Combining mitochondrial DNA sequences and morphological data to infer species boundaries: phylogeography of lanceheaded pitvipers in the Brazilian Atlantic forest, and the status of Bothrops pradoi (Squamata : Serpentes : Viperidae)

Phylogeographic studies using mitochondrial DNA sequence information are fr equently used as the principal source of evidence to infer species boundari es. However, a critical analysis of further evidence is essential to test w hether different haplotype clades identify different species. We demonstrat e a hypothesis-testing approach, using a combination of phylogeographic met hods, multivariate morphometrics and matrix association tests, to investiga te species boundaries in eastern Brazilian pitvipers conventionally assigne d to the species Bothrops leucurus and B. pradoi. Two basal haplotype clade s with partly overlapping geographical distributions are identified, which could either represent two partly sympatric species, or multiple haplotypes within one organismal lineage. We use partial Mantel matrix association te sts to verify whether generalized morphology, or any of four Supposedly dia gnostic characters for the two species, show any association with mtDNA var iation. Negative results lead to the conclusion that the haplotype clades d o not denote independently evolving organismal lineages, and do not constit ute separate species under any criterion.