Systematics of the greenside darter Etheostoma blennioides complex: Consensus from nuclear and mitochondrial DNA sequences

The phylogenetic systematic relationships of the enigmatic greenside darter Etheostoma blennioides complex are analysed using sequences from the mitochondrial (mt) DNA cytochrome b gene and nuclear S7 ribosomal protein intron 1 from putative members of the complex, close relatives, and outgroups (totaling 421 individuals). We compare results from Bayesian and maximum likelihood analysis approaches and a variety of rooting and taxon inclusion scenarios, and include all putative subspecies and intergrade taxa for a new comprehensive analysis. Results reveal that nuclear and mtDNA data congruently, under all scenarios and approaches tested, define a highly-supported restricted greenside darter complex comprising three putative subspecies: E. b. blennioides, E. b. pholidotum, and part of E. b. newmanii (excepting those from the Tennessee/Hiwassee River clade). Within this redefined E. blennioides, only a single putative subspecies - E. b. blennioides - is monophyletic in the mtDNA trees, and none are monophyletic in the nuclear DNA trees. Nuclear and mtDNA results support E. gutselli as a separate species and suggest that the Tennessee/Hiwassee River clade of “E. b. newmanii” also may constitute a separate species (provisionally “E. newmanii”), with neither being a part of our redefined E. blennioides complex. The nuclear DNA trees depict the two as highly-supported divergent clades, but the mtDNA results group them together as a single clade, indicating introgression. Future study with greater sample sizes in the southern watersheds, coupling morphological analyses with additional nuclear gene phylogenies, is recommended to further investigate the relationships within the greenside darter complex.