Delimitation and species discovery in the Profundulidae fish family: Using genetic, environmental and morphologic data to address taxonomic uncertainty.

The family Profundulidae includes some one of the most enigmatic freshwater fishes of Mesoamerica: despite many attempts, a robust phylogenetic framework to delimit species is lacking, mainly due to limited morphological variation within the group. The accumulation of molecular data of profundulid fishes has led to advances in the description of new taxa, but relatively less progress has been made estimating evolutionary and phylogenetic relationships for this fish family. Here, we adopt an integrative taxonomy approach including the use of nuclear and mitochondrial DNA sequences, morphometric and ecological data, to test species boundaries in profundulid fishes in the westernmost area of their known distribution range in the states of Guerrero and Oaxaca, Mexico. Using a combination of methods for species discovery and validation based on Bayesian gene tree topologies, our analyses support the delimitation of 15 valid species of profundulid fishes - a combination of previously described species validated by this study, the synonymy of unsupported taxa, and the description of two new species. Using species delimitation methods, examination of phenotypic variation, and ecological niche characterization, we also identify five potentially new lineages which require further evidence to be erected as new species. We demonstrate that the use of an integrative taxonomy approach provides a robust methodology to delimit species in a taxonomically complex group like Profundulidae. Accurate taxonomic and ecological information is crucial for the conservation of these microendemic fishes, as several species are endangered.