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Genetic relationship among four subspecies of cherry salmon (Oncorhynchus masou) inferred using AFLP
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Molecular Phylogenetics and Evolution 48 (2008) 776–781 www.elsevier.com/locate/ympev
Short Communication
Genetic relationship among four subspecies of cherry salmon (Oncorhynchus masou) inferred using AFLP Jin-Chywan Gwo a,*, Te-Hua Hsu a, Kuan-Hung Lin b, Yii-Cheng Chou c a
Department of Aquaculture, Taiwan National Ocean University, No. 2 Pei-Ning Road, Keelung 20224, Taiwan b Graduate Institute of Biotechnology, Chinese Culture University, Taipei 11119, Taiwan c Department of Medical Technology, Chung-Hwa University of Medical Technology, Tainan 717, Taiwan Received 6 December 2007; accepted 21 December 2007 Available online 3 January 2008
1. Introduction The Pacific salmons are comprised of at least seven species. These seven species are distributed in fresh water and in the sea, including most of the North Pacific rim and adjacent ocean regions, collectively extending across the Pacific (Sanford, 1990; Kato, 1991; Kiso, 1995). One of the seven known species, the cherry salmon (Oncorhynchus masou), also known as ‘‘Sakuramasu”, is confined to Asian rivers and coastal marine waters (Fig. 1). The four known extant subspecies of the O. masou complex (the cherry salmon complex) are Masu (O. masou masou), Amago (O. masou ishikawae), Biwa (O. masou subsp.) and the Formosa landlocked salmon (O. masou formosanus) (Kato, 1991; Kimura, 1990). The fluvial Formosa landlocked salmon is found only in the head waters of the Tachia River, Taiwan. This is the southernmost limit of the distribution range of salmonids (Kimura, 1990). Historically, the taxonomy and nomenclature of the O. masou complex has been greatly debated, in part because of the similar morphological and ecological traits shared by all four Masu salmon subspecies. The scientific name and systematic position of the Formosa landlocked salmon has been especially contested. In fact, its scientific name has been changed four times to date. Oshima (1919) originally referred to the Formosa landlocked salmon as Salmo saramao, in Japanese, but gave a different name––Salmo formosanus––in his English publication the same year (Jordan and Oshima, 1919). Upon further research, Oshima revised the scientific name of the Formosa landlocked salmon to O. formosanus (1934). In 1936, Oshima modified the scientific name once *
Corresponding author. Fax: +886 2 25093958. E-mail address: [email protected] (J.-C. Gwo).
1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.12.023
again to O. masou because he found no red spots on the sides of the body (Oshima, 1936). The Formosa landlocked salmon is a highly inbred population, with the existing population estimated at less than 1000 individuals (Gwo et al., 1999, 2007; Healy et al., 2001). Because it was listed as endangered by the Taiwan Government in 1984, defining the evolutionary unit in this species has become urgent to protect the remaining population. The scientific name and taxonomic position of the Formosa landlocked salmon is based on morphology and meristics (Jordan and Oshima, 1919; Oshima, 1919, 1934, 1936; Teng 1959; Behnke et al., 1962; Watanabe and Lin, 1985; Jan et al., 1990; Hosoya et al., 1992), on analysis of mt DNA (Numachi et al., 1990; Chung et al., 2006; Tzeng et al., 2006), or on sperm ultrastructure (Gwo et al., 1996, 1999). After comparing 25 meristic and morphometric characters of seven Oncorhynchus species, Jan et al. (1990) concluded that morphometric and meristic counts alone are insufficient to clarify the phylogeny of the Formosa landlocked salmon. Indeed, these four known extant subspecies of the O. masou complex exhibit little or no morphological difference (Watanabe and Lin, 1985; Jan et al. 1990). Further research in genetics, using molecular markers, may help to elucidate whether the Formosa landlocked salmon is one distinct subspecies of O. masou complex, or merely a separated local population of Masu salmon (O. masou masou). However, studies using several microsatellite DNA loci and mitochondrial control region sequences have failed to resolve relationships among the four subspecies of the cherry salmon complex (Tzeng et al., 2006; Gwo, unpublished data). Amplified fragment length polymorphism (AFLP) analysis has been widely applied to study species, strain and hybrid identification, gene mapping, linkage and genetic
J.-C. Gwo et al. / Molecular Phylogenetics and Evolution 48 (2008) 776–781
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Fig. 1. Sample collection sites. Amago salmon (O. masou ishikawae), Masu salmon (O. masou masou), Biwa salmon (O. masou subsp.), Formosa landlocked salmon (O. masou formosanus) and pink salmon (O. gorbuscha).
diversity of species and populations in a wide variety of organisms including fishes and shrimp (Liu and Cordes, 2004; Wang et al., 2004; Kakehi et al., 2005; Kassam et al., 2005). In this study, we used AFLP to study variation sites across the genome. We also compared the AFLP fingerprints derived from PCR amplification using seven pairs of selective primers for three Oncohynchus species–– rainbow trout (O. mykiss), pink salmon (O. gorbuscha), and cherry salmon (O. masou)––with cherry salmon further divided into the four subspecies, Amago (O. masou ishikawae), Biwa (O. masou subsp.), Masu (O. masou masou), and Formosa landlocked salmon (O. masou formosanus) to explore the applicability of AFLP technology in species/ subspecies identification and phylogenetic analysis. Our results demonstrated that the AFLP technique is indeed useful to resolve the phylogenetic relationships among these four subspecies.
2. Materials and methods 2.1. Sample collection and DNA extraction Nineteen wild population Formosa landlocked salmon, and six cultivated rainbow trout were collected from Chichawan Stream in the Shei-Pa National Park, Taiwan and from a private hatchery at Jinsan, Taiwan, respectively. Pink salmon, Amago salmon, Masu salmon, and Biwa salmon were obtained from the following locations in Japan: Hokkaido Kumagaya Station––wild Masu salmon (5 specimens); Pacific Ocean near Hokkaido––wild pink salmon (5 specimens); Inland Station of the National Research Institute of Aquaculture––cultivated stock Amago salmon (10 specimens) and Biwa salmon (3 specimens; Fig. 1). The specimens were either stored at 80 °C or preserved in 95% ethanol prior to analysis. Total DNA was
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J.-C. Gwo et al. / Molecular Phylogenetics and Evolution 48 (2008) 776–781
extracted from muscle (10–15 mg) using proteinase-K digestion and the phenol–chloroform–isoamyl alcohol method with slight modifications. DNA concentration was measured with an UV spectrophotometer. The quality of extracted DNA was assessed by 1.0% agarose gel electrophoresis with ethidium bromide. 2.2. AFLP reactions Procedures of AFLP analysis were essentially based on Wang et al. (2004). Fingerprint patterns were visualized on a 5% denaturing polyacrylamide gel using the silver staining method. DNA templates for AFLP reactions were generated by restriction digestion and ligation. Initially, about 100 ng of total DNA was digested with 5 U of EcoRI and Tru9 1 (Promega) in 1 NE buffer 2 at 37 °C for 2 h. To generate DNA templates for subsequent PCR amplification, the digested DNA fragments were ligated with 2.5 pmol of EcoRI and 25 pmol Msel adapters in a reaction mixture containing 0.25 mg BSA, 5 pmol ATP, 0.04 U T4 DNA ligase and 10 NE buffer 2 at 37 °C for 6 h. Preamplification PCR reactions were conducted using an Eppendorf mastercycler gradient with a pair of primers containing single selective nucleotides. Amplification was performed at an annealing temperature of 53 °C for 30 s. The 20 ll PCR product mixture was diluted 10-fold with distilled water and used as templates for the subsequent selective PCR amplification. The selective amplification was performed using seven pairs of primers, E-ACA/M-CAC, E-ACC/M-CAC, E-ACC/M-CAT, E-ACG/M-CAT, E-AAC/M-CAC, E-ACG/M-CAC, and E-ACA/M-CAA. 2.3. Electrophoresis and silver staining The PCR products were mixed with 10 ll AFLP loading buffer (99% formamide, 10 mM EDTA, 0.05%. bromophenol and 0.05% xylene cyanol). The product mixtures were denatured and concentrated at 90 °C for 25 min, and quickly cooled in an ice bath after denaturation. A 5% denaturing polyacrylamide gel (4.75% acrylamide, 0.25% bisacrylamide, 7 mol l 1 urea and 0.5 TBE) was prerun at 145 W for 30 min. Each well was loaded with 2.5 ll of sample. The gel was electrophoresed for 2.5 h in an ATTO (Type AE6155) DNA sequencing cell (38 50 cm) at 110 W and 50 °C. After electrophoresis, the gel was fixed in 1% ethanoic acid for at least 30 min. The gel was rinsed in distilled water and stained with a mixture of 0.1% silver nitrate and 0.007% benzene sulphonic acid for 30 min. The stained gel was rinsed again with distilled water and immersed in a developing solution (2.5% sodium carbonate, 0.037% formaldehyde and 0.002% sodium thiosulphate). The development was subsequently stopped with 1% ethanoic acid when bands were visible and reached desirable intensity. Band sizes were estimated using a standard AFLP
DNA ladder and analyzed using the Imaging Analyzing System (HP scanjet 5370c). 2.4. Data analysis AFLP bands were scored for presence (1) or absence (0), and transformed into a 0/1 binary character matrix. Fragments that could not be scored unambiguously were excluded from the analysis. The total number of individuals analyzed was 48, with three individuals for each species. The fragment was recognized using a sequence of 16 bp (Wang et al., 2004). Data were analyzed using a set of programs in the PHYLIP v3.6a3 Inference Package. The 0/1 matrix was converted into a distance matrix using the program RESTDIST. One thousand bootstrapped data sets were generated using SEQBOOT, and 1000 bootstrapped trees were constructed. A neighbor-joining (NJ) tree was constructed using the NEIGHBOR based on the matrix from RESTDIST, and redrawn using the TreeView program. 3. Results This study established AFLP fingerprints for 48 individuals from three Oncohynchus species––rainbow trout (O. mykiss), pink salmon (O. gorbuscha), and cherry salmon (O. masou)––with cherry salmon further divided into the four subspecies, Amago (O. masou ishikawae), Biwa (O. masou subsp.), Masu (O. masou masou), and Formosa landlocked salmon (O. masou formosanus). The seven pairs of primer sets yielded a total of 782 scorable bands (size range 100–350 bp). One hundred and fourteen bands (15%) were found in all species, with 77 (10%) scored in all individuals (i.e. monomorphic), and the other 37 polymorphic. The number of bands generated from a single individual ranged from 23 to 101, while a single species produced 337 (in O. gorbuscha) to 522 (in O. masou ishikawae) bands. Species differed in the levels of genetic diversity among individuals, with 11.5% in O. masou subsp. to 41.3% in O. masou ishikawae of the bands scored as polymorphic. The proportion of subspecies-specific markers to the total number of bands scored for each subspecies ranged from 0.20% (in O. masou ishikawae and O. masou masou) to 1.55% (O. masou formosanus). Many specific markers among Masu, rainbow and pink salmon were detected. Specific markers among the four O. masu complex subspecies were also detected, including one to ten markers (primer pair specific to Formosa landlocked salmon; Table 1). For example, there were ten fragments (117, 118, 118.5, 178.5, 180, 209, 217.5, 285, 297 and 348 bp) specific to Formosa landlocked salmon, five fragments (203, 206, 248.5, 260 and 280 bp) specific to Biwa salmon, and two fragments (194.5 and 281.5 bp) specific to Masu salmon (Table 1). The average intra-specific genetic distances ranged from 0.0292 (between individuals of O. masou formosanus) to 0.1026 (between individuals of O. masou ishikawae; Table 2). The average inter-specific
J.-C. Gwo et al. / Molecular Phylogenetics and Evolution 48 (2008) 776–781 Table 1 The subspecies-specific bands detected from AFLP analysis for seven selective-primer pairs in four subspecies of Oncorhynchus masou complex Amago
Masu
Biwa
Formosa
ACACAA-190
AAGCAA-281.5 AACCAC-194
AAGCAA-260 AAGCAA-248.5 ACCCAA-206 ACCCAT-280 ACCCAT-203
AAGCAA-217.5 ACACAA-178.5 ACGCAT-118.5 ACGCAT-348 ACGCAT-297 ACGCAT-118 ACGCAT-209 ACCCAC-285 ACCCAC-180 ACACAC-117
Species abbreviations: Amago (O. masou ishikawae); Biwa (O. masou subsp.); Masu (O. masou masou); Formosa (O. masou formosanus).
Table 2 The average intra-specific genetic distances among six Oncorhynchus species
Rainbow Amago Masu Biwa Formosa Pink
Rainbow
Amago
Masu
0.0646 0.6702 0.6686 0.6745 0.6748 0.7148
0.1026 0.1602 0.1735 0.2127 0.6929
0.0969 0.1772 0.1863 0.7124
Biwa
0.0416 0.2209 0.7060
Formosa
0.0292 0.7100
Pink
0.0333
Species abbreviations: Amago (O. masou ishikawae); Biwa (O. masou subsp.); Masu (O. masou masou); Pink (O. gorbuscha); Rainbow (Oncorhynchus mykiss); Formosa (O. masou formosanus).
distances ranged from 0.1602 (between O. masou ishikawae and O. masou masou) to 0.7148 (between O. gorbuscha and
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O. mykiss). Intra-specific variation of AFLP is clearly lower than the difference between species. Our tree recovers the predicted relationships among the outgroup taxa. Rainbow trout and pink salmon occupy their expected positions as sisters to the O. masou complex (Fig. 2). The high bootstrap values at each node indicate that this tree is robust. The NJ phenogram generated from the matrix of AFLP showed three main groupings: (1) O. mykiss (rainbow trout); (2) O. gorbuscha (pink salmon); and (3) O. masou complex. The species of O. masou complex were further divided into four subgroups: (1) O. masou formosanus (Formosa landlocked salmon); (2) O. masou masou (Masu salmon), (3) O. masou subsp. (Biwa salmon) and (4) O. masou ishikawae (Amago salmon). O. masou subsp. and O. masou ishikawae were clustered first, O. masou masou second, and then Formosa landlocked salmon were jointed to this cluster. Bootstrap analysis showed a high confidence of the nodes for O. masou complex. 4. Discussion The taxonomy and evolutionary relationships of the Formosa landlocked salmon to the other three species of the O. masou complex remains uncertain. Differences in scale morphology and the presence of red spots on the body are diagnostic characters for distinguishing among the four subspecies (Kimura, 1990; Hosoya et al., 1992). The Formosa landlocked salmon can be separated from the Japanese subspecies by having fewer vertebrae, anal fin rays, pectoral fin rays, higher body depth and several meristic characters (Behnke et al., 1962; Watanabe and
Fig. 2. The relationships among six Oncorhynchus species inferred from applying the neighbor-joining method to the data of AFLP banding profiles. Species abbreviations: A, Amago (O. masou ishikawae); B, Biwa (O. masou subsp.); M, Masu salmon (O. masou masou); P, Pink salmon (O. gorbuscha); R, Rainbow trout (O. mykiss); F, Formosa landlocked salmon (O. masou formosanus). Each number indicates the proportion of 1000 bootstrap samples in which a particular clade was found. Only numbers above 60 were given.
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J.-C. Gwo et al. / Molecular Phylogenetics and Evolution 48 (2008) 776–781
Lin, 1985; Jan et al., 1990). Many of these studies have suggested that the Formosa landlocked salmon is evolutionarily close to O. masou. Hosoya et al. (1992) suggested that the Formosa landlocked salmon is similar enough to cherry salmon (O. masou) to be regarded as a subspecies of the O. masou complex. This research indicated that the four subspecies of cherry salmon form a close evolutionary group, but did not provide a clear indication of the phylogenetic relationships. The present study identified markers that are unique for each subspecies of the O. masou complex. Studies using mitochondrial control region sequences have not led to conclusions regarding the species identification and relationships among the four subspecies of the O. masou complex (Tzeng et al., 2006). The conclusions from the AFLP approach also are consistent with those from numerical analyses of the general anatomy of the O. masou complex and the Formosa landlocked salmon (Oshima, 1919; Teng, 1959; Watanabe and Lin, 1985; Jan et al., 1990; Hosoya et al., 1992). Two complexes of forms are distinguished within the O. masou complex, i.e., the presence or absence of red spots on the body. Our data also show strong support (83% bootstrap) for the clustering of Biwa and Amago salmon. This is consistent with the fact that subgroup A (Amago and Biwa) and subgroup B (Masu and Formosa landlocked salmon) have consistent differences in coloration and differing morphology of the scales (Watanabe and Lin, 1985; Okazaki, 1986; Jan et al., 1990; Kimura, 1990; Hosoya et al., 1992). Based on these data, we share the same opinion expressed by Watanabe and Lin (1985) and Hosoya et al. (1992) that the Formosa landlocked salmon is not a local population of O. masou masou, and should be regarded as a new subspecies distinct from the rest of O. masou complex, i.e., Amago, Biwa, and Masu salmon. The most surprising discovery of the present study was that the AFLP distance data and evolutionary tree did not support the current view of cherry salmon complex evolution. Our tree supported the predicted relationships among the outgroup taxa; rainbow trout and pink salmon as sisters to the O. masou complex. DNA sequence analysis of the mitochondrial genome demonstrated that the lacustrine Biwa salmon is the oldest lineage of the O. masou complex in Japan, i.e., Amago, Biwa, and Masu salmon (Oohara and Okazaki, 1996; Mckay et al., 1998). Based on 10 kinds of 6 base-pair restriction endonuclease fragment pattern analyses of mtDNA, Numachi et al. (1990) speculated that the Formosa landlocked salmon originated from Japan Masu salmon distributed in the Sea of Japan, and reached Taiwan through the Tsushima channel 100– 800 thousand years ago when a cold current prevailed in the Sea of Japan (Fig. 1). Our tree rejects the hypothesis that the Formosa landlocked salmon arose from Masu salmon (O. masou masou) in Japan, but instead supports the view of Watanabe and Lin (1985) that the ancestor of the O. masou complex may have originated from an ancient lake in the northern part of the Amur River, in northern
China. The fish migrated down the Amur River, rode the cold currents moving southward through the Sea of Japan, passed the East China Seas, and finally became landlocked in Taiwan during the formation of the Lishan Basin in the post-glacial period. More cherry salmon specimens from Russia, Korea and China should be studied to confirm this proposal. It would be difficult to define species on the basis of mtDNA genes if the species had become isolated, or if there was hybridization (Albertson et al., 1999; Sullivan et al., 2004; Bensch and Akesson, 2005). Although AFLP is not commonly used in phylogenetic studies, it has been successfully used to resolve phylogenies and species boundaries in rapidly-evolving systems, including clades of Lake Malawi cichlids (Albertson et al., 1999; Kassam et al., 2005), a species flock of African electric fish (Sullivan et al., 2004) and six penaeid shrimp species (Wang et al., 2004). In this study, use of AFLP revealed the genetic relationships among the four subspecies of the O. masou complex. We conclude that, with appropriate precautions, AFLP is informative in distinguishing phylogenetic relationships. Our results once again highlight the potential power of AFLP to resolve complex species phylogenies among closely-related species. Acknowledgments We thank Mr. L.-Y. Liao, Drs. H. Mayama, H. Ohta, and H. Onozato for providing samples and Mr. Andrew Yeh for his help with English. We are grateful to Drs. W.-H. Li, and R.C. Burghardt for kindly reviewing the manuscript. The work was supported by Shei-Pa National Park, Ministry of the Interior, Taiwan. References Albertson, R.C., Markert, J.A., Danley, P.D., et al., 1999. Phylogeny of a rapidly evolving clade: the cichlid fishes of Lake Malawi, East Africa. Proceedings of the National Academy of Sciences of the United States of America 96, 5107–5110. Behnke, R.J., Koh, T.P., Needham, P.R., 1962. Status of the landlocked salmonid fishes of Formosa with a review of Oncorhynchus masou (Brevoort). Copeia 2, 400–407. Bensch, T., Akesson, M., 2005. Ten years of AFLP in ecology and evolution: why so few animals? Molecular Ecology 14, 2899– 2914. Chung, Y.H., Chang, H.W., Gwo, J.C., et al., 2006. Genetic relationship among the subspecies of Oncorhynchus masou determined by growth hormone genes. Poster #162 in Fourteenth Symposium on Recent Advances in Cellular and Molecular Biology, Taiwan, January 18–20, 2006 (abstract). Gwo, J.-C., Lin, X.-W., Gwo, H.H., et al., 1996. The ultrastructure of Formosan landlocked salmon, Oncorhynchus masou formosanus, spermatozoon (Teleostei; Salmoniformes; Salmonidae). Journal of Submicroscopic Cytology & Pathology 28, 33–40. Gwo, J.-C., Ohta, H., Okuzawa, K., et al., 1999. Cryopreservation of fish sperm from the endangered Formosan landlocked salmon (Oncorhynchus masou formosanus). Theriogenology 51, 569–582. Gwo, J.-C., Chen, M.-L., Ding, W.-H., et al., 2007. What inhibits the testicular development in Taiwan landlocked salmon (Oncorhynchus masou formosanus)? 5th International Conference on Marine Pollution
J.-C. Gwo et al. / Molecular Phylogenetics and Evolution 48 (2008) 776–781 and Ecotoxicology, June 3–7 at City University of Hong Kong, Hong Kong. Healy, M., Kline, P., Tsai, C.F., 2001. Saving the endangered Formosa landlocked salmon. Fisheries 26, 6–14. Hosoya, K., Chang, K.H., Numachi, K.I., 1992. Character examination of the basibranchial teeth of the Formosan salmon. Bulletin of the Institute of Zoology Academia Sinica 31, 213–220. Jan, R.Q., Jaung, L.C., Lin, Y.S., et al., 1990. A morphometric and meristic study of the landlocked salmon in Taiwan, in comparison with other members of the genus Oncorhynchus (Salmonidae). Bulletin of the Institute of Zoology Academia Sinica 29 (3 suppl.), 41–59. Jordan, D.S., Oshima, M., 1919. Salmo formosanus, a new trout from the mountain streams of Formosa. Proceedings of the Academy of Natural Sciences of Philadelphia 71, 122–124. Kakehi, Y., Nakayama, K., Watanabe, K., et al., 2005. Inheritance of amplified fragment length polymorphism markers and their utility in population genetic analysis of Plecoglossus altivelis. Journal of Fish Biology 66, 1529–1544. Kassam, D., Seki, S., Rusuwa, B., et al., 2005. Genetic diversity within the genus Cynotilapia and its phylogenetic position among Lake Malawi’s mbuna cichlids. African Journal of Biotechnology 4, 1195–1202. Kato, F., 1991. Life histories of masu and amago salmon (Oncorhynchus masou and Oncorhynchus rhodurus). In: Groot, C., Margolis, L. (Eds.), Pacific Salmon Life Histories. University of British Columbia Press, Vancouver, pp. 449–516. Kimura, S., 1990. On the type specimens of Salmo macrostoma, Oncorhynchus ishikawae and O. rhodurud. Bulletin of the Institute of Zoology Academia Sinica 29, 1–16. Kiso, K., 1995. The life history of masu salmon Oncorhynchus masou originated from rivers of the Pacific coast of Northern Honshu, Japan. Bulletin National Research Institute of Fishery Sciences 7, 1–188. Liu, Z.J., Cordes, J.F., 2004. DNA marker technologies and their applications in aquaculture genetics. Aquaculture 238, 1–37. McKay, S.J., Nakayama, I., Smith, M.J., et al., 1998. Genetic relationships between masu and amago salmon examined through sequence analysis of nuclear and mitochondrial DNA. Zoological Science 15, 971–979.
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Numachi, K.I., Kobayashi, T., Chang, K.H., et al., 1990. Genetic identification and differentiation of Formosan salmon, Oncorhynchus masou formosanus, by restriction analysis of mitochondrial DNA. Bulletin of the Institute of Zoology Academia Sinica 29 (3suppl), 61– 72. Okazaki, T., 1986. Genetic variation and population structure in masu salmon Oncorhynchus masou of Japan. Bulletin of the Japanese Society Scientific Fisheries 52, 1365–1376. Oohara, I., Okazaki, T., 1996. Genetic relationship among three subspecies of Oncorhynchus masou determined by mitochondrial DNA sequence analysis. Zoological Science 13, 189–198. Oshima, M., 1919. A new salmonid fish from Taiwan. Taiwan-nojihou 151, 14–16 (in Japanese). Oshima, M., 1934. Life-history and distribution of the freshwater salmons found in the waters of Japan. Proceedings of the 5th Pacific Science Congress of Canada 5, 3751–3773. Oshima, M., 1936. Ecological study on the masu of the Taiko River. Botany and Zoology 4, 1–13 (in Japanese). Sanford, C.P., 1990. The phylogenetic relationships of salmonid fishes. Bulletin of the British Museum Natural History (Zoology) 56, 145– 153. Sullivan, J.P., Lavoue, S., Arnegard, M.E., et al., 2004. AFLPs resolve phylogeny and reveal mitochondrial introgression within a species flock of African electric fish (Mormyroidea: Teleostei). Evolution 58, 825–841. Teng, F.T., 1959. On morphology and ecology of Formosa landlocked salmon. Bulletin of Taiwan Fisheries Research Institute 5, 77–82 (in Chinese). Tzeng, C.S., Lin, Y.S., Lin, S.M., et al., 2006. The phylogeography and population demographics of selected freshwater fishes in Taiwan. Zoological Studies 45, 285–297. Wang, Z.Y., Tsoi, K.H., Chu, K.H., 2004. Applications of AFLP technology in genetic and phylogenetic analysis of penaeid shrimp. Biochemical Systematics and Ecology 32, 399–407. Watanabe, M., Lin, Y., 1985. Revision of the salmonid fish in Taiwan. Bulletin of the Biogeographical Society of Japan 40, 75–85.
Molecular Phylogenetics and Evolution 48 (2008) 776–781 www.elsevier.com/locate/ympev
Short Communication
Genetic relationship among four subspecies of cherry salmon (Oncorhynchus masou) inferred using AFLP Jin-Chywan Gwo a,*, Te-Hua Hsu a, Kuan-Hung Lin b, Yii-Cheng Chou c a
Department of Aquaculture, Taiwan National Ocean University, No. 2 Pei-Ning Road, Keelung 20224, Taiwan b Graduate Institute of Biotechnology, Chinese Culture University, Taipei 11119, Taiwan c Department of Medical Technology, Chung-Hwa University of Medical Technology, Tainan 717, Taiwan Received 6 December 2007; accepted 21 December 2007 Available online 3 January 2008
1. Introduction The Pacific salmons are comprised of at least seven species. These seven species are distributed in fresh water and in the sea, including most of the North Pacific rim and adjacent ocean regions, collectively extending across the Pacific (Sanford, 1990; Kato, 1991; Kiso, 1995). One of the seven known species, the cherry salmon (Oncorhynchus masou), also known as ‘‘Sakuramasu”, is confined to Asian rivers and coastal marine waters (Fig. 1). The four known extant subspecies of the O. masou complex (the cherry salmon complex) are Masu (O. masou masou), Amago (O. masou ishikawae), Biwa (O. masou subsp.) and the Formosa landlocked salmon (O. masou formosanus) (Kato, 1991; Kimura, 1990). The fluvial Formosa landlocked salmon is found only in the head waters of the Tachia River, Taiwan. This is the southernmost limit of the distribution range of salmonids (Kimura, 1990). Historically, the taxonomy and nomenclature of the O. masou complex has been greatly debated, in part because of the similar morphological and ecological traits shared by all four Masu salmon subspecies. The scientific name and systematic position of the Formosa landlocked salmon has been especially contested. In fact, its scientific name has been changed four times to date. Oshima (1919) originally referred to the Formosa landlocked salmon as Salmo saramao, in Japanese, but gave a different name––Salmo formosanus––in his English publication the same year (Jordan and Oshima, 1919). Upon further research, Oshima revised the scientific name of the Formosa landlocked salmon to O. formosanus (1934). In 1936, Oshima modified the scientific name once *
Corresponding author. Fax: +886 2 25093958. E-mail address: [email protected] (J.-C. Gwo).
1055-7903/$ - see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.ympev.2007.12.023
again to O. masou because he found no red spots on the sides of the body (Oshima, 1936). The Formosa landlocked salmon is a highly inbred population, with the existing population estimated at less than 1000 individuals (Gwo et al., 1999, 2007; Healy et al., 2001). Because it was listed as endangered by the Taiwan Government in 1984, defining the evolutionary unit in this species has become urgent to protect the remaining population. The scientific name and taxonomic position of the Formosa landlocked salmon is based on morphology and meristics (Jordan and Oshima, 1919; Oshima, 1919, 1934, 1936; Teng 1959; Behnke et al., 1962; Watanabe and Lin, 1985; Jan et al., 1990; Hosoya et al., 1992), on analysis of mt DNA (Numachi et al., 1990; Chung et al., 2006; Tzeng et al., 2006), or on sperm ultrastructure (Gwo et al., 1996, 1999). After comparing 25 meristic and morphometric characters of seven Oncorhynchus species, Jan et al. (1990) concluded that morphometric and meristic counts alone are insufficient to clarify the phylogeny of the Formosa landlocked salmon. Indeed, these four known extant subspecies of the O. masou complex exhibit little or no morphological difference (Watanabe and Lin, 1985; Jan et al. 1990). Further research in genetics, using molecular markers, may help to elucidate whether the Formosa landlocked salmon is one distinct subspecies of O. masou complex, or merely a separated local population of Masu salmon (O. masou masou). However, studies using several microsatellite DNA loci and mitochondrial control region sequences have failed to resolve relationships among the four subspecies of the cherry salmon complex (Tzeng et al., 2006; Gwo, unpublished data). Amplified fragment length polymorphism (AFLP) analysis has been widely applied to study species, strain and hybrid identification, gene mapping, linkage and genetic
J.-C. Gwo et al. / Molecular Phylogenetics and Evolution 48 (2008) 776–781
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Fig. 1. Sample collection sites. Amago salmon (O. masou ishikawae), Masu salmon (O. masou masou), Biwa salmon (O. masou subsp.), Formosa landlocked salmon (O. masou formosanus) and pink salmon (O. gorbuscha).
diversity of species and populations in a wide variety of organisms including fishes and shrimp (Liu and Cordes, 2004; Wang et al., 2004; Kakehi et al., 2005; Kassam et al., 2005). In this study, we used AFLP to study variation sites across the genome. We also compared the AFLP fingerprints derived from PCR amplification using seven pairs of selective primers for three Oncohynchus species–– rainbow trout (O. mykiss), pink salmon (O. gorbuscha), and cherry salmon (O. masou)––with cherry salmon further divided into the four subspecies, Amago (O. masou ishikawae), Biwa (O. masou subsp.), Masu (O. masou masou), and Formosa landlocked salmon (O. masou formosanus) to explore the applicability of AFLP technology in species/ subspecies identification and phylogenetic analysis. Our results demonstrated that the AFLP technique is indeed useful to resolve the phylogenetic relationships among these four subspecies.
2. Materials and methods 2.1. Sample collection and DNA extraction Nineteen wild population Formosa landlocked salmon, and six cultivated rainbow trout were collected from Chichawan Stream in the Shei-Pa National Park, Taiwan and from a private hatchery at Jinsan, Taiwan, respectively. Pink salmon, Amago salmon, Masu salmon, and Biwa salmon were obtained from the following locations in Japan: Hokkaido Kumagaya Station––wild Masu salmon (5 specimens); Pacific Ocean near Hokkaido––wild pink salmon (5 specimens); Inland Station of the National Research Institute of Aquaculture––cultivated stock Amago salmon (10 specimens) and Biwa salmon (3 specimens; Fig. 1). The specimens were either stored at 80 °C or preserved in 95% ethanol prior to analysis. Total DNA was
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extracted from muscle (10–15 mg) using proteinase-K digestion and the phenol–chloroform–isoamyl alcohol method with slight modifications. DNA concentration was measured with an UV spectrophotometer. The quality of extracted DNA was assessed by 1.0% agarose gel electrophoresis with ethidium bromide. 2.2. AFLP reactions Procedures of AFLP analysis were essentially based on Wang et al. (2004). Fingerprint patterns were visualized on a 5% denaturing polyacrylamide gel using the silver staining method. DNA templates for AFLP reactions were generated by restriction digestion and ligation. Initially, about 100 ng of total DNA was digested with 5 U of EcoRI and Tru9 1 (Promega) in 1 NE buffer 2 at 37 °C for 2 h. To generate DNA templates for subsequent PCR amplification, the digested DNA fragments were ligated with 2.5 pmol of EcoRI and 25 pmol Msel adapters in a reaction mixture containing 0.25 mg BSA, 5 pmol ATP, 0.04 U T4 DNA ligase and 10 NE buffer 2 at 37 °C for 6 h. Preamplification PCR reactions were conducted using an Eppendorf mastercycler gradient with a pair of primers containing single selective nucleotides. Amplification was performed at an annealing temperature of 53 °C for 30 s. The 20 ll PCR product mixture was diluted 10-fold with distilled water and used as templates for the subsequent selective PCR amplification. The selective amplification was performed using seven pairs of primers, E-ACA/M-CAC, E-ACC/M-CAC, E-ACC/M-CAT, E-ACG/M-CAT, E-AAC/M-CAC, E-ACG/M-CAC, and E-ACA/M-CAA. 2.3. Electrophoresis and silver staining The PCR products were mixed with 10 ll AFLP loading buffer (99% formamide, 10 mM EDTA, 0.05%. bromophenol and 0.05% xylene cyanol). The product mixtures were denatured and concentrated at 90 °C for 25 min, and quickly cooled in an ice bath after denaturation. A 5% denaturing polyacrylamide gel (4.75% acrylamide, 0.25% bisacrylamide, 7 mol l 1 urea and 0.5 TBE) was prerun at 145 W for 30 min. Each well was loaded with 2.5 ll of sample. The gel was electrophoresed for 2.5 h in an ATTO (Type AE6155) DNA sequencing cell (38 50 cm) at 110 W and 50 °C. After electrophoresis, the gel was fixed in 1% ethanoic acid for at least 30 min. The gel was rinsed in distilled water and stained with a mixture of 0.1% silver nitrate and 0.007% benzene sulphonic acid for 30 min. The stained gel was rinsed again with distilled water and immersed in a developing solution (2.5% sodium carbonate, 0.037% formaldehyde and 0.002% sodium thiosulphate). The development was subsequently stopped with 1% ethanoic acid when bands were visible and reached desirable intensity. Band sizes were estimated using a standard AFLP
DNA ladder and analyzed using the Imaging Analyzing System (HP scanjet 5370c). 2.4. Data analysis AFLP bands were scored for presence (1) or absence (0), and transformed into a 0/1 binary character matrix. Fragments that could not be scored unambiguously were excluded from the analysis. The total number of individuals analyzed was 48, with three individuals for each species. The fragment was recognized using a sequence of 16 bp (Wang et al., 2004). Data were analyzed using a set of programs in the PHYLIP v3.6a3 Inference Package. The 0/1 matrix was converted into a distance matrix using the program RESTDIST. One thousand bootstrapped data sets were generated using SEQBOOT, and 1000 bootstrapped trees were constructed. A neighbor-joining (NJ) tree was constructed using the NEIGHBOR based on the matrix from RESTDIST, and redrawn using the TreeView program. 3. Results This study established AFLP fingerprints for 48 individuals from three Oncohynchus species––rainbow trout (O. mykiss), pink salmon (O. gorbuscha), and cherry salmon (O. masou)––with cherry salmon further divided into the four subspecies, Amago (O. masou ishikawae), Biwa (O. masou subsp.), Masu (O. masou masou), and Formosa landlocked salmon (O. masou formosanus). The seven pairs of primer sets yielded a total of 782 scorable bands (size range 100–350 bp). One hundred and fourteen bands (15%) were found in all species, with 77 (10%) scored in all individuals (i.e. monomorphic), and the other 37 polymorphic. The number of bands generated from a single individual ranged from 23 to 101, while a single species produced 337 (in O. gorbuscha) to 522 (in O. masou ishikawae) bands. Species differed in the levels of genetic diversity among individuals, with 11.5% in O. masou subsp. to 41.3% in O. masou ishikawae of the bands scored as polymorphic. The proportion of subspecies-specific markers to the total number of bands scored for each subspecies ranged from 0.20% (in O. masou ishikawae and O. masou masou) to 1.55% (O. masou formosanus). Many specific markers among Masu, rainbow and pink salmon were detected. Specific markers among the four O. masu complex subspecies were also detected, including one to ten markers (primer pair specific to Formosa landlocked salmon; Table 1). For example, there were ten fragments (117, 118, 118.5, 178.5, 180, 209, 217.5, 285, 297 and 348 bp) specific to Formosa landlocked salmon, five fragments (203, 206, 248.5, 260 and 280 bp) specific to Biwa salmon, and two fragments (194.5 and 281.5 bp) specific to Masu salmon (Table 1). The average intra-specific genetic distances ranged from 0.0292 (between individuals of O. masou formosanus) to 0.1026 (between individuals of O. masou ishikawae; Table 2). The average inter-specific
J.-C. Gwo et al. / Molecular Phylogenetics and Evolution 48 (2008) 776–781 Table 1 The subspecies-specific bands detected from AFLP analysis for seven selective-primer pairs in four subspecies of Oncorhynchus masou complex Amago
Masu
Biwa
Formosa
ACACAA-190
AAGCAA-281.5 AACCAC-194
AAGCAA-260 AAGCAA-248.5 ACCCAA-206 ACCCAT-280 ACCCAT-203
AAGCAA-217.5 ACACAA-178.5 ACGCAT-118.5 ACGCAT-348 ACGCAT-297 ACGCAT-118 ACGCAT-209 ACCCAC-285 ACCCAC-180 ACACAC-117
Species abbreviations: Amago (O. masou ishikawae); Biwa (O. masou subsp.); Masu (O. masou masou); Formosa (O. masou formosanus).
Table 2 The average intra-specific genetic distances among six Oncorhynchus species
Rainbow Amago Masu Biwa Formosa Pink
Rainbow
Amago
Masu
0.0646 0.6702 0.6686 0.6745 0.6748 0.7148
0.1026 0.1602 0.1735 0.2127 0.6929
0.0969 0.1772 0.1863 0.7124
Biwa
0.0416 0.2209 0.7060
Formosa
0.0292 0.7100
Pink
0.0333
Species abbreviations: Amago (O. masou ishikawae); Biwa (O. masou subsp.); Masu (O. masou masou); Pink (O. gorbuscha); Rainbow (Oncorhynchus mykiss); Formosa (O. masou formosanus).
distances ranged from 0.1602 (between O. masou ishikawae and O. masou masou) to 0.7148 (between O. gorbuscha and
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O. mykiss). Intra-specific variation of AFLP is clearly lower than the difference between species. Our tree recovers the predicted relationships among the outgroup taxa. Rainbow trout and pink salmon occupy their expected positions as sisters to the O. masou complex (Fig. 2). The high bootstrap values at each node indicate that this tree is robust. The NJ phenogram generated from the matrix of AFLP showed three main groupings: (1) O. mykiss (rainbow trout); (2) O. gorbuscha (pink salmon); and (3) O. masou complex. The species of O. masou complex were further divided into four subgroups: (1) O. masou formosanus (Formosa landlocked salmon); (2) O. masou masou (Masu salmon), (3) O. masou subsp. (Biwa salmon) and (4) O. masou ishikawae (Amago salmon). O. masou subsp. and O. masou ishikawae were clustered first, O. masou masou second, and then Formosa landlocked salmon were jointed to this cluster. Bootstrap analysis showed a high confidence of the nodes for O. masou complex. 4. Discussion The taxonomy and evolutionary relationships of the Formosa landlocked salmon to the other three species of the O. masou complex remains uncertain. Differences in scale morphology and the presence of red spots on the body are diagnostic characters for distinguishing among the four subspecies (Kimura, 1990; Hosoya et al., 1992). The Formosa landlocked salmon can be separated from the Japanese subspecies by having fewer vertebrae, anal fin rays, pectoral fin rays, higher body depth and several meristic characters (Behnke et al., 1962; Watanabe and
Fig. 2. The relationships among six Oncorhynchus species inferred from applying the neighbor-joining method to the data of AFLP banding profiles. Species abbreviations: A, Amago (O. masou ishikawae); B, Biwa (O. masou subsp.); M, Masu salmon (O. masou masou); P, Pink salmon (O. gorbuscha); R, Rainbow trout (O. mykiss); F, Formosa landlocked salmon (O. masou formosanus). Each number indicates the proportion of 1000 bootstrap samples in which a particular clade was found. Only numbers above 60 were given.
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Lin, 1985; Jan et al., 1990). Many of these studies have suggested that the Formosa landlocked salmon is evolutionarily close to O. masou. Hosoya et al. (1992) suggested that the Formosa landlocked salmon is similar enough to cherry salmon (O. masou) to be regarded as a subspecies of the O. masou complex. This research indicated that the four subspecies of cherry salmon form a close evolutionary group, but did not provide a clear indication of the phylogenetic relationships. The present study identified markers that are unique for each subspecies of the O. masou complex. Studies using mitochondrial control region sequences have not led to conclusions regarding the species identification and relationships among the four subspecies of the O. masou complex (Tzeng et al., 2006). The conclusions from the AFLP approach also are consistent with those from numerical analyses of the general anatomy of the O. masou complex and the Formosa landlocked salmon (Oshima, 1919; Teng, 1959; Watanabe and Lin, 1985; Jan et al., 1990; Hosoya et al., 1992). Two complexes of forms are distinguished within the O. masou complex, i.e., the presence or absence of red spots on the body. Our data also show strong support (83% bootstrap) for the clustering of Biwa and Amago salmon. This is consistent with the fact that subgroup A (Amago and Biwa) and subgroup B (Masu and Formosa landlocked salmon) have consistent differences in coloration and differing morphology of the scales (Watanabe and Lin, 1985; Okazaki, 1986; Jan et al., 1990; Kimura, 1990; Hosoya et al., 1992). Based on these data, we share the same opinion expressed by Watanabe and Lin (1985) and Hosoya et al. (1992) that the Formosa landlocked salmon is not a local population of O. masou masou, and should be regarded as a new subspecies distinct from the rest of O. masou complex, i.e., Amago, Biwa, and Masu salmon. The most surprising discovery of the present study was that the AFLP distance data and evolutionary tree did not support the current view of cherry salmon complex evolution. Our tree supported the predicted relationships among the outgroup taxa; rainbow trout and pink salmon as sisters to the O. masou complex. DNA sequence analysis of the mitochondrial genome demonstrated that the lacustrine Biwa salmon is the oldest lineage of the O. masou complex in Japan, i.e., Amago, Biwa, and Masu salmon (Oohara and Okazaki, 1996; Mckay et al., 1998). Based on 10 kinds of 6 base-pair restriction endonuclease fragment pattern analyses of mtDNA, Numachi et al. (1990) speculated that the Formosa landlocked salmon originated from Japan Masu salmon distributed in the Sea of Japan, and reached Taiwan through the Tsushima channel 100– 800 thousand years ago when a cold current prevailed in the Sea of Japan (Fig. 1). Our tree rejects the hypothesis that the Formosa landlocked salmon arose from Masu salmon (O. masou masou) in Japan, but instead supports the view of Watanabe and Lin (1985) that the ancestor of the O. masou complex may have originated from an ancient lake in the northern part of the Amur River, in northern
China. The fish migrated down the Amur River, rode the cold currents moving southward through the Sea of Japan, passed the East China Seas, and finally became landlocked in Taiwan during the formation of the Lishan Basin in the post-glacial period. More cherry salmon specimens from Russia, Korea and China should be studied to confirm this proposal. It would be difficult to define species on the basis of mtDNA genes if the species had become isolated, or if there was hybridization (Albertson et al., 1999; Sullivan et al., 2004; Bensch and Akesson, 2005). Although AFLP is not commonly used in phylogenetic studies, it has been successfully used to resolve phylogenies and species boundaries in rapidly-evolving systems, including clades of Lake Malawi cichlids (Albertson et al., 1999; Kassam et al., 2005), a species flock of African electric fish (Sullivan et al., 2004) and six penaeid shrimp species (Wang et al., 2004). In this study, use of AFLP revealed the genetic relationships among the four subspecies of the O. masou complex. We conclude that, with appropriate precautions, AFLP is informative in distinguishing phylogenetic relationships. Our results once again highlight the potential power of AFLP to resolve complex species phylogenies among closely-related species. Acknowledgments We thank Mr. L.-Y. Liao, Drs. H. Mayama, H. Ohta, and H. Onozato for providing samples and Mr. Andrew Yeh for his help with English. We are grateful to Drs. W.-H. Li, and R.C. Burghardt for kindly reviewing the manuscript. The work was supported by Shei-Pa National Park, Ministry of the Interior, Taiwan. References Albertson, R.C., Markert, J.A., Danley, P.D., et al., 1999. Phylogeny of a rapidly evolving clade: the cichlid fishes of Lake Malawi, East Africa. Proceedings of the National Academy of Sciences of the United States of America 96, 5107–5110. Behnke, R.J., Koh, T.P., Needham, P.R., 1962. Status of the landlocked salmonid fishes of Formosa with a review of Oncorhynchus masou (Brevoort). Copeia 2, 400–407. Bensch, T., Akesson, M., 2005. Ten years of AFLP in ecology and evolution: why so few animals? Molecular Ecology 14, 2899– 2914. Chung, Y.H., Chang, H.W., Gwo, J.C., et al., 2006. Genetic relationship among the subspecies of Oncorhynchus masou determined by growth hormone genes. Poster #162 in Fourteenth Symposium on Recent Advances in Cellular and Molecular Biology, Taiwan, January 18–20, 2006 (abstract). Gwo, J.-C., Lin, X.-W., Gwo, H.H., et al., 1996. The ultrastructure of Formosan landlocked salmon, Oncorhynchus masou formosanus, spermatozoon (Teleostei; Salmoniformes; Salmonidae). Journal of Submicroscopic Cytology & Pathology 28, 33–40. Gwo, J.-C., Ohta, H., Okuzawa, K., et al., 1999. Cryopreservation of fish sperm from the endangered Formosan landlocked salmon (Oncorhynchus masou formosanus). Theriogenology 51, 569–582. Gwo, J.-C., Chen, M.-L., Ding, W.-H., et al., 2007. What inhibits the testicular development in Taiwan landlocked salmon (Oncorhynchus masou formosanus)? 5th International Conference on Marine Pollution
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