Hidden diversity in the Andes: comparison of species delimitation methods in montane marsupials

Cryptic genetic diversity is a significant challenge for systematists faced with ever-increasing amounts of DNA sequence data. Computationally intensive coalescent-based analyses involving multiple unlinked loci are the only currently viable methods by which to assess the extent to which phenotypically similar populations (or metapopulations) are genetically distinct lineages. Although coalescent-based approaches have been tested extensively via simulations, few empirical studies have examined the impact of prior assumptions and dataset size on the ability to assess genetic isolation (evolutionary independence) using molecular data alone. Here, we consider the efficacy of two coalescent-based approaches (BPP and SpeDeSTEM) for testing the evolutionary independence of cryptic mtDNA haplogroups within three morphologically diagnosable species of Andean mouse opossums (Thylamys pallidior, T. sponsorius, and T. venustus). Fourteen anonymous nuclear loci, one X-linked nuclear intron, and one mitochondrial gene were analyzed for multiple individuals within each haplogroup of interest. We inferred individual gene trees for each locus and considered all of the nuclear loci jointly in a species-tree analysis. Using only the nuclear loci, we performed "species validation" tests for the cryptic mitochondrial lineages in SpeDeSTEM and BPP. For BPP, we also tested a wide range of prior assumptions, assessed performance of the rjMCMC algorithm, and examined how many loci were necessary to confidently delimit lineages. Results from BPP provided strong support for two independent evolutionary lineages each within T. pallidior, T. sponsorius, and T. venustus, whereas SpeDeSTEM results did not support splitting out mtDNA haplogroups as distinct evolutionary units. For most tests, BPP was robust to prior assumptions, although priors were shown to have an effect on both the strength of lineage recognition among T. venustus haplotypes and on the efficiency of the rjMCMC algorithm. Comparisons of results from datasets with different numbers of loci revealed that some cryptic lineages could be confidently delimited with as few as two loci.