Sharon A. Jansa

Phylogenetic Relationships and Classification of Didelphid Marsupials, an Extant Radiation of New World Metatherian Mammals

Abstract This report summarizes a decade of morphological and molecular research on the phylogenetic relationships of didelphid marsupials (opossums), a substantially intact radiation of New World metatherian mammals. We review the comparative morphology of Recent opossums, emphasizing those anatomical systems from which taxonomically useful information is available for the majority of living genera and species, namely the integument, cranium, and dentition. Morphological similarities and differences among didelphids and other plesiomorphic marsupials (caenolestids, microbiotheriids, dasyurids, and peramelids) are also described. These observations, representing evolved differences in diverse functional-morphological systems, together with karyotypic information gleaned from the literature, provide the basis for coding 129 phylogenetic characters that we scored for 44 ingroup and seven outgroup taxa. Published information about the size, internal organization, chromosomal location, and physiological properties of five nuclear genes (BRCA1, DMP1, IRBP, RAG1, vWF) sequenced for this study suggest that these loci are unlinked, exist as single copies, are active in different tissues, and encode protein products with widely divergent functions. All of the sequenced fragments are long (>900 bp), free of ingroup alignment ambiguities, and translate to open reading frame. Nucleotide data from a total of 7320 aligned sites were obtained from 43 ingroup and seven outgroup taxa. Separate parsimony, likelihood, and Bayesian analyses of these six data partitions (morphology + karyotypes, five genes) resulted in highly congruent estimates of didelphid phylogeny with few examples of conflict among strongly supported nodes. Analyses of concatenated sequences and combined (nonmolecular + sequence) datasets effectively summarize all of the common signal recovered from separate analyses: a completely resolved ingroup phylogeny with high support statistics at most nodes. Remaining problems (not conclusively resolved in this study) include the position of the ingroup root and the relationships of three genera (Chacodelphys, Cryptonanus, Tlacuatzin) within their respective suprageneric clades. The history of didelphid classification is reviewed, and all previous systems are found to contain nonmonophyletic groups. A revised phylogenetic classification consistent with our analytic results includes the following higher taxa: Glironiinae (for Glironia), Caluromyinae (Caluromys and Caluromysiops), Hyladelphinae (Hyladelphys), Didelphinae (Marmosini, Metachirini, Didelphini, and Thylamyini), Marmosini (Marmosa, Monodelphis, and Tlacuatzin), Metachirini (Metachirus), Didelphini (Chironectes, Didelphis, Lutreolina, and Philander), and Thylamyini (Chacodelphys, Cryptonanus, Gracilinanus, Lestodelphys, Marmosops, and Thylamys). The probable relationships of several Neogene fossil genera are also discussed. To facilitate identifications, all Recent genera are redescribed, representative crania are illustrated, and a key is provided.

PHYLOGENETIC STUDIES ON DIDELPHID MARSUPIALS II. NONMOLECULAR DATA AND NEW IRBP SEQUENCES: SEPARATE AND COMBINED ANALYSES OF DIDELPHINE RELATIONSHIPS WITH DENSER TAXON SAMPLING

Abstract In order to test the results of a previous study of didelphid marsupial phylogeny based on IRBP nuclear gene sequences (Jansa and Voss, 2000. Phylogenetic studies on didelphid marsupials I. Introduction and preliminary results from nuclear IRBP gene sequences. Journal of Mammalian Evolution 7: 43–77), we surveyed external, cranial, dental, and karyotypic characters among a more densely taxon-sampled didelphine ingroup. Separate maximum-parsimony analyses of these nonmolecular data and of a new (taxon-dense) IRBP matrix yielded superficially dissimilar strict-consensus topologies. However, no didelphine clade that was even moderately well supported by either separate analysis was contradicted by any equivalently well-supported clade in the other. Instead, all examples of taxonomic incongruence involved weak nodal support from one or both datasets. A maximum-likelihood analysis of the IRBP data produced a consensus topology that was completely congruent with, although slightly more resolved than, the maximum-parsimony consensus. A combined (simultaneous) maximum-parsimony analysis of both datasets (nonmolecular + IRBP) produced a consensus topology that closely resembled the results of analyzing IRBP separately. Most of the didelphine relationships previously reported by Jansa and Voss (op. cit.) are supported by these analytic exercises, with some notable exceptions. The taxon currently known as Marmosa canescens is conspicuously divergent from congeneric species and variously clusters with three different groups (“other Marmosa” + Micoureus, Monodelphis, or higher didelphines [= clade H of Jansa and Voss, op. cit.] ) in several parsimony-equivalent resolutions of a fourfold basal polytomy in the IRBP and combined-data consensus topologies. Even without canescens, however, the genus Marmosa is not demonstrably monophyletic. The nomenclatural consequences of these results are discussed, and a new genus is described for “Marmosa” canescens. Future analyses should test the monophyly of other speciose didelphine genera, but new sources of character data will be needed to offset the loss of resolution and decreased nodal support that are often caused by denser taxon sampling.

On the contents of Gracilinanus gardner and creighton, 1989, with the description of a previously unrecognized clade of small didelphid marsupials

Abstract Five nominal species of small didelphid marsupials previously referred to Gracilinanus differ conspicuously from the type species (G. microtarsus) and from all of the other valid taxa that we recognize as members of that genus (G. aceramarcae, G. agilis, G. dryas, G. emiliae, G. marica). These anomalous forms can be distinguished morphologically from Gracilinanus (in the strict sense just defined) by lacking maxillary palatal vacuities, a secondary foramen ovale, and a rostral process of the premaxillae; in addition, P3 is taller than P2, and accessory cusps are often present on C1. A new genus, Cryptonanus, is described to contain these forms, all of which are provisionally recognized as valid species: C. agricolai, C. chacoensis, C. guahybae, C. ignitus, and C. unduaviensis. Separate and combined phylogenetic analyses of nonmolecular data and nuclear gene sequences suggest that Cryptonanus and Gracilinanus (sensu stricto) are reciprocally monophyletic and closely related, although they were not consistently recovered as sister taxa in any analysis. Available specimen records document that Cryptonanus is widely distributed in mostly unforested tropical, subtropical, and temperate biomes south of the Amazon River (from ca. 7°S in the Brazilian state of Ceará to ca. 34°S in the Argentinian province of Buenos Aires), but significant range extensions could be expected from pitfall trapping in extralimital savanna landscapes. Scant field data suggest that species of Cryptonanus may often be associated with wet or seasonally inundated grasslands, an unusual habitat for small didelphids.

Tests for Positive Selection on Immune and Reproductive Genes in Closely Related Species of the Murine Genus Mus

We examine variation among species of Mus in four genes involved in reproduction and the immune response for evidence of positive selection: the sperm recognition gene Zp-3, the testis-determining locus Sry, the testicular cell surface matrix protein Tcp-1, and the immune system protein β2m. We use likelihood ratio tests in the context of a well-supported phylogeny to determine whether models that allow for positively selected sites fit the sequences better than models that assume purifying selection. We then apply a Bayesian approach to identify particular sites in each gene that have a high posterior probability of being under positive selection. We find no evidence of positive selection on the Tcp-1 gene, but for Zp-3, Sry, and β2m, models that allow for positively selected sites fit the sequences better than alternatives. For each of these genes, we identify sites that have a high (> 95%) posterior probability of being positively selected. For Zp-3, two of these sites occur near the sperm-binding region, while one occurs in a region whose functional role remains unstudied but where the pattern of change predicts functional importance. A single site in Sry shows an elevated rate of replacement substitution but occurs in a region of apparently little functional importance; therefore, relaxation of functional constraints may better explain the rapid evolution of this site. Three sites in β2m have a posterior probability > 50% of being under positive selection. While the functional role for two of these sites is unknown, the third is known to influence the ability of MHC class I molecules to present antigens to the immune system; therefore, the elevated rate of replacement substitutions at this site is consistent with selection acting to promote variability in immune system proteins.

Adaptive Evolution of the Venom-Targeted vWF Protein in Opossums that Eat Pitvipers

The rapid evolution of venom toxin genes is often explained as the result of a biochemical arms race between venomous animals and their prey. However, it is not clear that an arms race analogy is appropriate in this context because there is no published evidence for rapid evolution in genes that might confer toxin resistance among routinely envenomed species. Here we report such evidence from an unusual predator-prey relationship between opossums (Marsupialia: Didelphidae) and pitvipers (Serpentes: Crotalinae). In particular, we found high ratios of replacement to silent substitutions in the gene encoding von Willebrand Factor (vWF), a venom-targeted hemostatic blood protein, in a clade of opossums known to eat pitvipers and to be resistant to their hemorrhagic venom. Observed amino-acid substitutions in venom-resistant opossums include changes in net charge and hydrophobicity that are hypothesized to weaken the bond between vWF and one of its toxic snake-venom ligands, the C-type lectin-like protein botrocetin. Our results provide the first example of rapid adaptive evolution in any venom-targeted molecule, and they support the notion that an evolutionary arms race might be driving the rapid evolution of snake venoms. However, in the arms race implied by our results, venomous snakes are prey, and their venom has a correspondingly defensive function in addition to its usual trophic role.

Molecular Systematics of Mouse Opossums (Didelphidae: Marmosa): Assessing Species Limits using Mitochondrial DNA Sequences, with Comments on Phylogenetic Relationships and Biogeography

Abstract The genus Marmosa contains 15 currently recognized species, of which nine are referred to the subgenus Marmosa, and six to the subgenus Micoureus. Recent revisionary research based on morphological data, however, suggests that the subgenus Marmosa is more diverse than the currently accepted taxonomy indicates. Herein we report phylogenetic analyses of sequence data from the mitochondrial cytochrome-b gene representing 12 of the 14 morphologically defined taxa recently treated as valid species of Marmosa (Marmosa) in the aforementioned revisionary work. These data provide a basis for testing the monophyly of morphologically defined taxa in the subgenus Marmosa, and they afford the first opportunity to assess phylogenetic relationships among the majority of species currently referred to the genus. Ten of 11 species of Marmosa (Marmosa) represented by multiple sequences in our analyses were recovered as monophyletic. In contrast, our samples of M. mexicana were recovered as two deeply divergent haplogroups that were not consistently associated as sister taxa. Among other results, our analyses support the recognition of M. isthmica and M. simonsi as species distinct from M. robinsoni, and the recognition of M. macrotarsus and M. waterhousei as species distinct from M. murina. The validity of three other species long recognized as distinct (M. rubra, M. tyleriana, and M. xerophila) is also clearly supported by our results. Although cytochrome-b sequence data are not consistently informative about interspecific relationships in this study, we found strong support for several clades, including (1) the subgenus Micoureus; (2) a group comprised of Marmosa macrotarsus, M. murina, M. tyleriana, and M. waterhousei; (3) a group comprised of M. robinsoni and M. xerophila; and (4) a group comprising all of the species in the subgenus Marmosa that occur north and west of the Andes (M. isthmica, M. mexicana, M. robinsoni, M. simonsi, M. xerophila, and M. zeledoni). Our discovery of the latter clade suggests that the Andes may have played a major role in the early diversification of this speciose radiation of small Neotropical marsupials.

Base-Compositional Heterogeneity in the RAG1 Locus among Didelphid Marsupials: Implications for Phylogenetic Inference and the Evolution of GC Content

Although theoretical studies have suggested that base-compositional heterogeneity can adversely affect phylogenetic reconstruction, only a few empirical examples of this phenomenon, mostly among ancient lineages (with divergence dates > 100 Mya), have been reported. In the course of our phylogenetic research on the New World marsupial family Didelphidae, we sequenced 2790 bp of the RAG1 exon from exemplar species of most extant genera. Phylogenetic analysis of these sequences recovered an anomalous node consisting of two clades previously shown to be distantly related based on analyses of other molecular data. These two clades show significantly increased GC content at RAG1 third codon positions, and the resulting convergence in base composition is strong enough to overwhelm phylogenetic signal from other genes (and morphology) in most analyses of concatenated datasets. This base-compositional convergence occurred relatively recently (over tens rather than hundreds of millions of years), and the affected gene region is still in a state of evolutionary disequilibrium. Both mutation rate and substitution rate are higher in GC-rich didelphid taxa, observations consistent with RAG1 sequences having experienced a higher rate of recombination in the convergent lineages.

Species Limits and Phylogenetic Relationships in the Didelphid Marsupial Genus Thylamys Based on Mitochondrial DNA Sequences and Morphology

Abstract Species of the didelphid marsupial genus Thylamys, commonly known as fat-tailed mouse opossums, are broadly distributed in the open habitats of central and southern South America. In this report we examine species limits in the genus and infer phylogenetic relationships among Thylamys species using both molecular phylogenetic and morphological methods. We assessed species limits using a broad geographic sample of DNA sequences from the mitochondrial gene cytochrome b in conjunction with morphological character analysis, and we inferred phylogenetic relationships among species using the cytochrome-b dataset in addition to sequences from the mitochondrial genes cytochrome c oxidase subunit II and NADH dehydrogenase 2 for a representative subset of individuals. Based on the results of these analyses, we recognize Xerodelphys (new subgenus) for T. karimii and T. velutinus, and we recognize seven valid species in the nominotypical subgenus. The latter includes T. macrurus, T. pusillus, and two monophyletic species groups: the Elegans Group (T. elegans, T. pallidior, T. tatei) and the Venustus Group (T. sponsorius, T. venustus). Analysis of cytochrome-b sequences additionally reveals deep phylogeographic structuring in three species (T. pallidior, T. pusillus, T. venustus), each of which contains two or three robustly supported allopatric haplogroups. The existence of undescribed Peruvian forms of the Elegans Group is also plausibly indicated. We provide morphological diagnoses of all species recognized as valid in this report, summarize information about geographic distributions, comment on previous misidentifications, and briefly consider historical-biogeographic scenarios with a focus on dispersal events across the Andes.

The early diversification history of didelphid marsupials: a window into South America's "Splendid Isolation".

The geological record of South American mammals is spatially biased because productive fossil sites are concentrated at high latitudes. As a result, the history of mammalian diversification in Amazonia and other tropical biomes is largely unknown. Here we report diversification analyses based on a time‐calibrated molecular phylogeny of opossums (Didelphidae), a species‐rich clade of mostly tropical marsupials descended from a Late Oligocene common ancestor. Optimizations of habitat and geography on this phylogeny suggest that (1) basal didelphid lineages inhabited South American moist forests; (2) didelphids did not diversify in dry‐forest habitats until the Late Miocene; and (3) most didelphid lineages did not enter North America until the Pliocene. We also summarize evidence for an Early‐ to Middle‐Miocene mass extinction event, for which alternative causal explanations are discussed. To the best of our knowledge, this study provides the first published molecular‐phylogenetic evidence for mass extinction in any animal clade, and it is the first time that evidence for such an event (in any plant or animal taxon) has been tested for statistical significance. Potentially falsifying observations that could help discriminate between the proposed alternative explanations for didelphid mass extinction may be obtainable from diversification analyses of other sympatric mammalian groups.

PHYLOGENETIC RELATIONSHIPS OF THE MARSUPIAL GENUS HYLADELPHYS BASED ON NUCLEAR GENE SEQUENCES AND MORPHOLOGY

Abstract Phylogenetic analyses of sequence data from the nuclear gene encoding the interphotoreceptor retinoid–binding protein (Irbp) provide compelling evidence that the recently described Amazonian marsupial genus Hyladelphys is a didelphid and suggest that it occupies an internal branch separating the traditionally recognized subfamilies Caluromyinae and Didelphinae. Although this phylogenetic position also is supported by morphological character data, analyses of sequence data from the dentin matrix protein 1 gene (Dmp1) place Hyladelphys within the didelphine radiation. A parsimony analysis of the combined morphological and molecular data supports the Irbp-only results, but a partitioned Bayesian analysis of the combined gene data does not provide strong support for either placement. The implications of these results are discussed in terms of long-branch attraction, base-compositional bias, and other possibly confounding factors. Whether Hyladelphys is the sister group of Didelphinae or an independent lineage at some basal level within that subfamily, the absence of any close relationship to other Recent taxa is clearly indicated. We redescribe the genus based on new morphological character data and comment on the probable existence of undescribed taxa in addition to H. kalinowskii, the type species.