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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.

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.

The gopher tortoise (Gopherus polyphemus) has experienced dramatic population declines throughout its distribution in the southeastern United States and is federally listed as threatened in the area west of the Tombigbee and Mobile rivers. While there is molecular support for recognizing the listed portion of the range as genetically distinct, other research has suggested that additional population structure exists at both range-wide and regional scales. In this study, we sought to comprehensively define genetic population structure at both spatial scales by doubling the data available in terms of the number of sampling sites, individuals, and microsatellite loci compared to previously published work. We also compared patterns of genetic diversity, gene flow, and demographic history across the range. We collected 933 individuals from 47 sampling sites across the range and genotyped them for 20 microsatellite loci. Our range-wide analyses supported the recognition of five genetic groups (or regions) delineated by the Tombigbee and Mobile rivers, Apalachicola and Chattahoochee rivers, and the transitional areas between several physiographic province sections of the Coastal Plains (i.e., Eastern Gulf, Sea Island, and Floridian). We found genetic admixture at sampling sites along the boundaries of these genetically defined groups. We detected some degree of additional genetic structure within each of the five regions. Notably, within the range listed as threatened under the Endangered Species Act, we found some support for two additional genetic groups loosely delineated by the Pascagoula and Chickasawhay rivers, and we detected four more genetic groups within the Florida region that seemed to reflect the influence of the local physiography. Additionally, our range-wide analysis found the periphery of the range had lower levels of genetic diversity relative to the core. We suggest that the five main genetic groups delineated in our study warrant recognition as management units in terms of conservation planning. Intraregional population structure also points to the potential importance of other barriers to gene flow at finer spatial scales, although additional work is needed to better delineate these genetic groups.

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.

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.

Identifying geographic barriers that define genetic structure within a species is crucial in formulating an effective conservation plan. The identification of appropriate management units is critical for the protection and recovery of the gopher tortoise Gopherus polyphemus, which have declined across their entire range. Previous molecular work at various spatial scales has identified distinct population assemblages of the gopher tortoise. The goal of this study was to assess the genetic structure in gopher tortoises through a more complete sampling of the federally listed as threatened portion of the range and evaluate the extent of genetic isolation imposed by several potential geographic barriers. We sequenced a 712–base-pair portion of a mitochondrial gene (NADH dehydrogenase 4) for 322 individuals from 42 sites across the range. We found two major assemblages of haplotypes separated by a modest phylogenetic break (average uncorrected p distance = 0.015). The biogeographic barrier that best...

This study documents the ingestion of leaves and seeds by gopher tortoises (Gopherus polyphemus) in a relatively intact pine savanna habitat. Fifty-three species of seeds were identified. Species richness of seeds ingested was highest during spring and fall and was lowest during summer. Grass seed and foliage were consumed consistently by tortoises throughout the year. Despite being found in only 12% of all scats, Rubus seed comprised half of all seeds recovered. Based on seeds in scats, tortoises ingested the fruits of plants in the same proportions in which the plants occurred immediately around active burrows. Although some kinds of leaves were less prevalent in scats than predicted by their availability near active tortoise burrows, this may have occurred because these leaves were digested and, therefore unrecognizable in scats. The role of the gopher tortoise as a generalist herbivore and an opportunistic frugivore was confirmed.

This research examined the influence of tracked-vehicle use on gopher tortoises (Gopherus polyphemus) on Fort Benning, GA. Did tortoises on sites with heavy tracked-vehicle use differ from those in areas with limited tracked-vehicle use in: number of burrows used during the active season; rates at which female tortoises receive visitors; and number of males that visit each female? Data from an undisturbed off-post site with characteristics of old-growth longleaf pine forest were used for comparison. Similar numbers of burrows were used by both sexes in both the heavily disturbed and less-disturbed sites on Fort Benning, but more burrows per animal were used at the undisturbed comparison site. Significantly fewer total social visits were received by females in the more highly disturbed sites. In both the less-disturbed areas and the off-post site, many more different males visited each female. One conclusion drawn is that females on sites impacted by tracked-vehicle activities seem to have more limited options for mate choice and, therefore, are susceptible to problems associated with inbreeding and restricted gene flow. These susceptibilities are not desirable for population viability.

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.