Heredity (2002) 88, 466–473 2002 Nature Publishing Group All rights reserved 0018-067X/02 $25.00 www.nature.com/hdy Multiple origins of polyploidy in the phylogeny of southern African barbs (Cyprinidae) as inferred from mtDNA markers CS Tsigenopoulos1,4,PRa´b2, D Naran3 and P Berrebi1,5 1Laboratoire Ge´nome, Populations et Interactions, CNRS UMR 5000, cc063, Universite´ Montpellier 2, place E. Bataillon, 34095 Montpellier, cedex 05, France; 2Laboratory of Fish Genetics, Institute of Animal Physiology and Genetics, AS CR, 277 21 Libe˘chov, Czech Republic; 3JLB Smith Institute of Ichthyology, Private Bag 1015, Grahamstown 6140, South Africa The cyprinid genus Barbus, with more than 800 nominal spec- indicate that most of the lineages are incorrectly classified in ies, is an apparently polyphyletic assemblage to which a num- the genus ‘Barbus’. The southern African tetraploids probably ber of unrelated species, groups and/or assemblages have originated from southern African diploids. They constitute a been assigned. It includes species that exhibit three different monophyletic group distinct from tetraploids occurring in the ploidy levels: diploid, tetraploid and hexaploid. Several lineages Euro-Mediterranean region (Barbus sensu stricto). The ‘small’ of the family Cyprinidae constitute a major component of the African diploid species seem to be paraphyletic, while the ‘large’ African freshwater ichthyofauna, having about 500 species, and African hexaploid barbs species are of a single, recent origin fishes assigned to the genus ‘Barbus’ have the most species and form a monophyletic group. The evidence of multiple, inde- on the continent. We used complete sequences of the mito- pendent origins of polyploidy occurring in the African cyprinine chondrial cytochrome b gene in order to infer phylogenetic cyprinids thus provides a significant contribution to the knowl- relationships between diploid, tetraploid and hexaploid species edge on the systematic diversity of these fishes, and warrants of ‘Barbus’ occurring in southern Africa, the only region where a thorough taxonomic reorganization of the genus. representatives of all of the three ploidy levels occur. The results Heredity (2002) 88, 466–473. DOI: 10.1038/sj/hdy/6800080 Keywords: Barbus; Cyprinidae; mitochondrial DNA; cytochrome b; polyploidy Introduction Nevertheless polyploidy, when established in the past (generally several million years ago), obviously gives the Polyploidy is considered as an important evolutionary concerned lineage an added impetus in terms of spe- mechanism for diversification and speciation in eukary- ciation and dispersion. In fish in particular, the evolution- otes (Ohno, 1970; Soltis and Soltis, 1999). While poly- ary success of groups such as catostomids (Buth, 1983), ploidy by means of hybridization and/or genome salmonids (Allendorf and Thorgaard, 1984) and Barbus duplication has been documented to be an extremely sig- (Berrebi et al, 1996) can be understood as a consequence nificant evolutionary mechanism by botanists, zoologists of ancestral polyploidization. Polyploids are considered have not seriously considered its importance as an evol- to have more tolerance to ecological variation because the utionary process, mostly due to the lack of relevant data duplication of their genes provides metabolic flexibility (Dowling and Secor, 1997; Otto and Whitton, 2000). The (Uyeno and Smith, 1972; Otto and Whitton, 2000). relative paucity of polyploidy in animals has been In fish, published data-sets have shown, however, observed and first explained by its interference in sex that bisexual and/or unisexual polyploids occur in determination (Mu¨ller, 1925) and more precisely in sex representatives of several fish orders: Acipenseri- ratio (Basolo, 1994). For a review of additional factors formes, Salmoniformes, several families of ostariophy- contributing to the rarity of polyploidy in animals see san orders Cypriniformes (Cyprinidae, Catostomidae, Otto and Whitton (2000). Cobitidae) Siluriformes (Callichthyidae), Poeciliiformes (Poeciliidae) and Atheriniformes (Vasil’ev, 1985; Klink- hardt et al, 1995). One of the vertebrate groups where Correspondence: P Berrebi, Laboratoire Ecosyste`mes Lagunaires, CNRS polyploidy mechanisms have undoubtedly played a con- UMR 5119, cc093, Universite´ Montpellier 2, place E. Bataillon, 34095 spicuously significant role during its evolution is the fam- Montpellier, cedex 05, France. E-mail: berrebiȰcrit.univ-montp2.fr ily Cyprinidae (Ohno et al, 1967). 4Present address: Department of Genetics and Molecular Biotechn- The family Cyprinidae with more than 2000 species ology, Institute of Marine Biology of Crete (IMBC), PO Box 2214, (Nelson, 1994) is the most abundant and widespread of 71003 Heraklion, Greece all primary freshwater fish families across Europe, Asia, 5Present address: Laboratoire Ecosyste`mes Lagunaires, CNRS UMR 5119, cc093, Universite´ Montpellier 2, place E. Bataillon, 34095 Africa and North America. The cyprinid genus Barbus, Montpellier, cedex 05, France with more than 800 nominal species, is an apparently Received 9 July 2001; accepted 15 February 2002 polyphyletic assemblage to which a number of unrelated Multiple polyploidy in southern African barbs CS Tsigenopoulos et al 467 species, groups and/or assemblages have been assigned Materials and methods (Myers, 1960). In Africa, the fishes classified into the genus ‘Barbus’ are ubiquitous with more than 300 species Taxa examined (Le´veˆque and Daget, 1984; Skelton, 1988; Skelton et al, Table 1 shows the taxa used in the analysis, the collection 1991), distributed in all the African ichthyologic prov- sites and the chromosome numbers (when known). The inces (Roberts, 1975). The taxonomy of these fishes, fish were collected by electrofishing or nets. Figure 1 especially in Africa is still muddled (Skelton, 1988; shows the localities of a total of 47 selected cyprinid spec- Berrebi et al, 1996) because the morphological character- ies from Europe, Africa and Asia. The complete cyto- istics that have been used to group the species have very chrome b sequences of already karyotyped Euro–Medit- low systematic value. For example, the number of pairs erranean species of Barbus (Tsigenopoulos and Berrebi, of barbels and the presence/absence of ossified and/or 2000) as well as other cyprinid species recovered from serrated rays in the dorsal fin were considered as discrim- the GenBank/EMBL (see accession numbers in brackets inant characters, but in fact, they do not allow these spec- in Table 1) were included. In order to check variability ies to be classified into proper, systematically well- in each species and to test for misidentifications due to defined genera (Howes, 1987, 1991; Banarescu, 1992; PCR-generated errors, at least three individuals per taxon Berrebi et al, 1996). were analyzed when available. Phylogenetic trees were Recently, ploidy level has been proposed as a new key rooted using sequences of three species from the closely character for a reorganization of such a large group related cypriniform family Catostomidae. (Berrebi et al, 1996). Until now, three ploidy levels were According to Berrebi et al (1996), only the species recognized in African species of cyprinine cyprinids belonging to the genus Barbus sensu stricto are true Barbus, including the European and peri-Mediterranean tetra- (sensu Howes, 1991): diploid (about 50 chromosomes), ploid species. All the Barbus sensu lato species still cur- tetraploid (about 100 chromosomes), and hexaploid rently called Barbus have to be called ‘Barbus’ (in single (about 150 chromosomes) (Agne`se et al, 1990; Berrebi et quotation marks) until a taxonomic analysis elucidates al, 1990, 1996; Oellerman and Skelton, 1990; Golubtsov their systematic position. and Krysanov, 1993; Gue´gan et al, 1995; Ra´b et al, 1995; Naran, 1997). DNA extraction, polymerase chain reaction (PCR) The only non-Mediterranean African tetraploid species amplification and sequencing = with the chromosome numbers 2n 100 were recently Samples were either muscle preserved in 70% ethanol or identified through analyses of karyotypes among a num- frozen (at −80°C) protein extracts, which were already ber of endemic barb species, also classified into the genus available in the Montpellier laboratory from previous ‘Barbus’ (Naran, 1997) in the southern African region. allozyme studies (Agne`se et al, 1990; Berrebi et al, 1990). This discovery naturally poses the question of the mutual DNA extraction followed the Sambrook et al (1989) phylogenetic relationships with another tetraploid group method adapted in Tsigenopoulos and Berrebi (2000). of barbs inhabiting the Euro-Mediterranean region. The For PCR amplifications we used primers L15267 (5Ј- tetraploid status of the most widely distributed European AAT GAC TTG AAG AAC CAC CGT-3Ј), L15803 (5Ј- species B. barbus was first reported by Ohno et al (1967). TGA GGG GGA TTT TCA GTA GA-3Ј), H15891 (5Ј-GTT Recent studies (Ra´b and Collares-Pereira, 1995; Ra´b et al, TGA TCC CGT TTC GTG TA-3Ј) and H16461 (5Ј-CTT 1996) have suggested that this species is a member of a CGG ATT ACA AGA CC-34) (Briolay et al, 1998). The monophyletic lineage distributed mainly in Europe and L15803 primer was slightly modified from the original in adjacent regions of North-western Africa, the Middle definition. Numbers refer to the position of the 3Ј end of East and Caspian/Aral Seas (Berrebi, 1995; Berrebi et al, the primers in the complete mitochondrial DNA 1996). This hypothesis, initially based on morphological sequence of the carp Cyprinus