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Author's personal copy Molecular Phylogenetics and Evolution 52 (2009) 544–549 Contents lists available at ScienceDirect Molecular Phylogenetics and Evolution journal homepage: www.elsevier.com/locate/ympev Short Communication Molecular systematics of the Cyprinoidea (Teleostei: Cypriniformes), the world’s largest clade of freshwater fishes: Further evidence from six nuclear genes Wei-Jen Chen *, Richard L. Mayden Department of Biology, Saint Louis University, 3507 Laclede Ave., St. Louis, MO 63103, USA article info abstract Article history: Received 12 December 2008 Ó 2009 Elsevier Inc. All rights reserved. Available online 21 January 2009 Keywords: Cyprinidae Cyprinioidea Cypriniformes Psilorhynchus Tinca Leptobabrus Nuclear gene Phylogenomics Taxonomic chaos 1. Introduction Previous systematic analyses investigating monophyly and inter-relationships of the Cyprinidae have focused largely on mor- With over 210 genera and 2010 described species, the fam- phology or mitochondrial gene/genome sequences. Chen et al. ily Cyprinidae is currently the largest family of freshwater (1984) was the first such study to propose a ‘‘phylogenetic” fishes (Nelson, 2006). Over the years, the Cyprinidae has been hypotheses” of cyprinid inter-relationships based on morphologi- divided into different ‘‘groupings” for either taxonomic conve- cal data. Later Cavender and Coburn (1992) reanalyzed the data nience or to represent presumed natural groups; usually these matrix of Chen et al. (1984), recovering a tree of equal length but groupings have been recognized at or below the level of sub- of a different topology to that recovered by Chen et al. (1984) family (Cavender and Coburn, 1992; Howes, 1991; Nelson, (Fig. 1B). Cavender and Coburn (1992) also proposed an alternative 2006). Howes (1991) recognized seven such subgroupings of phylogeny for the Cyprinidae based on the analysis of their own 47 the Cyprinidae, including the Alburninae, Cyprininae, Rasbori- morphological characters (Fig. 1A). Despite these early morpholog- nae, Cultrinae, Acheilognathinae, Tincinae, Leuciscinae, and ical phylogenetic investigations of the Cyprinidae, some uncer- Gobioninae. Cavender and Coburn (1992) recognized two, the tainty regarding the basal lineage of cyprinds and the placement Cyprininae and Leuciscinae, the former including those cypri- of the enigmatic genus Tinca remains (Fig. 1). nids referred to as barbins, labeonins and cyprinins, and the Recent systematic investigations of the Cyprinidae have utilized later including those referred to as tincins, rasborins, gobionins, a molecular phylogenetic approach, with mitochondrial sequence acheilognathins, cultrins, xenocyprins, leucisins and phoxinins. data being most readily utilized (e.g., Cunha et al., 2002; Gilles The genus Psilorhynchus (a grouping of small, ventrally flat- et al., 2001; He et al., 2008a; Liu and Chen, 2003; Okazaki et al., tened fishes adapted for benthic life in fast flowing water) is 2001; Saitoh et al., 2006; Simons et al., 2003; Xiao et al., 2001; either considered to be the sole member of the family Zardoya and Doadrio, 1998). Although the taxonomic sampling of Psilorhynchidae (Conway and Mayden, 2007; Nelson, 2006; these studies was limited to certain subgroupings or to species Ramaswami, 1952) following Hora (1925) or as the sole mem- from geographic regions of interests to the authors each of these ber of the cyprinid subfamily Psilorhynchinae (Chen, 1981; investigations provided valuable insight into the evolution of these Nelson, 1994). morphologically diverse fishes. Two studies presented their hypotheses with the samplings covering approximately all cyprinid subfamilies (Gilles et al., 2001; Liu and Chen, 2003). Both of * Corresponding author. Fax: +1 314 977 365. these studies resolved Tinca as more closely related to members E-mail address: [email protected] (W.-J. Chen). 1055-7903/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2009.01.006 Author's personal copy W.-J. Chen, R.L. Mayden / Molecular Phylogenetics and Evolution 52 (2009) 544–549 545 phylogenetic position of the enigmatic genera Psilorhynchus (the stone carps), Tinca (the tench), and Leptobabrus (the mad barb or sultan fish) in relation to other cyprinid fishes. These enigmatic genera have received little attention from molecular systematists and have been difficult to place within the current cypriniform classification, likely because of morphological diver- gence. DNA sequence data were generated from six nuclear gene loci (RAG1, Rhodopsin, IRBP, EGR1, EGR2B, and EGR3). These gene markers have recently been shown to be phylogenetically informative in reconstructing the phylogenetic relationships of ray-finned fishes, particularly among fishes of the order Cyprin- iformes (Chen et al., 2008). 2. Materials and methods 2.1. DNA data collection A total of 54 samples were included for investigation. The ana- lytical dataset was composed of DNA sequences of 6 targeted nuclear loci obtained from 2 Psilorhynchus species, Tinca tinca, Leptobarbus hoevenii, 45 other diverse specimens of cyprinids from all recognized subfamily groups, and five outgroups from the superfamily Cobitoidea. Several sequences used in this study have been previously described in Mayden et al. (2008) and Chen et al. (2008). Methods for collecting new DNA data from the specimens and/or gene loci followed the procedures outlined in Chen et al. (2008). The GenBank accession numbers of corresponding gene sequences used in this study are listed in the Table 1. 2.2. Phylogenetic analysis Phylogenetic analyses were based on a partitioned Maximum Likelihood (ML) method and partitioned Bayesian approach (BA) Fig. 1. Previous (A–E) and present (F) hypotheses depicting relationships of major for two different types of character matrices as implemented in lineages within the Cyprinidae. (A) Cavender and Coburn’s (1992) hypothesis based the parallel version of RAxML (version 7.0.4) (Stamatakis, 2006) on 47 morphological characters; (B) Most-parsimonious tree found by Cavender and MrBayes (version 3.1.1) (Huelsenbeck and Ronquist, 2001), and Coburn (1992) based on morphological matrix of Chen et al. (1984); (C) Gilles et al.’s (2001) hypothesis based on 1374 aligned nucleotides from mt-DNA respectively. The first matrix was composed of all available charac- sequences of 16S, D-loop and cytochrome b genes; (D) Liu and Chen’s (2003) ters without employing a particular weighting scheme. As phylo- hypothesis based on 1051 aligned nucleotides from mt-DNA sequences of D-loop; genetic analyses of protein-coding genes can be biased from (E) Saitoh et al.’s (2006) hypothesis based on 14,563 aligned nucleotides from homoplasy at third codon positions due to multiple substitutions whole mt-genomic sequences; (F) Present hypothesis from this study based on 5733 aligned nucleotides from six nuclear genes with suggesting revised phyloge- in transitions (Saitoh et al., 2006) and/or because of base composi- netic classification for the family (see: discussion). The three enigmatic taxa are tion biases across taxa (Chen et al., 2003; Lockhart et al., 1994), a highlighted. Leuciscinae(-dae) here includes leuciscines and phoxinines. Following second matrix (partial RY-coding matrix) was prepared according results of previous studies of morphology and molecules, Cultrinae(-dae) should to the results obtained from absolute saturation tests (Philippe include cultrines, xenocyprines plus several other taxa suggested to be included to et al., 1994) and from v2 tests of base composition stationarity per- this group (see: discussion). formed using PAUPÃ-version 4.0b10 (Swofford, 2002). As outlined in our previous study, no clear saturation plateau on substitutions of the subfamilies Acheilognathinae, Gobioninae, and Cultrinae in transitions at the third codon position of six nuclear genes used than to members of the Rasborinae or Cyprininae (Fig. 1C and D). here was detected by comparing the sequences recovering all main More recently, using whole mitochondrial genomes, Saitoh et al. lineages of cypriniform species (see Fig. 2 in Chen et al., 2008). (2006) provided a robust phylogenic hypothesis for the main cyp- However, the tests of base composition stationarity revealed that riniform lineages for the first time. In their hypothesis, two recipro- the Rhodopsin dataset exhibits significant base composition bias cal monophyletic groups were resolved: Cobitoidea and across taxa when analyzed using variable sites only and the sites Cyprinoidea (or Cyprinidae). Their hypothetical relationships of at third codon position for the tests. Thus, we complied an opera- main cyprind clades were summarized in Fig. 1E. Even if a larger tional dataset in which the nucleotides A and G and the nucleotides number of characters (e.g. complete mitochondrial genome data) T and C at the third codon position of Rhodopsin were converted was used, it has limitations to reaching a resolution of certain rela- into purine (R) and pyrimidine (Y), respectively. tionships within the family and some of critical and/or ambigu- Search for optimal ML trees and Bayesian analyses were per- ously classified taxa such as Psilorhynchus and Leptobabrus were formed by a high performance cluster computing facility (with missing in the analysis (Fig. 1). Moreover, the resulting hypotheses 32 nodes) located at Saint Louis University. We used mixed model require further testing with more intensive taxonomic sampling analysis, which allows an individual model of nucleotide substitu- and

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