Phylogeny and Temporal Diversification of Darters ( Percidae : Etheostomatinae )

—Discussions aimed at resolution of the Tree of Life are most often focused on the interrelationships of major organismal lineages. In this study, we focus on the resolution of some of the most apical branches in the Tree of Life through exploration of the phylogenetic relationships of darters, a species-rich clade of North American freshwater fishes. With a near-complete taxon sampling of close to 250 species, we aim to investigate strategies for efficient multilocus data sampling and the estimation of divergence times using relaxed-clock methods when a clade lacks a fossil record. Our phylogenetic data set comprises a single mitochondrial DNA (mtDNA) gene and two nuclear genes sampled from 245 of the 248 darter species. This dense sampling allows us to determine if a modest amount of nuclear DNA sequence data can resolve relationships among closely related animal species. Darters lack a fossil record to provide age calibration priors in relaxed-clock analyses. Therefore, we use a near-complete species–sampled phylogeny of the perciform clade Centrarchidae, which has a rich fossil record, to assess two distinct strategies of external calibration in relaxed-clock divergence time estimates of darters: using ages inferred from the fossil record and molecular evolutionary rate estimates. Comparison of Bayesian phylogenies inferred from mtDNA and nuclear genes reveals that heterospecific mtDNA is present in approximately 12.5% of all darter species. We identify three patterns of mtDNA introgression in darters: proximal mtDNA transfer, which involves the transfer of mtDNA among extant and sympatric darter species, indeterminate introgression, which involves the transfer of mtDNA from a lineage that cannot be confidently identified because the introgressed haplotypes are not clearly referable to mtDNA haplotypes in any recognized species, and deep introgression, which is characterized by species diversification within a recipient clade subsequent to the transfer of heterospecific mtDNA. The results of our analyses indicate that DNA sequences sampled from single-copy nuclear genes can provide appreciable phylogenetic resolution for closely related animal species. A well-resolved near-complete species–sampled phylogeny of darters was estimated with Bayesian methods using a concatenated mtDNA and nuclear gene data set with all identified heterospecific mtDNA haplotypes treated as missing data. The relaxed-clock analyses resulted in very similar posterior age estimates across the three sampled genes and methods of calibration and therefore offer a viable strategy for estimating divergence times for clades that lack a fossil record. In addition, an informative rank-free clade-based classification of darters that preserves the rich history of nomenclature in the group and provides formal taxonomic communication of darter clades was constructed using the mtDNA and nuclear gene phylogeny. On the whole, the appeal of mtDNA for phylogeny inference among closely related animal species is diminished by the observations of extensive mtDNA introgression and by finding appreciable phylogenetic signal in a modest sampling of nuclear genes in our phylogenetic analyses of darters. [Ammocrypta; Crystallaria; divergence times; Etheostoma; mitochondrial DNA; Nothonotus; nuclear DNA; Percina; phylocode; phylogenetic classification; relaxed molecular clock; Teleostei. ] The diversity of freshwater fishes in eastern North America is unrivaled among temperate regions of the world (Briggs 1986; Lundberg et al. 2000). The ability to test evolutionary hypotheses that address the origin and timing of diversification of the species-rich North American freshwater fish fauna is dependent on the availability of resolved species-level phylogenetic hypotheses and robust estimates of divergence times. In addition, resolved phylogenetic trees with dense taxon sampling provide the ideal basis from which to construct species-level phylogenetic classifications (de Queiroz and Gauthier 1990; Cantino et al. 1999; Hillis and Wilcox 2005) and to test the veracity of the traditional prephylogenetic classifications that have historically served as the basis for comparative evolutionary studies. The challenge of estimating comprehensive phylogenies for the North American freshwater fish fauna is daunting because the two most species-rich clades, darters (Etheostomatinae) and minnows (Cyprinidae), contain an estimated 250 and 320 species (Mayden et al. 1992; Scharpf 2005, 2008), respectively, and previous inferences indicate that these clades exhibit a fairly recent history of diversification, which does not predate the Eocene (Dowling et al. 2002; Near and Keck 2005; Rüber et al. 2007; Hollingsworth and Near 2009). Appropriate data selection strategies are required to resolve the species-level phylogenetic relationships of such large and recent radiations (Wiens et al. 2005; McGuire et al. 2007), and the lack of a fossil record for a clade, as is the case in darters, leads to major challenges in attempting to estimate divergence times using relaxed molecular clock methods (Near and Benard 2004). Over the past few years, there has been progress in the development of phylogenetic hypotheses and molecular divergence time estimates for select North American freshwater fish clades. Near-complete species