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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.

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.

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.

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.

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.

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 .

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.

The classification of the American box turtles (Terrapene spp.) has remained enigmatic to systematists. Previous comprehensive phylogenetic studies focused primarily on morphology. The goal of this study was to re-assess the classification of Terrapene spp. by obtaining DNA sequence data from a broad geographic range and from all four recognized species and 11 subspecies within the genus. Tissue samples were obtained for all taxa except for Terrapene nelsoni klauberi. DNA was extracted, and the mitochondrial DNA (mtDNA) cytochrome b (Cytb) and nuclear DNA (nucDNA) glyceraldehyde-3-phosphate-dehydrogenase (GAPD) genes were amplified via polymerase chain reaction and sequenced. In addition, the mtDNA gene commonly used for DNA barcoding (cytochrome oxidase c subunit I; COI) was amplified and sequenced to calculate pairwise percent DNA sequence divergence comparisons for each Terrapene taxon. The sequence data were analyzed using maximum likelihood and Bayesian phylogenetic inference, a molecular clock, AMOVAs, SAMOVAs, haplotype networks, and pairwise percent sequence divergence comparisons. Terrapene carolina mexicana and T. c. yucatana formed a monophyletic clade with T. c. triunguis, and this clade was paraphyletic to the rest of T. carolina. Terrapene ornata ornata and T. o. luteola lacked distinction phylogenetically, and Terrapene nelsoni was confirmed to be the sister taxon of T. ornata. Terrapene c. major, T. c. bauri, and Terrapene coahuila were not well resolved for some of the analyses. The DNA barcoding results indicated that all taxa were different species (>2% sequence divergence) except for T. c. triunguis - T. c. mexicana and T. o. ornata - T. o. luteola. The results suggest that T. c. triunguis should be elevated to species status (Terrapene mexicana), and mexicana and yucatana should be included in this group as subspecies. In addition, T. o. ornata and T. o. luteola should not be considered separate subspecies. The DNA barcoding data support these recommended taxonomic revisions. Because conservation efforts are typically species-based, these results will be important for facilitating successful conservation management strategies.

The Western Lesser Siren, Siren intermedia nettingi, is a fully aquatic, paedomorphic salamander. The foraging ecology of S. i. nettingi is poorly understood in part because of its nocturnal, benthic nature. We examined sensory mediation of S. i. nettingi foraging behavior focusing on how chemical and visual stimuli may be used to find prey. Specifically, sirens were observed in a test chamber that gave them the option of associating with a control stimulus or either a visual or a chemical prey stimulus. Our paired t-tests indicate a significant difference between the treatment and control groups for chemical stimuli but not for visual stimuli, and this is confirmed by our one-sample t-tests that indicated a significant difference from 50% for chemical but not for visual stimuli. These results suggest that Western Lesser Sirens use chemical cues over visual cues when foraging. Our results are logical considering that sirens have noticeably reduced eyes and are thought to be nocturnal foragers. This study contributes to the limited natural history knowledge of an amphibian species that is often a top predator in the aquatic systems in which it is found.

To examine the prevalence of raised periodic limb movements of sleep (PLMS) index in children referred for polysomnography (PSG) and whether parental report of symptoms correlates with objective measurement during PSG.Records of children undergoing PSG from January 2006 to July 2006 were retrospectively reviewed. At their initial sleep clinic visit, parents had been asked whether their child was restless or moved their legs excessively during sleep. Their response to these questions was compared to the child's PLMS index (number of periodic limb movements per hour) during a full PSG. PLMS were scored according to internationally accepted criteria.Data were examined for 101 children (60 male) with mean age 6.5 years (range 1.2 to 17.6 years). Excessive leg movements were reported by parents in 50% and restlessness in 73%. A raised PLMS index (defined asor = 5 per hour) was noted in 10 cases (prevalence 10%). Asking parents about whether their child kicks their legs excessively in sleep had sensitivity 50%, specificity 51%, positive predictive value (PPV) 10%, negative predictive value (NPV) 90% and positive likelihood ratio (LR+) 1.02 when compared to objective analysis. Asking parents about whether their child is restless in sleep had sensitivity 70%, specificity 26%, PPV 9%, NPV 89% and LR+ 0.95.Asking parents about their child's symptoms is not an accurate predictor of raised PLMS index. We recommend that leg electromyography be used in all pediatric sleep studies to record PLMS.

The American box turtles (Terrapene spp.) consist of four historically recognized species based primarily on morphological data (Minx 1996): Terrapene carolina (Linnaeus), T. ornata (Agassiz), T. nelsoni (Stejneger), and T. coahuila (Schmidt & Owens). Each of these species is polytypic except for T. coahuila. Recently, the Placyk lab re-assessed the classification of Terrapene using molecular phylogenetic data, and suggested elevating a clade consisting of T. carolina triunguis (Agassiz), T. c. mexicana (Gray), and T. c. yucatana (Boulenger) to a distinct species: Terrapene mexicana ssp. (Martin et al. 2013). Our dataset included all Terrapene species and almost all of the subspecies (except for T. nelsoni klauberi), as well as large sample sizes and a wide geographic sampling for both mitochondrial (mt) and nuclear (nuc) DNA. The mtDNA Cytochrome b (Cytb) gene and a nucDNA intron from the Glyceraldehyde-3-phosphate-dehydrogenase (GAPD) gene were used for phylogenetic analyses, and indicated that the T. mexicana clade was highly divergent from the remainder of the original T. carolina clade. In addition, the Cytochrome c oxidase subunit 1 (COI) gene, which is used in many animals for DNA barcoding, was sequenced and used to calculate pairwise percent DNA sequence divergence between taxa. The barcoding data also strongly supported our suggested classification revisions. However, because possible introgression was detected for some of the individuals, T. mexicana was not recognized as a distinct species in a recent assessment of Testudines taxonomic updates (Fritz & Havas 2013). The purpose of this treatise is to suggest that the possible presence of introgression seen in Martin et al. (2013) does not warrant disregarding T. mexicana as a unique species.