Phylogenetic relationships of species of genus Pseudoxiphophorus have been only tackled in detail based on morphology so far. However, phylogenetic evidence based on molecular data is still lacking. In this study, we have used five molecular markers (mitochondrial cytb, 16S, atp6-8, and nuclear actB and S7) to reconstruct a robust, inclusive phylogeny of Pseudoxiphophorus. Our phylogenetic results strongly disagree with the main morphological hypothesis, and indicate different phylogenetic relationships among the recognized species of Pseudoxiphophorus. Pseudoxiphophorus jonesii is recovered as the sister group of all other Pseudoxiphophorus lineages, and this initial splitting may be associated to the extension of the Mexican Neovolcanic Plateau at the Punta del Morro site (event used to calibrate our dating analysis). The branch leading to all other Pseudoxiphophorus separated subsequently into two major groups, one comprising those lineages occurring in southern Mexico and Guatemala-Belize, and another with those lineages that extended further southwards to Honduras and Nicaragua. This event took place during the Pliocene, and is likely associated with periods of inundation of the Polochic-Motagua fault area. The Isthmus of Tehuantepec also appears to have been a strong biogeographic barrier triggering cladogenesis in Pseudoxiphophorus. Heterandria formosa (traditionally placed as sister to Pseudoxiphophorus) is not sharing the most recent common ancestor with Pseudoxiphophorus, and is recovered as more distantly related to them. Furthermore, Pseudoxiphophorus bimaculatus (the most cosmopolitan species) is also recovered as a polyphyletic assemblage that appears to comprise those Pseudoxiphophorus that have not been assigned to the other eight, more localized species. All this suggests that Pseudoxiphophorus needs a major taxonomic revision as a whole in order to incorporate all existing diversity.
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