Broad-Scale Phylogeography of the Palearctic Freshwater Fish Cottus

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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.

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

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). The G = 4.1189, A–T = 1.2555, C–G = 0.7870, C–T = 2.9193, nucleotide sequences have been submitted to the DDBJ/ and G–T = 1.000). In MP, NJ and ML analyses, the reli- EMBL/Genbank under Accession Nos. AB308477– ability of the internal branches was assessed by 1000 AB308537. bootstrap replicates. The Bayesian inference of phylogeny was done by using MrBayes 3.1.2 (Huelsenbeck and 2.3. Data analysis Ronquist, 2001) with the GTR + I + C model. The Mar- kov Chain Monte Carlo process was set for four chains Sequences were aligned using Clustal X (Thompson to run simultaneously for 10,000,000 generations, with et al., 1997) with default settings and were checked by sampling trees at every 100 generations. The first eye. Phylogenetic trees were constructed using maximum 25,000 trees were discarded in the computation of the parsimony (MP), neighbor-joining (NJ) and maximum majority-rule consensus tree. Posterior probabilities were likelihood (ML) methods implemented in PAUP* calculated by generating a 50% majority rule consensus 4.0b10 (Swofford, 2003). The MP analysis was done tree with the remaining trees. R. Yokoyama et al. / Molecular Phylogenetics and Evolution 48 (2008) 1244–1251 1247

3. Results an ancestral stock that was distributed throughout Eur- asian fresh water. The ancestral stock of the C. poecil- The sequencing of 116 individuals for C. poecilopus and opus complex had diverged into seven lineages in each 11 individuals for C. volki resulted in the length of 927– region by the end of the Pliocene (slow rate) or early- 932 bp, containing the entire control region (854–858 bp). mid Pleistocene (fast rate) (Table 2). In either case, isola- Forty-four and five haplotypes were found in C. poecilopus tion of these seven lineages would have been maintained and C. volki, respectively. Table 1 lists the geographic dis- during glacial cycles in the late Pleistocene that had a tributions of haplotypes. Most sampling sites had specific strong effect on freshwater fish distributions (e.g., Bernat- haplotypes. chez and Wilson, 1998). Together with the 24 control region sequences from 14 Cottus volki, endemic in southern Primorye, is distin- Eurasian Cottus species (Table 1), the alignment of the 73 guished from C. poecilopus by combination of several mor- sequences resulted in 945 bp with 231 parsimony informa- phological characters. The part of them is considered as tive sites. Because ML, MP, NJ and Bayesian analyses ancestral for the C. poecilopus complex (Shedko and Mir- resulted in similar topologies, the ML tree (ÀlnL = oshnichenko, 2007). Therefore, Shedko and Mir- 5618.66) is shown in Fig. 2. The haplotypes from the C. oshnichenko (2007) concluded that C. volki is a basal poecilopus complex comprised a monophyletic lineage with sister to C. poecilopus. In this study, using more compre- 69%, 86% and 93% bootstrap probabilities for ML, MP hensive samples, all phylogenetic reconstruction methods and NJ, respectively, and a 1.0 posterior probability for inferred that the C. volki lineage is a basal position to other the Bayesian analysis. The phylogeny of haplotypes lineages of C. poecilopus complex but with low statistical showed seven major lineages (lineages I–VI and the C. volki supports. C. volki lineage would be isolated initially from lineage) in the C. poecilopus complex (Fig. 2). The grouping the ancestral stock in the margin (southern Primorye) of of haplotypes corresponded to their geographic distribu- its distribution range. To resolve interrelationships of the tions: lineage I from Europe, lineage II from upper Irtysh, seven lineages, including the phylogenetic position of C. lineage III from Lena, lineage IV from Kolyma-Magadan, volki, we should analyze using datasets including further lineage V from upper Amur, lineage VI from middle Amur, phylogenetic information. Primorye and Sakhalin, and C. volki lineage from Pri- Lineage I consists of haplotypes from Odra River (Car- morye. In the C. poecilopus complex, the monophyly of pathian mountain stream). The distribution of C. poecil- each lineage was supported with high statistical values opus in Europe is restricted to two major regions, (>94% bootstrap and 1.0 posterior probabilities), except Scandinavia and Sudeten-Carpathia (Witkowski, 1979). for the C. volki lineage that had moderate statistical values The Carpathian populations including Odra and Vistula (>69% bootstrap and 0.97 posterior probabilities). All phy- (type locality of C. poecilopus) rivers are closely related logenetic methods showed that lineages V and VI formed a (Pas´ko and Mas´lak, 2003). Therefore, the samples in this sister group (>74% bootstrap probability and 1.0 posterior study would be regarded as representative of a main Euro- probability). The relationships among the other lineages in pean lineage (Carpathian lineage) and so is a true C. poecil- the C. poecilopus complex were not resolved confidently. opus lineage (Heckel, 1837). We could not determine if the Haplotypes of other Cottus species collected from the same Scandinavian populations correspond to the lineages of rivers or regions as C. poecilopus were distantly related to this study. Samples from Scandinavian populations should the C. poecilopus complex (Fig. 2). The mean pairwise dif- be included in the future study. ferences between lineages in C. poecilopus complex were The lineage II is in the upper Irtysh, a main tributary of the 3.4–5.6% (Table 2). Yokoyama and Goto (2005) estimated Ob River. We suggest that the upper Irtysh region is a refu- a rate of 1.0–2.4% per million years (MY) for a combined gium that enabled the survival of freshwater animals during data set of 12S rRNA and the control region. Assuming the Pleistocene. In glacial periods of the late Pleistocene, the linear relationship of mutation rate between 12S rRNA Barents-Kara ice sheet repeatedly formed along the conti- and control region, we obtained a rate of 1.4–3.4% per nental rim and blocked the outflow of the ancient Ob and MY for control region using the data set of Yokoyama Enisei rivers (e.g., Mangerud et al., 2004). As a result, the and Goto (2005). The rates 1.4% and 3.4% per MY were lower part of the Ob and Enisei rivers was widely covered used to estimate the age as slow and fast rates, respectively. with large ice-dammed lakes (West Siberian ice lake) during glacial periods. In even these disturbances, habitats of fresh- 4. Discussion water animals were maintained in the upper Irtysh. Alterna- tively, the West Siberian ice lake itself may be a refugium for 4.1. Major lineages in the C. poecilopus complex lineage II, as previously hypothesized in studies of west Sibe- rian and European fishes (e.g., Østbye et al., 2005). We could Allopatric distribution of each lineage and lack of not include samples from lower Ob and Enisei rivers that genetic mixing among lineages suggest a long-term isola- possibly originated from the West Siberian lake refugium. tion of the lineages in each river and region. The mono- In the future, the upper Irtysh refugium hypothesis should phyly of C. poecilopus complex within Eurasian Cottus be confirmed by comparing samples from lower Ob, Enisei fishes suggests that the regional lineages originated from and North Europe. 1248 R. Yokoyama et al. / Molecular Phylogenetics and Evolution 48 (2008) 1244–1251

100/100/100 CP1 Europe I 1.0 CP2 CP3 upper- 100/100/100 CP4 1.0 Irtysh II CP5 CP16 CP22 CP17 CP18 CP19 CP21 CP23 upper- CP26 Amur V CP30 CP24 CP27 90/98/100 CP28 1.0 CP29 CP20 CP25 74/84/87 1.0 CP31 CP32 CP37 middle- Cottus poecilopus CP33 98/98/99 Amur 1.0 CP34 complex CP35 CP36 VI CP38 CP39 Primorye CP40 CP41 69/86/93 CP42 55/61/56 1.0 CP43 Sakhalin 1.0 CP44 100/100/100 CP6 Lena 1.0 CP7 III CP8 CP10 CP9 Kolyma CP11 CP13 IV 97/100/100 CP14 Magadan 1.0 CP15 CP12 CV1 69/65/76 CV2 CV3 C. volki CV4 0.97 CV5 CCZ1 CCZ6 CCZ4 C. czerskii 100/100/100 CCZ2 1.0 CCZ3 88/82/72 CCZ5 1.0 85/99/100 C. reinii 95/100/100 97/98/98 1.0 C. pollux small egg type 1.0 1.0 98/95/99 C. pollux middle egg type 1.0 C. pollux large egg type 100/100/100 C. amblystomopsis 70/80/71 1.0 C. nozawae 1.0 82/92/91 C. koreanus 1.0 C. hangiongensis CSB1 CSB2 CSB3 C. sibiricus CSB4 CSB5 CSB6 C. gobio CCG1 C. cognatus C. kazika Trachidermus fasciatus 0.005 GTR+I+G

Fig. 2. Phylogenetic relationships based on control region sequences among Cottus poecilopus complex including C. volki, and other Cottus species from Eurasia. The tree represents the topology and branch lengths recovered in the maximum-likelihood analysis (ÀlnL = 5618.66). Numbers above and below the major branches indicate bootstrap percentages (>50%) of ML/MP/NJ analyses and posterior probabilities (>0.95) of Bayesian analysis, respectively.

Two individuals from middle course of the Lena River age III originated in the early Pleistocene–late Pliocene comprised lineage III. Based on its genetic diﬀerence, line- (2.43–3.5 MY ago by a slow rate or 1.0–1.44 MY ago by R. Yokoyama et al. / Molecular Phylogenetics and Evolution 48 (2008) 1244–1251 1249

Table 2 Mean pairwise differences (± standard deviation) between lineages below the diagonal and estimated divergence times (million years) above the diagonal I II III IV V VI C. volki I –– 3.86/1.59 3.14/1.29 2.57/1.06 3.36/1.38 3.64/1.50 2.64/1.09 II 0.054 ± 0.009 –– 2.93/1.21 3.64/1.50 2.64/1.09 3.57/1.47 3.36/1.38 III 0.044 ± 0.008 0.041 ± 0.007 –– 2.43/1.00 2.57/1.06 3.50/1.44 3.29/1.46 IV 0.036 ± 0.006 0.051 ± 0.007 0.034 ± 0.005 –– 2.71/1.12 3.50/1.44 2.57/1.06 V 0.047 ± 0.008 0.037 ± 0.006 0.036 ± 0.006 0.038 ± 0.007 –– 2.43/1.00 3.07/1.26 VI 0.051 ± 0.008 0.050 ± 0.007 0.049 ± 0.007 0.049 ± 0.007 0.034 ± 0.006 –– 4.00/1.59 C. volki 0.037 ± 0.006 0.047 ± 0.007 0.046 ± 0.007 0.036 ± 0.006 0.043 ± 0.007 0.056 ± 0.007 –– Divergence times are estimated using mutation rates of 1.4% and 3.4% per million years as slow and fast rates (slow/fast), respectively (see text for details). a fast rate). Weiss et al. (2006) reported the existence of endemic lineage T. grubii is distributed in the upper Amur endemic Thymallus lineages in the Lena River that have (Onon and Ingoda rivers), and the ‘‘orange-spotted gray- originated in the mid-Pliocene epoch (3.2 MY). The Lena ling” lineage is distributed in the middle-lower Amur and lineage of C. poecilopus and Thymallus must have survived adjacent rivers (Froufe et al., 2003, 2005). Froufe et al. in a refugium near the present middle reaches of the Lena (2003) proposed a split of the two lineages in the early River basin during glacial cycles in the Pleistocene, as sug- Pleistocene (1.4–1.6 MY ago). The phylogenetic pattern gested by Weiss et al. (2006). This similarity between the and time frame of divergence of Thymallus lineages in the two species suggests that a refugium would have existed Amur basin is very similar to those in the C. poecilopus in the Lena basin during the Pleistocene glacial cycle. complex. This similarity strongly suggests the existence of Haplotypes from Kolyma-Magadan region form line- common event(s) to isolate freshwater fish populations in age IV with large differences from other lineages. In the upper Amur region from the middle-lower Amur in many Holarctic animals, the Kolyma River is regarded the early Pleistocene. as the boundary or contact zone between Siberian and Generally, the phylogeography of Holarctic fishes in Beringian lineages (reviewed in Hewitt, 2004). Since Siberia show shallower divergence and simpler patterns C. poecilopus is absent in the Anadyr River and North (e.g., Bernatchez and Dodson, 1994; Brunner et al., America (Berg, 1949; Chereshnev, 1982) where Beringian 2001; Van Houdt et al., 2003). Siberian lamprey, Lethen- lineages should have dispersed (e.g., Bernatchez and Wil- teron reissneri complex shows few genetic differences son, 1998; Hewitt, 2004), considering Beringia as the between samples from Ob, Lena, Amur, Sakhalin and refugium for lineage IV seems unlikely. This suggests Japan (Yamazaki et al., 2006). These studies suggest the existence of a refugium for freshwater fishes in the extensive dispersal of Siberian fishes from a much Kolyma-Magadan area that differs from Beringian and reduced refugial source. Hewitt (2004) proposed that, other Siberian refugia. Although reconstruction of the even for cold-adapted species, life was hard in Central ice sheet history of northeastern Eurasia is controversial Asia during the ice age, providing few refugia. However, (e.g., Grosswald and Hughes, 2002), the refugium in the this study showed the existence of refugia in upper Kolyma-Magadan area was probably maintained during Irtysh, Lena and probably Kolyma-Magadan in Siberia Pleistocene glaciation cycles. Alternatively, lineage IV that enabled survival of boreal fishes during the Pleisto- might be part of the lineage that is widely distributed cene. The divergence pattern of C. poecilopus lineages throughout the Arctic rims of Eurasia, like Thymallus with respect to the river drainages that originated in late arcticus pallasii lineage (Weiss et al., 2006). To reveal Pliocene or early-mid Pleistocene is generally similar to the origin of lineage IV, further samples from the lower that of grayling Thymallus species (Froufe et al., 2005; part of rivers flowing to the Arctic Sea where the Pale- Weiss et al., 2006), suggesting common paleogeographic arctic lineage is possibly distributed (Froufe et al., effect(s) between Palearctic fishes. These results strongly 2005; Weiss et al., 2006) should be analyzed in the suggest a different evolutionary history of Palearctic future. fishes than by Holarctic fishes as generally accepted The most interesting result was the existence of two lin- (e.g., Bernatchez and Wilson, 1998). In future, compari- eages in the Amur River basin: the endemic lineage V in the son of other Palearctic fishes, such as Barbatula toni upper Amur (Onon and Ingoda rivers), and lineage VI in and Phoxinus species, would be fruitful to understand the middle Amur, Primorye and Sakhalin. The divergence biogeography in the north Eurasia. between the two lineages suggests that they split in 2.43 MY ago (slow rate) or 1 MY ago (fast rate). The upper 4.2. Implication for taxonomic problems Amur populations were isolated from the ancestral stock that was distributed throughout the paleo-Amur basin in The extent of sequence difference between seven lineages the early Pleistocene. Interestingly, the existence of sister in the C. poecilopus complex (3.4–5.6%) is relatively greater lineages in the Amur River basin was also reported for than those of C. gobio complex which consists of 14 species the grayling, Thymallus (Froufe et al., 2003, 2005). The (1.0–4.3%, Englbrecht et al., 2000; Volckaert et al., 2002). 1250 R. Yokoyama et al. / Molecular Phylogenetics and Evolution 48 (2008) 1244–1251

Our results suggest that each lineage corresponds to a tax- Teleostei: Cottidae) suggests a pre-Pleistocene origin of the major onomic category, probably the species rank. Lineage I is central European populations. Mol. Ecol. 9, 709–722. regarded as a representative of the true C. poecilopus line- Froufe, E., Knizhin, I., Koskinen, M.T., Primmer, C.R., Weiss, S., 2003. Identification of reproductively isolated lineages of Amur grayling age (Heckel, 1837). The sampling sites of lineages II and (Thymallus grubii Dybowski 1869): concordance between phenotypic III were in the same basins as the type localities of the nom- and genetic variation. Mol. Ecol. 12, 2345–2355. inal species, C. altaicus from upper Ob (Kaschenko, 1899) Froufe, E., Knizhin, I., Weiss, S., 2005. Phylogenetic analysis of the genus and C. kuznetzovi from Lena (Berg, 1903), respectively. Thymallus (grayling) based on mtDNA control region and ATPase 6 Samples of lineage V were obtained from the upper Amur genes, with inferences on control region constraints and broad-scale Eurasian phylogeography. Mol. Phylogenet. Evol. 34, 106–117. (Onon and Ingoda rivers), the type locality of C. szanaga Grosswald, M.G., Hughes, T.J., 2002. The Russian component of an (Dybowski, 1869). C. poecilopus populations from Arctic ice sheet during the last glacial maximum. Quatern. Sci. Rev. 21, Kolyma-Magadan region were morphologically different 121–146. from those of other regions (Chereshnev, 1982). Cheresh- Heckel, J.J., 1837. Ichthyologische Beitra¨ge zu den Familien der Cotto- nev (1982) proposed that it is an undescribed species, Cot- iden, Scorpaenoiden, Gobioiden und Cyprinoiden. Ann. Wien. Mus. Naturgesch. 2, 143–164. tus sp. Lineage IV would correspond to the Cottus sp. Hewitt, G.M., 2004. The structure of biodiversity––insights from molec- population of Chereshnev (1982). Due to geographical ular phylogeography. Front. Zool. 1, 4 doi:10.1186/1742-9994-1-4. proximities of our sample sites to the type localities of each Huelsenbeck, J.P., Ronquist, F., 2001. MRBAYES: Bayesian inference of of the nominal species, lineages I, II, III, IV and V would phylogeny. Bioinformatics 17, 754–755. correspond to C. poecilopus, C. altaicus, C. kuznetzovi, Cot- Kaschenko, N.F., 1899. Results of Altai Zoological Expedition in 1898, Vertebrates. Tomsk, pp. 138–158. tus sp. and C. szanaga, respectively. Therefore, we propose Mangerud, J., Jakobsson, M., Alexanderson, H., Astakhov, V., Clarke, taxonomic revision of the C. poecilopus complex, including G.K.C., Henriksen, M., Hjort, C., Krinner, G., Lunkka, J.-P., Mo¨ller, restoration of C. altaicus, C. kuznetzovi and C. szanaga, P., Murray, A., Nikolskaya, O., Saarnisto, M., Svendsen, J.I., 2004. and the description of a new species for the lineage IV. 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