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.     

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.     

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.     

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.     

Chaenothecopsis quintralis, a new species of calicioid fungus

Chaenothecopsis quintralis from southwestern Argentina is described and illustrated as a new species in the family Mycocaliciaceae. It has been found in three localities in the Andean Patagonian temperate forests, growing strictly on dung of an endemic marsupial Dromiciops gliroides. The new species is distinguished by the hemispherical, black capitulum of ascoma, the presence of asci with croziers, one-celled brown ascospores, and its fimicolous habitat. Analysis of partial nuclear large subunit rDNA (LSU) sequences showed that this taxon is within Mycocaliciales.     

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.     

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.     

The potential key seed-dispersing role of the arboreal marsupial Dromiciops gliroides

Marsupial seed dispersal is a rare phenomenon, although it may be ecologically significant in southern South America. The marsupial Dromiciops gliroides is endemic to the northern part of the temperate forest of South America. Here we describe the food habits and examine the potential role of D. gliroides as a seed disperser. We evaluated the diet of this marsupial in its natural habitat and in captivity. Dromiciops gliroides is omnivorous showing high consumption of a diversity of fruits. In captivity, D. gliroides consumed fruits from 80% of 22 native plant species we examined. Experiments conducted with fruits from two common understory shrubs show that seed passage through the digestive tract of D. gliroides enhances germination. Our results suggest that this species may have an important role as a seed disperser in the temperate forest of South America, which might offset a scarcity of frugivorous bird species.     

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.     

Evidence of early evolution of Australidelphia (Metatheria,Mammalia) in South America: phylogenetic relationships of the metatherians from the Late Palaeocene of Itaboraí (Brazil) based on teeth and petrosal bones

New metatherian petrosal bones from the mid to Late Palaeocene of Itaboraí, belonging to three morphotypes (VI, VII, and VII), are formally described and compared to fossil and extant taxa known by their auditory region. An attempt at assigning petrosal types to tooth‐based taxa from Itaboraí was made by combining parsimony and morphometric methods. The first large scale phylogenetic analysis of the Itaboraían metatherians, involving basicranial and dental characters in a larger number of taxa, is provided here and is at the basis of a systematic revision of the metatherians from Itaboraí. The combination of morphometric and cladistic analyses helps in understanding the affinities between the petrosals and the tooth‐based taxa. The metatherians from Itaboraí were taxonomically diverse, belonging to each of the most important radiations in marsupial evolutionary history (Didelphimorphia, Paucituberculata, Eometatheria). The inclusion of Palaeocene taxa in the crown group Marsupialia and above all in the Eometatheria radiation points to an early emergence of these clades in South America and corroborates the main molecular hypotheses. © 2010 The Linnean Society of London, Zoological Journal of the Linnean Society, 2010, 159 , 746–784.     

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.     

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.     

First combined cladistic analysis of marsupial mammal interrelationships

We combine osteological, dental, and soft tissue data with sequences from three nuclear and five mitochondrial genes, sampling all major living clades of marsupials plus several extinct taxa, to perform a simultaneous analysis of marsupial interrelationships. These data were analyzed using direct optimization and sensitivity analysis on a parallel supercomputing cluster, and compared with trees produced with conventional parsimony and likelihood algorithms using a static alignment. A major issue in marsupial phylogeny is the relationships among australidelphians. Optimal analyses using direct optimization and those based on the static alignment support the basal positions of peramelians (bandicoots) and Dromiciops ('monito del monte') within Australidelphia, and in all but one case these analyses support a monophyletic Eometatheria, a group consisting of all australidelphians excluding peramelians. Dromiciops is basal within Eometatheria in analyses that maximize congruence across partitions, including the equally weighted parameter set. The topologies resulting from direct optimization under all parameter sets show some differences, but all show a high degree of resolution. Direct optimization supports high-level clades supported by analyses of partitioned molecular (e.g., Notoryctes as sister group of Dasyuromorphia) and morphological (e.g., Diprotodontia) data.     

Cranial morphological variation of Dromiciops gliroides (Microbiotheria) along its geographical distribution in south-central Chile: A three-dimensional analysis

We analyzed the variation in cranial morphology of the marsupial Dromiciops gliroides along its distribution in south-central Chile. We evaluated whether the cranial morphological variation is congruent with the phylogeographic structure previously observed in this species. We built three-dimensional models of 69 crania on which we digitized 30 landmarks. We used standard geometric morphometric methods to extract and analyze the shape and size components of the crania. Our data showed a subtle but consistent cranial size and shape variation along the studied distributional range, suggesting a geographic variation pattern rather than a phylogeographic structuring. Indeed, our multivariate analyses recovered a subtle morphological differentiation between island and mainland populations, contrary to what is suggested by a former phylogeographic study. We detected that either the cranial size variation, as well as the insularity and the latitude could be important factors underlying the cranial shape changes. We suggest that an interplay of historical and contemporary processes could be shaping the morphological pattern observed in this marsupial.     

Suprafamilial relationships within marsupialia: Resolution and discordance from multidisciplinary data

Selected characters of the cranioskeletal, dental, neuroanatomical, reproductive, lymphoid, and developmental systems that are known for most or all extant marsupial families are reviewed and analyzed for their abilities to corroborate higher-level hypotheses of suprafamilial relationships among extant marsupials. In addition, relatively conservative nucleotides from the mitochondrial 12S rDNA gene (obtained from the recent study by Springeret al., J. Mammal. Evol. 2, 85–115, 1994) were incorporated into the same character analysis. The ancestral morphotype for each marsupial family and each character was reconstructed, using the reconstructed eutherian and therian morphotypes for outgroup comparisons. In addition, ontogenetic data, stratigraphic position of fossils, and form-functional considerations were used, whenever feasible, to assess character state polarity of anatomical traits. Despite missing data from some families and many genera, this preliminary and modified total evidence approach helps to identify several well corroborated higher taxa, including Diprotodontia, Vombatiformes, and Macropodoidea, and it provides modest support for Ameridelphia, Australidelphia, and Syndactyla. An important conclusion is that no single data set is capable of resolving all suprafamilial relationships among marsupials. Suggestions are also presented for future multidisciplinary approaches to help resolve several polychotomies that have remained resistant to phylogenetic analyses of single data sets.     

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.     

Spatial ecology of monito del monte (Dromiciops gliroides) in a fragmented landscape of southern Chile

Habitat loss is one of the most important causes of biodiversity loss in South American temperate rainforests, where many endemic species exist. Among these is the monito del monte (Dromiciops gliroides), an arboreal marsupial with restricted distribution, and the only extant species of the order Microbiotheria. Current knowledge about monito del monte habitat use and its responses to human disturbances is scarce. To help fill this gap we investigated its habitat use and selection patterns in a fragmented landscape in southern Chile. Monito del monte individuals were abundant in a large and a small fragment, but rare or undetected in forest strips. Using telemetry data from 12 neighboring individuals in a large fragment and 2 individuals in a small fragment, we estimated their mean home range size of 1.6 ha±0.6 (1SD). Monitos del monte had a spatial overlap among individuals of 50±4%. Tracked individuals used old- and second-growth habitats as available, did not use the prairie habitats, and strongly avoided the scrublands. In the large fragment we estimated a relative population density of 21±5 individuals/ha (mean±1SD), whereas in the small fragment it was of 19±6 individuals/ha. This is, to our knowledge, the first study of the spatial ecology of the monito del monte based on telemetry data, and evidence presented here could have conservation and planning implications, not only for the target species but also its habitat.     

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.     

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.     

Nuciruptor rubricae,a new Pitheciin seed predator from the Miocene of Colombia

A new genus and species of platyrrhine primate, Nuciruptor rubricae, are added to the increasingly diverse primate fauna from the middle Miocene of La Venta, Colombia. This species displays a number of dental and gnathic features indicating that it is related to living and extinct Pitheciinae (extant Callicebus, Pithecia, Chiropotes, Cacajao, and the Colombian middle Miocene Cebupithecia sarmientoi). Nuciruptor is markedly more derived than Callicebus but possesses a less derived mandibular form and incisor-canine complex than extant and extinct pitheciins (Cebupithecia, Pithecia, Chiropotes, and Cacajao), suggesting that it is a primitive member of the tribe Pitheciini within the larger monophyletic Pitheciinae. Nuciruptor has procumbent and moderately elongate lower incisors and low-crowned molars, suggesting that it was a seed predator, as are living pitheciins. Its estimated body size of approximately 2.0 kg places it within the size range of extant pitheciines. The dental and gnathic morphology of Nuciruptor clarifies several aspects of dental character evolution in Pitheciinae and makes it less likely that the enigmatic Mohanamico hershkovitzi (m. Miocene, Colombia) is a pitheciin. Am. J. Phys. Anthropol. 102:407–427, 1997. © 1997 Wiley-Liss, Inc.