Phylogenetic relationships among Boleosoma darter species (Percidae: Etheostoma) K.L. Heckman a , T.J. Near a,b , S.H. Alonzo a, * a Department of Ecology and Evolutionary Biology, Yale University, 165 Prospect Street, New Haven, CT 06511, USA b Peabody Museum of Natural History, Yale University, New Haven, CT, USA article info Article history: Received 12 February 2009 Revised 26 May 2009 Accepted 27 May 2009 Available online 31 May 2009 Keywords: Hybridization Molecular phylogeny Breeding color abstract Darters represent a species rich group of North American freshwater fishes studied in the context of their diverse morphology, behavior, and geographic distribution. We report the first molecular phylogenetic analyses of the Boleosoma darter clade that includes complete species sampling. We estimated the rela- tionship among the species of Boleosoma using DNA sequence data from a mitochondrial (cytochrome b) and a nuclear gene (S7 ribosomal protein intron 1). Our analyses discovered that the two Boleosoma spe- cies with large geographic distributions (E. nigrum and E. olmstedi) do not form reciprocally monophyletic groups in either gene trees. Etheostoma susanae and E. perlongum were phylogenetically nested in E. nigrum and E. olmstedi, respectively. While analysis of the nuclear gene resulted in a phylogeny where E. longimanum and E. podostemone were sister species, the mitochondrial gene tree did not support this relationship. Etheostoma vitreum was phylogenetically nested within Boleosoma in the mitochondrial DNA and nuclear gene trees. Our analyses suggest that current concepts of species diversity underesti- mate phylogenetic diversity in Boleosoma and that Boleosoma species likely provide another example of the growing number of discovered instances of mitochondrial genome transfer between darter species. Ó 2009 Elsevier Inc. All rights reserved. 1. Introduction Darters are a species rich clade of North American freshwater fishes. Extensive work on the taxonomy of darters has produced a classification that comprises 17 or 18 subgenera and as many as five genera (Keck and Near, 2008; Near and Keck, 2005; Page, 2000). Over the past several years, DNA sequence data have been used to investigate species-level relationships within darter clades (Keck and Near, 2008; Lang and Mayden, 2007; Near, 2002; Near et al., 2000; Porter et al., 2002; Porterfield et al., 1999). In some cases, relationships estimated in molecular phylogenies are incon- gruent with traditional taxonomic arrangements that are based on overall morphological similarities (Lang and Mayden, 2007; Near, 2002). While a growing number of molecular studies have investi- gated the evolutionary relationships among darters, there are no published studies that provide molecular phylogenies of Boleosoma that sample all the species in the clade. Boleosoma is an interesting darter clade for molecular phyloge- netic analysis, because a clear consensus does not exist regarding species level diversity in the clade (e.g., Cole, 1965; Cole, 1967; Shute, 1984; Starnes and Starnes, 1979). In addition, there are striking differences among these species in male nuptial coloration, a character routinely used in the diagnosis and differentiation of darter taxa (Page, 1983). Etheostoma nigrum, E. susanae, and E. olms- tedi males darken during the reproductive season, while in E. per- longum, E. longimanum, and E. podostemone reproductive males exhibit bright nuptial coloration (Lindquist et al., 1981; Page, 1983 pp. 89, 92). Boleosoma species also vary in the sizes of their geographic ranges. Etheostoma nigrum and E. olmstedi are found throughout a large portion of eastern North America (Figs. 1 and 2), while the other four Boleosoma species have relatively restricted geographic distributions (Figs. 1 and 2). A phylogenetic analysis using discretely coded morphological characters resulted in mono- phyly of the five sampled Boleosoma species; and within this clade were two monophyletic groups, a clade containing E. longimanum and E. podostemone, and a clade containing E. nigrum, E. olmstedi, and E. perlongum (Simons, 1992). There have been various opinions regarding the species level diversity in Boleosoma (Bailey and Gosline, 1955; Cole, 1957; Col- lette, 1965; Winn, 1958). Five species were recognized in an eval- uation of variation in external morphological characters (e.g., scale row and fin element counts, patterns of squamation), and these species were distributed among three species groups: (1) Etheos- toma olmstedi and E. nigrum, (2) E. longimanum and E. podostemone, and (3) E. perlongum (Cole, 1957). Based on the analysis of allozyme data and external morphological characters, E. perlongum has been argued to represent an ecomorph of E. olmstedi and not a distinct species (Shute, 1984); however, E. perlongum does exhibit dispa- rate male breeding coloration and life history characteristics when compared to E. olmstedi (Lindquist et al., 1981; Shute, 1984). Etheostoma susanae is another Boleosoma species with a complicated taxonomic history. Jordan and Swain (1883) described 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.05.027 * Corresponding author. Fax: +1 203 432 3854. E-mail addresses: Kellie.Heckman@gmail.com (K.L. Heckman), thomas.near@ Yale.edu (T.J. Near), suzanne.alonzo@Yale.edu (S.H. Alonzo). Molecular Phylogenetics and Evolution 53 (2009) 249–257 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev