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.
22Hybridization is differentially manifested across the genome, with observed introgression 23 representing a balance between selection and migration. The capacity to quantify introgression 24 and subsequently pinpoint the constituent genetic elements governing cross-species exchange has 25 been promoted by the unprecedented resolution of contemporary sequencing technologies.
26Furthermore, the availability of annotated reference genomes has allowed genomic patterns to be 27 associated with ecologically relevant phenotypes. We followed this pattern herein by harnessing 28 genomic resources to decipher the role of selection in shaping hybrid zones at the interface of 29 species-boundaries in North American box turtles (Terrapene). By so doing, we identified 30 adaptive divergence in genes related to immune system function and intrinsic thermal 31 adaptations. These, in turn, impact temperature-dependent sex determination and hypoxia 32 tolerance. Their patterns were then contrasted among inter-and intra-specific hybrid zones that 33 differed in a temporal and biogeographic context. Our results demonstrate that hybridization is 34 broadly apparent in Terrapene, but with varying levels of divergence at loci that impinge upon 35 thermal adaptation. These loci displayed signatures of adaptive introgression across intraspecific 36 boundaries, and do so despite a genome-wide selective trend against intergrades. By contrast, 37 interspecific comparisons at the same loci retained evidence of divergence. Importantly, 38 adaptations that shape species-boundaries in Terrapene not only underscore climatic boundaries 39 for these terrestrial ectotherms, but also bookmark their vulnerability to anthropogenic pressures. 40
Model-based approaches to species delimitation are constrained both by 22 computational capacities as well as by algorithmic assumptions that are frequently violated when 23 applied to biologically complex systems. An alternate approach, demonstrated herein, employs 24 machine learning (=ML) approaches from which species limits are derived without an explicit 25 definition of an underlying species model. By doing so, we demonstrate the capacity of these 26 approaches to designate phylogenomically and biologically relevant groups, using North 27 American box turtles (Terrapene spp.) as an example. Several different ML-based and traditional 28 species delimitation algorithms were invoked to parse a large SNP dataset derived from ddRAD 29 sequencing. Our results illuminate two major findings. First, more traditional model-based 30 approaches perform poorly, a likely reflection of systematic biases inherent in their formulation.
31Multispecies coalescent methods consistently over-split Terrapene, particularly given prior 32 evidence and our own phylogenetic results. Second, results from ML and clustering algorithms 33 consistently reiterated the presence of clades that were well-supported in prior species tree 34 analyses. In summary, we highlight both the strengths and limitations of ML algorithms, and in 35 doing so, explore appropriate approaches to data manipulation and model fit. Our study was 36 accomplished within the context of a well-characterized empirical system that allowed a direct 37 contrast between ML versus traditional approaches. It allowed the utility of ML-methods to be 38 underscored for species delimitation and serves as a study case from which guidelines implicit to 39 ML methods could be applied to other study systems. 40 41
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