Molecular phylogeny and evolution of internal fertilization in South American seasonal cynopoeciline killifishes.

Internal fertilization is a widespread mode of reproduction in chondrichthyans and tetrapods, but uncommon in actinopterygian fishes. In killifishes of the suborder Aplocheiloidei, internal fertilization is restricted to two genera, Campellolebias and Cynopoecilus, both containing species adapted to life in seasonal pools of subtropical South America and exhibiting elaborated inseminating structures. Phylogenetic studies involving these genera are scarce and limited to morphological characters and fragments of mitochondrial DNA sequences available for a few taxa, providing incongruent results and thus impeding hypotheses on the evolution of insemination and related morphological traits. We analyzed three nuclear loci (GLYT1, ENC1, Rho) for 13 aplocheiloid taxa obtaining the first well-supported phylogeny for cynopoecilines, thus providing a significant background to interpret evolutionary changes within the group. Like in killifishes of the suborder Cyprinodontoidei, the evolution of internal fertilization in aplocheiloids is associated with deep changes in the structure of male anal fin. The phylogenetic analyses indicate that internal fertilization corresponds to a single evolutionary event during the evolution of aplocheiloid killifishes. The analyses also indicate that male specialized muscle characters, comprising a muscular ejaculatory pump in the urogenital region and hypertrophied inclinatores and depressores anales, arose in the ancestor of the clade comprising Campellolebias and Cynopoecilus. On the other hand, anal fin specialized structures including the male inseminating tube of Campellolebias and the male inseminating fan of Cynopoecilus evolved independently in each genus.