This study examines the role that historical events have played in the diversification of members of the genus Ambloplites by estimating divergence times of clades within the genus. DNA sequences of the mitochondrial cytochrome b gene were used to develop phylogenetic hypotheses for Ambloplites using maximum parsimony and Bayesian methods. The timing of diversification within and between species of Ambloplites was estimated using nonparametric rate-smoothing and penalized likelihood methods calibrated by fossil dates. Monophyly of three of the four species was supported. The ages of the most recent common ancestors of species of Ambloplites estimated in this study confirm earlier estimates that in most instances, their origins occurred prior to the Pleistocene. A comparison of estimated divergence times of lineages to sea level fluctuations indicates a correspondence to extremely high or low sea stands throughout the Neogene Period. Populations of Ambloplites in the Ozarks and Ouachita highlands previously assigned to A. ariommus appear to have either been introgressed by A. rupestris or were misidentified as A. ariommus. Ambloplites rupestris displays low genetic divergence throughout its range and is consistent with a re-colonization of previously glaciated areas following the Pleistocene.

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