935 Asian-Aust. J. Anim. Sci. Vol. 25, No. 7 : 935 - 944 July 2012 www.ajas.info http://dx.doi.org/10.5713/ajas.2011.11500 Phylogeography and Population Genetic Structure of Amur Grayling Thymallus grubii in the Amur Basin Bo Ma, Tingting Lui1, Ying Zhang and Jinping Chen1,* Heilongjiang River Fisheries Research Institute, Chinese Academy of Fishery Sciences, Harbin 150070, China ABSTRACT: Amur grayling, Thymallus grubii, is an important economic cold freshwater fish originally found in the Amur basin. Currently, suffering from loss of habitat and shrinking population size, T. grubii is restricted to the mountain river branches of the Amur basin. In order to assess the genetic diversity, population genetic structure and infer the evolutionary history within the species, we analysised the whole mitochondrial DNA control region (CR) of 95 individuals from 10 rivers in China, as well as 12 individuals from Ingoda/Onon and Bureya River throughout its distribution area. A total of 64 variable sites were observed and 45 haplotypes were identified excluding sites with gaps/missing data. Phylogenetic analysis was able to confidently predict two subclade topologies well supported by maximum-parsimony and Bayesian methods. However, basal branching patterns cannot be unambiguously estimated. Haplotypes from the mitochondrial clades displayed local homogeneity, implying a strong population structure within T. grubii. Analysis of molecular variance detected significant differences among the different geographical rivers, suggesting that T. grubii in each river should be managed and conserved separately. (Key Words: Amur Grayling, Population Genetic Structure, Phylogeography, Mitochondrial DNA Control Region) INTRODUCTION zoogeographical faunal complexes in this area (Nicolsigy, 1960). In this region, Da xing an Mountain, Xiao xing an Dispersal ability is a key demographic force shaping Mountain and Chang bai Mountain surround the Northeast natural populations and maintaining evolutionary potential Plain, and the five main rivers that flow through this area, (Proctor et al., 2004). Species with limited dispersal Amur River, Sungari, Nenjiang, Yalu and Tumen River, abilities display clearer population genetic structure but have been repeatedly connected and disconnected with each lower genetic diversity than do species with a tendency other during the past million years. It has been proposed towards greater dispersal (Kark et al., 1999; Garcia-Ramos that the geological history and topographical complexity of et al., 1999; Bailey et al., 2007). The populations of this region would have an effect on species diversity. freshwater fish species from different river systems often However, little research has focused on this area, and there show significant genetic differentiation resulting from is no phylogeographic studies on terrestrial vertebrates or restricted gene flow, and also there is evidence that some freshwater species in this region. species show high levels of genetic differentiation between Amur grayling described at the specific level as populations within a river system (McGlashan et al., 2000). Thymallus grubii (Dybowski, 1869), have been maintained Consequently, changes in interconnectivity within and to support capture and commercial fishing in some between drainages have greatly influenced the distribution temperate countries in the northern hemisphere, and have of many fish species and their genetic structure. recently become the focus of conservation efforts in the Northeast China has a special and old complex animal North Pacific (Mikko et al., 2002). In China, Amur grayling fauna and high biodiversity. Based on the geographic is also a specific and economically important cold distribution, origin and evolution of fishes, there are six freshwater fish with extreme site fidelity and low dispersal capacity. It has only been recorded in the Amur basin and * Corresponding Author: Jinping Chen. Tel: +86-20-84183704, limited to the upper levels of some rivers, which makes the Fax: +86-20-89100920, E-mail: [email protected] 1 Guangdong Entomological Institute/South China Institute of species an ideal model for the study of palaeoclimatic Endangered Animals, Guangzhou, 510260, China. effects on population genetic structure and phylogeography. Submitted Dec. 22, 2011; Accepted Mar. 23, 2012; Revised Apr. 3, 2012 A recent study of T. grubii showed the existence of three 936 Ma et al. (2012) Asian-Aust. J. Anim. Sci. 25:935-944 diagnosable, reciprocally monophyletic, and most probably reproductively isolated lineages of grayling within the Amur drainage (Froufe et al., 2003), In this study, therefore, we examine the phylogeography of T. grubii and explore the possible mechanisms responsible for the current genetic pattern and to provide an insight into the historical legacy of zoogeographical faunal in Northeast China. MATERIALS AND METHODS Amur grayling (N = 95) were collected in 2004-2008 Figure 1. Sampling locations for Amur graying Thymallus grubii from 10 localities of ten rivers distributed throughout the across whole sampling range and detailed view of sampling sites with 1, 2 and 3. species’ range in the Amur basin in China (Figure 1, Table 1). Based on a comment by Antonov et al. (1996), fish was sequenced. The primers were described in Uiblein et al. first noted as to whether they had an obvious yellow-orange (2001). Amplification was performed in a total volume of ocellus-like spot on the posterior edge of the dorsal fin, and 50 l containing 25 mM MgCl2, 10 mM Tris-HCl, 1.5 mM to eliminate the possibility that these samples came from 2+ related individuals, we selected many patches from one Mg , 200 mol of each dNTP, 0.2 mol of each primer, 2.0 location. After, a small fin clip had been preserved in 96% U Hot start Taq DNA polymerase (QIAGEN), and 10 ng ethanol, whole fish were stored at -20 degrees. We also of genomic DNA. Thirty cycles were amplified on a Perkin- retrieved an additional 12 individual sequences collected Elmer Cetus 9700 DNA thermocycler with pre denaturing from throughout its distribution area in the Ingoda/Onon at 94C for 3 min; denaturing at 94C for 45 s, annealing at and Bureya Rivers from GenBank (http:// 55C for 45 s, and extension at 72C for 45 s; and a final 8 www.ncbi.nlm.nih.gov/Genbank/) for evolutionary analyses. min of extension at 72C. Positive (with DNA template) (Accession no. AY168390~AY168399, AY779010, and negative (with water) controls were used to check PCR AY779009) performance and contamination. The PCR products were purified using PCR purification Kit (Shanghai Bio-Tec, Ltd) Mitochondrial DNA analysis and sequenced with the ABI PRISM BigDye Terminator Total genomic DNA was extracted from the tissue Ready Reaction Kit (Applied Biosystems) and run on an samples using phenol-chloroform extraction (Taggart et al., ABI 377 genetic analyzer according to manufacturer’s 1992). The DNA precipitate was re-suspended in TE buffer protocol. To avoid the errors in sequencing, PCR then stored at -20C. The complete mitochondrial DNA amplifications of all samples were sequenced for both (mtDNA) control region (904 base pairs) and 200 base pairs strands. of flanking tRNA gene regions were amplified and Table 1. Sample locations including major river basin, geographical coordinates and the number of individuals analyzed for mtDNA variation No. of Map no. Population Basin Pop code Latitude Longitude individuals 1 Emur River Amur EM 4 52 55 122 47 2 Huma River Amur HM 16 52 18 124 42 3 Ingoda River* Amur Amu 10 51 20 113 26 Onon River* 48 75 110 25 4 Gen River Amur GE 4 50 46 121 31 5 Nuomin River NenSungariAmur NM 3 48 36 124 07 6 Kuile GanNenSungariAmur K 3 49 32 124 27 7 Gan River NenSungariAmur GH 18 49 42 124 35 8 Hailang River MudanSungariAmur HL 19 44 28 128 54 9 Yalu River Yalu YL 12 41 26 128 12 10 Songjiang River deuto-SungariSungariAmur SJ 8 42 10 127 30 11 Wudaobai River deuto-SungariSungariAmur WD 8 42° 16 128 25 12 Bureya* Amur Bur 2 51 55 134° 53 * Data is from Froufe et al. (2003a) and Froufe et al. (2003b). Ma et al. (2012) Asian-Aust. J. Anim. Sci. 25:935-944 937 Data analysis neutrality and demographic equilibrium. Because we were Sequences were aligned by using the CLUSTALX interested in discriminating between demographic software (1.83) and visually rechecked. Haplotypes were expansion and contraction, we chose two statistical tests, identified using DNASP (Rozas, 2003). Pairwise sequence each with particular sensitivity to one demographic scenario. differences among the haplotypes were calculated using Fu and Li’s D* is designed to detect an excess of old MEGA 2.1 (Kumar et al., 2001). For phylogenetic analysis, mutations, characteristic of a population that has we performed maximum-parsimony (MP) using the experienced a historical reduction in effective population program PAUP 4.0 (Swofford 2001) and MRBAYES 3.0 size (Fu, 1997). In contrast, Fu’s Fs is sensitive to an excess (Huelsenbeck and Ronquist, 2001). Modeltest 3.06 (Posada of recent mutations, a pattern typical of both a demographic and Crandall, 1998) was run to determine the appropriate expansion and a selective sweep (Fu, 1997; Ramos-Onsins model of molecular evolution in a likelihood ratio test and Rozas, 2002). D* was calculated in DNASP, Fs in framework, and Thymallus thymallus and T. tugarinae were Arlequin 2.0. used as out-groups. Gaps were treated as missing in parsimony analyses. Bootstrap analyses were performed RESULTS with 6,000 replicates and 1,000 full heuristic replicates for maximum parsimony. For Bayesian phylogenetic inference, mtDNA four Markov chain Monte Carlo (MCMC) were run for From 1,104 bp CR mtDNA sequence generated from 100,000 generations, sampling every 10 generations; the 107 individuals, 64 variable nucleotide sites were resolved, initial 5% of trees were discarded as burn-in, finally, a 50% including transversion (tv), transition (ts) and 16 insertion/ majority rule consensus tree was constructed. deletions, and 45 haplotypes were defined (Accession no.