Journal of Fish Biology (2002) 61, 929–944 doi:10.1006/jfbi.2002.2105, available online at http://www.idealibrary.com on Phylogenetic relationships of Eurasian and American cyprinids using cytochrome b sequences C. C*, N. M*, T. E. D†, A. G‡ M. M. C*§ *Centro de Biologia Ambiental, Departamento de Zoologia e Antropologia, Faculdade de Cieˆncia de Lisboa, Campo Grande, Bloco C2, 3 Piso. 1749-016 Lisboa, Portugal, †Department of Biology, Arizona State University, Tempe, Arizona 85287-1501, U.S.A. and ‡Laboratoire d’Hydrobiology, Universite´ de Provence, 1 Place Victor Hugo, 1331 Marseille, France (Received 30 January 2002, Accepted 6 August 2002) Neighbour-joining and parsimony analyses identified five lineages of cyprinids: (1) European leuciscins (including Notemigonus)+North American phoxinins (including Phoxinus phoxinus); (2) European gobionins+Pseudorasbora; (3) primarily Asian groups [cultrins+acheilognathins+ gobionins (excluding Abbotina)+xenocyprinins]; (4) Abbottina+Sinocyclocheilus+Acrossocheilus; (5) cyprinins [excluding Sinocyclocheilus and Acrossocheilus]+barbins+labeonins. Relationships among these lineages and the enigmatic taxa Rhodeus were not well-resolved. Tests of mono- phyly of subfamilies and previously proposed relationships were examined by constraining cytochrome b sequences data to fit previous hypotheses. The analysis of constrained trees indicated that sequence data were not consistent with most previously proposed relationships. Inconsistency was largely attributable to Asian taxa, such as Xenocypris and Xenocyprioides. Improved understanding of historical and taxonomic relationships in Cyprinidae will require further morphological and molecular studies on Asian cyprinids and taxa representative of the diversity found in Africa. 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved. Key words: Cyprinidae; molecular phylogeny; cytochrome b; monophyly of subfamilies. INTRODUCTION The Cyprinidae family is one of the most widespread, diverse families of freshwater fishes. It consists of >340 genera and 2000 species (Banarescu & Coad, 1991) and naturally occurs in almost all types of habitats on all continents except for Australia and South America (Banarescu & Coad, 1991; Howes, 1991). These features make it an excellent group for a diversity of biological studies. Previous morphological studies failed to reach a consensus regarding the number and monophyly of subfamilies (Fig. 1). Morphological studies (Chen et al., 1984; Howes, 1991; Cavender & Coburn, 1992) supported the division of Cyprinidae into two major lineages, but there was disagreement over taxonomic alignment. Chen et al. (1984) identified two lineages: the series Leuciscini (containing the subfamilies Danioninae, Leuciscinae, Cultrinae, Xenocyprinae, Gobioninae and Acheilognathinae) and series Barbini (consisting of the §Author to whom correspondence should be addressed. Tel.: +351 217500000 (ext.: 22314); fax: +351 217500028; email: [email protected] 929 0022–1112/02/010929+16 $35.00/0 2002 The Fisheries Society of the British Isles. Published by Elsevier Science Ltd. All rights reserved. 930 . . Cyprininae Gobioninae Rasborinae Alburninae Cultrinae Leuciscinae Acheilognathinae Barbinae Cyprininae Labeoninae Tincinae Danioninae Leuciscinae Cultrinae Xenocyprinae Gobioninae Acheilognathinae Barbelled Barbel- lacking Cyprinines Leuciscines Series Barbini Series Leuciscini (a) (b) Barbins Cyprinins Labeonins Rasborins Gobionins Acheilognathins Xenocyprins Cultrins Leuciscins Phoxinins Tincins Subfamily Cyprininae Subfamily Leuciscinae (c) F. 1. Hypothesized relationships among cyprinid groups based on morphological characters: (a) Chen et al. (1984);(b)Howes (1991); (c) Cavender & Coburn (1992). Tincinae, Barbinae, Cyprininae and Labeoninae). Howes (1991) identified the cyprinines (Cyprininae, Gobioninae and Rasborinae) and the leuciscines (Leuciscinae, Alburninae, Cultrinae and Acheilognathinae) based on the presence and absence (sporadic in some groups) of barbels. It was unclear, however, whether the presence of this character is plesiomorphic or apomorphic. Results presented by Cavender & Coburn (1992) partially agreed with those of Chen et al. (1984). They proposed that cyprinids fell in two subfamilies, Leuciscinae (including the phoxinins, leuciscins, cultrins, xenocyprins, acheilognathins, gobionins, rasborins and tincins) and Cyprininae (including the barbins, cyprinins and labeonins). The leuciscins included most Eurasian cyprinids and the monotypic North American genus Notemigonus with the remaining North American forms placed in the phoxinin lineage. 931 Cyprinids have recently been the focus of molecular studies addressing the evolutionary history of the family and the validity of its subfamilies (Zardoya & Doadrio, 1999; Tsigenopulos & Berrebi, 2000; Gilles et al., 2001; Machordom & Doadrio, 2001). Recently, phylogenetic studies of mainly European cyprinids based on the mitochondrial genes cytochrome b (cytb), D-loop and 16S rRNA sequences (Briolay et al., 1998; Gilles et al., 1998, 2001; Zardoya & Doadrio, 1998, 1999) presented results inconsistent with recognition of several of these subfamilies. Briolay et al. (1998) found a radiation of six subfamilies [Leuciscinae, including Alburninae sensu Howes (1991), Rasborinae, Gobioninae, Tincinae, Acheilognathinae, Cyprininae] rather than an early split between barbelled and barbel-lacking lineages, consistent with several origins of barbels during cyprinid evolution. In all morphological and molecular studies, the relative positions of Gobio and Tinca were unresolved, with these taxa considered as Leuciscinae, or referred to the subfamilies Gobioninae and Tincinae, respectively. Zardoya & Doadrio (1999) considered cyprinids to contain two subfamilies: Cyprininae (including barbins) and Leuciscinae (includ- ing cultrins, tincins, gobionins, phoxinins and alburnins+leuciscins). The results obtained by Gilles et al. (2001) are mostly in agreement with those of Briolay et al. (1998) and Zardoya & Doadrio (1999). This study, however, considered the Rasborinae as the most basal cyprinid subfamily and did not consider the Tincinae and the Acheilognathinae as a sister group of the Cyprininae. Some authors (Briolay et al., 1998; Gilles et al., 1998) included a few North American and Asian species, however, the restricted availability of sequences limited their ability to examine historical relationships. The present study is the first attempt to infer evolutionary relationships of cyprinids based on a consider- able number and diversity of representatives from genera representing diversity in Asia, Europe and North America. Sequences from the cyt b gene were used to estimate phylogenetic relationships and examine previously hypothesized relationships presented by Chen et al. (1984), Howes (1991) and Cavender & Coburn (1992), based on morphology. MATERIAL AND METHODS Several specimens of European (33), Asian (38), North American (18) cyprinids and outgroups [Crossostoma lacustre Steindachner, Balitoridae; Myxocyprinus asiaticus (Bleeker), Catostomidae] were sampled or were taken from published databases (Table I). Total DNA was extracted from fins preserved in absolute ethanol or from frozen muscle tissue using standard methods (Sambrook et al., 1989). Cyt b was amplified using primers reported elsewhere (Schmidt & Gold, 1993; Alves et al., 1997; Brito et al., 1997; Dowling & Naylor, 1997). Amplification conditions varied, but generally fit the following profile: 20–25 cycles of 1 min at 94 C, 1 min 48–50 C; 2 min at 72 C. Reactions were performed in 25–50 l volumes containing 25–50 ng of template DNA, and 0·5 of each primer. PCR products were purified with QIAquick PCR Purification Kit (Quiagen) or Millipore centrifugation tubes. Sequences were generated with an automated sequencer (Genome Express or ASU). In European sequences, both strands were sequenced to control sequence accuracy. In American sequences only one strand was sequenced, however, there was several hundred bases pairs overlap between fragments. DNA sequences were aligned manually using DNASIS (Macintosh ver. 2.0) and translated into amino acids using McClade v.3.08a (Maddison & Maddison, 1992). No T I. List of cyprinid species analysed in the present study. EMBL access numbers (*present study), specimens’ geographic origin (**data not available) and morphological classification considered by Chen et al. (1984), Howes (1991) and Cavender & Coburn (1992) are presented in this table Cavender & Chen et al. (1984) Howes (1991) Taxa EMBL no. Drainage Origin Coburn (1992) subfamilies subfamilies lineages Abbottina rivularis AF051856 ** Asia Gobioninae Gobioninae Gobionins Abramis brama Y10441 Saoˆne Europe Leuciscinae Leuciscinae Leuciscins Acheilognathus chankaensis AF051854 ** Asia Acheilognathinae Acheilognathinae Acheilognathins Acrocheilus alutaceus AF452076* Columbia North America Leuciscinae Leuciscinae Phoxinins Acrossocheilus yunnanensis AF051857 ** Asia Barbinae Cyprininae Cyprinins Alburnoides bipunctatus Y10445 Saoˆne Europe Leuciscinae Alburninae Leuciscins Alburnus alburnus Y10443 Rhoˆne Europe Leuciscinae Alburninae Leuciscins Alburnus filippi AF095602 Samur Europe Leuciscinae Alburninae Leuciscins Anaecypris hispanica AJ439881* Guadiana Europe Leuciscinae Leuciscinae Leuciscins Aulopyge hyegelii AF112133 Krka Europe Barbinae Cyprininae Barbins Barbus brachycephalus AF095603 Terek Europe Barbinae Cyprininae Barbins Barbus caninus AF112124 Astico Europe Barbinae Cyprininae