Molecular phylogeny, biogeography, and habitat preference evolution of marsupials

Marsupials exhibit great diversity in ecology and morphology. However, compared to their sister group, the placental mammals, our understanding of many aspects of marsupial evolution remains limited. We use 101 mitochondrial genomes and data from 26 nuclear loci to reconstruct a dated phylogeny including 97% of extant genera and 58% of modern marsupial species. This tree allows us to analyze the evolution of habitat preference and geographic distributions of marsupial species through time. We found a pattern of mesic-adapted lineages evolving to use more arid and open habitats, which is broadly consistent with regional climate and environmental change. However, contrary to the general trend, several lineages subsequently appear to have reverted from drier to more mesic habitats. Biogeographic reconstructions suggest that current views on the connectivity between Australia and New Guinea/Wallacea during the Miocene and Pliocene need to be revised. The antiquity of several endemic New Guinean clades strongly suggests a substantially older period of connection stretching back to the Middle Miocene, and implies that New Guinea was colonized by multiple clades almost immediately after its principal formation.

Evolution of a contagious cancer: epigenetic variation in devil facial tumour disease

The emergence of Devil Facial Tumour Disease (DFTD), a highly contagious cancer, is driving Tasmanian devils (Sarcophilus harrisii) to extinction. The cancer is a genetically and chromosomally stable clonal cell line which is transmitted by biting during social interactions. In the present study, we explore the Devil Facial Tumour (DFT) epigenome and the genes involved in DNA methylation homeostasis. We show that tumour cells have similar levels of methylation to peripheral nerves, the tissue from which DFTD originated. We did not observe any strain or region-specific epimutations. However, we revealed a significant increase in hypomethylation in DFT samples over time (p < 0.0001). We propose that loss of methylation is not because of a maintenance deficiency, as an upregulation of DNA methyltransferase 1 gene was observed in tumours compared with nerves (p < 0.005). Instead, we believe that loss of methylation is owing to active demethylation, supported by the temporal increase in MBD2 and MBD4 (p < 0.001). The implications of these changes on disease phenotypes need to be explored. Our work shows that DFTD should not be treated as a static entity, but rather as an evolving parasite with epigenetic plasticity. Understanding the role of epimutations in the evolution of this parasitic cancer will provide unique insights into the role of epigenetic plasticity in cancer evolution and progression in traditional cancers that arise and die with their hosts.

Evolution of sexual segregation in mammalian herbivores: kangaroos as marsupial models

Sexual segregation is best known in sexually dimorphic ungulates. Many hypotheses have been proposed to explain the evolution of sexual segregation in ungulates, but all are reducible to the influence of two factors: body size and sex-specific reproductive strategy. Definitive tests of these hypotheses are lacking in ungulates because these factors are confounded, all males being somewhat larger than females. Kangaroos represent a parallel radiation of terrestrial herbivores, but their populations are composed of a spectrum of adult body sizes, ranging from small males the same size as females to large males more than twice the size. We exploited this heteromorphism to assess the independent influences of size and sex in these ungulate analogues. We conducted a preliminary study of western grey kangaroos (Macropus fuliginosus) in north-western Victoria, Australia. Adult males predominately occupied grassland habitat, whereas females occurred mostly in lakebed, woodland and shrubland. Single-sex groups occurred more often than expected during the non-mating season. The diet of large males had the highest proportion of grass, and females had the least. These initial results indicate that both size and sex influence segregation in this species, confirming the worth of kangaroos as marsupial models for research into the evolution of sexual segregation.

The human sex-reversing ATRX gene has a homologue on the marsupial Y chromosome, ATRY: Implications for the evolution of mammalian sex determination

Mutations in the ATRX gene on the human X chromosome cause X-linked α-thalassemia and mental retardation. XY patients with deletions or mutations in this gene display varying degrees of sex reversal, implicating ATRX in the development of the human testis. To explore further the role of ATRX in mammalian sex differentiation, the homologous gene was cloned and characterized in a marsupial. Surprisingly, active homologues of ATRX were detected on the marsupial Y as well as the X chromosome. The Y-borne copy (ATRY) displays testis-specific expression. This, as well as the sex reversal of ATRX patients, suggests that ATRY is involved in testis development in marsupials and may represent an ancestral testis-determining mechanism that predated the evolution of SRY as the primary mammalian male sex-determining gene. There is no evidence for a Y-borne ATRX homologue in mouse or human, implying that this gene has been lost in eutherians and its role supplanted by the evolution of SRY from SOX3 as the dominant determiner of male differentiation.

Family-level relationships among the Australasian marsupial “herbivores” (Diprotodontia: Koala, wombats, kangaroos and possums)

The marsupial order Diprotodontia includes 10 extant families, which occupy all terrestrial habitats across Australia and New Guinea and have evolved remarkable dietary and locomotory diversity. Despite considerable attention, the interrelations of these families have for the most part remained elusive. In this study, we separately model mitochondrial RNA and protein-coding sequences in addition to nuclear protein-coding sequences to provide near-complete resolution of diprotodontian family-level phylogeny. We show that alternative topologies inferred in some previous studies are likely to be artifactual, resulting from branch-length and compositional biases. Subordinal groupings resolved herein include Vombatiformes (wombats and koala) and Phalangerida, which in turn comprises Petauroidea (petaurid gliders and striped, feathertail, ringtail and honey possums) and a clade whose plesiomorphic members possess blade-like premolars (phalangerid possums, kangaroos and their allies and most likely, pygmy possums). The topology resolved reveals ecological niche structuring among diprotodontians that has likely been maintained for more than 40 million years.

Reconstruction of the ancestral marsupial karyotype from comparative gene maps

BACKGROUND: The increasing number of assembled mammalian genomes makes it possible to compare genome organisation across mammalian lineages and reconstruct chromosomes of the ancestral marsupial and therian (marsupial and eutherian) mammals. However, the reconstruction of ancestral genomes requires genome assemblies to be anchored to chromosomes. The recently sequenced tammar wallaby (Macropus eugenii) genome was assembled into over 300,000 contigs. We previously devised an efficient strategy for mapping large evolutionarily conserved blocks in non-model mammals, and applied this to determine the arrangement of conserved blocks on all wallaby chromosomes, thereby permitting comparative maps to be constructed and resolve the long debated issue between a 2n=14 and 2n=22 ancestral marsupial karyotype. RESULTS: We identified large blocks of genes conserved between human and opossum, and mapped genes corresponding to the ends of these blocks by fluorescence in situ hybridization (FISH). A total of 242 genes was assigned to wallaby chromosomes in the present study, bringing the total number of genes mapped to 554 and making it the most densely cytogenetically mapped marsupial genome. We used these gene assignments to construct comparative maps between wallaby and opossum, which uncovered many intrachromosomal rearrangements, particularly for genes found on wallaby chromosomes X and 3. Expanding comparisons to include chicken and human permitted the putative ancestral marsupial (2n=14) and therian mammal (2n=19) karyotypes to be reconstructed. CONCLUSIONS: Our physical mapping data for the tammar wallaby has uncovered the events shaping marsupial genomes and enabled us to predict the ancestral marsupial karyotype, supporting a 2n=14 ancestor. Futhermore, our predicted therian ancestral karyotype has helped to understand the evolution of the ancestral eutherian genome.

Architectonic subdivisions of the amygdalar complex of a primitive marsupial (Didelphis aurita)

The architecture of the amygdaloid complex of a marsupial, the opossum Didelphis aurita, was analyzed using classical stains like Nissl staining and myelin (Gallyas) staining, and enzyme histochemistry for acetylcholinesterase and NADPH-diaphorase. Most of the subdivisions of the amygdaloid complex described in eutherian mammals were identified in the opossum brain. NADPH-diaphorase revealed reactivity in the neuropil of nearly all amygdaloid subdivisions with different intensities, allowing the identification of the medial and lateral subdivisions of the cortical posterior nucleus and the lateral subdivision of the lateral nucleus. The lateral, central, basolateral and basomedial nuclei exhibited acetylcholinesterase positivity, which provided a useful chemoarchitectural criterion for the identification of the anterior basolateral nucleus. Myelin stain allowed the identification of the medial subdivision of the lateral nucleus, and resulted in intense staining of the medial subdivisions of the central nucleus. The medial, posterior, and cortical nuclei, as well as the amygdalopiriform area did not exhibit positivity for myelin staining. On the basis of cyto- and chemoarchitectural criteria, the present study highlights that the opossum amygdaloid complex shares similarities with that of other species, thus supporting the idea that the organization of the amygdala is part of a basic plan conserved through mammalian evolution. (C) 2008 Elsevier Inc. All rights reserved.

Evolution of lactation : ancient origin and extreme adaptations of the lactation system

Lactation, an important characteristic of mammalian reproduction, has evolved by exploiting a diversity of strategies across mammals. Comparative genomics and transcriptomics experiments have now allowed a more in-depth analysis of the molecular evolution of lactation. Milk cell and mammary gland genomic studies have started to reveal conserved milk proteins and other components of the lactation system of monotreme, marsupial, and eutherian lineages. These analyses confirm the ancient origin of the lactation system and provide useful insight into the function of specific milk proteins in the control of lactation. These studies also illuminate the role of milk in the regulation of growth and development of the young beyond simple nutritive aspects.

Anthropogenic selection enhances cancer evolution in Tasmanian devil tumours

The Tasmanian Devil Facial Tumour Disease (DFTD) provides a unique opportunity to elucidate the long-term effects of natural and anthropogenic selection on cancer evolution. Since first observed in 1996, this transmissible cancer has caused local population declines by >90%. So far, four chromosomal DFTD variants (strains) have been described and karyotypic analyses of 253 tumours showed higher levels of tetraploidy in the oldest strain. We propose that increased ploidy in the oldest strain may have evolved in response to effects of genomic decay observed in asexually reproducing organisms. In this study, we focus on the evolutionary response of DFTD to a disease suppression trial. Tumours collected from devils subjected to the removal programme showed accelerated temporal evolution of tetraploidy compared with tumours from other populations where no increase in tetraploid tumours were observed. As ploidy significantly reduces tumour growth rate, we suggest that the disease suppression trial resulted in selection favouring slower growing tumours mediated by an increased level of tetraploidy. Our study reveals that DFTD has the capacity to rapidly respond to novel selective regimes and that disease eradication may result in novel tumour adaptations, which may further imperil the long-term survival of the world's largest carnivorous marsupial.

Identification of natural killer cell receptor genes in the genome of the marsupial Tasmanian devil (Sarcophilus harrisii)

Within the mammalian immune system, natural killer (NK) cells contribute to the first line of defence against infectious agents and tumours. Their activity is regulated, in part, by cell surface NK cell receptors. NK receptors can be divided into two unrelated, but functionally analogous superfamilies based on the structure of their extracellular ligand-binding domains. Receptors belonging to the C-type lectin superfamily are predominantly encoded in the natural killer complex (NKC), while receptors belonging to the immunoglobulin superfamily are predominantly encoded in the leukocyte receptor complex (LRC). Natural killer cell receptors are emerging as a rapidly evolving gene family which can display significant intra- and interspecific variation. To date, most studies have focused on eutherian mammals, with significantly less known about the evolution of these receptors in marsupials. Here, we describe the identification of 43 immunoglobulin domain-containing LRC genes in the genome of the Tasmanian devil (Sarcophilus harrisii), the largest remaining marsupial carnivore and only the second marsupial species to be studied. We also identify orthologs of NKC genesKLRK1, CD69, CLEC4E, CLEC1B, CLEC1A and an ortholog of an opossum NKC receptor. Characterisation of these regions in a second, distantly related marsupial provides new insights into the dynamic evolutionary histories of these receptors in mammals. Understanding the functional role of these genes is also important for the development of therapeutic agents against Devil Facial Tumour Disease, a contagious cancer that threatens the Tasmanian devil with extinction.

Differential temporal expression of milk miRNA during the lactation cycle of the marsupial tammar wallaby (Macropus eugenii)

Lactation is a key aspect of mammalian evolution for adaptation of various reproductive strategies along different mammalian lineages. Marsupials, such as tammar wallaby, adopted a short gestation and a relatively long lactation cycle, the newborn is immature at birth and significant development occurs postnatally during lactation. Continuous changes of tammar milk composition may contribute to development and immune protection of pouch young. Here, in order to address the putative contribution of newly identified secretory milk miRNA in these processes, high throughput sequencing of miRNAs collected from tammar milk at different time points of lactation was conducted. A comparative analysis was performed to find distribution of miRNA in milk and blood serum of lactating wallaby.

Role of marsupial tammar wallaby milk in lung maturation of pouch young

Background: Marsupials such as the tammar wallaby (M.Eugenii) have a short gestation (29.3 days) and at birth the altricial young resembles a fetus, and the major development occurs postnatally while the young remains in the mother's pouch. The essential functional factors for the maturation of the neonate are provided by the milk which changes in composition progressively throughout lactation (300 days). Morphologically the lungs of tammar pouch young are immature at birth and the majority of their development occurs during the first 100 days of lactation. Results: In this study mouse embryonic lungs (E-12) were cultured in media with tammar skim milk collected at key time points of lactation to identify factors involved in regulating postnatal lung maturation. Remarkably the embryonic lungs showed increased branching morphogenesis and this effect was restricted to milk collected at specific time points between approximately day 40 to 100 lactation. Further analysis to assess lung development showed a significant increase in the expression of marker genes Sp-C, Sp-B, Wnt-7b, BMP4 and Id2 in lung cultures incubated with milk collected at day 60. Similarly, day 60 milk specifically stimulated proliferation and elongation of lung mesenchymal cells that invaded matrigel. In addition, this milk stimulated proliferation of lung epithelium cells on matrigel, and the cells formed 3-dimensional acini with an extended lumen. Conclusions: This study has clearly demonstrated that tammar wallaby milk collected at specific times in early lactation contains bioactives that may have a significant role in lung maturation of pouch young.

The Genome Sequence of Taurine Cattle: A Window to Ruminant Biology and Evolution

To understand the biology and evolution of ruminants, the cattle genome was sequenced to about sevenfold coverage. The cattle genome contains a minimum of 22,000 genes, with a core set of 14,345 orthologs shared among seven mammalian species of which 1217 are absent or undetected in noneutherian (marsupial or monotreme) genomes. Cattle-specific evolutionary breakpoint regions in chromosomes have a higher density of segmental duplications, enrichment of repetitive elements, and species-specific variations in genes associated with lactation and immune responsiveness. Genes involved in metabolism are generally highly conserved, although five metabolic genes are deleted or extensively diverged from their human orthologs. The cattle genome sequence thus provides a resource for understanding mammalian evolution and accelerating livestock genetic improvement for milk and meat production.

The prion protein gene: Identifying regulatory signals using marsupial sequence

The function of the prion protein gene (PRNP) and its normal product PrPC is elusive. We used comparative genomics as a strategy to understand the normal function of PRNP. As the reliability of comparisons increases with the number of species and increased evolutionary distance, we isolated and sequenced a 66.5 kb BAC containing the PRNP gene from a distantly related mammal, the model Australian marsupial Macropus eugenii (tammar wallaby). Marsupials are separated from eutherians such as human and mouse by roughly 180 million years of independent evolution. We found that tammar PRNP, like human PRNP, has two exons. Prion proteins encoded by the tammar wallaby and a distantly related marsupial, Monodelphis domestica (Brazilian opossum) PRNP contain proximal PrP repeats with a distinct, marsupial-specific composition and a variable number. Comparisons of tammar wallaby PRNP with PRNPs from human, mouse, bovine and ovine allowed us to identify non-coding gene regions conserved across the marsupial-eutherian evolutionary distance, which are candidates for regulatory regions. In the PRNP 3' UTR we found a conserved signal for nuclear-specific polyadenylation and the putative cytoplasmic polyadenylation element (CPE), indicating that post-transcriptional control of PRNP mRNA activity is important. Phylogenetic footprinting revealed conserved potential binding sites for the MZF-1 transcription factor in both upstream promoter and intron/intron 1, and for the MEF2, MyTI, Oct-1 and NFAT transcription factors in the intron(s). The presence of a conserved NFAT-binding site and CPE indicates involvement of PrPC in signal transduction and synaptic plasticity. (c) 2004 Elsevier B.V. All rights reserved.