Species Delimitation and Phylogeography of Aphonopelma hentzi (Araneae, Mygalomorphae, Theraphosidae): Cryptic Diversity in North American Tarantulas

Theraphosidae): Cryptic Diversity in North American Tarantulas. PLoS ONE 6(10): e26207. doi:10.1371/journal.pone.0026207 Species Delimitation and Phylogeography of Aphonopelma hentzi (Araneae, Mygalomorphae, Theraphosidae): Cryptic Diversity in North American Tarantulas Chris A. Hamilton 0 Daniel R. Formanowicz 0 Jason E. Bond 0 Dirk Steinke, Biodiversity Insitute of Ontario - University of Guelph, Canada 0 1 Auburn University Museum of Natural History and Department of Biological Sciences, Auburn University , Auburn , Alabama, United States of America, 2 Department of Biology, The University of Texas at Arlington , Arlington, Texas , United States of America Background: The primary objective of this study is to reconstruct the phylogeny of the hentzi species group and sister species in the North American tarantula genus, Aphonopelma, using a set of mitochondrial DNA markers that include the animal ''barcoding gene''. An mtDNA genealogy is used to consider questions regarding species boundary delimitation and to evaluate timing of divergence to infer historical biogeographic events that played a role in shaping the present-day diversity and distribution. We aimed to identify potential refugial locations, directionality of range expansion, and test whether A. hentzi post-glacial expansion fit a predicted time frame. Methods and Findings: A Bayesian phylogenetic approach was used to analyze a 2051 base pair (bp) mtDNA data matrix comprising aligned fragments of the gene regions CO1 (1165 bp) and ND1-16S (886 bp). Multiple species delimitation techniques (DNA tree-based methods, a ''barcode gap'' using percent of pairwise sequence divergence (uncorrected pdistances), and the GMYC method) consistently recognized a number of divergent and genealogically exclusive groups. Conclusions: The use of numerous species delimitation methods, in concert, provide an effective approach to dissecting species boundaries in this spider group; as well they seem to provide strong evidence for a number of nominal, previously undiscovered, and cryptic species. Our data also indicate that Pleistocene habitat fragmentation and subsequent range expansion events may have shaped contemporary phylogeographic patterns of Aphonopelma diversity in the southwestern United States, particularly for the A. hentzi species group. These findings indicate that future species delimitation approaches need to be analyzed in context of a number of factors, such as the sampling distribution, loci used, biogeographic history, breadth of morphological variation, ecological factors, and behavioral data, to make truly integrative decisions about what constitutes an evolutionary lineage recognized as a ''species''. - Funding: This work was supported by the American Arachnological Society (AAS) (2009) Vincent Roth Fund for Systematic Research (http://www. americanarachnology.org) to C. Hamilton; NSF REVSYS #DEB0841610 Systematics and taxonomy of the tarantula spider genus Aphonopelma (Araneae, Mygalomorphae, Theraphosidae) (http://www.nsf.gov) to J. Bond. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. The spider genus Aphonopelma, the only genus of tarantula known to North America, is distributed throughout the southern third of the United States, west of the Mississippi River to California and down into the Central American Neotropics. Despite the charismatic nature of the group (large bodied and hairy), the taxonomy of Aphonopelma has received comparatively little attention and has been largely reliant upon sparse and sometimes poorly defined morphological data. Historically, three major efforts [24] have been undertaken to evaluate the taxonomic status, morphological character variation, and relationships of Aphonopelma, none of which employed an explicit phylogenetic approach. Unfortunately, much of the past descriptive work was based on only one or two specimens, and generally lacked consideration of the wide range of intraspecific variation noted within group. Morphology-based phylogenies of tarantulas and other related mygalomorph taxa seem to signal widespread problems in homoplasy among morphological characters [59]. Also, the quantitative or meristic features often used to evaluate relationships among these taxa may be problematic [10,11]. Not surprisingly, many arachnologists have lamented the present state of theraphosid taxonomy due to the morphological characters employed for diagnostic use [4,5,1216]. Raven [17] considered the family Theraphosidae a nomenclatural and taxonomic nightmare. Accurate delimitation of species boundaries is especially important in taxa where one or more morphological characters are uninformative or are in conflict. Hebert et al [18,19] suggest traditional taxonomy in morphologically conserved groups can lead to incorrect identifications and may fail to recognize cryptic taxa. Because many of the described tarantula species in the United States can be difficult to differentiate based on morphological characteristics alone, molecular data may prove to be an additional source of characters to evaluate taxonomy, species delineation, and to uncover cryptic species in this charismatic yet understudied group. The objective of the study presented herein is to reconstruct the phylogeny of the hentzi species group and sister species across Aphonopelma taxa distributed in the eastern portion of the southwest United States, using a set of mitochondrial DNA markers that include the animal barcoding gene [18,19]. We then employ these data, through the testing of multiple species delimitation techniques DNA tree-based methods, a barcode gap using percent of pairwise sequence divergence (uncorrected p-distances), and the GMYC method to consider questions regarding species boundary delimitation. Subsequently, we evaluate timing of divergence events for these delimited species to infer historical biogeographic events that have played a potential role in shaping the present-day diversity and distribution. The use of tree-based methods for species delimitation recognizes species as historical lineages [20] and has been shown to be effective when used in conjunction with uncorrected p-distance data outlined in Hebert et al [18,19] and in a number of other studies [2027]. We have chosen to make use of mitochondrial genes, as we are particularly interested in addressing the efficacy of cytochrome c oxidase subunit 1 (CO1) and ribosomal large subunit (16S) as species identification tools in both known and unknown groups within the genus Aphonopelma and other theraphosid taxa [18,19,28]. Mitochondrial genes have long served as preferred markers for phylogeographic and species-level phylogenetic analyses because of the speed with which they evolve -- mutations may reflect real substantive changes in metabolic pathways (e.g. cell res (...truncated)