Species delimitation in the grey zone: introgression obfuscates phylogenetic inference and species boundaries in a cryptic frog complex (Ranidae: Pulchrana picturata)

As molecular methods continue to elucidate genetic structure at increasingly finer resolutions, delimiting species in the grey zone of the speciation continuum is becoming more relevant in biodiversity research, especially in under-studied biodiversity hotspots such as Southeast Asia where new species are being described at an unprecedented rate. Obvious species at both ends of the speciation continuum have mostly been described and attention is now turning towards the \"grey zone:\" an intermediate stage in which species criteria are in conflict and boundaries between populations and species are less clear. This study demonstrates that widely-used criteria (phylogenetic placement, genetic divergence, phylogeny- and distance-based species delimitation methods) can overestimate species diversity/boundaries when introgression is present. However, a comprehensive species delimitation framework that considers spatial and genetic population structure, introgression, and the use of species delimitation methods based on parameter estimation, can provide a more accurate characterization of species boundaries in this grey zone. We applied this approach to a group of Southeast Asian frogs from the Pulchrana picturata Complex that exhibits continuous morphological variation and high genetic divergences. Results showed that introgression was rampant among Bornean populations, which led to phylogenetic discordance and an overestimation of species. We suspect that our results do not form an isolated case; and that introgression among cryptic populations, occurring continuously across a wide geographic area (e.g., the topographically complex island of Borneo, and Earths major continents), may be more common than previously thought.