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.

The phylogenomic and biogeographic history of the gartersnakes, watersnakes, and allies (Natricidae: Thamnophiini).

North American Thamnophiini (gartersnakes, watersnakes, brownsnakes, and swampsnakes) are an ecologically and phenotypically diverse temperate clade of snakes representing 61 species across 10 genera. In this study, we estimate phylogenetic trees using âˆ¼3,700 ultraconserved elements (UCEs) for 76 specimens representing 75% of all Thamnophiini species. We infer phylogenies using multispecies coalescent methods and time calibrate them using the fossil record. We also conducted ancestral area estimation to identify how major biogeographic boundaries in North America affect broadscale diversification in the group. While most nodes exhibited strong statistical support, analysis of concordant data across gene trees reveals substantial heterogeneity. Ancestral area estimation demonstrated that the genus Thamnophis was the only taxon in this subfamily to cross the Western Continental Divide, even as other taxa dispersed southward toward the tropics. Additionally, levels of gene tree discordance are overall higher in transition zones between bioregions, including the Rocky Mountains. Therefore, the Western Continental Divide may be a significant transition zone structuring the diversification of Thamnophiini during the Neogene and Pleistocene. Here we show that despite high levels of discordance across gene trees, we were able to infer a highly resolved and well-supported phylogeny for Thamnophiini, which allows us to understand broadscale patterns of diversity and biogeography.

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.

Phylogenomics Reveals Ancient Gene Tree Discordance in the Amphibian Tree of Life.

Molecular phylogenies have yielded strong support for many parts of the amphibian Tree of Life, but poor support for the resolution of deeper nodes, including relationships among families and orders. To clarify these relationships, we provide a phylogenomic perspective on amphibian relationships by developing a taxon-specific Anchored Hybrid Enrichment protocol targeting hundreds of conserved exons which are effective across the class. After obtaining data from 220 loci for 286 species (representing 94% of the families and 44% of the genera), we estimate a phylogeny for extant amphibians and identify gene tree-species tree conflict across the deepest branches of the amphibian phylogeny. We perform locus-by-locus genealogical interrogation of alternative topological hypotheses for amphibian monophyly, focusing on interordinal relationships. We find that phylogenetic signal deep in the amphibian phylogeny varies greatly across loci in a manner that is consistent with incomplete lineage sorting in the ancestral lineage of extant amphibians. Our results overwhelmingly support amphibian monophyly and a sister relationship between frogs and salamanders, consistent with the Batrachia hypothesis. Species tree analyses converge on a small set of topological hypotheses for the relationships among extant amphibian families. These results clarify several contentious portions of the amphibian Tree of Life, which in conjunction with a set of vetted fossil calibrations, support a surprisingly younger timescale for crown and ordinal amphibian diversification than previously reported. More broadly, our study provides insight into the sources, magnitudes, and heterogeneity of support across loci in phylogenomic data sets.[AIC; Amphibia; Batrachia; Phylogeny; gene tree-species tree discordance; genomics; information theory.].

Cryptic Patterns of Speciation in Cryptic Primates: Microendemic Mouse Lemurs and the Multispecies Coalescent.

Mouse lemurs (Microcebus) are a radiation of morphologically cryptic primates distributed throughout Madagascar for which the number of recognized species has exploded in the past two decades. This taxonomic revision has prompted understandable concern that there has been substantial oversplitting in the mouse lemur clade. Here, we investigate mouse lemur diversity in a region in northeastern Madagascar with high levels of microendemism and predicted habitat loss. We analyzed RADseq data with multispecies coalescent (MSC) species delimitation methods for two pairs of sister lineages that include three named species and an undescribed lineage previously identified to have divergent mtDNA. Marked differences in effective population sizes, levels of gene flow, patterns of isolation-by-distance, and species delimitation results were found among the two pairs of lineages. Whereas all tests support the recognition of the presently undescribed lineage as a separate species, the species-level distinction of two previously described species, M. mittermeieri and M. lehilahytsara is not supported-a result that is particularly striking when using the genealogical discordance index (gdi). Nonsister lineages occur sympatrically in two of the localities sampled for this study, despite an estimated divergence time of less than 1 Ma. This suggests rapid evolution of reproductive isolation in the focal lineages and in the mouse lemur clade generally. The divergence time estimates reported here are based on the MSC calibrated with pedigree-based mutation rates and are considerably more recent than previously published fossil-calibrated relaxed-clock estimates. We discuss the possible explanations for this discrepancy, noting that there are theoretical justifications for preferring the MSC estimates in this case. [Cryptic species; effective population size; microendemism; multispecies coalescent; speciation; species delimitation.].

A spatial genomic approach identifies time lags and historical barriers to gene flow in a rapidly fragmenting Appalachian landscape.

The resolution offered by genomic data sets coupled with recently developed spatially informed analyses are allowing researchers to quantify population structure at increasingly fine temporal and spatial scales. However, both empirical research and conservation measures have been limited by questions regarding the impacts of data set size, data quality thresholds and the timescale at which barriers to gene flow become detectable. Here, we used restriction site associated DNA sequencing to generate a 2,140 single nucleotide polymorphism (SNP) data set for the copperhead snake (Agkistrodon contortrix) and address the population genomic impacts of recent and widespread landscape modification across an ~1,000-km2 region of eastern Kentucky, USA. Nonspatial population-based assignment and clustering methods supported little to no population structure. However, using individual-based spatial autocorrelation approaches we found evidence for genetic structuring which closely follows the path of a historically important highway which experienced high traffic volumes from c. 1920 to 1970 before losing most traffic to a newly constructed alternative route. We found no similar spatial genomic signatures associated with more recently constructed highways or surface mining activity, although a time lag effect may be responsible for the lack of any emergent spatial genetic patterns. Subsampling of our SNP data set suggested that similar results could be obtained with as few as 250 SNPs, and a range of thresholds for missing data exhibited limited impacts on the spatial patterns we detected. While we were not able to estimate relative effects of land uses or precise time lags, our findings highlight the importance of temporal factors in landscape genetics approaches, and suggest the potential advantages of genomic data sets and fine-scale, spatially informed approaches for quantifying subtle genetic patterns in temporally complex landscapes.

Long-distance dispersal, ice sheet dynamics and mountaintop isolation underlie the genetic structure of glacier ice worms.

Disentangling the contemporary and historical factors underlying the spatial distributions of species is a central goal of biogeography. For species with broad distributions but little capacity to actively disperse, disconnected geographical distributions highlight the potential influence of passive, long-distance dispersal (LDD) on their evolutionary histories. However, dispersal alone cannot completely account for the biogeography of any species, and other factors-e.g. habitat suitability, life history-must also be considered. North American ice worms ( Mesenchytraeus solifugus) are ice-obligate annelids that inhabit coastal glaciers from Oregon to Alaska. Previous studies identified a complex biogeographic history for ice worms, with evidence for genetic isolation, unexpectedly close relationships among geographically disjunct lineages, and contemporary migration across large (e.g. greater than 1500 km) areas of unsuitable habitat. In this study, we analysed genome-scale sequence data for individuals from most of the known ice worm range. We found clear support for divergence between populations along the Pacific Coast and the inland flanks of the Coast Mountains (mean FST = 0.60), likely precipitated by episodic ice sheet expansion and contraction during the Pleistocene. We also found support for LDD of ice worms from Alaska to Vancouver Island, perhaps mediated by migrating birds. Our results highlight the power of genomic data for disentangling complex biogeographic patterns, including the presence of LDD.

Microbial assemblages reflect environmental heterogeneity in alpine streams.

Alpine streams are dynamic habitats harboring substantial biodiversity across small spatial extents. The diversity of alpine stream biota is largely reflective of environmental heterogeneity stemming from varying hydrological sources. Globally, alpine stream diversity is under threat as meltwater sources recede and stream conditions become increasingly homogeneous. Much attention has been devoted to macroinvertebrate diversity in alpine headwaters, yet to fully understand the breadth of climate change threats, a more thorough accounting of microbial diversity is needed. We characterized microbial diversity (specifically Bacteria and Archaea) of 13 streams in two disjunct Rocky Mountain subranges through 16S rRNA gene sequencing. Our study encompassed the spectrum of alpine stream sources (glaciers, snowfields, subterranean ice, and groundwater) and three microhabitats (ice, biofilms, and streamwater). We observed no difference in regional (γ) diversity between subranges but substantial differences in diversity among (ß) stream types and microhabitats. Within-stream (α) diversity was highest in groundwater-fed springs, lowest in glacier-fed streams, and positively correlated with water temperature for both streamwater and biofilm assemblages. We identified an underappreciated alpine stream type-the icy seep-that are fed by subterranean ice, exhibit cold temperatures (summer mean <2°C), moderate bed stability, and relatively high conductivity. Icy seeps will likely be important for combatting biodiversity losses as they contain similar microbial assemblages to streams fed by surface ice yet may be buffered against climate change by insulating debris cover. Our results show that the patterns of microbial diversity support an ominous trend for alpine stream biodiversity; as meltwater sources decline, stream communities will become more diverse locally, but regional diversity will be lost. Icy seeps, however, represent a source of optimism for the future of biodiversity in these imperiled ecosystems.

Extensive variability in the gut microbiome of a highly-specialized and critically endangered lemur species across sites.

Deforestation continues to jeopardize Malagasy primates as viable habitats become smaller, more fragmented, and more disturbed. This deforestation can lead to changes in diet, microhabitat, and gene flow between populations of endangered species, and it remains unclear how these changes may affect gut microbiome (GM) characteristics. The black-and-white ruffed lemur (Varecia variegata), which is among Madagascar's most threatened lemur species, provides a critical model for understanding the relationships between historical and on-going deforestation (habitat disturbance), feeding ecology, and GM composition and diversity. We studied four populations inhabiting two rainforests (relatively pristine vs. highly disturbed) in southeastern Madagascar. We conducted full-day focal animal behavioral follows and collected fecal samples opportunistically across a three-month period. Our results indicate that lemurs inhabiting sites characterized by habitat disturbance and low dietary diversity exhibited reduced gut microbial alpha diversity. We also show that these same factors were associated with high community dissimilarity using weighted and unweighted UniFrac metrics. Finally, an indicator species analysis showed that the most pristine site was characterized by an abundance of methanogenic archaea. While it is impossible to disentangle the relative contributions of each confounding variable presented by our sampling design, these results provide crucial information about GM variability, thereby underscoring the importance of monitoring endangered species at the population-level.

Geogenetic patterns in mouse lemurs (genus Microcebus) reveal the ghosts of Madagascar's forests past.

Phylogeographic analysis can be described as the study of the geological and climatological processes that have produced contemporary geographic distributions of populations and species. Here, we attempt to understand how the dynamic process of landscape change on Madagascar has shaped the distribution of a targeted clade of mouse lemurs (genus Microcebus) and, conversely, how phylogenetic and population genetic patterns in these small primates can reciprocally advance our understanding of Madagascar's prehuman environment. The degree to which human activity has impacted the natural plant communities of Madagascar is of critical and enduring interest. Today, the eastern rainforests are separated from the dry deciduous forests of the west by a large expanse of presumed anthropogenic grassland savanna, dominated by the Family Poaceae, that blankets most of the Central Highlands. Although there is firm consensus that anthropogenic activities have transformed the original vegetation through agricultural and pastoral practices, the degree to which closed-canopy forest extended from the east to the west remains debated. Phylogenetic and population genetic patterns in a five-species clade of mouse lemurs suggest that longitudinal dispersal across the island was readily achieved throughout the Pleistocene, apparently ending at ∼55 ka. By examining patterns of both inter- and intraspecific genetic diversity in mouse lemur species found in the eastern, western, and Central Highland zones, we conclude that the natural environment of the Central Highlands would have been mosaic, consisting of a matrix of wooded savanna that formed a transitional zone between the extremes of humid eastern and dry western forest types.

Estimation of contemporary effective population size and population declines using RAD sequence data.

Large genomic data sets generated with restriction site-associated DNA sequencing (RADseq), in combination with demographic inference methods, are improving our ability to gain insights into the population history of species. We used a simulation approach to examine the potential for RADseq data sets to accurately estimate effective population size (N e) over the course of stable and declining population trends, and we compare the ability of two methods of analysis to accurately distinguish stable from steadily declining populations over a contemporary time scale (20 generations). Using a linkage disequilibrium-based analysis, individual sampling (i.e., n ≥ 30) had the greatest effect on N e estimation and the detection of population size declines, with declines reliably detected across scenarios ~10 generations after they began. Coalescent-based inference required fewer sampled individuals (i.e., n = 15), and instead was most influenced by the size of the SNP data set, with 25,000-50,000 SNPs required for accurate detection of population trends and at least 20 generations after decline began. The number of samples available and targeted number of RADseq loci are important criteria when choosing between these methods. Neither method suffered any apparent bias due to the effects of allele dropout typical of RAD data. With an understanding of the limitations and biases of these approaches, researchers can make more informed decisions when designing their sampling and analyses. Overall, our results reveal that demographic inference using RADseq data can be successfully applied to infer recent population size change and may be an important tool for population monitoring and conservation biology.

Genomic data detect corresponding signatures of population size change on an ecological time scale in two salamander species.

Understanding the demography of species over recent history (e.g. <100 years) is critical in studies of ecology and evolution, but records of population history are rarely available. Surveying genetic variation is a potential alternative to census-based estimates of population size, and can yield insight into the demography of a population. However, to assess the performance of genetic methods, it is important to compare their estimates of population history to known demography. Here, we leveraged the exceptional resources from a wetland with 37 years of amphibian mark-recapture data to study the utility of genetically based demographic inference on salamander species with documented population declines (Ambystoma talpoideum) and expansions (A. opacum), patterns that have been shown to be correlated with changes in wetland hydroperiod. We generated ddRAD data from two temporally sampled populations of A. opacum (1993, 2013) and A. talpoideum (1984, 2011) and used coalescent-based demographic inference to compare alternate evolutionary models. For both species, demographic model inference supported population size changes that corroborated mark-recapture data. Parameter estimation in A. talpoideum was robust to our variations in analytical approach, while estimates for A. opacum were highly inconsistent, tempering our confidence in detecting a demographic trend in this species. Overall, our robust results in A. talpoideum suggest that genome-based demographic inference has utility on an ecological scale, but researchers should also be cognizant that these methods may not work in all systems and evolutionary scenarios. Demographic inference may be an important tool for population monitoring and conservation management planning.

Species discovery and validation in a cryptic radiation of endangered primates: coalescent-based species delimitation in Madagascar's mouse lemurs.

Implementation of the coalescent model in a Bayesian framework is an emerging strength in genetically based species delimitation studies. By providing an objective measure of species diagnosis, these methods represent a quantitative enhancement to the analysis of multilocus data, and complement more traditional methods based on phenotypic and ecological characteristics. Recognized as two species 20 years ago, mouse lemurs (genus Microcebus) now comprise more than 20 species, largely diagnosed from mtDNA sequence data. With each new species description, enthusiasm has been tempered with scientific scepticism. Here, we present a statistically justified and unbiased Bayesian approach towards mouse lemur species delimitation. We perform validation tests using multilocus sequence data and two methodologies: (i) reverse-jump Markov chain Monte Carlo sampling to assess the likelihood of different models defined a priori by a guide tree, and (ii) a Bayes factor delimitation test that compares different species-tree models without a guide tree. We assess the sensitivity of these methods using randomized individual assignments, which has been used in bpp studies, but not with Bayes factor delimitation tests. Our results validate previously diagnosed taxa, as well as new species hypotheses, resulting in support for three new mouse lemur species. As the challenge of multiple researchers using differing criteria to describe diversity is not unique to Microcebus, the methods used here have significant potential for clarifying diversity in other taxonomic groups. We echo previous studies in advocating that multiple lines of evidence, including use of the coalescent model, should be trusted to delimit new species.

The influence of locus number and information content on species delimitation: an empirical test case in an endangered Mexican salamander.

Perhaps the most important recent advance in species delimitation has been the development of model-based approaches to objectively diagnose species diversity from genetic data. Additionally, the growing accessibility of next-generation sequence data sets provides powerful insights into genome-wide patterns of divergence during speciation. However, applying complex models to large data sets is time-consuming and computationally costly, requiring careful consideration of the influence of both individual and population sampling, as well as the number and informativeness of loci on species delimitation conclusions. Here, we investigated how locus number and information content affect species delimitation results for an endangered Mexican salamander species, Ambystoma ordinarium. We compared results for an eight-locus, 137-individual data set and an 89-locus, seven-individual data set. For both data sets, we used species discovery methods to define delimitation models and species validation methods to rigorously test these hypotheses. We also used integrated demographic model selection tools to choose among delimitation models, while accounting for gene flow. Our results indicate that while cryptic lineages may be delimited with relatively few loci, sampling larger numbers of loci may be required to ensure that enough informative loci are available to accurately identify and validate shallow-scale divergences. These analyses highlight the importance of striking a balance between dense sampling of loci and individuals, particularly in shallowly diverged lineages. They also suggest the presence of a currently unrecognized, endangered species in the western part of A. ordinarium's range.

Go west: Population genomics reveals unexpected population fluctuations and little gene flow in Western hemisphere populations of the predatory lady beetle, Hippodamia convergens.

Hippodamia convergens-the convergent lady beetle, has been used extensively in augmentative biological control of aphids, thrips, and whiteflies across its native range in North America, and was introduced into South America in the 1950s. Overwintering H. convergens populations from its native western range in the United States are commercially collected and released across its current range in the eastern USA, with little knowledge of the effectiveness of its augmentative biological control. Here we use a novel ddRADseq-based SNP/haplotype discovery approach to estimate its range-wide population diversity, differentiation, and recent evolutionary history. Our results indicate (1) significant population differentiation among eastern USA, western USA, and South American populations of H. convergens, with (2) little to no detectable recent admixture between them, despite repeated population augmentation, and (3) continued recent population size expansion across its range. These results contradict previous findings using microsatellite markers. In light of these new findings, the implications for the effectiveness of augmentative biological control using H. convergens are discussed. Additionally, because quantifying the non-target effects of augmentative biological control is a difficult problem in migratory beetles, our results could serve as a cornerstone in improving and predicting the efficacy of future releases of H. convergens across its range.

Concatenation and concordance in the reconstruction of mouse lemur phylogeny: an empirical demonstration of the effect of allele sampling in phylogenetics.

The systematics and speciation literature is rich with discussion relating to the potential for gene tree/species tree discordance. Numerous mechanisms have been proposed to generate discordance, including differential selection, long-branch attraction, gene duplication, genetic introgression, and/or incomplete lineage sorting. For speciose clades in which divergence has occurred recently and rapidly, recovering the true species tree can be particularly problematic due to incomplete lineage sorting. Unfortunately, the availability of multilocus or "phylogenomic" data sets does not simply solve the problem, particularly when the data are analyzed with standard concatenation techniques. In our study, we conduct a phylogenetic study for a nearly complete species sample of the dwarf and mouse lemur clade, Cheirogaleidae. Mouse lemurs (genus, Microcebus) have been intensively studied over the past decade for reasons relating to their high level of cryptic species diversity, and although there has been emerging consensus regarding the evolutionary diversity contained within the genus, there is no agreement as to the inter-specific relationships within the group. We attempt to resolve cheirogaleid phylogeny, focusing especially on the mouse lemurs, by employing a large multilocus data set. We compare the results of Bayesian concordance methods with those of standard gene concatenation, finding that though concatenation yields the strongest results as measured by statistical support, these results are found to be highly misleading. By employing an approach where individual alleles are treated as operational taxonomic units, we show that phylogenetic results are substantially influenced by the selection of alleles in the concatenation process.

Parallel tagged amplicon sequencing reveals major lineages and phylogenetic structure in the North American tiger salamander (Ambystoma tigrinum) species complex.

Modern analytical methods for population genetics and phylogenetics are expected to provide more accurate results when data from multiple genome-wide loci are analysed. We present the results of an initial application of parallel tagged sequencing (PTS) on a next-generation platform to sequence thousands of barcoded PCR amplicons generated from 95 nuclear loci and 93 individuals sampled across the range of the tiger salamander (Ambystoma tigrinum) species complex. To manage the bioinformatic processing of this large data set (344 330 reads), we developed a pipeline that sorts PTS data by barcode and locus, identifies high-quality variable nucleotides and yields phased haplotype sequences for each individual at each locus. Our sequencing and bioinformatic strategy resulted in a genome-wide data set with relatively low levels of missing data and a wide range of nucleotide variation. structure analyses of these data in a genotypic format resulted in strongly supported assignments for the majority of individuals into nine geographically defined genetic clusters. Species tree analyses of the most variable loci using a multi-species coalescent model resulted in strong support for most branches in the species tree; however, analyses including more than 50 loci produced parameter sampling trends that indicated a lack of convergence on the posterior distribution. Overall, these results demonstrate the potential for amplicon-based PTS to rapidly generate large-scale data for population genetic and phylogenetic-based research.

Species tree reconstruction of a poorly resolved clade of salamanders (Ambystomatidae) using multiple nuclear loci.

The analysis of diverse data sets can yield different phylogenetic estimates that challenge systematists to explain the source of discordance. The mole salamanders (family Ambystomatidae) are a classic example of this phylogenetic conflict. Previous attempts to resolve the ambystomatid species tree using allozymic, morphological, and mitochondrial sequence data have yielded different estimates, making it unclear which data source best approximates ambystomatid phylogeny and which ones yield phylogenetically inaccurate reconstructions. To shed light on this conflict, we present the first multi-locus DNA sequence-based phylogenetic study of the Ambystomatidae. We utilized a range of analyses, including coalescent-based methods of species-tree estimation that account for incomplete lineage sorting within a locus and concordance-based methods that estimate the number of sampled loci that support a particular clade. We repeated these analyses with the removal of individual loci to determine if any locus has a disproportionate effect on our phylogenetic results. Collectively, these results robustly resolved many deep and relatively shallow clades within Ambystoma, including the placement of A. gracile and A. talpoideum as the sister clade to a clade containing all remaining ambystomatids, and the placement of A. maculatum as the sister lineage to all remaining ambystomatids excluding A. gracile and A. talpoideum. Both Bayesian coalescent and concordance methods produced similar results, highlighting strongly supported branches in the species tree. Furthermore, coalescent-based analyses that excluded loci produced overlapping species-tree posterior distributions, suggesting that no particular locus--including mtDNA--disproportionately contributed to our species-tree estimates. Overall, our phylogenetic estimates have greater similarity with previous allozyme and mitochondrial sequence-based phylogenetic estimates. However, intermediate depths of divergence in the ambystomatid species tree remain unresolved, potentially highlighting a region of rapid species radiation or a hard polytomy, which limits our ability to comment on previous morphologically-based taxonomic groups.

Molecular phylogenetic reconstruction of the endemic Asian salamander family Hynobiidae (Amphibia, Caudata).

The salamander family Hynobiidae contains over 50 species and has been the subject of a number of molecular phylogenetic investigations aimed at reconstructing branches across the entire family. In general, studies using the greatest amount of sequence data have used reduced taxon sampling, while the study with the greatest taxon sampling has used a limited sequence data set. Here, we provide insights into the phylogenetic history of the Hynobiidae using both dense taxon sampling and a large mitochondrial DNA sequence data set. We report exclusive new mitochondrial DNA data of 2566 aligned bases (with 151 excluded sites, of included sites 1157 are variable with 957 parsimony informative). This is sampled from two genic regions encoding a 12S-16S region (the 3' end of 12S rRNA, tRNA(VAI), and the 5' end of 16S rRNA), and a ND2-COI region (ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), the origin for light strand replication--O(L), tRNA(Cys), tRNAT(Tyr), and the 5' end of COI). Analyses using parsimony, Bayesian, and maximum likelihood optimality criteria produce similar phylogenetic trees, with discordant branches generally receiving low levels of branch support. Monophyly of the Hynobiidae is strongly supported across all analyses, as is the sister relationship and deep divergence between the genus Onychodactylus with all remaining hynobiids. Within this latter grouping our phylogenetic results identify six clades that are relatively divergent from one another, but for which there is minimal support for their phylogenetic placement. This includes the genus Batrachuperus, the genus Hynobius, the genus Pachyhynobius, the genus Salamandrella, a clade containing the genera Ranodon and Paradactylodon, and a clade containing the genera Liua and Pseudohynobius. This latter clade receives low bootstrap support in the parsimony analysis, but is consistent across all three analytical methods. Our results also clarify a number of well-supported relationships within the larger Batrachuperus and Hynobius clades. While the relationships identified in this study do much to clarify the phylogenetic history of the Hynobiidae, the poor resolution among major hynobiid clades, and the contrast of mtDNA-derived relationships with recent phylogenetic results from a small number of nuclear genes, highlights the need for continued phylogenetic study with larger numbers of nuclear loci.

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.