Sergia Lucens) Between Japanese and Taiwanese Populations

Contributions to Zoology, 82 (3) 123-130 (2013)

Genetic and morphological differentiation in the Sakura shrimp (Sergia lucens) between Japanese and Taiwanese populations

Hideyuki Imai1, 4, Yukio Hanamura2, Jin-Hua Cheng3 1 Laboratory of Marine Biology and Coral Reef Studies, Faculty of Science, University of the Ryukyus, Nishihara, Okinawa 903-0213, Japan 2 National Research Institute of Fisheries Science, Fisheries Research Agency, Kanazawa-ku, Yokohama 236-8648, Japan 3 Tungkang Biotechnology Research Center, Taiwan Fisheries Research Institute, Pingtung 928, Taiwan 4 E-mail: [email protected]

Keywords: gene flow, genetic diversification, morphology, stock identification

Abstract Introduction

The Sakura shrimp Sergia lucens is a remarkable meso-pelagic The genetic population structures of aquatic animals crustacean species, which is harvested for human consumption are often correlated with varying levels of dispersal in in restricted geographical areas of Taiwan and Japan in the northwestern Pacific. Nucleotide sequence analysis of the mito- their species, which are closely associated with swim- chondrial DNA control region was conducted to investigate the ming ability, type/duration of larval stages, or water levels of genetic variability and differentiation between Japa- currents. In the north Pacific region, from tropical to nese and Taiwanese populations of S. lucens. The latter half of temperate areas, the Kuroshio Current plays an impor- the control region, which contained 589 nucleotide sites, was tant role in the distribution of marine organisms. For sequenced using DNA extracted from 178 individuals from the two geographical regions. Analyses yielded 162 haplotypes, and example, the Japanese eel Anguilla japonica Temmink the amount of genetic variability within the populations as shown and Schlegel, 1847 does not show any genetic differen- by the levels of haplotype and nucleotide diversity revealed that tiation among Taiwanese, Chinese and Japanese popu- the two populations included in this study have higher levels of lations (Taniguchi and Numachi, 1978; Minegishi et al., diversity than intraspecific variation previously reported inother 2012). On the other hand, some species, such as the crustaceans. Our AMOVA and pairwise Fst results indicated the existence of significant genetic differences between the Bowed fiddler crabUca arcuata (De Haan, 1833), Big- Japanese and Taiwanese populations. Morphological analyses fin reef squid Sepioteuthis spp. and Golden rabbitfish also revealed minor but notable differences between the two Siganus guttatus (Bloch, 1787), show genetic differ- groups. These findings suggest that postulated gene flow of ences among populations (Aoki et al., 2008a, b; Imai S. lucens is unlikely to occur across the isolated Japanese and and Aoki, 2012; Iwamoto et al., 2012). Taiwanese populations, and that each population completes their life history in a restricted geographical area. The Sakura shrimp Sergia lucens (Hansen, 1922) is a commercially important species that is found in the same Kuroshio region. In the past, based on limited Contents numbers of specimens, genetic differences were reported between Japanese and Taiwanese populations (Omori Introduction ...... 123 et al., 1988), despite the two populations supposedly be- Material and methods ...... 124 ing connected by the Kuroshio Current. Recently, the Genetic analysis ...... 124 number of Sakura shrimp caught in Japan has de- Morphological analysis ...... 125 creased. It is important to understand this species’ pop- Results ...... 125 Discussion ...... 127 ulation structure for appropriate resource management. Acknowledgements ...... 128 Further compounding potential management prob- References ...... 128 lems of this species, the species name for the Sakura

Downloaded from Brill.com10/06/2021 05:01:41PM via free access 124 Imai et al. – Genetic diversification ofSergia lucens shrimp has not yet been definitively decided. Article of Omori et al. (1988) are under question, in this study, 15.1 of the International Code of Zoological Nomen- the mitochondrial DNA of S. lucens collected from the clature (ICZN, 2000; see also Article 11.5.1 concern- Japanese and Taiwanese coastal waters were analyzed ing “conditional proposals” made before 1961) gives together with comparative morphology to more clearly priority to the designation Sergia kishinouyei (Naka- understand the population structure of this commer- zawa and Terao, 1915) over S.lucens, although the lat- cially important species. ter name has been popularly used by researchers since Gordon (1935), who provided a detailed account of the species (cf. Omori, 1969). Sergestes phosphoreus Material and methods Kishinouye, 1928 has also been used to refer to the Sakura shrimp, but the validity of this name is ques- Genetic analysis tioned (see Vereshchaka, 2000). To avoid taxonomic confusion, herein we use S. lucens until the nomencla- Samples were obtained by trawl fishing at three loca- ture issues of the Sakura shrimp are formally resolved. tions in Japan and Taiwan, which represent the main Sergia lucens is found from Suruga Bay, Sagami fishing grounds ofS . lucens (Fig. 1). In total, 178 spec- Bay, and the mouth of Tokyo Bay in Japan, and the imens were examined for sequence analysis of mito- coastal waters of Tungkang as well as off the east coast chondrial control region: 70 from Suruga Bay (35º06'N, of Taiwan (Omori, 1969, 1995; Holthuis, 1980; Omori 138º38'E) (collected in 1999), 61 from Tungkang et al., 1988; Isshiki and Tajima, 1992; Lee et al., 1996). (22º20'N, 120º20'E) and 47 from Gueishan Island wa- Furthermore, Vereshchaka (2000) recorded this spe- ters (24º51'N, 121º55'E), Taiwan (collected in 2003 and cies from off Borneo and New Guinea. This shrimp 2010). All samples were stored at -40ºC until use. has been commercially exploited in Suruga Bay and Total crude DNA was extracted from the pereopod Tungkang, where the annual yields average around muscle of the frozen specimens using proteinase K, 2,000 and 100 tons, respectively (Omori, 1989; Fukui phenol-chloroform, and TNES-8 M urea buffer (Imai et al., 2004). Several biological aspects of this shrimp, et al., 2004). The mitochondrial (mt) DNA control re- including the spawning activity, egg abundances, lar- gion was then amplified by polymerase chain reaction val development, swimming behaviour, and popula- (PCR) using the designed primers shrimp-slCR-f tion size fluctuation, have been reported in Suruga Bay (Huzita, 1959; Kosaka et al., 1969; Omori, 1971; Omori and Ohta, 1981; Tsukui, 1987; Bishop et al., 1989; Mu- ranaka and Jo, 1990; Hirai et al., 1992; Omori and Seino, 1993; Isshiki, 1996). In fact, more than 200 research articles, most of them in Japanese, dealing with this shrimp have been published over the last century (Kubota et al., 1995). Omori et al. (1988) conducted a comparative taxonomic study on individuals collected from Suruga Bay and Tungkang waters. These authors did not find morphological differences discriminating the two populations, suggesting that they could be a single species, although they observed differences in spawning season and isoelectrophoretic patterns of water-soluble proteins. However, as this study examined only five individuals per population, no definitive conclusions could be reached in this study. Since this study, no further information regarding the population structure of S. lucens has become available. Genetic markers such as the mitochondrial DNA control region sequence have been shown to be effective in identifying population structures, and therefore genetic data can contribute to resource management and conservation. As the validity of the research results Fig. 1. Sampling sites of Sergia lucens in Japan and Taiwan.

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(5’-GAAATAGTAAACTGTAAGTT-3’) and sakura- Results tRNA (5’-TCTATCCTATCAAGATAGCCC-3’). All reactions were carried out in a 50 µl total volume con- In total, 589 base pairs (bp) from the control region taining 1 µl of total crude DNA, 25 pM each primer, were sequenced for 178 individuals from the three 2.5 U of ExTaqTM DNA polymerase (Takara Bio), 5 µl sampling locations. One hundred and sixty-two haplo- of each dNTP, 1 × buffer, and dH2O. A GeneAmp types and 149 variable sites were identified (On-line 9700 thermal cycler (Applied Biosystems) was used Supplementary Material, Table S1), and nearly all of with the following parameters: 94ºC (60 s) followed the sequenced individuals had a unique haplotype (60 by 30 cycles of denaturation at 94.5ºC (30 s), anneal- haplotypes from Suruga Bay, 40 haplotypes from Gue- ing at 40.5ºC (30 s), and extension at 72ºC (60 s). PCR ishan and 59 haplotypes from Tungkang). However, products were purified with Exo-Sap-IT (Usb) and shared haplotypes were detected in four cases: haplo- cycle-sequenced using a BigDye Terminator Kit and type 1, seven individuals from Suruga Bay and two an ABI 3700 genetic analyzer (Applied Biosystems). from Gueishan; haplotype 2, one from Suruga Bay, The sequences obtained in this research were depos- three from Gueishan and one from Tungkang; haplo- ited in the DNA Data Bank of Japan (DDBJ) under type 92, one from Suruga Bay and one from Gueishan Accession Numbers AB846666 to AB846827. and haplotype 122, one from Gueishan and one from All sequences were initially aligned using ClustalX Tungkang. Relationships between haplotypes were ver.1.83.1 (Thompson et al., 1997). A median-joining represented on a network tree, which did not show any network of haplotypes was constructed using the Net- geographical structuring (Fig. 3). work ver. 4.6.1.1 (Bandelt et al., 1999) including all The haplotype and nucleotide diversities of the three individuals. All calculations [haplotype diversity (h; geographical groups ranged from 0.991 to 1.000 and Nei, 1987), nucleotide diversity (π; Tajima, 1983), Fst from 1.083% to 1.131%, respectively (Table 1). AMOVA value and 10,000 times Fst permutation test (Reynolds in the three populations also showed a significant Φst et al., 1983), an exact test based on 10,000 steps in a value of 0.0126 (P < 0.001), indicating significant het- Markov chain (Raymond and Rousset, 1995), and the erogeneity in at least one of the pairwise comparisons. analysis of the molecular variance (AMOVA) (Ex- The pairwise Fst value ranged from 0.0179 to 0.0205, coffieret al., 1992) were processed with Arlequin (Ex- differing significantly between Japan and Taiwan P( < coffieret al., 2005). A sequential Bonferroni test (Rice, 0.001); however, the values between Gueishan and 1989) was used to correct multiple tests of the hypoth- Tungkang indicated no significant genetic differences. esis, for which the pairwise Fst statistics did not differ Significant population subdivisions were detected be- from zero. Gene flow N( em) was estimated using the tween the Japanese and Taiwanese groups (Suruga Bay relationship Nem = [(1/Fst) - 1]/2 (Hudson et al., 1992). vs. Gueishan, and Suruga Bay vs. Tungkang; P < 0.05,

To investigate the population history of S. lucens, a Bonferroni adjustment), while the Nem value between mismatch distribution based on pairwise differences pairwise comparisons was 22.96 (Suruga Bay vs. Gue- in the mtDNA control region sequences was conduct- ishan + Tungkang waters). Tajima’s D and Fu and Li’s D ed. In addition, to check for deviations from neutrality, tests were also performed to determine neutrality. Sig- two different D tests (Tajima, 1989; Fu and Li, 1993) nificant negative values were obtained from all sam- were performed using Arlequin. pling sites by both tests (Table 1). The distribution of the pairwise number of differences in the control region Morphological analysis haplotypes fits an expansion model, with a smooth wave predicted for each location that had undergone a demo- Thirty-seven specimens of both sexes (8.4-10.4 mm in graphic expansion (Fig. 4). The isolation time between carapace length) from the two aforementioned sites in the Japanese and Taiwanese populations was estimated Taiwan (Feb. 2003; July 2010) and 18 specimens (10.0- to be 305,000 years, based on the observed nucleotide 13.0 mm) from Suruga Bay (May 2003) were used for divergence between them and a mutation rate of 19%/ the examination of a relative distance of the photo- MY, taken from the mtDNA control region of penaeid phores on the antennal scale (Fig. 2). The measure- shrimps (McMillen-Jackson and Bert, 2003). ments were made using an ocular-micrometer mount- To support the differences observed in genetic analy- ed on a bi-ocular microscope. In addition, the structure ses, some morphological features of S. lucens collected of the petasma was examined for male specimens (16 from Japan and Taiwan were examined (Fig. 5). These individuals from Japan and 42 from Taiwan). analyses showed that the relative distance of the three

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Fig. 2. Antennal scale of Sergia lucens and measurement of photophore distance: male 10.3 mm in carapace length from Suruga Bay, Japan (A) and male 10.3 mm from off Gueishan, Taiwan (B). Fig. 3. Haplotype network for 162 haplotypes of Sergia lucens. White, dark and black circles indicated haplotypes among lo- calities. The size of the circles is proportional to the haplotype frequencies. Lengths of the line showed relative to the number of mutations between haplotypes.

Fig. 5. Comparison in relative distance between the photophores on the antennal scale of Sergia lucens in specimens collected from Japanese and Taiwanese waters.

photophores on the antennal scale differed significantly between the two populations (t-test = 2.414, p < 0.05), with the Japanese specimens having a mid-photophore placed more posteriorly than the Taiwanese ones. The male petasma also showed minor variations in Fig. 4. Observed pairwise differences (bars) and the expected mismatch distributions under the sudden expansion model its form between the two groups, with approximately (line) of haplotypes detected in the Japanese and Taiwanese 21% of Taiwanese specimens having a small but dis- populations of Sergia lucens. tinct spine-like projection at the base of the lobus ter-

Downloaded from Brill.com10/06/2021 05:01:41PM via free access Contributions to Zoology, 82 (2) – 2013 127 minalis as compared to less than 7% in the Japanese cate a lack of ongoing gene flow in S. lucens. Al- specimens (Fig. 6). though Omori et al. (1988) mentioned that Japanese and Taiwanese S. lucens were morphologically indis- tinguishable, in our examination minor but apprecia- Discussion ble differences were found in the relative distance of photophores on the antennal scale. The shape of the Genetic diversity, as shown by haplotype diversity, petasma also slightly differed between the two popu- was found to be very high in S. lucens. The values lations as the males from Taiwan had a tendency to generated in this study are comparable with those de- have a developed projection at the base of the lobus rived from the mtDNA control region sequences for terminalis of the petasma (Fig. 6; Table 2). the crustaceans Farfantepenaeus aztecus (Ives, Following these findings, we presume the isolation 1891), Litopenaeus setiferus (Linnaeus, 1767) and time between the Japanese and Taiwanese popula- Panulirus penicillatus (Olivier, 1791) (McMillen- tions to be 305,000 years (middle of the Pleistocene) Jackson and Bert, 2003; Abdullah et al., in press). based on nucleotide divergence estimation time (19%/ The reported value for haplotype diversity of penaeid MY) for penaeid shrimps (Kimura, 1996; McMillen- shrimp and spiny lobster were 0.996 and 1.000, re- Jackson and Bert, 2003). The edge of the Chinese spectively (Tzeng, 2007; Abdullah et al., in press), continent, which carries several large rivers, was which is almost same to our study (ranging from once much closer to the Okinawa Trough. Sergia lu- 0.991 to 1.000). The nucleotide diversity of penaeid cens may require large rivers and ridges on the deep shrimp (McMillen-Jackson and Bert, 2003) was sea floor to sustain a large-sized population (Omoriet higher than the value of 5.4% obtained in this study. al., 1988). Thus, it may be that there used to be sev- A high level of genetic diversity in the mtDNA con- eral small populations along the continental shelf, trol region could be due to a large population size. among which gene flow most likely was maintained. Omori et al. (1988) conducted a comparative ge- However, now the main populations are distributed in netic study of S. lucens for individuals collected from central Japan and Taiwan at the edge of the continen- Suruga Bay, Japan and Tungkang waters, Taiwan, and tal shelf in isolation to each other. found that the two populations differed in isoelectro- The neutrality of mutations in the mtDNA control phoretic patterns of water-soluble proteins, implying region was rejected on the basis of Tajima’s D and Fu limited gene flow between them. The present study, and Li’s D tests. The significant negative values ob- based on the significance of isolation indicated by tained using these tests suggest that the Japanese and pairwise Fst statistical analysis by AMOVA, also sug- Taiwanese populations have experienced a popula- gested a clear isolation between the Japanese and Tai- tion expansion during their geographic history. The wanese populations. observed unimodal mismatched frequency distribu- A similar disjunctive distribution has been ob- tion pattern also matched the predicted distribution served in other crustaceans: i.e., Portunus tritubercu- under a model of population expansion. This uni- latus (Miers, 1876) by Imai et al. (1999) and Imai and modal pattern has also been observed in other crusta- Numachi (2002), Farfantepenaeus aztecus by Mc- ceans, including Euphausia superba Dana, 1850, F. Millen-Jackson and Bert (2003), Alpheus djeddensis aztecus, F. duorarum (Burkenroad, 1939), and P. (Coutiere, 1897) by Thompson et al. (2005), Parape- hardwickii (see Zane et al., 1998; McMillen-Jackson naeopsis hardwickii (Miers, 1878) by Tzeng (2007), and Bert, 2003; Tzeng, 2007). and Uca arcuata by Aoki et al. (2008b). The Kuro- Recently, Vereshchaka (2000) recorded a small shio Current runs along the eastern coasts of Taiwan number of S. lucens from off Borneo and New Guin- and Japan at a rate of nearly 50 million m3/s (Taira, ea, which notably expanded the known geographical 1997; Ujiié et al., 2003). This strong northward cur- range of this species. Due to the disjunctive distribu- rent seemingly enhances the larval transportation. tion of this species, coupled with the genetic differ- However, for some previously studied species (e.g., ences observed in this study between the Japanese Uca arcuata, Sepioteuthis spp. and Siganus guttatus), and Taiwanese populations, S. lucens is likely to be no ongoing gene flow was found between the Japa- formed by several small breeding units implying high nese and Taiwanese populations (Aoki et al., 2008a, genetic variability. Further research into the relation- b; Imai and Aoki, 2012; Iwamoto et al., 2012). Addi- ships between these units is needed for effective con- tionally, the mtDNA data of this study clearly indi- servation of this commercially important species.

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Fig. 6. Variation in form of petasma of Sergia lucens as indicated in the part of circle: male (10.3 mm in carapace length) from Suruga Bay, Japan, with no additional projection at the basal part of the lobus terminalis showing a typical from (entire part of left petasma, posterior view) (A); and male (10.0 mm) with a carinate swelling (distal part of right petasma, posterior view) (B); male from Taiwan (10.3 mm) with a distinct projection (entire part of right petasma, posterior view) (C): a, pars astringens; e, pars externa; lc, lobus terminals; lc. lobus connectens; pv, processus ventalis. Male with each form of petasma (A, B, C) was categorized as to III, I, and II in Table 2.

Table 1. Sampling locality, haplotype diversity (h), nucleotide diversity (π), and Tajima’s D and Fu and Li’s D test results for the Japanese and Taiwanese populations. *P < 0.005. sample locality sampling size h ± SD π ± SD Tajima’s D Fu and Li’s D

Suruga Bay, Japan 70 0.991 ± 0.01 1.096 ± 0.58 -2.40* -11.30* Gueishan waters, N Taiwan 47 0.995 ± 0.01 1.331 ± 0.70 -2.03* -2.38* Tungkang waters, S Taiwan 61 1.000 ± 0.00 1.299 ± 0.68 -2.31* -11.32*

Table 2. Percentage occurrence of males with an additional pro- tute for help in sampling at the Tungkang fish market; Ms. jection at the base of the lobus terminals of the petasma. Basal Kaori Hirouchi for her assistance in preparing the manuscript part of lobus terminals (encircled part): I, smooth (typical form); and Mr. Abdullah Fadry Muhamad for his research assistance. II, carinate swelling; III, distinct projection. We specially thank Asst. Prof. Tetsuo Yoshino of the Univer- sity of the Ryukyus and Dr. Makoto Omori of the Akajima category Suruga Bay Taiwan Marine Science Laboratory for some suggestions on species nomenclature. We also specially thank Asst. Prof. James Davis I 50.0 39.4 Reimer of the University of the Ryukyus for checking the man- II 43.7 39.4 uscript’s English. We thank two anonymous reviewers for their III 6.3 21.2 critical and editorial comments. This study was partially sup- ported by a Grant-in-Aid for Young Scientists (B15780136) total N 16 33 and the 21st Century COE program at the University of the Ryukyus from the Ministry of Education, Culture, Sports, Sci- ence and Technology, Japan.

Acknowledgements References

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On-line Supplementary Information (SI)

S1. Variable sites in the 162 haplotypes found in mtDNA control region of 178 Sergia lucens. The numbers above sequences correspond to the positions of the polymorphic sites. Dots indicate an identical nucleotide at the position relative to haplotype 1.

Downloaded from Brill.com10/06/2021 05:01:41PM via free access S1. Variable sites in the 162 haplotypes found in mtDNA control region of 178 Sergia lucens. The numbers above sequences correspond to the positions of the polymorphic sites. Dots indicate an identical nucleotide at the position relative to haplotype 1.

Nucleotide Sites Nucleotide Sites No. of individuals 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 2 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 3 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 5 Suruga South North Haplo- 1 4 6 6 6 7 7 7 9 0 0 0 0 0 1 1 1 1 1 2 2 2 2 2 2 3 4 4 5 5 5 5 5 5 5 6 6 6 6 6 7 7 8 8 8 8 9 9 9 9 0 0 1 1 1 2 2 2 2 2 3 3 3 4 9 9 9 3 3 3 4 4 5 5 5 5 5 6 6 7 7 8 8 8 9 9 0 0 1 2 2 2 3 3 3 3 4 4 4 4 4 5 5 5 5 5 6 6 6 7 8 9 9 0 0 0 0 0 0 0 0 1 1 1 1 1 1 1 1 2 2 2 2 2 2 3 3 3 3 3 3 4 4 4 4 5 5 5 5 5 6 6 6 6 7 7 7 7 7 7 7 8 8 8 Bay Taiwan Taiwan types 0 1 4 5 8 4 8 9 1 4 5 7 8 9 0 2 4 6 8 2 3 4 5 8 9 3 8 9 0 4 5 6 7 8 9 4 5 6 7 8 8 9 0 1 2 3 0 1 2 7 0 6 0 8 9 0 1 2 4 9 0 1 2 0 6 7 8 2 7 9 0 4 1 2 3 4 6 2 7 4 8 0 1 2 0 9 1 2 6 3 4 5 2 3 8 9 2 3 4 6 9 0 2 5 7 9 0 3 8 1 8 4 5 0 1 2 3 4 5 6 9 0 2 3 4 5 6 7 8 0 1 3 4 5 6 2 3 4 5 6 7 1 2 4 8 0 3 4 5 8 0 1 2 3 1 2 3 4 5 8 9 1 2 4 1 T T A G A A T T A C A T A T T T A T T A G C T T A A T A A C T T C A A C T A A G T T G C T T A A T A A A A A A T T A C A A T T T G A A G T T A A T C T A G T A C A A A A T G A T C T T C T T G G A A C A A T C A C T T A A T A A T A T C C T T A A T C G A A G T G A T T A A A T A A C G A A T C T T T G A A T G A C T T T A A A T T T A 7 2 2 ...... T ...... T . . . . . A ...... C ...... 1 1 3 3 ...... T ...... T ...... G ...... T . . . . . A ...... C ...... T ...... C ...... 1 4 ...... T ...... A ...... T . . . . . A . G ...... C ...... 1 5 ...... T . C ...... T . . . . . A ...... A . . . . C ...... G ...... T ...... 1 6 ...... T ...... T ...... T . . . . . A ...... T . C ...... A ...... 1 7 ...... T ...... T . . . . . A ...... C ...... G ...... A ...... C ...... 1 8 ...... T . . T ...... C ...... T . . . . . A ...... C ...... G . . . G ...... C ...... 1 9 ...... T ...... A ...... G ...... T . . . . . A . G ...... T . . . . . A . G . . . . A ...... C ...... T ...... G . G C . . . A . . . A ...... C ...... 1 10 ...... C ...... T ...... T . . . . . A ...... C ...... 1 11 ...... T . . . . G ...... T . . . . . A ...... C . . . . . G ...... C ...... G ...... C ...... 1 12 ...... T ...... T . . . C . A ...... C ...... G ...... A ...... 1 13 ...... T ...... C ...... T . . . . . A ...... C . . G ...... 1 14 ...... G . . . T ...... T ...... T . . . . . A ...... C ...... T ...... A ...... 1 15 ...... T ...... T . . . . . A . G ...... C ...... C ...... A ...... 1 16 ...... T . C ...... T . . . . . A . G ...... G . . . . G ...... C ...... G ...... 1 17 ...... T ...... T G . . . . A ...... C ...... C ...... C ...... G ...... A ...... 1 18 ...... T ...... C ...... T . . . . . A ...... C . G ...... G ...... G ...... 1 19 ...... C ...... 2 20 . . . . G ...... T ...... T . . . . . A ...... C ...... G . G ...... 1 21 ...... T ...... T . . . . . A ...... C . . . . . C . . . G ...... A ...... 1 22 ...... T ...... G ...... T . . . . . A ...... C ...... A ...... 1 23 ...... T ...... T . . . . . A ...... C ...... C . 1 24 ...... T ...... T . . . . . A ...... C ...... T T ...... 1 25 ...... T ...... T . . . C . A ...... C ...... G ...... 1 26 ...... A ...... T ...... T . . . . . A . . A ...... C ...... C ...... 1 27 ...... T ...... C ...... T . . . . . A ...... G ...... C ...... 1 28 ...... T ...... T . . . . . A ...... C ...... G . G ...... 1 29 ...... T ...... C ...... T . . . . . A ...... C ...... C ...... 1 30 ...... T ...... T . . . . . A ...... G ...... C ...... G . G ...... 1 31 ...... T ...... T . . . . . A ...... C ...... G ...... 1 32 ...... C ...... T ...... T . . . . . A ...... C ...... 1 33 ...... T ...... T . . . . . A . . . . C ...... C ...... T ...... G . . . . . 1 34 ...... T . . . . . T ...... T . C ...... T . . . . . A ...... C . G ...... 1 35 ...... T ...... T . . . . . A ...... C ...... C ...... C ...... 1 36 ...... T ...... T . . . . . A ...... A . . . . . C . . G ...... 1 37 ...... T . C ...... T . . . . . A ...... C ...... A ...... C ...... C ...... 1 38 ...... T . C ...... T . . . . . A ...... C ...... T ...... T ...... 1 39 ...... T ...... T ...... G . . . . . T . . . . . A ...... C ...... G ...... C ...... 1 40 ...... T ...... T . . . . . A ...... C ...... G ...... G ...... 1 41 ...... T ...... T . . . . . A ...... C ...... T ...... 1 42 ...... T ...... G ...... T . . . . . A ...... T ...... A ...... 1 43 ...... T ...... T . . . . . A ...... C ...... C ...... T ...... 1 44 ...... G . . . T ...... A ...... T . . . . . A . . . . C ...... C ...... C ...... 1 45 ...... T ...... T ...... T . G . . . A ...... G . . . G ...... A . . . . . C ...... C ...... G . . . . T ...... C ...... 1 46 ...... C ...... T . . T ...... G ...... T . . . . . A G . A . . G . . T C ...... G . . . . . C . A . . . . C . T . T ...... T ...... C ...... G ...... 1 47 ...... G . . . T ...... T ...... T . . . . . A ...... C ...... C ...... 1 48 ...... T ...... T ...... T . . . . . A ...... G ...... C . T ...... 1 49 ...... T ...... T . . . . . A ...... C . . G ...... T ...... 1 50 ...... G . . . T ...... G ...... T . . . . . A . . . . C . G ...... C ...... T ...... C ...... G ...... 1 51 ...... T ...... T . . . . . A ...... G . C ...... C ...... 1 52 . . . . . G ...... T C ...... A ...... T . . . . . A ...... C ...... G ...... G . . . G ...... 1 53 ...... T ...... T . . . . . A ...... G ...... C ...... A ...... 1 54 ...... T ...... A ...... T . . . . . A ...... C ...... G . . . C . T C ...... T ...... 1 55 ...... A . C . . . . . T ...... T . . . . . A ...... C ...... C ...... 1 56 ...... G . . . T . . . . . A ...... C ...... G ...... C ...... 1 57 ...... G . . . T ...... T ...... T ...... G . . C ...... C ...... 1 58 ...... C . . . . . T ...... G T . . . . . A ...... C ...... 1 59 ...... T ...... G . . . . T ...... C . . T . . . . . A ...... G ...... C ...... 1 60 ...... C ...... T . . T ...... A . . . T . . . G ...... T . . . . . A ...... T ...... G . . . . . C . A . . . . C . T . T ...... T ...... A . . . C ...... 1 61 ...... C ...... T ...... T . . . . . A ...... G ...... C ...... G ...... 1 62 ...... T ...... G . . . T ...... T . . . . . A G ...... G ...... C ...... T ...... 1 63 ...... T ...... T . . . . . A . . . . C ...... C ...... 1 64 ...... C ...... T . C ...... G ...... T . . . . . A ...... C ...... C ...... T ...... 1 65 ...... T ...... A ...... T . . . . . A ...... C ...... G ...... 1 66 ...... G ...... T ...... T . . . . . A ...... C C ...... C ...... G ...... T ...... T ...... 1 67 ...... T ...... T . . . . . A ...... G ...... C ...... 1 68 ...... T ...... C ...... T . . . . . A ...... C ...... T ...... 1 69 ...... G . . . T . . . . . T ...... T ...... T . . . . . A ...... C ...... 1 70 ...... G ...... T ...... G ...... T . . . . . A ...... C ...... 1 71 ...... T ...... T ...... A ...... G ...... T . . . . . A ...... T ...... G . . . . . C . A . . . . C . T . T ...... T G ...... A . . . C . . . . C . . . 1 72 ...... T ...... T . . C . . A . . . C ...... C ...... C ...... C ...... T . G . . G ...... 1 73 ...... T . T ...... T . . . . . A ...... C ...... A ...... C ...... 1 74 ...... T . . T ...... G . . . T . . . . . A ...... C ...... 1 75 ...... G T C ...... T . . . . . A . . . . . G ...... C . . . T ...... 1 76 ...... T . C ...... G . . . . T . . . . . A G . . C . . . . . C ...... C ...... T ...... 1 77 ...... T ...... T . . . . . A G ...... C ...... T ...... 1 78 ...... T ...... C . C . T ...... T . . . . . A ...... C . . . T . . . . C ...... C ...... 1 79 ...... T . C ...... T . . . . . A ...... C ...... T ...... G ...... C ...... 1 80 ...... T ...... G ...... T . . . . . A ...... C ...... T ...... 1 81 ...... T ...... T . . . . . A ...... G ...... G ...... G ...... 1 82 ...... T ...... G ...... T . . . . . A ...... C ...... C ...... A ...... 1 83 . C ...... C . C ...... C ...... T . . . C . . C ...... T . . . . . A ...... C ...... C . . . T ...... G ...... 1 84 C C ...... C . C ...... C ...... C . . C ...... C ...... 1 85 . C ...... T . . . C ...... T . . . . . A ...... C ...... G ...... 1 86 . C ...... C . C ...... C ...... T . C . C ...... T . . . . . A ...... A . . . . C ...... G ...... 1 87 ...... T ...... G ...... T . . . . . A ...... C ...... T ...... T ...... 1 88 ...... T ...... T . . . . . A ...... C ...... C ...... 1 89 ...... T ...... C . . T . . . . . A ...... C ...... T T ...... 1 90 . . . . G ...... T C ...... C . . . . . G ...... T . . . . . A ...... C ...... G ...... 1 91 ...... T ...... T . . . . . A ...... C ...... C ...... 1 92 ...... T ...... T ...... T . . . . . A ...... C ...... 1 1 93 ...... T ...... T . . . . . A ...... G . C ...... 1 94 ...... T ...... T . . . . . A ...... T ...... C ...... 1 95 ...... T ...... T . . . . . A ...... C ...... C ...... C . . 1 96 ...... C ...... T ...... T . . . . . A ...... C ...... 1 97 ...... T ...... T . . . . . A ...... T ...... C ...... T . . . . . C ...... C ...... 1 98 ...... T ...... T . . . . . A ...... C ...... C ...... A ...... 1 99 ...... G ...... T ...... T . . . . . A . . . . C . . . . C ...... T ...... C ...... A ...... C ...... 1 100 ...... T . . . C ...... T ...... T . . . . . A ...... C ...... T ...... 1 101 ...... T ...... C ...... T . . . . . A ...... C ...... T ...... 1 102 ...... G . . . . 1 103 ...... T ...... T . . . . . A ...... C ...... C ...... 1 104 ...... T ...... T . . . . . A ...... C ...... C ...... 1 105 ...... T . . . . G ...... T . . . . . A ...... C ...... C ...... G ...... 1 106 ...... T ...... T . . . . . A ...... A ...... C ...... 1 107 ...... T ...... G ...... T . . . . . A ...... T ...... A ...... 1 108 ...... G ...... T ...... T . . . . . A ...... G C ...... 1 109 ...... T ...... A . . . . . C ...... T . . . . . A ...... C ...... C ...... 1 110 ...... T ...... G ...... T . . . . . A ...... C ...... T . . . A ...... 1 111 ...... T ...... T . . . . . A ...... C ...... T ...... C . 1 112 ...... T ...... T ...... T . . . . . A ...... G ...... C ...... 1 113 ...... T ...... T . . . . . A ...... A ...... C ...... C ...... 1 114 ...... T ...... T . . . . . A ...... C ...... G ...... G . . C ...... 1 115 ...... G ...... T ...... G ...... T . . . . . A ...... C ...... C . . G ...... 1 116 . . . . G ...... T ...... T ...... T . . . . . A ...... C . . . . C . . . C ...... G . G . . . G A ...... G ...... 1 117 ...... T ...... T ...... T . . . . . A ...... C ...... G ...... 1 118 ...... C ...... G . . . . T ...... A ...... T . . . . . A ...... A . G ...... C ...... T ...... C G ...... G ...... T ...... 1 119 ...... T ...... T . . . . . A ...... C ...... C ...... T ...... A ...... 1 120 ...... T ...... T . . . . . A ...... C . T ...... C ...... 1 121 ...... T ...... T . . . . . A ...... C ...... C ...... G ...... 1 122 ...... G . . . T ...... T ...... T . . . . . A ...... C ...... 1 1 123 ...... G . . . . T ...... T . . . . . A ...... C ...... C ...... 1 124 ...... T ...... C . . T . . . . . A ...... C ...... G ...... 1 125 ...... T ...... A C ...... T . . . . . A ...... C ...... C ...... T ...... G ...... G ...... 1 126 ...... T ...... T . . . . . A ...... C ...... C ...... G ...... 1 127 ...... T ...... T . . . . . A ...... C ...... G ...... 1 128 ...... C . . T ...... A ...... T . . . . . A ...... C ...... C ...... 1 129 ...... A . . . G . . . T ...... T ...... T . . . . . A ...... G . . . C ...... A ...... 1 130 ...... T ...... T . . . . . A ...... C ...... G ...... 1 131 ...... T ...... T . . . . . A . G ...... C ...... 1 132 ...... T . . . . . T ...... T . . . . A A G ...... C ...... 1 133 ...... T ...... G ...... T . . . . . A ...... C . . . T ...... G 1 134 ...... T C C ...... T . . . . . A . . A ...... C ...... C ...... C . . . . . C . 1 135 ...... T ...... T ...... C . . T . . . . . A ...... C ...... 1 136 ...... T ...... G . . . T . . . . . A ...... 1 137 ...... T . C ...... T . . . . . A ...... C ...... 1 138 ...... T . . T ...... A ...... G ...... T . . . . . A G ...... T ...... G . . . . . C . A . . . . C . T . T ...... G ...... T ...... C ...... 1 139 ...... T ...... A ...... G ...... T . . . . . A ...... T . G ...... G . . . . . C . A . . . . C . T . T ...... G ...... C ...... T G ...... C . . . . C . . . 1 140 ...... T ...... T . . . . . A ...... G ...... T . . . . G ...... 1 141 ...... T ...... C . T . . . . . A ...... A ...... C . G ...... C ...... C ...... C ...... 1 142 ...... T ...... T ...... T . . . . C A ...... C ...... T T ...... C ...... T . . . . G ...... 1 143 ...... T ...... T . . . . . A ...... C ...... T ...... 2 144 ...... T ...... C ...... T ...... T . . . . . A ...... C ...... 1 145 ...... T ...... A ...... G ...... T . . . . . A G ...... T . G ...... G . . . . . C . A . . . . C . T . T ...... G ...... T G ...... C . . . . C . . . 1 146 ...... G ...... T ...... T . . . . . A ...... C . . . T ...... A ...... 1 147 ...... T ...... T . . . . . A ...... C ...... T ...... C . C ...... T ...... A ...... 1 148 ...... T ...... G . . . T . . . . . A ...... A ...... C ...... T ...... C ...... 1 149 . . . A ...... T ...... A ...... G ...... T . . . . . A ...... T . G . . G ...... G . . . . . C . A . . . . C . T . T ...... G ...... T G G . . . C . . . . . A ...... G . C . . . 1 150 ...... T ...... T . . . . . A ...... C ...... G ...... 1 151 ...... T . . . G . . . G ...... G ...... T . . . . . A ...... C ...... C ...... G ...... 1 152 ...... T ...... T . . . . . A ...... C ...... T ...... G 1 153 ...... T ...... T . . . . . A ...... C . . . T ...... T ...... G ...... A . C ...... 1 154 ...... T ...... T . . . . . A ...... T . C . . G ...... T ...... 1 155 ...... T ...... T . . . . . A ...... G ...... C ...... T ...... C ...... 1 156 ...... T ...... T . . . . . A ...... C ...... G ...... T ...... G ...... 1 157 ...... C ...... T C ...... G T . . . . . A ...... C ...... A ...... 1 158 ...... T ...... A ...... T . . . . . A ...... C ...... 1 159 ...... G . . . T ...... T ...... T . . . . . A ...... C ...... C ...... 1 160 . . G ...... T ...... T . . C . . A ...... C ...... C ...... 1 161 ...... T ...... T . . . . . A ...... C ...... A ...... C ...... 1 162 ...... A ...... T ...... T . . C . . A ...... C ...... C ...... C ...... C ...... A ...... 1

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