits at Riversleigh the climate and vegetation of the site was still rainforest but as time passed the site became progressively drier and the Nothofagus-type forests were replaced with Eucalyptus dominated forest. The new animals reflect these changes: apart from a variety of carnivorous and insectivorous species, similar to the Murgon fossils, there are now present herbivores of various kinds, which were not present 30 million years before. There are koala-like animals adapted to feeding on Eucalyptus, the ancestors of ringtail possums, striped possums and Burramys-like possums, as well as the ancestors of rat kangaroos, the forerunners of the grass eaters of the late Miocene. Whether these various groups evolved in response to the new food resource of grasses, eucalypts and other nectar rich plant species cannot be resolved until more is known about the 30 million year period after Murgon. No longer present are any placental mammals, except bats, which could have reached Australia from the north. The brief appearance at Murgon of a possible placental has not persisted. Was Tingamara really a placental, which left no descendants, or was it an aberrant marsupial? If it were indeed a placental, this raises the interesting question of why placental mammals failed to become established in Australia when they flourished through the Tertiary in South America. Darwin’s ideas and alternatives Darwin (1859) and others before and since him have assumed that the adaptive radiation of marsupials occurred in Australia because they were protected from competition with placentals by the isolation of Australia through the early Tertiary. If Tingamara is indeed a placental mammal, this argument loses much of its force and we must ask why the placentals did not survive in Australia after its separation from Antarctica, while marsupials did? One possibility is that marsupials prevailed in Australia because they were pre-adapted to the Australian


environment that developed after the separation from Antarctica about 45–38 million years ago (Woodburne and Case 1996). These climatic changes could have presented a critical challenge to the mammals isolated on the Australian continent, which were still all very small creatures. As already discussed, two features distinguish living marsupials from living placentals and may have pre-adapted them for survival in Australia: their lower metabolic rate and their manner of reproduction. If either or both of these features were important in the survival of marsupials in Australia, it suggests that Darwin was not right on this matter and that marsupials were preadapted for the special conditions of low fertility soils and uncertain climates. In subsequent chapters we will examine the evidence for this idea.

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Chapter 2


A neonatal brushtail possum; pen and ink sketch by AG Lyne.


R

eproduction is a costly business for any mammal, but essential for survival of the species. For females it demands the transfer of nutrients to the young throughout the dependent period of its development and growth, whether this be by way of yolk, uterine secretions or milk; for males it demands the expenditure of much energy in establishing territory, repulsing rival males and seeking oestrous females; and for the young the stage when it begins to leave its mother is the most vulnerable time of its life. There is, therefore, a high premium on synchronising the most demanding stages of reproduction with the most favourable times of the year. But most reproductive functions have a long leadtime: it takes from 20 to 60 days to mature an egg for ovulation, 80 days to fashion a sperm capable

Figure 2.1: Distribution of births and time of pouch exit (arrow) for brushtail possum, Trichosurus vulpecula, tammar wallaby, Macropus eugenii, and the western grey kangaroo, Macropus fuliginosus, and pattern of rainfall on Kangaroo Island, to show that the young of all three species leave the pouch at the most favourable time of the year. For the tammar this is when the daily consumption of milk is highest. Milk consumption after Cork and Dove (1989).

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of fertilising the egg, and much longer from fertilisation to independence of the offspring. So to match the most vulnerable or costly part of the reproductive process to the most favourable time of the year the first steps in the process must take place many months before the critical event. For mammals the most vulnerable period is when the young are weaned and this usually coincides with the most favourable period of the year. Because lactation in marsupials is long, mating and birth may occur many months before, at an unfavourable season. For instance, the brown antechinus, Antechinus stuartii, gives birth in winter but the young are weaned three months later in late spring when there is abundant insect prey; the brushtail possum, Trichosurus vulpecula, with a five month pouch life gives birth in autumn and the young leaves the pouch in late spring, while tammar wallabies, Macropus eugenii, give birth in mid summer at the hottest, driest time of the year when they are in a poor state of nutrition, but again the young one emerges from the pouch eight months later in spring when its demands for milk are highest and there is abundant new feed after winter rains (Fig. 2.1). How is this timing accomplished? In most habitats the abundance of food varies through the year, whether this be new plant growth in spring after winter rains, the seasonal abundance of insects in a forest or fruiting times in tropical rainforest. If these episodes of food abundance occur regularly each year, mammals that time their reproductive cycle to coincide with it will leave more offspring than competitors that do not. The most widely used predictor in temperate regions of the world is the changing length of the day, which is longest at mid summer and shortest at mid winter. Some species respond to a shortening day length and are termed ‘short-day breeders’ while others respond to increasing day length and are termed ‘long-day breeders’. In the examples given above, male and female brown antechinus come into breeding condition after mid winter and presumably respond to increasing day length (see Chapter 4), whereas brushtail possums and tammars are responding to declining day length after mid summer (see Chapters 7 and 9). In very few marsupials has the response to changing day length been experimentally tested, so inferences about its importance in synchronising breeding must be made with caution. For instance, where the brown antechinus and the dusky antechinus, Antechinus swainsonii, occur together both breed after the winter solstice but the latter species begins five weeks earlier than the former: where the two species live in separate places, both breed at the same time (Dickmann 1982). Clearly, other factors as well as photoperiod are involved (see Chapter 4). Brushtail possums came into oestrus two months early when the normal changes in day length were experimentally advanced (Gemmell and Sernia 1992), but in New Zealand the actual onset of breeding in the autumn varies from year to year in response to other factors, such as an abundant seed set in southern beech trees (Brockie 1992) (see Chapter 7). Similarly, Virginia opossums, Didelphis virginiana, kept in Philadelphia by Farris (1950) were induced to breed in mid winter, two months earlier than normal, by increasing the hours of light in October to mimic the increasing day length that naturally occurs after the winter solstice on 21 December. At this latitude the Virginia opossum is a seasonal breeder but further south in the tropics, where the change in day length is slight, its close relative, the common opossum, Didelphis marsupialis, breeds for most of the year and it is unlikely that change in day length is used to predict favourable times to come. Many tropical species, such as the woolly opossum, Caluromys philander, begin to breed at the end of the annual dry season and their first young leave the pouch when fruit is abundant, while subsequent litters born after the main fruiting season do not survive (Atramentowicz 1982) (see Chapter 3). Conversely, in central Australia, where the rainfall is unpredictable, changes in day length cannot predict favourable times to come and the desert kangaroos have evolved an opportunistic breeding strategy, which is responsive to breaking rains (see Chapter 9). Details of the various breeding strategies that marsupials display will be considered in the later chapters. In this chapter we consider the common features of marsupial reproduction: the


production of mature gametes by males and females at the same time so that fertilisation can occur; the development of the embryo during pregnancy and the extraordinary process of marsupial birth; the changing composition of milk during the lengthy lactation; and the development of the young marsupial to independence. The tammar is the main example against which other species are compared: this is because more is known about reproduction in this species than in any other marsupial.

From gametogenesis to fertilisation Marsupials have between 10 and 32 chromosomes, comprising between 4 and 15 pairs of autosomes and a single pair of sex chromosomes (see Table 1.4). At each division of a body cell, the double strand of DNA of each chromosome separates into its component strands and each reconstitutes the complementary half, so that the two new cells each have a full complement of chromosomes (mitosis). In the transformation of a cell into a gamete (gametogenesis to produce a gamete, or sex cell), however, a two-step division takes place (meiosis): at the first division each chromosome lines up with its partner, the DNA strands of both separate, entwine with complementary strands of the other pair, then separate and go to opposite poles of the dividing cell; at the second division each pair of chromosomes separates, so that each daughter cell receives half the autosomes and one sex chromosome. Since female mammals generally have two X chromosomes, each egg has one X chromosome and since each male mammal has an X and a Y chromosome, half the sperm carry an X chromosome and half carry a Y chromosome. At fertilisation each gamete brings half the autosomes and one sex chromosome to the union, so the sex of the new individual is determined by the sex chromosome carried by the fertilising sperm. Oogenesis: production of the egg from stem cell to ovulation The stem cells that will ultimately give rise to eggs, called primordial germ cells, are set aside at a very early stage in the formation of an embryo and subsequently migrate to the site of the future gonad: in the tammar the primordial germ cells have been identified in the day 17 embryo (Ullmann et al 1997) and have reached the ovary by the time the young female is born on day 26. During their migration and after they reach the ovaries they undergo many mitotic cell divisions to give rise to half a million germ cells by two months after birth; and this is followed by the commencement of meiosis (Alcorn and Robinson 1983). This is typical of all mammals: for instance, in the human female fetus the peak of three million germ cells is reached in the second trimester of pregnancy and has declined to one million by birth; no more are formed after this time and so the number steadily declines through life. The other remarkable thing about the germ cells in the mammalian ovary is that after they enter the first phase of meiosis, when the DNA strands have unravelled, they remain in this state, called the primary oocyte, until after leaving the ovary at ovulation, which may be years later. For long-lived species, such as humans, this can be hazardous because the unravelled chromosomes are more susceptible to damage from radiation and so the incidence of genetic abnormalities increases with increasing age of the mother. In the tammar each oocyte grows to a maximum diameter of 0.12 mm and secretes an outer coat of special proteins around itself, called the zona pellucida. Four or five layers of cells surround the zona pellucida, growing on a basement membrane that isolates the oocyte from the blood circulation of the female; the whole structure is called a primary follicle. Further growth of the follicle, leading to ovulation, depends on hormones secreted by the pituitary gland during the oestrous cycle of the adult female (see Ovulation). The cells of the follicle wall secrete a fluid so that the oocyte lies in a central fluid-filled cavity, the antrum, and

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Figure 2.2: Changes during the oestrous cycle of the brushtail possum, Trichosurus vulpecula, in the corpus luteum and uterine glands and their correlation with progesterone concentration and relaxin activity in the peripheral circulation. Lipid vesicles in the corpus luteal cells contain precursors of progesterone, which are converted to progesterone at day 8–12 before being secreted into the adjacent capillaries. The small dense bodies, which appear later and are associated with the Golgi, probably release relaxin between day 12 and 16. The uterine glands begin to synthesise material by day 4 and maximum release into the lumen occurs between day 8 and 12, after which the exhausted cells are replaced by an underlying epithelium, newly formed from stromal cells. Redrawn from Shorey and Hughes (1973a, b).


in the tammar the whole structure expands to a maximum diameter of 4 mm: it is now called a Graafian follicle, after the Dutch anatomist, Reinier de Graaf, who first described it in the ovary of the rabbit. It is not clear how the selection of follicles that will grow in each cycle is made but the number varies from species to species. In the Virginian opossum and in the Tasmanian devil, Sarcophilus harrisii, up to 50 follicles may grow and ovulate at once, whereas in the kangaroos and brushtail possum very few grow and it is rare for more than one of these to ovulate at one time. In the latter species ovulation occurs in the opposite ovary to the last ovulation, which suggests that there is some local influence in each ovary that stops other follicles from growing when a Graafian follicle is present. This particular influence does not apply in species that ovulate many eggs each time from both ovaries. Ovulation At ovulation the outer wall of the Graafian follicle ruptures, the fluid and the contained oocyte are extruded into the oviduct, where cilia on the surface cells and contractions of the wall move it on to meet the awaiting sperm. Up to the moment of ovulation the oocyte nucleus still contains four sets of chromosomes but ovulation triggers the final process of maturation: at the first division two sets of chromosomes remain in the egg cell and two sets are discarded with a little cytoplasm as the first polar body; the second division only begins when a sperm penetrates the egg membrane (see Fertilisation) and the chromosomes then reduce to a single set ready for fusion with the sperm nucleus, while the other set are discarded as the second polar body. The corpus luteum After ovulation the remains of the follicle left behind in the ovary collapse, the cells enlarge and change their function from nourishing the oocyte and secreting oestrogen, to secreting progesterone, the hormone of pregnancy. A network of fine capillaries and lymph ducts grow among them so that a highly vascular, compact sphere is formed, which is called the corpus luteum, or yellow body, from the colour it acquires as it ages; and its cells are now known as luteal cells (Fig. 2.2). Although the corpus luteum is an inconspicuous little object on the surface of the ovary, it is profoundly important in marsupial reproduction. In the brushtail possum and the tammar it is essential at the beginning and at the end of pregnancy and at the onset of lactation; also in its presence further ovulation is inhibited and in kangaroos it is involved in the phenomenon of embryonic diapause (see Embryonic diapause). It synthesises and secretes at least two hormones: progesterone and relaxin. Progesterone is synthesised on membrane stacks inside the cell and passes easily through the outer cell membrane into the blood circulation, where it can be measured in the blood leaving the ovarian vein and in the general circulation (Fig. 2.2). In the blood it is bound to a protein, called sex hormone binding globulin, which selectively binds to receptors on the cell membranes of target tissues, such as the uterine glands and later the mammary glands, where it releases the progesterone into the cell. The protein hormone relaxin also occurs in the luteal cells of the brushtail possum and the tammar and its concentration rises to a maximum in the middle of the oestrous cycle of the brushtail possum (Fig. 2.2) and at the end of pregnancy in the tammar. The luteal cells become crammed with exceedingly small bodies 200 µm in diameter, budded off the Golgi apparatus, which contain the relaxin (Parry et al 1997b). This was shown by an elegant technique in which luteal cells were incubated with an antibody to relaxin that had been labelled with colloidal gold. Under the the electron microscope it could be seen that gold particles were concentrated in the tiny granules and nowhere else in the cells. By two days after birth both the granules and

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the associated gold particles had almost all gone, presumably released into the blood circulation at birth. The oestrous cycle in marsupials To breed successfully a female marsupial has to resolve two different functions: to prepare the genital tract to receive and convey sperm to the vicinity of ripe eggs; and to prepare the genital tract to secrete egg coats and nourish the developing embryo to full term. For all species of Antechinus and a few other species there is a single protracted phase of oestrus lasting for one or two weeks before ovulation occurs; and this is followed by a luteal phase controlled by progesterone from the corpus luteum. During the long oestrus the female mates with several males and the accumulated sperm survive in special crypts in the oviducts, awaiting the arrival of unfertilised eggs: in the luteal phase the fertilised eggs complete their development. If the female fails to conceive during the prolonged oestrus phase, she fails to become pregnant for that year. Most female marsupials, however, have more than one oestrous event in a year and the interval between one oestrus and the next is called an oestrous cycle. In the tammar this interval is 28 days; in the brushtail possum, 25 days. In other species the length of the oestrous cycle ranges from 21 days in bandicoots (ie Isoodon macrourus), and some rat kangaroos (ie Bettongia), to 25 days in opossums (ie Marmosa robinsoni and Didelphis virginiana) to 45 days in the eastern grey kangaroo (Macropus giganteus). Two phases can be distinguished in the marsupial oestrous cycle: the pro-oestrous phase during the growth of the Graafian follicle leading to oestrus and ovulation, and the luteal phase controlled by the newly formed corpus luteum. In the Virginia opossum, the brushtail possum and most marsupials, pregnancy is accommodated in the luteal phase, which is about two-thirds of the length of the oestrous cycle. After the young are born their sucking suppresses the next pro-oestrous phase and the subsequent oestrus and ovulation. By contrast, in kangaroos and rat kangaroos the length of pregnancy is about the same length as the oestrous cycle, so that oestrus occurs a few hours after the birth of the young, when the female is able to become pregnant again. However, if the newborn young finds a teat and suckles, development of the newly formed corpus luteum and the new embryo are suspended until the end of lactation: this phenomenon is called embryonic diapause (see Embryonic diapause). In all marsupials that have been studied the events in the ovaries and the reproductive tract during the oestrous cycle follow a similar sequence, albeit at different rates. What follows is the sequence in the tammar with some reference to the brushtail possum. Hormonal control of ovulation and pregnancy Four hormones are involved in the process of follicle growth and ovulation and two more in pregnancy. The pituitary gland, which lies directly underneath the midbrain, secretes two hormones, each from a separate cell type. Follicle stimulating hormone (FSH) stimulates follicle growth, expansion of the fluid-filled antrum and secretion of oestrogen by the follicle cells before ovulation. Luteinising hormone (LH) is also important in follicle maturation but its main role is to induce ovulation and the transformation of the cells of the follicle wall into luteal cells that secrete progesterone. Oestrogen, in turn, affects the concentration of pituitary hormones in the circulation. At low concentrations oestrogen inhibits the secretion of LH and FSH – a negative feedback effect – but at high concentration it induces a massive outpouring of LH – a positive feedback effect. The secretion of the two pituitary hormones is also controlled by another hormone, called Gonadotrophin releasing hormone (GnRH), which is synthesised by nerve cells in the brain. It is secreted in regular pulses of about three per hour, which causes the pituitary hormones to likewise be secreted in pulses, especially LH. Both the pulse rate and the amount of hormone


released into the blood stream are important factors in stimulating the ovary to grow follicles and secrete oestrogen. GnRH has not been measured directly in any marsupial but its role can be inferred from three experiments in tammars. When female tammars were injected with GnRH a pulse of LH was detected in the blood within one hour. Furthermore, after removal of the ovaries of female tammars the levels of both FSH and LH rose, indicating the removal of oestrogen negative feedback. The pattern of LH in these tammars was pulsatile with a frequency of 1 pulse every two hours. When injected with low doses of oestrogen, equivalent to levels in intact females, there was a rapid reduction in both hormones – the negative feedback, while higher doses equivalent to oestrous levels caused a very large pulse of LH 20 h after the injection – the positive feedback effect (Horn et al 1985). If the pituitary gland is surgically removed from a tammar, no follicles grow in the ovaries and those that have already begun, go no further (Hearn 1975). A similar effect has been induced in female tammars that were immunised against GnRH (Short et al 1985), so that the pituitary is unable to secrete FSH and LH. The appearance of the ovary under both these experimental conditions looks very like the anoestrous ovaries of species that have a short breeding season, such as the greater glider, Petauroides volans, and the brushtail possum, or red kangaroos during a severe drought, so it is likely that their pituitary hormones are at low levels at these times. Presumably the onset of the breeding season in these species begins when the pulse rate of GnRH secretion from the brain increases and the pituitary gland secretes FSH, which stimulates some follicles to begin their final growth. In the anoestrous red kangaroo the response to breaking rains after a drought is dramatic: in three studies the females had growing follicles and some had come into oestrus 14 days after the rains began (see Chapter 9). The process is slower in the greater glider, taking one month from the start of changes in the ovary to the time of oestrus and ovulation (see Chapter 7). This complex interaction of hormones secreted by the brain, the pituitary gland and the ovary provide the means for each species to adapt its investment in reproduction to the most favourable times of the year. Hormone changes during the oestrous cycle in the tammar wallaby Changes in progesterone and oestrogen in the tammar have been measured in blood samples taken at daily or shorter intervals during pregnancy or the oestrous cycle (Fig. 2.3). Four or five days before birth and oestrus the concentration of progesterone in circulation is high, oestrogen and LH undetectable and FSH rising. At birth, a few hours before oestrus, the progesterone level falls to one-quarter of its former level while the oestrogen level rises to reach its peak level of 15–20 pg/mL at oestrus (see Fig. 2.12). Coincident with, or eight hours after the oestrogen peak, there is a large, transient, pulse of LH, and this is followed 24 h later by ovulation. By sampling blood from the veins draining each ovary (Fig. 2.3) it is clear that the progesterone comes from the ovary bearing the corpus luteum of pregnancy and the oestrogen comes from the other ovary bearing the Graafian follicle destined to ovulate imminently (Harder et al 1984). For the first five days after ovulation the concentration of progesterone in the circulation is low, less than 0.2 ng/mL, and then on day 6 or 7 there is a brief rise in concentration to 0.5 ng/mL (Fig. 2.3). On about day 10 a gradual rise begins to reach a relatively high concentration (about 0.5 ng/mL), which persists to the end of the cycle, when there is a return to the basal level. The first rise results from an increased rate of secretion of progesterone by luteal cells and the second, prolonged rise is due to the increase in the number and size of the luteal cells themselves, not to a change in the rate of progesterone secretion. Neither the secretion rate nor the subsequent growth was affected by removing the pituitary gland, which shows that the corpus luteum, once formed after ovulation, has an independent

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Brushtail possum

Post-partum uterus Non-pregnant

uterus

Figure 2.3: Central panel, changing levels of progesterone in the general circulation of the tammar wallaby, Macropus eugenii, through pregnancy, showing the early pulse and later substantial rise in late pregnancy and rapid fall at birth. Side panels, showing the large output of progesterone from the corpus luteum on one ovary through late pregnancy, and the large output of oestrogen from the Graafian follicle on the other in the post partum period. Data from Harder et al (1985), TyndaleBiscoe et al (1986).

life. How the small luteal cells can independently increase and then decrease their rate of progesterone synthesis and secretion remains unknown. And more remarkable is that they do this while simultaneously dividing and beginning to enlarge. No other endocrine cell is known to do this. At the end of the oestrous cycle the luteal cells cease to secrete progesterone and shrink in size and the corpus luteum becomes a functionless scar in the ovary. Changes in the vaginal complex during the oestrous cycle During the pro-oestrous phase, while the follicles are maturing, the vaginal complex enlarges, the lining thickens and the two lateral vaginae open. At oestrus the vaginal complex becomes several times larger than in the quiescent state and fluid is secreted into the lumen by the glandular lining of the median vagina and by the shedding of lining cells: the appearance of these cells in a swab taken from the genital tract provides evidence of oestrus. The great size of the vaginal complex in oestrous kangaroos led early observers to think that it is the site of sperm storage: but this not so, since sperm do not remain for more than a few days in the female tract of tammars and other kangaroos. However, in tammars that mate with several males during their brief oestrus, the vaginal complex becomes grossly distended with more than 100 mL of seminal fluid, some of which may coagulate to form a plug. The significance of this is not clear, but the fact that the prostate glands of males enlarge greatly during the main breeding period (see Breeding strategies of male marsupials) when the vaginal apparatus of females is enlarged, indicates some complementary function, either in sperm competition or to aid the transport of spermatozoa by stimulating the uterus and oviduct to undergo regular peristaltic contractions during this time. After oestrus the vaginal complex shrinks, while the two uteri enlarge and become more vascular and swollen.


Uterine changes during the oestrous cycle The enlargement of the uteri is due to an increase in the number of cells and their transformation to active secretion. The cells divide during and after oestrus and, in the brushtail possum, this appears to be at a greater rate in the uterus contiguous to the ovary bearing the Graafian follicle or new corpus luteum. Subsequently, when the cells enlarge, this uterus is larger than the other because of the greater number of cells. In the brushtail possum and the tammar each ovarian vein forms a network of small branches that are intimately associated with branches of the uterine artery of the same side (Lee and O’Shea 1977, Towers et al 1986), so that oestrogen, arising in the Graafian follicle, can probably be conveyed directly from the ovary to the contiguous uterus, without being diluted in the general circulation. Later in the cycle in the tammar secretions from the uterus associated with the new corpus luteum consistently contain greater amounts of protein than from the opposite uterus. This may be important for nourishing the developing embryo, which is carried in this uterus. As the corpus luteum grows and progesterone in circulation increases, the gland cells of the uterus are transformed from cuboidal cells with large central nuclei to elongate columnar

Figure 2.4: Protein components, separated by electrophoresis, of maternal blood serum and uterine secretions, and fetal yolk sac, allantoic and amniotic fluid during pregnancy in the tammar wallaby, Macropus eugenii. After Renfree (1973a).

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cells with small basal nuclei. This characteristic form has been termed the luteal phase because progesterone induces it. In the brushtail possum it extends from day 4 (when progesterone begins to increase in the blood) to day 18. Shorey and Hughes (1973a) showed by using the electron microscope that the region around the nucleus of each cell is undergoing active protein synthesis (Fig. 2.2). After day 8 synthetic activity declines and the accumulated secretion pours from the apices of the cells and may even include cell components. This material continues to flow into the lumen of the uterus for several days, reaching a peak at day 12 and declining thereafter. This secretion is essential for the early development of the embryo. Uterine gland cells of the tammar in the luteal phase show a similar appearance under the electron microscope. In this species the secretion has been analysed (Renfree 1973a). It contains glucose and protein, the latter being composed almost entirely of albumin and pre-albumin fractions (Fig. 2.4), whereas the slower moving proteins, characteristic of blood serum, are not detectable. This supports the evidence from electron microscopy that uterine fluid is a product of active secretion rather than an exudate of lymph or serum, as was formerly thought. By day 18 in the brushtail possum, when birth occurs, the gland and epithelial cells are exhausted and dying. Meanwhile undifferentiated cells in the uterus, having assembled under the basement membrane, establish contact with each other and form a new basement membrane beneath the old one. When this is complete the old epithelium sloughs away into the lumen of the uterus where it is absorbed and the new layer becomes a new epithelium in readiness for the next cycle (Fig. 2.2) (Shorey and Hughes 1973a). A similar regeneration occurs in other species; and in some dasyurids, such as the kowari, Dasycercus byrnei (Fletcher 1989), the breakdown at the end of the luteal phase leads to bleeding at the time when birth occurs in the pregnant animal. The saga of the sperm As in the female tammar, the primordial germ cells of the male have migrated to the future testis by the day of birth, where they undergo many mitotic divisions, and become arranged in the wall of the seminiferous tubules of the testis. But their transformation into sperm does not begin until the young male reaches sexual maturity between one and two years later. At sexual maturity these cells undergo waves of mitotic divisions at intervals of 16 days for the rest of the animal’s life (Jones 1989). The wall of the seminiferous tubule comprises two kinds of cell: the spermatogonia that give rise to sperm, and the Sertoli cells that support them. The first divisions increase the number of cells destined to become sperm from the spermatogonia that remain on the wall of the tubule and give rise to the next wave of sperm. This process in the tammar takes 26 days and is followed by meiosis, which takes a further 21 days. Unlike in the female, where each primary oocyte gives rise to one egg and two polar bodies, each spermatocyte gives rise to four spermatozoa, two bearing 7 autosomes and an X chromosome and two bearing 7 autosomes and a Y chromosome. Supported by the surrounding Sertoli cells, these cells now transform into spermatozoa: the nucleus condenses and changes shape; the Golgi apparatus migrates to a position in front of the nucleus and becomes the acrosome; the mitochondria are rearranged into a spiral around the flagellum to provide the energy for swimming; and the Sertoli cells absorb the surplus cytoplasm. In the tammar and brushtail possum this process takes a further 25 days, very similar to placental mammals. At its completion the young sperm detaches from its supporting Sertoli cell, and begins its journey along the length of the seminiferous tubule to the epididymis. The whole process to this point has taken 72 days. The epididymis is a highly convoluted tube, 35 m long in the tammar, and it is while it passes along this tube that the sperm acquires its final form, characteristic of the particular species: in the American marsupials it is in the epididymis that pairs of sperm become attached


to each other by their respective acrosomes (see Fig. 1.6). In the tammar and brushtail possum the sperm take 13 days to reach the end of the epididymis, travelling at about 11 cm/h. Although the spermatozoa can now move and are capable of fertilisation, they can remain here immotile for up to 28 days or until ejaculated at copulation, or shed in the urine. Thus, it takes 85 days to fashion a sperm capable of fertilisation and it can remain in storage for one month. The caudal region of the epididymis lies at the lower pole of the testis and is, therefore, nearest to the bottom of the scrotum. For some reason, not understood, the core body temperature in most mammals is too high for survival of sperm and, if the testes are experimentally returned to the body cavity, or the scrotum is heated, spermatogenesis is disrupted and sperm in the epididymis lose their potential for fertilisation. In a variety of marsupials that have been examined the temperature of the testis and epididymis is about 5oC lower than the core body temperature. In the tammar this is achieved by a counter current heat exchange system in the neck of the scrotum (Setchell and Waites 1969). The testicular artery of the tammar divides into about 150 branches that are intimately associated with the similarly subdivided testicular vein of 50 branches, so that heat is transferred from the warm arterial blood, entering the scrotum, to the cooler venous blood returning to the body. In other marsupials the number of branches is highest in large species, less in small species and there is none in the marsupial mole, Notoryctes

Figure 2.5: Changes in weight of prostate glands of the brushtail possum, Trichosurus vulpecula, and the tammar wallaby, Macropus eugenii, associated with the main and subsidiary periods of the year when females are in oestrus. After Gilmore (1969) and Inns (1982).

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typhlops, which carries the testes in the abdominal wall. This arrangement for cooling the testes is quite different from the arrangement in placental mammals and is further evidence that the evolution of the scrotum in marsupials was independent of that in placentals. Prostate gland The prostate in marsupial males is a glandular outgrowth of the wall of the urethra through which sperm and urine pass to the exterior. Secretions from the prostate glands provide the carbohydrate fuel for the sperm in the female reproductive tract. The size of the prostate varies seasonally in some species, such as the brown antechinus, and in others, such as the brushtail possum and the tammar the size is maximal when females are in oestrus during the breeding season (Fig. 2.5). As in other mammals the prostate is stimulated by testosterone secreted by the testes and this hormone is elevated during the breeding season. Hormonal control of reproduction in the male marsupial Only in the tammar has the role of the pituitary in reproduction been examined. After its removal spermatogenesis ceased, the testosterone-secreting cells shrank and all sperm were lost from the epididymis after two months (Hearn 1975). Likewise, the prostate gland shrank in the same way as it does after castration, indicating that the testes were unable to secrete testosterone in the absence of the pituitary. Conversely when intact male tammars were injected with the brain hormone GnRH the concentration of the pituitary hormone LH rose in the circulation and testosterone was also elevated (Lincoln 1978). From the appearance of the tissues in other species we can infer that pituitary hormones, likewise, stimulate them leading up to the breeding season. Breeding strategies of male marsupials Because of the long period required to produce sperm, male marsupials have three possible strategies for ensuring that they are able to deliver sperm capable of fertilisation when females are in oestrus. First, they can produce sperm continuously and be capable of fertilisation at all times of the year. Second, they can respond directly to the changes in the females; or third, they can independently respond to seasonal changes and only produce sperm during a brief breeding season, synchronised to the breeding season of females. Continuous breeders The adult males of all species of kangaroo, wallaby and rat kangaroo are continuously fertile, as are many species of opossum and the larger arboreal marsupials, such as the brushtail possum and koala, Phascolarctos cinereus. Since male marsupials produce about 25 million sperm each day, there is some cost in this strategy and, under extremely arid conditions, sperm production in kangaroos may be impaired (see Chapter 9). However, a larger cost may be the production of sufficient seminal plasma, mainly from the prostate. In tammars and brushtail possums the males are continuously fertile but the size of the prostate gland is enlarged only during peak breeding, which for the possum is April to May (Gilmore 1969) and for the tammar is January to March (Inns 1982). In both species there is a second, smaller increase in prostate weight in September to October, which coincides with the time when young females of the year come into first oestrus (Fig. 2.5). Both peaks in prostate weight of the tammar were associated with elevated testosterone in the circulation (Inns 1982), which only occurs if the males are associating with females (see Fig. 9.21). Thus, the males are responding to changes taking place in the females as they come into breeding condition.


Seasonal breeders A second strategy, seen in highly seasonal breeders, such as the greater glider, is for the testes to be shut down for most of the year and for sperm to be produced only during the short breeding season of the female. Because of the long lead time needed to produce mature sperm, the males must respond to some external signal, rather than to the immediate condition of the females. In greater gliders the testes begin to enlarge in mid January and spermatogenesis takes place from then until the end of February, with mature sperm being produced only between mid March and mid May (Smith 1969). The ovaries of the females increase in mass from mid March, when pair formation and copulation occurs, and young are born in April to May. After this time the testes of the males shrink and they cease to produce sperm, so females that fail to conceive at the first oestrus of the year do not have another opportunity to become pregnant. This highly synchronised breeding season results in a single, even-aged cohort of young emerging from the pouch in September and becoming independent of their mothers by January (see Chapter 7). The most likely signal that the males respond to is change in day length after the summer solstice, but this has not been investigated in this species. Single cycle breeders The ultimate strategy is seen in all the species of Antechinus and Phascogale in which there is only one wave of spermatogenesis, resulting in a single cohort of sperm (Kerr and Hedger 1983). The onset of spermatogenesis is probably triggered by a photoperiod signal (although this has not been investigated critically) so that the sperm complete their development in the epididymis, and the prostate reaches maximum size, at precisely the same time as females come into oestrus. Within days of the females becoming pregnant all the males die. Males taken into captivity and nursed through the post mating period lived for another year but never produced any more sperm because their seminiferous tubules were totally degenerate (Woolley 1966). Some of the small didelphids of South America have a similarly brief period of sperm production and males only contribute to one breeding season (Pine et al 1985, Lorini et al 1994) but it is not clear that the males are as strictly programmed for one wave of spermatogenesis as the dasyurids are. Copulation and the fate of sperm in the female tract The tammar female will accept the male for a brief period of about eight hours at the time when a mature Graafian follicle is present in one ovary, oestradiol is at a peak level and progesterone is in decline. The essential hormonal conditions that elicit oestrus may be the change in the ratio of the two hormones (Figs 2.3, 2.12). As the female comes into oestrus several males follow her persistently, with the dominant male keeping a close guard and repulsing other males. This male is the first to copulate but other males will do so over the next several hours. A similar pattern has been described in the Virginia opossum, where the largest male mates first and guards the female for several hours after (see Chapter 3). Since ovulation in the tammar does not occur until about 30 h after the end of oestrus the sperm of all the males that mate will be available to fertilise the egg. So, what is the fate of sperm in the female reproductive tract after copulation? In the tammar the sperm enter the cervix within one hour of the first mating and some have reached the oviducts one to four hours later, others arriving over the next 24 h (Tyndale-Biscoe and Rodger 1978). The question of whether sperm travel the distance by their own efforts or are carried by peristaltic contractions of the tract is still debated. In a dilute medium the sperm of bandicoots and mouse opossums do not move forward. However, when placed in medium of the viscosity of the genital tract secretions, the sperm of both species showed directional movement. In the case of bandicoots the rate of

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movement was 0.45 mm/s (Taggart 1994), which is quite fast enough to cover the length of the tract in the time available. Nevertheless, many sperm do not make the distance because they go into the blind ends of uterine glands. Since ovulation occurs about 30 h after the end of oestrus, many sperm will already be at the top of the oviduct when the egg enters it. This is important because the oviduct secretes a second protein coat around the egg soon after it enters the oviduct, which is impenetrable to sperm. Thus, there is only a very brief opportunity for the sperm to penetrate the zona pellucida after ovulation and only those sperm that are present will be able to engage with the egg surface and fertilise it. In the gray short-tailed opossum, Monodelphis domestica, Moore (1996) showed that there is only about 15 minutes after the egg reaches the oviduct when sperm can attach to the zona pellucida, before the new layer to which the sperm cannot attach covers the egg. The same is true of the Virginia opossum (Rodger and Bedford 1982a) and probably all marsupials. Therefore, fertilisation must always occur at the top of the Fallopian tube. Whose sperm fertilises the egg? Because male tammars are larger than females and compete vigorously for access to the female it has been supposed that sperm of the dominant male, which mates first, will reach the egg first. Peter Temple-Smith and colleagues investigated this question in captive tammars (Ewen et al 1993) by comparing minisatellite DNA profiles from the competing males and the subsequent progeny, to identify the father of each one. The results did not support the supposition: there was no correlation between the order of mating and the sire of the young produced. Similar results have subsequently been reported in other captive tammars but no one has repeated their work in wild animals, which would be necessary to be certain that the most dominant male has no selective advantage in the paternity stakes. In antechinus oestrus lasts for several days and ovulation does not occur until the end of that time. During oestrus a female will copulate with up to four different males, so sperm must survive for much longer than in species with a brief period of oestrus. Large numbers of sperm accumulate in the oviducts, where they are packed together in ordered ranks in special crypts (Breed 1994). Unlike in the tammar, many eggs are shed at ovulation in antechinus, which again raises the question of the paternity of the resulting litter. Because they only have this one time in their brief lives to leave offspring there is strong competition among males to mate with oestrous females. Unlike other marsupials and placentals, the number of sperm produced by male antechinus are surprisingly low – less than half a million per ejaculate – and the number declines through the brief breeding season as the limited supply is used up. Nevertheless, the transport of sperm through the female tract is much more efficient than in other species, so that the number of sperm found in the oviduct of a mated female are similar to those of other species (Taggart and Temple-Smith 1991). When the paternity of the resulting litters was determined by genomic DNA profiles it showed that several males share paternity of the litter, with the last to mate leaving the largest number of offspring (Shimmin et al 2000, Kraaijeveld-Smit et al 2002).

Life before birth – fertilisation to parturition Fertilisation The process of fertilisation has now been described in five species of marsupial: the Virginia opossum, the gray short-tailed opossum, the brushtail possum, the brushtailed bettong, Bettongia penicillata, and the fat-tailed dunnart, Sminthopsis crassicaudata, and the details are similar in all of them (Rodger and Bedford 1982b, Baggott and Moore 1990, Breed 1996).


The sperm attaches to the zona pellucida by its acrosomal face and this causes the acrosome to release four enzymes, which dissolve the proteins of the zona pellucida and enable the sperm to push through (Fig. 2.6) (Breed 1996). As soon as the head of the first sperm is through the zona pellucida it attaches to the outer membrane of the egg, setting off a reaction in the outer part of the egg that prevents any other sperm from attaching. It also provokes the egg to complete its second maturation division, extruding the second polar body and reducing the egg nucleus to a single set of chromosomes, ready to meet the complementary set from the sperm nucleus.

Figure 2.6: The moment of fertilisation in the short-tailed opossum, Monodelphis domestica. The head of the sperm is burrowing into the outer matrix of the zona pellucida of the oocyte. From Breed (1996) Reproduction, Fertility and Development 6, 627, fig 16d, with permission of CSIRO Publishing.

Only the head and midpiece of the sperm pass into the egg, the outer membrane and the tail being discarded. In the sperm nucleus, which until now has been very compact, the chromosomes separate in preparation for their pas de deux with the chromosomes of the egg nucleus. Each chromosome meets its pair from the other parent and becomes intertwined with it on the spindle. Each then replicates itself and one full set of the new chromosomes separates to each

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pole of the spindle. The egg cytoplasm divides between the two sets and the first division of the newly fertilised egg is completed. All of this activity takes place in the first 24 h after ovulation, as the egg is carried along the oviduct and enters the uterus. This is much faster than in placental mammals, where the first four or five divisions occur in the oviduct, and the embryo enters the uterus three to five days after ovulation as a ball of about 60 cells.

Figure 2.7: Early development of the tammar wallaby, Macropus eugenii: (a) the mature ovarian oocyte, surrounded by the zona pellucida; (b) the fertilised egg in the oviduct, surrounded by the mucoid layer and engulfed sperm; (c) the 4-cell stage; (d) the12-cell stage in the uterus, surrounded by the shell membrane; (e) an early formed blastocyst on day 6; and (f) a blastocyst in diapause. From own collection.


As the fertilised egg passes along the oviduct it is covered by the second egg coat, in which many sperm become trapped. Then, upon entering the uterus on the second day after ovulation, the egg is covered by a third coat of keratin, secreted by the cells of the uterus (Fig. 2.7). These two egg coats are homologous to the albumin and soft shell membrane of a snake’s egg and of the eggs of platypus and echidna. No placental mammal has a shell membrane but a few species, such as the rabbit and hare, do have the middle mucoid coat. Early development of the marsupial embryo The early development of five species of marsupial is known in detail and of several others less completely (see Tyndale-Biscoe and Renfree 1987, Selwood 1992, Renfree and Lewis 1996). Cleavage and blastocyst formation The marsupial egg contains material, which has been called yolk, although it is not clear that it is the same as the yolk of a hen’s egg. During the first cleavage division it is extruded from the cells into the surrounding space where it forms either a discrete body (in dasyurids and peramelids) or a diffuse mass (in didelphids). In the diprotodonts (eg honey possum, Tarsipes rostratus, greater glider, brushtail possum, tammar and eastern grey kangaroo) there is no evidence of a separate yolk mass and the first two cleavage divisions result in a tetrad of cells. As further cleavage divisions occur the cells become flattened against the inner surface of the zona, which acts as a scaffold, to form a hollow sphere of 60–80 cells, the blastocyst (Fig. 2.7). With the electron microscope it can be seen that these flattened cells are joined at their margins to each other by junctional complexes, which effectively close off the space inside, containing the yolk sac fluid, from the surrounding environment (Renfree and Lewis 1996). Thus, all subsequent movement of substances must be through the cells themselves. In the tammar all the blastocyst cells appear to be identical, with no hint of where the embryo proper will form. However, in dasyurids, the position of the yolk body gives the blastocyst a polarity, with smaller cells near it and larger cells further away. Yousef and Selwood (1996) traced these individual cells through later development in Sminthopsis and Antechinus and showed that the small cells eventually give rise to the embryo proper and the remaining cells become the fetal membranes. This confirmed the views of Hill (1910), who described the development of the eastern quoll, Dasyurus viverrinus, and surmised that the unequal sized cells might have different developmental fates. This finding brings marsupial development closer to the placental pattern (Johnson and Selwood 1996). In most placentals the first cleavage divisions give rise to a solid ball of cells called a morula (Latin for mulberry) without a central cavity containing yolk, and junctional complexes form only between the outside cells. The cells trapped inside become the embryonic stem cells that give rise to the embryo proper and the outside cells give rise to the placenta. Thus, the true embryo is set apart from the supporting tissues from a very early stage of development, whereas in marsupials it had been supposed that the embryo is differentiated much later. Now it seems that early development may not be so different. Indeed, there are some placentals, such as the elephant shrew, Elephantulus myurus, (van der Horst 1942), in which the fertilised egg enters the uterus in early cleavage and becomes a hollow sphere without an apparent presumptive embryo, just like diprotodont marsupials do. The blastocyst is the stage of development that the embryo can reach in both groups of mammals on the resources carried in the egg at fertilisation: further development depends absolutely on nourishment provided by the enclosing uterus. In placentals the embryo hatches from the zona pellucida at this stage and the outer trophoblast cells attach to, or actually invade, the uterus by a process called implantation. By contrast, in marsupials the blastocyst remains

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enclosed by the three egg coats and it grows by absorption of uterine secretions across the blastocyst wall. If the uterine glands are not in a secretory condition, further development cannot take place. Embryonic diapause In a wide variety of marsupials and placentals the blastocyst has the ability to enter a state of dormancy, called embryonic diapause, during which cell division and growth either cease or continue at a very slow pace until an appropriate signal from the mother is received. The signal may be a rise in an ovarian hormone or a specific growth-promoting substance secreted by the uterus. Embryonic diapause occurs in all kangaroos, wallabies and rat kangaroos, except the western grey kangaroo, Macropus fuliginosus, Lumholtz’s tree kangaroo, Dendrolagus lumholtzi, and the musky rat kangaroo, Hypsiprymnodon moschatus (see Chapter 9), and it occurs in pygmy possums, the feathertail glider, Acrobates pygmaeus, and the honey possum (see Chapter 6). Even in the dasyurids, which do not undergo diapause, there is an important change during the formation of the blastocyst: whereas it is possible to grow embryos through the early cleavage divisions in culture, it is not yet possible to carry development through to the blastocyst stage, although blastocysts recovered after this critical period will readily grow on in culture (Selwood and Young 1983). A similar block has been observed in the mouse and it is possible that in all mammals there is a change in the metabolic pathway on the completion of cleavage divisions and the beginning of cell growth. The way in which diapause is controlled in marsupials has been thoroughly investigated only in the tammar wallaby and the results are examined in the following two sections. Initiation and maintenance of embryonic diapause Tammar females come into oestrus within a few hours of giving birth and, if the newborn attaches to a teat and is suckled, the new pregnancy is delayed during lactation. If the young in the pouch dies, or is experimentally removed, the corpus luteum begins to grow, the delayed pregnancy resumes and birth occurs 26–27 days later. So to address the first question of when diapause begins, we can compare the progress of early pregnancy in females that are carrying a new young in the pouch with females that are not. There is no discernable difference in the rate of cleavage up to day 8, at which time the blastocyst has formed and consists of 80–100 cells (Fig. 2.7f). In lactating females no further change occurs in the blastocyst, the corpus luteum does not grow, the early pulse of progesterone does not occur and the uterine glands remain small. By contrast, in the non-lactating female the new corpus luteum grows, the transient early pulse of progesterone in the circulation occurs on day 7, the uterine glands enlarge and become secretory, and cell division continues in the blastocyst, which increases in size, so that by day 12 it is 1 mm in diameter and growing fast. To test the role of the corpus luteum, Sharman and Berger (1969) surgically removed it from several non-lactating tammars on day 2 after oestrus, when the fertilised egg would just have reached the uterus and begun to divide. When they examined the tammars 10 days later all had quiescent blastocysts, whereas unoperated females on the same day had enlarged blastocysts. From this they concluded that the fertilised egg can reach the blastocyst stage without the corpus luteum but it can go no further. How long can a blastocyst remain dormant and not die? In the tammar the blastocyst remains in diapause throughout lactation and then for some months after, only resuming its interrupted development after the summer solstice, when the corpus luteum grows and progesterone levels increase in the circulation (see Chapter 9). Indeed, blastocysts can survive for several months in


ovariectomised females, which suggests that diapause is a passive state from which the blastocyst must be awakened by a special signal. The end of diapause, first steps in reactivation Removing the pouch young starts a train of events in the ovaries, the uterus and the dormant embryo that culminates in birth 26–27 days later (Fig. 2.8). During days 1–3 the process can be reversed if the young is returned to the pouch and resumes sucking: after this day the blastocyst reactivates irreversibly and either develops to full term or expands briefly and then dies (K Gordon et al 1988). Days 1–3 encompass the time it takes for the corpus luteum to resume its own development, which is evident on day 4 when the luteal cells begin to divide and enlarge. Progesterone secretion increases and the transient pulse occurs on day 5, 6 or 7. Diapause -----

Reactivation

Embryo metabolism

anaerobic glycolosis

glucose oxidation

Embryo development

none

cells resume division

increased protein synthesis

blastocyst expansion begins

Endometrium no change

increased protein synthesis

luteal phase in glands

release of uterine secretion

Corpus luteum

reversible if sucking resumes

cells divide, large pulse of low level of elevated elevated sharp fall in progesterone progesterone progesterone progesterone progesterone progesterone increases

Effect of removing corpus luteum

blocks embryo reactivation reactivates, then dies

Days after removing pouch young

1

2

3

4

5

glucose uptake increases shell membrane breaks down

embryo goes to term

6

7

fetus begins to secrete cortisol

birth

blocks blocks birth mammary and lactation gland development 8

9

10

18

19

23

24

25

26

Figure 2.8: Summary of changes after diapause in the tammar wallaby, Macropus eugenii: the series of changes in the corpus luteum, the endometrium of the uterus, and in the metabolism and growth of the blastocyst after the sucking inhibition is lifted by removing the pouch young (RPY), and the effects of removing the corpus luteum on successive days after RPY.

When the corpus luteum was removed on day 4 the blastocyst resumed its development but then died; but when the corpus luteum was removed on or after day 6, development went to full term but birth did not occur (Sharman and Berger 1969). Thus, the corpus luteum is necessary for the first six days of active pregnancy and for parturition but is not required for the whole process of growth and differentiation of the embryo. Very similar results have been found in the quokka, Setonix brachyurus, the Virginia opossum, the brushtail possum and the long-nosed potoroo, Potorous tridactylus (Tyndale-Biscoe and Renfree 1987). In all these species the essential factor for gestation to continue is that the uterus is in the luteal phase at the time of operation: once established, its secretions are adequate to initiate and maintain development of the embryo to full term. If the blastocyst awaits a signal from the stimulated uterus to reactivate, what is the nature of that signal? It cannot be the abundant secretions of the luteal uterus because they do not occur until later, so it must be something more subtle coming from the corpus luteum. The first

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changes in the reawakened blastocyst have been the subject of much research and point to what the signal may be. The first sign that the blastocyst is reactivating occurs on day 4 when the first mitotic figures are seen in its cells as they resume division; and on day 5 the first changes in the synthesis of nuclear and cytoplasmic RNA occur (Moore 1978, Shaw and Renfree 1986), the precursor to new protein synthesis (Fig. 2.8). Cell division and protein synthesis require energy, which comes from carbohydrate metabolism in the blastocyst. During diapause the main pathway of glucose metabolism is anaerobic glycolysis (Spindler et al 1998) with a high ratio of adenosine triphosphate to adenosine diphosphate (ATP:ADP), which may inhibit the enzymes of oxidative metabolism. Between day 3 and day 4 after RPY this changes dramatically: ATP stores become depleted so that the ATP:ADP ratio falls significantly by day 3 and the contribution of glucose oxidation to ATP production rises significantly by day 4. This means that the capacity of the blastocyst to generate energy from each molecule of glucose increases nearly 20-fold between day 0 and day 5. This is followed by a 10-fold increase in uptake of glucose by day 10, reflecting the profound changes in the metabolism of the growing blastocyst (Pike 1981) and the increasing demands for energy to fuel the active transport of uterine secretion across the blastocyst wall as it expands. Without the increased resources of uterine secretions, including glucose, expansion must fail, as it does when the corpus luteum is removed before day 6. The findings of Spindler et al (1998) show that the first signal from the corpus luteum to the blastocyst must occur on day 3, coinciding with its own release from the quiescent state. Is this signal a change in the concentration of progesterone coming directly to the uterus via the network of small branches of the ovarian vein and the uterine artery, mentioned earlier (Fig. 2.3), or is it a specific growth factor secreted by the uterus? Geoff Shaw (1996) has described the increase in the number of proteins in uterine secretion when it is stimulated with oestrogen or progesterone and one such factor, platelet activating factor (PAF) was found to be elevated on day 3 in tammars (Kojima et al 1993), but it is not known whether this factor is involved in blastocyst reactivation. Several other polypeptide growth factors could also be candidates but at present none has been identified. So the question as to the nature of the early signal remains unanswered, but the critical time for it has been narrowed down to early on day 3. Once reactivated and with an adequate supply of nutrients from uterine secretions, the embryo continues without further delay to completion of pregnancy: indeed, in the tammar the time from the early pulse of progesterone to birth is constant at 22 days. Development of the fetus The blastocyst increases in size by absorption of fluid and by a rapid increase in the number of cells: by day 10 it is a translucent vesicle 5–10 mm in diameter. It then develops a polarity that shapes its future. In one hemisphere some cells become detached from the outer layer and lie free inside and spread around the inside of the vesicle forming a rather loose network held together by thin strands. This becomes the inner layer of the two-layered blastocyst and its cells are destined to become the linings of the gut, lungs and bladder – the so-called endoderm. The first formed endoderm cells induce the overlying cells to differentiate into an oval plate of thick cuboidal cells, which will become the outer layer of the embryo – the ectoderm, which gives rise to the skin, brain and eyes. As the blastocyst continues to expand the outer cells of the yolk sac become very thin but still retain complete attachment with each other. The cells of the future embryo, however, do not expand so that this area becomes more distinct and is clearly visible in living vesicles as an oval plate (Fig. 2.9a, Plate 3). The embryo now develops a bilateral symmetry with the appearance of a midline groove and knot of cells at one end, in the same manner as does the chicken egg at 18 h of incubation. As in


the chicken, these are the site of a remarkable migration of cells from the outside between the two existing layers to form the third or mesoderm layer, which will give rise to blood, muscle and bone. At the same time the notochord is formed and over it develops the neural tube from which develops the brain vesicles and spinal cord. From the lateral mesoderm differentiate the somites, which will later form the trunk muscles; these first appear on day 16 in the tammar embryo (Fig. 2.9a, Plate 3) and on day 17 the primordial germ cells can be seen at the periphery of the somites. On day 19 the great vessels of the heart appear and soon after they begin to pulse; and the limb buds appear. Between days 19 and 26 the embryo completes the development of all the organ systems necessary for life outside the uterus. This development is entirely dependent on the functions of the fetal membranes that surround it and form the placental connection to the uterus that provides its nourishment, respiration and excretion. Egg coats, fetal membranes and placental transport Possible functions of the egg coats The three egg coats consist of the inner zona pellucida, laid down by the oocyte while in the ovary, the mucoid coat laid down in the oviduct and the keratinous shell membrane secreted by the uterine cells. As already mentioned, an important role for the zona pellucida is to act as a scaffold for the early development of the blastocyst before its cells have made strong connections to each other, but it is not clear what function the other two coats have in marsupials: the zona pellucida and mucoid coat disappear as the embryo enlarges and they may provide nutrients at this stage. By contrast, the shell membrane remains intact for more than three-quarters of the length of pregnancy, as the fertilised egg transforms into a fluid-filled vesicle and developing embryo. During the enormous expansion of the embryonic vesicle the shell membrane becomes extremely thin but remains intact, so much so that the vesicle can be rolled out of the uterus and grown in culture without damage. Since the shell membrane is intact uterine secretions must be able to pass through it for synthesis into new tissues. In the last few days before birth, proteolytic enzymes secreted by the fetal membranes of the embryo break down the shell membrane (Denker and Tyndale-Biscoe 1986), and an intimate connection is made between the membranes and the uterine lining to form the yolk sac placenta. This pattern holds for all marsupials: the period before breakdown of the shell membrane ranges from 8 to 23 days, and much longer when embryonic diapause occurs, whereas after the shell membrane breaks down the rest of gestation is brief, varying from four to five days in bandicoots and opossums to 8–10 days in macropods. The reason for the persistence of the shell membrane has long been a puzzle. One idea is that it protects the developing embryo from exposure to maternal antibodies that would otherwise be directed at the embryo’s proteins, which are genetically different from the mother’s. The amount of time after the shell membrane breaks down and the fetal tissues are exposed is sufficiently short to avoid such a reaction by the mother’s immune system and consequent rejection of the fetus. Evolutionary biologists have suggested that this was the key difference between marsupials and placental mammals: placental mammals evolved a way to avoid maternal rejection of the fetus, which opened the way for prolonged gestation whereas marsupials did not and were constrained to have a very brief gestation and deliver very immature young (Amoroso and Perry 1975). One way to test this idea is to sensitise the female marsupial to the antigens of the prospective father, by giving her two successive skin grafts from him: foreign skin is very antigenic, and since the fetus will inherit paternal antigens, this should provoke a strong reaction at the end of gestation. When we did this with tammars the females developed very strong immune responses to the male, rejecting the second skin graft within a few days, as was to be expected

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(Laplante et al 1969). Nevertheless, they subsequently conceived a succession of offspring with the male to whom they were sensitised, and some females produced young to the same male for five years without any reduction in their fertility (Walker and Tyndale-Biscoe 1978, Rodger et al 1985). These experiments therefore do not support the idea that the shell membrane protects the female from male transplantation antigens and leaves open the question of what the function of the shell membrane is. Another possible function is that it is a resilient container during the formation of the fetal membranes, rather as the zona pellucida is for the early development of the blastocyst. While

Figure 2.10: Fetal membranes in four marsupials to show the different proportions of the yolk sac, allantois and amnion in late pregnancy. From the top they are the quokka, Setonix brachyurus, eastern quoll, Dasyurus viverrinus, koala, Phascolarctos cinereus, and long-nosed bandicoot, Perameles nasuta. Thin lines represent ectoderm, dashed lines, endoderm, and thick lines, mesoderm. After Tyndale-Biscoe and Renfree (1987).


studying tammar embryonic vesicles growing in a test tube, from which the shell membrane had been removed, we made a curious discovery: instead of the amnion growing over and enclosing the embryo, as occurs in the uterus, the folds of amnion remained on each side of the embryo like two large cushions. Without the constraint of the shell membrane the folds of the amnion were apparently unable to press the embryo down into the yolk sac and close over it and so the embryo remained exposed. It is interesting that in all marsupials the amnion envelops the fetus before the shell membrane breaks down. Development of the yolk sac, amnion and allantois While the main form of the embryo is being laid down, mesoderm is spreading out beyond the limits of the embryo plate, until it extends nearly half way around the yolk sac. However, it never reaches the whole way, so that the marsupial yolk sac wall is two layered in its lower half and three layered in its upper pole (Fig. 2.10). As blood vessels develop only from the mesoderm, the three-layered part becomes the vascular yolk sac (Fig. 2.9b, Plate 3) and the main respiratory organ of the fetus, while the two-layered, non-vascular yolk sac is the main route of absorption of uterine secretions: together these comprise the yolk sac placenta of marsupials. Within the limits of the embryo and for a short distance beyond, the mesoderm splits into two layers, one being applied to the ectoderm and the other to the endoderm. The space between these layers is the coelom or body cavity. Folds of the outer layer now rise up at the head end and sides of the embryo and enshroud it within two membranes, an outer chorion and an inner amnion, which encloses a fluid-filled space, the amniotic cavity in which the developing fetus now lies (Fig. 2.9c, Plate 3, Fig. 2.10). Once again, this manner of forming the amnion and chorion closely resembles birds, reptiles and monotremes and in all of them the embryo is contained within a shell membrane when the amnion is formed. In all reptiles, birds and mammals (amniote animals) another sac grows behind the yolk sac: this is the allantois (Fig. 2.9c, Plate 3), which in birds, reptiles and monotremes becomes the main respiratory surface for the embryo in the egg. In placental mammals the allantois retains this function and also assumes the major nutritive role as the fetal component of the definitive, allantoic placenta. In most marsupials, however, the allantois is an inconspicuous sac with a modest vascular supply buried in the enfolding yolk sac (Fig. 2.10); and its main function is to store urine excreted by the fetal kidneys in the last days of gestation. In a few species, such as the koala, it is larger and in the bandicoots it becomes highly vascular, its cells fuse with cells of the uterus, and it forms an allantoic placenta, which functions for the last three days of gestation (Fig. 2.10). In summary, marsupials have developed the yolk sac as the main organ of exchange between the fetus and the mother (a yolk sac placenta) and the placental mammals have developed the allantois as the main organ of exchange (the allantoic placenta). Notwithstanding this main distinction, some placental mammals, such as the rabbit, have a yolk sac placenta in the early part of gestation and some marsupials have an allantoic placenta in the last days of gestation. Placental transport in the tammar In the tammar, as we have seen, the protein constituents of the uterine secretion differ from serum in several particulars, which is further evidence that it is not a simple exudate but is the product of active secretion (Fig. 2.4). The outer surface of the yolk sac wall of the tammar is seen under the electron microscope to carry a thick weft of microvilli with pinocytotic vesicles at their bases (Tyndale-Biscoe and Renfree 1987). Mitochondria are numerous, as also is rough endoplasmic reticulum, all of which indicate transport and metabolism of uterine secretions. Later, when the shell membrane breaks down, the yolk sac membrane has direct contact with the uterine epithelium and the microvilli of the two tissues interdigitate and establish an intimate

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contact: although younger embryos can be rolled unharmed from the opened uterus, this is not possible after the shell membrane has gone. This intimate contact of the tissues brings the fetal and maternal blood vessels very close together, thereby facilitating exchange between them. These histological changes are reflected in changes in the constituents of the yolk sac fluid. Functions of the yolk sac placenta During the pre-attachment phase, nutrient and respiratory requirements must be met by exchanges with uterine secretions across the yolk sac membrane and at this stage the concentrations of glucose and urea in yolk sac fluid resemble those in maternal serum (Fig. 2.11) (Renfree 1973b). The protein concentration in yolk sac fluid is much lower than in serum and is so similar to uterine secretion, which comprises just albumin and pre-albumin (Fig. 2.4), so it is very likely that the great increase in the volume of the yolk sac at this stage is caused by direct uptake of uterine secretion across the yolk sac membrane. After attachment there are big changes in the composition of yolk sac fluid. The protein contents now resemble those of serum, although there has been no change in the composition of the uterine secretion, the concentration of glucose increases steadily (Fig. 2.11) and the colour of yolk sac fluid changes from clear, through straw to yellow by full term. The yellow colour results from breakdown products of haemoglobin and reflects the development of the fetal liver in which this takes place. By contrast allantoic and amniotic fluids remain clear and contain the same limited number of

Figure 2.11: Glucose and urea content of yolk sac fluid (O) and allantoic fluid (O) through pregnancy in the tammar wallaby, Macropus eugenii. Shaded regions represent the range of concentrations in maternal serum. After Renfree (1973b).


proteins as formerly (Fig. 2.4). Glucose concentration in the allantois is low but the urea concentration increases progressively to four times that of the other compartments and of fetal serum: this coincides with the development of the fetal kidneys as excretory organs. The marked changes in the yolk sac fluid after attachment could result from greater ease of transport of maternal proteins across the contiguous circulations, or they may reflect the growing maturity of the fetal systems, such as the liver and kidneys. The evidence supports the second conclusion: when maternal proteins, labeled with an isotope of iodine, were injected into the maternal circulation in late pregnancy almost none of the label was later detected in the yolk sac; and iron-binding proteins, called transferrins in yolk sac fluid have a different mobility to transferrins in maternal serum, indicating that they have been synthesised by the fetus itself. Thus, fetal membranes in the final days of gestation are actively controlling the transfer of substances found in the fluid compartments, reflecting the growing autonomy of fetus as it reaches the time to be born.

Parturition The birth of a marsupial is an extraordinary phenomenon. The newborn is so small and so undeveloped that it belies belief that it could be anything but passive in the events that take it from the uterus, through the temporary birth canal and thence from the cloaca to the pouch and attachment to a teat. For as long as it has been known many explanations have been entertained for how marsupial parturition is achieved. Even after Hartman (1920) established that the young make the journey to the pouch unaided by the mother, direct involvement of the young in the process of parturition was still discounted. About 30 years ago we were still convinced that no special endocrine changes were associated with parturition: now we know that the tiny fetus is as much involved in the events leading to its birth as is the lamb or the human baby. Role of the corpus luteum in parturition The single most important organ in the preparation of the female tammar for birth is the corpus luteum of pregnancy. If the corpus luteum is surgically removed after day 6, the pregnancy will continue through to full term but the fetus is not born: the fetus leaves the uterus but is then impounded in the median vagina, where it dies. If the corpus luteum is removed on or after day 23 (of the 26-day gestation), however, the fetus is born and reaches the pouch but dies one day later, presumably because the mammary gland is unprepared for lactation (see Preparation of the mammary glands, or mammogenesis) (Young and Renfree 1979, Harder et al 1984). Progesterone is the main hormone that prepares the genital tract and the mammary gland, since injections of progesterone will substitute for the absent corpus luteum. Without progesterone the temporary canal through which the young must pass after it leaves the uterus is small or absent, whereas progesterone softens the tissues, making it possible for the young to pass through, still enclosed in its fetal membranes. This is an important point because it was earlier thought that the young might force its way through, using its well-developed arms. Clearly it is unable to do this while still in the amnion. The other hormone secreted by the corpus luteum is relaxin, but its role in preparing the genital tract is still unclear. The peak production of relaxin is three days before birth and it declines rapidly after birth, so the role is inferred (Tyndale-Biscoe 1981, Parry et al 1997b). Relaxin may act with progesterone to soften and dilate the cervix, as it does in other mammals, and it may be involved in preparing the birth canal, but critical experiments to test this have not been done. Because in the tammar and other macropods, birth is followed after a few hours by oestrus, oestrogen might also play a role in birth. However, removal of the Graafian follicle, the source

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of oestrogen at this time, during late pregnancy did not prevent parturition at the normal time, although it did abolish post-partum oestrus (Harder et al 1985). This result confirms the earlier work of Newsome (1964), who observed that red kangaroos could give birth in the absence of growing follicles, and consolidates Poole’s (1975) study of grey kangaroos that can give birth without ever having a post-partum oestrus. Release of the young from the uterus The first hint that the marsupial fetus might be involved in determining the time of its birth came from a study of hybrids between the two species of grey kangaroos by Kirsch and Poole (1972). The pregnancy of the eastern grey kangaroo, Macropus giganteus, is 36 days while that of the western grey kangaroo, Macropus fuliginosus, is 31 days. However, when eastern grey females were crossed with western grey males, pregnancy was 34 days, indicating that the genetic status of the hybrid fetus had shortened it. When the female hybrids grew up and were themselves mated to western grey males, their pregnancy was further shortened to 31.6 days. In tammars Merchant (1979) discovered that the interval from oestrus to post-partum oestrus in pregnant tammars was significantly shorter than the interval from oestrus to oestrus when the same tammars were not pregnant, which further implicated the fetus and/or the placenta in controlling the events around birth. More direct evidence of the fetal role came later when it became possible, simultaneously, to measure six hormones in circulation around the time of birth in tammars, compared to the same hormones circulating at the equivalent time in non-pregnant tammars. These results clearly showed that the pattern and occurrence of all the hormones differed between the two states. Progesterone falls rapidly at the time of birth but gradually over several more days in the non-pregnant cycle. Large but highly transient peaks of the hormones prolactin, prostaglandin and mesotocin occur close to the time of birth (Fig. 2.12), whereas none of these hormones is detectable in the non-pregnant tammar at the equivalent time. Since these hormones are associated with parturition in placental mammals it was interesting to find that they are also associated with parturition in the tammar. Within one hour of giving birth the female tammar comes into oestrus, which is associated with high levels of oestrogen and is followed 24 hours later by a sharp peak of LH, followed by ovulation. In the non-pregnant female the same sequence is followed but two to three days later. The questions are the respective roles of each hormone and how the fetus is involved in their secretion. One way to test the importance of the fetus is experimentally to advance its development ahead of its mother’s. Clark (1968) did this in red kangaroos by injecting lactating females with progesterone, which mimicked the action of the corpus luteum and reactivated their diapausing blastocysts (Fig. 2.8); 10 days later she removed the young from the pouches of their mothers, which initiated development of the corpus luteum in their ovaries. If the mother determines the time of birth, the young should not have been born until 31 days after removing the pouch young, whereas if the fetus determines the time it should have been born 10 days sooner: the latter result occurred. This design was repeated some years later in tammars when the several hormones were also measured (Tyndale-Biscoe et al 1988). As with red kangaroos, the tammars gave birth early and the pre-partum pulse of prolactin occurred at the time of birth, three days earlier than in control animals, again showing that the fetus is influencing the hormonal events. There is now good evidence on how the tammar fetus influences the cascade of events around the time of birth (Fig. 2.13). Briefly, the pituitary gland in the head of the fetus stimulates the fetal adrenal gland to produce the hormone cortisol, which in turn causes a massive release of prostaglandin from the yolk sac placenta and endometrium of the uterus. This in turn


Figure 2.12: Profiles of six hormones in circulation around the time of parturition in the tammar wallaby, Macropus eugenii. The precipitate fall in progesterone and the very brief pulses of prostaglandin, mesotocin and prolactin only occur in the presence of a fetus and not in nonpregnant tammars at the same time after oestrus. From Shaw and Renfree (2001) Reproduction, Fertility and Development 13, 657, with permission of CSIRO Publishing.

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provokes the release of prolactin and mesotocin from the mother’s pituitary gland. Together the prostaglandin and the mesotocin stimulate contractions in the uterine muscle, and the fetus is expelled through the cervix into the median vagina and thence to the outside. The prostaglandin also provokes the whole repertoire of the birth position and behaviour by the mother and the mesotocin and prolactin prepare the mammary glands for the start of lactation (for details see Boxes 2.1–2.4).

Figure 2.13: Diagram to show how the hormones in Figure 2.12 interact during birth. While the corpus luteum prepares the cervix and birth canal for passage of the young, and the mammary glands for lactation, it is the fetus that starts the whole cascade of events, through four steps of amplification: fetal pituitary; fetal adrenal; placenta and endometrium; and maternal pituitary and brain.


Box 2.1: Role of prostaglandins during birth Shaw (1983) showed that prostaglandins stimulate contractions in the uterus of the tammar, these contractions become more marked as the time of birth is approached and are larger in the pregnant than in the non-pregnant uterus. Shaw et al (1999) showed that two kinds of prostaglandins are synthesised in the placenta and endometrium in late pregnancy. They cultured pieces of each tissue in vitro and measured the production of the active forms and their metabolite, PGFM. The active form of prostaglandin, PGF2_, is produced in the endometrium of the pregnant uterus from day 18 and the rate increases three-fold by the day of birth. It is also produced in the non-pregnant uterus but does not increase in a consistent way and its contribution to the total output of prostaglandins is probably negligible. However, the production of PGF2_ by the fetal membranes is considerably higher and increases at a faster rate. The production rate in none of these tissues is affected by mesotocin or the cortisol-like hormone, dexamethasone, so the inference is that neither the posterior pituitary nor the fetal adrenal gland are involved in the increased rate of production. PGF2_ is metabolised to another form called PGFM, which is the form that is measured in the circulation around the time of birth. PGFM is not produced by the fetal membranes and only to a very small extent by the endometrium, so the origin of the PGFM in the blood is not clear. It is possible that the PGF2_ is released into the circulation and is metabolised to PGFM in the lungs. Whatever the process, PGF2_ is essential for parturition to occur and for the female to adopt the birth position, because both events are prevented when pregnant tammars are injected with an antagonist of PGF2_, indomethacin, from day 24 to 28 (Renfree et al 1994). PGF2_ also induces the release of prolactin from the maternal pituitary (Hinds et al 1990) and causes the precipitous fall in progesterone from the corpus luteum (Renfree et al 1994).

Box 2.2: Role of mesotocin during birth Mesotocin is a peptide molecule of nine amino acids, very similar to the pituitary hormone oxytocin secreted by the posterior pituitary of placental mammals. Indeed, it only differs by one amino acid and has the same functions in kangaroos that oxytocin does in placentals, namely to stimulate contractions in the uterine muscle and stimulate milk let down. This is supported by the results of surgically removing the posterior pituitary from pregnant tammars: they failed to give birth and the fetuses were found dead in the uterus (Hearn 1974). Mesotocin is found in the posterior pituitary of marsupials (Bathgate et al 1995) and there is a marked pulse of mesotocin at parturition (Parry et al 1996), which almost certainly comes from the maternal pituitary. The link to the uterus at parturition is supported by the presence, in the muscle tissue surrounding the pregnant uterus, of specific receptors that bind mesotocin (Parry et al 1997a). The concentration of these receptors was higher in the pregnant than in the non-pregnant uterus and reached a maximum just before the time of birth, suggesting that the presence of the fetus may stimulate synthesis of the receptor protein. Renfree et al (1996b) tested the role of mesotocin by infusing an antagonist of its receptors into tammars on the last three days of pregnancy. The effect was to delay birth but not prevent it taking place. These separate pieces of information all support a role for mesotocin at the time of birth by stimulating contractions of the gravid uterus and so releasing the fetus for the start of its journey to the pouch. As the peak of mesotocin in circulation occurs after the young has been born, it may also be important in the initiation of lactation.

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Box 2.3: Role of prolactin during birth The maternal pituitary also secretes the transient pulse of prolactin but its timing is clearly influenced by the fetus, since it only occurs in pregnant tammars and was advanced when the fetuses were experimentally advanced. However, its role is still unclear: when first discovered it was thought to be the signal that initiated birth (Hinds and Tyndale-Biscoe 1985) but when Fletcher et al (1990) tested this by treating pregnant tammars with the prolactin antagonist, bromocriptine, which suppressed the pulse, birth was not delayed but the young died soon after birth. Like mesotocin, the main role of the prolactin pulse may be after birth, preparing the mammary gland for imminent lactation.

Box 2.4: Role of the fetus during birth Despite its tiny size and immaturity, the tammar fetus has surprisingly well-differentiated pituitary and adrenal glands. Leatherland and Renfree (1983) described cells in the pituitary four days before birth, that appeared to be capable of secreting hormones, such as adrenocorticotrophic hormone (ACTH), which controls the activity of the adrenal cortex. The adrenal cortex of the tammar fetus was suspected to be active many years ago when Catling and Vinson (1976) measured cortisol in the blood of two full-term fetuses. Shaw et al (1996) tested the importance of cortisol in initiating parturition by treating pregnant tammars with a related steroid, dexamethazone, which caused them to deliver their young a day earlier than control tammars treated with saline. While the young were significantly lighter than the controls the interesting point was that the other hormones, PGFM, prolactin and progesterone, underwent their normal sequence of changes a day earlier than the control tammars. This strongly suggested that cortisol from the fetus was acting as the signal to start the cascade of events leading to parturition. Now Ingram et al (1999) have demonstrated that the source of cortisol at parturition is indeed the fetus: from day 24 to day 26 the adrenal cortex of the fetus increases in size and the content of cortisol to a peak just before birth, as does the concentration of cortisol in yolk sac, allantoic fluid and fetal blood plasma. More importantly, the production of cortisol by fetal adrenal tissue in culture was increased three-fold in the presence of ACTH or prostaglandin (PGE2). This was the evidence needed to show that the fetus is capable of timing its own delivery.

We can now try to put these several pieces together in the order in which they occur in the living tammar (Fig. 2.13). While the cervix and birth canal are being softened by elevated progesterone and relaxin from the maternal corpus luteum, the fetal pituitary is maturing sufficiently to secrete ACTH, the fetal adrenal is enlarging and beginning to synthesise cortisol, and the yolk sac placenta is synthesising prostaglandin PGF2_. Cortisol secretion accelerates towards the day of birth under the influence of fetal ACTH and prostaglandin. Fetal cortisol may influence the release of prostaglandins from the yolk sac and endometrium, which starts muscle contractions of the uterine wall and the evacuation of the fetus. The rapid rise of PGFM in maternal circulation as birth occurs may cause the immediate release of mesotocin from the mother’s posterior pituitary gland, which enhances those contractions but is not essential for birth. PGFM also induces the prolactin pulse from the mother’s anterior pituitary gland and the fall in progesterone from the declining corpus luteum. This sequence and the several interactions are very similar to the events that occur at parturition in the best-studied placental mammals – sheep and humans. In both these placental species the fetal pituitary and adrenal cortex are central in the onset of parturition, through their interplay with placental and uterine prostaglandins and oxytocin. But in both species


the fetus is far more advanced in its development at birth than a tammar is, and it is easier to comprehend that it could have well-developed hormonal controls in place. It has been much harder to appreciate that a fetus of 200–400 mg is equally capable of controlling its own birth, and in the same way. Now consider that the tammar at birth is one of the largest marsupial neonates (see Fig. 1.2). Do all marsupial neonates have the same capacity to control the time of their birth? The young of dasyurids weigh less than 20 mg at birth and those of the honey possum weigh 4 mg! What little has been found out about these matters in other species of marsupial seems to be in accord with the tammar: bandicoots, brushtail possums and northern quolls, Dasyurus hallucatus, have well-developed pituitaries and adrenals at birth (Gemmell and Nelson 1988), the cells of which contain secretory granules similar to those in hormone-secreting glands. In the bandicoot a large pulse of PGFM occurs around the time of birth (Gemmell et al 1980) and injections of prostaglandin induce birth behaviour in several species. This points to the likelihood that fetuses of other marsupials are similar to the tammar fetus in being able to control events at birth. But if one thinks the achievements of the prenatal marsupial are remarkable, consider what it does after it is born. Maternal behaviour during parturition Birth has now been observed and described for many species of marsupial and the story of its discovery often told. In most species the young makes its way from the cloaca to the pouch by its own efforts within minutes and the mother does not directly help it. However, she does adopt a particular posture and pattern of behaviour that aid the young in its journey. Detailed observations of the entire behaviour leading up to birth in the red kangaroo and the tammar wallaby have been made and both recorded on film. In the tammar the sequence of events can be related to the hormonal changes outlined (see Parturition) (see Renfree et al 1989 for an illustrated account). The pouches of non-lactating kangaroos and tammars contain a brown, dry scale and in the last week of pregnancy this is removed by the female putting her muzzle into the pouch, while holding the sides open with her forepaws. The pouch appears clean and moist and a small bud develops on the end of each teat. Non-pregnant kangaroos at the same stage of the cycle also clean the pouch in the same way. Since in both states this is the stage of the oestrous cycle when the progesterone level is elevated and before the levels of other hormones have risen, it is likely that increased progesterone provokes this behaviour. While cleaning the pouch the female may adopt the so-called birth posture, in which she sits on the butt of the tail with the tail and hind legs extended forward, so that the cloaca is directed upwards. In the hour or so preceding birth, pregnant animals adopt this posture much more frequently than non-pregnant animals and the intensity of pouch cleaning increases greatly. At the same time the animal begins to lick the cloaca as well. The physiological state of the pregnant female also changes: alertness is lost, shivering and whole body spasms occur and she is undisturbed by outside events; she can be picked up without showing alarm and when released will immediately resume the birth posture and persistently lick between the cloaca and the pouch. The discovery that PGF2_ would induce this intense behaviour was made by chance while trying to work out the respective roles of prolactin and PGF2_ for the rapid decline of progesterone at the end of pregnancy (Hinds et al 1990). Non-pregnant females at the end of the oestrous cycle were injected with PGF2_ and almost immediately went into the full repertoire of birth position and behaviour and remained like that until the decline of hormone in the blood (Fig. 2.14). Other females injected with prolactin showed no change in behaviour. Subsequently Shaw (1990) showed that the intensity and duration of the behaviour was directly related to the level of prostaglandin in circulation and that it could be induced in females that had never been

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Figure 2.14: Two non-pregnant female tammar wallabies, Macropus eugenii, that adopted the birth position a few minutes after receiving an injection of prostaglandin into a tail vein.

pregnant and, more remarkably, in adult male tammars. The males adopted the birth position and intently licked the scrotum and genital region! These results show that birth behaviour is not learned but is ‘hard wired’ in the brain. In other marsupials that have since been tested PGF2_ has the same effect on birth behaviour. Indeed, this may be a very ancient function of PGF2_, since it also induces spawning behaviour in fish and sexual behaviour in a wide range of other vertebrates. From the less complete observations on other marsupials birth appears to be much the same as for the red kangaroo and tammar wallabies. The quokka, brushtail possum, koala and Virginia opossum adopt the same posture as the red kangaroo and tammar but grey kangaroos give birth in a standing position. In the small dasyurids, Antechinus swainsonii and Dasycercus byrnei, and in the northern quoll, the female stands with its hips raised so that the young travel down from the cloaca to the teats: in the latter species two gelatinous strands are ejected from the uteri in which the emerging young pass towards the pouch area, where each one grasps a hair and wriggles to a teat (Nelson and Gemmell 2003). In the northern brown bandicoot, Isoodon macrourus, the female lays on her side with the upper leg raised and the cloaca brought close to the backward opening pouch in such a manner that when the young appear they almost fall into the pouch, still attached by the elongated umbilical chord (Gemmell et al 1999). In the kangaroo and tammar, the onset of birth is heralded by a flow of fluid at the cloaca from the ruptured yolk sac. This is immediately licked up and is followed, in the red kangaroo, by the appearance of the intact allantois, which may fall to the ground. Then the young emerges head first, still enclosed in the fluid-filled amnion, from which it now frees itself with its swinging, clawed arms. In the tammar the young also emerges in the amnion but it is followed by the allantois and the remaining yolk sac membranes, trailing away from the umbilicus, from which it breaks free, partly aided by the mother’s persistent licking. If the mother is undisturbed, the young orientates itself towards the pouch and rapidly moves there by grasping the fur in its claws


and by alternate movements of the forelimbs. At the same time the head turns from side to side, so that the muzzle describes an arc. Within a few minutes, usually less than five, it has gained the lower lip of the pouch and thence passes from view. If the mother has not been suckling a previous offspring, all four teats are available, otherwise three are. These have developed a small bud at the tip, which the young now draws into its mouth. For two days after birth the young can be removed from one teat and placed on another and it will suckle successfully, but as time passes this fails because only the suckled gland secretes milk (see Journey of a lifetime – adaptations for reaching and attaching to a teat). Similarly, young transferred to the budded teat of a non-pregnant female will attach and be suckled successfully until weaned. In tammars it is possible, experimentally, to place a newborn young on each of the four teats and all four young will grow for a few weeks, showing that all the teats have the potential to support a young at the time of birth. Of course, in other species that have multiple young at birth, all the teats will usually be occupied. In some species, such as the Virginia opossum, the number of young born often exceeds the number of teats and those that fail to grasp a teat die. Journey of a lifetime – adaptations for reaching and attaching to a teat The marsupial at birth is a marvelous composite of embryonic structures and precociously developed functional organs (Figs 2.9d, Plate 3, Fig. 2.15). The head, shoulders and forelimbs are relatively large but the hind legs are mere paddles, and the hips and tail are small. The ears and eyelids are shut but the mouth and tongue are large, the nostrils are open and the blood vessels beneath the skin are prominent. These precociously developed organs enable it to reach the pouch unaided, to respire, to attach to a teat and gain nourishment from the mammary gland. Locomotion – forelimb skeleton, musculature and innervation The forelimbs and shoulders are well developed and the digits are armed with sharp recurved claws, with which the newborn grasps the mother’s fur. These claws are extensions of the outer keratin layer of the skin and are shed a few days later: they are not the definitive fingernails, which appear later in development. The skeleton of the shoulder region is a single cartilage, comprising the future scapula, glenoid, coracoid and sternum, which provide firm support for the forearm during the journey to the pouch (Fig. 2.15b) (Klima 1987). This is similar to the arrangement in adult monotremes and reptiles. A week after birth the coracoid separates from the sternum and is reduced to a small nubbin, so that the scapula is only loosely joined to the sternum by the clavicle, as in adult placentals and marsupials. One hundred years ago, before it was appreciated that the young travels unaided to the pouch, this anatomy was seen as a vestige from the reptilian ancestry of marsupials, rather than a functional adaptation. However, the relatively large coracoids do not support large coracobrachial muscles, as these bones do in monotremes and reptiles: the muscles of the shoulder and forelimb of the newborn marsupial have the adult mammalian form and are innervated by a large brachial nerve (Fig. 2.15a). The muscles of the neck and thorax are also well developed, and the young animal carries out a series of alternate lateral contractions, starting at the head and ending with the forelimbs. What controls these movements? Langworthy (1925) showed that the movements continued normally after he removed the cerebral hemispheres of newborn Virginia opossums, so the control centre must be in the brain stem or spinal cord. The same probably holds for other marsupials, since few of the neurons that will constitute the cerebral cortex have migrated there at the time of birth (Reynolds and Saunders 1988). Furthermore, the special tract of fibres that carries signals from the cerebral cortex to the spinal cord, called the pyramidal tract, does not develop until the week 5 after birth in the opossum (Ward 1954). So how is this complex and highly coordinated series of

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Figure 2.15: Anatomy of the newborn marsupial: (a) diagram of the head and forearm of a newborn tammar wallaby, Macropus eugenii, to show the innervation of the nose by cranial nerve I; the innervation of the lips, jaws and tongue by the trigeminal ganglion (cranial nerves V and VII); and the innervation of the forearm by the brachial plexus (redrawn after Hughes et al 1989); (b) the shoulder girdle of the newborn brushtail possum, Trichosurus vulpecula, to show how it is united with the sternum on the day of birth but has separated from it 14 days later (redrawn after Klima 1987).

movements controlled? Is it entirely controlled at the level of the brachial plexus in the spinal cord, or are higher centres in the brain stem involved? Ho (1997) and Ho and Stirling (1998) have investigated the neural control of the alternating opening and closing of the hands and movement of the forelimbs in the newborn tammar, which they call clock-like and which is retained in the later stages of pouch life. While the young tammar was attached to a teat and warm the movements ceased but if it was cooled or removed from the teat, the clock-like alternating movement resumed. When one limb of the newborn was


restrained the movement of the other became irregular, which indicates that there is coordination of the two sides in the spinal cord. The information about this is probably conveyed to the brachial plexus via sensory nerves from stretch receptors in the forelimbs. However, information about the limbs cannot go further because sensory nerve connections from the brachial plexus to the brain stem have not developed at this stage. When Ho isolated the brain stem and spinal cord of the newborn tammar in a water bath the alternate firing of the left and right brachial motor nerves continued, thereby showing that control of rhythmic limb movement is located in the spinal cord. In addition, motor nerve connections from the brain stem to the brachial plexus can modulate the movements of the limbs. This means that the information needed to find the pouch and attach to a teat can be provided from sensory input from the mouth. Hughes et al (1989) strikingly demonstrated this with freely held newborn tammars: when their lips were gently stroked the clock-like movements immediately ceased. Sense organs that function at birth – smell, touch and balance The behaviour of the newborn marsupial suggests that three sensory systems are functional at birth, namely smell, touch and balance. The evidence for a sense of smell or touch is the way marsupial young redirect their movements when they reach the edge of the pouch and enter it. This indicates a response to the texture of the naked, moist skin in the pouch or to a particular smell emanating from the pouch. Sensory cells, called olfactory knobs, have been described in the nasal epithelium of three species (Gemmell and Rose 1989) and well-developed nerve fibres connect to the relatively large olfactory lobes of the brain (Hill and Hill 1955). Touch receptors, called Merkel cells, with associated nerve cells, have also been described in the skin surrounding the mouth of the newborn of six species, representing five families (Gemmell et al 1988, Hughes and Hall 1988). The trigeminal nerve plexus (cranial nerve V), a prominent feature of the newborn (Fig. 2.15a), conveys sensory information from the mouth, lips and tongue to the brain stem. Its motor fibres, together with those from cranial nerve VII, control the movements involved in sucking and breathing (Hughes et al 1989). The trigeminal plexus enters the brain stem close to the motor fibres that pass down to the brachial plexus, so it is highly likely that it is involved in the coordination of the sensory input from the nose and lips and the movements of the trunk and forelimbs. Hughes and Hall (1988) and Hughes et al (1989) called this complex system the ‘locomotor generator’, which is most highly developed in the newborn tammar, less developed in the newborn brushtail possum and least developed in the newborn quoll. Since much less is known about newborn marsupials other than the tammar, it is not possible to correlate the anatomical level of development with neonatal behaviour. For instance, is the less developed condition of the northern quoll associated with Nelson and Gemmell’s (2003) observations that the newborn, still enclosed in the amnion, are aided in their journey to the pouch by the gelatinous strands from the mother, rather than making the journey to the teat by their own efforts? They have noted that, unlike tammar newborn, these tiny creatures cannot repeat the journey if returned to the cloaca. Does the newborn have a sense of gravity? The question of whether the newborn marsupial has a sense of gravity has been more contentious. Because the young one usually climbs upwards to enter the pouch it was thought that it must orientate according to gravity. However, the first studies of the Virginia opossum led McCrady (1938) to conclude that neither the utricle nor semicircular canals of the inner ear were sufficiently developed to provide any information on gravity to the newborn. He suggested that the young orientates itself by the peculiar geometry of its body at birth, the passive hind

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part of the body hanging down between the two large forelimbs. Cannon et al (1976) supported McCrady’s observations in experiments with newborn quokkas. They found that the young invariably orientated themselves upwards and, when the mother was laid on her side, so that the opening of the pouch was not directly above the young, it passed within 10 mm of the pouch and went on towards the mother’s chest. They concluded from this that olfaction is not important, and that gravity is the sole influence directing the young to the pouch, although they did not know how gravity was being sensed. Since then the structure of the inner ear of the newborn brushtail possum, brushtailed bettong and northern quoll have been studied with the electron microscope (Gemmell and Nelson 1989b, Gemmell and Rose 1989) and all three species have a well-differentiated sensory, ciliated epithelium on the inner surface of the vesicle that will become the inner ear, and lying on the epithelium is a cluster of crystalline granules or otoliths. This structure is probably capable of providing information to the newborn marsupial about gravity and thus position, although the neural connection to the brain has not been established. Adaptations for sucking – lips and hyoid musculature The buccal cavity is large and the tongue is large and muscular. After the young becomes attached to the apical bud the teat expands inside the mouth and the lips and tongue grow around it, so that the young becomes firmly attached and can be removed only with difficulty. This arrangement also allows the young to develop a negative pressure in the buccal cavity by depressing the floor of the chamber with the hyoid muscles and so suck milk from the teat. The large trigeminal nerve supplies these muscles also (Fig. 2.15a). Griffiths and Slater (1988) demonstrated that newborn tammars and red kangaroos are able to suck, by offering them a fine pipette filled with warm water or milk and held so that the pipette was below the mouth of the young: all these young animals sucked up 20–30 mg of fluid in about two to three minutes and the fluid in the pipette exhibited motions that indicated that the young were sucking. This disposed of an old belief that the marsupial young is unable to suck and that the milk is forced into it by contractions of a special muscle, the ilio-marsupialis, which penetrates the mammary gland. However, when this muscle was electrically stimulated, no milk was expressed from the teat (Enders 1966, Griffiths and Slater 1988). This muscle has a different function, which will be discussed later. Swallowing milk – epiglottis and stomach Because the young is permanently attached to the teat for several weeks it might be thought that the flow of milk to the gullet would interfere with breathing, especially as the air from the nostrils must cross the gullet to reach the trachea. However, newborn marsupials have a special arrangement that avoids this: at the back of the mouth the epiglottis is large and extends through the soft palate so that the glottis opens into the nasopharynx. The buccal cavity extends around each side of it and communicates with the oesophagus, so that the young can feed and breathe simultaneously. The stomach and duodenum are in an advanced stage of development at birth and so is the pancreas. By contrast, the small intestine and colon are not. Respiration via skin and lungs: surfactant and the role of cortisol Baudinette et al (1988b) have studied the metabolism of the newborn tammar as it makes its first journey. The effort required to reach the pouch is considerable and the young has energy reserves sufficient for little more than one journey. As in any newborn mammal the first requirement for respiration is the switch from placental exchange to air, and the rearrangement of the great vessels entering and leaving the heart to convey oxygenated blood from the lungs instead of from the placenta. This involves the separation of the right and left halves of the heart by the


formation of a wall between the two atria and between the two ventricles. It also involves closure of the connecting artery (ductus arteriosus) between the pulmonary artery, taking blood to the lungs, and the dorsal aorta taking blood to the rest of the body. In placentals, such as a baby, these three events occur immediately after birth, at the first breath; if they do not, the baby goes blue through lack of oxygenated blood. In marsupials the process is slower and in the tammar closure is not complete until day 3 (Runciman et al 1995). As the young tammar is born, blood flow from the yolk sac placenta ceases and blood flow to the lungs increases, as they fill with air. Since the circulation to the lungs is not separate from that to the rest of the body during the first hours after birth, mixing of oxygenated blood from the lungs with blood from the rest of the body must occur until day 3. Is this difference from placental mammals because the young of marsupials are so small that respiratory efficiency is less important after birth, or is it because there is another route for gas exchange – the skin? The newly born brushtail possum, kangaroo and tammar are a fiery red colour on the first day after birth and they have very conspicuous subcutaneous blood vessels (Fig. 2.9d, Plate 3) and a moist skin. John Shield (pers. comm. in Richardson and Russell 1969) noted that newborn quokkas paled in an atmosphere low in oxygen and recovered their bright red colour when returned to an oxygen-rich atmosphere. If respiration can take place across the skin as well as the lungs, there is clearly an advantage in allowing the blood to be mixed in the heart. During the journey to the pouch, when the demands for oxygen are great and the lungs are filling with air for the first time, gas exchange across the skin may well be important, especially in the smallest marsupials. Baudinette et al (1988a) discount the importance of this in the tammar for two reasons: by day 3 after birth the distance between the air and the blood in vessels under the skin was 100 times greater than the distance across the lung epithelium (100

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10 Prolactin-receptor concentration

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Figure 2.18: Lactation in the tammar wallaby, Macropus eugenii, is controlled by the hormone prolactin and specific receptors for prolactin on cells of the mammary glands: (a) after an initial pulse of prolactin at birth the concentration of the hormone remains low throughout Phase 2, rises in Phase 3 and declines when the young is weaned. (b) The concentration of prolactin receptors in all four mammary glands (6) rises through Phase 1, under the influence of progesterone, and then declines after birth in all except the suckled mammary gland (O), in which the receptor concentration rises to a maximum at the end of Phase 2 and then plateaus through Phase 3. After Tyndale-Biscoe and Renfree (1987)

Sucking increases the target instead of the hormone In the tammar the concentration of prolactin receptors on the cells of the sucked gland increase rapidly after attachment of the young (Stewart 1984). One week after birth the weight of the sucked gland had doubled and the receptor concentration per cell had also doubled. By contrast


the adjacent non-sucked glands had shrunk to one-fifth their former weight and the prolactin receptor concentration had halved. Thus, one week after birth there is a 40-fold difference in prolactin receptor concentration between the sucked gland and the non-sucked glands (Fig. 2.18b). Fostering additional newborn young onto the other teats showed that this is entirely due to the presence of the young on the teat, because all four mammary glands developed equally and produced milk. Thus, in the tammar, and by inference all marsupials, the sucking stimulus of the newborn young increases the local target for prolactin without increasing the secretion rate of prolactin from the pituitary. This explains why only the glands to which young attach lactate. It is yet another way in which the tiny newborn marsupial redirects its mother’s physiology for its own survival. Phase 2 lactation in the tammar lasts until about day 200, during which time the young first voluntarily relinquishes the teat, its eyes open and it is becoming physiologically independent (Fig. 2.17). Late stage lactation Phase 3 of marsupial lactation is equivalent to the whole postnatal phase of lactation of placentals, during which prolactin is elevated and the young are growing rapidly to independence (see Growing up and leaving the pouch). The lactating mammary gland grows with the suckling In both the tammar (Stewart 1984) and the brushtail possum (Smith et al 1969) the weight of the mammary gland increases three to four-fold in late lactation and remains at this size until the young is weaned, when it rapidly shrinks. The sucking stimulus still maintains lactation but the concentration of prolactin receptors does not increase any more after 85 days, although because of the larger size of the gland, the total number of receptors is substantially higher. More importantly, from about day 150 the level of prolactin in the circulation increases and this is directly due to the sucking stimulus of the young. When the young is experimentally removed the prolactin level falls to a low concentration, and after the young is returned to the pouch and sucking resumes, the prolactin level again increases. This response is the same as that seen in placental mammals, where the frequency of sucking episodes is the main factor in maintaining elevated prolactin in circulation. Further evidence for the importance of the sucking stimulus rather than milk withdrawal in late lactation comes from the Virginia opossum. Lactation normally ceases at about 90 days when the young begin to feed actively outside the pouch. By fostering a second litter 60 days old to a weaning mother, Reynolds (1952) was able to maintain lactation for 154 days. The mammary glands first regressed to the size appropriate to the younger litter and then began to enlarge again slowly in response to their increasing demands. The persistent sucking of the younger litter, although withdrawing less milk, was yet more effective in maintaining lactation than the infrequent sucking of the older litter. In two other examples the activities of the young determined the length of lactation. A pouch young of the swamp wallaby, Wallabia bicolor, was fostered into the pouch of a red kangaroo (Merchant and Sharman 1966) where it grew at a faster rate than normal swamp wallabies. However, it left the pouch at 267 days, which is the normal pouch life for a swamp wallaby but 30 days longer than that of a red kangaroo. Similarly, a grey kangaroo young, fostered into a red kangaroo, was retained in the pouch for 374 days, or 135 days longer than a red kangaroo young would have stayed. What causes the young to leave the pouch is discussed later (see Growing up and leaving the pouch). The accelerated growth of the swamp wallaby is particularly interesting because its rate of growth was equal to that of young red kangaroos and greatly exceeded that of normal swamp

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wallabies, and it reached sexual maturity much before them. Similarly, tammar young growing in large mothers grew faster than those growing in the pouches of small mothers (Merchant 1989), and the female young reached puberty earlier. Both observations suggest that the condition of the mother is a significant factor in the growth and development of the young. How mesotocin helps Dairy farmers call it let down. It is the preliminary response of the cow that allows the milk to flow easily and be drawn from the teat. In the cow this is known to involve the release of the hormone oxytocin from the pituitary gland into the circulation where it stimulates contractions of the muscle fibres that surround the alveoli of the mammary gland. The normal trigger for this signal is the butting of the calf but other external events associated with milking can also elicit release of oxytocin. Like the cow, the lactating mammary glands of marsupials and monotremes are highly responsive to oxytocin and within minutes of an injection of oxytocin milk begins to flow from the teat of the lactating gland. However, in most marsupials the hormone responsible for this is not oxytocin but the related molecule, mesotocin. Lincoln and Renfree (1981) examined the response to oxytocin in agile wallabies, Macropus agilis, during Phase 2 and 3 of lactation, by measuring the pressure in the teat ducts of the lactating glands. Since agile wallabies, like red kangaroos, can suckle two young of different ages concurrently, Lincoln and Renfree (1981) were able to measure the response to the same dose of oxytocin at two stages of lactation simultaneously. They were interested in how a dose of oxytocin that could stimulate the small gland at the start of lactation did not flood the pouch with milk from the mammary gland at Phase 3 of lactation. They found that the small gland is more sensitive to oxytocin than the late stage gland and responded maximally at doses that would not elicit a response from the mature gland. Indeed, they concluded that the background level of oxytocin is enough to stimulate the early stage gland sufficiently for the small young one to obtain the small volume of milk it requires. Conversely, the mammary gland supporting the older young requires a higher level of oxytocin to release the mature milk and this is induced by the vigorous sucking stimulus of the older young.

Composition of marsupial milk Because the milk of marsupials must support the young from its tiny size at birth until it is an independent animal, the composition changes profoundly through lactation (Fig. 2.19). At first the milk is a dilute fluid containing more sugars than fats, while at the end of lactation it is rich in fats and proteins but contains little sugar. Not only do the major components change, the constituent sugars, fats, proteins, salts and minerals also change in relation to the needs of the developing young. The milks of several species have now been sampled through lactation, and the sequence of changes is the same in all of them, although the time course differs in relation to diet and life history of each species. Monotremes are similar to marsupials in this matter. These profound changes through lactation in marsupials, and monotremes, are quite different from those in placental lactation. Apart from the first few days, when colostrum is produced in some species, the constituents and volume of placental milks change very little through lactation when compared to the large changes in all marsupials. Conversely, the differences between species of placental mammals in the concentration of milk and its constituents are much greater than between different marsupials, from the very dilute milks of horse and human (10% solids) to the highly concentrated fatty milks of seals (78% solids). We will now look at the composition of marsupial milk in more detail before considering the development of the young (see How the young marsupial becomes independent). Once again the tammar is the main species against which others will be compared.


Figure 2.19: The changing composition of milk through lactation in the tammar wallaby, Macropus eugenii; (a) total of carbohydrates, lipids and proteins as g/100 mL milk; (b) major components of the whey proteins; (c) daily energy intake calculated as product of milk intake (Fig. 2.1) and energy content of milk components, with area under the curve representing the total energy exported by the mother tammar through lactation. After Nicholas (1988) and Cork and Dove (1989).

Carbohydrates in marsupial milk Lactose, composed of one molecule of glucose and one of galactose, is the main sugar of placental milk but it is a very transient component of marsupial milk: during days 1–4 of lactation, lactose is the only sugar but thereafter its concentration rapidly declines. For the next 200 days tammar milk contains high concentrations of several oligosaccharides of increasing size, most composed of one molecule of lactose and from one to seven molecules of galactose. After 210 days these oligosaccharides disappear from the milk and the only carbohydrates are galactose and glucose in very low concentration. The question that exercised people in the 1970s, when this was first discovered, was why the simple sugars are packaged in the milk as large molecules that then have to be broken down in the young animal?

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Part of the answer may be that the early milk of marsupials contains higher concentrations of sodium and potassium ions than late stage milk or placental milk (Green et al 1980). The changeover occurs at about the time when the kidneys of the young develop the ability to concentrate urine (see Water economy and kidney function). Throughout lactation in the tammar the osmotic pressure of the milk remains the same as the plasma and tissues of the mother (ie it is isotonic). If it were higher than the maternal tissues, it would require energy to drive the secretion against the higher osmotic pressure of the milk. Since one molecule of a simple sugar exerts the same osmotic pressure as a large oligosaccharide molecule, secreting the sugars as large molecules keeps the milk isotonic and reduces the energy required to transport it across the mammary cell walls, while providing the young animal with high levels of carbohydrate and essential salt. Second, were the large sugar molecules hydrolysed in the gut, the increased osmotic pressure would draw water into the gut and cause diarrhoea. Instead, the oligosaccharides may be transported into the intestinal cells by pinocytosis (meaning cell drinking), ingestion of droplets containing the sugar molecules and maternal antibodies in the milk. The enzyme that breaks down the oligosaccharides to galactose and glucose, acid beta-galactosidase, occurs inside the cells lining the intestinal wall of the pouch young but not in the lumen of the gut. Once inside the cell, the droplets discharge their contents, and the complex sugars are broken down into their constituents, glucose and galactose, which enter the circulation and provide the main energy needs of the young (Messer and Green 1979, Messer et al 1989). Lipids in marsupial milk The early milk of the tammar is low in lipids, which comprise about 5 g/100 mL; this rises to 24 g/100 mL by the end of lactation (Fig. 2.19a). The change from carbohydrate to fat as the main source of energy in the milk coincides with the first emergence of the young from the pouch and its accelerated growth and increasing metabolic rate (Fig. 2.17). In dasyurids the same changes occur in the milk when the young are left in a nest. For the tammar the changes are even more dramatic when converted to daily energy intake (Fig. 2.19c). This reaches a peak of 668 kJ/day at day 260, with lipid contributing more than 70%: such a large increase could not be supplied by carbohydrate. The most abundant component in the lipid fraction is triglyceride (Griffiths et al 1972, Green 1984, Green and Merchant 1988) but traces of phospholipids, cholesterol and free fatty acids are also found. More than half the triglyceride in early milk of tammars, red kangaroos and quolls is saturated palmitic acid (C16:0), which declines after about day 70. Palmitic acid may be important in the synthesis of surfactants, which as we saw earlier, are required for oxygen transport across the lung. In the second half of lactation the predominant triglyceride is unsaturated oleic acid (C18:1), also characteristic of placental milk. Oleic acid is important in the synthesis of myelin sheaths around nerve fibres during the second half of pouch life (see Box 2.5). Proteins in marsupial milk The protein component of milk of all marsupials studied increases through lactation but not as dramatically as do lipids (Fig. 2.19b). In the tammar the two major protein fractions are casein and whey, which both increase gradually (Green and Renfree 1982, Nicholas 1988). However, for whey this gradual increase hides large changes in the individual proteins, and more subtle changes in the component amino acids (Renfree et al 1981). Of the whey proteins, alphalactalbumin remains constant throughout lactation while serum albumin increases during Phase 2 of lactation and declines in Phase 3. Transferrin, the protein that carries iron, increases substantially in Phase 3 but the most remarkable feature of this phase of lactation is an unique protein, late lactation protein (LLP), which first appears at 180 days and rapidly becomes the major component of the whey proteins (Nicholas 1988). Its secretion coincides with the rise


in concentration of prolactin, which as we saw is due to the sucking stimulus of the advanced pouch young. However, secretion of LLP cannot be induced in mammary gland tissue during early lactation by raising the concentration of prolactin experimentally and expression of the gene for LLP is only induced between 200 and 240 days (Trott et al 2002), so it is probable that the protein can be synthesised only after the gland has passed through the earlier phases of lactation. Collett and Joseph (1994) showed by in situ hybridisation that gene expression for alpha-lactalbumin and for LLP occurred in the same mammary epithelial cells at the changeover period in mid lactation, which supports the idea of developmental change in the secretory cells themselves. Similar whey proteins appear in the milk of the quokka, red kangaroo and grey kangaroos, coinciding with the time when the young emerges from the pouch and begins to eat herbage. A similar protein is not found in the milks of non-macropod species, such as the brushtail possum and the common ringtail possum, Pseudocheirus peregrinus, nor in the carnivorous eastern quoll. One idea is that it has a dietary role in kangaroos and wallabies when they change from milk to the fermentation of herbage in the forestomach (see Growing up and leaving the pouch). This is supported by the observation that an LLP-like protein does not appear in the blood of the young tammar or quokka (Jordan and Morgan 1968). The gamma-globulin fraction of whey milk of marsupials contains all three classes of immunoglobulins, namely IgG, IgM and IgA. In the tammar IgG from the mother appears in the earliest milk and in the serum of pouch young in the first two days after birth (Deane et al 1990), which may confer passive immunity on the young animal. In the quokka gamma-globulins remain constant throughout lactation at levels below those in the mother’s serum and it is thought that they are derived from the maternal blood by selective transfer, as in placentals (see First phase of pouch life – acquiring immune competence). In the brushtail possum IgA is only high at the beginning of lactation, while IgG only rises in late lactation (Adamski and Demmer 2000). Inorganic elements and salts in marsupial milk One major difference between marsupials and placental mammals is the means of transporting iron and copper to the young. In placentals, such as humans and cattle, copper and iron are actively transferred across the placenta, so that the newborn has levels in the blood (bound to the serum protein transferrin) and in the liver much higher than those in the mother’s blood. The milk contains very little of these elements, so that the levels in the suckling fall progressively until weaning. Although there are transferrin proteins in the yolk sac fluid of the tammar (Fig. 2.4), it is impossible for the marsupial at birth to contain adequate stores of copper or iron to maintain it until it is weaned. Indeed, in the young quokka the levels of both elements rise progressively during pouch life, as do the transferrin proteins in the plasma: these elements are obtained from the milk. During the first 170 days the concentration of iron in the milk is about five times (Kaldor and Ezekiel 1962) and of copper, three times, the concentration of maternal plasma and then falls to maternal plasma levels when the young quokka begins to emerge from the pouch and eat grass. The same pattern occurs in the tammar, red necked wallaby, Macropus rufogriseus, common wombat and eastern quoll (Green 1984, Green and Merchant 1988). In all these species the fall to lower levels coincides with the time, in each species, when the young first leaves the pouch. Like other macropods, quokkas can suckle two young differing in age by about 170 days, so such an animal must be transporting a far higher concentration of iron and copper across the newly suckled gland than across the other. Whatever the transport mechanisms are they must be intrinsic to the gland and be a function of the stage of development of the gland, just as for LLP.

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Other minerals show opposite patterns of secretion in milk. For calcium, phosphorus and to a lesser extent magnesium, the concentrations in late stage milk rise to double the concentrations in early milk (Green and Merchant 1998), perhaps reflecting the increasing requirements for these elements in bone growth, which is accelerating. Sodium and potassium concentrations change during lactation in all marsupials so far examined (Green and Merchant 1988), with high levels of sodium in early milk declining to low levels after about 170 days, and potassium being the opposite. The changeover coincides with the maturation of the young animal’s kidneys (see Water economy and kidney function). Conclusions about marsupial milk The clear pattern that emerges from the foregoing is that there are profound differences in almost all constituents between the early milk that supports the very immature pouch young and the later milk that supports the young when it is becoming independent of its mother and growing fast. It suggests that lactation in marsupials, and probably monotremes, is a much more complex process than lactation in placental mammals. This conclusion is supported by the extraordinary phenomenon of concurrent, asynchronous lactation in kangaroos and wallabies. As mentioned earlier, in several species of marsupial, which have successive litters, the second litter attach to teats unused by the previous litter, after the first litter have weaned. However, in many species of kangaroo and wallaby the first young continues to feed from the elongated teat on the much-enlarged gland, while the succeeding newborn attaches to one of the three remaining teats. Thus, the female simultaneously secretes from adjacent mammary glands two kinds of milk, differing widely in volume, energy content and all the component parts that represent the beginning and the end of lactation. The broad outline of the endocrine control of this is now understood but many of the more subtle changes that take place are certainly not. Some of the changes, such as the appearance of LLP or the secretion of iron and copper, seem to be due to an intrinsic sequence of cellular changes in the mammary gland itself, not directly controlled by pituitary hormones.

How the young marsupial becomes independent Three stages of postnatal development At birth the young tammar weighs about 350 mg, less than 0.008% of its adult weight. It remains continuously attached to one teat until 100 days, during which time it reaches a body weight of about 100 g. Although the overall rate of growth appears to be slow, the instantaneous growth rate – the daily increment as a proportion of total size – is high. Between 100 and 200 days big changes occur: the eyes and ears open, the young tammar can stand on its feet, it is finely furred, it can concentrate urine and maintain a constant body temperature (Fig. 2.17). After 200 days its instantaneous growth rate accelerates again and it begins to leave the pouch and eat grass. It is weaned off milk by 300 days and, if female, then comes into first oestrus. All marsupials follow this sequence, albeit with different intervals and sizes. It is convenient to divide pouch life of a marsupial into these three phases. Phase 1 is, broadly, about developing an immune system and a functional nervous system, Phase 2 about controlling physiological functions of the body, and Phase 3 about physical growth, sexual maturity and independence. For the mother Phase 1 is not energetically costly but Phases 2 and 3 are very demanding.


Phase 1 of pouch life – acquiring immune competence Foreign organisms or proteins entering the body of an adult mammal elicit a defence response, which involves the proliferation of special cells by the lymphoid tissues of the body and the production of specific immunoglobulins, known as antibodies. These appear in the gammaglobulin fraction of the serum and react specifically with the foreign matter or antigen to neutralise it. In placental mammals the ability to mount an antibody reaction develops at about the time of birth, coincident with the differentiation of the lymphoid tissue of the thymus. Before this stage the animal or fetus is tolerant to foreign tissues and proteins and moreover in subsequent life will remain tolerant to the same proteins if encountered again. During this early period the fetus or newborn receives immunoglobulins from the mother, either across the placenta, as in the rabbit and human, or through the milk, as in cattle and horses, in which the first milk, or colostrum, is enriched with maternal immunoglobulins. The newborn marsupial is far more immature than the newborn placental and enters an environment, either in a pouch or on the belly of its mother that is certainly not sterile, and bacteria colonise the gut from an early age (Yadav et al 1972): how then does it survive? As already noted, immunoglobulins provided by the mother are present in yolk sac fluid of the tammar and they are also secreted in very early stage milk of the quokka (Yadav and Eadie 1973), the euro, Macropus robustus (Deane and Cooper 1984), the tammar (Deane et al 1990) and the brushtail possum (Adamski and Demmer 2000). This continues in all four species until the young first relinquishes the teat. Throughout this period the maternal proteins are absorbed unchanged across the gut epithelium, along with the oligosaccharides mentioned earlier, and presumably they provide protection to the immature young during this lengthy period: this was demonstrated in the quokka and the short-tailed opossum by immunising the mothers with specific bacterial antigens and subsequently finding antibodies specific to the bacterial antigens in the serum of the young (Old and Deane 2000). The immune system of the young animal develops much earlier than in placental mammals and this has been most thoroughly studied in the Virginia opossum and four other species. No lymphoid tissue is present at birth in any species but within the first week after birth lymphocytes appear in the thymus, which is the first lymphoid tissue to develop. The thymus of the Virginia opossum consists of a pair of structures lying near the base of the aortic arch and on the day of birth consists of undifferentiated embryonic cells (Block 1964). Within a day or so of birth, the first lymphocytes and the first lymph nodes appear and by day 17 the spleen also contains differentiated lymphoid tissue; plasma cells and secondary lymph nodes appear by day 60. The development of lymphoid tissue was grossly affected by removing the thymus at day 7 (Miller et al 1965): the number of small and medium lymphocytes was reduced and they failed to appear in the spleen, in which myeloid tissue persisted and increased. This suggests that the thymus is important in the origin and maintenance of lymphoid tissue and the suppression of myeloid tissue. All polyprotodont marsupials have a single thoracic thymus, like the opossum but the diprotodont marsupials, such as the brushtail possum, tammar and quokka have a superficial thymus in the neck as well, and this becomes the dominant lymphoid tissue in these species. The thymus has differentiated into a distinct cortex and inner medulla by day 14 and its adult structure was fully developed by day 120 in the tammar (Basden et al 1997). The development of immune competence has been tested in the Virginia opossum and the quokka by the response of the young to skin grafts and to infection with bacteria. Before day 6 young opossums injected with a bacterial suspension or infected by a dirty wound do not produce antibody, do not show an inflammatory reaction and rapidly succumb to the infection (Rowlands et al 1964); but after this age reaction to infection progressively increases and antibody can be detected in progressively higher amounts, while wounds rapidly heal.

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Similarly, the young opossum less than 10 days old will accept a foreign skin graft (Laplante et al 1969), retain it permanently and, if later challenged with a second graft from the same donor, will not reject it either. However, after day 12 a foreign skin graft was rapidly rejected. These experiments show that immunological tolerance is lost and immune competence is achieved in the opossum within the first two weeks of pouch life, an extraordinarily early stage of development that is far in advance of the young of placental mammals: however, it is an equivalent time after birth. The quokka also develops immune competence at an early stage of pouch life, so it is very likely that precocious development of immune competence is common to all marsupials and is an important adaptation for survival outside the mother’s body. Phase 1 of pouch life – getting wired up For the first 120 days after birth (Phase 1) the brain is the fastest growing part of the body and the first part to differentiate into its final form. The separate parts of the brain grow at different rates and the change to a slower rate also varies (Renfree et al 1982). Thus, the olfactory lobes grow fastest and the rate slows at 120 days, while the brain stem grows slowest and does not change until 180 days. The gross changes in the brain reflect changes to the internal structure of the brain. At birth the brain stem and olfactory lobes are partly differentiated but the cerebral hemispheres and the cerebellum, so important in adult life, are small, undifferentiated and lack neural connection to or from the brain stem. Also, only some of the 12 cranial nerves have made connection to the brain, notably I, V and VII, and possibly VIII. As we saw earlier, these are the parts of the nervous system that control the movements of the young as it travels to the pouch and attaches to a teat. The major developments during the first 100 days are the differentiation of the eye, ear and whiskers and the cerebral cortex. Also during this first period the connection of the remaining cranial nerves to the brain, and the relay systems that link the several parts of the brain to each other and to the spinal cord, take place. In placental mammals all of this development takes place before birth, whereas in marsupials it takes place in the pouch. This accessibility of the young has allowed detailed studies on the developing functions of the nervous system (Box 2.5). The visual system The eyes begin as paired outgrowths from the front end of the brain. When they make contact with the outer layers of the head they invaginate to become two-layered cups. The inner layer of each cup becomes the light sensitive retina and the outer layer becomes the pigment layer of the eye. The other parts of the eye, such as the lens, form from the overlying layers of the head. Differentiation of the retina of the tammar and the quokka has been described in detail and both follow a similar pattern (Dunlop et al 1988, Mark 1997, Marotte and Sheng 2000). The retinal cells divide and differentiate into three layers of interconnected sensory neurones. The outermost layer comprises the photosensitive receptors, the second layer are interneurones, which modify and accentuate various aspects of the light signal before passing it on to the innermost layer, the ganglion cells. Each ganglion cell sends an axon to the brain along the optic nerve. Cell division begins just before birth and continues until day 100, after which very few neurones are formed. There is, however, considerable loss of cells by spontaneous death during this period, a feature common to the differentiation of other parts of the nervous system as well. In both species the first axons from the retinal ganglion cells begin their growth along the optic nerve (II) at birth. By day 12 they have reached the two main visual centres in the brain, the superior colliculus (SC) in the midbrain, and the lateral geniculate nucleus (LGN) of the forebrain. Those that go to the superior colliculus synapse with motor neurones that later will control eye and neck movements and thus the direction of gaze and attention. Those ganglion cell axons that reach


Box 2.5: Neurones: what they are and what they do The building blocks of the nervous system are specialised cells called neurones and supporting cells called glia. Neurones come in many shapes and sizes but all comprise a central cell body containing the nucleus and one long tubular outgrowth called an axon or nerve fibre, and many short, much-branched outgrowths, called dendrites. The cell body and dendrites receive chemical signals from other neurones or from sensory organelles, which initiate an electrical disturbance of the cell membrane called a nerve impulse. The nerve impulse travels out along the axon to its end, where the axon forms a special contact with the dendrites or cell body of another neurone, which in turn excites another neurone, and so on. Eventually the nerve impulse reaches a specialised motor neurone, whose axon terminates on a muscle fibre and initiates a muscle contraction. The contact between neurones is called a synapse and an individual neurone may have up to 100 000 synapses with other neurones. By far the largest number of neurones form connections only with each other and are called interneurones. Interneurones are of two sorts, excitatory and inhibitory, depending on the specialisation of the synapses made by their axons. Excitatory synapses start nerve impulses and inhibitory ones prevent or block them. Interneuronal connections form specific circuits, which are characteristic of the species and organ, and are set up during development before they become functional. The working of this immensely complex system depends on the precision of the anatomical circuitry and the physiological balance between excitation and inhibition. Sensory neurones, and those interneurones to which they are connected, convey signals from sense organs, such as touch and temperature receptors in the skin, photoreceptors in the eye, stretch receptors in muscle or chemoreceptors in the nose and mouth, to the brain. They are also called afferent nerves because the impulse is towards the brain. Motor neurones convey nerve impulses away from the brain or spinal cord, and are called efferent nerves. The axons of motor neurones form synapses with muscle fibres and their signal excites the muscle cell to contract. The simplest circuit in mammals is the stretch reflex, in which stretch sensitive sensory nerves in muscle form synapses in the spinal cord with the motor neurone serving the same muscle. Stretching the muscle sends impulses up the sensory axons to the dendrites of the motor neurone, which transmits them back to the muscle, causing it to contract to its stable length. Usually, however, there is one or more interneurone between the sensory and motor neurones. Complex behaviour, such as walking, requires an intrinsic rhythm to be set up by a specialised set of interneurones in the spinal cord, called a pattern generator, the operation of which may be modified by sensory neurones and which gives out coordinated impulses to the motor neurones. The arm movements of the newborn marsupial, described earlier, are an example of the integrative actions of the nervous system. The speed of transmission of a neural signal depends on the diameter of the axon and the number of synapses across which it passes. It is also much faster if the axon is covered in a fatty sheath called a myelin sheath formed by the wrappings of membranes of supporting glia cells. Myelin sheaths develop during development of the central nervous system.

the LGN, however, synapse with a second relay of neurones that grow from the LGN into the roof of the forebrain, later to become the cerebral cortex, which they reach by day 15. In common with all mammals, most of the axons from the retina of one eye connect to the centres on the opposite side of the brain. This large crossover site is called the optic chiasm, a prominent feature on the lower surface of the brain. It is thought that crossover of the optic nerves compensates for the inversion of the image in the retina and so brings it into register with information coming from the body, especially the hands. Despite this very early establish-

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ment of the main nerve tracts in the brain, none is functional for many weeks. During this long period other axons from the retina follow the first ones and synapses form at the relay stations. In addition, efferent neurones in the cortex send axons down to the LGN by day 40 and to the superior colliculus by day 85. Part of the delay is because the efferent neurones that form layers 5 and 6 of the cortex involved in vision do not reach their own final positions until about day 36 (see The cerebral cortex). So what is the significance of this long, drawn out process? Mark’s (1997) idea is that in their early migration from the retina the first axons are responding to spatial cues in the matrix of the brain and follow pre-ordained pathways that take them to the SC and the LGN. Here they are positioned in patterns that accurately represent the pattern of their cell bodies in the retina of the opposite eye. He showed that the outgrowing axons would reach the normal position even after the eye from which they arose had been rotated 180°. Once the basic plan is established by the pioneer axons, others follow the same course. The linking up of the several elements takes place when the ends of the axons make multiple synaptic connections with other neurones, and signals can begin to pass from the eye to the cortex. The earliest age at which neural signals can be recorded from the cortex of the tammar, in response to stimuli of the optic nerve, is on day 46 and from the SC on day 39. In addition signals from descending neurones from the cortex to the SC can first be picked up at day 130. Thus, the neural connections between the eye and the brain have assumed their full capacity at the same time as the eyes open for the first time at 140 days. The auditory system The main studies of this system in marsupials have been done on the gray short-tailed opossum, the northern quoll and the brushtail possum. While the sequence of events is similar in each species, the times for development differ. The following description is for the quoll (Aitkin 1998). While the parts of the inner ear, concerned with detecting gravity, are developed to some degree at the time of birth, the cochlea, which detects sound, is absent: it develops during pouch life at the same time as the auditory pathways within the brain are being established. As with vision, the auditory pathway involves several relays from the auditory nerve that enters the brain stem to the part of the cerebral cortex concerned with hearing. The first neurones in the brain stem arise within a few days of birth and their axons cross to the opposite side of the brain stem, like the crossover in the visual pathway. The next set of neurones arises during days 7–22 and their axons grow out to the roof of the midbrain, the inferior colliculus (IC). During the same time other neurones arise in the thalamus and their axons reach the cerebral cortex, where the six layers are being formed by migration, as in the visual cortex. This is complete by day 42 in the quoll. While the main neural network is established early in pouch life, the synaptic connections between the several sets of neurones take much longer to form. For the first 45 days the number of synapses increases slowly but after this age they increase rapidly to a maximum by day 70. The change in rate coincides with the opening of the ears and the exposure of the eardrum to external sound at day 63, and it has been suggested that the sounds received actually stimulate the formation of synapses. By this age the young quoll responds to loud noise and by day 80 hearing acuity and sensitivity have attained the adult state. This sequence of events occurs earlier in the shorttailed opossum, at about day 25 when it is first left in a nest. In the tammar, the first response to sound was detected at day 114, considerably later than in the smaller species (Liu et al 1997). Whiskers Whiskers are a good example of the sensory input from the body surface – the somatosensory system. They are highly specialised hairs around which are grouped several kinds of recep-


tor cells: in the tammar, about 200 sensory fibres serve each whisker. In the adult animal they respond to bending of the hair shaft by anything that touches it, different receptors being tuned to different directions. Development of whisker innervation in the tammar takes the first three months of pouch life and is a three-stage process (Waite and Weller 1988, Waite et al 1998). Whisker buds appear at birth with outgrowth of the whisker itself occurring at day 35. The cell bodies of their sensory neurones lie just outside the brainstem in the trigeminal ganglion (cranial nerve V). Each

Figure 2.20: (a) The surface pattern of whiskers on the right muzzle of the 91-day tammar wallaby pouch young, Macropus eugenii and (b) the corresponding spatial representation of whiskers A and E in the somatosensory cortex of the left cerebral hemisphere After Waite et al (1998), photos by PME Waite.

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neurone gives rise to a long dendron that connects with the receptor cells on the whisker, and to an axon that enters the brain stem. The axons synapse with a second set of interneurones that send axons from the brain stem to the opposite thalamus region of the brain. Here, third stage neurones grow out from the thalamus and enter the developing cerebral cortex. This three-stage nerve framework is in place by day 15 after birth but the highly complex synaptic arrangement at each relay station does not become functionally connected until day 72. During this time the axons make many synapses with dendrites of the neurones of the next relay in a highly characteristic pattern, called a barrel. Each barrel comprises the cell bodies of the next relay arranged in a circle, with their dendrites pointing inwards and the ends of the axons of the previous relay in synaptic interaction. In the brain stem the barrels appear at day 40 and those in the thalamic relay appear at day 55. The final relay form synapses in layer 4 of the developing cerebral cortex, where they form the definitive barrel, at around day 80–90 (Fig. 2.20b). The actual whiskers have appeared by day 35 and are now connected, through the three relays, to the cerebral cortex. Each barrel in the cortex corresponds to an individual whisker on the opposite side of the snout of the young tammar and, remarkably, the pattern of the barrels in the cortex is the same as the pattern of the whiskers on the snout (Fig. 2.20a). In the brushtail possum when single whisker follicles were experimentally removed before day 50, the corresponding position in the cortex lacked a barrel, which suggests that the differentiation of each barrel is controlled in some way by the developing whisker, or the first signals emanating from it. The cerebral cortex All the sensory input from the body is projected onto the cortex in specific sites, which can be mapped. If one looks at a section taken through the cerebral hemispheres of any mammal, the cell bodies of the neurones are arranged in six distinct layers, each with a separate function. The neurones in layer 4 receive axons ascending from lower centres in the brain, while neurones in layers 5 and 6 send axons to relay stations in the lower brain and, hence, to the motor neurones of the cranial nerves and the spinal cord. Likewise the centres that control motor function can also be mapped to the surface of the cortex. Layers 2, 3 and 5 are connected to other neurones in the same and in other layers of the cortex. The two halves of the cerebral cortex are connected by transverse bundles of axons arising from neurones in layers 3 and 5. In marsupials the most important of these interhemispheric tracts is the anterior commissure, with 20 million fibres in the adult tammar (Shang et al 1997). In placentals the anterior commissure is small and another structure, the corpus callosum, carries the main traffic from one cerebral hemisphere to the other, with up to 200 million fibres in the human brain. At birth, the cerebral cortex of placental mammals already has six layers of neurones, but in marsupials the cortex consists of only two embryonic layers: the six layers of the mature cortex will be formed during the first 70 days after birth. This has enabled researchers to study the formation of the cerebral cortex and the development of the neural connections between the six layers much more easily in marsupials than in placentals. The main technique used is to label the nucleus of dividing cells by incorporating a radioactive marker into one of the bases in the DNA molecule. Uridine or thymidine labelled with tritium (3H) is the usual marker, which is injected into the animal at a known age and, as the cells divide, the radiolabelled base is incorporated into the DNA of the newly formed neurone. If the cell does not divide again after being labelled in this way, the signal from the cell is of maximum strength but if it divides, the label is shared between the daughter cells and the signal becomes weaker with each division. By injecting a series of tammar pouch young of increasing age up to 70 days, it is possible to identify the age at


which cells reach each of the six layers of the cerebral cortex and cease to divide (Reynolds and Saunders 1988). The neurones migrate to their final positions from the formative layer of cells lining the inner surface of the brain. The first cells to divide migrate to layer 6 during days 16–28, wheras the successively later born cells migrate through the lower layers to their positions further out. Each layer is established about 10 days after the last so that all the layers can be recognised by day 120 (Marotte and Sheng 2000). After day 120 no further neurones are formed from the basal layer and the young animal now has its final complement of neurones that will suffice for the rest of its life. From day 120 the cerebral cortex is ready to coordinate the activities of the young tammar. Connections between the cortex and the underlying centres of the brain take place during the same period that the six layers are forming, with axons progressively reaching the furthest, layer 1 (Mark and Marotte 1992). Axons from the eye, the whiskers and the ear make synaptic connection mainly in layer 4, while the efferent neurones reside in layers 5 and 6. Overproduction of neurones in early development of the brain and subsequent cell death is common to the establishment of all sensory pathways in the brain. For instance, in the tammar the number of axons in the anterior commissure peaks at 60 million by day 140 and then declines to the adult number of 20 million (Ashwell et al 1996). It is supposed that this allows for the final sculpting of the tracts and connections between the different parts of the brain by experience and repeated use. Myelinisation of fibres of the anterior commissure begins at day 160 and is completed by day 300 (Ashwell et al 1996). This is when oleic acid, which is an important component in the synthesis of myelin, becomes one of the main lipids in the milk. The pyramidal tract The pyramidal tract is a large tract of motor neurones that convey signals direct from the cerebral cortex to the spinal cord. The cells are in layers 5 and 6 of the cerebral cortex and their axons pass down to the brain stem, where they cross over to the opposite side and synapse with the ventral column of the spinal cord. The pyramidal tract first appears at day 30–40 in the Virginia opossum. It is especially important in species that use their hands, since its role is to convey motor control from the cerebral cortex directly to the forearm and hand. It is well developed in the brushtail possum and tammar but not in dasyurids. In addition, there are other relays that connect the cerebellum to the spinal cord, which are involved in the control of movement and position, as well as the direct motor tracts that connect the cerebral cortex to the limbs and trunk via the spinal cord. Maturity of the nervous system is shown by the ability of the young tammar to hear at day 114, to see at day 140, to call and to stand at about day 200 and to hop at day 210. Phase 2 of pouch life: becoming physiologically independent Phase 2 of development in the tammar lasts from about day 100 to day 200. It begins when the young first relinquishes the teat and lasts until it makes its first excursion from the pouch and begins to nibble grass. During this phase it develops control of its body temperature, the thyroid gland becomes functional as the fur thickens, and the kidney matures so that the young can concentrate its urine and conserve water. By day 200 it is physiologically independent of the mother (Fig. 2.17). Thermoregulation, respiration and metabolism The ability to maintain body temperature at a constant level regardless of the surrounding air temperature has allowed birds and mammals to occupy a wide range of habitats and for the body to function at a high rate (see Chapter 1). But it comes at a high cost in terms of food intake,

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and the costs are inversely related to body size, the smallest species having the highest relative food requirements and oxygen consumption (Table 1.2). For the very small young of birds and mammals, maintaining a constant body temperature would require a very high input of energy, because of their small size. Instead, they rely on the mother’s body heat, either during incubation in a nest, in the uterus or in the pouch. For the young marsupial the pouch is an environment with a high stable humidity and temperature and, in species with a closed pouch, a carbon dioxide concentration of about 3% (Hulbert 1988). Virginia opossum, pouch young

69 days

94 days

81 days

75 days

3 84 days Virginia opossum Oxygen consumption (ml/g/h)

94

Quokka

2 94 days 153 days

178 days

1

64 days 100 days

Figure 2.21: Development of thermoregulation in the Virginia opossum, Didelphis virginiana, and the quokka, Setonix brachyurus. Top panel. In very young opossums body temperature falls rapidly to the low ambient temperature but at 81 days thermal stability has begun and is fully established at 94 days. Lower panel. Oxygen consumption of young opossums and quokkas at a range of ambient temperatures: before thermoregulation has developed oxygen consumption is directly related to the ambient temperature but as thermoregulation is established the pattern reverses, so that oxygen consumption is then highest at the lowest ambient temperature. After Reynolds (1952) and Shield (1966).


If removed from the pouch during the first half of pouch life, the body temperature of the young marsupial rapidly changes to the surrounding temperature. If this is a low temperature, the animal becomes torpid and may cease to move, but will recover as quickly if returned to the warmth of the pouch. As it grows older the fall in body temperature after removal from the pouch is slower and, in the second phase of pouch life, the young may hold a steady temperature for several hours, before falling to ambient (Fig. 2.21). Finally, at about 94 days, when the young Virginia opossum first emerges from the pouch, it can maintain a stable body temperature against an ambient gradient as well as the adult animal can: now it shivers in response to low temperature and pants and licks its arms when exposed to a high temperature. Maintaining the temperature of the body above or below its surroundings requires the animal to generate or dissipate its own heat and to have sufficient insulation to prevent heat loss. The necessary adaptations for this develop during Phase 2 of pouch life in the marsupial. The daily energy intake from milk in the tammar young increases steeply from 200 days to a peak at 269 days, which dramatically reflects its increasing energy needs and the increasing burden for the mother of supporting it (see Lipids in milk). By measuring the oxygen consumed by an animal in a closed system, we can assess its net expenditure of energy at a given temperature. The pouch young of opossums, quokkas and tammars have been subjected to this procedure. The oxygen consumption of tammars and quokkas less than 100 days, and of opossums less than 60 days old, increases directly with an increase in the ambient temperature up to 35°C. However, as older animals begin to control their body temperature this is reversed: they now consume more oxygen when held at a low temperature than when held at a high temperature (ie oxygen consumption is inversely proportional to ambient temperature, Fig. 2.21). In opossums and quokkas held at low temperatures, the young that were just becoming furred consumed more oxygen than older animals with a full pelage of under-fur and guard hairs, presumably because the older ones had better insulation. In the quokka the ability to shiver develops at 120 days (Shield 1966), and in the tammar at 150 days, which is the age in each species when thermoregulation is beginning. Conversely, when tammar young were held at an ambient temperature of 37.5°C, all showed a rise in body temperature but only those about 200 days old displayed panting and licking of their fur, whereas younger ones did not (Janssens and Rogers 1989). The major organs involved in the development of thermoregulation and water conservation in the young marsupial are the thyroid, liver and kidney, and the hormones that control the functions of each one. We will now consider the development of each of these systems in the tammar. The thyroid gland Mammals from which the thyroid gland has been removed are unable to respond to cold temperatures by increasing their metabolic rate. The thyroid synthesizes two hormones, thyroxine and tri-iodothyronine, which both contain iodine as a component of the molecule. Hence, the uptake of radioactively labelled iodine can be used to measure thyroid activity. When secreted into the circulation the general effect of these hormones is to stimulate glucose oxidation and protein synthesis. The way that the thyroid hormones stimulate endogenous heat production is by uncoupling oxidative phosphorylation and so channeling energy into heat rather than into synthesis of adenosine triphosphate (ATP). Because the development of thermoregulation occurs during pouch life in marsupials, it was inferred that thyroid function would also develop at this time. In tammar pouch young the thyroid tissue has differentiated into the characteristic follicles by day 75, but secretion did not accumulate in the follicles until day 180 (Setchell 1974). This coincided with a sharp rise in the uptake of iodine by thyroid tissue, reflecting the onset of active synthesis of thyroxin and tri-iodothyronine (Fig. 2.22). At the same time the level of iodine

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loosely bound to serum proteins fell, again indicating that the thyroid was sequestering all of the injected iodide. Both hormones rose to peak concentrations in the blood between day 160 and 180 and had dropped to adult values by day 300 (Janssens et al 1997). This transient peak may be associated with the higher oxygen consumption of the finely furred young before it is fully insulated. The importance of the thyroid gland for the development of thermoregulation in the tammar was critically shown by Setchell (1974), who surgically removed the thyroid glands of young tammars before day 140. These young subsequently failed to grow at the normal rate, to differentiate and grow a full coat of fur, and were unable to respond to low temperature by increased oxygen consumption, like normal young can do.

Tammar

Figure 2.22: Thyroid development in the tammar wallaby, Macropus eugenii. Uptake of the radioactive isotope of iodine, as Na131I, by the thyroid gland, liver and blood plasma of pouch young from 60 to 220 days. After Setchell (1974).

The increased metabolism of the older young requires a substrate to provide the energy for oxidation. At first this comes from the abundant sugars in the milk but after 180 days it comes from the increasing amount of lipids and proteins in the milk (Fig. 2.19). Liver function Enzymes in the liver control the release of the energy substrates. Complex sugars, proteins and lipids in the milk are digested in the intestine to simple sugars, amino acids and short and longchain fatty acids and pass directly to the liver. The liver is the main clearing house for the products


of digestion, storing some and releasing others, so that the level of each in the blood remains fairly constant. Glucose is the main sugar arriving from the intestine and excess glucose is stored in the liver as glycogen. Under the influence of specific enzymes the process can be reversed when glucose is released into the blood stream. In well-fed pouch young the level of glycogen in the liver is generally high but after 24 h off the teat the glycogen is much reduced. The ability of the liver to maintain blood glucose at a constant level by storing or releasing glucose as required develops during Phase 1 of pouch life, while carbohydrate is plentiful in the milk. Towards the end of Phase 1 lipids become more abundant and these enter the blood stream as short-chain fatty acids. They are converted in the liver either to acetate (2-carbon) and enter the citric acid or Krebs cycle, generating energy in the form of ATP, or to propionate (3-carbon), which results in amino acids and glucose. If carbohydrate reserves are insufficient to maintain blood glucose levels, tissue and dietary protein may be converted to glucose by a process called gluconeogenesis and the amino groups appear in the blood as ammonia or urea. This can be detected in unfed pouch young by the rise in ammonia in the blood as the liver responds to the lowered blood glucose by converting protein. During the transition that starts at day 200 the liver enzymes responsible for using milk carbohydrates decline to a fraction of their former level while the enzymes responsible for gluconeogenesis increase several fold (Wilkes and Janssens 1988, Janssens and Rogers 1989). This reflects the profound changes in the constituents of the milk and the increasing demands of the young animal as it becomes physiologically independent, and begins to eat grass. Water economy and kidney function For very small pouch young the high humidity of the pouch may be important, as mentioned earlier, to help it to respire through the skin during the first few days of pouch life. However, any water loss across the skin is more than balanced by the continuous sucking of the dilute early milk. More important is the loss of water through the immature kidneys, which cannot concentrate urine. When denied milk, young tammars can lose up to 12.5% of their body mass from this cause, but will recover when given milk (Wilkes and Janssens 1988). Passing dilute urine could potentially cause a severe loss of water from the mother–young unit and, in the dry conditions of inland Australia this could compromise their survival. However, it does not happen because the lactating tammar, and probably the females of all kangaroos, ingests all the wastes produced by the young in the pouch. Indeed, a small young will not normally void urine or faeces unless the mother licks its cloaca. This response thus conserves precious water through the concentrating abilities of the mother’s kidneys. This remarkable behaviour was discovered in tammars by labeling the water ingested by the mother with the deuterium isotope (2H20) and labeling water injected into the young with the tritium isotope (3H20). By sampling the blood of mother and young at a later time the tritium label was found in the mother and the deuterium label in the milk and in the blood of the young one (Dove et al 1989). Not only does this behaviour conserve water, it also protects the young from bacterial infections in its gut: any bacteria in the faeces of the young that enter the mother’s body induce an immune response in her and antibodies specific to the pathogen later pass back to the young in the immunoglobulins of the milk, effectively neutralising the infection. This phenomenon is known to occur in cattle and probably also plays a protective role in human infants living in unhygienic conditions. While water recycling is important in the early phases of pouch life, the young must be able to regulate its own urine concentration before the end of pouch life: this requires a functional kidney and hormonal controls. The kidney is composed of hundreds of blind tubules, called nephrons, all connected at their open ends to the ureter, which conveys the urine to the bladder for eventual voiding. The closed

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end of each nephron is expanded into a cup containing a network of blood capillaries, called a glomerulus, where water, urea and electrolytes pass from the blood into the tubules. The fluid then begins a slow passage along the tubule, where selective reabsorption of water and sodium ions takes place, and urea is progressively concentrated. The development of the kidney and its functions have been studied in three marsupials, the opossum (Krause et al 1979), the quokka (Bentley and Shield 1962) and the tammar (Wilkes and Janssens 1988). For the first week after birth the fetal kidneys are the functional excretory organs, as they were before birth (see Development of the yolk sac, amnion and allantois and Fig. 2.11). By day 20 of pouch life the fetal kidneys have shrunk and have been replaced by the definitive kidneys. For the first 100 days in the quokka and for 140 days in the tammar, new nephrons are being formed in the cortex of the kidney but not thereafter, so that the ratio of kidney mass: body mass declines to the adult value of about 0.6%. At the same time the glomeruli at the closed ends of the nephrons begin to enlarge, which means that filtration across the glomerular membrane from the blood increases. In the tammar this flow is 1 mL/kg body mass per minute at day 2000 Urine

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Figure 2.23: The development of kidney function during pouch life in the tammar wallaby, Macropus eugenii: (a) urine concentrating ability of the kidney begins after day 200 when the concentration, or osmolality, of the urine can greatly exceed the osmolality of the blood plasma; (b) before day 200 the young tammar excretes ammonia in its dilute urine but after this age it excretes waste nitrogen as urea. After Wilkes and Janssens (1988) and Janssens and Messer (1988).


140 increasing five-fold by day 250 (Wilkes and Janssens 1988). Second, the nephrons increase greatly in length, becoming much coiled in the cortex and with a long loop extending into the inner medulla of the kidney. Now the concentration of urine begins, as a result of the selective reabsorption of sodium in the cortex and of water in the medulla. The main factor that prevents the young tammar from concentrating its urine earlier is the immaturity of the kidney tubules. The hormone that controls fluid retention, vasopressin, is produced by the pituitary gland from a very early age but the kidney tubules cannot respond to it until after day 200 (Fig. 2.23a). As the kidneys develop the ability to concentrate urine, the excretion of nitrogenous waste changes from ammonia to urea. While the urine is very dilute in early pouch life ammonia can be excreted as it is, just as it can in fish and frogs that have no shortage of water with which to dilute it. However, ammonia is highly toxic when concentrated so, as the urine of the growing pouch young becomes progressively concentrated, ammonia in the urine declines and urea increases as the main nitrogenous waste (Fig. 2.23b). From the earliest stages the concentration of sodium ions in the blood plasma exceeds potassium ions, the normal ratio being 15:1. This ratio is necessary for the proper functioning of nerve conduction and muscle contraction and is achieved in the adult animal by selective reabsorption of sodium ions across the kidney tubules, so that the ratio of the two ions in the urine is about 1:1. Before 120 days in the quokka and 140 days in the tammar the kidney cannot reabsorb sodium, which is lost in the dilute urine. After this age in both species sodium is conserved and potassium excreted at a higher rate. As mentioned earlier, the early milk of both species has elevated concentrations of sodium, which falls to a low level when the kidney develops the capacity to reabsorb sodium. Presumably the higher concentration in early milk replaces the sodium lost in the urine, which is then recycled back to the mother when she takes in the urine of the pouch young. One consequence of the need to secrete sodium at high concentration in the early milk is the secretion of carbohydrate as long-chain sugars, as discussed earlier (see Carbohydrates in marsupial milk). Phase 3 of pouch life: growing up and leaving the pouch At 200 days the young tammar is able to maintain a steady body temperature but its preferred temperature, when it uses the least amount of oxygen, is several degrees lower than the temperature in the closed pouch. It needs to dissipate heat but it cannot do this inside the pouch and so it begins to put its head out to cool off. With time more of its body is exposed until it leaves the pouch entirely and begins to hop about and feed on grass. It leaves the pouch permanently at about 250 days, earlier if raised by a large female with more abundant milk, and it is fully weaned by about 300 days. The young tammar continues to take milk for several more weeks after first putting its head out of the pouch, with peak milk intake at 240 days, but herbage progressively forms a greater proportion of its diet. This is a critical period, as the diet changes from rich fatty milk to a diet of grass and herbs to meet the increasing energy demands and increasing independence of the young animal. The herbage must be fermented in the forestomach before being digested in the intestine. Bacteria and protozoa ferment the carbohydrate from the vegetation, especially the cellulose, to volatile fatty acids, and synthesise bacterial protein from urea recycled to the stomach (see Chapter 9). How do these microbes invade the forestomach? Croft (1981b) observed that red kangaroo young at foot would lick the lips of the mother for extended bouts of several minutes, investigating food items in mother’s mouth but more often taking saliva dripping from her mouth. Since this behaviour only occurs in young being weaned, it is possible that its main function is the transfer of microbes to the young. Young koalas and wombats display an

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analogous behaviour during weaning when they take matter from the mother’s cloaca, which has come from the caecum and is quite different from normal faeces (see Chapters 7 and 8, respectively). In these species the hind gut, rather than the forestomach, is the site of bacterial fermentation. The transition from milk to a diet of grass is complex for all species of kangaroo. The decline in complex sugars in milk is further accelerated by the arrival of bacteria, which consume any remaining carbohydrate. Instead the bacteria produce volatile fatty acids by their anaerobic metabolism of cellulose and this becomes the main source of energy for the tammar, requiring different liver enzymes. The change in diet may actually induce the changes in energy metabolism, with gluconeogenesis becoming the main pathway for the production of glucose in the blood. As mentioned earlier, several enzymes that catalyse this pathway show much increased activity in the liver after day 200. It is at this stage of lactation that LLP becomes a major component of the milk of all species of kangaroo that have been investigated (see Proteins in marsupial milk). It is not clear what the role of LLP is in kangaroos but the coincidence of its appearance at the time of transition from milk to a grass diet that is fermented in the forestomach, suggests that it may be important in facilitating the transition. One idea is that it is a source of dietary protein that can be hydrolysed to glucose by gluconeogenesis during the transition to bacterial protein (Janssens and Messer 1988), but this may be too simple an explanation for the function of a complex protein. Another important factor at this transition period, especially for desert kangaroos, is the increased need for water by the young animal as it becomes physiologically independent. Because of its small mass its SMR could be expected to be twice as high as its mother’s: it is actually three times as high because of its rapid growth rate, and its body temperature and evaporative heat loss are also significantly higher, especially at the highest ambient temperatures in summer (Munn and Dawson 2001), so its need for water much exceeds that of adults in the same environment. During hot, dry conditions this puts a great demand on the newly weaned red kangaroo for water, energy and protein. When conditions are good these needs are adequately met from the pasture and milk is not required as a supplement: however, if the pasture quality is low in protein and water, milk can supplement this if the mother can still supply it (Munn and Dawson 2003a), but under such conditions she usually cannot. Hence, the high mortality of young at foot among desert kangaroos (see Chapter 9). Growth in the young tammar accelerates after it leaves the pouch. At 200 days it weighs about 0.5 kg, 100 days later it is 2 kg and at one year it is 3 kg (Janssens et al 1997). By this age the growth of females has slowed down and they enter their first oestrus before they are one year old, whereas the young males continue to grow for another year and do not become sexually mature until the beginning of their third year (Williamson et al 1990). This pattern of sexual dimorphism in growth and sexual maturity is seen in all macropod species over 5 kg and in the large opossums. In the smaller wallabies and rat kangaroos, however, there is no difference between the sexes in final body size.

Relationship of reproductive processes to body size While this chapter has focussed largely on the development of the tammar wallaby, the general sequence of events, though not their duration, holds for other species that have been studied. Russell (1982) reviewed the available information on 56 species, representing all major families of marsupials, and showed that all parameters, such as litter size, neonatal size, development stages during early life and the length of lactation, correlate with adult body mass and the pattern of maternal care. Thus, species that leave the litter of young in a nest at an early stage of develop-


ment, before the eyes open, such as dasyurids and small didelphids, have a shorter lactation than kangaroos that carry their young to an advanced stage of development in the pouch but, within each family, the correlation with maternal size is close. Likewise, within families, the weight of the whole litter at weaning correlates closely with maternal body mass but the weight of a single young at weaning does not. From this it follows that smaller species, which invariably have more than one young in a litter, make a larger investment proportionately in reproduction than do larger species. Russell (1982) expressed maternal investment as the weight of the litter at weaning as a percentage of maternal body weight, and it varies from over 300% for the smallest dasyurids to less than 30% for the largest kangaroos, wombats and the koala: it is not, therefore, surprising that the smallest species leave their young in a nest from a relatively early stage of development. Energetics of reproduction Another way to assess the cost of reproduction is to compare the food consumed, the time spent foraging, or the field metabolic rate of lactating and non-lactating females, all of which will be considered in Chapters 3, 4 and 6. For other species the total investment by the female has been measured as the product of the amount of milk produced through lactation and its changing composition. Cork and Dove (1989) provide an interesting comparison between the tammar

Figure 2.24: A comparison of the investment in pregnancy and lactation (expressed as metabolisable energy, ME) in 500 kg dairy cows, 50 kg ewes, 9 kg koalas, Phascolarctos cinereus, 5 kg tammar wallabies, Macropus eugenii and 1 kg common ringtail possums, Pseudocheirus peregrinus. To compare species of very different body mass, time from conception to weaning has been converted to ‘metabolic days’ (d/kg0.25). The three marsupials from different environments and life styles make similar energy investments in reproduction (MJ/kg under the curves) as do the two very different placental mammals, but the investment is differently distributed throughout reproduction. After Cork and Dove (1989), Krockenberger (1993) and Munks and Green (1997).

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and sheep and cattle (Fig. 2.24): while sheep and cattle make a large investment in reproduction over a relatively short time, tammars make their investment over a much longer time but the total investment, adjusted for body mass, is almost the same in all three species. Subsequent studies on the common ringtail possum (Munks and Green 1997) and the koala (Krockenberger 1993) show the same pattern as the tammar, which supports the idea that this may be a common feature of marsupial reproductive investment: minute investment in gestation, low investment in early lactation and major investment in late lactation. Because the investment in pregnancy and the first half of lactation is small and extended in time, the female marsupial can terminate it in unfavourable conditions with a minimal loss of reproductive investment, and the early phase can be repeated again without much cost. Desert kangaroos have exploited this in their superb adaptation to uncertain conditions and their opportunistic reproductive strategy (see Chapter 9). By contrast the placental female, once committed to pregnancy is unable to terminate without a serious loss of investment. In temperate climates with highly predictable times of food abundance this is not a significant risk, compared to the great advantage that accrues from the rapid exploitation of abundant resources: in the Australian environment of infertile soils and uncertain climate, however, the highly seasonal reproductive strategy of the sheep leads to severe loss in years when rains do not come and is thus not sustainable in the long term. McNab (1986) considers that marsupials in South America and Australia are competitively equal to their placental counterparts where the resources are limited and mammals need to be parsimonious in energy expenditure: where the resources available permit high rates of energy expenditure marsupials are competitively at a disadvantage compared to ecologically equivalent placentals. These ideas will recur in subsequent chapters as we consider the reproductive strategies of different species in various environments.

Chapter 3


Pre-European terracotta opossum from Colombia.


S

eventy-six species of marsupial live in South America and Central America, from sea level to the subalpine zone at 4200 m and from the tropics to the cool temperate climate of southern Chile and Patagonia: most are forest dwellers, living on fruits, insects and other small animals. Although widespread, they comprise only 7% of all mammal species of the American tropical region, being far outnumbered by bats (46%) and rodents (27%). In size they range from 10 g to 2 kg and resemble one another in body form, diet and life history (Fig. 3.1, Plates 4 and 5). Nevertheless, the four extant families are not closely related, each having a separate ancestry back to the early Tertiary period: they are the living twigs of a very old tree. One twig consists of a single species from southern Chile, which has closer affinities with Australasian marsupials than with the other American species (see Chapter 1); the second twig comprises seven species of shrew opossums in the northern Andes and southern Chile; the third twig comprises five species of woolly opossums and the remaining 63 species comprise the large opossums and mouse opossums, most of which live in the tropical rainforests, and a few in the grasslands and two in the southern cone. It was not always thus: before the American continents became united 3 million years ago, South America had a more diverse marsupial fauna, which included large carnivores up to 200 kg.

A brief history of marsupials in South America Origins When South America became separated from North America at the close of the Cretaceous period, 65 million years ago, the few small mammals – placental and marsupial – isolated there evolved independently for the next 35 million years. The marsupials filled the ecological niche of small insectivore, shared the niche of large carnivore with birds, and also edged into the niche of small rodent. Conversely, placentals in South America became large herbivores of various kinds but none became a large carnivore. Unlike in Australia, there is an abundant and continuous fossil history dating back to the very beginning of the Palaeocene epoch in South America, so that the lineages of the present day marsupials can be traced through the whole span of the Tertiary. In one extraordinarily rich fossil site at Tiupampa, Bolivia (see Fig. 1.12), formerly dated to late Cretaceous but now considered to be very early Tertiary, 11 species of marsupials and seven species of placental are known from teeth, skulls and even whole skeletons (Marshall and de Muizon 1988, Goin 2003). Among the marsupials, all of which were between the size of a rat and a small cat, are representatives of the major lineages that will become dominant in later periods: the opossum-like didelphids, very similar in size and dentition to the living species; a microbiotheriid, the presumed ancestor of Dromiciops; and another species that could be the ancestor of the shrew opossums, or caenolestids. In addition, there is a species of borhyaenid, the family that was to become abundant later as the large marsupial carnivores. Thus, from the earliest fossil site in South America the lineages that would predominate for the next 65 million years were already established (see Fig. 1.13). In the Palaeocene and Eocene epochs, which encompass the first 25 million years of the Tertiary, 24 genera of marsupials representing six distinct families are known (Marshall et al 1990). The one group that had teeth adapted to eating plants, the polydolopodids, did not survive past the Eocene but the carnivorous species persisted. Polidolopodid and microbiotheriid species have recently been discovered in a middle Eocene formation on the Antarctic (formerly Palmer) Peninsula (Goin et al 1999): all are small and none of the larger borhyaenid carnivores have been found there. Total isolation of South America ended in the Oligocene epoch, about 30 million years ago, when the earliest rodents and primates entered the southern continent (Marshall et al 1979).

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These were the ancestors of today’s capybaras, Hydrochaeris, and guinea pigs, Cavia, and New World monkeys (Platyrhinni). How they came is still conjectural: presumably by way of an island chain from North America or, possibly, from Africa while that continent was much closer to South America. They may have displaced some of the smaller arboreal marsupials or even prevented marsupials from evolving into the small to medium herbivore niches that they came to dominate thereafter. This was the time in Australia when the great expansion of marsupials into arboreal and browsing niches took place. In the Miocene epoch (23–5 million years ago) the shrew opossums (caenolestids) and the large carnivorous borhyaenids, became abundant, both being represented by eight genera. The borhyaenids flourished right through to the Pleistocene epoch (3 million years ago), when they all became extinct; the caenolestids also declined to a tiny remnant of their former numbers and now only persist as seven species in the high Andes and southern Chile. In the Pliocene epoch, small placental carnivores, the ancestors of the coati, Nasua nasua, and kinkajou, Potos flavus, also reached South America and their arrival may have been a factor in the decline and extinction of the borhyaenids. At the same time as these two groups declined the didelphids, forerunners of today’s opossums, increased in number of species, and the present day genera began to appear (see Fig. 1.13). The smaller species, such as Marmosa and Monodelphis, made their first appearance in the Miocene, Lutreolina, Thylamys and Philander somewhat later in the Pliocene, but the other species, including the largest species of opossum, Didelphis, do not appear until the Pleistocene, just 2 million years ago. The apparent ‘relay’ of various carnivorous marsupials through time in South America, with borhyaenids replaced first by large carnivorous birds and later by placental carnivores, and the rise and diversification of smaller carnivorous opossums, is paralleled in Australia, where the early carnivores were large thylacinids that were replaced in the last five million years by the smaller dasyurids (Marshall 1982, Reig et al 1987) (see Chapter 4). Marsupial top carnivores Borhyaenids were the large mammalian carnivores of the continent for more than 20 million years: they had short limbs and were probably not fast pursuit hunters like wolves but more like badgers or wolverines. Their teeth were very similar to those of the Australian thylacine, Thylacinus cynocephalus, so much so that they were at one time thought to be closely related: but by the Miocene there was no land connection with Australia. The thylacine is now known to be closely related to dasyurids (Krajewski et al 1997), so this is a case of independent convergence in adaptations for predation and eating flesh. But the most bizarre convergence to arise from borhyaenids in South America were the sabre-tooth carnivores that flourished in the Pliocene (5 million years ago), at the same time as placental sabre-tooth tigers, Barbourofelis and Smilodon, were living in North America. Thylacosmilus atrox was a large carnivore with a pair of huge upper canines that sprang from roots lying on either side of the nose and swept across the lower jaw, bearing against a large flange of bone and a tiny pair of lower canines; it had no incisor teeth but a normal complement of premolars and molars (Fig. 3.2). The enormous canines had very thin enamel and had open roots, so that they could grow continuously through life. While its teeth and skull resembled those of the sabre-tooth tigers of North America, Thylacosmilus did not have a tiger’s feet and limbs: instead it had short, strong forelimbs, which suggest that it was not a pursuit hunter but ambushed its prey and stabbed it to death. Its ability to stab was much helped by its enormous (67°) gape. Churcher (1985) estimates that it could have stabbed a body of 400 mm diameter, an animal the size of a deer. The prey available to it was a variety of large placental herbivores, some with long necks like giraffes and others with trunks and some that could run like horses; none was related to the modern animals that they superficially resembled but had evolved independently in South America, along with the carnivorous marsupials. Both


the variety of prey species and the marsupials that hunted them disappeared just before or soon after the continent became joined to North America about 3 million years ago. Opinions differ on whether Thylacosmilus was displaced by the placental sabre-tooth tigers coming in from the north, were starved out as their prey were extinguished by the new predators, or disappeared before the great interchange from other causes unknown.

Figure 3.2: Evolutionary convergence of sabre-tooth predators of South and North America from the Pliocene epoch: (a) the marsupial Thylacosmilus atrox; (b) the North American felid Barbourofelis fricki. Note the enormous gape of the marsupial species. After Churcher (1985).

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One other family of marsupials appeared in the Pliocene, which were different from all other marsupials in South America: Argyrolagus were small, hopping forms, with very short forelegs and elongated hind legs and the toes reduced to the 3rd and 4th and partially fused together. They also had a reduced number of teeth, with only five cheek teeth and one incisor in each jaw, and all open rooted like rodents’ teeth. They were remarkably similar to present day hopping mice but became extinct in the Pleistocene about 2 million years ago. The great American faunal interchange The greatest change to the mammals of South America occurred when the isthmus of Panama arose from the sea in the Pliocene, 2.5–3 million years ago: this provided a continuous land link between the two American continents for the first time in more than 60 million years (Marshall et al 1979). There had been a few exchanges before this, with sloths, Bradypodidae, entering North America, and small procyonid carnivores, such as the coati and the kinkajou entering South America, but once the land connection was established a flood of species moved south and a lesser number of species moved north: this is called the great American faunal interchange. Those placental mammals that went south evolved into new species of all kinds in the southern environments, especially in the rainforests but also in the higher altitudes and in the southern grasslands. It is not clear how much this invasion affected the indigenous marsupials of the forest but the large carnivorous marsupials became extinct at or before this time. One group of placental mammals that did not penetrate further than northern Colombia were the shrews and moles, Order Insectivora, which are represented in North America by 16 species in 10 genera: their absence further south may be because the small insectivore niche was already fully occupied by many small to medium marsupials, especially the shrew opossums. From South America a smaller number of indigenous mammals, both marsupial and placental, moved north into Central America and a few, like the the armadillo, Dasypus novemcinctus, and the Virginia opossum, Didelphis virginiana, extended far into the northern continent. Relationships of living American marsupials The fossil history shows that the four families of present day marsupials are not closely related and this has been corroborated by recent evidence from DNA hybridisation and DNA sequence comparisons (see Fig. 1.10) (Kirsch and Palma 1995, Kirsch et al 1997, Jansa and Voss 2000, Patton and Costa 2003). On these criteria the Microbiotheriidae, represented today by Dromiciops, and the Didelphidae separated more than 60 million years ago, during the Palaeocene. Likewise, the differences between the caenolestids and the didelphids put their separation at about 50 million years ago. Both these figures agree well with the evidence from the Tiupampa fossils that these three families were already distinct then.

Natural regions of South America There are four major habitats of the continent (Fig. 3.3). Almost all of the northern two-thirds is the Brazilian zone, comprising the Orinoco and Amazon River basins, from the eastern slopes of Colombia and Peru, across all of Venezuela and Guiana to the eastern highlands of Brazil; it also extends into Central America. The dominant vegetation of this vast region is tropical rainforest in the central part and semiarid grasslands (llanos) on the periphery. It is bounded on the west by the Andean cordillera, which extends all the way to Patagonia in the far south; and on the east by the dry highlands of Brazil, from the Gran Chaco to the Cerrados and Caatinga in the north east. Along the Atlantic seaboard of Brazil there was a coastal forest, the Restinga, now largely cleared for agriculture. The extensive grassland pampas of Argentina extends to latitude 40°S;


beyond this is the dry rainshadow region of Patagonia to the east of the southern Andes. Most marsupial species dwell in the northern rainforests, while a few species live in the drier regions of the Caatinga and the southern pampas; only 10 species live in the cool temperate regions of the Andes and the far south, and their collective distributions leave much of the southern cone devoid of marsupials. Thus, despite their long occupation of South America, few of its marsupials have

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Figure 3.3: Major vegetation regions of South America, separated by lines and with the high altitude regions shaded. Sites (O) where research mentioned in the text has been conducted; major Tertiary sites (V) where fossil marsupials have been found. After Patterson and Pascual (1972).

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Life of Marsupials

truly adapted to life in a cool temperate environment (McNab 1986). This is curious because most of the sites of fossil marsupials from the distant past are located in the south or west, away from the present centre of distribution (Fig. 3.3). This may merely be because the tropical rainforest is not a suitable site for fossilisation or to recover fossils from. Alternatively, it may indicate that there have been considerable changes in the climate of the southern sites since the fossils were laid down and that the southern cone and western Antarctica was the centre from which mammals spread northwards into the rest of the continent and also into Australia. Indeed, the great Argentinian palaeontologist, Carol Ameghino, who discovered many of the fossil sites, postulated this more than 100 years ago and was derided by other scientists for it.

The cool temperate marsupials of South America The shrew opossums, Caenolestidae, and the sole member of the Microbiotheriidae are restricted to the cool temperate region but only two species of the Didelphidae occur in it. High altitude species The climate above 3000 m in the Andes is cold and wet and the vegetation is sometimes described as ‘elfin forest’: it consists of low, dense shrubs covered in mosses and ferns beneath which are tunnels and galleries made by the small mammals that live there. This is the parãmos, the home of the obscure silky shrew opossum, Caenolestes fuliginosus, and four related species. They are obscure because they are very difficult to catch and almost impossible to study alive. The few people who have attempted to study them have been frustrated because none has lived for more than a few days in captivity, and no long series of animals have been collected from one site so as to understand their ecology, behaviour or reproduction. From the first two specimens collected during the 19th century, however, it was recognised that these animals were very different from all the other marsupials of South America because of the pair of large forward directed incisors in the lower jaw, which resemble those of the Diprotodontia in Australia. These and other features of the skull pointed to their close relationship to fossil marsupials from the early Tertiary Casamayoran formation in Patagonia (Fig. 3.3). The name given to the first species reflected this: Caeno is derived from a Greek word meaning new or modern and lestes (a thief) is a suffix often applied to small predaceous fossil marsupials, so the name means a modern member of an ancient group. Since the first two specimens were collected from the northern end of the Andean range in Colombia and Venezuela, other species of Caenolestes have been described from the parãmos of Colombia, Ecuador and northern Peru, and another species, Lestoros inca, from southern Peru (Fig. 3.4). These species live at altitudes between 1800 and 4200 m; the only other caenolestid is the Chilean shrew opossum, Rhyncholestes raphanurus, which lives at a lower altitude but in a similar habitat in southern Chile and is separated from the northern species by the desert regions of northern Chile and Peru. Caenolestes fuliginosus The best known species is the silky shrew opossum (Fig. 3.1h, Plate 5), the first to be discovered (Osgood 1921). Based on stomach contents of recently caught specimens, its diet consists of a variety of insects but when captive animals were presented with live rats they killed them by stabbing with their procumbent lower incisors and then bit off pieces of the head and body with the cheek teeth (Kirsch and Waller 1979). At the end of its meal the silky shrew opossum rubbed its muzzle on the cage floor to remove blood and washed off the rest by licking the forepaws and rubbing them over the snout. This suggests that they probably eat a variety of


small vertebrates and invertebrates. Only four sets of data on reproduction of the silky shrew opossum have been collected, all from the parãmos of Colombia, by different people and in separate years (Tyndale-Biscoe and Renfree 1987). Adult males had enlarged testes and very large prostate glands in all months, and far more males were captured than females on each occasion. One pregnant female with three embryos was obtained in February 1910, another female at oestrus with four large follicles was collected on 30 April 1971 and four lactating females, without attached young, were collected in August–September 1969. Females do not have a pouch and

10 N

10 N

Caenolestes 0N

0N

10 S

10 S

Lestoros inca

20 S

20 S

Thylamys elegans

30 S

30 S

Dromiciops gliroides 40 S

40 S

Rhyncholestes raphanurus Lestodelphys halli 50 S

50 S

Figure 3.4: Distribution of marsupials that live in the cool temperate regions of the Andes and the southern cone of the continent. After Hershkovitz (1972), Hunsaker (1977), Patterson and Pascual (1972) and Streilein (1982b).

111

112

Life of Marsupials

only have four teats in the inguinal region, three or four of which were enlarged in the lactating females. Taken together, these scattered observations point to a seasonal breeding, beginning in February and ending in September. If this is correct, it implies one small litter a year and a very slow rate of development. High latitude species Rhyncholestes raphanurus Unlike the other caenolestids, the Chilean shrew opossum is found from sea level to about 1000 m from latitude 40°30'S to 43°30'S in southern Chile and on the island of Chiloe. It occurs in a range of habitats including sub-Antarctic rainforest, and is most frequently caught on the ground in dense cover or near den sites. Its diet includes earthworms, insects, seeds and fungi. The snout is very narrow and the pair of long stout incisors in the lower jaw may be used, as forceps, to pull worms and other prey from crevices. At the back of the mouth there are two flaps of skin, as in other caenolestids, past which the food is directed onto the molars. In animals collected in late summer and autumn the base of the tail was enlarged with stored fat, while other animals collected after winter had thin tails. This fat storage is not seen in other caenolestids and has been interpreted as evidence that these shrew opossums may undergo winter torpor or hibernation (Patterson and Gallardo 1987). Temple-Smith (1986) had evidence that males live for only one breeding season and that females produce a single litter of up to seven young (there are seven teats but no pouch). Thylamys elegans The elegant fat-tailed opossum, which weighs about 30 g, has an extensive distribution on the western side of the Andes from 14°S in Peru to 36°S in Chile, north of Rhyncholestes (Fig. 3.4). They have a very small home range of less than one hectare and they build nests of hair and leaves in dens in rocks or trees or in the burrows of guinea pigs. Like the other southern species, their tails fatten in winter, reaching maximum thickness in August when they enter torpor (Palma 1997). There is no evidence that they share nests during torpor, as do small hibernating possums in Australia (see Chapter 6). Females have 11–13 teats but no pouch and the breeding season begins in September, in early spring, and extends to the following March, during which time two litters are produced. In one 10-year study in south-western Chile the population fluctuated from peaks of 20/ha in three of the years to a minimum of less than 3/ha in the intervening years (Lima et al 2001). These marked changes correlated tightly with the prevailing rainfall, associated with the events of the Southern Oscillation Index, or El Niño. Because the habitat where the species lives is semi-arid thorn scrub, rainfall is critical to the abundance of insect food on which the species depends: larval insects rich in energy become available in the winter and support the growth of adults prior to the onset of reproduction, while adult insects in spring and summer provide the protein requirements of lactation and the growth of the young. Conversely, in years of low rainfall population increase is small because reproduction is reduced and the maturation of young animals is slowed down. During the dry seasons adult opossums survive by undergoing torpor and drawing on reserves in the fat deposited in their tails, and reproduction ceases. Lestodelphys halli The Patagonian opossum is closely related to Thylamys elegans, but larger, and is the only didelphid marsupial that lives south of 40°S (Fig. 3.4). It is known from fewer than 10 specimens, collected in Patagonia between 33°S and 48°S, the most southerly locality of any marsupial in South America (Marshall 1977) and indeed the world, since it is further south than most of Tasmania. Little is known about it but it is thought to prey on small mammals and birds: it has


larger canines than other didelphids in a shorter and more massive jaw, its feet are stronger than those of Thylamys and it has exceptionally large auditory bullae, the part of the skull that encloses the inner ear, and presumably contributes to acute hearing. In these respects it more closely resembles the smaller dasyurids of Australia, such as Phascogale and Dasyurus (see Chapter 4). Like the other southern species its tail becomes fattened before winter. Dromiciops gliroides The monito del monte, or little monkey of the mountains, Dromiciops gliroides, is the sole living representative of the Family Microbiotheriidae. Fossils species included in this family are known from the earliest formations of the Tertiary of South America, going back 60 million years. In addition, fossils that may be related to Dromiciops have been found at Murgon, Queensland, in eastern Australia that are 55 million years old. This marsupial holds an unique place in the history of marsupials because several important features link it more closely to Australasian marsupials than to South American species (see Chapter 1): it does not have paired sperm like all didelphids and caenolestids and its DNA has greater affinity to the diprotontid marsupials than to the other South American groups. Curiously, Oldfield Thomas anticipated this affinity in 1894, when he named it Dromiciops because of its close resemblance to the Australian pygmy possum, Dromicia, and –ops meaning ‘appearance of ’ (Marshall 1978a). (Please note that Dromicia is now called Cercartetus, see Chapter 6). The two species do look superficially alike: both are less than 30 g body mass, both have short fat tails and both hibernate in the winter months in a nest. However, Dromiciops has more than one pair of incisors in the lower jaw and it does not have fused toes on its hind feet, like its Australian namesake. Dromiciops occurs only in south-central Chile from Concepcion (36°S) to the island of Chiloe and east to the Andean border with Argentina (Fig. 3.4). In this region it lives in bamboo thickets of the moist, cool temperate forests, which share many plant species with the same forest type of Tasmania and New Zealand. Like most other South American marsupials its diet consists of insects, especially beetle larvae and pupae, but in captivity it will eat a variety of other food, including fruit, oats, potato and minced meat. It constructs a nest 200 mm in diameter from the water repellent leaves of the bamboo, which protects it from rain. The nest chamber is reached through a small opening and the outside is covered with moss, which provides excellent camouflage (Mann 1958). Nests may be shared by a pair of animals and are also used by breeding females for the suckling young. Animals have been found torpid in a nest as have animals held in traps but the degree to which they hibernate in winter is still unclear. Females have a well-developed forward directed pouch containing four teats, again like Australasian marsupials. The scattered observations on reproduction by nine different people since 1893 indicate that breeding begins in the southern spring in November and ends in May as the first snow falls. The litter size in the pouch ranges from 1 to 4; after they leave the pouch the young stay together in the well-formed nest and older young may be carried on the mother’s back. It is not clear from these observations whether a female can produce more than one litter in a year, or what the life span of the adults is. Conclusion These four marsupial species from the extreme southern cone of the continent show similar adaptations for low temperatures and food shortages in winter: all are small, store fat in the base of the tail, and can enter torpor and fairly prolonged hibernation. Since they are from three distinct families of American marsupials, each with a long separate ancestry, these common features of their biology are examples of adaptive convergence to an extreme environment. The only other American marsupial that has adapted to a cool temperate climate is the Virginia opossum in North America (see The Virginia opossum goes north).

113

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Life of Marsupials

The neotropical marsupials Family Didelphidae This is the largest family of American marsupials. The dentition is common to all species: adults have 50 teeth, which comprise five pairs of incisors in the upper jaw and four pairs in the lower, followed by a pair of prominent canines in each jaw then three pairs of premolars and four pairs of molars (Fig. 3.5). The dentition of sharp pointed simple teeth reflects the generalised diet of insects, small vertebrates and soft plant tissue. The sequence of eruption of the premolars and molars and their subsequent wear has been used in several species to determine the age of specimens caught in the field and so understand the rate of turnover of the population. For species of Didelphis the seven dental age classes have been related to actual age (Fig. 3.5). The full complement of teeth is achieved at about 9 months (class 4) and the subsequent three classes are

Figure 3.5: The upper premolar and molar teeth of a series of the common opossum, Didelphis marsupialis, to show the eruption of successive molars, the replacement of the deciduous premolar (dP3) with the permanent P3, and the wear on the cusps of the oldest animals that can be used to tell the relative age of the specimens’ and the differential growth of the upper canines in males and females after sexual maturity at dental class 4. After Tyndale-Biscoe and MacKenzie (1976).


more than 10 months old. Males grow to a larger size than females and have larger canines, these differences appearing at sexual maturity. The 63 species in the family Didelphidae are grouped on size and associated characters into three subfamilies (Table 3.1), which have had separate lineages for about 12 million years (Kirsch and Palma 1995, Kirsch et al 1997). Table 3.1: Genera of American marsupials From Kirsch and Waller (1979), Harder and Fleck (1997), Kirsch and Palma (1995), Fonseca et al (2003). Family

Genus

No. of chromosomes (N)

Microbiotheriidae

DromiciopsA

14

Caenolestidae

Caenolestes

14

5

20–30

Lestoros

14

1

20–30

Rhyncholestes

14

1

20–30

Marmosa

14

9

10–130

Micoureus

14

4

80–150

Monodelphis

18

16

20–150

Thylamys

14

5

20–40 76

Didelphidae

Subfamily

Marmosinae

Thylamyinae

?

1

14

11

50–80

Gracilinanus

?

6

20–30

Metachirus

14

1

300–750

ChironectesA

22

1

650

22

4

500–5000

Lutreolina

22

1

500–800

A

Philander

22

4

200–660

CaluromysA

14

3

200–400

Caluromysiops

?

1

[200]B

Glironinae

Glironia

?

1

[

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