Whole Genomes Reveal Evolutionary Relationships and Mechanisms Underlying Gene-Tree Discordance in Neodiprion Sawflies.

Rapidly evolving taxa are excellent models for understanding the mechanisms that give rise to biodiversity. However, developing an accurate historical framework for comparative analysis of such lineages remains a challenge due to ubiquitous incomplete lineage sorting and introgression. Here, we use a whole-genome alignment, multiple locus-sampling strategies, and summary-tree and SNP-based species-tree methods to infer a species tree for eastern North American Neodiprion species, a clade of pine-feeding sawflies (Order: Hymenopteran; Family: Diprionidae). We recovered a well-supported species tree that-except for three uncertain relationships-was robust to different strategies for analyzing whole-genome data. Nevertheless, underlying gene-tree discordance was high. To understand this genealogical variation, we used multiple linear regression to model site concordance factors estimated in 50-kb windows as a function of several genomic predictor variables. We found that site concordance factors tended to be higher in regions of the genome with more parsimony-informative sites, fewer singletons, less missing data, lower GC content, more genes, lower recombination rates, and lower D-statistics (less introgression). Together, these results suggest that incomplete lineage sorting, introgression, and genotyping error all shape the genomic landscape of gene-tree discordance in Neodiprion. More generally, our findings demonstrate how combining phylogenomic analysis with knowledge of local genomic features can reveal mechanisms that produce topological heterogeneity across genomes.

Ancient DNA indicates a century of overhunting did not reduce genetic diversity in Pacific Walruses (Odobenus rosmarus divergens).

Pacific Walruses (Odobenus rosmarus divergens [Illiger 1815]) are gregarious marine mammals considered to be sentinels of the Arctic because of their dependence on sea ice for feeding, molting, and parturition. Like many other marine mammal species, their population sizes were decimated by historical overhunting in the nineteenth and twentieth centuries. Although they have since been protected from nearly all commercial hunting pressure, they now face rapidly accelerating habitat loss as global warming reduces the extent of summer sea ice in the Arctic. To investigate how genetic variation was impacted by overhunting, we obtained mitochondrial DNA sequences from historic Pacific Walrus samples in Alaska that predate the period of overhunting, as well as from extant populations. We found that genetic variation was unchanged over this period, suggesting Pacific Walruses are resilient to genetic attrition in response to reduced population size, and that this may be related to their high vagility and lack of population structure. Although Pacific Walruses will almost certainly continue to decline in number as the planet warms and summer sea ice is further reduced, they may be less susceptible to the ratcheting effects of inbreeding that typically accompany shrinking populations.

Disentangling morphology and genetics in two voles (Microtus pennsylvanicus and M. ochrogaster) in a region of sympatry.

Species in recent, rapid radiations can be difficult to distinguish from one another due to incomplete sorting of traits, insufficient time for novel morphologies to evolve, and elevated rates of hybridization and gene flow. The vole genus Microtus (58 spp.) is one such system where all three factors are likely at play. In the central United States, the prairie vole, Microtus ochrogaster, and the eastern meadow vole, M. pennsylvanicus, occur in sympatry and can be distinguished on the basis of molar cusp patterns but are known to be exceptionally difficult to distinguish using external morphological characters. Using a combination of morphometrics, pelage color analyses, and phylogenetics, we explored which traits are most effective for species identification and whether these same traits can be used to identify the subspecies M. o. ohionensis. While we were able to identify six traits that differed significantly between M. ochrogaster and M. pennsylvanicus, we also found substantial measurement overlap which limits the utility of these traits for species identification. The subspecies M. o. ohionensis was particularly difficult to distinguish from M. p. pennsylvanicus, and we did not find any evidence that this subspecies forms a distinct genetic clade. Furthermore, the full species M. ochrogaster and M. pennsylvanicus did not form reciprocal clades in phylogenetic analyses. We discuss several possible reasons for these patterns, including unrecognized variation in molar cusp patterns and/or localized hybridization. Overall, our results provide useful information that will aid in the identification of these species and subspecies in the future, and provides a case study of how genetics, morphometrics, and fur color analyses can be used to disentangle signatures of evolutionary history and hybridization.

A Pervasive History of Gene Flow in Madagascar's True Lemurs (Genus Eulemur).

In recent years, it has become widely accepted that interspecific gene flow is common across the Tree of Life. Questions remain about how species boundaries can be maintained in the face of high levels of gene flow and how phylogeneticists should account for reticulation in their analyses. The true lemurs of Madagascar (genus Eulemur, 12 species) provide a unique opportunity to explore these questions, as they form a recent radiation with at least five active hybrid zones. Here, we present new analyses of a mitochondrial dataset with hundreds of individuals in the genus Eulemur, as well as a nuclear dataset containing hundreds of genetic loci for a small number of individuals. Traditional coalescent-based phylogenetic analyses of both datasets reveal that not all recognized species are monophyletic. Using network-based approaches, we also find that a species tree containing between one and three ancient reticulations is supported by strong evidence. Together, these results suggest that hybridization has been a prominent feature of the genus Eulemur in both the past and present. We also recommend that greater taxonomic attention should be paid to this group so that geographic boundaries and conservation priorities can be better established.

Lemur Gut Microeukaryotic Community Variation Is Not Associated with Host Phylogeny, Diet, or Habitat.

Identifying the major forces driving variation in gut microbiomes enhances our understanding of how and why symbioses between hosts and microbes evolved. Gut prokaryotic community variation is often closely associated with host evolutionary and ecological variables. Whether these same factors drive variation in other microbial taxa occupying the animal gut remains largely untested. Here, we present a one-to-one comparison of gut prokaryotic (16S rRNA metabarcoding) and microeukaryotic (18S rRNA metabarcoding) community patterning among 12 species of wild lemurs. Lemurs were sampled from dry forests and rainforests of southeastern Madagascar and display a range of phylogenetic and ecological niche diversity. We found that while lemur gut prokaryotic community diversity and composition vary with host taxonomy, diet, and habitat, gut microeukaryotic communities have no detectable association with any of these factors. We conclude that gut microeukaryotic community composition is largely random, while gut prokaryotic communities are conserved among host species. It is likely that a greater proportion of gut microeukaryotic communities comprise taxa with commensal, transient, and/or parasitic symbioses compared with gut prokaryotes, many of which form long-term relationships with the host and perform important biological functions. Our study highlights the importance of greater specificity in microbiome research; the gut microbiome contains many "omes" (e.g., prokaryome, eukaryome), each comprising different microbial taxa shaped by unique selective pressures.

Ultraconserved elements improve resolution of marmot phylogeny and offer insights into biogeographic history.

Marmots (Marmota spp.) comprise a lineage of large-bodied ground squirrels that diversified rapidly in the Pleistocene, when the planet quickly transitioned to a drier, colder, and highly seasonal climate-particularly at high latitudes. Fossil evidence indicates the genus spread from North America, across Beringia, and into the European Alps over the course of only a few million years, beginning in the late Pliocene. Marmots are highly adapted to survive long and severely cold winters, and this likely favored their expansion and diversification over this time period. Previous phylogenetic studies have identified two major subgenera of marmots, but the timing of important speciation events and some species relationships have been difficult to resolve. Here we use ultraconserved elements and mitogenomes, with samples from all 15 extant species, to more precisely retrace how and when marmots came to inhabit a vast Holarctic range. Our results indicate marmots arose in North America in the Early Miocene (∼16.3 Ma) and dispersed across the Bering Land Bridge in the Pliocene (∼3-4 Ma); in addition, our fossil-calibrated timeline is suggestive of the rise and spread of open grasslands as being particularly important to marmot diversification. The woodchuck (M. monax) and the Alaska marmot (M. broweri) are found to be more closely related to the Eurasian species than to the other North American species. Paraphyly is evident in the bobak marmot (M. bobak) and the hoary marmot (M. caligata), and in the case of the latter the data are highly suggestive of a second, cryptic species in the Cascade Mountains of Washington.

Significant effects of host dietary guild and phylogeny in wild lemur gut microbiomes.

Mammals harbor diverse gut microbiomes (GMs) that perform critical functions for host health and fitness. Identifying factors associated with GM variation can help illuminate the role of microbial symbionts in mediating host ecological interactions and evolutionary processes, including diversification and adaptation. Many mammals demonstrate phylosymbiosis-a pattern in which more closely-related species harbor more similar GMs-while others show overwhelming influences of diet and habitat. Here, we generated 16S rRNA sequence data from fecal samples of 15 species of wild lemurs across southern Madagascar to (1) test a hypothesis of phylosymbiosis, and (2) test trait correlations between dietary guild, habitat, and GM diversity. Our results provide strong evidence of phylosymbiosis, though some closely-related species with substantial ecological niche overlap exhibited greater GM similarity than expected under Brownian motion. Phylogenetic regressions also showed a significant correlation between dietary guild and UniFrac diversity, but not Bray-Curtis or Jaccard. This discrepancy between beta diversity metrics suggests that older microbial clades have stronger associations with diet than younger clades, as UniFrac weights older clades more heavily. We conclude that GM diversity is predominantly shaped by host phylogeny, and that microbes associated with diet were likely acquired before evolutionary radiations within the lemur families examined.

Geography is more important than life history in the recent diversification of the tiger salamander complex.

The North American tiger salamander species complex, including its best-known species, the Mexican axolotl, has long been a source of biological fascination. The complex exhibits a wide range of variation in developmental life history strategies, including populations and individuals that undergo metamorphosis; those able to forego metamorphosis and retain a larval, aquatic lifestyle (i.e., paedomorphosis); and those that do both. The evolution of a paedomorphic life history state is thought to lead to increased population genetic differentiation and ultimately reproductive isolation and speciation, but the degree to which it has shaped population- and species-level divergence is poorly understood. Using a large multilocus dataset from hundreds of samples across North America, we identified genetic clusters across the geographic range of the tiger salamander complex. These clusters often contain a mixture of paedomorphic and metamorphic taxa, indicating that geographic isolation has played a larger role in lineage divergence than paedomorphosis in this system. This conclusion is bolstered by geography-informed analyses indicating no effect of life history strategy on population genetic differentiation and by model-based population genetic analyses demonstrating gene flow between adjacent metamorphic and paedomorphic populations. This fine-scale genetic perspective on life history variation establishes a framework for understanding how plasticity, local adaptation, and gene flow contribute to lineage divergence. Many members of the tiger salamander complex are endangered, and the Mexican axolotl is an important model system in regenerative and biomedical research. Our results chart a course for more informed use of these taxa in experimental, ecological, and conservation research.

Speciation and gene flow in two sympatric small mammals from Madagascar, Microgale fotsifotsy and M. soricoides (Mammalia: Tenrecidae).

Madagascar's shrew tenrecs (Mammalia: Tenrecidae; Microgale, Nesogale) represent an excellent system for studying speciation. Most species are endemic to the island's eastern humid forests, a region renowned for high levels of biodiversity and a high rate of in situ diversification. We set out to understand the speciation dynamics in a clade of recently described taxa: Microgale fotsifotsy and M. soricoides, which have nearly identical distributions in the moist evergreen forest, and M. nasoloi, which occurs in the western dry deciduous forest. A phylogenetic analysis using mitochondrial DNA data recovered two distinct clades of M. fotsifotsy: a south clade that is sister to, and broadly sympatric with, M. soricoides, and a north clade that is sister to the dry-forest and distantly allopatric species M. nasoloi. To better understand this result, we analysed cranioskeletal measurements and performed demographic analyses using nuclear sequence data from ultraconserved elements. Nuclear data did not support a sister relationship between M. soricoides and the south clade of M. fotsifotsy but did demonstrate introgression between these clades, which probably explains the discordance between nuclear and mitochondrial phylogenies. Demographic analyses also revealed the absence of gene flow between the north and south clades of M. fotsifotsy. Morphometric data revealed several major differences between M. soricoides and M. fotsifotsy, as well as more subtle differences between the two clades of M. fotsifotsy. In light of these results, we treat the south clade of M. fotsifotsy as a new candidate species. Our findings demonstrate the utility of integrating multiple data types to understand complex speciation histories, and contribute to a growing body of evidence that species diversity on Madagascar is underestimated.

Speciation, gene flow, and seasonal migration in Catharus thrushes (Aves:Turdidae).

New World thrushes in the genus Catharus are small, insectivorous or omnivorous birds that have been used to explore several important questions in avian evolution, including the evolution of seasonal migration and plumage variation. Within Catharus, members of a clade of obligate long-distance migrants (C. fuscescens, C. minimus, and C. bicknelli) have also been used in the development of heteropatric speciation theory, a divergence process in which migratory lineages (which might occur in allopatry or sympatry during portions of their annual cycle) diverge despite low levels of gene flow. However, research on Catharus relationships has thus far been restricted to the use of small genetic datasets, which provide limited resolution of both phylogenetic and demographic histories. We used a large, multi-locus dataset from loci containing ultraconserved elements (UCEs) to study the demographic histories of the migratory C. fuscescens-minimus-bicknelli clade and to resolve the phylogeny of the migratory species of Catharus. Our dataset included more than 2000 loci and over 1700 variable genotyped sites, and analyses supported our prediction of divergence with gene flow in the fully migratory clade, with significant gene flow among all three species. Our phylogeny of the genus differs from past work in its placement of C. ustulatus, and further analyses suggest historic gene flow throughout the genus, producing genetically reticulate (or network) phylogenies. This raises questions about trait origins and suggests that seasonal migration and the resulting migratory condition of heteropatry is likely to promote hybridization not only during pairwise divergence and speciation, but also among non-sisters.

Caught in the act: Incipient speciation across a latitudinal gradient in a semifossorial mammal from Madagascar, the mole tenrec Oryzorictes hova (Tenrecidae).

Madagascar is one of the world's foremost biodiversity hotspots, yet a large portion of its flora and fauna remains undescribed and the driving forces of in situ diversification are not well understood. Recent studies have identified a widespread, latitudinally structured phylogeographic pattern in Madagascar's humid-forest mammals, amphibians, reptiles, and insects. Several factors may be driving this pattern, namely biogeographic barriers (i.e., rivers or valleys) or past episodes of forest contraction and expansion. In this study, we describe the phylogeographic structure of the small, semifossorial mammal Oryzorictes hova, one of Madagascar's two species of mole tenrec, found throughout Madagascar's eastern humid forest belt, from high-elevation montane forest to low-elevation forests, as well as disturbed habitat such as rice fields. Using one mitochondrial locus, four nuclear loci, and 31 craniomandibular measurements, we identified three distinct populations of O. hova associated with the northern, central, and southern regions of the island. We found little evidence of gene flow among these populations, so we treated each population as a potential species. We validated species limits using two Bayesian methods: BP&P, employing only DNA sequence data, and iBPP using both DNA and morphological data, and we assessed whether these methods are susceptible to producing false positive errors. Molecular and morphological data support the recognition of each of the three populations of O. hova as distinct species, but formal species descriptions will require additional data from type specimens. This study illustrates the importance of using integrative datasets, multiple methodological approaches, and extensive geographic sampling for species delimitation and adds evidence for a widespread phylogeographic pattern in Madagascar's humid forest taxa.

Multiple Loci and Complete Taxonomic Sampling Resolve the Phylogeny and Biogeographic History of Tenrecs (Mammalia: Tenrecidae) and Reveal Higher Speciation Rates in Madagascar's Humid Forests.

The family Tenrecidae (tenrecs) is one of only four extant terrestrial mammal lineages to have colonized and diversified on Madagascar. Over the last 15 years, several studies have disagreed on relationships among major tenrec lineages, resulting in multiple reinterpretations of the number and timing of historical transoceanic dispersal events between Africa and Madagascar. We reconstructed the phylogeny of Tenrecidae using multiple loci from all recognized extant species and estimated divergence timing using six fossil calibrations within Afrotheria. All phylogenetic analyses strongly support monophyly of the Malagasy tenrecs, and our divergence timing analysis places their colonization of the island at 30-56 Ma. Our comprehensive phylogeny supports three important taxonomic revisions that reflect the evolutionary history of tenrecs: (1) we formally elevate the African otter shrews to their own family Potamogalidae, thereby rendering extant Tenrecidae entirely endemic to Madagascar; (2) we subsume the semiaquatic genus Limnogale within the shrew tenrec genus Microgale; and (3) we re-elevate the two largest-bodied shrew tenrecs, Microgale dobsoni and Microgale talazaci, to the genus Nesogale Thomas (1918) Finally, we use recently summarized habitat data to test the hypothesis that diversification rates differ between humid and arid habitats on Madagascar, and we compare three common methods for ancestral biogeographic reconstruction. These analyses suggest higher speciation rates in humid habitats and reveal a minimum of three and more likely five independent transitions to arid habitats. Our results resolve the relationships among previously recalcitrant taxa, illuminate the timing and mechanisms of major biogeographic patterns in an extraordinary example of an island radiation, and permit the first comprehensive, phylogenetically consistent taxonomy of Madagascar's tenrecs.