JapaneseJapaneseSociety Society ofSystematicof Systematic Zoology

Species Diversity, 2009,14, 157-164

Molecular Evidence for the Taxonomic Status of an Unde-

scribed Species of Dasyatis (Chondrichthyes: Dasyatidae)

from Japan

Naoki Yagishitai, Keisuke Furumitsu2 and Atsuko Yamaguchi2

' ftzstitutefor East China Sea Research, AJLigasaki Uitiversity, 1551-7 Tairamachi,

IVtlgasaki, as1-2213 .llipan E-mait:yagi@nagasaki-u,ac.ip 2Laboratoi), ofharine Zbolqg],, FaculCy ofFisheries, AJtxgasaki Vniversily, 1-14Bunltyo-machi, Nagasahi, 852-8521 Jlipan

(Received 13 March 2009; Accepted 4 June 2009)

Specimens of an undescribed species ofDasyatis stingray (D, sp.) were col- lected firom Nagasaki and Kagoshima Prefectures, Japan. These specimens are more similar to D. akojei (MUIIer and Henle, 1841), D. Iaevigata Chu, 1960, and D. izuensis Nishida and Nakaya, 1988 than they are to other con- geners obtained from the western North Pacific Ocean. The genetic differ- ences among D. sp,, D. ahojei, D. Iaevigata, D, izuensis, and two other con- generic species, D. matsubarai Miyosi, 1939 and D, kuhlii (MUIIer and Henle, 1841), were investigated on the basis of partial sequences (621 bp) of the mito- chondrial cytochrome oxidase subunit I (COI) gene. All three specimens of D. sp, that were analyzed showed identical haplotypes and formed a well-di- verged clade in the phylogenetic analysis. The pairwise sequence differences between D, sp. and the five other species of Dasyatis ranged from 7.09% to

15.30t/a. These values almost equalled, or exceeded, the interspecific difft)r- ences among the latter five species [range, 5.82% (D. akojei vs D, izuensis) to 16.43% (D. kuhlii vs D, izuensis, and D. kuhlii vs D. Iaevigata)]. Our findings indicate that D. sp, is a reproductively isolated and distinct species. Key Words: Dasyatis, evolutionary distance, mitochondrial DNA, cy- tochrome oxldase subunit I,

Introduction

The genus Dasyatis (Chondrichthyes: Dasyatidae) comprises at least 38 species of stingray around the world (Nelson 2006), and 16 of these inhabit the western North Pacific Ocean (Nishida and Nakaya 1990; Zhu and Meng 2001; Aonuma and Yoshino 2002), Furumitsu et al. (2006) recentiy reported an undescribed species of Dasyatis (D. sp.) frem Ariake Bay, Japan, which is characterized by a unique com- bination of morphological characters. These authors also collected a specimen of D, sp. (sensu Furumitsu et al. 2006) from Kasasa, Kagoshima, Japan, Dasyatis sp, (sensu Furumitsu et al, 2006; Fig. 1) has a diamond-shaped disc, three to nine oral papMae, 15-22 intestinal valve turns, and a tail with both ventral and dorsal folds; it does not have a band of small denticles on its prespinal region, a scutellate spine on its tail, nor any characteristic spots on the dorsal side of its

NII-Electronic Library Service JapaneseJapaneseSociety Society ofSystematicof Systematic Zoology

158 Naoki Yagishita et al.

disc (Furumitsu and Yamaguchi unpublished). Thus, it is more simllar to D. akojei (Mtiller and Henle, 1841), D laevigata Chu, 1960, and D. izuensis Nishida and Nakaya, 1988 than it is to the other congeners identified from the western Nerth Pacific. However, D. sp. dilfers firom these three species in several respects (Ituru- mitsu and Yamaguchi unpublished). Adults of D, sp. have a longitudinal row of small tubercles midway along the midline of dersal surface of the disc (Fig. IB); in contrast, no such tubercles are present in D. Iaevtgata, Dasyatis sp. has no tuber- cles on its prespinal region (Fig. IC) whereas adults of D. akojei have large tuber- cles there. A black ventral fold with a white rim can be observed on the tail of D. sp. (Fig. ID), while a white ventral foId with no rim is present on the tail of D. izuensis, Furthermore, D. sp. is characterized by the presence of a furrow (Fig, IF, G) in the center of the ventral suiface of the disc posterior to the fifth branchial openings (Fig. IE). This fuiTow may resemble two dots (Fig, IF) or a straight 1ine (Fig. IG). A groeve or furrow is present at this same location in the Japanese D. matsubarai Miyosi, 1939 and the Brazilian D. dypostigma Santos and Carvalho, 2004 (Santos and Carvalho 2004); however, the groove or ftnrrow in these two species is w-shaped, unlike the furrow in D. sp. In order to clarify the taxonemic status of D. sp., we investigated the genetic differenees among D. sp. and some other spcies of Dasyatis on the basis of partial sequences of the cytochrome oxidase subunit I (COI) gene from the mitochondrial (mt) DNA.

Materials and Methods

We determined and compared the DNA sequences isolated from three speci- mens of Dasyatis sp, (sensu Furumitsu et aL 2006), six specimens of D. akojei, two of D, laevigata, two ofD. izuensts, two ofD, rnatsubarai, and one specimen ofD. huhlii (MUIIer and Henie, 1841), We conducted a phylogenetic analysis with Hirnantura .fl]i Jordan and Seale, 1906 and ll gerrardi (Gray, 1851) (Dasyatidae) included as outgroups in order to root the tree on the basis of the phylogeny presented by Rosenberger (2001). The specimens examined are deposited in the National Mu- seum of Nature and Science, Tokye (NSMT), with the exception of E[ foi and HL gerrardi, the DNA sequences of which were obtatned from the DDBJfEMBL/Gen- Bank database (EU398839 and EU398840, respectively; Ward et al. 20e5). Total DNA was extiracted using the QIAamp DNA Mini Kit (Qiagen K. K,) firom frozen-preserved muscle tissue, according to the manufaeturer's protocol. We per- formed a polymerase chain reaction (PCR) and amplified approximately 650 base pairs (bp) of the COI gene firom the mtDNA. The PCR was per fbrmed using the fbl- lewing primers (Ward et al. 2005): FishF2 (5'-TCGACTAATCATAAAGATATCG- GCAC-3') and FishR2 (5'-ACTTCAGGGTGACCGAAGAATCAGAA-3'). The PCRs were carried out using a Dice PCR Thermal Cycler (Takara Bio), each with 10uL of reaction solution containing 10ng template DNA, 0.2gM of each primer, O.2mM dNTP mixture, lxPCR buffer [10mM Tris-HCI

NII-Electronic Library Service JapaneseJapaneseSociety Society ofSystematicof Systematic Zoology

COI analysis of Dasyatis sp. t'rom Japan 159

umww-ww '$ t ' - ' ･ ･ , ,, .,em..,. $･igi$re:ig.xet･i::･-･ee,..., lj.,XmeligeIFeeliimaee,ige

Fig. 1, Dasyatis sp. (sensu Furumitsu et al. 2006>, with two morphological variations of the furrow in the center of the ventral surface of the disc posterior to the tifth branchial open- ings. A-F, NSMT-P 93295, 500 mm in disc width (DW), Kasasa, Kagoshima, Japan; G, NSMT-P 93297, 32emm DW, Ushibuka, Kumamoto, Japan, A, Dorsal view; B, center of midline of dor- sal sur:face of disc; C, prespinal region; D, ventral fold of tail; E, ventral view; F, furrow re- sembling two dots; G, furrow resembling a straight line. Arrow in E indicates location of ftur- row, Scale bars: 10mm,

NII-Electronic Library Service JapaneseJapaneseSociety Society ofSystematicof Systematic Zoology

160 Naoki Yagishita et aL

IYe agarose gel electrophoresis. The PCR products, purMed by ExoSap-IT (GE Healthcare Bio-Sciences Corp.), were directiy sequenced on an ABI Prism 3100 or 3130 DNA sequencer using the BigDye Terminator Cycle Sequencing Kit ver, 3.1 (Applied Biosystems) and the primers used in the PCR. The DNA sequences were aligned with CLUSTAL W ver. 1.8 (Thompson et al. 1994). Phylogenetic relationships were estimated by the neighborjoining (NJ) method (Saitou and Nei 1987), using PHYLIP ver. 3,6 (Felsenstein 1995), Evolution- ary distances were calculated using Kimura's two-parameter model (Kimura 1980), The tiransition/transversion bias ratio was assumed to be 6:1 en the basis of the ob- served ratio. Confidence values were established by bootstrapping analysis (Felsenstein 1985) with 1000 replications,

Materials examined. Dasyatis sp. (sensu Furumitsu et aL 2006): NSMT-P 93295, Kasasa, Kagoshima, Japan, with a furrow resembling two dots; NSMT-P 93296, Shimabara, Nagasaki, Japan, with a fuiTow resembling two dets; NSMT-P 93297, Ushibuka, Kumamoto, Japan, with a furrow resembling a straight line. Dasyatis akojei: NSMT-P 9I833, Fukue Island, Nagasaki, Japan; NSMT-P 91837, Maizuru, Kyoto, Japan; NSMT-P 91841, Ushibuka, Kumamoto, Japan; NSMT-P 91844, 91845, Kasasa, Kagoshima, Japan; NSMT-P 91846, Xiamen, China, Dasyatis laevigata: NSMT-P 91823, Ushibuka, Ku- mamoto, Japan; NSMT-P 93294, Shimabara, Nagasaki, Japan. Dasyatts izuensis: NSMT-P 91830, Shimabara, Nagasaki, Japan; NSMT-P 91831, Kasasa, Kagoshima, Japan. Dasyatis matsubarai: NSMT-P 91827, 91828, Maizuru, Kyoto, Japan. Dasy- atis huhlii: NSMT-P 91858, Ishigaki Island, Okinawa, Japan.

Results

The partial sequences of the COI gene obtained firom three specimens of Dasy- atis sp. (sensu Furumitsu et al, 2006) and 13 specimens of the five other investigated species of Dasyatis have been deposited in DDBJ/EMBL/GenBank (accession num- bers AB485677-AB485692), These sequences included 621 bp, 190 of which were variable, The numbers of nucleotide differences and evolutionary distances are shown in Table 1. AII three specimens of D. sp, showed identical haplotypes (DaspOl). Of the six specimens of D. akojei, five had the same haplotype (DaakOl), which difL fered from the hapletype (Daak02) of the remaining specimen by one substitution. The twe specimens of D. Iaevigata exhibited different haplotypes (DalaOl and Dala02), which diffbred from each other by one substitution, The two specimens of D. izuensis shared a commen haplotype (DaizOl). The two specimens of D. matsu- barai exhibited different haplotypes (DamaOl and Dama02), which differed from each other by two substitutions. The pairwise sequence diEferences within each species of Dasyatis

NII-Electronic Library Service Japanese SooietySociety of SystematicSystematio Zoology

COI analysis of D α sy αtis sp ． from Japan 161

蝋 − 瑁 O 卜 卜 O ぬ 煽 り 寸 の 卜 の ゜ っ ゜っ 寸 の 旬 ℃ ゜っ 瑟 の ゜っ H 霧 霧 ＼ 劬 耘 ＼ ＼ ＼ ＼ ＼ ＼ ＼ ＼ ＼ 1 O O O 一 Ok O 卜 』 〇 諱 卜 卜 卜 躊 寓 紹 卜 卜 O

qg 韓

ワ ・鴇 【（ 霞 鳶 ゆ ゆ 一 H 咽 寸 剛 崘 ゆ 』 り 輻 む 丶 0 う o つ α つ ¢ つ 孕 つ φつ e つ ゜う 90bo 遣 ＼ ＼ ＼ ＼ ＼ ＼ ＼ ＼ ＼ ヨ ミ ゆ H ． 菖 鴇 k 卜 q つ 寸 Ooc O 一 卜 ト C つ ○つ cG ¢ 9 嵩 ト 応 〇 自 局 州 ℃ 隠 ． 卜 〉 丶 鴫 ⇒9eq α ⊃ 訓 飼 卜 H ≧ 9e コ N 唱 心 N σつ ○つ 〇 出 ＼ ＼ ＼ ＼ 雪 嵩 ＼ ＼ ＼ ＼ 一 O ． 邸 5 軸 α う O O 的 ○ つ ゆ ． ． （ 吋 O O O8N コ ． 【∩ 卜 ト 卜 卜 卜 ト ト ）⊇ 一 OON 〔 口 鬥 の O 山 Q 角 邸 卜 p つ 。 の α O 笛 丶 d ＼ ＼ っ H 口 ［ 9 ＼ ミ ミ q ［ 雪 c9 ¢ q O O O ＼ °っ °⊃ 扇 ． ． ， 邸 ゜っ 需 崘 專 葛 口 軸 豁 O O OOQ 例 鴇 ∩ 認 祠 軸 Ok 【 娼 口 あ づ ◎ ρ ρ コ Q 口 〉 剃 O 。 っ 捻州 一 嵩 霧 斜 ゴ乃 一 自 d り 日 口 鷲 ． ． ， O O O O 冨 』 口 再 』 5 鼕 一 9り ゴ ． 〇 畠 面 而 OH σ旁 5 っ 的 σっ 〉 q の OO cDH ○つ あ HO ＼ ミ ＼ ＼ ← 謡 ヨ ＼ ミ N 諞 O °o の ． ． ． お の 鳴 ， 岡 °っ ゜っ 蹟 鑢 專 O O O O Φ ミ 〔 認 O 自 【 而 勅 ＝ 物 幅り ‘ ） ρ ＝ N つ◎ αっ ． 逵 ゜っ 頃 Φ 囚 罵 一 認 N O σ O q 動 一 ＼ ＼ ミ ＼ ＼ の 嵩 邸 O O 曾 一 〇 鬥 ゜畑 8，， ． ， ． ． の一 N ［〔 総 幅 o っ ゜っ 守 專 0 O O O O O 冒 口 総 ． 鴇 眉 唱 Q ＝ 囲 舳 σ っ の σ蔭 訂 口 ヨ 臼 臼 D ⇒ H H お HN 再 們 OQ 寵 OO く ＼ ＼ O 目 一 H ． 嗣 ． ． ≧ ＞ ， ， ， 門』 ∩ 等 お 鋸 O1O O O O O O O ロBq

口 O ー 鳥 隔 − 弓 』 ρ ‘ 日 軸 。⊃ も も O 。。． 諂 們 ≧ 鴇 qっ 。っ O ρ 喬 O ＝ 雪 N 扇 ρ Od ． ． ， ． ． O O O O O H 職 ρ 、 囚 叨 の ． 啗 qo O q 囮 叨 O 卜 卜 噂 ← 甸 σつ 叭 h 口 冖 ヨ 鬯 OO 帽 漏 H Φ 一 〔 ＼ 〕 肖 尸 一 囚 四 目 邸 〇 ＼ ＼ ． ． ， ． ． H ⊇ 祠 8 O ． ， 〔 O O O O O Oc 口 O 働自 H 【 寸 崘 硲 一 自 ， つ O 目 あ 靨 図 HH 寸 ○っ ゆ O O OH 口 』 因 角 ト 卜 N O 邸 邸 O O O ＼ ＝ 肖 O ロ ． O ． 円 ． 刳 ， ． ＞ 瑁 qO の ， ． ， g 日O 〔 ＞ O O O O O ［ の つ O O O 口 ｝』 鋒 O の 』 帽 ρ ゜ づ 周 ρ ρ “ bD 一 名 寸 馬 。 っ ＼ 〇 卜 醜 O 90 ． な お 缶 の 〉 自 O O 目 靄 口 ． ， ． ． ． ． O ． 菴 O O O O （ ． O O 覃 OoDc も 卸艮 q 届 鳶 O 自 口 門 【 邸 邸 一 O 一 寸 尊っ 寸 Q っ 鳴 qO 出 』 H OO 卜 苺 N O 』 gD 邸 HO 円 祠 H 自 O O O 円 釦 謂 。ゴ ． ． ． ． ． ． ． 鯛 ⇒ お ， ． 【 哨 O O O O O O O O O O O 口 ∩ 醗 祠 O ＝ の ． O 隔 図 』 の 』 【 ） 泊 稽 』 鬥 卜 O OHH O ゆ 鴎 二 ρ 弱 ． 恥 の も 鴎 卜 【 口 £ の ． ooO 〔 OH 卜 o っ H 口 H 含 一 ミ 房 円 。っ Dっ 〕 唱 自 ゜5 O O ヨ 囲 O 自 コ 同 国 民 ， O づ 弓 一 跨 ゜っ O ．． ． ．， ． ， ．． ． ， ． ． q り ◎ 【〔 O O O O O O O O O O O O O 9 日 q Φ 一 ［ づ q 邸 』 ［ q 一Φ O ≧ リロ 9 州輯 二 〇 Φ り 的 〇 α （ 的ρ Φ Ho 毎 一 50 』 O Φ つ 婿 O 〇 邸 角 り 葺 （ Q つ 邸 φ 口 円 （ 邸 目 畆 ＼ q 一 矧 ） ・湿 o O 拐 』 コ Φ I （ （ （ （ （ 一 q 州 Oo 一 目 σ ｝ 卜 o つ ○⊃ 邸 輔 も OO 鴫 （ do ≧ 凧 σ つ 寸 NOo つ 囚 の ←円 囗 目 qOb 匿 cqo ○⊃ 」 09H o つ ○つ o ⇒ ○⊃ の 』 爵 q h H マ 鬥 寓 同 掲 鬥 円 同 N 汐 q つ 幻 為 而 寸 ◎ ロ 遣 ¢ ρ ← o つ 羽 刺 』 O 口 ， kOQ 丶 用 ＼ 唱 自 幽 一 山 O 山 山 山 山 山 N 邸 嚀 ＝ 一〇 q − −紬 ， の − − − ・む う O 日 日 − 唱 ← 自 白e 』 ］ ］ 缶 N k 類 ゴ 農 ［ 、 』 O ［ − 卜 ［臼 臼 臼 ぐ つ k 寸 屑ρ自 5 ゜， 剃 （ り HO 一 ｝ 邸 の 」 卜 糾 賄 遣 O O 縄 謎 0 つ O つ 円 Φ 〇 尸 卜 馬 鴇 HO NO ← 跫 丶 尊 O 畄 O Z ≧ 8 Σ o う O Σ 匿 匿 め 芝 芝 N ≧ e つ 」 Q つ 目 d 一 鞫 急 鋤 8N 鴇 而 師 ρ ＞ ． 毎 の 当 QeZ Q3H 革 の の Qつ QQ の 縄 コ QD7 黛 」 鳴 』 国 qO ◎ 8 智 OO 邸 邸 囲 嵩 唱 O O 一 嵩 O ひつ つ ρ ＼ °。 応 吋 7 ブ 一 7 日 日 鳶 図 O Z 圉 Φ ［ 【 の NN 「 宮 Z ［ h の b 竜 巴 ｝ 邸 価 応 ） に ） 邸 ）起 O ） 芍 顛 ρ 募 而 ） GD 耄 ） ）丶罵 ） 葛 建 ミ ⇒ 劬 冖 O 一 の 宀 Z ．〔 ． ，〔 描 Φ 旧 邸 ⇔ ロ ．∩ O ．O ∩ ） 【 O ∩ 【 国 国 づ β〔 語 〔 【 冖 ρ 応 2 6 尊 q q Q q 図 q 窺 国 邸 【〔 ≧ ［］q 〉 堯 詔ρ

一 NII-ElectronicN 工 工 Eleotronio Library Service JapaneseJapaneseSociety Society ofSystematicof Systematic Zoology

162 Naoki Yagishita et al.

Hitnantura fai

H. gefvardi

NsMT-pgtess Dakuol Z Dasyotis kuhtii

fooNSMT-P9d823 DalaOl D. Iaevigata NSMT-P93294 DaEa02 a

NSMT-P 93295 100 aoo NSMT-P 93296 DaspOl D. sp. NSMT-P93297

toe NSMT-P9d83e Dalzol D.Izuensis 58 NSMT-P9tS31 ] g

100 NSMT-P9t827 DamaOl D. matsubarai 69 N5MT-P91828 Dama02 g

NSMT-P91833 ijsias

76 NSMT-P S1837

NSMTtP 9d844 OaakOl too NSMT-P91845 D. akojei

O.I NSMTtP9d846

NSMT-P9t841 Daak02

Fig. 2. Neighbor-joining tree t'or Dasyatis sp, (sensu Furumitsu et al. 2006), D. akcijei, D. Iaevigata, D. izuensis, D. matsubarai, D, kuhlii, and two outgroup species, based on 621 bp of the COI gene ebtained from the mitochondrial DNA, Distances were corrected for multiple substitutions by Kimura's two-parameter model (Kimura 1980). The number at each branch indicates the bootstrap probability obtained firom 1000 repllcations. The haplotypes and the registration numbers of the specimens correspond te those listed in Table 1.

and D. kuhlii vs D, laevigata), with a mean difference of 11.55gih. The NJ tree derived from the distance matrix (Table 1) is shown in Fig. 2. The species of Dasyatis, including D. sp., reciprocally constituted a monophyletic group, with each clade being supported by a bootstrap probability (BP> of 1009ra. Dasyatis sp. fbrmed a monophyletic group with D. izuensis, D. matsubarai, and D, akojei with a BP of 580/o.

NII-Electronic Library Service JapaneseJapaneseSociety Society ofSystematicof Systematic Zoology

COI analysis ef Dasyatis sp. from Japan 163

Discussion

The range ef pairwise sequence differences between Dasyatis sp. (sensu Furu- mitsu et al. 2006) and the five other Dasyatis species (7.09-15.30%) examined here almost equalled, or even exceeded, the range of differences among the five other species of Dasyatis (5.82-16.439g"). The clade constituted of the three specimens of D, sp, that exhibited identical haplotypes was well separated from the clade com- prising D. iiuensis, D. matsubarai, and D. akojei (69?/e BP; Fig, 2). These molecular findings and the morphological distinctness of D, sp. indicate that D. sp, is repro- ductively isolated from the other species of Dasyatis and therefore represents an independent species. We intend to publish its fbumal description elsewhere. The lack of sequence differences between the specimens having diffbrent furrow shapes (Fig. IF, G) implies that these morphological differences merely represent intraspecific variation.

Acknowledgements

We are gratefu1 to S. Yoshida and the other fisherrnen of the Shimabara Fish- ermen's Cooperative Association and the members of our Iaboratory at Nagasaki University for collecting specimens. We also thank Y. Natsukari, Nagasaki Univer- sity, for providing the facilities required in this study. This research was sup- ported by a Grant-in-Aid for Scientific Research (no. 20580205) from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

References

Aonuma, Y. and Yoshino, T. 2002. Dasyatidae. Pp, 177-182. ini Nakabo, T, (Ed.) Fishes of Jlipan with Pictorial Keys to the Species. E)zglish Edition. Tokai University Press, Tokyo. Felsenstein, J, 1985. Confidence limits on phylogenies: an approach using the bootstrap. Evo-

lution 39: 783-791. Felsenstein, J, 1995. PHYLIP (phylogeny inference package), ver. 3.6, University of Washing- ton, Seattle. Furumitsu, K., Yamaguchi, A. and Nishida, K. 2006, Taxonomy and biology of the yantai stingray, Dasyatis Zaevigata. Kaiyo Monthly, Extra 45: 32-36. [In Japanese] Kimura, M. 1980. A simple method for estimating evolutionary rates of base substitutions through comparative studies of nucleotide sequences. Journal of Molecular Evolution 16: 111-120. Nelson, J. S, 2006, Fishes ofthe WOrld. Fburth Edition. John Wiley & Sons, New York, xix+601 pp.Nishida, K. and Nakaya, K, 1990. Taxonomy of the genus Dasyatts (Elasmobranchii, Dasyati- didae) firom the Nerth Pacific. Pp. 327-346, Ini Pratt Jr., H, L., Gruber, S. H, and Taniuchi, T, (Eds) Elasmobranchs as Living Resources: Advances in the Biotogy, Ecology, S,stemat- ics, and the Status of the Fisheries. NOAA Technical Report NMFS 90. United States De- partment of Cornmerce, Seattle. Rosenberger, L. J. 2001. Phylogenetic relationships within the stingray genus Dasyatis CChon-

drichthyes: Dasyatidae). Copeia 2001: 615-627.

NII-Electronic Library Service JapaneseJapaneseSociety Society ofSystematicZoologyof Systematic Zoology

164 Naokl Yagishita et al.

Saitou, N. and Nei, M, 1987. The neighborjoining method: a new method fbr reconstructing phylogenetie trees, Molecular Biology and Evolution 4: 406-425, Santos, H. R. S. and Carvalho, M. R. de. 2004. Description of a new species of whiptailed stingray from the southwestern Atlantic Ocean (Chondrichthyes, Myliobatiformes, Dasy- atidae). Boletim do Museu Nacional. Nova Serie, Zoologia 516: 1-24. Thompson, J. D., Higgins, D, G, and Gibson, T. J, 1994. CLUSTAL W: improving the sensitiv- ity of progressive mu]tiple sequence alignment through sequence weighting, posMon- specific gap penalties and weight matrix choiee. Nucleic Acids Research 22: 4673-4680. Ward, R. D,, Zemlak, T. S., Innes, B. H., Last, P. R. and Hebert, P. D. N. 2005. DNA barcoding Australia's fish species. Philosophical Transactions of the Royal Society of London, Se- ries B, Biological Sciences 360: 1847-1857. Zhu, Y, and Meng, Q. (Eds) 2001. Fauna Sinica. Qyclostornata, Chondrtchthyes. Science Press, Beijing, xv+552 pp. [In Chinese]

NII-Electronic Library Service