Molecular phylogeny of European and African Barbus and their West Asian relatives in the Cyprininae (Teleostei: Cypriniformes) and orogenesis of the Qinghai-Tibetan Plateau

WANG Jing 1 3 4 WU XiaoYun 1 2 3 4 CHEN ZiMing 0 1 3 YUE ZhaoPing 0 1 3 MA Wei 1 3 4 CHEN ShanYuan 1 3 4 XIAO Heng 0 1 3 MURPHY Robert W 1 2 3 5 ZHANG YaPing 1 2 3 4 ZAN RuiGuang 0 1 3 LUO Jing 1 3 4 0 School of Life Sciences, Yunnan University , Kunming 650091, China 1 The Cyprininae sensu Cavender and Coburn [1] contains three tribes: Cyprinini, Labeonini, and Barbini (including 2 State Key Laboratory of Genetic Resources and Evolution and Yunnan Laboratory of Molecular Biology of Domestic Animals, Kunming Institute of Zoology, Chinese Academy of Sciences , Kunming 650223, China 3 Barbus, cyprininae, molecular clock , Qinghai-Tibetan Plateau 4 Laboratory of Conservation and Utilization of Bio-resources and Key Laboratory for Animal Genetic Diversity and Evolution of High Education in Yunnan Province, School of Life Sciences, Yunnan University , Kunming 650091, China 5 Centre for Biodiversity and Conservation Biology, Department of Natural History, Royal Ontario Museum , 100 Queen's Park, Toronto, ON , M5S 2C6, Canada The phylogenetic relationships of European and African Barbus and their West Asian relatives in Cyprininae remain largely unresolved. Consequently, little is known about the drivers of their evolution, including the possible association of uplifting of the Qinghai-Tibetan Plateau (QTP) with the early divergence of the subfamily. We use complete sequence data of the mitochondrial DNA gene encoding the protein cytochrome b (Cytb) to hypothesize the phylogeny of 85 species belonging to 47 genera in the Cyprininae plus 6 species from the Leuciscinae. We employ 6 other species from Cypriniformes as outgroup taxa and estimate divergence times. Our results indicate that European Barbus sensu stricto lineage including Aulopyge shares a common ancestor with specialized and highly specialized schizothoracins and the genera Cyprinion and Scaphiodonichtys. The common ancestor appears to have originated in the Qinghai-Tibetan Plateau (QTP) region about 19.4-17.8 Ma. Barbus sensu stricto lineage appears to have originated about 16.6-15.5 Ma. Small to medium sized African Barbus sensu lato appear to have had an Oriental origin about 19.1-15.3 Ma and are closely related to Asian Puntius. West Asian Carasobarbus lineage including large African Barbus sensu lato might have originated about 9.94 Ma, also in Oriental Realm and has a close relationship to Asian Neolissochilus and Tor. The large-sized Barbus sensu lato appear to have diverged from Carasobarbus about 7.7 Ma. Finally, the Cyprininae appear to have radiated rapidly into nine lineages and many sublineages from about 27.8 to 17.8 Ma, close to the time of the second-stage tectonic movements of the QTP. Our analyses provide evidence that the uplifting of the QTP drove early diversification of the Cyprininae. Our extensive sampling of species involving all of the important areas results in clear evolutionary scenario for the Cyprininae. - the schizothoracines). It is one of the two largest lineages within Cyprinidae and was named series Barbini by some Chinese authors [24]. The subfamily has an extensive distribution in Eurasia and Africa and its members share similar morphological characteristics, such as the third neural The Author(s) 2013. This article is published with open access at Springerlink.com spine being a single plate without a dissociative (unbounded) supraneural and the anal fin having five branched rays. Morphology indicates monophyly of this group of fishes [4]. Genus Barbus, one of numerous genera forming the Cyprininae, once included several hundred species spread widely across Asia, Europe, and Africa. Thought to be a polyphyletic assemblage of species, it was often referred to as Barbus sensu lato [5], hereafter termed barbs. Nowadays, Asian species of barbs are in other genera (e.g. Puntius, Acrossocheilus, Tor) except for West Asian forms [68]. In Europe, all species of barbs and some species from North Africa and West Asia are in Barbus sensu stricto including subgenera Barbus and Luciobarbus [6,912]. The genera Aulopyge and Capoeta belong to the Barbus sensu stricto lineage and the Barbus sensu stricto respectively [9,13]. Africa barbs comprise 60.5% of the 477 African cyprinids [14] and these occur in two groups: large size and smallmedium size [5,13,14]. The large fishes now belong to West Asian Carasobarbus lineage and West Asia now has appears to have one species only of barbs [13]. Taxonomists do not present consistent viewpoints on the relationships of species of Euro-Mediterranean Barbus and African barbs, as well as for Cyprininae from China, Southeast Asia, and South Asia [6,15]. Previous molecular phylogenetic studies of Cyprinidae and Cypriniformes evaluate too few species to obtain an overall suite of relationships for the group, though these studies achieve their major objectives [1634]. Herein, we hypothesize the evolutionary relationships of European and African Barbus, their West Asian relatives, and Cyprininae in China, Southeast Asia, and South Asia. We assess 85 species belonging to 47 genera of Cyprininae as well as six species of Leuciscinae and five outgroup taxa. We estimate divergence times and investigate the biogeography of Barbus. We explore the possibility that orogenesis of the Qinghai-Tibetan Plateau (QTP) drove the early radiation of the Cyprininae. To maximize taxonomic representation, we employ data from the mitochondrial DNA (mtDNA) gene encoding cytochrome b (Cytb) because this marker is widely used in phylogenetic studies of these fishes and it is a useful marker for studying higher-level relationships of teleosts [10,13,16,19,20,3539]. Materials and methods Sample collection and DNA extraction Thirty one species were sampled de novo mainly from Yunnan, China (Table 1). Tissue samples were preserved in 95% ethanol and voucher specimens were deposited in the Zoological Museum of Yunnan University. Total DNA was extracted from muscle tissues using the standard phenol-chloroform extraction method. Sequences of cytb from 67 species were downloaded from GenBank (Accession numbers were listed as Table 1). PCR amplification and sequencing The complete sequences of Cytb were amplified with the primers L14724, L14737, and H15915 [33,40]. PCR amplifications were carried out in 50 L reaction mixture containing 5 L 10PCR buffer (TaKaRa, Japan), 0.2 mmol L1 dNTPs, 0.2 mol L1 each primer, with 1.5 U Taq DNA polymerase (TaKaRa) and approximately 50100 ng genomic DNA. Reactions involved 33 cycles, each including denaturation at 94C for 1 min, annealing at 52C for 1min, and extension at 72C for 1 min. PCR products were purified with Gel Extraction Mini Kit (Waston BioTechnologies). PCR products were sequenced in an ABI Prism 3730 (Applied Biosystems) automatic sequencer. Sequencing used both the PCR primers and internal primers (L15138, L15286, L15519, H15374, and H15560) [40,41]. All sequences were aligned using the MegAlign implemented in DNAStar 6.0 (DNASTAR, (...truncated)