COMPARATIVE PATTERNS OF CRANIOFACIAL DEVELOPMENT IN EUTHERIAN AND METATHERIAN MAMMALS

The sequence of differentiation of major elements of the skeletal, muscular and nervous systems of the head is examined in developmental series of five eutherian (placental) and four metatherian (marsupial) mammals. The analysis identifies the elements that are conserved across the Theria, those that are unique to the Metatheria and to the Eutheria, and those that are variable. It is shown that although there are slight shifts in the sequence of development within the somatic tissues of the head, the primary difference between marsupial and placental mammals involves the timing and rate of differentiation of structures of the central nervous system (CNS) relative to a specific subset of structures of the cranial skeleton and musculature. In eutherians, CNS morphogenesis is well underway before the somatic tissues of the head begin differentiation. In metatherians, CNS development is delayed considerably and certain elements of the skeletal and muscular systems are advanced. It is concluded that the developmental differences between marsupial and placental mammals are best explained by the interaction of several processes including neurogenesis as a potential rate-limiting step, the developmental requirements of somatic elements, and the extremely short period of organogenesis of marsupial mammals. Several other issues, including the way that these data may be applied to determine the primitive therian developmental condition, and the use of comparative developmental data to address basic questions on morphogenetic processes, are discussed.     

Unusually similar patterns of antibody V segment diversity in distantly related marsupials

A pattern of coevolution between the V gene segments of Ig H and L chains has been noted previously by several investigators. Species with restricted germline V(H) diversity tend to have limited germline V(L) diversity, whereas species with high levels of germline V(H) diversity have more diverse V(L) gene segments. Evidence for a limited pool of V(H) but diverse V(L) gene segments in a South American opossum, Monodelphis domestica, is consistent with this marsupial being an exception to the pattern. To determine whether M. domestica is unique or the norm for marsupials, the V(H) and V(L) of an Australian possum, Trichosurus vulpecula, were characterized. The Ig repertoire in T. vulpecula is also derived from a restricted V(H) pool but a diverse V(L) pool. The V(L) gene segments of T. vulpecula are highly complex and contain lineages that predate the separation of marsupials and placental mammals. Thus, neither marsupial follows a pattern of coevolution of V(H) and V(L) gene segments observed in other mammals. Rather, marsupial V(H) and V(L) complexity appears to be evolving divergently, retaining diversity in V(L) perhaps to compensate for limited V(H) diversity. There is a high degree of similarity between the V(H) and V(L) in M. domestica and T. vulpecula, with the majority of V(L) families being shared between both species. All marsupial V(H) sequences isolated so far form a common clade of closely related sequences, and in contrast to the V(L) genes, the V(H) likely underwent a major loss of diversity early in marsupial evolution.     

Relationships Among the Families and Orders of Marsupials and the Major Mammalian Lineages Based on Recombination Activating Gene-1

Controversies remain over the relationships among several of the marsupial families and between the three major extant lineages of mammals: Eutheria (placentals), Metatheria (marsupials), and Prototheria (monotremes). Two opposing hypotheses place the marsupials as either sister to the placental mammals (Theria hypothesis) or sister to the monotremes (Palimpsest or Marsupionta hypothesis). A nuclear gene that has proved useful for analyzing phylogenies of vertebrates is the recombination activation gene-1 (RAG1). RAG1 is a highly conserved gene in vertebrates and likely entered the genome by horizontal transfer early in the evolution of jawed vertebrates. Phylogenetic analyses were performed on RAG1 sequences from seven placentals, 28 marsupials, and all three living monotreme species. Phylogenetic analyses of RAG1 sequences support many of the traditional relationships among the marsupials and suggest a relationship between bandicoots (order Peramelina) and the marsupial mole (order Notoryctemorphia), two lineages whose position in the phylogenetic tree has been enigmatic. A sister relationship between South American shrew opossums (order Paucituberculata) and all other living marsupial orders is also suggested by RAG1. The relationship between the three major groups of mammals is consistent with the Theria hypothesis, with the monotremes as the sister group to a clade containing marsupials and placentals.     

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.     

Biting through constraints: cranial morphology, disparity and convergence across living and fossil carnivorous mammals

Carnivory has evolved independently several times in eutherian (including placental) and metatherian (including marsupial) mammals. We used geometric morphometrics to assess convergences associated with the evolution of carnivory across a broad suite of mammals, including the eutherian clades Carnivora and Creodonta and the metatherian clades Thylacoleonidae, Dasyuromorphia, Didelphidae and Borhyaenoidea. We further quantified cranial disparity across eutherians and metatherians to test the hypothesis that the marsupial mode of reproduction has constrained their morphological evolution. This study, to our knowledge the first to extensively sample pre-Pleistocene taxa, analysed 30 three-dimensional landmarks, focused mainly on the facial region, which were digitized on 130 specimens, including 36 fossil taxa. Data were analysed with principal components (PC) analysis, and three measures of disparity were compared between eutherians and metatherians. PC1 showed a shift from short to long faces and seemed to represent diet and ecology. PC2 was dominated by the unique features of sabre-toothed forms: dramatic expansion of the maxilla at the expense of the frontal bones. PC3, in combination with PC1, distinguished metatherians and eutherians. Metatherians, despite common comparisons with felids, were more similar to caniforms, which was unexpected for taxa such as the sabre-toothed marsupial Thylacosmilus. Contrary to previous studies, metatherian carnivores consistently exhibited disparity which exceeded that of the much more speciose eutherian carnivore radiations, refuting the hypothesis that developmental constraints have limited the morphological evolution of the marsupial cranium.     

Evolution of parasitism in mammal-associated mites of the Psoroptidia group (Acari: Astigmata)

The distribution of parasitic mites of the Psoroptidia group on mammals was analyzed. Nearly all the mammal-associated Psoroptidia belong to the paraphyletic superfamily Sarcoptoidea. Mites of the family complex Psoroptidae (Lobalgidae, Psoroptidae, and Paracoroptinae) shifted from birds to parasitism on placental mammals independently from each other. Mites of the Sarcoptidae complex, comprising all the other mammal-associated Psoroptidia, originated from the common stalk of Psoroptidia independently. They are widely represented on both marsupial and placental mammals and are primarily or secondarily absent on Monotremata.     

Use of helium-oxygen to examine the effect of cold acclimation on the summit metabolism of a marsupial, Dasyuroides byrnei

To determine whether marsupial mammals increase their metabolic capabilities during cold acclimation, the metabolism of both warm and cold acclimated Dasyuroides byrnei was examined by exposure to cold in a helium-oxygen atmosphere. Mean values of heat production and conductance were significantly higher in a helium-oxygen atmosphere than in air. Body temperature did not change until metabolic capacity was exhausted. Both cold and warm acclimated groups could maintain a metabolic scope of 10-11 times the basal or standard level for this species. Such a metabolic scope is much higher than levels recorded for placental mammals. At very low ambient temperatures cold acclimated D. byrnei could sustain a high level of heat production longer than could warm acclimated animals. While there are some similarities between marsupial mammals and placental mammals in their responses to cold acclimation, an increase in maximum metabolism, as reported for placentals, does not seem to occur in marsupials.     

Molecular phylogeny of living xenarthrans and the impact of character and taxon sampling on the placental tree rooting

Extant xenarthrans (armadillos, anteaters and sloths) are among the most derived placental mammals ever evolved. South America was the cradle of their evolutionary history. During the Tertiary, xenarthrans experienced an extraordinary radiation, whereas South America remained isolated from other continents. The 13 living genera are relics of this earlier diversification and represent one of the four major clades of placental mammals. Sequences of the three independent protein-coding nuclear markers alpha2B adrenergic receptor (ADRA2B), breast cancer susceptibility (BRCA1), and von Willebrand Factor (VWF) were determined for 12 of the 13 living xenarthran genera. Comparative evolutionary dynamics of these nuclear exons using a likelihood framework revealed contrasting patterns of molecular evolution. All codon positions of BRCA1 were shown to evolve in a strikingly similar manner, and third codon positions appeared less saturated within placentals than those of ADRA2B and VWF. Maximum likelihood and Bayesian phylogenetic analyses of a 47 placental taxa data set rooted by three marsupial outgroups resolved the phylogeny of Xenarthra with some evidence for two radiation events in armadillos and provided a strongly supported picture of placental interordinal relationships. This topology was fully compatible with recent studies, dividing placentals into the Southern Hemisphere clades Afrotheria and Xenarthra and a monophyletic Northern Hemisphere clade (Boreoeutheria) composed of Laurasiatheria and Euarchontoglires. Partitioned likelihood statistical tests of the position of the root, under different character partition schemes, identified three almost equally likely hypotheses for early placental divergences: a basal Afrotheria, an Afrotheria + Xenarthra clade, or a basal Xenarthra (Epitheria hypothesis). We took advantage of the extensive sampling realized within Xenarthra to assess its impact on the location of the root on the placental tree. By resampling taxa within Xenarthra, the conservative Shimodaira-Hasegawa likelihood-based test of alternative topologies was shown to be sensitive to both character and taxon sampling.     

A molecular and evolutionary study of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata

Beta-globin gene families in eutherians (placental mammals) consist of a set of four or more developmentally regulated genes which are closely linked and, in general, arranged in the order 5'-embryonic/fetal genes- adult genes-3'. This cluster of genes is proposed to have arisen by tandem duplication of ancestral beta-globin genes, with the first duplication occurring 200 to 155 MYBP just prior to a period in mammalian evolution when eutherians and marsupials diverged from a common ancestor. In this paper we trace the evolutionary history of the beta-globin gene family back to the origins of these mammals by molecular characterization of the beta-globin gene family of the Australian marsupial Sminthopsis crassicaudata. Using Southern and restriction analysis of total genomic DNA and bacteriophage clones of beta-like globin genes, we provide evidence that just two functional beta-like globin genes exist in this marsupial, including one embryonic- expressed gene (S.c-epsilon) and one adult-expressed gene (S.c-beta), linked in the order 5'-epsilon-beta-3'. The entire DNA sequence of the adult beta-globin gene is reported and shown to be orthologous to the adult beta-globin genes of the North American marsupial Didelphis virginiana and eutherian mammals. These results, together with results from a phylogenetic analysis of mammalian beta-like globin genes, confirm the hypothesis that a two-gene cluster, containing an embryonic- and an adult-expressed beta-like globin gene, existed in the most recent common ancester of marsupials and eutherians. Northern analysis of total RNA isolated from embryos and neonatals indicates that a switch from embryonic to adult gene expression occurs at the time of birth, coinciding with the transfer of the marsupial from a uterus to a pouch environment.      

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.     

Exceptionally high conservation of the MHC class I-related gene, MR1, among mammals

The major histocompatibility complex (MHC) class I-related gene, MR1, is a non-classical MHC class IA gene and is encoded outside the MHC region. The MR1 is responsible for activation of mucosal-associated invariant T (MAIT) cells expressing semi-invariant T cell receptors in the presence of bacteria, but its ligand has not been identified. A unique characteristic of MR1 is its high evolutionary conservation of the α1 and α2 domains corresponding to the peptide-binding domains of classical MHC class I molecules, showing about 90 % amino acid identity between human and mouse. To clarify the evolutionary history of MR1 and identify more critically conserved residues for the function of MR1, we searched for the MR1 gene using jawed vertebrate genome databases and isolated the MR1 cDNA sequences of marsupials (opossum and wallaby). A comparative genomic analysis indicated that MR1 is only present in placental and marsupial mammals and that the gene organization around MR1 is well conserved among analyzed jawed vertebrates. Moreover, the α1 and α2 domains, especially in amino acid residues presumably shaping a ligand-binding groove, were also highly conserved between placental and marsupial MR1. These findings suggest that the MR1 gene might have been established at its present location in a common ancestor of placental and marsupial mammals and that the shape of the putative ligand-binding groove in MR1 has been maintained, probably for presenting highly conserved component(s) of microbes to MAIT cells.     

Why are There Fewer Marsupials than Placentals? On the Relevance of Geography and Physiology to Evolutionary Patterns of Mammalian Diversity and Disparity

Placental mammals occupy a larger morphospace and are taxonomically more diverse than marsupials by an order of magnitude, as shown by quantitative and phylogenetic studies of several character complexes and clades. Many have suggested that life history acts as a constraint on the evolution of marsupial morphology. However, the frequent circumvention of constraints suggests that the pattern of morphospace occupation in marsupials is more a reflection of lack of ecological opportunity than one of biases in the production of variants during development. Features of marsupial physiology are a potential source of biases in the evolution of the group; these could be coupled with past macroevolutionary patterns that followed conditions imposed by global temperature changes. This is evident at the K/Pg boundary and at the Eocene/Oligocene boundary. The geographic pattern of taxonomic and morphological diversity in placental clades mirrors that of extant placentals as a whole versus marsupials: placentals of northern origin are more diverse those of southern one and include the clades that are outliers in taxonomic (rodents and bats) and ecomorphological (whales and bats) richness.     

Evolution of parasitism in mammal-associated mites of the group Psoroptidia (Acari:Astigmata)

Host-parasite relationships of mammals and astigmatan mites (Acariformes: Astigmata) belonging to the parvorder Psoroptidia are analyzed. The absolute majority of mammal-associated psoroptidians belongs to the paraphyletic superfamily Sarcoptoidea. Mites of the family complex Psoroptidae (Lobalgidae, Psoroptidae, and Paracoroptinae) shifted from birds to placental mammals independently from each other. Mites of the family complex Sarcoptidae, including all other sarcoptoid families, derived from the common stalk of Psoroptidia independently from the Psoroptid complex. Mites of the sarcoptid complex shifted from nidicoly in mammalian nests to the permanent parasitism on these hosts. They are widely represented on both marsupial and placental mammals and are absent on Monotremata.     

Scaling of the marsupial middle ear and its functional significance

The marsupial middle ear performs an anatomical impedance matching for acoustic energy travelling in air to reach the cochlea. The size of the middle ear sets constraints for the frequencies transmitted. For generalized placental mammals, it has been shown that the limit for high-frequency hearing can be predicted on the basis of middle ear ossicle mass, provided that the ears fulfil requirements of isometry. We studied the interspecific size variation of the middle ear in 23 marsupial species, with the following measurable parameters: skull mass, condylobasal length, ossicular masses for malleus, incus and stapes, tympanic membrane area, oval window area, and lever arm lengths for malleus and incus. Our results show that the middle ear size grows with negative allometry in relation to body size and that the internal proportions of the marsupial middle ear are largely isometric. This resembles the situation in placental mammals and allows us to use their isometric middle ear model to predict the high-frequency hearing limit for marsupials. We found that the isometry model predicts the high-frequency hearing limit for different marsupials well, indicating that marsupials can be used as auditory models for general therian mammalian hearing. At very high frequencies, other factors, such as the inner ear, seem to constrain mammalian hearing.     

Conservation throughout mammalia and extensive protein-encoding capacity of the highly repeated DNA long interspersed sequence one

We report an investigation of the structure, evolutionary history, and function of the highly repeated DNA family named Long Interspersed Sequence One (L1). Hybridization studies show, first, that L1 is present throughout marsupial and placental mammalian orders. Second, L1 is more homologous within these species than between them, which suggests that it has undergone concerted evolution within each mammalian lineage. Third, on the whole L1 diverges in accordance with the fossil record. This suggests that it arose in each lineage rather by inheritance from a common ancestral family, which was present in the progenitor to mammals, than by cross-species transmission. Alignment of 1.6 X 10(3) bases of primate and mouse L1 DNA sequences shows a predominance of silent mutations within aligned long open reading frames, indicating that at least this part of L1 has produced functional protein. The observation of additional long open reading frames in further unaligned DNA sequences suggests that a minimum of 3.2 X 10(3) bases or at least half of the L1 structure is a protein-coding sequence. Thus L1, which contains about 100,000 members in mouse, is by far the most repetitive family of which a subset comprises functional protein-encoding genes. The ability of the putative protein-encoding regions of mouse L1 to hybridize to L1 homologs throughout the Mammalia implies that these sequences have been subject to conservative selection upon protein function in all mammalian lineages, rather than in a few. L1 is therefore a highly repeated family of genes with both a widespread and an ancient history of function in mammals.     

A VpreB3 homologue in a marsupial, the gray short-tailed opossum, Monodelphis domestica

A VpreB surrogate light (SL) chain was identified for the first time in a marsupial, the opossum Monodelphis domestica. Comparing the opossum VpreB to homologues from eutherian (placental mammals) and avian species supported the marsupial gene being VpreB3. VpreB3 is a protein that is not known to traffic to the cell surface as part of the pre-B cell receptor. Rather, VpreB3 associates with nascent immunoglobulin chains in the endoplasmic reticulum. Homologues of other known SL chains VpreB1, VpreB2, and λ5, which are found in eutherian mammals, were not found in the opossum genome, nor have they been identified in the genomes of nonmammals. VpreB3 likely evolved from earlier gene duplication, independent of that which generated VpreB1 and VpreB2 in eutherians. The apparent absence of VpreB1, VpreB2, and λ5 in marsupials suggests that an extracellular pre-B cell receptor containing SL chains, as it has been defined in humans and mice, may be unique to eutherian mammals. In contrast, the conservation of VpreB3 in marsupials and its presence in nonmammals is consistent with previous hypotheses that it is playing a more primordial role in B cell development.     

Metabolic physiology of the numbat (Myrmecobius fasciatus)

The numbat (Myrmecobius fasciatus) is unique amongst marsupials as it is exclusively diurnal, feeds only on termites and is semi-fossorial. This study examines the thermal and metabolic physiology of the numbat to determine if its physiology reflects its phylogeny, diet and semi-fossorial habit. Numbats (mean adult body mass 552 g) were able to regulate body temperature at ambient temperatures of 15-30 degrees C, with a body temperature at thermoneutrality (30 degrees C) of 34.1 degrees C. The thermoneutral body temperature was not significantly different from that predicted for an equivalent-sized marsupial. Basal metabolic rate, measured at 30 degrees C, was 0.389 +/- 0.025 ml O(2) g(-1) h(-1), and was slightly but not significantly lower at 82.5% of that predicted for a typical marsupial of equivalent body mass. Metabolic rate increased with decreasing ambient temperatures below 30 degrees C. Patterns of metabolic cycling observed for completely inactive numbats at ambient temperatures below 30 degrees C are likely to be related to sleep phase. Wet thermal conductance of 1.94 J g(-1) h(-1) degrees C(-1) (at 30 degrees C) was 131% of that predicted for a marsupial. Evaporative water loss of the numbat remained constant below the thermoneutral zone (<30 degrees C) at approximately 0.6 ml g(-1) h(-1), only 47.4% of that predicted for a marsupial. It increased to 1.01 +/- 0.16 ml g(-1) h(-1) at an ambient temperature of 32.5 degrees C. The thermal and metabolic physiology of the numbat is generally similar to that expected for other marsupials, and is also comparable to that of termitivorous placental mammals. Thus the reduction in body temperature and basal metabolic rate of placental termitivores is a "marsupial-like" low energy turnover physiology, and the numbat being a marsupial already has an appropriate physiology to survive exclusively on a low energy diet of termites.     

Australia's first fossil marsupial mole (Notoryctemorphia) resolves controversies about their evolution and palaeoenvironmental origins

Fossils of a marsupial mole (Marsupialia, Notoryctemorphia, Notoryctidae) are described from early Miocene deposits in the Riversleigh World Heritage Area, northwestern Queensland, Australia. These represent the first unequivocal fossil record of the order Notoryctemorphia, the two living species of which are among the world's most specialized and bizarre mammals, but which are also convergent on certain fossorial placental mammals (most notably chrysochlorid golden moles). The fossil remains are genuinely 'transitional', documenting an intermediate stage in the acquisition of a number of specializations and showing that one of these-the dental morphology known as zalambdodonty-was acquired via a different evolutionary pathway than in placentals. They, thus, document a clear case of evolutionary convergence (rather than parallelism) between only distantly related and geographically isolated mammalian lineages-marsupial moles on the island continent of Australia and placental moles on most other, at least intermittently connected continents. In contrast to earlier presumptions about a relationship between the highly specialized body form of the blind, earless, burrowing marsupial moles and desert habitats, it is now clear that archaic burrowing marsupial moles were adapted to and probably originated in wet forest palaeoenvironments, preadapting them to movement through drier soils in the xeric environments of Australia that developed during the Neogene.     

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.