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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 (ILS) and introgression. Here, we use a whole-genome alignment, multiple locus-sampling strategies, and summary-tree and single nucleotide polymorphism-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 ILS, 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., (© The Author(s) 2024. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

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., Competing Interests: The authors declare that there are no conflicts of interest., (© 2023 The Authors. Evolutionary Applications published by John Wiley & Sons Ltd.)

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., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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 ∼3,700 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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

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., (© 2022. The Author(s).)

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., Competing Interests: The authors declare no competing interest.

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.]., (© The Author(s) 2020. Published by Oxford University Press, on behalf of the Society of Systematic Biologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

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.]., (© The Author(s) 2020. Published by Oxford University Press, on behalf of the Society of Systematic Biologists.)

Biological control-the use of organisms (e.g., nematodes, arthropods, bacteria, fungi, viruses) for the suppression of insect pest species-is a well-established, ecologically sound and economically profitable tactic for crop protection. This approach has served as a sustainable solution for many insect pest problems for over a century in North America. However, all pest management tactics have associated risks. Specifically, the ecological non-target effects of biological control have been examined in numerous systems. In contrast, the need to understand the short- and long-term evolutionary consequences of human-mediated manipulation of biological control organisms for importation, augmentation and conservation biological control has only recently been acknowledged. Particularly, population genomics presents exceptional opportunities to study adaptive evolution and invasiveness of pests and biological control organisms. Population genomics also provides insights into (1) long-term biological consequences of releases, (2) the ecological success and sustainability of this pest management tactic and (3) non-target effects on native species, populations and ecosystems. Recent advances in genomic sequencing technology and model-based statistical methods to analyze population-scale genomic data provide a much needed impetus for biological control programs to benefit by incorporating a consideration of evolutionary consequences. Here, we review current technology and methods in population genomics and their applications to biological control and include basic guidelines for biological control researchers for implementing genomic technology and statistical modeling.

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-km 2 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., (© 2020 John Wiley & Sons Ltd.)

Sensory gene families are of special interest for both what they can tell us about molecular evolution and what they imply as mediators of social communication. The vomeronasal type-1 receptors (V1Rs) have often been hypothesized as playing a fundamental role in driving or maintaining species boundaries given their likely function as mediators of intraspecific mate choice, particularly in nocturnal mammals. Here, we employ a comparative genomic approach for revealing patterns of V1R evolution within primates, with a special focus on the small-bodied nocturnal mouse and dwarf lemurs of Madagascar (genera Microcebus and Cheirogaleus, respectively). By doubling the existing genomic resources for strepsirrhine primates (i.e. the lemurs and lorises), we find that the highly speciose and morphologically cryptic mouse lemurs have experienced an elaborate proliferation of V1Rs that we argue is functionally related to their capacity for rapid lineage diversification. Contrary to a previous study that found equivalent degrees of V1R diversity in diurnal and nocturnal lemurs, our study finds a strong correlation between nocturnality and V1R elaboration, with nocturnal lemurs showing elaborate V1R repertoires and diurnal lemurs showing less diverse repertoires. Recognized subfamilies among V1Rs show unique signatures of diversifying positive selection, as might be expected if they have each evolved to respond to specific stimuli. Furthermore, a detailed syntenic comparison of mouse lemurs with mouse (genus Mus) and other mammalian outgroups shows that orthologous mammalian subfamilies, predicted to be of ancient origin, tend to cluster in a densely populated region across syntenic chromosomes that we refer to as a V1R "hotspot.", (© The Author(s) 2020. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Systems of genetic sex determination and the homology of sex chromosomes in different taxa vary greatly across vertebrates. Much progress remains to be made in understanding systems of genetic sex determination in non-model organisms, especially those with homomorphic sex chromosomes and/or large genomes. We used reduced representation genome sequencing to investigate genetic sex determination systems in the salamander family Cryptobranchidae (genera Cryptobranchus and Andrias ), which typifies both of these inherent difficulties. We tested hypotheses of male- or female-heterogamety by sequencing hundreds of thousands of anonymous genomic regions in a panel of known-sex cryptobranchids and characterized patterns of presence/absence, inferred zygosity, and depth of coverage to identify sex-linked regions of these 56 gigabase genomes. Our results strongly support the hypothesis that all cryptobranchid species possess homologous systems of female heterogamety, despite maintenance of homomorphic sex chromosomes over nearly 60 million years. Additionally, we report a robust, non-invasive genetic assay for sex diagnosis in Cryptobranchus and Andrias which may have great utility for conservation efforts with these endangered salamanders. Co-amplification of these W-linked markers in both cryptobranchid genera provides evidence for long-term sex chromosome stasis in one of the most divergent salamander lineages. These findings inform hypotheses about the ancestral mode of sex determination in salamanders, but suggest that comparative data from other salamander families are needed. Our results further demonstrate that massive genomes are not necessarily a barrier to effective genome-wide sequencing and that the resulting data can be highly informative about sex determination systems in taxa with homomorphic sex chromosomes., (Copyright © 2019 Hime et al.)

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., (© 2019 Wiley Periodicals, Inc.)

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 (β) 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., (© 2019 John Wiley & Sons Ltd.)

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 F ST = 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.

In the past few decades, population genetics and phylogeographic studies have improved our knowledge of connectivity and population demography in marine environments. Studies of deep-sea hydrothermal vent populations have identified barriers to gene flow, hybrid zones, and demographic events, such as historical population expansions and contractions. These deep-sea studies, however, used few loci, which limit the amount of information they provided for coalescent analysis and thus our ability to confidently test complex population dynamics scenarios. In this study, we investigated population structure, demographic history, and gene flow directionality among four Western Pacific hydrothermal vent populations of the vent limpet Lepetodrilus aff. schrolli . These vent sites are located in the Manus and Lau back-arc basins, currently of great interest for deep-sea mineral extraction. A total of 42 loci were sequenced from each individual using high-throughput amplicon sequencing. Amplicon sequences were analyzed using both genetic variant clustering methods and evolutionary coalescent approaches. Like most previously investigated vent species in the South Pacific, L . aff.  schrolli showed no genetic structure within basins but significant differentiation between basins. We inferred significant directional gene flow from Manus Basin to Lau Basin, with low to no gene flow in the opposite direction. This study is one of the very few marine population studies using >10 loci for coalescent analysis and serves as a guide for future marine population studies., Competing Interests: None declared.

Closely related taxa with dissimilar morphologies are often considered to have diverged via natural selection favoring different phenotypes. However, some studies have found these scenarios to be paired with limited or no genetic differentiation. Desmognathus quadramaculatus and D. marmoratus are sympatric salamander species thought to represent a case of ecological speciation based on distinct morphologies, but the results of previous studies have not resolved corresponding patterns of lineage divergence. Here, we use genome-wide data to test this hypothesis of ecological speciation. Population structure analyses partitioned individuals geographically, but not morphologically, into two adjacent regions of western North Carolina: Pisgah and Nantahala. Phylogenetic analyses confirmed the nominal species are nonmonophyletic and resolved deep divergence between the two geographic clusters. Model-testing overwhelmingly supported the hypothesis that lineage divergence followed geography. Finally, ecological niche modeling showed that Pisgah and Nantahala individuals occupy different climatic niches, and geographic boundaries for the two lineages correspond to differences in precipitation regimes across southern Appalachia. Overall, we reject the previous hypothesis of ecological speciation based on microhabitat partitioning. Instead, our results suggest that there are two cryptic lineages, each containing the same pair of morphotypes., (© 2018 The Author(s). Evolution © 2018 The Society for the Study of Evolution.)

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.

In alpine regions worldwide, climate change is dramatically altering ecosystems and affecting biodiversity in many ways. For streams, receding alpine glaciers and snowfields, paired with altered precipitation regimes, are driving shifts in hydrology, species distributions, basal resources, and threatening the very existence of some habitats and biota. Alpine streams harbour substantial species and genetic diversity due to significant habitat insularity and environmental heterogeneity. Climate change is expected to affect alpine stream biodiversity across many levels of biological resolution from micro- to macroscopic organisms and genes to communities. Herein, we describe the current state of alpine stream biology from an organism-focused perspective. We begin by reviewing seven standard and emerging approaches that combine to form the current state of the discipline. We follow with a call for increased synthesis across existing approaches to improve understanding of how these imperiled ecosystems are responding to rapid environmental change. We then take a forward-looking viewpoint on how alpine stream biologists can make better use of existing data sets through temporal comparisons, integrate remote sensing and geographic information system (GIS) technologies, and apply genomic tools to refine knowledge of underlying evolutionary processes. We conclude with comments about the future of biodiversity conservation in alpine streams to confront the daunting challenge of mitigating the effects of rapid environmental change in these sentinel ecosystems., (© 2017 Cambridge Philosophical Society.)

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., (© 2016 John Wiley & Sons Ltd.)

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., (© 2016 John Wiley & Sons Ltd.)

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.

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., (© 2016 John Wiley & Sons Ltd.)

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., (Published by Elsevier Inc.)

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.

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., (© 2012 Blackwell Publishing Ltd.)

Biological invasions have been accelerated by a variety of human activities. Propagule pressure, the number of introduced individuals and independent introductions, is probably to be influenced by these human activities and may be an important factor for successful range expansion in new environments. We tested whether the current distribution of the predatory ladybeetle Coccinella septempunctata in the introduced range (USA) is the result of multiple historical human introductions or natural range expansion from the first established populations in the USA. To test this hypothesis, we compared historical records of propagule size, propagule number, specific introduction locations and the date of each introduction, with estimates of genetic variation in mitochondrial DNA (cytochrome oxidase I). Our results indicated that genetic diversity in the introduced range was positively correlated with historical records of propagule size and number and negatively correlated with distance to nearest introduction point, suggesting that multiple human releases were successful. Higher genetic diversity in populations found near introduction points suggest that initial founder effects were limited, but lower genetic diversity found farther from introduction points is probably the result of serial founder effects during secondary range expansion. These results suggest that the current distribution of C. septempunctata in the introduced range is the result of a combination of human releases and short-range expansion from multiple established populations in the introduced range., (© 2012 Blackwell Publishing Ltd.)

Here I advocate the utility of Bayesian concordance analysis as a mechanism for exploring the magnitude and source of phylogenetic signal in concatenated mitogenomic phylogenetic studies. While typically applied to the study of independently evolving gene trees, Bayesian concordance analysis can also be applied to linked, but individually analyzed, gene regions using a prior probability that reflects the expectation of similar phylogenetic reconstructions. For true branches in the mitogenomic tree, concordance factors should represent the number of gene regions that contain phylogenetic signal for a particular clade. As a demonstration of the application of Bayesian concordance analysis to empirical data, I analyzed two different salamander (Hynobiidae and Plethodontidae) mitogenomic data sets using a gene-based partitioning strategy. The results revealed many strongly supported clades in the concatenated trees that have high concordance factors, permitting the inference that these are robustly resolved through phylogenetic signal distributed across the mitogenome. In contrast, a number of strongly supported clades in the concatenated tree received low concordance factors, indicating that their reconstruction is either driven primarily by phylogenetic signal in a small number of gene regions, or that they are inconsistent reconstructions influenced by properties of the data that can produce inaccurate trees (e.g., compositional bias, selection, etc.). Exploration of the Bayesian joint posterior distribution of trees highlighted partitions that contribute phylogenetic information to similar clade reconstructions. This approach was particularly insightful in the hynobiid data, where different combinations of genes were identified that support alternative tree reconstructions. Concatenated analysis of these different subsets of genes highlighted through Bayesian concordance analysis produced strongly supported and contrasting trees, demonstrating the potential for inconsistency in concatenated mitogenomic phylogenetics. The overall results presented here suggest that Bayesian concordance analysis can serve as an effective exploration of the influence of different gene regions in mitogenomic (and other organellar genomic) phylogenetic studies., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Background: The increased use of multi-locus data sets for phylogenetic reconstruction has increased the need to determine whether a set of gene trees significantly deviate from the phylogenetic patterns of other genes. Such unusual gene trees may have been influenced by other evolutionary processes such as selection, gene duplication, or horizontal gene transfer., Results: Motivated by this problem we propose a nonparametric goodness-of-fit test for two empirical distributions of gene trees, and we developed the software GeneOut to estimate a p-value for the test. Our approach maps trees into a multi-dimensional vector space and then applies support vector machines (SVMs) to measure the separation between two sets of pre-defined trees. We use a permutation test to assess the significance of the SVM separation. To demonstrate the performance of GeneOut, we applied it to the comparison of gene trees simulated within different species trees across a range of species tree depths. Applied directly to sets of simulated gene trees with large sample sizes, GeneOut was able to detect very small differences between two set of gene trees generated under different species trees. Our statistical test can also include tree reconstruction into its test framework through a variety of phylogenetic optimality criteria. When applied to DNA sequence data simulated from different sets of gene trees, results in the form of receiver operating characteristic (ROC) curves indicated that GeneOut performed well in the detection of differences between sets of trees with different distributions in a multi-dimensional space. Furthermore, it controlled false positive and false negative rates very well, indicating a high degree of accuracy., Conclusions: The non-parametric nature of our statistical test provides fast and efficient analyses, and makes it an applicable test for any scenario where evolutionary or other factors can lead to trees with different multi-dimensional distributions. The software GeneOut is freely available under the GNU public license.

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.

Fruit bats of the genus Pteropus occur throughout the Austral-Asian region west to islands off the eastern coast of Africa. Recent phylogenetic analyses of Pteropus from the western Indian Ocean found low sequence divergence and poor phylogenetic resolution among several morphologically defined species. We reexamine the phylogenetic relationships of these taxa by using multiple individuals per species. In addition, we estimate population genetic structure in two well-sampled taxa occurring on Madagascar and the Comoro Islands (P. rufus and P. seychellensis comorensis). Despite finding a similar pattern of low sequence divergence among species, increased sampling provides insight into the phylogeographic history of western Indian Ocean Pteropus, uncovering high levels of gene flow within species.

Background: Speciation begins when populations become genetically separated through a substantial reduction in gene flow, and it is at this point that a genetically cohesive set of populations attain the sole property of species: the independent evolution of a population-level lineage. The comprehensive delimitation of species within biodiversity hotspots, regardless of their level of divergence, is important for understanding the factors that drive the diversification of biota and for identifying them as targets for conservation. However, delimiting recently diverged species is challenging due to insufficient time for the differential evolution of characters--including morphological differences, reproductive isolation, and gene tree monophyly--that are typically used as evidence for separately evolving lineages., Methodology: In this study, we assembled multiple lines of evidence from the analysis of mtDNA and nDNA sequence data for the delimitation of a high diversity of cryptically diverged population-level mouse lemur lineages across the island of Madagascar. Our study uses a multi-faceted approach that applies phylogenetic, population genetic, and genealogical analysis for recognizing lineage diversity and presents the most thoroughly sampled species delimitation of mouse lemur ever performed., Conclusions: The resolution of a large number of geographically defined clades in the mtDNA gene tree provides strong initial evidence for recognizing a high diversity of population-level lineages in mouse lemurs. We find additional support for lineage recognition in the striking concordance between mtDNA clades and patterns of nuclear population structure. Lineages identified using these two sources of evidence also exhibit patterns of population divergence according to genealogical exclusivity estimates. Mouse lemur lineage diversity is reflected in both a geographically fine-scaled pattern of population divergence within established and geographically widespread taxa, as well as newly resolved patterns of micro-endemism revealed through expanded field sampling into previously poorly and well-sampled regions.

Background: Species are viewed as the fundamental unit in most subdisciplines of biology. To conservationists this unit represents the currency for global biodiversity assessments. Even though Madagascar belongs to one of the top eight biodiversity hotspots of the world, the taxonomy of its charismatic lemuriform primates is not stable. Within the last 25 years, the number of described lemur species has more than doubled, with many newly described species identified among the nocturnal and small-bodied cheirogaleids. Here, we characterize the diversity of the dwarf lemurs (genus Cheirogaleus) and assess the status of the seven described species, based on phylogenetic and population genetic analysis of mtDNA (cytb + cox2) and three nuclear markers (adora3, fiba and vWF)., Results: This study identified three distinct evolutionary lineages within the genus Cheirogaleus. Population genetic cluster analyses revealed a further layer of population divergence with six distinct genotypic clusters., Conclusion: Based on the general metapopulation lineage concept and multiple concordant data sets, we identify three exclusive groups of dwarf lemur populations that correspond to three of the seven named species: C. major, C. medius and C. crossleyi. These three species were found to be genealogically exclusive in both mtDNA and nDNA loci and are morphologically distinguishable. The molecular and morphometric data indicate that C. adipicaudatus and C. ravus are synonymous with C. medius and C. major, respectively. Cheirogaleus sibreei falls into the C. medius mtDNA clade, but in morphological analyses the membership is not clearly resolved. We do not have sufficient data to assess the status of C. minusculus. Although additional patterns of population differentiation are evident, there are no clear subdivisions that would warrant additional specific status. We propose that ecological and more geographic data should be collected to confirm these results.

Lemurs and the other strepsirrhine primates are of great interest to the primate genomics community due to their phylogenetic placement as the sister lineage to all other primates. Previous attempts to resolve the phylogeny of lemurs employed limited mitochondrial or small nuclear data sets, with many relationships poorly supported or entirely unresolved. We used genomic resources to develop 11 novel markers from nine chromosomes, representing approximately 9 kb of nuclear sequence data. In combination with previously published nuclear and mitochondrial loci, this yields a data set of more than 16 kb and adds approximately 275 kb of DNA sequence to current databases. Our phylogenetic analyses confirm hypotheses of lemuriform monophyly and provide robust resolution of the phylogenetic relationships among the five lemuriform families. We verify that the genus Daubentonia is the sister lineage to all other lemurs. The Cheirogaleidae and Lepilemuridae are sister taxa and together form the sister lineage to the Indriidae; this clade is the sister lineage to the Lemuridae. Divergence time estimates indicate that lemurs are an ancient group, with their initial diversification occurring around the Cretaceous-Tertiary boundary. Given the power of this data set to resolve branches in a notoriously problematic area of primate phylogeny, we anticipate that our phylogenomic toolkit will be of value to other studies of primate phylogeny and diversification. Moreover, the methods applied will be broadly applicable to other taxonomic groups where phylogenetic relationships have been notoriously difficult to resolve.

We examine phylogenetic relationships among salamanders of the family Salamandridae using approximately 2700 bases of new mtDNA sequence data (the tRNALeu, ND1, tRNAIle, tRNAGln, tRNAMet, ND2, tRNATrp, tRNAAla, tRNAAsn, tRNACys, tRNATyr, and COI genes and the origin for light-strand replication) collected from 96 individuals representing 61 of the 66 recognized salamandrid species and outgroups. Phylogenetic analyses using maximum parsimony and Bayesian analysis are performed on the new data alone and combined with previously reported sequences from other parts of the mitochondrial genome. The basal phylogenetic split is a polytomy of lineages ancestral to (1) the Italian newt Salamandrina terdigitata, (2) a strongly supported clade comprising the "true" salamanders (genera Chioglossa, Mertensiella, Lyciasalamandra, and Salamandra), and (3) a strongly supported clade comprising all newts except S. terdigitata. Strongly supported clades within the true salamanders include monophyly of each genus and grouping Chioglossa and Mertensiella as the sister taxon to a clade comprising Lyciasalamandra and Salamandra. Among newts, genera Echinotriton, Pleurodeles, and Tylototriton form a strongly supported clade whose sister taxon comprises the genera Calotriton, Cynops, Euproctus, Neurergus, Notophthalmus, Pachytriton, Paramesotriton, Taricha, and Triturus. Our results strongly support monophyly of all polytypic newt genera except Paramesotriton and Triturus, which appear paraphyletic, and Calotriton, for which only one of the two species is sampled. Other well-supported clades within newts include (1) Asian genera Cynops, Pachytriton, and Paramesotriton, (2) North American genera Notophthalmus and Taricha, (3) the Triturus vulgaris species group, and (4) the Triturus cristatus species group; some additional groupings appear strong in Bayesian but not parsimony analyses. Rates of lineage accumulation through time are evaluated using this nearly comprehensive sampling of salamandrid species-level lineages. Rate of lineage accumulation appears constant throughout salamandrid evolutionary history with no obvious fluctuations associated with origins of morphological or ecological novelties.

Delimiting the boundaries of species involved in radiations is critical to understanding the tempo and mode of lineage formation. Single locus gene trees may or may not reflect the underlying pattern of population divergence and lineage formation, yet they constitute the vast majority of the empirical data in species radiations. In this study we make use of an expressed sequence tag (EST) database to perform nuclear (nDNA) and mitochondrial (mtDNA) genealogical tests of species boundaries in Ambystoma ordinarium, a member of an adaptive radiation of metamorphic and paedomorphic salamanders (the Ambystoma tigrinum complex) that have diversified across terrestrial and aquatic environments. Gene tree comparisons demonstrate extensive nonmonophyly in the mtDNA genealogy of A. ordinarium, while seven of eight independent nuclear loci resolve the species as monophyletic or nearly so, and diagnose it as a well-resolved genealogical species. A differential introgression hypothesis is supported by the observation that western A. ordinarium localities contain mtDNA haplotypes that are identical or minimally diverged from haplotypes sampled from a nearby paedomorphic species, Ambystoma dumerilii, while most nDNA trees place these species in distant phylogenetic positions. These results provide a strong example of how historical introgression can lead to radical differences between gene trees and species histories, even among currently allopatric species with divergent life history adaptations and morphologies. They also demonstrate how EST-based nuclear resources can be used to more fully resolve the phylogenetic history of species radiations.

Adaptive radiations have served as model systems for quantifying the build-up of species richness. Few studies have quantified the tempo of diversification in species-rich clades that contain negligible adaptive disparity, making the macroevolutionary consequences of different modes of evolutionary radiation difficult to assess. We use mitochondrial-DNA sequence data and recently developed phylogenetic methodologies to explore the tempo of diversification of eastern North American Plethodon, a species-rich clade of woodland salamanders exhibiting only limited phenotypic disparity. Lineage-through-time analysis reveals a high rate of lineage accumulation, 0.8 species per million years, occurring 11-8 million years ago in the P. glutinosus species group, followed by decreasing rates. This high rate of lineage accumulation is exceptional, comparable to the most rapid of adaptive radiations. In contrast to classic models of adaptive radiation where ecological niche divergence is linked to the origin of species, we propose that phylogenetic niche conservatism contributes to the rapid accumulation of P. glutinosus-group lineages by promoting vicariant isolation and multiplication of species across a spatially and temporally fluctuating environment. These closely related and ecologically similar lineages persist through long-periods of evolutionary time and form strong barriers to the geographic spread of their neighbours, producing a subsequent decline in lineage accumulation. Rapid diversification among lineages exhibiting long-term maintenance of their bioclimatic niche requirements is an under-appreciated phenomenon driving the build-up of species richness.

Salamanders of the genus Ambystoma are a unique model organism system because they enable natural history and biomedical research in the laboratory or field. We developed Sal-Site to integrate new and existing ambystomatid salamander research resources in support of this model system. Sal-Site hosts six important resources: 1) Salamander Genome Project: an information-based web-site describing progress in genome resource development, 2) Ambystoma EST Database: a database of manually edited and analyzed contigs assembled from ESTs that were collected from A. tigrinum tigrinum and A. mexicanum, 3) Ambystoma Gene Collection: a database containing full-length protein-coding sequences, 4) Ambystoma Map and Marker Collection: an image and database resource that shows the location of mapped markers on linkage groups, provides information about markers, and provides integrating links to Ambystoma EST Database and Ambystoma Gene Collection databases, 5) Ambystoma Genetic Stock Center: a website and collection of databases that describe an NSF funded salamander rearing facility that generates and distributes biological materials to researchers and educators throughout the world, and 6) Ambystoma Research Coordination Network: a web-site detailing current research projects and activities involving an international group of researchers. Sal-Site is accessible at http://www.ambystoma.org.

Phylogenetic relationships among salamander families illustrate analytical challenges inherent to inferring phylogenies in which terminal branches are temporally very long relative to internal branches. We present new mitochondrial DNA sequences, approximately 2,100 base pairs from the genes encoding ND1, ND2, COI, and the intervening tRNA genes for 34 species representing all 10 salamander families, to examine these relationships. Parsimony analysis of these mtDNA sequences supports monophyly of all families except Proteidae, but yields a tree largely unresolved with respect to interfamilial relationships and the phylogenetic positions of the proteid genera Necturus and Proteus. In contrast, Bayesian and maximum-likelihood analyses of the mtDNA data produce a topology concordant with phylogenetic results from nuclear-encoded rRNA sequences, and they statistically reject monophyly of the internally fertilizing salamanders, suborder Salamandroidea. Phylogenetic simulations based on our mitochondrial DNA sequences reveal that Bayesian analyses outperform parsimony in reconstructing short branches located deep in the phylogenetic history of a taxon. However, phylogenetic conflicts between our results and a recent analysis of nuclear RAG-1 gene sequences suggest that statistical rejection of a monophyletic Salamandroidea by Bayesian analyses of our mitochondrial genomic data is probably erroneous. Bayesian and likelihood-based analyses may overestimate phylogenetic precision when estimating short branches located deep in a phylogeny from data showing substitutional saturation; an analysis of nucleotide substitutions indicates that these methods may be overly sensitive to a relatively small number of sites that show substitutions judged uncommon by the favored evolutionary model.

We report on a study that extended mitochondrial transcript information from a recent EST project to obtain complete mitochondrial genome sequence for 5 tiger salamander complex species (Ambystoma mexicanum, A. t. tigrinum, A. andersoni, A. californiense, and A. dumerilii). We describe, for the first time, aspects of mitochondrial transcription in a representative amphibian, and then use complete mitochondrial sequence data to examine salamander phylogeny at both deep and shallow levels of evolutionary divergence. The available mitochondrial ESTs for A. mexicanum (N=2481) and A. t. tigrinum (N=1205) provided 92% and 87% coverage of the mitochondrial genome, respectively. Complete mitochondrial sequences for all species were rapidly obtained by using long distance PCR and DNA sequencing. A number of genome structural characteristics (base pair length, base composition, gene number, gene boundaries, codon usage) were highly similar among all species and to other distantly related salamanders. Overall, mitochondrial transcription in Ambystoma approximated the pattern observed in other vertebrates. We inferred from the mapping of ESTs onto mtDNA that transcription occurs from both heavy and light strand promoters and continues around the entire length of the mtDNA, followed by post-transcriptional processing. However, the observation of many short transcripts corresponding to rRNA genes indicates that transcription may often terminate prematurely to bias transcription of rRNA genes; indeed an rRNA transcription termination signal sequence was observed immediately following the 16S rRNA gene. Phylogenetic analyses of salamander family relationships consistently grouped Ambystomatidae in a clade containing Cryptobranchidae and Hynobiidae, to the exclusion of Salamandridae. This robust result suggests a novel alternative hypothesis because previous studies have consistently identified Ambystomatidae and Salamandridae as closely related taxa. Phylogenetic analyses of tiger salamander complex species also produced robustly supported trees. The D-loop, used in previous molecular phylogenetic studies of the complex, was found to contain a relatively low level of variation and we identified mitochondrial regions with higher rates of molecular evolution that are more useful in resolving relationships among species. Our results show the benefit of using complete genome mitochondrial information in studies of recently and rapidly diverged taxa.

Plethodon shermani comprises a series of geographically disjunct populations occupying high-elevation mountain isolates. These populations hybridize at their borders with salamanders of the Plethodon glutinosus species complex, and past range expansions inferred from Pleistocene climatic cycles may have increased the possible genetic interactions between P. shermani and species of the P. glutinosus complex. Because mitochondrial DNA haplotypes often show introgression across species borders, we survey mtDNA variation for evidence of past and ongoing genetic interactions between P. shermani, its close relative Plethodon cheoah, and species of the P. glutinosus complex. Ongoing hybridization with the P. glutinosus-complex species Plethodon teyahalee is accompanied by extensive mitochondrial introgression in some Unicoi populations of P. shermani, but it has little genetic impact on P. shermani populations outside hybrid zones at three other isolates (Tusquitee, Wayah Bald, Standing Indian). Some Unicoi populations of P. shermani exhibit mtDNA evidence of past hybridization with diverse lineages from P. aureolus and P. glutinosus. The Tusquitee isolate of P. shermani is also characterized by mtDNA haplotypes most closely related to Plethodon aureolus and P. glutinosus, presumably introduced by past genetic contact with these species or with introgressed populations of Unicoi P. shermani. The mtDNA variation in sampled populations of the Wayah Bald and Standing Indian isolates of P. shermani appears largely unaffected by ongoing hybridization. Principal components analyses of allozymic data indicate that P. shermani isolates collectively form a genetically homogeneous unit clearly demarcated from species with which they have had current or past genetic interactions. Rapid mtDNA introgression associated with transient contacts between P. shermani and other species permits a fine-level resolution of evolutionary lineages not evident from allozymic data.

Background: Establishing genomic resources for closely related species will provide comparative insights that are crucial for understanding diversity and variability at multiple levels of biological organization. We developed ESTs for Mexican axolotl (Ambystoma mexicanum) and Eastern tiger salamander (A. tigrinum tigrinum), species with deep and diverse research histories., Results: Approximately 40,000 quality cDNA sequences were isolated for these species from various tissues, including regenerating limb and tail. These sequences and an existing set of 16,030 cDNA sequences for A. mexicanum were processed to yield 35,413 and 20,599 high quality ESTs for A. mexicanum and A. t. tigrinum, respectively. Because the A. t. tigrinum ESTs were obtained primarily from a normalized library, an approximately equal number of contigs were obtained for each species, with 21,091 unique contigs identified overall. The 10,592 contigs that showed significant similarity to sequences from the human RefSeq database reflected a diverse array of molecular functions and biological processes, with many corresponding to genes expressed during spinal cord injury in rat and fin regeneration in zebrafish. To demonstrate the utility of these EST resources, we searched databases to identify probes for regeneration research, characterized intra- and interspecific nucleotide polymorphism, saturated a human - Ambystoma synteny group with marker loci, and extended PCR primer sets designed for A. mexicanum / A. t. tigrinum orthologues to a related tiger salamander species., Conclusions: Our study highlights the value of developing resources in traditional model systems where the likelihood of information transfer to multiple, closely related taxa is high, thus simultaneously enabling both laboratory and natural history research.

Phylogenetic relationships among salamandrids of the "true" salamander clade are investigated using 2019 aligned base positions (713 parsimony informative) of 20 mitochondrial DNA sequences from the genes encoding ND1 (subunit one of NADH dehydrogenase), tRNA(Ile), tRNA(Gln), tRNA(Met), ND2, tRNA(Trp), tRNA(Ala), tRNA(Asn), tRNA(Cys), tRNA(Tyr), and COI (subunit I of cytochrome c oxidase), plus the origin for light-strand replication (O(L)) between the tRNA(Asn) and the tRNA(Cys) genes. Parsimony analysis produces a robust phylogenetic estimate for the relationships of the major groups of "true" salamanders. Strong support is provided for the sister taxon relationship of Chioglossa and Mertensiella caucasica and for the placement of Salamandra and Mertensiella luschani as sister taxa. These relationships suggest two vicariant events between Europe and Anatolia caused by the formation of seaways in the Mediterranean Basin. Molecular divergence indicates an Early Miocene separation of Chioglossa and M. caucasica and a Late Miocene separation of Salamandra and M. luschani. The traditional phylogenetic hypothesis of a monophyletic Mertensiella is statistically rejected, indicating that southwestern and northeastern Anatolian populations have separate historical biogeographic origins. Therefore, we recommend placement of M. luschani in the genus Salamandra. Within M. luschani, six highly divergent lineages showing 7.6 to 10.1% pairwise sequence divergence are identified. Tests using four-taxon subsamples suggest that these lineages diverged nearly simultaneously in the Late Miocene, approximately 6 to 8 million years ago, when extensive uplifting of Anatolia occurred in response to the Arabian collision., (Copyright 2001 Academic Press.)

We use a comparative analysis of partial cytochrome b sequences to evaluate the evolutionary forces shaping wide-scale phylogeographic patterns of all three North American softshell turtles (Apalone ferox, A. mutica, and A. spinifera). The overall phylogeographic patterns are concordant with results from both extensive regional studies of southeastern species, implicating historical vicariant processes during the Pliocene and Pleistocene, and investigations of more northerly distributed species, indicating a bottleneck effect of recent dispersal into postglacial habitat. We also resolved a novel, shared genetic break between northern-western and southeastern populations within both A. mutica and A. spinifera, demonstrating the value of using widespread taxa to evaluate both regional and wider scale phylogeographic patterns. The extensive phylogenetic structure and sequence divergences within both A. mutica and A. spinifera contrast sharply with most previous studies of turtles and with the hypothesis that turtles in general have slow rates of mtDNA evolution., (Copyright 2000 Academic Press.)