Sex determination in marsupials: evidence for a marsupial-eutherian dichotomy

In this paper, we review briefly the current state of knowledge about sexual differentiation in eutherian mammals, and then describe the situation in detail in two marsupial species: the North American opossum and the tammar wallaby. The conventional explanation for the genesis of all male somatic sexual dimorphisms in mammals is that they are a consequence of the systemic action of testicular hormones. In the absence of testes, the embryo will develop a female phenotype. We present evidence for the tammar wallaby that calls into question the universal applicability of this hormonal theory of mammalian sexual differentiation. We have shown that extensive somatic sexual dimorphisms precede by many days the first morphological evidence of testicular formation, which does not occur until around the third day of pouch life. Male foetuses, and pouch young on the day of birth, already have a well-developed gubernaculum and processus vaginalis, paired scrotal anlagen, and a complete absence of mammary anlagen, whereas female foetuses and newborn pouch young have a poorly developed gubernaculum and processus vaginalis, no scrotal anlagen, and well-developed mammary anlagen. Because it seems unlikely that the male gonad could begin hormone secretion until after the Sertoli and Leydig cells are developed, our results strongly suggest that some sexually dimorphic somatic characteristics develop autonomously, depending on their genotype rather than the hormonal environment to which they are exposed. We have been able to confirm the hormonal independence of the scrotum, pouch and mammary gland by administering testosterone propionate daily by mouth to female pouch young from the day of birth; although the Wolffian duct was hyperstimulated, there was no sign of scrotal development, or pouch or mammary inhibition. When male pouch young were treated with oestradiol benzoate in a similar fashion, there was hyperstimulation of the Müllerian duct and inhibition of testicular migration and development, but no sign of scrotal inhibition or pouch or mammary development. Our results in the tammar wallaby are consistent with the earlier studies on the opossum, whose significance was not appreciated at the time. Further evidence in support of this hormonal independence comes from earlier studies of spontaneously occurring intersexes in several species of marsupial, including the opossum and the tammar wallaby. An XXY individual had intra-abdominal testes and complete masculinization of the male reproductive tract internally, but externally there was a pouch and mammary glands and no scrotum.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ossification heterochrony in the therian postcranial skeleton and the marsupial-placental dichotomy.

Postcranial ossification sequences in 24 therian mammals and three outgroup taxa were obtained using clear staining and computed tomography to test the hypothesis that the marsupial forelimb is developmentally accelerated, and to assess patterns of therian postcranial ossification. Sequence rank variation of individual bones, phylogenetic analysis, and algorithm-based heterochrony optimization using event pairs were employed. Phylogenetic analysis only recovers Marsupialia, Australidelphia, and Eulipotyphla. Little heterochrony is found within marsupials and placentals. However, heterochrony was observed between marsupials and placentals, relating to late ossification in hind limb long bones and early ossification of the anterior axial skeleton. Also, ossification rank position of marsupial forelimb and shoulder girdle elements is more conservative than that of placentals; in placentals the hind limb area is more conservative. The differing ossification patterns in marsupials can be explained with a combination of muscular strain and energy allocation constraints, both resulting from the requirement of active movement of the altricial marsupial neonates toward the teat. Peramelemorphs, which are comparatively passive at birth and include species with relatively derived forelimbs, differ little from other marsupials in ossification sequence. This suggests that ossification heterochrony in marsupials is not directly related to diversity constraints on the marsupial forelimb and shoulder girdle.     

OSSIFICATION HETEROCHRONY IN THE THERIAN POSTCRANIAL SKELETON AND THE MARSUPIAL–PLACENTAL DICHOTOMY

Postcranial ossification sequences in 24 therian mammals and three outgroup taxa were obtained using clear staining and computed tomography to test the hypothesis that the marsupial forelimb is developmentally accelerated, and to assess patterns of therian postcranial ossification. Sequence rank variation of individual bones, phylogenetic analysis, and algorithm-based heterochrony optimization using event pairs were employed. Phylogenetic analysis only recovers Marsupialia, Australidelphia, and Eulipotyphla. Little heterochrony is found within marsupials and placentals. However, heterochrony was observed between marsupials and placentals, relating to late ossification in hind limb long bones and early ossification of the anterior axial skeleton. Also, ossification rank position of marsupial forelimb and shoulder girdle elements is more conservative than that of placentals; in placentals the hind limb area is more conservative. The differing ossification patterns in marsupials can be explained with a combination of muscular strain and energy allocation constraints, both resulting from the requirement of active movement of the altricial marsupial neonates toward the teat. Peramelemorphs, which are comparatively passive at birth and include species with relatively derived forelimbs, differ little from other marsupials in ossification sequence. This suggests that ossification heterochrony in marsupials is not directly related to diversity constraints on the marsupial forelimb and shoulder girdle.     

Severe Loss of Suitable Climatic Conditions for Marsupial Species in Brazil: Challenges and Opportunities for Conservation

A wide range of evidences indicate climate change as one the greatest threats to biodiversity in the 21st century. The impacts of these changes, which may have already resulted in several recent species extinction, are species-specific and produce shifts in species phenology, ecological interactions, and geographical distributions. Here we used cutting-edge methods of species distribution models combining thousands of model projections to generate a complete and comprehensive ensemble of forecasts that shows the likely impacts of climate change in the distribution of all 55 marsupial species that occur in Brazil. Consensus projections forecasted range shifts that culminate with high species richness in the southeast of Brazil, both for the current time and for 2050. Most species had a significant range contraction and lost climate space. Turnover rates were relatively high, but vary across the country. We also mapped sites retaining climatic suitability. They can be found in all Brazilian biomes, especially in the pampas region, in the southern part of the Brazilian Atlantic Forest, in the north of the Cerrado and Caatinga, and in the northwest of the Amazon. Our results provide a general overview on the likely effects of global climate change on the distribution of marsupials in the country as well as in the patterns of species richness and turnover found in regional marsupial assemblages.     

Marsupial--mouse cell hybrids containing fragments of the marsupial X chromosome.

Hybrids were obtained from fusions of HPRT-deficient mouse fibroblasts and marsupial lymphocytes. These hybrids retained no identifiable marsupial chromosomes, but all expressed the marsupial form of HPRT. Half the clones also expressed marsupial PGK-A, and half of these also marsupial G6PD; no other marsupial allozyme markers were detected. Since G6PD is known to be sex linked in these species, we conclude that Hpt and Pgk-A are also located on the X chromosome and the markers lie in the order Hpt-Pgk-A-Gpd.     

Cone pigments in a North American marsupial, the opossum (Didelphis virginiana)

Only two of the four cone opsin gene families found in vertebrates are represented in contemporary eutherian and marsupial species. Recent genetic studies of two species of South American marsupial detected the presence of representatives from two of the classes of cone opsin genes and the structures of these genes predicted cone pigments with respective peaks in the ultraviolet and long-wavelength portions of the spectrum. The Virginia opossum (Didelphis virginiana), a profoundly nocturnal animal, is the only marsupial species found in North America. The prospects for cone-based vision in this species were examined through recordings of the electroretinogram (ERG), a commonly examined retinal response to photic stimulation. Recorded under flickering-light conditions that elicit signals from cone photoreceptors, the spectral sensitivity of the opossum eye is well accounted for by contributions from the presence of a single cone pigment having peak absorption at 561–562 nm. A series of additional experiments that employed various chromatic adaptation paradigms were conducted in a search for possible contributions from a second (short-wavelength sensitive) cone pigment. We found no evidence that such a mechanism contributes to the ERG in this marsupial.     

Evaluation of Ostrinia nubilalis (Lepidoptera: Crambidae) neonate preferences for corn and weeds in corn

Choice tests were conducted to determine feeding preferences of European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), neonates for 15 species of plants. Percentage of neonates accepting (found on) each leaf disc after 24 h was measured using choice tests. Initially, nine species of plants were evaluated. The following year, 10 plant species were evaluated during O. nubilalis first generation and 11 species during the second generation. Pennsylvania smartweed, Polygonum pennsylvanicum (L.), had the highest percentage of neonates accepting leaf discs in both years. Other plants with high acceptance rates included swamp smartweed, Polygonum amphibium L.; velvetleaf, Abutilon theophrasti Medicus; cocklebur, Xanthium strumarium L.; and yellow foxtail, Setaria glauca (L.). Corn, Zea mays L., consistently had low percentages of neonates accepting leaf discs along with common waterhemp, Amaranthus rudis Sauer. Implications these results may have on O. nubilalis host plant selection in central Iowa's corn dominated landscape are considered.     

Comparative phylogeography and demographic history of two marsupials of the Atlantic Forest in eastern Brazil

The Atlantic Forest biodiversity hotspot in eastern South America has been the focus of several phylogeographic studies concerning relationships between populations and areas and how taxa respond to environmental changes. We infer and compare the demographic and biogeographic histories of two didelphid marsupial species, Gracilinanus microtarsus and Marmosops incanus, from the Atlantic Forest of eastern Brazil to determine how these species responded to environmental changes over time, using mitochondrial and nuclear DNA sequences. We found great intraspecific genetic divergence in both species and a strong geographic structure related to similar and spatially cohesive groups within each species. These groups are consistent with the same topographical barriers, such as mountains and river valleys. Intraspecific clades are very old, dating back to a period of tectonic activities in the Neogene (5.39–8.57 Mya). Changes in the environment over the last 7 million years lead to fairly concordant demographic changes in both marsupial species, including population expansion during the last glacial maximum (ca. 21,000 years ago) or last interglacial (ca. 120,000 years ago) or both. These results do not fit the Pleistocene refuge hypothesis as an explanation of the historical biogeography and diversification of both species in the Atlantic Forest, but are compatible with the Atlantis Forest hypothesis.     

Cranial mechanics compared in extinct marsupial and extant African lions using a finite-element approach

Few species have generated more or longer running controversy than Australia's extinct marsupial lion Thylacoleo carnifex ( Owen, 1859 ). Over the last century and a half, feeding behaviours as disparate as osteophagy and specialized herbivory have been suggested and T. carnifex has been placed in both phalangeriform and vombatiform clades. Phylogenetic placement remains uncertain, but broad consensus has been achieved regarding diet, with all recent authors agreeing that T. carnifex was a carnivore. However, the marsupial lion's extraordinary cranial and dental morphologies remain without clear analogy, leaving many questions unanswered regarding how this most atypical mammalian predator killed its prey. Here I apply a rapidly emerging new approach in comparative biology, finite-element analysis, to the examination of cranial mechanics in T. carnifex . Comparisons are made with an extant lion Panthera leo (Linnaeus, 1758) under simulations designed to model stress distributions imposed by biting (intrinsic loads) and interaction with struggling prey (extrinsic loads). Modelling that approximates the 3-D architecture of jaw adductors suggests that both the placental and marsupial lions could generate considerably greater bite forces than has been predicted using 2-D approaches, but with relatively greater forces in the marsupial. The distribution of cranial stress is in many respects similar in both species, but results from simulations of extrinsic forces suggest that the marsupial was particularly well adapted to resist the high stresses that would be expected in dealings with relatively large prey. On the other hand, relatively high stress recorded in the rostrum of T. carnifex under intrinsic loadings suggests that it may have deployed a very different modus operandi , wherein the carnassial teeth played an active role in effecting a kill.     

Peace-Making in Marsupials: The First Study in the Red-Necked Wallaby (Macropus rufogriseus)

The issue of reconciliation has been widely investigated in many eutherian mammal species. Nevertheless, no data are available for marsupial mammals. Indeed, the majority of reports focus on group dynamics from an ecological and reproductive perspective, but no study has investigated them from a social point of view. We observed the red-necked wallaby colony (Macropus rufogriseus) hosted at the Tierparc Zoo Berlin (Germany) and collected data on aggressive and post-conflict interactions between group members. We found that the phenomenon of reconciliation is present in the study species (mean group CCT 27.40% ±8.89% SE). Therefore, we demonstrated, for the first time, the occurrence of reconciliation in a gregarious marsupial mammal. Post-conflict reunion was not affected by the relationship quality between individuals (friendship or kinship) but it was fine-tuned according to the aggression intensity. For example, low intensity conflicts were reconciled whereas high intensity ones were not. Reconciliation reduced anxiety-related scratching in both of the former opponents and limited further attacks towards the victim during the post-conflict period. These findings suggest that the red-necked wallaby, like many eutherian species, can evaluate the costs of reconciliation and engage in peace-making behavior in the right contexts, in order to maximize its pay-offs.     

Elimination of micronuclear specific DNA sequences early in anlagen development.

After conjugation in Tetrahymena thermophila, the old macronuclei degenerate, and new macronuclei (anlagen) develop. During anlagen development a number of DNA sequences found in the micronuclear genome (micronuclear limited sequences) are eliminated from the anlagen. A cloned copy of a repetitive micronuclear limited sequence has been used to determine the developmental stage at which micronuclear limited sequences are eliminated. DNAs from anlagen of various developmental stages were examined by Southern analysis. It was found that micronuclear limited sequences are present in 4C anlagen and essentially absent in 8C and 16C anlagen. The precipitous loss of these sequences in the 8C anlagen rules out under-replication as the mechanism for the loss and suggests that these sequences are specifically degraded early during anlagen development.     

Unexpected oocyte growth after follicular antrum formation in four marsupial species.

During examination of maturing preovulatory marsupial oocytes we noted that oocyte diameters were invariably about 50% greater than the figures reported in earlier histological studies. As all previous investigations were limited to small follicles (at most 25% the size of the ovulating follicle), the present study was initiated to examine oocyte growth during the whole period of follicular development. Oocyte and follicle diameters were measured for three Australian (Trichosurus vulpecula, Macropus eugenii and Bettongia penicillata--fresh nonfixed material) and one American marsupial species (Monodelphis domestica--histological sections) in which multiple follicle development had been induced by exogenous gonadotrophin treatment. In all species oocytes were obtained from follicles ranging from pre-antral to immediately pre-ovulatory (maximum follicle sizes obtained were: T. vulpecula, 4.5 mm; M. eugenii, 4.3 mm; B. penicillata, 2.5 mm; M. domestica, 0.7 mm). In two of the species (T. vulpecula and B. penicillata) ovulated oocytes were also examined. In T. vulpecula and M. eugenii oocytes were found to achieve much greater diameters than previously reported from histological studies of small follicles (< 0.8 mm) and similar patterns of growth were found in the other two species. In the four species oocytes reached diameters about two to three times that found for eutherian mammals. It was concluded that the marsupial oocyte continued to grow after formation of the follicular antrum and that, although the rate of oocyte growth slowed in larger follicles, it continued into the period immediately before ovulation. In B. penicillata the largest oocytes were obtained after ovulation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Phase Contrast Imaging Reveals Low Lung Volumes and Surface Areas in the Developing Marsupial

Marsupials are born with immature lungs when compared to eutherian mammals and rely, to various extents, on cutaneous gas exchange in order to meet metabolic requirements. Indeed, the fat-tailed dunnart is born with lungs in the canalicular stage of development and relies almost entirely on the skin for gas exchange at birth; consequently undergoing the majority of lung development in air. Plane radiographs and computed tomography data sets were acquired using phase contrast imaging with a synchrotron radiation source for two marsupial species, the fat-tailed dunnart and the larger tammar wallaby, during the first weeks of postnatal life. Phase contrast imaging revealed that only two lung sacs contain air after the first hour of life in the fat-tailed dunnart. While the lung of the tammar wallaby was comparatively more developed, both species demonstrated massive increases in air sac number and architectural complexity during the postnatal period. In addition, both the tammar wallaby and fat-tailed dunnart had lower lung volumes and parenchymal surface areas than were expected from morphometrically determined allometric equations relating these variables to body mass during the neonatal period. However, lung volume is predicted to scale with mass as expected after the neonatal marsupial reaches a body mass of ∼1 g and no longer relies on the skin for gas exchange. Decreased lung volume in the marsupial neonate further supports the maxim that cutaneous gas exchange occurs in the marsupial neonate because the respiratory apparatus is not yet capable of meeting the gas exchange requirements of the newborn.     

Prenatal bias in sex ratios in a marsupial,Antechinus agilis.

Biased sex ratios of young of birds and mammals clearly occur and may have an adaptive significance, but we rarely know the stage at which a bias is generated, or the mechanism. If a bias is generated prior to birth, studies of marsupials may be insightful because gestation is short and neonates are relatively undifferentiated. This study investigated whether biased sex ratios in Antechinus agilis are generated in the brief period between birth and the attachment of young to the mother''s teats. When all young born, or just pouch young, or supernumerary young were considered, litters were strongly biased towards females (0.32 males), and there was no significant difference across the groups, so a bias is generated before birth in this species. Evidence from counts of corpora lutea suggests that embryo loss during gestation cannot account fully for the level of bias observed. Therefore, prefertilization mechanisms must contribute to the generation of sex-biased litters in this marsupial.     

The marsupial placenta: a phylogenetic analysis

The structure, physiology, and endocrinology of the yolk sac placenta of different marsupial groups is compared and phylogenetically analyzed to provide information on placental characters in the marsupial stem species. We conclude that the marsupial stem species possessed a functional yolk sac placenta. Histotrophic nutrition by uterine secretion decreased during late pregnancy and at least half of the yolk sac was vascularized at the time of shell coat rupture. Due to yolk sac fusion, the larger part of the avascular, bilaminar yolk sac could not serve as a placenta at late gestation in the polyovular marsupial stem species. The bilaminar yolk sac gained a relatively greater importance for nutrition in monovular australidelphians. In macropodids a greater proportion of the yolk sac remained bilaminar at the time of shell coat rupture than in the stem species. Another derived feature of macropodids is the sustained plasma progesterone synthesis that is in turn responsible for an extended secretory phase of the uterus and a lengthened gestation. The placenta of the marsupial stem species was probably capable of metabolising histo- and hemotrophes. Recognition of pregnancy during early stages of development is a derived character of macropodids that we suggest did not occur in the marsupial stem species. However, birth and birth behaviour were apparently induced by prostaglandins in the marsupial stem species. Although the yolk sac formed the definitive placenta, it is likely that the allantois provided a supplementary placental function in the marsupial stem species, but that the role of the allantois became progressively less important during the evolution of marsupial placentation.     

Active generation glands present in neonates of some Cordylid Lizards: A case study of Cordylus macropholis (Sauria: Cordylidae)

Generation glands are holocrine epidermal glands occurring on the ventral aspect of the thigh of cordylid lizards. In most species these glands seemingly start to differentiate with the onset of sexual maturity, but macroscopic signs of generation gland activity were noted in neonates of the large-scaled girdled lizard, Cordylus macropholis. The glands of neonatal, subadult, and adult individuals were examined microscopically using standard histological techniques. The glands of the five neonatal specimens examined, including both males and females, all had the same basic structure and displayed two layers of mature glandular material. In subadult and adult specimens, the number of layers varied from seven to nine. The structure of the generation glands of C. macropholis is similar to that of the few other Cordylus species that have been described to date. They are of the protruding kind with multiple mature glandular generations. Juveniles of an additional 12 cordylid species have been examined for the presence of active generation glands. Active glands were found to be present in neonates of C. tasmani and C. tropidosternum, both of which are, like C. macropholis, terrestrial species. In C. cordylus and C. coeruleopunctatus, active generation glands are absent in neonates, but differentiate soon after birth. In other cordylid species, generation glands apparently differentiate only with the onset of sexual maturity. J. Morphol. 235:177–182, 1998. © 1998 Wiley-Liss, Inc.     

Comparative genetic structure reflects underlying life histories of three sympatric small mammal species in continuous forest of south-eastern Australia

There are strong links between habitat fragmentation, population size and genetic structure. However, to fully understand the long‐term effects of fragmentation on population viability, it is necessary to first understand the relationship between life history traits and genetic characteristics in un‐fragmented habitats. This is best done through comparing patterns of genetic diversity in sympatric species, since relative data may be maximally informative. We compared genetic structure and diversity among three sympatric small mammals – of which two were marsupial species – over a small spatial scale (<4 km) using microsatellite genotypic data from individuals sampled from four grids spaced in a linear fashion. Expected heterozygosity was high for all three species (average H_e range: 0.781–0.886), but the smallest species had significantly higher genetic diversity (both heterozygosity and allelic diversity) than the two larger species, possibly due to it having; 1) large effective population size and little overlap in generations, and/or 2) high mutation rates in large alleles. Genotypic isolation by distance (measured as relatedness) was detected only at the smallest distance of 750 m, for any species. In the two marsupial species F_st, relatedness and percentage of individuals assigned to site of capture were significantly lower in males than in females, corroborating previous reports of male‐biased dispersal. However, sex‐biased dispersal was not detected for the third species (the native bush rat Rattus fuscipes), and presence of significant heterozygous deficits suggested this resulted in inbreeding within kin‐structured demes. We speculate that habitat fragmentation will have differing effects on population dynamics, social organisation and mating systems for the two marsupial species compared to the native murid rodent, due to their differing population structure and divergent inbreeding avoidance mechanisms.     

Comparative study of the antioxidant defence systems in the erythrocytes of australian marsupials and monotremes

A comparison of the erythrocyte (RBC) antioxidant metabolites and enzymes in nine marsupial and two monotreme species was carried out. Reduced glutathione (GSH) concentrations were comparable with those reported for other marsupial and eutherian species. An important finding was that the erythrocytes of the southern hairy nosed wombat regenerated GSH faster than the erythrocytes from its close relative, the common wombat. The activities of glutathione-S-transferase, NADH-methaemoglobin reductase, Superoxide dismutase, and glutathione peroxidase (GSH-Px), showed similar levels and extents of variation as those observed in other marsupial and eutherian species. Catalase activities in the marsupials were lower than those measured in the two monotreme species and much lower than those reported in eutherian species. A negative correlation, significant at P < 0.05, was observed between GSH-Px and catalase activities in the RBC of the marsupials. Since both these enzymes “detoxify” H₂O₂, there appears to be a reciprocal relationship between the activities of these enzymes in marsupial RBC     

Doublecortin is expressed in articular chondrocytes

Articular cartilage and cartilage in the embryonic cartilaginous anlagen and growth plates are both hyaline cartilages. In this study, we found that doublecortin (DCX) was expressed in articular chondrocytes but not in chondrocytes from the cartilaginous anlagen or growth plates. DCX was expressed by the cells in the chondrogenous layers but not intermediate layer of joint interzone. Furthermore, the synovium and cruciate ligaments were DCX-negative. DCX-positive chondrocytes were very rare in tissue engineered cartilage derived from in vitro pellet culture of rat chondrosarcoma, ATDC5, and C3H10T1/2 cells. However, the new hyaline cartilage formed in rabbit knee defect contained mostly DCX-positive chondrocytes. Our results demonstrate that DCX can be used as a marker to distinguish articular chondrocytes from other chondrocytes and to evaluate the quality of tissue engineered or regenerated cartilage in terms of their "articular" or "non-articular" nature.