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Hybridization is a complicated, oft-misunderstood process. Once deemed unnatural and uncommon, hybridization is now recognized as ubiquitous among species. But hybridization rates within and among communities are poorly understood despite the relevance to ecology, evolution and conservation. To clarify, we examined hybridization across 75 freshwater fish communities within the Ozarks of the North American Interior Highlands (USA) by single nucleotide polymorphism (SNP) genotyping 33 species ( N = 2865 individuals; double-digest restriction site-associated DNA sequencing (ddRAD)). We found evidence of hybridization (70 putative hybrids; 2.4% of individuals) among 18 species-pairs involving 73% (24/33) of study species, with the majority being concentrated within one family (Leuciscidae/minnows; 15 species; 66 hybrids). Interspecific genetic exchange—or introgression—was evident from 24 backcrossed individuals (10/18 species-pairs). Hybrids occurred within 42 of 75 communities (56%). Four selected environmental variables (species richness, protected area extent, precipitation (May and annually)) exhibited 73–78% accuracy in predicting hybrid occurrence via random forest classification. Our community-level assessment identified hybridization as spatially widespread and environmentally dependent (albeit predominantly within one diverse, omnipresent family). Our approach provides a more holistic survey of natural hybridization by testing a wide range of species-pairs, thus contrasting with more conventional evaluations.

Genetic differentiation among local groups of individuals, i.e., genetic β-diversity, is a key component of population persistence related to connectivity and isolation. However, most genetic investigations of natural populations focus on a single species, overlooking opportunities for multispecies conservation plans to benefit entire communities in an ecosystem. We present an approach to evaluate genetic β-diversity within and among many species and demonstrate how thisriverscape community genomicsapproach can be applied to identify common drivers of genetic structure. Our study evaluated genetic β-diversity in 31 co-distributed native stream fishes sampled from 75 sites across the White River Basin (Ozarks, USA) using SNP genotyping (ddRAD). Despite variance among species in the degree of genetic divergence, general spatial patterns were identified corresponding to river network architecture. Most species (N=24) were partitioned into discrete sub-populations (K=2–7). We used partial redundancy analysis to compare species-specific genetic β-diversity across four models of genetic structure: Isolation by distance (IBD), isolation by barrier (IBB), isolation by stream hierarchy (IBH), and isolation by environment (IBE). A significant proportion of intraspecific genetic variation was explained by IBH (x□ =62%), with the remaining models generally redundant. We found evidence for consistent spatial modularity in that gene flow is higher within rather than between hierarchical units (i.e., catchments, watersheds, basins), supporting the generalization of the Stream Hierarchy Model. We discuss our conclusions regarding conservation and management and identify the 8-digit Hydrologic Unit (HUC) as the most relevant spatial scale for managing genetic diversity across riverine networks.

Phenotypic traits modulate the fate of species interactions with one another and the environment; thus, traits directly shape the past, present, and future evolutionary trajectories of populations. As such, distinct species-specific responses to a shifting environment are widely documented in the form of distinct genetic signatures, i.e., genetic diversity, reflecting differential responses over time. While the link between genetic diversity and phenotypic traits is seemingly fundamental, it has been challenging to establish unequivocally. Across an exemplar freshwater fish metacommunity, we employ phenotypic traits to test if they are significantly related to observed genetic patterns among species. Associated traits were then used to construct trait-based predictive models of genetic diversity. We collected representative constituents of a freshwater fish community (N=31 species) sampled across 75 sites within the White River Basin (Ozark Plateau, USA). For each species, we derived three genetic diversity indices (=HS/HT/G”ST) from SNP data (N=2,000 loci) and assessed 28 phenotypic traits related to morphology, life history, and ecology. We identified a series of traits (N=2–5, depending upon the index) strongly associated with facets of genetic diversity. These were subsequently applied in predictive models that explained 31–68% of the genetic variability across species, suggesting a potential utility as an imputation tactic for data-deficient species. Our approach effectively linked species-specific traits with genetic diversity within and among populations, thereby further clarifying correlations between contemporary ecological processes, as modulated by species traits, and long-term evolutionary trajectories.

Climate change inevitably leaves behind a genetic footprint within phylogeographic legacies of affected species, as individuals are driven to either disperse to track suitable conditions or adaptin situ. One potential consequence is the possibility of hybridization among species, as both geographic ranges and adaptive landscapes shift. The admixture resulting from these newly formed ‘contact zones’ has various outcomes, to include the creation of new lineages. Interpreting these within the context of historic climate change provides clues necessary to predict biotic responses (and thus evolutionary trajectories) as a function of contemporary shifts. Herein, we dissect historic contact zones for Massasaugas (Viperidae;Sistrurusspp.) within two distinct North American regions (southwestern United States and Central Great Plains) using ddRAD sequencing. We identified fine-scale but previously unrecognized population structure within the southwestern contact zone, where we detected contemporary intergradation between Prairie and Desert massasaugas (S. tergeminus tergeminus, andS. t. edwardsii,respectively), with primary divergence indicated by demographic model selection. Within the Central Great Plains, we found evidence for historic secondary contact via Quaternary climatic cycles, subsequently followed by range expansion at the suture zone separatingS. tergeminusandS. catenatus. Extant Missouri populations represent ancestral/relictual vestiges of this earlier hybridization, isolated between the eastern terminus ofS. t. tergeminusand the western edge ofS. catenatus. Our results illustrate how abrupt climate change has driven ancestral hybridization, cryptic diversity, and range dynamism withinSistrurus.

Control methods that target specific traits of an invasive species can produce results contrary to the aims of management. If targeted phenotypes exhibit heritability, then it follows that the invasive species could evolve greater resistance to the applied control measures over time. Additional complications emerge if those traits targeted by control are also inversely related to reproductive success. Given this, prudent considerations for invasive species management are to quantify the heritability of traits selected through control measures and gauge their relationship with reproductive success. Herein we provide a case study utilizing long-term field data and a multi-generational pedigree of an experimentally-closed population of brown treesnakes (N = 426; Boiga irregularis) on Guam. We employed an “animal model” to estimate the narrow-sense heritability (h2) for annual body condition, a trait related to both susceptibility to a primary tool used for brown treesnake control (i.e., live-lure traps) and annual reproductive success. Annual body condition displayed significant heritability [h2 = 0.149 (95% highest posterior density interval: 0.059–0.220)]. Considering a negative effect of body condition on susceptibility to trap capture but positive effect on reproductive success, significant heritability of body condition suggests the potential for live-lure traps to lose efficacy over time while also eliciting an undesirable effect on brown treesnake fecundity. Our results highlight the potential for negative repercussions that can stem from management actions, while also serving to underscore the evolutionary implications that are often overlooked but subsumed within invasive species control.

Approximately 100 years ago, unregulated harvest nearly eliminated white-tailed deer (Odocoileus virginianus) from eastern North America, which subsequently served to catalyze wildlife management as a national priority. An extensive stock-replenishment effort soon followed, with deer broadly translocated among states as a means of re-establishment. However, an unintended consequence was that natural patterns of gene flow became obscured and pre-translocation signatures of population structure were replaced. We applied cutting-edge molecular and biogeographic tools to disentangle genetic signatures of historical management from those reflecting spatially heterogeneous dispersal by evaluating 35,099 single nucleotide polymorphisms (SNPs) derived via reduced-representation genomic sequencing from 1,143 deer sampled state-wide in Arkansas. We then employed Simpson’s diversity index to summarize ancestry assignments and visualize spatial genetic transitions. Using sub-sampled transects across these transitions, we tested clinal patterns across loci against theoretical expectations of their response under scenarios of recolonization and restricted dispersal. Two salient results emerged: (A) Genetic signatures from historic translocations are demonstrably apparent; and (B) Geographic filters (major rivers; urban centers; highways) now act as inflection points for the distribution of this contemporary ancestry. These results yielded a state-wide assessment of contemporary population structure in deer as driven by historic translocations as well as ongoing processes. In addition, the analytical framework employed herein to effectively decipher extant/historic drivers of deer distribution in Arkansas are also applicable for other biodiversity elements with similarly complex demographic histories.

Model-based approaches that attempt to delimit species are hampered by computational limitations as well as the unfortunate tendency by users to disregard algorithmic assumptions. Alternatives are clearly needed, and machine-learning (M-L) is attractive in this regard as it functions without the need to explicitly define a species concept. Unfortunately, its performance will vary according to which (of several) bioinformatic parameters are invoked. Herein, we gauge the effectiveness of M-L-based species-delimitation algorithms by parsing 64 variably-filtered versions of a ddRAD-derived SNP data set collected from North American box turtles (Terrapene spp.). Our filtering strategies included: (i) minor allele frequencies (MAF) of 5%, 3%, 1%, and 0% (= none), and (ii) maximum missing data per-individual/per-population at 25%, 50%, 75%, and 100% (= no filtering). We found that species-delimitation via unsupervised M-L impacted the signal-to-noise ratio in our data, as well as the discordance among resolved clades. The latter may also reflect biogeographic history, gene flow, incomplete lineage sorting, or combinations thereof (as corroborated from previously observed patterns of differential introgression). Our results substantiate M-L as a viable species-delimitation method, but also demonstrate how commonly observed patterns of phylogenetic discordance can seriously impact M-L-classification.

As anthropogenic changes continue to ecologically stress wildlife, obtaining measures of gene flow and genetic diversity are crucial for evaluating population trends and considering management and conservation strategies for small, imperiled populations. In our study, we conducted a molecular assessment to expand on previous work to elucidate patterns of diversity and connectivity in the remaining disjunct Eastern Massasauga Rattlesnake (Sistrurus catenatus) hibernacula in Illinois. We assayed genetic data for 327 samples collected during 1999–2015 from the Carlyle Lake study area across 21 microsatellite loci. We found hibernacula formed distinct genetic clusters corresponding to the three main study areas (Dam Recreation Areas, Eldon Hazlet State Park, and South Shore State Park). Genetic structuring and low estimates of dispersal indicated that connectivity among these study areas is limited and each is demographically independent. Hibernacula exhibited moderate levels of heterozygosity (0.60–0.73), but estimates of effective population size (5.2–41.0) were low and track census sizes generated via long-term mark-recapture data. Hibernacula at Carlyle Lake, which represent the only Eastern Massasauga remaining in Illinois, are vulnerable to future loss of genetic diversity through lack of gene flow as well as demographic and environmental stochastic processes. Our work highlights the need to include population-level genetic data in recovery planning and suggests that recovery efforts should focus on managing the three major study areas as separate conservation units in order to preserve and maintain long-term adaptive potential of these populations. Specific management goals should include improving connectivity among hibernacula, maintaining existing wet grassland habitat, and minimizing anthropogenic sources of mortality caused by habitat management (e.g., mowing, prescribed fire) and recreational activities. Our molecular study provides additional details about demographic parameters and connectivity at Carlyle Lake that can be used to guide recovery of Eastern Massasauga in Illinois and throughout its range.

Species are indisputable units for biodiversity conservation, yet their delimitation is fraught with both conceptual and methodological difficulties. A classic example is the taxonomic controversy surrounding the Gila robusta complex in the lower Colorado River of southwestern North America. Nominal species designations were originally defined according to weakly diagnostic morphological differences, but these conflicted with subsequent genetic analyses. Given this ambiguity, the complex was re-defined as a single polytypic unit, with the proposed “threatened” status under the U.S. Endangered Species Act of two elements being withdrawn. Here we re-evaluated the status of the complex by utilizing dense spatial and genomic sampling (n = 387 and >22 k loci), coupled with SNP-based coalescent and polymorphism-aware phylogenetic models. In doing so, we found that all three species were indeed supported as evolutionarily independent lineages, despite widespread phylogenetic discordance. To juxtapose this discrepancy with previous studies, we first categorized those evolutionary mechanisms driving discordance, then tested (and subsequently rejected) prior hypotheses which argued phylogenetic discord in the complex was driven by the hybrid origin of Gila nigra. The inconsistent patterns of diversity we found within G. robusta were instead associated with rapid Plio-Pleistocene drainage evolution, with subsequent divergence within the “anomaly zone” of tree space producing ambiguities that served to confound prior studies. Our results not only support the resurrection of the three species as distinct entities but also offer an empirical example of how phylogenetic discordance can be categorized within other recalcitrant taxa, particularly when variation is primarily partitioned at the species level.

The persistence of an invasive species is influenced by its reproductive ecology, and a successful control program must operate on this premise. However, the reproductive ecology of invasive species may be enigmatic due to factors that also limit their management, such as cryptic coloration and behavior. We explored the mating and reproductive ecology of the invasive Brown Treesnake (BTS: Boiga irregularis) by reconstructing a multigenerational genomic pedigree based on 654 single nucleotide polymorphisms for a geographically closed population established in 2004 on Guam (N = 426). The pedigree allowed annual estimates of individual mating and reproductive success to be inferred for snakes in the study population over a 14‐year period. We then employed generalized linear mixed models to gauge how well phenotypic and genomic data could predict sex‐specific annual mating and reproductive success. Average snout–vent length (SVL), average body condition index (BCI), and trappability were significantly related to annual mating success for males, with average SVL also related to annual mating success for females. Male and female annual reproductive success was positively affected by SVL, BCI, and trappability. Surprisingly, the degree to which individuals were inbred had no effect on annual mating or reproductive success. When juxtaposed with current control methods, these results indicate that baited traps, a common interdiction tool, may target fecund BTS in some regards but not others. Our study emphasizes the importance of reproductive ecology as a focus for improving BTS control and promotes genomic pedigree reconstruction for such an endeavor in this invasive species and others., Our study details the use of genomic tools (i.e., double‐digest restriction site‐associated DNA sequencing) to develop novel genomic resources (>6,000 SNPs) for a high‐profile and invasive vertebrate pest (i.e., Brown Treesnake). We used a subset of these SNPs to reconstruct a multigenerational genomic pedigree for a wild population of Brown Treesnakes from which we inferred patterns and predictors of annual mating and reproductive success, and we juxtaposed phenotypes of individuals with high annual mating and reproductive success against those of commonly trapped individuals to assess existing management efficacy. Our results promote the use of genomic pedigree reconstruction to elucidate the reproductive ecology of invasive species with cryptic coloration and behaviors, so as to inform and improve management.

Computation of Patterson’s D-statistic and its five-taxon derivatives are important phylogenetic methods for the quantification of reticulated evolution, yet are limited in application by the lack of a single, comprehensive program to efficiently perform all necessary calculations from common phylogenetic and population genetic program file formats. To increase accessibility for a broad range of researchers, we present a user-friendly program (COMP-D) that provides flexibility for incorporating heterozygous sites, implements multiple statistical methods, and aggregates results from multiple tests. Program augmentations also facilitate the detection of population-level introgression. COMP-D provides a threefold increase in speed relative to comparable software. It is implemented in C++ and released under the GNU General Public License v3.0. Source code is available for Linux/Mac OS X from: https://github.com/stevemussmann/Comp-D_MPI.

(225)Biodiversity in southwestern North America has a complex biogeographic history involving tectonism interspersed with climatic fluctuations. This yields a contemporary pattern replete with historic idiosyncrasies often difficult to interpret when viewed from through the lens of modern ecology. TheAspidoscelis tigris(Tiger Whiptail) complex (Squamata: Teiidae) is one such group in which potential taxonomic boundaries have been confounded by a series of complex biogeographic processes that have defined the evolution of the clade. To clarify this situation, we first generated multiple taxonomic hypotheses, which were subsequently tested using mitochondrial DNA sequences (ATPase 8 and 6) evaluated across 239 individuals representing five continental members of this complex. To do so, we evaluated the manner by which our models parsed phylogenetic and biogeographic patterns. We found considerable variation among species ‘hypotheses’, which we suggest in part reflects inflated levels of inter-population genetic divergence caused by historical demographic expansion and contraction cycles. Inter-specific boundaries withA. marmoratusjuxtaposed topographically with the Cochise Filter Barrier that separates Sonoran and Chihuahuan deserts (interpreted herein as case of ‘soft’ allopatry). Patterns of genetic divergence were consistent across the Cochise Filter Barrier, regardless of sample proximity. Surprisingly, this also held true for intraspecific comparisons that spanned the Colorado River. These in turn suggest geomorphic processes as a driver of speciation in thetigriscomplex, with intraspecific units governed locally by demographic processes.HIGHLIGHTSPhylogeographies of vertebrates within the southwestern deserts of North America have been shaped by climatic fluctuations imbedded within broad geomorphic processes.The resulting synergism drives evolutionary processes, such as an expansion of within-species genetic divergence over time. Taxonomic inflation often results (i.e., an increase in recognized taxa due to arbitrary delineations), such as when morphological divergences fail to juxtapose with biogeographic hypotheses.However, isolated groups can be discriminated within-species by mapping genetic variability onto geographic distances. This approach can often diagnose ‘hard’ barriers to dispersal, or alternatively, strong selection acting against hybridization. On the other hand, elevated genetic divergences among groups less-isolated would underscore isolation-by-distance (i.e., an increase in genetic differentiation concomitant with geographic distance).The biogeographic patterns we identified in Tiger Whiptail are largely synonymous with those found in other regional species, particularly given the geomorphic separation of Mohave and Sonoran deserts by the Colorado River, and Sonoran/ Chihuahuan deserts by the Cochise Filter Barrier.Our results for the Tiger Whiptail complex broaden and extend the context within which polytypic species are conserved and managed, particularly those that reflect an incongruence among molecular and morphological standards.

The recurrence of similar evolutionary patterns within different habitats often reflects parallel selective pressures acting upon either standing or independently occurring genetic variation to produce a convergence of phenotypes. This interpretation (i.e. parallel divergences within adjacent streams) has been hypothesized for drainage-specific morphological ‘ecotypes’ observed in polyploid snowtrout (Cyprinidae: Schizothorax ). However, parallel patterns of differential introgression during secondary contact are a viable alternative hypothesis. Here, we used ddRADseq ( N = 35 319 de novo and N = 10 884 transcriptome-aligned SNPs), as derived from Nepali/Bhutanese samples ( N = 48 each), to test these competing hypotheses. We first employed genome-wide allelic depths to derive appropriate ploidy models, then a Bayesian approach to yield genotypes statistically consistent under the inferred expectations. Elevational ‘ecotypes’ were consistent in geometric morphometric space, but with phylogenetic relationships at the drainage level, sustaining a hypothesis of independent emergence. However, partitioned analyses of phylogeny and admixture identified subsets of loci under selection that retained genealogical concordance with morphology, suggesting instead that apparent patterns of morphological/phylogenetic discordance are driven by widespread genomic homogenization. Here, admixture occurring in secondary contact effectively ‘masks’ previous isolation. Our results underscore two salient factors: (i) morphological adaptations are retained despite hybridization and (ii) the degree of admixture varies across tributaries, presumably concomitant with underlying environmental or anthropogenic factors.

Statistical methods that quantify individual movements have limited applicability within dendritic ecological networks (DENs), to include riverscapes. We promote an approach herein by deriving an analytical framework (=DistNet and ResistNet) that more appropriately quantifies differentiation by meshing individual DEN segments with fitted distances and annotated environmental features. We first explored the spatial arrangement of diversity in a river network by fitting pairwise distances against graph edges. We then derived effective resistance models (e.g., as a composite of an array of possible environmental covariates) by using a genetic algorithm for heuristic model selection, invoking circuit theory (CircuitScape) to form a graph-based complement to ResistanceGA (a landscape ecology package). Although numerous pairwise distance metrics can be employed, we utilized genetic distances encompassing 13,218 ddRAD loci from N=762 Speckled Dace (Cyprinidae: Rhinichthys osculus) sampled at 78 Colorado River localities (µ=9.77/site). When distances for each locus were fitted into the network, we found intraspecific divergences due to stream reaches were greater than expected when distances alone. In fitting model-averaged ‘effective resistance’ models to the same network, we showed the manner by which a host of anthropogenic and environmental variables contribute to patterns of connectivity underlying riverscape genetic differentiation. Our framework represents a contemporary approach to deriving metapopulation/ metacommunity structure within DENs, in that it allows: (a) The extension of projections into unsampled temporal/spatial components; (b) Comparisons to be made among species and/or drainages, both of which benefit multi-species management; and (c) the quantification of locus-specific patterns, from which adaptive hypotheses could then be derived. “The intimate relation of hydrological conditions to fish life is proverbial; to be hopelessly or dangerously maladjusted to one’s environment is to be a ‘fish out of water’” (Hubbs 1941).

Demographics of co-occurring species can often be diagnosed through population genomic analyses of single nucleotide polymorphisms (SNPs). These data can define population structure, gene flow, and candidate regions in the genome that potentially reflect local adaptations. They can also gauge if populations are demographically ‘open’ or ‘closed’ (i.e., with global or local recruitment). We derived SNPs from double-digest restriction-site associated DNA (ddRAD) to test the demographics of commercially important bonefish Albula vulpes (N=117) and queen conch Lobatus gigas (N=60) from two northeast Caribbean Basin islands (Grand Bahama to the north and Eleuthera to the south). Specifically, we tested the hypothesis that the strong west-to-east current in the Great Bahama Canyon is a vicariant barrier separating the two islands. We conducted Bayesian assignment tests on putatively neutral (A. vulpes=36,206 SNPs; L. gigas=64,863) and highly-differentiated outlier datasets (A. vulpes=123 and 79 SNPs; L. gigas=88 and 51, respectively). For bonefish, results diagnosed asymmetrical gene flow and north-south differentiation, as potentially driven by adult mobility and easterly currents. However, both analyses indicated genetic structure in conch, substantiating the vicariant hypothesis. These results provide templates for future research endeavors with these impacted species. Outlier loci, for example, can potentially place populations of each within a demographic continuum, rather than within a dichotomous ‘open/closed’ framework, as well as diagnose ‘source’ and ‘sink’ populations, as herein. These methodologies can then be applied to co-distributed species with similar but less well understood ecologies so as to evaluate basin-wide trends in connectivity and local adaptation.

Applying quantifiable metrics to validate the success of restoration efforts is crucial for ongoing management programs in anthropogenically fragmented habitats. Estimates of dispersal can provide such baseline data because they measure not only the extent to which restored patches are colonized and interconnected, but also their metapopulation source/sink dynamics. In this context, we estimated dispersal and population connectivity among prairie (Microtus ochrogaster; N=231) and meadow vole (M. pennsylvanicus; N=83), sampled from eight restored plots at five tallgrass prairie sites embedded within the agricultural matrix of midwestern North America. Our expectation was that extensive distances separating these restored habitats (i.e., 48–246 km) would spatially isolate vole metapopulations, resulting in significant genetic differentiation. We first used molecular taxonomy to validate the field-identifications of all sampled individuals, then used pairwiseFSTderived from 15 microsatellite DNA loci to estimate genetic connectivity among the species-delimited study populations. Metapopulation stability was gauged by assessing migration rates and deriving effective population sizes (Ne). We also calculated relatedness values (r) as a potential surrogate for contact in prairie vole, a primary vector for Lyme disease. Molecular species-assignments contravened field-identifications in 25% of samples (11 prairie/67 meadow) and identified two instances of species-hybridization (0.6%). Local effects (i.e., population crash/drought) were manifested at two sites, as documented by significant temporal declines inNeandr. Overall, high migration rates and non-significant (10/15) pairwiseFSTvalues underscored elevated metapopulation connectivity. A single site that recorded five significantFSTvalues also displayed significantr-values indicating the inadvertent sampling of closely related individuals. This highlights the close social groupings among cooperatively-breeding prairie vole that can exacerbate Lyme disease transmission. Thus, while elevated population connectivity aligns with prairie restoration goals, it also reinforces a need in adaptive management to evaluate environmental matrices for their permeability to vector-borne diseases.

The Himalayan uplift, a tectonic event of global importance, seemingly disseminated aquatic biodiversity broadly across Asia. But surprisingly, this hypothesis has yet to be tested. We do so herein by sequencing 1,140 base-pair of mtDNA cytochrome-b for 72 tetraploid Nepalese/Bhutanese Snowtrout (Schizothorax spp.), combining those data with 67 GENBANK® sequences (59 ingroup/8 outgroup), then reconstructing phylogenetic relationships using maximum likelihood/ Bayesian analyses. Results indicate Snowtrout originated in Central Asia, dispersed across the Qinghai-Tibetan Plateau (QTP), then into Bhutan via south-flowing tributaries of the east-flowing Yarlung-Tsangpo River (YLTR). The headwaters of five large Asian rivers provided dispersal corridors into southeast Asia. South of the Himalaya, the YLTR transitions into a westward-flowing Brahmaputra River that facilitated successive colonization of Himalayan drainages: First Bhutan, then Nepal, followed by far-western drainages subsequently captured by the Indus River. We found greater species-divergences across rather than within-basins, implicating vicariant evolution as a driver. The Himalaya is a component of the “third-pole” [Earth’s largest (but rapidly shrinking) glacial reservoir outside the Arctic/Antarctic]. Its unique aquatic biodiversity must not only be recognized (as herein) but also conserved through broad, trans-national collaborations. Our results effectively contrast phylogeography with taxonomy as a necessary first step in this process.The Himalaya is the most extensive and recently evolved mountain system on Earth (length=2400km; width=240km; elevation=75-8800m), with a global significance underscored by its large-scale lithospheric, cryospheric, and atmospheric interactions [1]. These have not only driven global climate, but also defined the cultural and biological endemism of the region [2]. Massive, tectonically derived mountain chains such as the Alps and the Himalaya are hypothesized as being fundamental to the formation of global biodiversity gradients via vicariance and local adaptation, with a significantly stronger signal in terrestrial rather than aquatic systems [3]. Here we test how orogeny (the deformation and folding of Earth’s crust by lateral compression) has contributed to the diversification of freshwater fishes broadly across Asia. We do so by evaluating the phylogeography of an endemic high-elevation fish, the Snowtrout (Schizothorax: Cyprinidae).

Patterns of multi-locus differentiation (i.e., genomic clines) often extend broadly across hybrid zones and their quantification can help diagnose how species boundaries are shaped by adaptive processes, both intrinsic and extrinsic. In this sense, the transitioning of loci across admixed individuals can be contrasted as a function of the genome-wide trend, in turn allowing an expansion of clinal theory across a much wider array of biodiversity. However, computational tools that serve to interpret and consequently visualize ‘genomic clines’ are limited. Here, we introduce the ClinePlotR R-package for visualizing genomic clines and detecting outlier loci using output generated by two popular software packages, bgc and Introgress. ClinePlotR bundles both input generation (i.e, filtering datasets and creating specialized file formats) and output processing (e.g., MCMC thinning and burn-in) with functions that directly facilitate interpretation and hypothesis testing. Tools are also provided for post-hoc analyses that interface with external packages such as ENMeval and RIdeogram. Our package increases the reproducibility and accessibility of genomic cline methods, thus allowing an expanded user base and promoting these methods as mechanisms to address diverse evolutionary questions in both model and non-model organisms.

Chronic-wasting disease (CWD) is a prion-derived fatal neurodegenerative disease that has affected wild cervid populations on a global scale. Susceptibility has been linked unambiguously to several amino acid variants within the prion protein gene (PRNP). Quantifying their distribution across landscapes can provide critical information for agencies attempting to adaptively manage CWD. Here we attempt to further define management implications of PRNP polymorphism by quantifying the contemporary geographic distribution (i.e., phylogeography) of PRNP variants in hunter-harvested white-tailed deer (WTD; Odocoileus virginianus, N=1433) distributed across Arkansas (USA), including a focal spot for CWD since detection of the disease in February 2016. Of these, PRNP variants associated with the well-characterized 96S non-synonymous substitution showed a significant increase in relative frequency among older CWD-positive cohorts. We interpreted this pattern as reflective of a longer life expectancy for 96S genotypes in a CWD-endemic region, suggesting either decreased probabilities of infection or reduced disease progression. Other variants showing statistical signatures of potential increased susceptibility, however, seemingly do so as an artefact of population structure. We also showed marked heterogeneity across the landscape in the prevalence of ‘reduced susceptibility’ genotypes. This may indicate, in turn, that differences in disease susceptibility among WTD in Arkansas are an innate, population-level characteristic that is detectable through phylogeographic analysis.

The tips in the tree of life serve as foci for conservation and management, yet clear delimitations are masked by inherent variance at the species-population interface. Analyses using thousands of nuclear loci can potentially sort inconsistencies, yet standard categories applied to this parsing are themselves potentially conflicting and/or subjective [e.g., DPS (distinct population segments); DUs (Diagnosable Units-Canada); MUs (management units); SSP (subspecies); Evolutionarily Significant Units (ESUs)]. One potential solution for consistent categorization is to create a comparative framework by accumulating statistical results from independent studies and evaluating congruence among data sets. Our study illustrates this approach in speckled dace (Cyprinidae:Rhinichthys osculus) endemic to two basins (Owens and Amargosa) in the Death Valley ecosystem (DVE). These fish persist in the Mojave Desert as isolated Pleistocene-relicts and are of conservation concern, but lack formal taxonomic descriptions/designations. Double-digest RAD (ddRAD) methods identified 14,355 SNP loci across 10 populations (N=140). Species delimitation analyses [multispecies coalescent (MSC) and unsupervised machine learning (UML)] delineated four putative ESUs.FSToutlier loci (N=106) were juxtaposed to uncover the potential for localized adaptations. We detected one hybrid population that resulted from upstream reconnection of habitat following contemporary pluvial periods, whereas remaining populations represent relics of ancient tectonism within geographically-isolated springs and groundwater-fed streams. Our study offers three salient conclusions: A blueprint for a multi-faceted delimitation of conservation units; a proposed mechanism by which criteria for intraspecific biodiversity can be potentially standardized; and a strong argument for the proactive management of critically-endangered DVE fishes.

Model-based approaches that attempt to delimit species are hampered by computational limitations as well as the unfortunate tendency by users to disregard algorithmic assumptions. Alternatives are clearly needed, and machine-learning (M-L) is attractive in this regard as it functions without the need to explicitly define a species concept. Unfortunately, its performance will vary according to which (of several) bioinformatic parameters are invoked. Herein, we gauge the effectiveness of M-L-based species-delimitation algorithms by parsing 64 variably-filtered versions of a ddRAD-derived SNP dataset collected from North American box turtles (Terrapenespp.). Our filtering strategies included: (A) minor allele frequencies (MAF) of 5%, 3%, 1%, and 0% (=none), and (B) maximum missing data per-individual/per-population at 25%, 50%, 75%, and 100% (=no filtering). We found that species-delimitation via unsupervised M-L impacted the signal-to-noise ratio in our data, as well as the discordance among resolved clades. The latter may also reflect biogeographic history, gene flow, incomplete lineage sorting, or combinations thereof (as corroborated from previously observed patterns of differential introgression). Our results substantiate M-L as a viable species-delimitation method, but also demonstrate how commonly observed patterns of phylogenetic discordance can seriously impact M-L-classification.

Hybridization is well recognized as a driver of speciation, yet it often remains difficult to parse phylogenomically in that post-speciation gene flow frequently supersedes an ancestral signal. Here we examined how interactions between recombination and gene flow shaped the phylogenomic landscape of red wolf to create non-random retention of introgressed ancestry. Our re-analyses of genomic data recapitulate fossil evidence by demonstrating red wolf was indeed extant and isolated prior to more recent admixture with other North American canids. Its more ancient divergence, now sequestered within low-recombinant regions on the X-chromosome (i.e., chromosomal ‘refugia’), is effectively masked by multiple, successive waves of secondary introgression that now dominate its autosomal ancestry. These interpretations are congruent with more theoretical explanations that describe the manner by which introgression can be localized within the genome through recombination and selection. They also tacitly support the large-X effect, i.e., the manner by which loci that contribute to reproductive isolation can be enriched on the X-chromosome. By contrast, similar, high recombinant regions were also found as enriched within very shallow gene trees, thus reflecting post-speciation gene flow and a compression of divergence estimates to 1/20thof that found in recombination ‘cold spots’. Our results effectively reconcile conflicting hypotheses regarding the impact of hybridization on evolution of North American canids and support an emerging framework within which the analysis of a phylogenomic landscape structured by recombination can be used to successfully address the macroevolutionary implications of hybridization.

The Eastern Massasauga (Sistrurus catenatus) is a small, grassland-dependent rattlesnake species declining throughout its native range, and is thus a species of high conservation priority. In Illinois, only a single population remains of a once widespread distribution. We documented genetic diversity in this population over a ten-year period and assessed levels of heterozygosity, allelic diversity, inbreeding (FIS), and effective population size (Ne). Neither heterozygosity nor levels of inbreeding differed significantly among periods. We identified 21 alleles that occurred in a single time period, some of which may have been lost from the population given our estimated detection probability of 93%. Effective population size (Ne) was numerically small and showed a decreasing trend through time. Despite small population size and a lack of connectivity, there was no significant decline in genetic diversity over the ten-year study. Aspects of life history, coupled with a preference for a historically patchy ...

Multi-species approaches provide valuable insight for conservation planning, yet most studies focus on only one species while generalizing across taxa. Here, we employed 5–14 microsatellite DNA loci to evaluate population genetic patterns and future vulnerability for a freshwater turtle assemblage distributed across north-eastern Illinois. Focal species (Emydoidea blandingii, Clemmys guttata, Chrysemys picta and Chelydra serpentina) differ in conservation status as well as in ecological and life-history traits, which modulate gene flow across heterogeneous landscapes. We hypothesized (1) common and more ubiquitous species (C. picta and C. serpentina) would exhibit higher levels of genetic connectivity compared to species more restricted in distributions and with an elevated conservation status (E. blandingii and C. guttata) and (2) endangered species exhibit a greater loss of future genetic diversity. We found that genetic patterns varied considerably among co-distributed species. Endangered species had lower levels of genetic diversity and gene flow, more pronounced genetic structure and a higher risk of genetic drift compared to common species, thus supporting our hypotheses. The observed patterns are potentially attributable to life-history and ecological traits and will affect the long-term viability of the four species within a modified north-eastern Illinois landscape. Our study is an important first step for understanding how landscape features and species-specific traits interact to affect gene flow and population genetic structure within altered landscapes. It also underscores how multi-species approaches can be informative for conservation actions.

Reduced-representation genomic methods are an invaluable data acquisition tool for conservation geneticists, yet a priori estimates of locus recovery are difficult for non-model organisms. We present a simple in silico approach (FRAGMATIC) that predicts locus recovery in ddRAD sequencing which utilizes genomic data for related organisms. Its applicability was tested by quantifying prediction accuracy versus genetic distances across five non-model organisms and reference genomes for related organisms of varying phylogenetic distance. We additionally examined sensitivity of the method using one organism (Danio rerio) with an available genome. FRAGMATIC supports population genomic projects in non-model species by providing a priori estimates of targeted ddRAD loci that, in turn, will curb wasted sequencing effort and optimize cost-efficiency. Validation shows that while predictive error is minimized when applied to a closely related reference genome, in silico estimates may also be robust to deeper (e.g. within-family) relationships, although weak correlation suggests that specific characteristics of genome architecture may be more predictive than genetic distance. This indicates that a more extensive exploration of genomes, including a broader taxonomic scope (e.g. beyond vertebrates), may be informative. All code is freely available at: https://github.com/tkchafin/fragmatic .

Despite its imperative, biodiversity conservation is chronically underfunded, a deficiency that often forces management agencies to prioritize. Single-species recovery thus becomes a focus (often with socio-political implications), whereas a more economical approach would be the transition to multi-targeted management (= MTM). This challenge is best represented in Midwestern North America where biodiversity has been impacted by 300+ years of chronic anthropogenic disturbance such that native tall-grass prairie is now supplanted by an agroecosystem. Here, we develop an MTM with a population genetic metric to collaboratively manage three Illinois upland gamebirds: common pheasant (Phasianus colchicus; pheasant), northern bobwhite quail (Colinus virginianus; quail), and threatened-endangered (T&E) greater prairie chicken (Tympanuchus cupido pinnatus; prairie chicken). We first genotyped our study pheasant at 19 microsatellite DNA loci and identified three captive breeding stocks (N = 143; IL Department of Natural Resources) as being significantly bottlenecked, with relatedness >1st-cousin (μR = 0.158). ‘Wild’ (non-stocked) pheasant [N = 543; 14 Pheasant-Habitat-Areas (PHAs)] were also bottlenecked, significantly interrelated (μR = 0.150) and differentiated (μFST = 0.047), yet distinct from propagation stock. PHAs that encompassed significantly with larger areas also reflected greater effective population sizes (μNE = 43; P

The delimitation of species boundaries, particularly those obscured by reticulation, is a critical step in contemporary biodiversity assessment. It is especially relevant for conservation and management of indigenous fishes in western North America, represented herein by two species with dissimilar life histories codistributed in the highly modified Colorado River (i.e., flannelmouth sucker, Catostomus latipinnis; bluehead sucker, C. (Pantosteus) discobolus). To quantify phylogenomic patterns and examine proposed taxonomic revisions, we first employed double‐digest restriction site‐associated DNA sequencing (ddRAD), yielding 39,755 unlinked SNPs across 139 samples. These were subsequently evaluated with multiple analytical approaches and by contrasting life history data. Three phylogenetic methods and a Bayesian assignment test highlighted similar phylogenomic patterns in each, but with considerable difference in presumed times of divergence. Three lineages were detected in bluehead sucker, supporting elevation of C. (P.) virescens to species status and recognizing C. (P.) discobolus yarrowi (Zuni bluehead sucker) as a discrete entity. Admixture in the latter necessitated a reevaluation of its contemporary and historic distributions, underscoring how biodiversity identification can be confounded by complex evolutionary histories. In addition, we defined three separate flannelmouth sucker lineages as ESUs (evolutionarily significant units), given limited phenotypic and genetic differentiation, contemporary isolation, and lack of concordance (per the genealogical concordance component of the phylogenetic species concept). Introgression was diagnosed in both species, with the Little Colorado and Virgin rivers in particular. Our diagnostic methods, and the agreement of our SNPs with previous morphological, enzymatic, and mitochondrial work, allowed us to partition complex evolutionary histories into requisite components, such as isolation versus secondary contact., Phylogenomic patterns and proposed taxonomic revisions were examined in two species with dissimilar life histories codistributed in the highly modified Colorado River, the flannelmouth sucker (Catostomus latipinnis) and the bluehead sucker (C. Pantosteus discobolus). We utilized ddRAD along with several phylogenetic, population genetic, and species delimitation approaches to examine the phylogeny, detect hybridization, and introgression, and test proposed taxonomic revision. Our diagnostic methods, and the alignment of our SNPs with previous morphological, enzymatic, and mitochondrial work, allowed us to partition complex evolutionary histories into requisite components, such as isolation versus secondary contact.

Hybridization occurs differentially across the genome in a balancing act between selection and migration. With the unprecedented resolution of contemporary sequencing technologies, selection and migration can now be effectively quantified such that researchers can identify genetic elements involved in introgression. Furthermore, genomic patterns can now be associated with ecologically relevant phenotypes, given availability of annotated reference genomes. We do so in North American box turtles (Terrapene) by deciphering how selection affects hybrid zones at the interface of species boundaries and identifying genetic regions potentially under selection that may relate to thermal adaptations. Such genes may impact physiological pathways involved in temperature-dependent sex determination, immune system functioning, and hypoxia tolerance. We contrasted these patterns across inter- and intra-specific hybrid zones that differ temporally and biogeographically. We demonstrate hybridization is broadly apparent inTerrapene, but with observed genomic cline patterns corresponding to species boundaries at loci potentially associated with thermal adaptation. These loci display signatures of directional introgression within intra-specific boundaries, despite a genome-wide selective trend against intergrades. In contrast, outlier loci for inter-specific comparisons exhibited evidence of being under selection against hybrids. Importantly, adaptations coinciding with species boundaries inTerrapeneoverlap with climatic boundaries and highlight the vulnerability of these terrestrial ectotherms to anthropogenic pressures.

Species is an indisputable unit for biodiversity conservation, yet their delimitation is fraught with both conceptual and methodological difficulties. A classic example is the taxonomic controversy surrounding the Gila robusta complex in the lower Colorado River of southwestern North America. Nominal species designations were originally defined according to weakly diagnostic morphological differences that conflicted with traditional genetic analyses. Consequently, the complex was re-defined as a single polytypic unit, with the proposed ‘threatened’ status of two being withdrawn at the federal level. Here, we utilized dense spatial and genomic sampling (N=387 and >22k loci) to re-evaluate the status of the complex, based on SNP-based coalescent and polymorphism-aware phylogenetic models. In doing so, all three species were supported as evolutionarily independent lineages, despite widespread phylogenetic discordance. To understand this discrepancy with past studies, we categorized evolutionary mechanisms driving discordance. We tested (and subsequently rejected) prior hypotheses suggesting that phylogenetic discord in the complex was hybridization-driven. Instead, we found the G. robusta complex to have diverged within the ‘anomaly zone’ of tree space and, as such, have accumulated inconsistent patterns of diversity which have confounded prior studies. After extending these analyses with phylogeographic modeling, we propose that this is reflective of a rapid radiation promoted by Plio-Pleistocene tectonism. Our results not only support resurrection of the three species as distinct entities, but also offer an empirical example of how phylogenetic discordance can be categorized in other recalcitrant taxa where variation is primarily partitioned at the species-level.

Many species have evolved or currently coexist in sympatry due to differential adaptation in a heterogeneous environment. However, anthropogenic habitat modifications can either disrupt reproductive barriers or obscure environmental conditions which underlie fitness gradients. In this study, we evaluated the potential for an anthropogenically-mediated shift in reproductive boundaries that separate two historically sympatric fish species (Gila cyphaandG. robusta) endemic to the Colorado River Basin using ddRAD sequencing of 368 individuals. We first examined the integrity of reproductive isolation while in sympatry and allopatry, then characterized hybrid ancestries using genealogical assignment tests. We tested for localized erosion of reproductive isolation by comparing site-wise genomic clines against global patterns and identified a breakdown in the drainage-wide pattern of selection against interspecific heterozygotes. This, in turn, allowed for the formation of a hybrid swarm in one tributary, and asymmetric introgression where species co-occur. We also detected a weak but significant relationship between genetic purity and degree of consumptive water removal, suggesting a role for anthropogenic habitat modifications in undermining species boundaries. In addition, results from basin-wide genomic clines suggested that hybrids and parental forms are adaptively non-equivalent. If so, then a failure to manage for hybridization will exacerbate the long-term extinction risk in parental populations. These results reinforce the role of anthropogenic habitat modification in promoting interspecific introgression in sympatric species by relaxing divergent selection. This, in turn, underscores a broader role for hybridization in decreasing global biodiversity within rapidly deteriorating environments.

Habitat modification and introduced species are impacting native fishes in western North America, a situation now exacerbated by drought and anthropogenic water acquisition. One species of concern, the Bluehead Sucker Catostomus discobolus in the Bonneville basin of Utah, Idaho, and Wyoming, has experienced sharp population declines potentially augmented by hybridization with native Utah Sucker C. ardens. To evaluate this situation, we sequenced three diagnostic nuclear DNA loci and two mitochondrial genes across 81 Bluehead Suckers, 74 of which were collected in a basinwide genetic assessment and 7 from a targeted morphological evaluation. Combined, we detected 14 hybrids from the single site in the Bear River and the three sites in the Weber River that hosts the three largest remaining populations in the basin. All individuals sampled as putative hybrids were confirmed as F1 hybrids, ascertaining efficacy of field-based morphological identifications. Hybridization may be especially problematic f...

Population genetic data are an important standard with which to gauge demography and gene flow within and among biodiversity units, and are often gathered on species of conservation concern. Yet an exclusive focus on ‘conservation immediacy' can also have negative consequences. For example, it can shift monitoring efforts away from more abundant and/or widely distributed clades and, by so doing, promote a more myopic approach to conservation and management. A current example concerns North American pitvipers (Viperidae: Crotalinae), within which listed species of Crotalus and Sistrurus receive considerable population genetic attention whereas broadly distributed Agkistrodon is largely overlooked. To address this disparity, we used 22 polymorphic tetra-nucleotide microsatellite loci to explore genetic structure, diversity, and relatedness in a Connecticut population of Copperhead (A. contortrix). Three admixed genetic clusters were identified across five winter dens, with overall and sex-specific relatedne...

Motivation It is a non-trivial task to identify and design capture probes (‘baits’) for the diverse array of targeted-enrichment methods now available (e.g. ultra-conserved elements, anchored hybrid enrichment, RAD-capture). This often involves parsing large genomic alignments, followed by multiple steps of curating candidate genomic regions to optimize targeted information content (e.g. genetic variation) and to minimize potential probe dimerization and non-target enrichment. Results In this context, we developed MrBait, a user-friendly, generalized software pipeline for identification, design and optimization of targeted-enrichment probes across a range of target-capture paradigms. MrBait is an open-source codebase that leverages native parallelization capabilities in Python and mitigates memory usage via a relational-database back-end. Numerous filtering methods allow comprehensive optimization of designed probes, including built-in functionality that employs BLAST, similarity-based clustering and a graph-based algorithm that ‘rescues’ failed probes. Availability and implementation Complete code for MrBait is available on GitHub (https://github.com/tkchafin/mrbait), and is also available with all dependencies via one-line installation using the conda package manager. Online documentation describing installation and runtime instructions can be found at: https://mrbait.readthedocs.io. Supplementary information Supplementary data are available at Bioinformatics online.