This article was downloaded by: [University of Bath] On: 13 February 2014, At: 12:19 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK

Journal of Natural History Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnah20 Taxonomic implications from phylogenetic relationships of subspecies of Schizopygopsis malacanthus (Pisces: ) based on sequence analysis of cytochrome b and mitochondrial DNA control region Chunjin Yu a , Zhaobin Song a & Bisong Yue a a Key Laboratory of Bio‐resources and Eco‐environment (Ministry of Education) , College of Life Sciences , Sichuan University , Chengdu 610064, P. R. China Published online: 16 Mar 2007.

To cite this article: Chunjin Yu , Zhaobin Song & Bisong Yue (2006) Taxonomic implications from phylogenetic relationships of subspecies of Schizopygopsis malacanthus (Pisces: Cyprinidae) based on sequence analysis of cytochrome b and mitochondrial DNA control region, Journal of Natural History, 40:44-46, 2569-2576, DOI: 10.1080/00222930601129463 To link to this article: http://dx.doi.org/10.1080/00222930601129463

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Taxonomic implications from phylogenetic relationships of subspecies of Schizopygopsis malacanthus (Pisces: Cyprinidae) based on sequence analysis of cytochrome b and mitochondrial DNA control region

CHUNJIN YU, ZHAOBIN SONG & BISONG YUE

Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610064, P. R. China

(Accepted 20 November 2006)

Abstract Phylogenetic relationships of three subspecies of Schizopygopsis malacanthus, S. m. malacanthus, S. m. chengi, and S. m. baoxingensis, were investigated based on the mitochondrial DNA control region and the cytochrome b gene. Phylogenetic analysis indicated that the three subspecies did not cluster as one monophyletic group; S. m. malacanthus clustered into one clade, while S. m. chengi and S. m. baoxingensis clustered into another. Genetic distances between S. m. malacanthus and the other two subspecies were either very close to or larger than those between S. m. malacanthus and some other species of Schizopygopsis. There was very small genetic distance between S. m. chengi and S. m. baoxingensis. The results suggested S. m. chengi should be split from S. malacanthus into a separate species, Schizopygopsis chengi (Fang); S. m. baoxingensis should be regarded as a subspecies, S. c. baoxingensis (Fu, Ding et Ye), of Schizopygopsis chengi.

Keywords: Pisces, Cyprinidae, Schizopygopsis malacanthus, subspecies, phylogenetic relationship, cytochrome b, mtDNA control region

Introduction Downloaded by [University of Bath] at 12:19 13 February 2014 The fishes of the genus Schizopygopsis Steindachner, 1866 (subfamily Schizothoracinae, family Cyprinidae) are found in cool and cold water in high mountain rivers, streams and lakes, and feed on insects and algae. There are eight species of Schizopygopsis in China (Yue 2000), but only three of them, S. malacanthus Herzenstein, 1891, S. kialingensis Tsao et Tun, 1962, and S. pylzovi Kessler, 1876, are found in Sichuan Province (Ding 1994). Schizopygopsis malacanthus, a species distributed in the upper reaches of the Yangtze River, has three subspecies, S. malacanthus malacanthus Herzenstein, 1891, S. m. chengi (Fang), 1936, and S. m. baoxingensis Fu, Ding et Ye, 1994 (Ding 1994). Fang (1936) originally

Correspondence: Zhaobin Song, Key Laboratory of Bio-resources and Eco-environment (Ministry of Education), College of Life Sciences, Sichuan University, Chengdu 610064, P. R. China. Email: [email protected] Published 29 December 2006 ISSN 0022-2933 print/ISSN 1464-5262 online # 2006 Taylor & Francis DOI: 10.1080/00222930601129463 2570 C. Yu et al.

described S. m. chengi as Chuanchia chengi, and Tsao and Tun (1962) revised it as a subspecies of S. malacanthus as its characters agreed with those of the genus Schizopygopsis, and were similar to those of S. malacanthus. Schizopygopsis m. baoxingensis was described by Fu et al. as a new subspecies of S. malacanthus (Ding 1994). These three subspecies have many common and overlapping morphological characters. For example, the dorsal fin spines are very weak, and the origin of the pelvic fin is opposite to the position of the fourth or fifth branched ray of the dorsal fin (Ding 1994). However, they still have some obvious differences in morphology. For example, the number of gill rakers in the first arch of S. m. baoxingensis is much lower than in the other two (Ding 1994). Schizopygopsis m. chengi differs from S. m. malacanthus in that the dorsal spine is comparatively weak, and the anal scaly sheath terminates further anteriorly (Tsao & Tun 1962). Also, these three subspecies have allopatric distributions, with S. m. malacanthus distributed in the Jinsha River system and Yalong River system, S. m. chengi distributed in the middle and upper reaches of the Dadu River, and S. m. baoxingensis distributed in the East River, a tributary of the upper reaches of the Baoxing River, which belongs to the Qingyi River system (Ding 1994) (Figure 1). They inhabit different water systems, and therefore do not interbreed, and the introgressive hybridization between them is impossible. A phylogenetic analysis of these three subspecies is necessary to test whether classification based on morphological characters is corroborated. It can also help determine the origin of S. m. baoxingensis, which has a very limited distribution range. Molecular tools have been widely used to resolve the of fishes. The mitochondrial DNA control region is particularly useful because it has a high rate of Downloaded by [University of Bath] at 12:19 13 February 2014

Figure 1. Collection locations of the different samples used in this study & Schizopygopsis malacanthus malacanthus (Jinsha River); N Schizopygopsis malacanthus malacanthus (Yalong River); 7 Schizopygopsis malacanthus chengi; m Schizopygopsis malacanthus baoxingensis; XSchizopygopsis kialingensis. Taxonomic implications from phylogenetic relationships of Schizopygopsis malacanthus 2571

evolution and huge variation (Meyer 1993; Hillis et al. 1996). Saccone et al. (1987) suggested that the mtDNA control region could solve different evolutionary problems through research at different taxonomic levels. Liu et al. (2002) reported that the CSB domain of the control region was highly conserved, making it suitable for phylogenetic studies in . Cytochrome b is one of the genes encoded in the mitochondria, and one of the most extensively used in vertebrate phylogenetic studies. In cyprinid fishes, cyt b has been used widely for phylogenetic analyses and in biogeography studies (Tsigenopoulos & Berrebi 2000; Kotlik & Berrebi 2002). In this study, we analyzed the phylogenetic relationships of these three subspecies based on the mtDNA control region and the cyt b gene, and discussed their taxonomic status. This is the first molecular analysis on this group of fish.

Materials and methods Schizopygopsis m. malacanthus were collected from the Litang River, a tributary of the Yalong River, in Litang County, and from the upper reaches of the Shuiluo River, a tributary of the Jinsha River, in Litang and Daocheng Counties; S. m. chengi were collected from Rangtang region of the Dadu River; S. m. baoxingensis were collected from the East River, a tributary of the Baoxing River located in upper reaches of the Qingyi River; S. kialingensis, another species of Schizopygopsis in the Yangtze River system, were collected from Jiuzhaigou region located in the upper reaches of the Jialing River and used for comparative analysis. Fins or muscle of fish were sampled and stored in anhydrous ethanol immediately after capture. All collection locations are shown in Figure 1. The specimens used in this study were from six S. m. malacanthus, two S. m. chengi, two S. m. baoxingensis and two S. kialingensis, and were lodged in the Key Laboratory of Bio-resources and Eco- environment, College of Life Sciences, Sichuan University, China. Total DNA was extracted using the phenol-chloroform method (Sambrook & Russell 2002) with some modifications. Polymerase chain reaction (PCR) was applied to amplify the sequences of cyt b gene and mtDNA control region. The primer sets L14724 (GACTTGAAAAACCACCGTTG)andH15915(CTCCGATCTCCGGATTACAAGAC) (Xiao et al. 2001) were used for amplifying the cyt b gene, and the primer sets DL1 (ACCCCTGGCTCCCAAAGC) and DH2 (ATCTTAGCATCTTCAGTG) (Liu et al. 2002) were used for amplifying the mtDNA control region. PCR was performed with an initial denaturation step at 94uC for 4 min followed by 35 cycles at 94uC for 1 min, 55uC for 1 min, and 72uC for 1 min with a final extension at 72uC for 10 min. The amplified Downloaded by [University of Bath] at 12:19 13 February 2014 fragments were purified with an EZNA Gel Extraction Kit (Omega Bio-Tek, USA) following the manufacturer’s instructions. The purified fragments were ligated into pMD 18-T vector (Takara, Dalian, China). The transformation of E. coli JM109 cells was followed by recombinant plasmid purification. The sequencing was performed by Sunbiotech Biotechnologies Company. All sequences are available from GenBank under accession nos. DQ533777–DQ533800. The cyt b gene sequences of S. younghusbandi Regan, 1905 (accession no. AY463501), S. sp. (accession no. AY463502), S. pylzovi (accession nos. AY608654–AY608656) were downloaded from the GenBank. All these sequences were used for estimating genetic distances among species or subspecies, and constructing the phylogenetic trees. All nucleotide sequences were aligned with Clustal X 1.8 multiple-alignment program (Thompson et al. 1997) and refined manually. Sequence polymorphisms were analyzed, genetic distances between species or subspecies were estimated and Neighbor-Joining trees 2572 C. Yu et al.

for all individuals were constructed according to the Kimura two-parameter model (Kimura 1980) using Mega version 3.1 (Kumar et al. 2004). At the same time, Maximum Parsimony trees were constructed using Mega version 3.1 in order to confirm the results. prenanti (Tchang), 1930, a species of another genus in Schizothoracinae, was chosen as outgroup in all analyses. The cyt b gene (accession no. AY608649) and mtDNA control region sequence (accession no. DQ201967) of this species were downloaded from the GenBank.

Results The cyt b and mtDNA control region sequences of the 10 individuals of S. malacanthus were analyzed. After being aligned according to the conserved region, the total sites of the control region were 940 bp, of which six were deletions and 34 were variable sites (five singleton variable sites and 29 parsimony informative sites). The average base composition for the control region was 32.2% thymine, 21.2% cytosine, 31.5% adenine and 15.1% guanine. The overall transition/transversion (Ts/Tv) ratio of the control region was 3.9. After being aligned according to the conserved region, the total sites of cyt b were 1140 bp, of which 94 were polymorphic sites (19 singleton variable sites and 75 parsimony informative sites). The average base composition for cyt b was 30.4% thymine, 26.9% cytosine, 25.5% adenine and 17.2% guanine. The Ts/Tv ratio was 6.2. Based on control region analysis, the genetic distances between S. m. malacanthus and the other two subspecies were 0.0277¡0.0053 and 0.0290¡0.0055, while the distance was only 0.0130¡0.0034 between S. m. malacanthus and S. kialingensis. There was much less divergence (0.0038¡0.0019) between S. m. baoxingensis and S. m. chengi. The maximum pairwise divergence value observed among all samples was 0.0313¡0.0057 between S. m. baoxingensis and S. kialingensis (Table I). According to cyt b gene analysis, the genetic distances between S. m. malacanthus and the other two subspecies were 0.0694¡0.0078 and 0.0659¡0.0075, which were larger than those between S. m. malacanthus and S. kialingensis, S. younghusbandi, and S. sp. The genetic distance between S. m. baoxingensis and S. m. chengi was only 0.0057¡0.0019, which was much lower than those between these two subspecies and some other species of Schizopygopis (Table II). Neighbor-Joining trees were constructed using the control region and cyt b and bootstrapped with 1000 replicates. From the tree of control region, S. m. malacanthus from the Yalong and Jinsha Rivers clustered together and then clustered with S. kialingensis into one clade. However, S. m. baoxingensis and S. m. chengi clustered together into another Downloaded by [University of Bath] at 12:19 13 February 2014 clade with 100% bootstrap value (Figure 2). The tree of cyt b was composed of four clades. S. m. malacanthus and S. kialingensis clustered into one clade and then clustered together with S. younghusbandi and S. sp.

Table I. Mean genetic distance (lower-left) and standard error (upper-right) of the mtDNA control region between species or subspecies in genus Schizopygopsis.

Species or subspecies Smm Smc Smb Sk Smm 0.0053 0.0055 0.0034 Smc 0.0277 0.0019 0.0056 Smb 0.0290 0.0038 0.0057 Sk 0.0130 0.0285 0.0313

Smb, S. m. baoxingensis; Smc, S. m. chengi; Smm, S. m. malacanthus; Sk, S. kialingensis Taxonomic implications from phylogenetic relationships of Schizopygopsis malacanthus 2573

Table II. Mean genetic distance (lower-left) and standard error (upper-right) of the cyt b between species or subspecies in genus Schizopygopsis.

Species or subspecies Smm Smc Smb Sk Sp Sy Ssp Smm 0.0078 0.0075 0.0041 0.0075 0.0059 0.0063 Smc 0.0694 0.0019 0.0084 0.0069 0.0083 0.0084 Smb 0.0659 0.0057 0.0085 0.0071 0.0082 0.0083 Sk 0.0218 0.0734 0.0729 0.0083 0.0066 0.0071 Sp 0.0709 0.0567 0.0562 0.0778 0.0075 0.0079 Sy 0.0420 0.0736 0.0711 0.0438 0.0671 0.0032 Ssp 0.0470 0.0786 0.0760 0.0504 0.0719 0.0133

Sp, S. pylzovi; Sy, S. younghusbandi; Ssp, S. sp.; other abbreviations are as same as in Table I.

However, S. m. baoxingensis and S. m. chengi clustered into another clade, and then clustered together with S. pylzovi (Figure 3). The trees showed that the three subspecies did not cluster into one clade. The phylogenetic relationships between S. m. malacanthus and the other two subspecies were remote, even farther than those between S. m. malacanthus and some other species of Schizopygopsis used for comparison in this study. However, S. m. baoxingensis and S. m. chengi were most closely related. Maximum Parsimony trees had the same topological shape as the N-J tree.

Discussion According to the primary common taxonomic characters, the dorsal fin spines were very weak, and the origin of the pelvic fin was opposite to the position of the fourth or fifth branched ray of the dorsal fin; in morphology, S. m. malacanthus, S. m. chengi and S. m. baoxingensis were regarded as three subspecies (Tsao & Tun 1962; Ding 1994). Although those taxonomic studies based on morphological data for S. malacanthus were reasonable, there were still some obvious differences in morphology of the subspecies. For example, Downloaded by [University of Bath] at 12:19 13 February 2014

Figure 2. Neighbor-Joining tree based on the mtDNA control region of species or subspecies in genus Schizopygopsis. The bootstrap values above 50% of 1000 replicates shown at nodes. 2574 C. Yu et al.

Figure 3. Neighbor-Joining tree based on the mitochondrial cyt b gene of species or subspecies in genus Schizopygopsis. The bootstrap values above 50% of 1000 replicates shown at nodes.

there were four differences in morphological taxonomy when S. m. baoxingensis was compared to S. m. malacanthus (Ding 1994). Moreover, molecular data may reflect phylogenetic relationships of fishes more objectively than morphological characters (He et al. 2004). From molecular analysis of the three subspecies of S. malacanthus, and comparison with other species of Schizopygopsis based on the cyt b gene and the mtDNA control region, we found different phylogenetic relationships compared to those relation- ships based on morphology. The genetic distances between S. m. malacanthus and the other two subspecies (S. m. chengi and S. m. baoxingensis) were very large, even larger than those between S. m. malacanthus and some other species of Schizopygopsis. However, there was a very small genetic distance between S. m. chengi and S. m. baoxingensis. The phylogenetic trees showed that the three subspecies did not cluster as one monophyletic group. Schizopygopsis m. malacanthus clustered into a clade including S. kialingensis and some other species of Schizopygopsis, while S. m. chengi and S. m. baoxingensis clustered into another clade. This suggests a very distant molecular phylogenetic relationship between S. m.

Downloaded by [University of Bath] at 12:19 13 February 2014 malacanthus and the other two subspecies. The same result was inferred from the analysis of the cyt b gene and the mtDNA control region. Our results suggest that S. m. chengi and S. m. baoxingensis should not be regarded as subspecies of S. malacanthus. However, S. m. chengi and S. m. baoxingensis are still two subspecies based on their molecular phylogeny. Schizopygopsis malacanthus should be regarded as a species without subspecies; S. m. chengi should be split from S. malacanthus as a separate species, Schizopygopsis chengi (Fang); S. m. baoxingensis should be regarded as a subspecies, S. c. baoxingensis (Fu, Ding et Ye), of S. chengi. Of course, in the future further validation of the sterility and development incompatibilities of hybrid individuals between S. m. malacanthus and S. m. chengi or S. m. baoxingensis would be helpful for understanding the phylogenetic status and the taxonomic revision among the three subspecies inferred from the mtDNA analyses in this study. Schizopygopsis m. baoxingensis is found in a 60 km long stream above the Yanjing region of the East River, a tributary of the upper reaches of the Baoxing River, which is part of the Taxonomic implications from phylogenetic relationships of Schizopygopsis malacanthus 2575

Qingyi River system (Song 2006). Thus it has a very limited range. Schizopygopsis m. baoxingensis and S. m. chengi are not found downstream of the connection of the Qingyi River and the Dadu River, which converge to form the Minjiang River at Leshan in Sichuan Province. At the same time these two subspecies are also isolated by the Jiajin Mountain in the upper limit of their distribution. Therefore, they have no opportunity to interbreed. However, these two subspecies have a very close phylogenetic relationship based on cyt b gene and mtDNA control region analysis. It seems reasonable to consider that S. m. baoxingensis originated from S. m. chengi as a result of stream capture or some other geological event in the Jiajin Mountain area. Stream capture is the process whereby a stream easily deepening its valley in soft rock can cut headward across a drainage divide to capture a portion of a neighboring stream that is working away slowly in harder rock. Stream capture events have provided dispersal opportunities for several species of freshwater fish in various drainages. Webb (2002) suggested that stream capture has played a significant role in the diversification of Allodontichthys, Cyprinodontiformes. Minckley et al. (1986) summarized several examples of these events in Oregon and Californian streams. The headward erosion of the Baoxing River could cut across the watershed to capture a portion of a neighboring stream, the Dawei River, the tributary in upper reaches of the Dadu River in Xiaojin County, Sichuan Province, on other side of the Jiajin Mountain. In this way, some S. m. chengi individuals were isolated from the Dadu River water system, and eventually evolved as S. m. baoxingensis in the upper reaches of the Qingyi River system. This divergence between S. m. chengi and S. m. baoxingensis might also be the result of stream connectivity. Some geological events, such as a landslide or earthquake, might have formed natural dams from the Dawei River. Schizopygopsis m. chengi individuals could have been locked in these temporary water areas. These water areas were then connected to the Baoxing River through subsequent geological movements; the fish could then spread into this water system and evolve as S. m. baoxingensis. From this study S. m. chengi and S. m. baoxingensis have a consanguineous evolutionary relationship. They became isolated and evolved until reproductive isolation occurred, the result of geographic isolation. Schizopygopsis m. baoxingensis is an endemic subspecies to the Baoxing River in China. Song (2006) showed that this fish was only found in the upper reaches of the Baoxing River. This subspecies should have special value in species conservation of fishes in China, and might have potential value in studies of geological movement in the Jiajin Mountain area. However, the construction of many hydropower stations along the East River would affect this subspecies and may even result in its extinction (Song 2006). Due to the very

Downloaded by [University of Bath] at 12:19 13 February 2014 limited research on this subspecies before Song (2006), legal protection still cannot be afforded to it. It is necessary to add this subspecies into the China Species Red List as vulnerable, thus it would be protected efficiently in the future.

Acknowledgments This work was funded by WWF small grant (CN0861.01 (3073)). We would like to thank Zhirong Zheng, Danzhou Shen and Chunlin He for their help in sample collecting, and Jun Song for his help during the experiments. We are grateful to Professor Wenxuan Cao for his helpful suggestions during the experiments and manuscript preparation, Dr. Huanzhang Liu and two anonymous reviewers for their helpful comments on the manuscript, and Emily H. King for her revision of English writing. 2576 C. Yu et al.

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