Zoological Studies 45(2): 149-156 (2006) Molecular Identification of Two Sibling Species of Puntius in Taiwan Chia-Hao Chang1, Yi-Ta Shao2, and Hsiao-Wei Kao1,* 1Department of Life Science, National Chung Hsing University, Taichung, Taiwan 402, R.O.C. 2Institute of Cellular and Organismic Biology, Academia Sinica, Nankang, Taipei, Taiwan 115, R.O.C. (Accepted November 25, 2005) Chia-Hao Chang, Yi-Ta Shao, and Hsiao-Wei Kao (2006) Molecular identification of two sibling species of Puntius in Taiwan. Zoological Studies 45(2): 149-156. Puntius fish from Taiwan and South China were collect- ed and analyzed. Specimens from the northern and central Taiwan were characterized by the absence of bar- bels. On the contrary, specimens from the southern Taiwan and South China were characterized by the pres- ence of barbels. These characteristics morphologically match P. snyderi and P. semifasciolatus respectively (Oshima 1919). We hypothesized that there were two species of Puntius in Taiwan. To test this hypothesis, we amplified and sequenced the cytochrome b gene from fish specimens of China and Taiwan. Phylogenetic analysis revealed the existence of two major clades of Puntius fishes with an average genetic distance of 0.12 between them. The fish specimens from southern Taiwan were clustered with P. semifasciolatus of China, but fish specimens from northern and central Taiwan were clustered together. The estimated evolutionary rate of cytochrome b gene for Puntius was 0.368% per million yrs (MY). Thus the divergence time between P. snyderi , and P. semifasciolatus was about 26.93 million years ago (MYA) and the divergent time between Taiwan s and , China s P. semifasciolatus was about 4.40 MYA. Taken together, our results supported the existence of two species of Puntius fishes in Taiwan. P. snyderi is distributed in northern and central Taiwan, while P. semifasci- olatus is in southern Taiwan. http://zoolstud.sinica.edu.tw/Journals/45.2/149.pdf Key words: Cytochrome b gene, Freshwater fish, Molecular clock, Phylogenetic tree. Puntius snyderi is a freshwater cyprinid fish only described P. semifasciolatus in Taiwan. In discovered by Oshima when he collected the their books, its distribution ranged from the north- freshwater fishes in Taiwan in 1915-1917. It was ern and central parts of Taiwan to the southern mainly distributed in northern and central Taiwan part of western Taiwan. Obviously, this geograph- (Oshima 1919) in contrast to another species, P. ic distribution was a combination of the areas semifasciolatus that was distributed in Pingtung, where P. snyderi and P. semifasciolatus were southern Taiwan (Oshima 1919, Chen 1969). The once respectively reported. major morphological difference between these two During collections of freshwater fish of Taiwan fishes is the presence of a pair of maxillary barbels and China, we noticed the existence of two major in P. semifasciolatusi but their absence in P. sny- forms of Puntius fish in Taiwan. Fish from central deri. Because of similarities of their morphology Taiwan were characterized by the absence of bar- and distribution, the absence of official collecting bels and a smaller number of horizontal black records of P. snyderi since 1927, and the disap- bands (crossbars), but fish from the southern pearance of the holotype of P. snyderi, P. snyderi Taiwan and China had one pair of barbels and had was regarded as a synonym of P. semifasciolatus a greater number of and thinner crossbars (Fig. 1). by (Shen 1984). The issue of species validity was Because these characteristics respectively also reflected in the books of (Tzeng 1986), (Shen matched those reported for P. semifasciolatus and et al. 1993), and (Chen and Feng 1999) which P. snyderi, we hypothesized that there were two *To whom correspondence and reprint requests should be addressed. Current address: Department of Life Science, National Chung Hsing University, 250, Kuo Kuang Road, Taichung, Taiwan 402, R.O.C. Tel: 886-04-22840416 ext. 111 or 112. E-mail: [email protected] 149 150 Zoological Studies 45(2): 149-156 (2006) species of Puntius in Taiwan. Puntius were also downloaded from GenBank and Mitochondrial DNA sequences are frequently analyzed because P. semifasciolatus had been utilized for inferring phylogenetic relationships placed in all of these genera at some point in the among organisms, because they have the proper- past (Günther 1868, Oshima 1919, Herre and ties of large copy number, faster evolutionary rate, Myers 1931). We finally estimated the divergence maternal inheritance, smaller molecular weight, time between and among the specimens from and a lack of introns (Brown et al. 1979, Moritz et Taiwan and China in order to trace the speciation al. 1987). Among genes, the mitochondrial event. cytochrome b has been widely used in various ver- tebrates as a genetic marker for species-level identification (Johns and Avise 1998). For this rea- MATERIALS AND METHODS son, we also chose cytochrome b as a genetic marker to resolve the relationships among the fish Sampling of specimens specimens we collected. We collected Puntius fishes from Taiwan and China. Morphological Because of habitat destruction or water pollu- characters of specimens either collected by us or tion, Puntius specimens were only collected at four deposited in museums of Taiwan were examined. locations in Taiwan (Fig. 2), and one location in Phylogenetic trees were constructed using the China (Fig. 2) in this study, although extensive cytochrome b gene. In addition, cytochrome b efforts were also made on the island of Kinmen sequences of Barbus, Capoeta, Linichthys, and (Fig. 2). We collected the P. snyderi at Sanyi in (A) (B) P. snyderi (Sanyi) P. snyderi (Tsaotun) (C) (D) P. semifascioiatus (Huadu) P. semifascioiatus (Huadu) (E) (F) P. semifascioiatus (Meinong) P. semifascioiatus (Wanluan) Fig. 1. Crossbar pattern of Puntius semifasciolatus and P. snyderi from different collection locales. (A) Puntius snyderi in Sanyi, Taiwan; (B) P. snyderi in Tsaotun, Taiwan; (C) P. semifasciolatus in Huadu, China; (D) P. semifasciolatus in Huadu, China; (E) P. semi- fasciolatus in Meinong, Taiwan; (F) P. semifasciolatus in Wanluan, Taiwan. Chang et al. -- Puntius in Taiwan 151 northern Taiwan and Tsaotun in central Taiwan, of P. semifasciolatus from Xiulu and Cenjia in and P. semifasciolatus in Meinong and Wanluan in Hainan (NTUM01985, NTUM01988) and Penghu southern Taiwan. Southern, northern, and central (NTUM00429) (Fig. 2) deposited in the Depart- Taiwan were distinguished according to (Chen and ment of Life science, National Taiwan University Feng 1999). Fish specimens were also collected were also examined. in Huadu, Guangdong in South China. Fish speci- mens were either collected by traps or fishing. For Crude DNA extraction DNA extraction, a piece of muscle tissue was excised and placed in 95% alcohol for DNA extrac- Specimens for each sampling sites were cho- tion. The remaining part of the specimen was sen for DNA extraction. Crude DNA extraction fixed in 30% formalin, then stored in the 70% alco- was followed the Gentra-DNA extraction protocol hol for morphometric measurements. Specimens (Gentra system, Minneapolis, MN55441, USA). 120°00' 121°00' 122°00' Northern Taiwan 25 00' Kinmen ° Huadu Hainan Sanyi Central Taiwan Tsaotun 24°00' Eastern Taiwan Penghu Southern Meinong 23°00' Taiwan Green Island Wanluan Western Orchid Island Hengchun 22°00' peninsula Fig. 2. Locations of sampling sites of Puntius snyderi and P. semifasciolatus, and the zoogeographical distribution regions of freshwa- ter fishes in Taiwan. 152 Zoological Studies 45(2): 149-156 (2006) Crude DNA samples were stored at -20 C. A likelihood (ML) model. The maximum likelihood fragment of 1140 bp of the mitochondrial° tree was constructed by the PAUP* program cytochrome b gene was amplified by polymerase (Swofford 2001) with the most-suitable model. chain reaction (Applied Biosystems 2700, USA). Because Puntius is among the most-primitive Briefly, each 100µl PCR reaction contain about 10 group in the subfamily Barbina, it is inappropriate ng template DNA, 10µl 10x reaction buffer, 8µl to choose species of the same genus or other dNTP mix (2.5 mM dNTP each), 25µmol of each genera in this subfamily as an outgroup. Although specific primer, Cyto1 (5'-GTTATTCAACTACAA- fossil record have revealed that the Cyprininae GAACTAC-3') and Cyto2 (5'-TTTAGAC- branched out the earliest among the other three TAAGCTACTAGGGCA-3'), 2.5 U of Taq poly- closely related subfamilies of the Barbinae, merase (TaKaRa Taq®, Otsu, Shiga, 520-2193, Schizothoracinae, and Labeoninae (Chen et al. Japan), and distilled water. Thermal cycling began 1998), this is yet unsupported by any phylogenetic with a single denaturation step at 94 C for 4 min, analysis. For this reason, we constructed an then 35 cycles were performed consisting° of unrooted tree. Statistical support of the tree was denaturation at 94 C for 1 min, annealing at 55-65 examined by bootstrapping 250 times. Second, C for 1 min, and° extension at 72 C for 1 min. we utilized the MrBayes program vers. 3.0 soft- Finally,° a single extension step at 72°C for 10 min ware (Ronquist and Huelsenbeck 2003) to conduct was utilized to complete the extension° of DNA Bayesian inferences of phylogeny. In this analy- fragments. PCR products were purified with PCR sis, sequences were analyzed using first, second, DNA Fragments Extraction Kit (Geneaid DF100, and third codons (Yang et al. 1998), respectively. Taipei, Taiwan). Approximately 50 ng of double- Probabilistic inference using the method of Markov strand PCR product was used in the cycle Chains of Monte Carlo (MCMC) was set to 2 x 106 sequencing reactions with the same primers fol- times and burn-in was set to 10,000. lowing the protocol of ABI PRISM BigDye The evolutionary rate of the molecular clock sequencing Kit (PE Biosystems, Foster City, was calibrated using Tammura-Nei distance. We CA94404, USA). Reaction products were elec- used two methods to estimate the evolutionary trophoresed on an ABI model 3100 version 3.7 rate of cytochrome b gene.