A Phylogeny and Timescale for Marsupial Evolution Based on Sequences for Five Nuclear Genes

Even though marsupials are taxonomically less diverse than placentals, they exhibit comparable morphological and ecological diversity. However, much of their fossil record is thought to be missing, particularly for the Australasian groups. The more than 330 living species of marsupials are grouped into three American (Didelphimorphia, Microbiotheria, and Paucituberculata) and four Australasian (Dasyuromorphia, Diprotodontia, Notoryctemorphia, and Peramelemorphia) orders. Interordinal relationships have been investigated using a wide range of methods that have often yielded contradictory results. Much of the controversy has focused on the placement of Dromiciops gliroides (Microbiotheria). Studies either support a sister-taxon relationship to a monophyletic Australasian clade or a nested position within the Australasian radiation. Familial relationships within the Diprotodontia have also proved difficult to resolve. Here, we examine higher-level marsupial relationships using a nuclear multigene molecular data set representing all living orders. Protein-coding portions of ApoB, BRCA1, IRBP, Rag1, and vWF were analyzed using maximum parsimony, maximum likelihood, and Bayesian methods. Two different Bayesian relaxed molecular clock methods were employed to construct a timescale for marsupial evolution and estimate the unrepresented basal branch length (UBBL). Maximum likelihood and Bayesian results suggest that the root of the marsupial tree is between Didelphimorphia and all other marsupials. All methods provide strong support for the monophyly of Australidelphia. Within Australidelphia, Dromiciops is the sister-taxon to a monophyletic Australasian clade. Within the Australasian clade, Diprotodontia is the sister taxon to a Notoryctemorphia + Dasyuromorphia + Peramelemorphia clade. Within the Diprotodontia, Vombatiformes (wombat + koala) is the sister taxon to a paraphyletic possum group (Phalangeriformes) with kangaroos nested inside. Molecular dating analyses suggest Late Cretaceous/Paleocene dates for all interordinal divergences. All intraordinal divergences were placed in the mid to late Cenozoic except for the deepest splits within the Diprotodontia. Our UBBL estimates of the marsupial fossil record indicate that the South American record is approximately as complete as the Australasian record. The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Nuclear gene sequences provide evidence for the monophyly of australidelphian marsupials

Relationships among the seven extant orders of marsupials remain poorly understood. Most classifications recognize a fundamental split between Ameridelphia, which contains the American orders Didelphimorphia and Paucituberculata, and Australidelphia, which contains four Australasian orders (Dasyuromorphia, Diprotodontia, Notoryctemorphia, and Peramelina) and the South American order Microbiotheria, represented by Dromiciops gliroides. Ameridelphia and Australidelphia are each supported by key morphological characters with dichotomous character states. To date, molecular studies indexing all marsupial orders have reported inconclusive results. However, several studies have suggested that Dromiciops is nested within Australidelphia. This result has important implications for understanding the biogeographic history of living marsupials. To address questions in higher-level marsupial systematics, we sequenced portions of five nuclear genes (Apolipoprotein B gene; Breast and Ovarian cancer susceptibility gene 1; Recombination activating gene 1; Interphotoreceptor retinoid binding protein gene; and von Willebrand factor gene) for representatives of all orders of marsupials, as well as placental outgroups. The resulting 6.4kb concatenation was analyzed using maximum parsimony, distance methods, maximum likelihood, and Bayesian methods. tests were used to examine a priori hypotheses. All analyses provided robust support for the monophyly of Australidelphia (bootstrap support=99-100%; posterior probability=1.00). Ameridelphia received much lower support, although this clade was not rejected in statistical tests. Within Diprotodontia, both Vombatiformes and Phalangeriformes were supported at the 100% bootstrap level and with posterior probabilities of 1.00.     

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.     

An Analysis of Marsupial Interordinal Relationships Based on 12S rRNA, tRNA Valine, 16S rRNA, and Cytochrome b Sequences

The basal split among living marsupials is traditionally placed between the cohorts Ameridelphiaand Australidelphia. Ameridelphia includes all American forms excepting the South AmericanDramicuipx gliroidex (Order Microbiotheria). Australidelphia includes all Australasian taxaplus Dromiciops glinmles. DNA data support Eometatheria Dromiciaps + Diprotodontia +Dasyuromorphia + Notoryctemorphia) but do not resolve the position of bandicoots, whetherwith other australidelphians or with ameridelphians. Also, the most robust molecular trees (DNAhybridization, multigene studies) exhibit minimal branch subdivision and raise the possibility ofartit'actual associations owing to long branch attraction. We analyzed data sets that consistedof complete sequences tor four niitochondrial genes (cytochrome b, 12S rRNA, tRNA valine,16S rRNA). One data set included 14 marsupial taxa. A second data set included 14 marsupialsas well as outgroup sequences (one monolreme; 20 placentals). Phylogenetic analyses includedparsimony, minimum evolution, maximum likelihood, and quartet puzzling. When phylogeneticanalyses were restricted to just the marsupial sequences, there was 75 to 96% boostrap supportfor the separation of Ameridelphia versus Australidelphia. This suggests that either one orboth of these groups are monophyletic. Also, there was 71 to 98% bootstrap support for theseparation of Eometatheria versus Ameridelphia + Peramelina. Nonmonophyly of several a prioriclades was accepted by at least some statistical tests including the following: Diprotodontia+ Peramelina, Notoryctemorphia + Peramelina, Diprotodonlia + Notoryctemorphia, and themonophyly of Australasian marsupials. With the inclusion of outgroup sequences, there wasreduced bootstrap support for associations among marsupial orders and statistical tests failed toreject all interordinal associations that were tested.     

Molecular systematics of marsupials based on the rRNA 12S mitochondrial gene: the phylogeny of didelphimorphia and of the living fossil microbiotheriid Dromiciops gliroides thomas

Nucleotide sequence data from the mitochondrial 12S rRNA gene were used to evaluate the phylogenetic relationships among the major groups of didelphimorph and paucituberculatan marsupials from South America, the microbiotheriid Dromiciops gliroides, and representatives of four orders of Australasian marsupials. Based on approximately 800 bp in 18 genera, we conclude that the didelphids constitute a monophyletic group with large-sized forms differentiated from small opossums, while Caluromys constitutes the sister taxon to didelphids. The peramelid Isoodon was recovered as the sister taxon to the paucituberculatans Caenolestes and Rhyncholestes, although it is in an uncertain phylogenetic position within the marsupial tree. Dromiciops was recovered as a well-differentiated lineage from South American opossums within the Australidelphian radiation of metatherians that include dasyurid, diprotodontian, and notoryctemorph marsupials.     

A morphological analysis of marsupial mammal higher-level phylogenetic relationships

Phylogenetic relationships among marsupial taxa have proven to be more complex than the simple grouping of species by continent. Recent marsupials are distributed across the New World, Australia, New Guinea, and certain neighboring islands. Morphological characteristics of various groups bridge different geographical areas. We investigated the origin of these characteristics by assembling a morphological data matrix consisting of a new suite of 149 postcranial characters and incorporated a series of previously published data on the craniodental (76 characters) and soft tissue (5 characters) anatomy. Twenty‐one marsupial terminal taxa representing all the major radiations of marsupials and 10 outgroups, most of which are exceptionally well‐preserved fossils such as Vincelestes, Ukhaatherium, and a few basal metatherian taxa, were investigated. A maximum parsimony analysis was conducted, resulting in one most parsimonious tree. Relationships among outgroups are congruent with current understanding of mammalian phylogeny. All currently accepted marsupial orders were recovered by the analysis. We confirmed previous results showing the South American “monito del monte”Dromiciops nested within the Australasian radiation. Within this australidelphian clade, Dromiciops was closely allied with the Diprotodontia. The South American paucituberculates appeared more closely related to the Australidelphia than to the American Didelphimorphia. The marsupial mole Notoryctes and the Peramelia were closely allied to each other and in turn were the sister group of the Dromiciops plus Diprotodontia clade. This pattern of relationships left Dasyuromorphia as the most basal offshoot of the Australidelphia. Whereas this tree topology recovers some signal that had been detected by previous studies, morphological and/or molecular, some novel hypotheses are also supported.     

Molecular Systematics of Marsupials Based on the rRNA 12S Mitochondrial Gene: The Phylogeny of Didelphimorphia and of the Living Fossil Microbiotheriid Dromiciops gliroides Thomas

Nucleotide sequence data from the mitochondrial 12S rRNA gene were used to evaluate the phylogenetic relationships among the major groups of didelphimorph and paucituberculatan marsupials from South America, the microbiotheriid Dromiciops gliroides, and representatives of four orders of Australasian marsupials. Based on approximately 800 bp in 18 genera, we conclude that the didelphids constitute a monophyletic group with large-sized forms differentiated from small opossums, while Caluromys constitutes the sister taxon to didelphids. The peramelid Isoodon was recovered as the sister taxon to the paucituberculatans Caenolestes and Rhyncholestes, although it is in an uncertain phylogenetic position within the marsupial tree. Dromiciops was recovered as a well-differentiated lineage from South American opossums within the Australidelphian radiation of metatherians that include dasyurid, diprotodontian, and notoryctemorph marsupials.     

Marsupial relationships and a timeline for marsupial radiation in South Gondwana

Recent marsupials include about 280 species divided into 18 families and seven orders. Approximately 200 species live in Australia/New Guinea. The remaining species inhabit South America with some of these secondarily ranging into North America. In this study, we examine marsupial relationships and estimate their divergences times using complete mitochondrial (mt) genomes. The sampling, which includes nine new mtDNAs and a total number of 19 marsupial genomes, encompasses all extant orders and 14 families. The analysis identified a basal split between Didelphimorphia and remaining orders about 69 million years before present (MYBP), while other ordinal divergences were placed in Tertiary times. The monotypic South American order Microbiotheria (Dromiciops gliroides, Monito del Monte) was solidly nested among its Australian counterparts. The results suggest that marsupials colonized Australia twice from Antarctica/South America and that the divergence between Microbiotheria and its Australian relatives coincided with the geological separation of Antarctica and Australia. Within Australia itself, several of the deepest divergences were estimated to have taken place close to the Eocene/Oligocene transition.     

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.     

Molecular Phylogeny and Evolution of the Neurotrophins from Monotremes and Marsupials

We have investigated the phylogenetic relationships of monotremes and marsupials using nucleotide sequence data from the neurotrophins; nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3). The study included species representing monotremes, Australasian marsupials and placentals, as well as species representing birds, reptiles, and fish. PCR was used to amplify fragments encoding parts of the neurotrophin genes from echidna, platypus, and eight marsupials from four different orders. Phylogenetic trees were generated using parsimony analysis, and support for the different tree structures was evaluated by bootstrapping. The analysis was performed with NGF, BDNF, or NT-3 sequence data used individually as well as with the three neurotrophins in a combined matrix, thereby simultaneously considering phylogenetic information from three separate genes. The results showed that the monotreme neurotrophin sequences associate to either therian or bird neurotrophin sequences and suggests that the monotremes are not necessarily related closer to therians than to birds. Furthermore, the results confirmed the present classification of four Australasian marsupial orders based on morphological characters, and suggested a phylogenetic relationship where Dasyuromorphia is related closest to Peramelemorphia followed by Notoryctemorphia and Diprotodontia. These studies show that sequence data from neurotrophins are well suited for phylogenetic analysis of mammals and that neurotrophins can resolve basal relationships in the evolutionary tree. Received: 27 January 1997 / Accepted: 20 March 1997     

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.     

On the existence of non‐microbiotherian Australidelphian marsupials (Diprotodontia) in the Eocene of Patagonia

A diverse assemblage of extinct mammals of early–middle Eocene age (Ypresian–Lutetian boundary) come from the Patagonian localities of La Barda and Laguna Fría around Paso del Sapo in northwestern Chubut Province (Argentina). Metatherians are well represented, mostly by dental remains of ‘Didelphimorphia’, Paucituberculata, Sparassodonta, Microbiotheria, and Polydolopimorphia. Here we analyse three calcanea and one astragalus referable to the same, indeterminate taxon, from La Barda, showing the fusion of their ectal and sustentacular facets. This facet arrangement characterizes the Australidelphia, a marsupial clade represented by Microbiotheria from South America and Antarctica, plus all Australasian lineages. Other australidelphian features shown by these tarsals include: in the calcanea, a reduced peroneal process and a tripartite cuboid facet; in the astragalus, a round, very small astragalar head in relation to the body; a convex trochlea with a half for the tibia and half, in slightly lower position, for the fibula. Their size and other anatomical features suggest that the new tarsals cannot be referred to the Microbiotheria. Phylogenetic analysis suggests that the La Barda taxon lies within the Australidelphia, and that it is either closely related or belongs to, the Diprotodontia. The existence of advanced australidelphians in Patagonia adds further evidence of the close relationship between Patagonia, Antarctica, and Australia during the Late Cretaceous – early Palaeogene.     

Phylogenetic studies of marsupials based on phosphoglycerate kinase DNA sequences

Phosphoglycerate kinase sequences were obtained for 313 aligned bases of 41 individuals from 39 marsupial species. In contrast to previous molecular analyses, the relationships suggested by these data show a high level of congruence with morphologically defined orders and families. Four main monophyletic lineages are recognizable. These are the monogeneric orders Microbiotheria (Dromiciops australis) and Notoryctemorphia (Notoryctes typhlops), a grouping of the American orders Didelphimorphia and Paucituberculata, and the Australasian species other than N. typhlops. Within the Australasian lineage, there are again four main monophyletic groups; the Dasyuridae, two peramelemorph (bandicoot) lineages (one comprised of pseudogene sequences) and the Diprotodontia. This topology is not greatly affected by the exclusion of pseudogenes except that a clade of syndactylous species (Peramelemorphia plus Diprotodontia) is recovered. Two other peramelemorph pseudogenes have inserts of about 1 kb with high levels of similarity to LINE 1 elements. The Diprotodontia is notable for its relative lack of intersequence variation in comparison to the Dasyuromorphia.     

The ecology and evolution of the monito del monte,a relict species from the southern South America temperate forests

The arboreal marsupial monito del monte (genus Dromiciops, with two recognized species) is a paradigmatic mammal. It is the sole living representative of the order Microbiotheria, the ancestor lineage of Australian marsupials. Also, this marsupial is the unique frugivorous mammal in the temperate rainforest, being the main seed disperser of several endemic plants of this ecosystem, thus acting as keystone species. Dromiciops is also one of the few hibernating mammals in South America, spending half of the year in a physiological dormancy where metabolism is reduced to 10% of normal levels. This capacity to reduce energy expenditure in winter contrasts with the enormous energy turnover rate they experience in spring and summer. The unique life history strategies of this living Microbiotheria, characterized by an alternation of life in the slow and fast lanes, putatively represent ancestral traits that permitted these cold‐adapted mammals to survive in this environment. Here, we describe the ecological role of this emblematic marsupial, summarizing the ecophysiology of hibernation and sociality, updated phylogeographic relationships, reproductive cycle, trophic relationships, mutualisms, conservation, and threats. This marsupial shows high densities, despite presenting slow reproductive rates, a paradox explained by the unique characteristics of its three‐dimensional habitat. We finally suggest immediate actions to protect these species that may be threatened in the near future due to habitat destruction and climate change.     

A New Marsupial from the Early Eocene Tingamarra Local Fauna of Murgon, Southeastern Queensland: A Prototypical Australian Marsupial?

Djarthia murgonensis, a new genus and species of marsupial from the early Eocene Tingamarra Local Fauna of Murgon in southeastern Queensland, is described on the basis of dental material. The combination of marsupial synapomorphies and symplesiomorphies present in D. murgonensis suggests phylogenetic placement within either Didelphidae or Australidelphia. Tarsal morphology, fundamental to the concepts of Ameridelphia and Australidelphia respectively, is not yet known for this taxon. Consequently, it cannot be assigned to either clade with confidence. If this taxon is australidelphian, it constitutes support for the hypothesis that the common ancestor of the Australian marsupial radiation was didelphoid-like in dental features. Some previous authors have contended that marsupial faunas of South America and Australia are manifestly distinct, excepting for the australidelphian affinity of South American microbiotheres. However, because tarsal anatomy is unknown in some generalized Australian fossil taxa, including D. murgonensis, and character analysis reveals that no synapomorphies of the dentition unequivocally define either Ameridelphia or Australidelphia to the exclusion of the other, we consider this interpretation to be premature. In short, available evidence neither supports nor refutes the argument of distinct South American and Australasian marsupial faunas. A further ramification is the need to reconsider the phylogenetic position of Ankotarinja tirarensis and Keeuna woodburnei. These central Australian fossil taxa might be referred to either Australidelphia or Ameridelphia, and it is recommended that both be treated as Marsupialia incertae sedis until further material comes to light.     

Mitochondrial genomes of a bandicoot and a brushtail possum confirm the monophyly of australidelphian marsupials.

Recent molecular analyses suggest that the position of bandicoots is the major difficulty in determining the root of the tree of extant marsupials. To resolve this, we analyse mitochondrial genome sequences of a bandicoot (Isoodon macrourus) and a brushtail possum (Trichosurus vulpecula) together with the previously available marsupial mitochondrial genomes, the Virginia opossum (Didelphis virginiana) and the wallaroo (Macropus robustus). Analyses of mitochondrial protein-coding and RNA genes strongly support the bandicoot as sister to the wallaroo and the brushtail possum. This result, combined with other recent molecular analyses, confirms the monophyly of Australidelphia (Australasian marsupials plus Dromiciops from South America). Further, RY coding was found to nullify AGCT coding nucleotide composition bias.     

Cranial Anatomy of the Earliest Marsupials and the Origin of Opossums

Background

The early evolution of living marsupials is poorly understood in part because the early offshoots of this group are known almost exclusively from jaws and teeth. Filling this gap is essential for a better understanding of the phylogenetic relationships among living marsupials, the biogeographic pathways that led to their current distribution as well as the successive evolutionary steps that led to their current diversity, habits and various specializations that distinguish them from placental mammals.

Methodology/Principal Findings

Here we report the first skull of a 55 million year old peradectid marsupial from the early Eocene of North America and exceptionally preserved skeletons of an Oligocene herpetotheriid, both representing critical groups to understand early marsupial evolution. A comprehensive phylogenetic cladistic analysis of Marsupialia including the new findings and close relatives of marsupials show that peradectids are the sister group of living opossums and herpetotheriids are the sister group of all living marsupials.

Conclusions/Significance

The results imply that North America played an important role in early Cenozoic marsupial evolutionary history and may have even been the center of origin of living marsupials and opossums. New data from the herpetotheriid postcranium support the view that the ancestral morphotype of Marsupialia was more terrestrial than opossums are. The resolution of the phylogenetic position of peradectids reveals an older calibration point for molecular estimates of divergence times among living marsupials than those currently used.     

Ontogenetic and phylogenetic transformations of the vomeronasal complex and nasal floor elements in marsupial mammals

Histological sections and three-dimensional reconstructions of section-series were used to document the anatomy of the vomeronasal complex and other aspects of the ethmoidal region in representatives of 13 families and six orders of marsupial mammals, including for the first time Microbiotheria. The changes during growth of several features were examined in ontogenetic series. Marsupials are very conservative in comparison with eutherians regarding the vomeronasal complex. All have a vomeronasal organ and a nasopalatine duct, have no nasopalatine duct cartilage, have no (or just an incipient) palatine cartilage, and the overall construction of the nasal floor is uniform across species. Most features examined show a high degree of homoplasy (e.g. presence of glandular ridges, isolated dorsal process of the paraseptal cartilage), and their systematic value is confined to low taxonomic levels. Significant ontogenetic changes occur in features usually discussed in the systematic/taxonomic literature. Amongst the didelphids examined, Caluromys philander shows several autapomorphies. It is hypothesized that the opening of the VNO into the upper end of the nasopalatine duct was present in the marsupial groundplan. Most marsupials have a large and horizontal anterior transverse lamina, the plesiomorphic condition, which becomes oblique in diprotodontians. Some features are autapomorphies of well-supported monophyletic groups of marsupials, e.g. the conspicuous internasal communication of perameliformes and the 'tube-like' or ring-shaped paraseptal cartilage of vombatiformes. An outer bar joining the middle (and not the dorsal-most portion) of the paraseptal cartilage characterizes Australasian marsupials and Dromiciops, with the exclusion of perameliformes, and evolved independently in Caluromys philander.     

Phylogenetic analysis of diprotodontian marsupials based on complete mitochondrial genomes

Australidelphia is the cohort, originally named by Szalay, of all Australian marsupials and the South American Dromiciops. A lot of mitochondria and nuclear genome studies support the hypothesis of a monophyly of Australidelphia, but some familial relationships in Australidelphia are still unclear. In particular, the familial relationships among the order Diprotodontia (koala, wombat, kangaroos and possums) are ambiguous. These Diprotodontian families are largely grouped into two suborders, Vombatiformes, which contains Phascolarctidae (koala) and Vombatidae (wombat), and Phalangerida, which contains Macropodidae, Potoroidae, Phalangeridae, Petauridae, Pseudocheiridae, Acrobatidae, Tarsipedidae and Burramyidae. Morphological evidence and some molecular analyses strongly support monophyly of the two families in Vombatiformes. The monophyly of Phalangerida as well as the phylogenetic relationships of families in Phalangerida remains uncertain, however, despite searches for morphological synapomorphy and mitochondrial DNA sequence analyses. Moreover, phylogenetic relationships among possum families (Phalangeridae, Petauridae, Pseudocheiridae, Acrobatidae, Tarsipedidae and Burramyidae) as well as a sister group of Macropodoidea (Macropodidae and Potoroidae) remain unclear. To evaluate familial relationships among Dromiciops and Australian marsupials as well as the familial relationships in Diprotodontia, we determined the complete mitochondrial sequence of six Diprotodontian species. We used Maximum Likelihood analyses with concatenated amino acid and codon sequences of 12 mitochondrial protein genomes. Our analysis of mitochondria amino acid sequence supports monophyly of Australian marsupials+Dromiciops and monophyly of Phalangerida. The close relatedness between Macropodidae and Phalangeridae is also weakly supported by our analysis.