Phylogenetic relationships in the genus Cyprinella (Actinopterygii: Cyprinidae) based on mitochondrial and nuclear gene sequences

Members of the genus Cyprinella are abundant and broadly distributed cyprinids in central and eastern rivers of North America. Currently, 30 species are placed in this genus, including seven species restricted to Mexico and three barbeled species formerly placed in different genera (Hybopsis, Erimonax). Phylogenetic analyses were performed for all recognized species of Cyprinella plus species from 14 related genera using complete nucleotide sequences of one mitochondrial (cytochrome b) and one nuclear (Rag1) gene. Cyprinella revealed high levels of genetic divergence between species in both genes compared with species from other related genera examined. Analyses of mitochondrial sequences from 98 specimens of Cyprinella revealed the gene trees of some species as non-monophyletic (i.e., C. venusta, C. lepida, C. nivea, C. lutrensis, C. whipplei). Mitochondrial analyses recovered nine well-supported species groups within a main clade inclusive of most species of Cyprinella, although resolution regarding the monophyly of the genus or its relationships with other genera was poorly resolved, except for C. callistia and C. monacha, two species that are apparently not part of the genus. Analyses of nuclear gene sequences from selected distinctive mitochondrial clades also recovered these nine clades and resulted in shorter branches but greater resolution at more basal nodes. Nuclear data also rejected the inclusion of C. monacha in Cyprinella and indicated that Cyprinella is not monophyletic if Tampichthys and Codoma are recognized as valid genera. A major Clade A (formed by Cyprinella, Tampichthys, Codoma) is more closely related to Pimephales, Opsopoeodus, Hybognathus and some Notropis than to Luxilus and Lythrurus as previously hypothesized. Although mitochondrial data are highly informative phylogenetically, our research revealed fundamental conflicts among mitochondrial and nuclear data for several species of Cyprinella. Interestingly, nuclear trees recovered some species resolved as polyphyletic with mitochondrial variation as monophyletic. While we remain highly supportive of phylogenetic studies using mitochondrial genes and genomes, this study does highlight the utility of nuclear data by identifying issues of possible hybridization or retained ancestral polymorphisms and lineage sorting that can yield erroneous hypothesis in studies using only mit-DNA analyses, while shedding invaluable light on the phylogeny of this controversial group of shiners.