Combining next-generation sequencing and mtDNA data to uncover cryptic lineages of Mexican highland frogs

Recently, molecular studies have uncovered significant cryptic diversity in the Mexican Highlands, leading to the description of many new endemic species. DNA approaches to this kind of species discovery have included both mitochondrial DNA (mtDNA) sequencing and multilocus genomic methods. While these marker types have often been pitted against one another, there are benefits to deploying them together, as linked mtDNA data can provide the bridge between uncovering lineages through rigorous multilocus genomic analysis and identifying lineages through comparison to existing mtDNA databases. Here, we apply one class of multilocus genomic marker, ultraconserved elements (UCEs), and linked mtDNA data to a species complex of frogs (Sarcohyla bistincta) found in the Mexican Highlands. We generated data from 1,891 UCEs, which contained 1,742 informative SNPs for S. bistincta and closely related species and captured mitochondrial genomes for most samples. Genetic analyses based on both whole loci and SNPs agree there are numerous distinct and divergent lineages within S. bistincta. The SNP-based species tree provides the most conservative estimate of 8 well-supported lineages in three major clades. Having linked mtDNA data allowed us to tap into the large number of mtDNA sequences available on GenBank and identify one of these lineages as an already-described species, S. pentheter. One identified clade (containing 2 of the 8 lineages) was 10% divergent in mtDNA and paraphyletic with other S. bistincta, making this clade a clear candidate for species status. Phylogenies from UCEs and mtDNA mostly agreed in their topologies, but differed in that mtDNA suggested a more complex evolutionary history perhaps influenced by gene flow between some neighboring lineages. Our study demonstrates that the Mexican Highlands still hold substantial undescribed diversity. Combining multilocus genomic data with linked mtDNA data is a useful approach for identifying potential new species and associating them with already described taxa, which is especially important in groups with undescribed subadult phenotypes, where geographic ranges are unclear, or where phenotypes are conserved.