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.

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.

Influence of atrazine on the scalation of Marcy's checkered gartersnake, Thamnophis m. marcianus (Baird and Girard, 1853).

Atrazine is one of the most commonly used herbicides in the United States. Despite the effectiveness of atrazine in eliminating broadleaf and grassy weeds, there has been growing concern over the potential impacts this chemical may have on non-target organisms. Little research has been conducted on the exposure of reptiles to this chemical. Our study examined the effects of environmentally relevant concentrations of atrazine on the scalation of Marcy's checkered gartersnake (Thamnophis m. marcianus). Our results indicate that atrazine exposure influences scalation, in particular, cranial scale counts. In addition, this alteration of morphology happens during embryological development as the result of the environment the mother was raised in. Further research on additional species and developmental exposure of atrazine and how it influences fitness of reptiles is required.

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.

Evolutionary persistence of chemically elicited ophiophagous antipredator responses in gartersnakes (Thamnophis sirtalis).

The ability to detect and respond to potential predators is key for the survival of individuals, but this ability is sometimes lost via relaxation of antipredator behavior when prey species are separated from predators. Adult and predator-naïve neonate gartersnakes (Thamnophis sirtalis) from mainland and insular sites where they do and do not occur with ophiophagous (snake-eating) snakes were tested to determine if responses to such predators have been lost, reduced, or retained, and what might be causing differences in such responses. Our data indicate that, overall, adult snakes from populations syntopic with ophiophagous milksnakes are more responsive to chemical stimuli from milksnakes than adults from areas where they are not syntopic with milksnakes, whereas there were few differences with neonate gartersnakes. Experiments with neonates with or without periodic ophiophagous snake chemical experience over several weeks showed that gartersnakes with such experience became more responsive to ophiophagous cues rather than habituating to them. Such evidence of both genetic and experiential factors underlying the antipredator responses to ophiophagous snakes indicates that antipredator responses have persisted despite separation of predator and prey.

Distinctiveness in the face of gene flow: hybridization between specialist and generalist gartersnakes.

Patterns of divergence and polymorphism across hybrid zones can provide important clues as to their origin and maintenance. Unimodal hybrid zones or hybrid swarms are composed predominantly of recombinant individuals whose genomes are patchworks of alleles derived from each parental lineage. In contrast, bimodal hybrid zones contain few identifiable hybrids; most individuals fall within distinct genetic clusters. Distinguishing between hybrid swarms and bimodal hybrid zones can be important for taxonomic and conservation decisions regarding the status and value of hybrid populations. In addition, the causes of bimodality are important in understanding the generation and maintenance of biological diversity. For example, are distinct clusters mostly reproductively isolated and co-adapted gene complexes, or can distinctiveness be maintained by a few 'genomic islands' despite rampant gene flow across much of the genome? Here we focus on three patterns of distinctiveness in the face of gene flow between gartersnake taxa in the Great Lakes region of North America. Bimodality, the persistence of distinct clusters of genotypes, requires strong barriers to gene flow and supports recognition of distinct specialist (Thamnophis butleri) and generalist (Thamnophis radix) taxa. Concordance of DNA-based clusters with morphometrics supports the hypothesis that trophic morphology is a key component of divergence. Finally, disparity in the level of differentiation across molecular markers (amplified fragment length polymorphisms) indicates that distinctiveness is maintained by strong selection on a few traits despite high gene flow currently or in the recent past.

Post-glacial recolonization of the Great Lakes region by the common gartersnake (Thamnophis sirtalis) inferred from mtDNA sequences.

Pleistocene events played an important role in the differentiation of North American vertebrate populations. Michigan, in particular, and the Great Lakes region, in general, were greatly influenced by the last glaciation. While several hypotheses regarding the recolonization of this region have been advanced, none have been strongly supported. We generated 148 complete ND2 mitochondrial DNA (mtDNA) sequences from common gartersnake (Thamnophis sirtalis) populations throughout the Great Lakes region to evaluate phylogeographic patterns and population structure and to determine whether the distribution of haplotypic variants is related to the post-Pleistocene retreat of the Wisconsinan glacier. The common gartersnake was utilized, as it is believed to have been one of the primary vertebrate invaders of the Great Lakes region following the most recent period of glacial retreat and because it has been a model species for a variety of evolutionary, ecological, behavioral, and physiological studies. Several genetically distinct evolutionary lineages were supported by both genealogical and molecular population genetic analyses, although to different degrees. The geographic distribution of the majority of these lineages is interpreted as reflecting post-glacial recolonization dynamics during the late Pleistocene. These findings generally support previous hypotheses of range expansion in this region.

Prey detection by vomeronasal chemoreception in a plethodontid salamander.

While chemoreception is involved in a wide variety of salamander behaviors, the chemosensory system that mediates specific behaviors is rarely known. We investigated the role of the vomeronasal system (VNS) in foraging behavior of the red-backed salamander (Plethodon cinereus) by manipulating salamanders' abilities to detect nonvolatile chemical cues emitted by potential prey. Subjects received one of three treatments: (1) impaired vomeronasal system, (2) sham manipulation, and (3) no manipulation. The role of the VNS in mediating foraging on motile prey (Drosophila melanogaster) was investigated under three light conditions (bright, dim, dark). Salamanders with impaired VNSs foraged less efficiently than either of the other experimental groups by displaying the longest latency to attack and the lowest rate of prey capture, especially in the absence of visual cues. A second experiment utilized freshly killed prey to determine whether the VNS takes on added importance in the absence of visual or tactile cues associated with moving prey. Animals with impaired VNSs showed a decreased foraging efficiency on stationary prey under both dark and light conditions. In addition, a mark-recapture study of VNS-impaired and sham salamanders in the field also indicated that salamanders with impaired VNSs consumed fewer stationary prey compared to shams. The study indicates that the VNS plays a substantial role in the foraging behavior of the plethodontid salamander, P. cinereus.