An evaluation of sampling effects on multiple DNA barcoding methods leads to an integrative approach for delimiting species: a case study of the North American tarantula genus Aphonopelma (Araneae, Mygalomorphae, Theraphosidae).

The North American tarantula genus Aphonopelma provides one of the greatest challenges to species delimitation and downstream identification in spiders because traditional morphological characters appear ineffective for evaluating limits of intra- and interspecific variation in the group. We evaluated the efficacy of numerous molecular-based approaches to species delimitation within Aphonopelma based upon the most extensive sampling of theraphosids to date, while also investigating the sensitivity of randomized taxon sampling on the reproducibility of species boundaries. Mitochondrial DNA (cytochrome c oxidase subunit I) sequences were sampled from 682 specimens spanning the genetic, taxonomic, and geographic breadth of the genus within the United States. The effects of random taxon sampling compared traditional Neighbor-Joining with three modern quantitative species delimitation approaches (ABGD, P ID(Liberal), and GMYC). Our findings reveal remarkable consistency and congruence across various approaches and sampling regimes, while highlighting highly divergent outcomes in GMYC. Our investigation allowed us to integrate methodologies into an efficient, consistent, and more effective general methodological workflow for estimating species boundaries within the mygalomorph spider genus Aphonopelma. Taken alone, these approaches are not particularly useful - especially in the absence of prior knowledge of the focal taxa. Only through the incorporation of multiple lines of evidence, employed in a hypothesis-testing framework, can the identification and delimitation of confident species boundaries be determined. A key point in studying closely related species, and perhaps one of the most important aspects of DNA barcoding, is to combine a sampling strategy that broadly identifies the extent of genetic diversity across the distributions of the species of interest and incorporates previous knowledge into the "species equation" (morphology, molecules, and natural history).