ClineHelpR: an R package for genomic cline outlier detection and visualization.

BACKGROUND: 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, and users must often write custom, relatively complex code to do so. RESULTS: Here, we introduce the ClineHelpR R-package for visualizing genomic clines and detecting outlier loci using output generated by two popular software packages, bgc and Introgress. ClineHelpR 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. CONCLUSIONS: 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. Furthermore, the ClineHelpR extended functionality can evaluate genomic clines in the context of spatial and environmental features, allowing users to explore underlying processes potentially contributing to the observed patterns and helping facilitate effective conservation management strategies.

Taxonomic Uncertainty and the Anomaly Zone: Phylogenomics Disentangle a Rapid Radiation to Resolve Contentious Species (Gila robusta Complex) in the Colorado River.

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.

Spatial population genetics in heavily managed species: Separating patterns of historical translocation from contemporary gene flow in white-tailed deer.

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 pretranslocation 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 1143 deer sampled statewide 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 re-colonization 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 statewide 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 is also applicable for other biodiversity elements with similarly complex demographic histories.

The choices we make and the impacts they have: Machine learning and species delimitation in North American box turtles (Terrapene spp.).

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.

Age structuring and spatial heterogeneity in prion protein gene (PRNP) polymorphism in white-tailed deer.

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 reflect 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.

Contrasting signatures of introgression in North American box turtle (Terrapene spp.) contact zones.

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 intraspecific hybrid zones that differ temporally and biogeographically. We demonstrate hybridization is broadly apparent in Terrapene, 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 intraspecific boundaries, despite a genome-wide selective trend against intergrades. In contrast, outlier loci for interspecific comparisons exhibited evidence of being under selection against hybrids. Importantly, adaptations coinciding with species boundaries in Terrapene overlap with climatic boundaries and highlight the vulnerability of these terrestrial ectotherms to anthropogenic pressures.

Hybridization drives genetic erosion in sympatric desert fishes of western North America.

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 cypha and G. 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 or expanding historically limited introgression. In addition, results from basin-wide genomic clines suggested that hybrids and parental forms are adaptively nonequivalent. 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.

Sequence-based molecular phylogenetics and phylogeography of the American box turtles (Terrapene spp.) with support from DNA barcoding.

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.

Parental symptom report and periodic limb movements of sleep in children.

STUDY OBJECTIVES: 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. METHODS: 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. RESULTS: 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 as > or = 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. CONCLUSIONS: 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.