Pronounced declines in heavy metal burdens of Minnesotan mammals over the last century.

Humans have drastically altered the ecology of heavy metals, which can have negative effects on animal development and neural functioning. Many species have shown the ability to adapt to anthropogenic increases in metal pollution, but such evolutionary responses will depend on the extent of metal variation over space and time. For terrestrial vertebrates, it is unclear how metal exposure has changed over time: some studies suggest metal content peaked with the enactment of policies controlling lead emissions, while other studies suggest metal levels peaked at least a century earlier. We used 162 specimens of four mammal species (a mouse, shrew, bat, and squirrel) to ask how metal content of the fur and skin has changed over a 90-year time period, and impacts on individual performance (body size and cranial capacity). Using ICP-MS, we show that for lead, cadmium, copper, and chromium, there were significant declines in metal content in mammal tissue over the 90-year time period, with lead levels five times lower now than in the early 1900s. Importantly, metal content began to drop well before the pollution regulation of the 1970s. Effects of time greatly outweighed any effects of an individual living near a human population center. Surprisingly, there were no effects of body metal content on body size, and only manganese was negatively related to relative cranial capacity. Taken together, these results suggest that present day populations of mammals are experiencing levels of heavy metal exposure that are less stressful than they were 100 years ago. In addition, temporal decreases in metal loads likely partly reflect global patterns of pollution decline that affect atmospheric metal deposition rather than local point sources of exposure.

Ancestrally Reconstructed von Willebrand Factor Reveals Evidence for Trench Warfare Coevolution between Opossums and Pit Vipers.

Opossums in the tribe Didelphini are resistant to pit viper venoms and are hypothesized to be coevolving with venomous snakes. Specifically, a protein involved in blood clotting (von Willebrand factor [vWF] which is targeted by snake venom C-type lectins [CTLs]) has been found to undergo rapid adaptive evolution in Didelphini. Several unique amino acid changes in vWF could explain their resistance; however, experimental evidence that these changes disrupt binding to venom CTLs was lacking. Furthermore, without explicit testing of ancestral phenotypes to reveal the mode of evolution, the assertion that this system represents an example of coevolution rather than noncoevolutionary adaptation remains unsupported. Using expressed vWF proteins and purified venom CTLs, we quantified binding affinity for vWF proteins from all resistant taxa, their venom-sensitive relatives, and their ancestors. We show that CTL-resistant vWF is present in opossums outside clade Didelphini and likely across a wider swath of opossums (family Didelphidae) than previously thought. Ancestral reconstruction and in vitro testing of vWF phenotypes in a clade of rapidly evolving opossums reveal a pattern consistent with trench warfare coevolution between opossums and their venomous snake prey.

A new genus and species of shrew-like mouse (Rodentia: Muridae) from a new center of endemism in eastern Mindanao, Philippines.

The Philippine archipelago hosts an exceptional diversity of murid rodents that have diversified following several independent colonization events. Here, we report the discovery of a new species of rodent from Mt. Kampalili on eastern Mindanao Island. Molecular and craniodental analyses reveal this species as a member of a Philippine "New Endemic" clade consisting of Tarsomys, Limnomys, and Rattus everetti (tribe Rattini). This new species of "shrew-mouse" is easily distinguished from its relatives in both craniodental and external characteristics including a long, narrow snout; small eyes and ears; short, dark, dense fur dorsally and ventrally; stout body with a tapering, visibly haired tail shorter than head and body length; stout forepaws; bulbous and nearly smooth braincase; narrow, tapering rostrum; short incisive foramina; slender mandible; and narrow, slightly opisthodont incisors. This new genus and species of murid rodent illustrates that murids of the tribe Rattini have exhibited greater species and morphological diversification within the Philippines than previously known and provides evidence that Mt. Kampalili represents a previously unrecognized center of mammalian endemism on Mindanao Island that is deserving of conservation action.

Ancestrally reconstructed Von Willebrand Factor (vWF) reveals evidence for trench warfare coevolution between opossums and pit vipers

Opossums in the tribe Didelphini are resistant to pit viper venoms and are hypothesized to be coevolving with venomous snakes. Specifically, a protein involved in blood clotting (von Willebrand Factor, which is targeted by snake venom C-type Lectins, or CTLs), has been found to undergo rapid adaptive evolution in Didelphini. Several unique amino acid changes in vWF could explain their resistance; however, experimental evidence that these changes disrupt binding to venom CTLs was lacking. Furthermore, without explicit testing of ancestral phenotypes to reveal the mode of evolution, the assertion that this system represents an example of coevolution rather than non-coevolutionary adaptation remains unsupported. Using expressed vWF proteins and purified venom CTLs, we quantified binding affinity for vWF proteins from all resistant taxa, their venom-sensitive relatives, and their ancestors. We show that CTL resistant vWF is present in opossums outside clade Didelphini and likely across a wider swath of opossums (family Didelphidae) than previously thought. Ancestral reconstruction and in vitro testing of vWF phenotypes in a clade of rapidly evolving opossums reveals a pattern consistent with trench warfare coevolution between opossums and their venomous snake prey.

Resistance of South American opossums to vWF-binding venom C-type lectins.

Opossums in the clade Didelphini are well known to be resistant to snake venom due to endogenous circulating inhibitors which target metalloproteinases and phospholipases. However, the mechanisms through which these opossums cope with a variety of other damaging venom proteins are unknown. A protein involved in blood clotting (von Willebrand Factor) has been found to have undergone rapid adaptive evolution in venom-resistant opossums. This protein is a known target for a subset of snake venom C-type lectins (CTLs), which bind it and then induce it to bind platelets, causing hemostatic disruption. Several amino acid changes in vWF unique to these opossums could explain their resistance; however, experimental evidence that these changes disrupt venom CTL binding was lacking. We used platelet aggregation assays to quantify resistance to a venom-induced platelet response in two species of venom-resistant opossums (Didelphis virginiana, Didelphis aurita), and one venom-sensitive opossum (Monodelphis domestica). We found that all three species have lost nearly all their aggregation response to the venom CTLs tested. Using washed platelet assays we showed that this loss of aggregation response is not due to inhibitors in the plasma, but rather to the failure of either vWF or platelets (or both) to respond to venom CTLs. These results demonstrate the potential adaptive function of a trait previously shown to be evolving under positive selection. Surprisingly, these findings also expand the list of potentially venom tolerant species to include Monodelphis domestica and suggest that an ecological relationship between opossums and vipers may be a broader driver of adaptive evolution across South American marsupials than previously thought.

Dietary morphology of two island-endemic murid rodent clades is consistent with persistent, incumbent-imposed competitive interactions.

A lineage colonizing a geographic region with no competitors may exhibit rapid diversification due to greater ecological opportunity. The resultant species diversity of this primary-colonizing (incumbent) clade may limit subsequent lineages' ability to persist unless these non-incumbent lineages are ecologically distinct. We compare the diversity in diet-related mandibular morphology of two sympatric murid rodent clades endemic to Luzon Island, Philippines-incumbent Phloeomyini and secondary-colonizing Chrotomyini-to the mandibular morphological diversity of Sahul Hydromyini, the sister clade of Chrotomyini and the incumbent murid lineage on the supercontinent of Sahul. This three-clade comparison allows us to test the hypothesis that incumbent lineages can force persistent ecological distinction of subsequent colonists at the time of colonization and throughout the subsequent history of the two sympatric clades. We find that Chrotomyini forms a subset of the diversity of their clade plus Sahul Hydromyini that minimizes overlap with Phloeomyini. We also infer that this differentiation extends to the stem ancestor of Chrotomyini and Sahul Hydromyini, consistent with a biotic filter imposed by Phloeomyini. Our work illustrates that incumbency has the potential to have a profound influence on the ecomorphological diversity of colonizing lineages at the island scale even when the traits in question are evolving at similar rates among independently colonizing clades.

Resistance of South American opossums to vWF-binding venom C-type lectins

Opossums in the clade Didelphini are well known to be resistant to snake venom due to endogenous circulating inhibitors which target metalloproteinases and phospholipases. However, the mechanisms through which these opossums cope with a variety of other damaging venom proteins are unknown. A protein involved in blood clotting (von Willebrand Factor) has been found to have undergone rapid adaptive evolution in venom-resistant opossums. This protein is a known target for a subset of snake venom C-type lectins (CTLs), which bind it and then induce it to bind platelets, causing hemostatic disruption. Several amino acid changes in vWF unique to these opossums could explain their resistance; however, experimental evidence that these changes disrupt venom CTL binding was lacking. We used platelet aggregation assays to quantify resistance to a venom-induced platelet response in two species of venom-resistant opossums (Didelphis virginiana, Didelphis aurita), and one venom-sensitive opossum (Monodelphis domestica). We found that all three species have lost nearly all their aggregation response to the venom CTLs tested. Using washed platelet assays we showed that this loss of aggregation response is not due to inhibitors in the plasma, but rather to the failure of either vWF or platelets (or both) to respond to venom CTLs. These results demonstrate the potential adaptive function of a trait previously shown to be evolving under positive selection. Surprisingly, these findings also expand the list of potentially venom tolerant species to include Monodelphis domestica and suggest that an ecological relationship between opossums and vipers may be a broader driver of adaptive evolution across South American marsupials than previously thought

Resistance of South American opossums to vWF-binding venom C-type lectins

Opossums in the clade Didelphini are well known to be resistant to snake venom due to endogenous circulating inhibitors which target metalloproteinases and phospholipases. However, the mechanisms through which these opossums cope with a variety of other damaging venom proteins are unknown. A protein involved in blood clotting (von Willebrand Factor) has been found to have undergone rapid adaptive evolution in venom-resistant opossums. This protein is a known target for a subset of snake venom C-type lectins (CTLs), which bind it and then induce it to bind platelets, causing hemostatic disruption. Several amino acid changes in vWF unique to these opossums could explain their resistance; however, experimental evidence that these changes disrupt venom CTL binding was lacking. We used platelet aggregation assays to quantify resistance to a venom-induced platelet response in two species of venom-resistant opossums (Didelphis virginiana, Didelphis aurita), and one venom-sensitive opossum (Monodelphis domestica). We found that all three species have lost nearly all their aggregation response to the venom CTLs tested. Using washed platelet assays we showed that this loss of aggregation response is not due to inhibitors in the plasma, but rather to the failure of either vWF or platelets (or both) to respond to venom CTLs. These results demonstrate the potential adaptive function of a trait previously shown to be evolving under positive selection. Surprisingly, these findings also expand the list of potentially venom tolerant species to include Monodelphis domestica and suggest that an ecological relationship between opossums and vipers may be a broader driver of adaptive evolution across South American marsupials than previously thought

Tempo and mode of mandibular shape and size evolution reveal mixed support for incumbency effects in two clades of island-endemic rodents (Muridae: Murinae).

Existing radiations in a spatially limited system such as an oceanic island may limit the ecological opportunity experienced by later colonists, resulting in lower macroevolutionary rates for secondary radiations. Additionally, potential colonists may be competitively excluded by these incumbent (resident) species, unless they are biologically distinct (biotic filtering). The extant phenotypic diversity of secondary colonists may thus be impacted by lower rates of phenotypic evolution, exclusion from certain phenotypes, and transitions to new morphotypes to escape competition from incumbent lineages. We used geometric morphometric methods to test whether the rates and patterns of mandibular evolution of the Luzon "old endemic" rodent clades, Phloeomyini and Chrotomyini, are consistent with these predictions. Each clade occupied nearly completely separate shape space and partially separate size space. We detected limited support for decelerating and clade-specific evolutionary rates for both shape and size, with strong evidence for a shift in evolutionary mode within Chrotomyini. Our results suggest that decelerating phenotypic evolutionary rates are not a necessary result of incumbency interactions; rather, incumbency effects may be more likely to determine which clades can become established in the system. Nonincumbent clades that pass a biotic filter can potentially exhibit relatively unfettered evolution.

Diversification rates of the "Old Endemic" murine rodents of Luzon Island, Philippines are inconsistent with incumbency effects and ecological opportunity.

Diversity-dependent cladogenesis occurs when a colonizing lineage exhibits increasing interspecific competition as it ecologically diversifies. Repeated colonization of a region by closely related taxa may cause similar effects as species within each lineage compete with one another. This may be particularly relevant for secondary colonists, which could experience limited diversification due to competition with earlier, incumbent colonists over evolutionary time. We tested the hypothesis that an incumbent lineage may diminish the diversification of secondary colonists in two speciose clades of Philippine "Old Endemic" murine rodents-Phloeomyini and Chrotomyini-on the relatively old oceanic island of Luzon. Although phylogenetic analyses confirm the independent, noncontemporaneous colonization of Luzon by the ancestors of these two clades, we found no support for arrested diversification in either. Rather, it appears that diversification of both clades resulted from constant-rate processes that were either uniform or favored the secondary colonists (Chrotomyini), depending on the method used. Our results suggest that ecological incumbency has not played an important role in determining lineage diversification among Luzon murines, despite sympatric occurrence by constituent species within each lineage, and a substantial head start for the primary colonists.

Venom Resistance as a Model for Understanding the Molecular Basis of Complex Coevolutionary Adaptations.

SynopsisVenom and venom resistance are molecular phenotypes widely considered to have diversified through coevolution between predators and prey. However, while evolutionary and functional studies on venom have been extensive, little is known about the molecular basis, variation, and complexity of venom resistance. We review known mechanisms of venom resistance and relate these mechanisms to their predicted impact on coevolutionary dynamics with venomous enemies. We then describe two conceptual approaches which can be used to examine venom/resistance systems. At the intraspecific level, tests of local adaptation in venom and resistance phenotypes can identify the functional mechanisms governing the outcomes of coevolution. At deeper evolutionary timescales, the combination of phylogenetically informed analyses of protein evolution coupled with studies of protein function promise to elucidate the mode and tempo of evolutionary change on potentially coevolving genes. We highlight case studies that use each approach to extend our knowledge of these systems as well as address larger questions about coevolutionary dynamics. We argue that resistance and venom are phenotypic traits which hold exceptional promise for investigating the mechanisms, dynamics, and outcomes of coevolution at the molecular level. Furthermore, extending the understanding of single gene-for-gene interactions to the whole resistance and venom phenotypes may provide a model system for examining the molecular and evolutionary dynamics of complex multi-gene interactions.

Why the honey badger don't care: Convergent evolution of venom-targeted nicotinic acetylcholine receptors in mammals that survive venomous snake bites.

Honey badgers (Mellivora capensis) prey upon and survive bites from venomous snakes (Family: Elapidae), but the molecular basis of their venom resistance is unknown. The muscular nicotinic cholinergic receptor (nAChR), targeted by snake α-neurotoxins, has evolved in some venom-resistant mammals to no longer bind these toxins. Through phylogenetic analysis of mammalian nAChR sequences, we show that honey badgers, hedgehogs, and pigs have independently acquired functionally equivalent amino acid replacements in the toxin-binding site of this receptor. These convergent amino acid changes impede toxin binding by introducing a positively charged amino acid in place of an uncharged aromatic residue. In venom-resistant mongooses, different replacements at these same sites are glycosylated, which is thought to disrupt binding through steric effects. Thus, it appears that resistance to snake venom α-neurotoxin has evolved at least four times among mammals through two distinct biochemical mechanisms operating at the same sites on the same receptor.

The impact of Quaternary climate oscillations on divergence times and historical population sizes in Thylamys opossums from the Andes.

Climate oscillations during the Quaternary altered the distributions of terrestrial animals at a global scale. In mountainous regions, temperature fluctuations may have led to shifts in range size and population size as species tracked their shifting habitats upslope or downslope. This creates the potential for both allopatric speciation and population size fluctuations, as species are either constrained to smaller patches of habitat at higher elevations or able to expand into broader areas at higher latitudes. We considered the impact of climate oscillations on three pairs of marsupial species from the Andes (Thylamys opossums) by inferring divergence times and demographic changes. We compare four different divergence dating approaches, using anywhere from one to 26 loci. Each pair comprises a northern (tropical) lineage and a southern (subtropical to temperate) lineage. We predicted that divergences would have occurred during the last interglacial (LIG) period approximately 125 000 years ago and that population sizes for northern and southern lineages would either contract or expand, respectively. Our results suggest that all three north-south pairs diverged in the late Pleistocene during or slightly after the LIG. The three northern lineages showed no signs of population expansion, whereas two southern lineages exhibited dramatic, recent expansions. We attribute the difference in responses between tropical and subtropical lineages to the availability of 'montane-like' habitats at lower elevations in regions at higher latitudes. We conclude that climate oscillations of the late Quaternary had a powerful impact on the evolutionary history of some of these species, both promoting speciation and leading to significant population size shifts.

Molecular phylogeny of short-tailed opossums (Didelphidae: Monodelphis): taxonomic implications and tests of evolutionary hypotheses.

Short-tailed opossums (genus Monodelphis) represent one of the most speciose clades of New World marsupials, with 26 currently recognized species that collectively range from eastern Panama to northern Argentina. Here we present the first phylogenetic analyses of the genus based on dense taxonomic sampling and multiple genes. From most sampled species we obtained >4800bp of DNA sequence from one mitochondrial gene (CYTB), two autosomal exons (IRBP exon 1, BRCA1 exon 11), one autosomal intron (SLC38 intron 7), and one X-linked intron (OGT intron 14). Maximum-parsimony, maximum-likelihood and Bayesian analyses of these data strongly support the monophyly of Monodelphis and recover six major clades within the genus. Additionally, our analyses support previous suggestions that several nominal taxa are synonyms of other species (M. "sorex" of M. dimidiata, M. "theresa" of M. scalops, M. "rubida" and M. "umbristriata" of M. americana, and M. "maraxina" of M. glirina). By contrast, four unnamed lineages recovered by our analyses may represent new species. Reconstructions of ancestral states of two discrete characters-dorsal pelage color pattern and habitat-suggest that the most recent common ancestor of Monodelphis was uniformly colored (with unpatterned dorsal pelage) and inhabited moist forest. Whereas some dorsal pelage patterns appear to have evolved homoplastically in Monodelphis, dorsal stripes may have had a unique historical origin in this genus.

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.

Hidden diversity in the Andes: comparison of species delimitation methods in montane marsupials.

Cryptic genetic diversity is a significant challenge for systematists faced with ever-increasing amounts of DNA sequence data. Computationally intensive coalescent-based analyses involving multiple unlinked loci are the only currently viable methods by which to assess the extent to which phenotypically similar populations (or metapopulations) are genetically distinct lineages. Although coalescent-based approaches have been tested extensively via simulations, few empirical studies have examined the impact of prior assumptions and dataset size on the ability to assess genetic isolation (evolutionary independence) using molecular data alone. Here, we consider the efficacy of two coalescent-based approaches (BPP and SpeDeSTEM) for testing the evolutionary independence of cryptic mtDNA haplogroups within three morphologically diagnosable species of Andean mouse opossums (Thylamys pallidior, T. sponsorius, and T. venustus). Fourteen anonymous nuclear loci, one X-linked nuclear intron, and one mitochondrial gene were analyzed for multiple individuals within each haplogroup of interest. We inferred individual gene trees for each locus and considered all of the nuclear loci jointly in a species-tree analysis. Using only the nuclear loci, we performed "species validation" tests for the cryptic mitochondrial lineages in SpeDeSTEM and BPP. For BPP, we also tested a wide range of prior assumptions, assessed performance of the rjMCMC algorithm, and examined how many loci were necessary to confidently delimit lineages. Results from BPP provided strong support for two independent evolutionary lineages each within T. pallidior, T. sponsorius, and T. venustus, whereas SpeDeSTEM results did not support splitting out mtDNA haplogroups as distinct evolutionary units. For most tests, BPP was robust to prior assumptions, although priors were shown to have an effect on both the strength of lineage recognition among T. venustus haplotypes and on the efficiency of the rjMCMC algorithm. Comparisons of results from datasets with different numbers of loci revealed that some cryptic lineages could be confidently delimited with as few as two loci.

Snake-venom resistance as a mammalian trophic adaptation: lessons from didelphid marsupials.

Mammals that prey on venomous snakes include several opossums (Didelphidae), at least two hedgehogs (Erinaceidae), several mongooses (Herpestidae), several mustelids, and some skunks (Mephitidae). As a group, these taxa do not share any distinctive morphological traits. Instead, mammalian adaptations for ophiophagy seem to consist only in the ability to resist the toxic effects of snake venom. Molecular mechanisms of venom resistance (as indicated by biochemical research on opossums, mongooses, and hedgehogs) include toxin-neutralizing serum factors and adaptive changes in venom-targeted molecules. Of these, toxin-neutralizing serum factors have received the most research attention to date. All of the toxin-neutralizing serum proteins discovered so far in both opossums and mongooses are human α1B-glycoprotein homologs that inhibit either snake-venom metalloproteinases or phospholipase A(2) myotoxins. By contrast, adaptive changes in venom-targeted molecules have received far less attention. The best-documented examples include amino-acid substitutions in mongoose nicotinic acetylcholine receptor that inhibit binding by α-neurotoxins, and amino-acid substitutions in opossum von Willebrand factor (vWF) that are hypothesized to weaken the bond between vWF and coagulopathic C-type lectins. Although multiple mechanisms of venom resistance are known from some species, the proteomic complexity of most snake venoms suggests that the evolved biochemical defences of ophiophagous mammals are likely to be far more numerous than currently recognized. Whereas most previous research in this field has been motivated by the potential for medical applications, venom resistance in ophiophagous mammals is a complex adaptation that merits attention from comparative biologists. Unfortunately, evolutionary inference is currently limited by ignorance about many relevant facts that can only be provided by future research.

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.

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.

The pattern and timing of diversification of Philippine endemic rodents: evidence from mitochondrial and nuclear gene sequences.

The 22 genera and 64 species of rodents (Muridae: Murinae) distributed in the Philippine Islands provide a unique opportunity to study patterns and processes of diversification in island systems. Over 90% of these rodent species are endemic to the archipelago, but the relative importance of dispersal from the mainland, dispersal within the archipelago, and in situ differentiation as explanations of this diversity remains unclear, as no phylogenetic hypothesis for these species and relevant mainland forms is currently available. Here we report the results of phylogenetic analyses of the endemic Philippine murines and a wide sampling of murine diversity from outside the archipelago, based on the mitochondrial cytochrome b gene and the nuclear-encoded IRBP exon 1. Analysis of our combined gene data set consistently identified five clades comprising endemic Philippine genera, suggesting multiple invasions of the archipelago. Molecular dating analyses using parametric and semiparametric methods suggest that colonization occurred in at least two stages, one ca. 15 Mya, and another 8 to 12 million years later, consistent with the previous recognition of "Old" and "New" endemic rodent faunas. Ancestral area analysis suggests that the Old Endemics invaded landmasses that are now part of the island of Luzon, whereas the three New Endemic clades may have colonized through either Mindanao, Luzon, or both. Further, our results suggest that most of the diversification of Philippine murines took place within the archipelago. Despite heterogeneity between nuclear and mitochondrial genes in most model parameters, combined analysis of the two data sets using both parsimony and likelihood increased phylogenetic resolution; however, the effect of data combination on support for resolved nodes was method dependent. In contrast, our results suggest that combination of mitochondrial and nuclear data to estimate relatively ancient divergence times can severely compromise those estimates, even when specific methods that account for rate heterogeneity among genes are employed. [Biogeography; divergence date estimation; mitochondrial DNA; molecular systematics; Murinae; nuclear exon; Philippines; phylogeny.].