Available online at www.sciencedirect.com Molecular Phylogenetics and Evolution 48 (2008) 1244–1251 www.elsevier.com/locate/ympev Short Communication Broad-scale phylogeography of the Palearctic freshwater fish Cottus poecilopus complex (Pisces: Cottidae) Ryota Yokoyama a,*, Valentina G. Sideleva b, Sergei V. Shedko c, Akira Goto a a Field Science Center for Northern Biosphere, Hokkaido University, Minatocho 3-1-1, Hakodate, Hokkaido 041-8611, Japan b Zoological Institute, Russian Academy of Sciences, Saint Petersburg 199034, Russia c Institute of Biology and Soil Sciences, Far East Division, Russian Academy of Sciences, Vladivostok 690022, Russia Received 30 January 2008; accepted 6 February 2008 Available online 14 February 2008 1. Introduction The alpine bullhead, Cottus poecilopus (Pisces: Cottidae), is a typical Palearctic fish species widely distributed in Eur- Because freshwater fishes only use freshwater drain- ope, major rivers flowing into the Arctic Sea from Scandina- ages for their colonization, phylogeographic patterns of via to Chaun, Amur River, Primorye and Sakhalin (Berg, them have been interpreted in conjunction with physical 1949). Previous studies of Cottus species in Europe (C. gobio) evidence of historical drainage patterns. The phylogeog- and Japan (C. nozawae) contributed to the reconstruction of raphy of freshwater fishes has an important role in the formation of ichthyofauna in respective regions (e.g., understanding the formation of regional biodiversity. In Englbrecht et al., 2000; Sˇlechtova´ et al., 2004; Volckaert North America and Europe, numerous studies of phylo- et al., 2002; Yokoyama and Goto, 2002). Therefore, widely geography of freshwater fishes have clarified lineage dis- distributed fish, C. poecilopus is a suitable species to study tributions and colonization histories of them with the phylogeography of animals in northern Eurasia. In the reference to drastic disturbance during glacial cycles in previous studies, C. poecilopus is shown to be a highly poly- Pleistocene (reviewed in Bernatchez and Wilson, 1998; morphic (Yokoyama and Goto, 2005) and polytypic taxon Hewitt, 2004). In contrast to North America and Eur- (so-called C. poecilopus complex). The endemic species ope, few studies have been on fishes in Siberia and C. volki from southern Primorye, formerly known as C. poe- northeastern Asia. Phylogeographic patterns of Siberian cilopus volki, is the sister species of C. poecilopus and fishes were partially discussed in studies of Holarctic regarded as a member of C. poecilopus complex (Shedko fishes, such as of Arctic charr (Brunner et al., 2001), and Miroshnichenko, 2007). Since comprehensive studies whitefish (Bernatchez and Dodson, 1994) and burbot of C. poecilopus complex using either molecular or morpho- (Van Houdt et al., 2003). In these studies, populations logical data are scarce, the taxonomy of C. poecilopus com- in Siberia comprise a single lineage (Siberian lineage) plex remains uncertain. Molecular approaches to study the that has less diversity among populations. These studies C. poecilopus complex, including C. volki, will be useful to suggest simpler phylogeography of fishes in Siberia investigate their phylogeographic patterns and to identify despite their geographical width. On the other hand, the taxonomic unit. the Arctic grayling, Thymallus arcticus, is genetically In this study, the phylogeography of the C. poecilopus polymorphic in Siberia, suggesting the existence of sev- complex in Eurasia is inferred from mitochondrial DNA eral refugia in Siberia (Froufe et al., 2003, 2005; Weiss control region sequences. Samples were obtained from et al., 2006). General patterns of freshwater fish phyloge- major drainage systems or regions across its range. The ography in Siberia seem to be far from resolved. phylogenetic position of C. poecilopus complex among other Cottus species in Eurasia is inferred by integration with the previous study (Yokoyama and Goto, 2005). * Corresponding author. Fax: +81 138 40 5537. Based on the phylogenetic relationships, implications for E-mail address: yokoryo@fish.hokudai.ac.jp (R. Yokoyama). taxonomic problems are briefly discussed. 1055-7903/$ - see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2008.02.002 R. Yokoyama et al. / Molecular Phylogenetics and Evolution 48 (2008) 1244–1251 1245 2. Materials and methods ki from Primorye were included in the analysis as part of the C. poecilopus complex. We used specimens representing con- 2.1. Samples generic species from the Eurasian continent (Table 1). Addi- tionally, sequences of the other Eurasian cottoid species were Cottus poecilopus samples were collected from Europe, taken from DDBJ/EMBL/Genbank (Table 1) and also Ob River, Lena River, Kolyma River, Amur River, Maga- included in the following analysis. Sequences of Trachider- dan region, Primorye region and Sakhalin Island across its mus fasciatus and C. kazika were used as outgroups, accord- major distribution area (Table 1, Fig. 1). Specimens of C. vol- ing to Yokoyama and Goto (2005). Table 1 Sample locations including site number, number of individuals analyzed, and haplotypes detected Species Site No. Site Drainage (region) N Haplotypes detected (n) Cottus poecilopus complex Cottus poecilopus 1 Dura stream Odra River 11 CP1(7), CP2(4) 2 Jurablikha River Irtysh River, Ob River 6 CP3(3), CP4(2), CP5(1) 3 Yakutsk Lena River 2 CP6(1), CP7(1) 4 Kolyma River Kolyma River 5 CP8(2), CP9(1), CP10(1), CP11(1) 5 Rech’ka River (Magadan region) 4 CP12(4) 6 Dukcha River (Magadan region) 5 CP13(3), CP14(1), CP15(1) 7 Ola River (Magadan region) 4 CP13(4) 8 Agutsua River Onon River, Amur River 8 CP16(1), CP17(5), CP18(1), CP19(1) 9 Bukukun River Onon River, Amur River 5 CP17(3), CP19(1), CP20(1) 10 Enda River Onon River, Amur River 10 CP17(5), CP20(1), CP21(1), CP22(1), CP23(2) 11 Kyra River Onon River, Amur River 5 CP17(3), CP19(1), CP24(1) 12 Ingoda River Ingoda River, Amur River 5 CP17(3), CP19(1), CP25(1) 13 Nikishika River Ingoda River, Amur River 5 CP19(1), CP26(2), CP27(1), CP28(1) 14 Chita Ingoda River, Amur River 8 CP19(1), CP27(3), CP28(1), CP29(2), CP30(1) 15 Bagbos River Amur River 3 CP31(2), CP32(1) 16 Polovinka River Amur River 8 CP33(3), CP34(2), CP35(3) 17 Manoma River Anui River, Amur River 2 CP37(2) 18 Kamen River Matai River, Amur River 5 CP33(3), CP34(1), CP36(1) 19 Maksimovka River (Primorye) 6 CP38(2), CP39(2), CP40(2) 20 Koppi River (Primorye) 5 CP41(5) 21 Sakhalin (Sakhalin) 4 CP42(1), CP43(1), CP44(2) C. volki 22 Serebryanka River (Primorye) 2 CV5(2) 23 Kievka River (Primorye) 5 CV4(5) 24 Avvakumovka River (Primorye) 4 CV1(2), CV2(1), CV3(1) Eurasian cottoids C. sibiricus 25 Irtysh River Ob River 3 CSB1(1), CSB2(1), CSB3(1) 26 Yakutsk Lena River 10 CSB4(6), CSB5(2), CSB6(2) C. cognatus 27 Kanchalan River (Chukotka) 1 CCG1(1) 28 Velikayar River (Chukotka) 1 CCG1(1) 29 Anadyr River (Chukotka) 1 CCG1(1) C. czerskii 30 Avvakumovka River (Primorye) 1 CCZ2(1) 31 Kievka River (Primorye) 4 CCZ2(2), CCZ4(1), CCZ6(1) 32 Litovka River (Primorye) 3 CCZ2(3) 33 Barbashevka River (Primorye) 4 CCZ2(2), CCZ3(1), CCZ5(1) Sedanka River (Primorye) AB059350a C. gobio Radunia River Visla River AB188168b C. pollux large egg type Kinu River (Honshu, Japan) AB188158b C. pollux middle egg type Amanogawa River (Hokkaido, Japan) AB188159b C. pollux small egg type Inabe River (Honshu, Japan) AB188160b C. reinii Lake Biwa (Honshu, Japan) AB188161b C. hangiongensis Moheji River (Hokkaido, Japan) AB188163b C. koreanus Kongnim River Namhan River (Korea) AB188167b C. amblystomopsis Tokotan River (Hokkaido, Japan) AB188162b C. nozawae Otofuke River (Hokkaido, Japan) AB059335a C. kazika Gakko River (Honshu, Japan) AB188157b Trachidermus fasciatus Fukanomi River (Kyushu, Japan) AB188172b N: number of individuals analyzed; n: number of individuals sharing a given haplotype. a Yokoyama and Goto (2002). b Yokoyama and Goto (2005). 1246 R. Yokoyama et al. / Molecular Phylogenetics and Evolution 48 (2008) 1244–1251 27 28 29 Kolyma R. 1 4 5, 6, 7 Lena R. 3 Ob R. 26 21 15,17 20 Amur R.16,18 19 12,13,14 22 8,9, 23,24 25 2 10,11 30,31 32,33 Fig. 1. Geographic distribution of sampling sites: Cottus poecilopus (closed circles), C. volki (open circles), other Cottus species (closed triangles). The shaded and dotted areas on the map indicate the distribution area of C. poecilopus and C. volki, respectively. Locality numbers correspond to those in Table 1. 2.2. DNA extraction, PCR amplification, and sequencing using heuristic searches with TBR branch swapping and random addition of taxa. The Akaike information crite- Genomic DNA was extracted from fin or muscle tissue rion implemented in ModelTest 3.7 (Posada and Crand- using the method described in Yokoyama and Goto all, 1998) was used to determine the best fitting model of (2005). The mitochondrial control region was amplified molecular evolution and parameter values for the follow- using primers L-Thr and H12Sr5 (Yokoyama and Goto, ing analysis. The general time reversible (GTR) model 2002). In some cases, CotL1 (Sˇlechtova´ et al., 2004) was with proportion of sites assumed to be invariable (I, used instead of L-Thr. The PCR conditions followed 0.42) and variable sites assumed to follow a discrete Yokoyama and Goto (2002). The PCR products were puri- gamma distribution (C, 0.63) was selected as the best fied by Exo-SAP IT (Amersham Pharmacia). Sequencing fitting model. NJ tree was constructed by using the was performed by using primers CotL1 and H12Sr5, and GTR + I + C model. The ML analysis was done using internal primers LCCR and H16498m (Yokoyama and heuristic algorithm with the GTR + I + C model and Goto, 2002). All sequencing reactions were prepared by estimated parameters (estimated nucleotide frequencies: using the BigDye Terminator v3.1 Cycle Sequencing Kit A = 0.2988, C = 0.2139, G = 0.1753, and T = 0.3120; (Applied Biosystems) and were analyzed by using an ABI nucleotide substitution rate matrix: A–C = 0.8425, A– PRISM 310 Genetic Analyzer (Applied Biosystems).