Constraints on the morphological evolution of marsupial shoulder girdles

Throughout their evolutionary histories, marsupial mammals have been taxonomically and morphologically less diverse than their sister taxa the placentals. Because of this, it has been proposed that the evolution of marsupials has been constrained by the functional requirements of their mode of reproduction. Marsupials give birth after short gestation times to immature neonates that immediately crawl, under the power of their precociously developed shoulder girdles, to the teat where they attach and complete their early development. Using a novel approach incorporating adult and embryological morphological data, this study is the first to both: (1) statistically support adult patterns of morphological divergence consistent with the constraint hypothesis, and (2) identify ontogenetic patterns of morphological change that demonstrate that the constraint was responsible, at least in part, for their formation. As predicted by the marsupial constraint, the shoulder girdles of adult marsupials are less diverse than those of adult placentals, and adult marsupial scapulae are less morphologically diverse than adult marsupial pelves. Furthermore, marsupials that complete an extensive crawl to the teat are restricted to a common pattern of ontogenetic scapular shape change, strongly supporting the hypothesis that the morphological development of the marsupial scapula has been limited evolutionarily by its obligate role in the crawl to the teat. Because this study establishes that ontogenetic and evolutionary morphological change is correlated within mammalian scapulae, it is probable that the marsupial constraint also restricted the morphological divergence of the scapula over evolutionary time by limiting ontogenetic change in the scapula. These findings, coupled with the importance of the shoulder girdle in mammalian locomotor specialization, support the conclusion that the low morphological diversity of marsupial forms over evolutionary time could be directly due to the constraint on marsupial morphological evolution caused by the functional requirements of the crawl to the teat.     

Embryonic growth rates of marsupials with a note on monotremes

Earlier workers suggest that marsupial embryonic growth rates are slower than those of many eutherians and that there is little correlation between marsupial gestation lengths and their weight at birth. Previously, this latter observation has been explained as being due to the considerable variability in duration of the initial slow phase of marsupial embryonic growth. The latter phase of pregnancy has always been regarded as rapid and highly uniform in all marsupials. However, this review shows that there can be considerable variation in growth rate during this 'fast' phase and also that marsupials have similar rates of embryonic growth to most eutherians. Development within the monotreme egg may proceed at a similar rate to intra-uterine growth in therian mammals.     

The development of the immune tissues in marsupial pouch young

Current knowledge of the development of the marsupial immune system, particularly in the context of lymphoid tissue development and the appearance of lymphocytes, has been examined and limitations identified. While primary lymphoid tissues like the thymus have been extensively studied, secondary lymphoid tissues such as the spleen and lymph nodes have been examined to a lesser extent, partly due to the difficulty of macroscopically identifying these structures, particularly in very small neonates. In addition, little research has been conducted on the mucosal‐associated lymphoid tissues; tissues that directly trap antigens and play an important role in the maturity of adaptive immune responses. Research on the development of the marsupial immune tissues to date serves as a solid foundation for further research, particularly on the mechanisms behind the development of the immune system of marsupials. With the recent sequencing and annotation of whole marsupial genomes, the current wealth of sequence data will be essential in the development of marsupial specific reagents, including antibodies, that are required to widen our specific knowledge of the complex marsupial immune system and its development. J. Morphol. 275:822–839, 2014. © 2014 Wiley Periodicals, Inc.     

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.     

Radiation of extant marsupials after the K/T boundary: evidence from complete mitochondrial genomes

The complete mitochondrial (mt) genomes of five marsupial species have been sequenced. The species represent all three South American orders (Didelphimorphia, Paucituberculata, and Microbiotheria). Phylogenetic analysis of this data set indicates that Didelphimorphia is a basal marsupial lineage followed by Paucituberculata. The South American microbiotherid Dromiciops gliroides (monito del monte) groups with Australian marsupials, suggesting a marsupial colonization of Australia on two occasions or, alternatively, a migration of an Australian marsupial lineage to South America. Molecular estimates suggest that the deepest marsupial divergences took place 64-62 million years before present (MYBP), implying that the radiation of recent marsupials took place after the K/T (Cretaceous/Tertiary) boundary. The South American marsupial lineages are all characterized by a putatively non-functional tRNA for lysine, a potential RNA editing of the tRNA for asparagine, and a rearrangement of tRNA genes at the origin of light strand replication.     

DNA phylogeny of the marsupial wolf resolved.

The phylogenetic position of the recently extinct marsupial ''wolf'', or thylacine (Thylacinus cynocephalus), has been a source of contention in mammalian systematics for nearly a century. Thylacines were endemic to Australasia, but possessed striking anatomical similarities to Oligo-Miocene borhyaenid marsupials of South America. At issue has been whether these features are indicative of common ancestry or convergent adaptation to carnivory. Recent morphological studies have supported both conclusions. Although current marsupial classifications group thylacines with Australian dasyuromorphians, this putative clade is characterized by mostly primitive morphological features. Attempts to determine thylacine affinities with ancient protein and DNA analyses have supported, but not resolved, a dasyuromorphian placement. We report 1546 bp of mitochondrial DNA sequence (from cytochrome b and 12S rRNA genes) and 841 bp of nuclear protamine gene sequence from the thylacine and representatives of all or most other marsupial orders. Phylogenetic analysis of these sequences shows unambiguously that thylacines are members of Dasyuromorphia, and suggests a late Oligocene or very early Miocene divergence of familial lineages.     

Phylogenetic relationships of the thylacine (Mammalia: Thylacinidae) among dasyuroid marsupials: evidence from cytochrome b DNA sequences.

DNA sequences from the mitochondrial cytochrome b gene were obtained from a museum specimen of the presumed extinct thylacine (Thylacinus cynocephalus) and were compared with homologous sequences from 13 representatives of the Australian marsupial family Dasyuridae. The relationship of the thylacine to dasyurids has been suggested by previous anatomical and molecular studies, but its position within the dasyuroid radiation has not been addressed with genetic data. Phylogenetic analysis of the sequences reported here suggests that the thylacine is a sister group to Dasyuridae and lends support to the hypothesis that Thylacinus represents an ancient Australian marsupial lineage. Relationships with Dasyuridae support the results of other recent molecular studies, particularly in showing the affinities of endemic New Guinean subfamilies with larger Australian clades.     

Tracking Marsupial Evolution Using Archaic Genomic Retroposon Insertions

The Australasian and South American marsupial mammals, such as kangaroos and opossums, are the closest living relatives to placental mammals, having shared a common ancestor around 130 million years ago. The evolutionary relationships among the seven marsupial orders have, however, so far eluded resolution. In particular, the relationships between the four Australasian and three South American marsupial orders have been intensively debated since the South American order Microbiotheria was taxonomically moved into the group Australidelphia. Australidelphia is significantly supported by both molecular and morphological data and comprises the four Australasian marsupial orders and the South American order Microbiotheria, indicating a complex, ancient, biogeographic history of marsupials. However, the exact phylogenetic position of Microbiotheria within Australidelphia has yet to be resolved using either sequence or morphological data analysis. Here, we provide evidence from newly established and virtually homoplasy-free retroposon insertion markers for the basal relationships among marsupial orders. Fifty-three phylogenetically informative markers were retrieved after in silico and experimental screening of ∼217,000 retroposon-containing loci from opossum and kangaroo. The four Australasian orders share a single origin with Microbiotheria as their closest sister group, supporting a clear divergence between South American and Australasian marsupials. In addition, the new data place the South American opossums (Didelphimorphia) as the first branch of the marsupial tree. The exhaustive computational and experimental evidence provides important insight into the evolution of retroposable elements in the marsupial genome. Placing the retroposon insertion pattern in a paleobiogeographic context indicates a single marsupial migration from South America to Australia. The now firmly established phylogeny can be used to determine the direction of genomic changes and morphological transitions within marsupials.     

Marsupial MHC class II beta genes are not orthologous to the eutherian beta gene families

The major histocompatibility complex (MHC) class II DRB, DQB, DPB, and DOB gene clusters are shared by different eutherian orders. Such an orthologous relationship is not seen between the beta genes of birds and eutherians. A high degree of uncertainty surrounds the evolutionary relationship of marsupial class II beta sequences with eutherian beta gene families. In particular, it has been suggested that marsupials utilize the DRB gene cluster. A cDNA encoding an MHC class II beta molecule was isolated from a brushtail possum mesenteric lymph node cDNA library. This clone is most similar to Macropus rufogriseus DBB. Our analysis suggests that all known marsupial beta-chain genes, excluding DMB, fall into two separate clades, which are distinct from the eutherian DRB, DQB, DPB, or DOB gene clusters. We recommend that the DAB and DBB nomenclature be reinstated. DAB and DBB orthologs are not present in eutherians. It appears that the marsupial and eutherian lineages have retained different gene clusters following gene duplication events early in mammalian evolution.     

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.     

The origin of the Australasian marsupial fauna and the phylogenetic affinities of the enigmatic monito del monte and marsupial mole.

Alternative hypotheses in higher-level marsupial systematics have different implications for marsupial origins, character evolution, and biogeography. Resolving the position of the South American monito del monte (Order Microbiotheria) is of particular importance in that alternate hypotheses posit sister-group relationships between microbiotheres and taxa with disparate temporal and geographic distributions: pediomyids; didelphids; dasyuromorphians; diprotodontians; all other australidelphians; and all other marsupials. Among Australasian marsupials, the placement of bandicoots is critical; competing views associate bandicoots with particular Australasian taxa (diprotodontians, dasyuromorphians) or outside of a clade that includes all other Australasian forms and microbiotheres. Affinities of the marsupial mole are also unclear. The mole is placed in its own order (Notoryctemorphia) and sister-group relationships have been postulated between it and each of the other Australasian orders. We investigated relationships among marsupial orders by using a data set that included mitochondrial and nuclear genes. Phylogenetic analyses provide support for the association of microbiotheres with Australasian marsupials and an association of the marsupial mole with dasyuromorphs. Statistical tests reject the association of diprotodontians and bandicoots together as well as the monophyly of Australasian marsupials. The origin of the paraphyletic Australasian marsupial fauna may be accounted for by (i) multiple entries of australidelphians into Australia or (ii) bidirectional dispersal of australidelphians between Antarctica and Australia.     

The evolution of marsupial and monotreme chromosomes

Marsupial and monotreme mammals fill an important gap in vertebrate phylogeny between reptile-mammal divergence 310 million years ago (mya) and the eutherian (placental) mammal radiation 105 mya. They possess many unique features including their distinctive chromosomes, which in marsupials are typically very large and well conserved between species. In contrast, monotreme genomes are divided into several large chromosomes and many smaller chromosomes, with a complicated sex chromosome system that forms a translocation chain in male meiosis. The application of molecular cytogenetic techniques has greatly advanced our understanding of the evolution of marsupial chromosomes and allowed the reconstruction of the ancestral marsupial karyotype. Chromosome painting and gene mapping have played a vital role in piecing together the puzzle of monotreme karyotypes, particularly their complicated sex chromosome system. Here, we discuss the significant insight into karyotype evolution afforded by the combination of recently sequenced marsupial and monotreme genomes with cytogenetic analysis, which has provided a greater understanding of the events that have shaped not only marsupial and monotreme genomes, but the genomes of all mammals.     

A New European Marsupial Indicates a Late Cretaceous High-Latitude Transatlantic Dispersal Route

The first record of an undoubted opossum-like marsupial from the Mesozoic of Europe indicates an invasion from North America at the end of Late Cretaceous (Maastrichtian). The new 66.1 million-year-old marsupial, Maastrichtidelphys meurismeti n. gen., n. sp., represented by a right upper molar, comes from the type Maastrichtian of The Netherlands. The Maastricht marsupial exhibits affinities with earlier (early Maastrichtian) North American herpetotheriids providing definitive evidence of a high-latitude North Atlantic dispersal route between North America and Europe during the latest Cretaceous. Previously, the first major interchange for marsupials was thought to have occurred nearly 10 million years later in the Eocene. The occurrence of this new marsupial in Europe implies that at some time during the latest Cretaceous, sea level and climatic conditions must have been sufficiently favorable to allow for such a high-latitude dispersal. The fragmentary remains of hadrosaurid and theropod dinosaurs, as well as boid snakes from northwestern Europe which have affinities with North American taxa help substantiate assumptions made by the occurrence of the herpetotheriid marsupial in Maastricht.     

Isolation and characterization of melanopsin (Opn4) from the Australian marsupial Sminthopsis crassicaudata (fat-tailed dunnart)

Melanopsin confers photosensitivity to a subset of retinal ganglion cells and is responsible for many non-image-forming tasks, like the detection of light for circadian entrainment. Recently, two melanopsin genes, Opn4m and Opn4x, were described in non-mammalian vertebrates. However, only one form, Opn4m, has been described in the mammals, although studies to date have been limited to the placentals and have not included the marsupials. We report here the isolation and characterization of an Opn4 gene from an Australian marsupial, the fat-tailed dunnart (Sminthopsis crassicaudata), and present evidence which suggests that the Opn4x gene was lost before the placental/marsupial split. In situ hybridization shows that the expression of Opn4 in the dunnart eye is restricted to a subset of ganglion cells, a pattern previously reported for rodents and primates. These Opn4-positive cells are randomly distributed across the dunnart retina. We also undertook a comparative analysis with the South American marsupial, the grey short-tailed opossum (Monodelphis domestica), and two placental mammals, mouse and human. This approach reveals that the two marsupials show a higher sequence identity than that seen between rodents and primates, despite separating at approximately the same point in time, some 65-85 Myr ago.     

Gene mapping in marsupials and monotremes. II. Assignments to the X chromosome of dasyurid marsupials

Somatic cell hybrids have been obtained between HPRT Chinese hamster cells and cells from several dasyurid marsupial species. These hybrids show the extensive loss of marsupial chromosomes characteristic of the majority of marsupial-eutherian somatic cell hybrids. Although all of the hybrids expressed the selected marsupial marker, HPRT, the only other markers observed were PGK, GLA, and G6PD, consistent with the conservation of X-linked genes extending to this major group of marsupials. Counterselection confirmed the synteny of PGK and GLA with HPRT, whereas G6PD showed decreased concordance.     

Studies on metatherian sex chromosomes. XII. Sex-linked inheritance and probable paternal X-inactivation of alpha-galactosidase A in Australian marsupials

An investigation of genetic variation in the electrophoretic mobility of the enzyme alpha-galactosidase A (EC 3.2.1.22) has been carried out for 33 species of Australian metatherian (marsupial) mammals. The results are compatible with the enzyme being sex-linked in macropodids (kangaroos and wallabies) and probably in dasyurids (marsupial 'mice', etc.), as it is in eutherian (placental) mammals. The results also suggest that the mode of dosage compensation for this locus is the same as for other sex-linked loci in kangaroos, i.e. paternal X inactivation, rather than the random X inactivation system of eutherian mammals. The bearing of the enzyme mobility data on phylogenetic relationships among macropodid species is discussed.     

In silico identification of opossum cytokine genes suggests the complexity of the marsupial immune system rivals that of eutherian mammals

Background

Cytokines are small proteins that regulate immunity in vertebrate species. Marsupial and eutherian mammals last shared a common ancestor more than 180 million years ago, so it is not surprising that attempts to isolate many key marsupial cytokines using traditional laboratory techniques have been unsuccessful. This paucity of molecular data has led some authors to suggest that the marsupial immune system is 'primitive' and not on par with the sophisticated immune system of eutherian (placental) mammals.

Results

The sequencing of the first marsupial genome has allowed us to identify highly divergent immune genes. We used gene prediction methods that incorporate the identification of gene location using BLAST, SYNTENY + BLAST and HMMER to identify 23 key marsupial immune genes, including IFN-γ, IL-2, IL-4, IL-6, IL-12 and IL-13, in the genome of the grey short-tailed opossum (Monodelphis domestica). Many of these genes were not predicted in the publicly available automated annotations.

Conclusion

The power of this approach was demonstrated by the identification of orthologous cytokines between marsupials and eutherians that share only 30% identity at the amino acid level. Furthermore, the presence of key immunological genes suggests that marsupials do indeed possess a sophisticated immune system, whose function may parallel that of eutherian mammals.     

Chicken GnRH II occurs together with mammalian GnRH in a South American species of marsupial (Monodelphis domestica)

Two molecular forms of gonadotropin-releasing hormone (GnRH) were demonstrated in hypothalamic extracts of M. domestica using high performance liquid chromatography and radioimmunoassay with specific GnRH antisera. One form eluted in the same position as synthetic mammalian GnRH and was quantified equally by two mammalian GnRH antisera, while the second form coeluted with synthetic chicken GnRH II and was quantified equally with two chicken GnRH II antisera. The finding of chicken GnRH II in a South American species of marsupial, which has previously been reported in some Australian species of marsupial and in species of Aves, Reptilia, Amphibia, Osteichthyes and Chondrichthyes, supports our hypothesis that this widespread structural variant may represent an early evolved and conserved form of GnRH.     

A 33 kDa molecular marker of sperm acrosome differentiation and maturation in the tammar wallaby (Macropus eugenii)

This study was undertaken to identify potential molecular markers of acrosomal biogenesis and post-testicular maturation in marsupials, using the tammar wallaby as a model species. A two-step sperm extraction procedure yielded two protein extracts of apparent acrosomal origin and a tail extract. The extracts were analysed by SDS-PAGE under reducing conditions. Several prominent polypeptide bands (45, 38 and 33 kDa) appeared common to both acrosomal extracts. Antiserum raised against the 33 kDa polypeptide from the inner acrosomal membrane matrix (IAMM) extract showed immunoreactivity with 45, 38 and 33 kDa polypeptides in both acrosomal extracts, indicating that the 33 kDa polypeptide was related to the proteins in the 45 and 38 kDa bands. Therefore, the antiserum was used as a molecular probe. Indirect immuno-fluorescence indicated that the acrosome was the major location of the 33 kDa polypeptide. This contention was confirmed by ultrastructural study: immunogold labelling indicated that the 33 kDa polypeptide associated with acrosomal matrix components throughout acrosomal development in the testes and throughout post-testicular maturation in the epididymis. The label clearly delineated the changing morphology of the maturing marsupial acrosome. This is the first study to use immunocytochemical techniques to chart testicular and post-testicular development of any sperm organelle in a marsupial. As a result of this study, a 33 kDa molecular marker of marsupial acrosome differentiation and maturation has been identified. It may be possible to chart similar events in other marsupial species and identify opportunities for manipulating fertility.