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DNA barcoding is a useful tool for the identification of marine fishes from Japan
Biochemical Systematics and Ecology 39 (2011) 31–42
Contents lists available at ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
DNA barcoding is a useful tool for the identification of marine fishes from Japan Jun-Bin Zhang a, b, *, Robert Hanner b a
Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China b Department of Integrative Biology, University of Guelph, Guelph ON, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 April 2010 Accepted 21 December 2010 Available online 22 January 2011
In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Keywords: DNA barcoding Marine fishes Japan
1. Introduction DNA barcoding, which was advocated by Hebert et al. (2003a,b), seeks to facilitate identifying the increasing number of unfamiliar taxa in biological conservation and biodiversity surveys, based on sequence diversity within a short and standardized gene region (Hebert et al., 2003a; Marshall, 2005). So far, scientists from over 30 different countries have participated in this international plan. For coordinating the collection data of specimens and performing data analysis with barcode data, the website Barcode of Life Data Systems (BOLD) (http://www.boldsystems.org) has been established (Ratnasingham and Hebert, 2007). BOLD is an accessible database that aids in management, analysis, dissemination, and searching of DNA barcodes. Fish Barcode of Life Initiative (FISH-BOL), as a campaign of the International Barcode of Life Project (iBOL), is to build up a standardized database of reference sequences for all fishes. With regard to fishes, the target DNA segment of 652 base-pairs near 50 end of the mitochondrial cytochrome oxidase subunit I gene is strongly proposed (Hebert et al., 2003a). These sequences are derived from voucher specimens preserved in the museums all around the world. Storage information (taxonomic identification, catalogue number and institution storing) and collection data (collector, collection date and location with longitude-latitude coordinates) are required to inject specimen records when creating projects for them. In addition, DNA barcodes (sequences of at least 500 bps and trace files) need to be uploaded to projects in FISH-BOL after finishing PCR amplification and sequencing. Thus, any problem concerning morphological identification can be solved by searching the relative data in BOLD or sending enquiries for checking voucher specimens preserved in natural history collections. Assignment of specimens to known species can be achieved through the comparison of genetic similarity with the
* Corresponding author. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China. Tel.: þ86 21 61900421. E-mail address: [email protected] (J.-B. Zhang). 0305-1978/$ – see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.12.017
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Table 1 Voucher and sequence information for these 229 specimens. Process IDs are sequence numbers of voucher specimens in Barcode of Life Database (www. barcodinglife.org), and Voucher IDs are voucher numbers in National Research Institute of Fisheries Science, Japan. Order
Family
Genus/Species
Voucher ID
Process ID
Anguilliformes
Congridae
Ariosoma meeki
Atheriniformes
Ophichthidae Synaphobranchidae Atherinidae
Conger japonicus Pisodonophis zophistius Synaphobranchus kaupii Hypoatherina valenciennei
Aulopiformes
Alepisauridae Synodontidae
Alepisaurus ferox Saurida elongata
Beloniformes
Exocoetidae
Hirundichthys oxycephalus
Belonidae
Ablennes hians
Exocoetidae Hemiramphidae
Cheilopogon pinnatibarbatus Hyporhamphus sajori
Berycidae Holocentridae Chimaeridae Clupeidae
Beryx splendens Sargocentron spiniferum Hydrolagus barbouri Engraulis japonicus Etrumeus teres
NRIFS:fish:HAA6 NRIFS:fish:HAA7 NRIFS:fish:MANG1 NRIFS:fish:BUM9 NRIFS:fish:IRAN1 NRIFS:fish:TGI1 NRIFS:fish:TGI2 NRIFS:fish:MIZU1 NRIFS:fish:TKE6 NRIFS:fish:TKE7 NRIFS:fish:TKE8 NRIFS:fish:HTB1 NRIFS:fish:HTB2 NRIFS:fish:DAT6 NRIFS:fish:DAT7 NRIFS:fish:HTBU1 NRIFS:fish:SDLG1 NRIFS:fish:SUJL1 NRIFS:fish:BERX1 NRIFS:fish:ISI6 NRIFS:fish:KHGZ1 NRIFS:fish:KTK1 NRIFS:fish:AMDK1 NRIFS:fish:AGM1 NRIFS:fish:KNOS1 NRIFS:fish:KNOS2 NRIFS:fish:SAPP2 NRIFS:fish:SAPP1 NRIFS:fish:MIW5 NRIFS:fish:MIW4 NRIFS:fish:MIW2 NRIFS:fish:MIW1 NRIFS:fish:KBG2 NRIFS:fish:KBG1 NRIFS:fish:KTK2 NRIFS:fish:95 KL NRIFS:fish:ISG35 NRIFS:fish:KNDR1 NRIFS:fish:HIMD1 NRIFS:fish:HANS1 NRIFS:fish:MADR1 NRIFS:fish:HONT1 NRIFS:fish:HGDR1 NRIFS:fish:EZIA1 NRIFS:fish:WAPL1 NRIFS:fish:NEZZ1 NRIFS:fish:KIAK1 NRIFS:fish:NUT6 NRIFS:fish:NUT7 NRIFS:fish:NUT8 NRIFS:fish:ISS1 NRIFS:fish:BBR1 NRIFS:fish:NIZ5 NRIFS:fish:NIZ4 NRIFS:fish:SK8 NRIFS:fish:SK9 NRIFS:fish:SK10 NRIFS:fish:KSI1 NRIFS:fish:TGD7 NRIFS:fish:TGD8 NRIFS:fish:TGD9 NRIFS:fish:NBD2 NRIFS:fish:NBD1 NRIFS:fish:NENB6 NRIFS:fish:NENB7 NRIFS:fish:NZGP2 NRIFS:fish:BJAP1 NRIFS:fish:TGE1
ABFJ241-07 ABFJ242-07 ABFJ138-06 ABFJ240-07 ABFJ130-06 ABFJ032-06 ABFJ172-06 ABFJ132-06 ABFJ234-07 ABFJ235-07 ABFJ236-07 ABFJ063-06 ABFJ064-06 ABFJ238-07 ABFJ239-07 ABFJ141-06 ABFJ154-06 ABFJ153-06 ABFJ182-06 ABFJ006-06 ABFJ147-06 ABFJ098-06 ABFJ156-06 ABFJ155-06 ABFJ202-07 ABFJ203-07 ABFJ177-06 ABFJ163-06 ABFJ171-06 ABFJ170-06 ABFJ081-06 ABFJ080-06 ABFJ087-06 ABFJ086-06 ABFJ099-06 ABFJ214-07 ABFJ245-07 ABFJ144-06 ABFJ109-06 ABFJ108-06 ABFJ131-06 ABFJ157-06 ABFJ143-06 ABFJ118-06 ABFJ129-06 ABFJ148-06 ABFJ128-06 ABFJ228-07 ABFJ229-07 ABFJ230-07 ABFJ037-06 ABFJ103-06 ABFJ039-06 ABFJ038-06 ABFJ247-07 ABFJ248-07 ABFJ249-07 ABFJ079-06 ABFJ257-07 ABFJ258-07 ABFJ259-07 ABFJ060-06 ABFJ059-06 ABFJ217-07 ABFJ218-07 ABFJ197-07 ABFJ113-06 ABFJ111-06
Beryciformes Chimaeriformes Clupeiformes
Konosirus punctatus Sardinella zunasi Sardinops melanostictus
Spratelloides gracilis
Gadidae
Engraulis japonicus Elops hawaiensis Megalops cyprinoides Antimora microlepis Coryphaenoides longifilis Coryphaenoides nasutus Gadus macrocephalus
Lamniformes Lophiiformes Myxiniformes
Macrouridae Moridae Gadidae Lamnidae Lophiidae Myxinidae
Nezumia proxima Physiculus maximowiczi Theragra chalcogramma Lamna ditropis Lophius litulon Eptatretus burgeri
Osmeriformes Osmeriformes Perciformes
Osmeridae Microstomatidae Acanthuridae
Salangichthys ishikawae Microstoma ashne Prionurus scalprum
Acropomatidae
Synagrops japonicus
Apogonidae
Apogon endekataenia Apogon lineatus
Elopiformes Gadiformes
Engraulidae Elopidae Megalopidae Moridae Macrouridae
Apogon semilineatus
Blenniidae Bramidae
Petroscirtes breviceps Brama japonica Taractes rubescens
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
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Table 1 (continued ) Order
Family
Genus/Species
Voucher ID
Process ID
Caesionidae
Pterocaesio digramma
Callionymidae
Repomucenus valenciennei
Carangidae
Alectis ciliaris
NRIFS:fish:ISI7 NRIFS:fish:ISI8 NRIFS:fish:HTTN1 NRIFS:fish:HTTN2 NRIFS:fish:ITHJ1 NRIFS:fish:ITHJ2 NRIFS:fish:GGAJ1 NRIFS:fish:GGAJ2 NRIFS:fish:MARJ1 NRIFS:fish:ON8 NRIFS:fish:MAJ1 NRIFS:fish:MAJ2 NRIFS:fish:OKAJ1 NRIFS:fish:IBD7 NRIFS:fish:MEDA1 NRIFS:fish:KUMD1 NRIFS:fish:TAK1 NRIFS:fish:TAK10 NRIFS:fish:TAK11 NRIFS:fish:SIR2 NRIFS:fish:SIR3 NRIFS:fish:HSKB1 NRIFS:fish:UM1J5 NRIFS:fish:OKT2 NRIFS:fish:OKT1 NRIFS:fish:MHZ1 NRIFS:fish:KMJ7 NRIFS:fish:KMJ8 NRIFS:fish:BIG1 NRIFS:fish:ITHZ1 NRIFS:fish:DIYG1 NRIFS:fish:DIYG2 NRIFS:fish:ISK2 NRIFS:fish:ISK1 NRIFS:fish:ISK5 NRIFS:fish:ISK3 NRIFS:fish:KOSH2 NRIFS:fish:ISI33 NRIFS:fish:MEJ10 NRIFS:fish:ISZ9 NRIFS:fish:ISI31 NRIFS:fish:KYS1 NRIFS:fish:TUMK1 NRIFS:fish:SSH2 NRIFS:fish:SSH1 NRIFS:fish:SZK1 NRIFS:fish:HIG1 NRIFS:fish:HIRG7 NRIFS:fish:HIRG8 NRIFS:fish:OKH1 NRIFS:fish:OKH2 NRIFS:fish:OKHG6 NRIFS:fish:ISI10 NRIFS:fish:ISI14 NRIFS:fish:ISI2 NRIFS:fish:ISI13 NRIFS:fish:ISI32 NRIFS:fish:ISI12 NRIFS:fish:ISI15 NRIFS:fish:ISI3 NRIFS:fish:ISI11 NRIFS:fish:ITFD1 NRIFS:fish:ISI37 NRIFS:fish:AKAM1 NRIFS:fish:BOR2 NRIFS:fish:BOR1.1 NRIFS:fish:HMJ6 NRIFS:fish:HOM9 NRIFS:fish:HBRU1 NRIFS:fish:TG1J5
ABFJ007-06 ABFJ008-06 ABFJ160-06 ABFJ178-06 ABFJ200-07 ABFJ201-07 ABFJ207-07 ABFJ208-07 ABFJ204-07 ABFJ246-07 ABFJ061-06 ABFJ062-06 ABFJ181-06 ABFJ268-07 ABFJ127-06 ABFJ107-06 ABFJ040-06 ABFJ194-07 ABFJ195-07 ABFJ075-06 ABFJ076-06 ABFJ114-06 ABFJ050-06 ABFJ072-06 ABFJ071-06 ABFJ035-06 ABFJ270-07 ABFJ271-07 ABFJ033-06 ABFJ165-06 ABFJ198-07 ABFJ199-07 ABFJ074-06 ABFJ073-06 ABFJ224-07 ABFJ169-06 ABFJ159-06 ABFJ023-06 ABFJ168-06 ABFJ232-07 ABFJ021-06 ABFJ093-06 ABFJ158-06 ABFJ097-06 ABFJ096-06 ABFJ090-06 ABFJ077-06 ABFJ219-07 ABFJ220-07 ABFJ082-06 ABFJ083-06 ABFJ221-07 ABFJ009-06 ABFJ013-06 ABFJ002-06 ABFJ012-06 ABFJ022-06 ABFJ011-06 ABFJ014-06 ABFJ003-06 ABFJ010-06 ABFJ205-07 ABFJ026-06 ABFJ187-07 ABFJ101-06 ABFJ100-06 ABFJ227-07 ABFJ231-07 ABFJ140-06 ABFJ048-06
Caranx sexfasciatus Decapterus akaadsi Megalaspis cordyla Trachurus japonicus
Cheilodactylidae
Uraspis helvola Psenopsis anomala Hyperoglyphe japonica Icichthys lockingtoni Goniistius zonatus
Coryphaenidae
Coryphaena hippurus
Echeneidae Embiotocidae
Remora osteochir Ditrema temminckii Neoditrema ransonnetii
Gerreidae
Acanthogobius flavimanus Amblychaeturichthys sciistius
Centrolophidae
Chaenogobius castaneus Cryptocentrus filifer Gerres japonicus Haemulidae
Kyphosidae
Parapristipoma trilineatum
Plectorhinchus cinctus Plectorhinchus orientalis Girella punctata Kyphosus vaigiensis
Labridae
Halichoeres poecilopterus Choerodon azurio Pteragogus flagellifer
Lateolabracidae Leiognathidae
Lateolabrax japonicus Leiognathus nuchalis
Leiognathus rivulatus
Lethrinidae
Lutjanidae
Malacanthidae Mugilidae
Nomeidae Pentacerotidae
Gnathodentex aureolineatus Gymnocranius elongatus Lethrinus harak Lethrinus semicinctus Lethrinus mahsena Lethrinus nebulosus Monotaxis grandoculis Etelis carbunculus Lutjanus bohar Lutjanus monostigma Lutjanus quinquelineatus Branchiostegus japonicus Mugil cephalus Upeneus japonicus Cubiceps baxteri Psenes pellucidus Evistias acutirostris
(continued on next page)
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Table 1 (continued ) Order
Family
Genus/Species
Voucher ID
Process ID
Polynemidae Pomacanthidae
Polydactylus plebeius Amblyglyphidodon leucogaster Chaetodontoplus septentrionalis Chromis notatus notatus Cookeolus boops Argyrosomus argentatus
NRIFS:fish:TUB10 NRIFS:fish:ISI28 NRIFS:fish:PM1 NRIFS:fish:SM1J5 NRIFS:fish:TKK1 NRIFS:fish:SIRG1 NRIFS:fish:GIRG2 NRIFS:fish:TSG2 NRIFS:fish:TSG1 NRIFS:fish:MSB2 NRIFS:fish:MSB1 NRIFS:fish:WAHO1 NRIFS:fish:KURM7 NRIFS:fish:KURM8 NRIFS:fish:ISI40 NRIFS:fish:ISI43 NRIFS:fish:ISI16 NRIFS:fish:ISI5 NRIFS:fish:HMD7 NRIFS:fish:ISI39 NRIFS:fish:KISS1 NRIFS:fish:KISS2 NRIFS:fish:MTG1 NRIFS:fish:CDI1 NRIFS:fish:MADA1 NRIFS:fish:SHAK1 NRIFS:fish:HED6 NRIFS:fish:ISI1 NRIFS:fish:KAM1 NRIFS:fish:KAM2 NRIFS:fish:GNP2 NRIFS:fish:DUB1 NRIFS:fish:TACU3 NRIFS:fish:TACU2 NRIFS:fish:SWT1 NRIFS:fish:KTGG1 NRIFS:fish:GEK7 NRIFS:fish:GEK8 NRIFS:fish:HME1 NRIFS:fish:HME2 NRIFS:fish:TGB7 NRIFS:fish:TGB8 NRIFS:fish:TGB9 NRIFS:fish:SOHH1 NRIFS:fish:MIGG1 NRIFS:fish:HIRG1 NRIFS:fish:AKGR1 NRIFS:fish:IGR1 NRIFS:fish:ISGR1 NRIFS:fish:MGR2 NRIFS:fish:MGR1 NRIFS:fish:SSU7 NRIFS:fish:SSU8 NRIFS:fish:SSU9 NRIFS:fish:AKE1 NRIFS:fish:TOKB1 NRIFS:fish:HACH5 NRIFS:fish:HACH6 NRIFS:fish:AIKK1 NRIFS:fish:HOTU1 NRIFS:fish:KMKK1 NRIFS:fish:HOKE1 NRIFS:fish:SKBN1 NRIFS:fish:KUSA1 NRIFS:fish:YGO1 NRIFS:fish:KOC2 NRIFS:fish:WANG1 NRIFS:fish:AKDK1 NRIFS:fish:ISI20 NRIFS:fish:ONKS5
ABFJ244-07 ABFJ020-06 ABFJ078-06 ABFJ047-06 ABFJ110-06 ABFJ166-06 ABFJ180-06 ABFJ058-06 ABFJ057-06 ABFJ066-06 ABFJ065-06 ABFJ134-06 ABFJ222-07 ABFJ223-07 ABFJ028-06 ABFJ029-06 ABFJ015-06 ABFJ005-06 ABFJ233-07 ABFJ027-06 ABFJ161-06 ABFJ179-06 ABFJ152-06 ABFJ186-06 ABFJ211-07 ABFJ164-06 ABFJ243-07 ABFJ001-06 ABFJ184-06 ABFJ185-06 ABFJ030-06 ABFJ116-06 ABFJ206-07 ABFJ183-06 ABFJ174-06 ABFJ119-06 ABFJ264-07 ABFJ265-07 ABFJ092-06 ABFJ176-06 ABFJ260-07 ABFJ261-07 ABFJ262-07 ABFJ135-06 ABFJ121-06 ABFJ136-06 ABFJ106-06 ABFJ036-06 ABFJ175-06 ABFJ085-06 ABFJ084-06 ABFJ254-07 ABFJ255-07 ABFJ256-07 ABFJ146-06 ABFJ117-06 ABFJ215-07 ABFJ216-07 ABFJ125-06 ABFJ145-06 ABFJ124-06 ABFJ112-06 ABFJ120-06 ABFJ123-06 ABFJ067-06 ABFJ068-06 ABFJ212-07 ABFJ122-06 ABFJ017-06 ABFJ190-07
Priacanthidae Sciaenidae
Argyrosomus macrocephalus Scombridae
Scomber australasicus
Scombropidae
Acanthocybium solandri Scombrops gilberti
Serranidae
Epinephelus coioides Epinephelus macrospilos
Siganidae Sillaginidae
Plectropomus leopardus Tosana niwae Siganus guttatus Sillago japonica
Sparidae
Evynnis japonica Pagrus major
Pleuronectiformes
Sphyraenidae
Rhabdosargus sarba Sphyraena forsteri Sphyraena pinguis
Stichaeidae Tetragonuridae Trichiuridae
Dictyosoma burgeri Tetragonurus cuvieri Trichiurus lepturus
Xiphiidae Zoarcidae Cynoglossidae
Xiphias gladius Bothrocarina microcephala Cynoglossus interruptus
Paralichthyidae
Paralichthys olivaceus Pseudorhombus pentophthalmus
Pleuronectidae
Cleisthenes pinetorum Dexistes rikuzenius Glyptocephalus stelleri Hippoglossoides dubius Kareius bicoloratus Pleuronectes herzensteini
Rajiformes Scorpaeniformes
Soleidae
Heteromycteris japonicus
Dasyatidae Agonidae
Dasyatis akajei Podothecus sachi Apistus carinatus
Cottidae Cyclopteridae Hemitripteridae Hexagrammidae Liparidae
Gymnocanthus intermedius Aptocyclus ventricosus Hemitripterus villosus Pleurogrammus azonus Careproctus rastrinus Liparis tanakai Cociella crocodila Platycephalus indicus Inegocia guttata Ebinania vermiculata Dendrochirus zebra Scorpaenopsis cirrosa
Platycephalidae
Psychrolutidae Scorpaenidae
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
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Table 1 (continued ) Order
Siluriformes Squaliformes Tetraodontiformes
Family
Genus/Species
Sebastidae
Sebastes inermis
Plotosidae Somniosidae Squalidae Molidae Monacanthidae
Sebastolobus macrochir Plotosus lineatus Somniosus pacificus Squalus suckleyi Mola mola Stephanolepis cirrhifer
Ostraciidae Tetraodontidae
Zeiformes
Thamnaconus modestus Ostracion immaculatus Canthigaster rivulata Lagocephalus wheeleri Takifugu poecilonotus Canthigaster rivulata Arothron hispidus Triacanthus biaculeatus Zenopsis nebulosa
Triacanthidae Zeidae
Voucher ID
Process ID
NRIFS:fish:ONKS4 NRIFS:fish:Nee1 NRIFS:fish:MB1J5 NRIFS:fish:KINK1 NRIFS:fish:GOZ2 NRIFS:fish:ONDZ1 NRIFS:fish:ABRZ1 NRIFS:fish:MNB1 NRIFS:fish:KW2J5 NRIFS:fish:KAW4 NRIFS:fish:KW1J5 NRIFS:fish:UMZH1 NRIFS:fish:HF1J5 NRIFS:fish:KTM1 NRIFS:fish:SIFU1 NRIFS:fish:KMFU1 NRIFS:fish:KITM6 NRIFS:fish:KITM7 NRIFS:fish:ISI17 NRIFS:fish:GIMA1 NRIFS:fish:KGMD1
ABFJ189-07 ABFJ054-06 ABFJ053-06 ABFJ102-06 ABFJ031-06 ABFJ149-06 ABFJ150-06 ABFJ213-07 ABFJ052-06 ABFJ188-07 ABFJ051-06 ABFJ133-06 ABFJ055-06 ABFJ088-06 ABFJ191-07 ABFJ209-07 ABFJ225-07 ABFJ226-07 ABFJ016-06 ABFJ162-06 ABFJ126-06
sequence database. FISH-BOL provides a platform for ichthyologists to collaborate more closely, but also greatly accelerate species identification and discovery (Swartz et al., 2008). In this study, a data set composed of 229 sequences of mitochondrial cytochrome oxidase subunit I gene (COI) from 158 species, some of which are represented by multiple sequences, was employed to elucidate the rationale of DNA barcoding and test its accuracy in species identification. 2. Material and methods 2.1. Fish specimens and DNA extraction Fish specimens were captured with a trawl at 5 localities in South Japan (35 000 N 139 300 E; 24 000 N 124 000 E; 35 250 N 139 500 E; 34 400 N 138 200 E; 23 000 N 124 000 E) in 2005 and 2006. Vouchers were deposited in National Research Institute of Fisheries Sciences, Japan. Genomic DNA was extracted according to the protocol for Barcode of Life (Ivanova et al., 2006). 2.2. PCR amplification Fragments of the 50 region of mitochondrial COI gene were initially amplified with VF2_t1/FR1d_t1 primers (Ivanova et al., 2007), and the failed ones were tried again with primers FishF2_t1/FR1d_t1. These primers are described as follows: FishF2_t1: *50 TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC 30 . VF2_t1: 50 TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC 30 . FishR2_t1: *50 CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA 30 . FR1d_t1: 50 CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA 30 . *M13 tails were underlined. PCRs were performed in 96-well plates using MastercyclerÒ Eppendorf gradient thermal cyclers. The reaction mixture, which consists of 825 ml water, 125 ml 10buffer, 62.5 ml MgCl2 (25 mM), 6.25 ml dNTP (10 mM), 6.25 ml each primer (0.01 mM) and 6.25 ml Taq DNA polymerase (5 U/ml), was prepared for each plate, and 10.5 ml mixture was added to each well with 2 ml genomic DNA. PCR procedures were an initial step of 2 min at 95 C and 35 cycles of 30 s at 94 C, 40 s at 52 C, and 1 min at Table 2 Genetic divergences (percentage, K2P distance) within various taxonomic levels. Comparisons within
Taxa
Number of comparisons
Mean (%)
Minimum (%)
Maximum (%)
S.E.a
Species Genus Family Order Class
158 144 93 21 2
91 48 220 7588 16372
0.3 17.6 18.5 23.2 24.6
0 8.4 3.8 12.1 11.5
4.0b 22.9 27.7 31.7 34.8
0.05 0.47 0.37 0.03 0.02
a b
Standard error. Pterocaesio digramma.
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J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
Fig. 1. The distribution of pairwise K2P genetic distance a. within species; b. within genus; c. within family; d. within order; e. within class.
72 C, followed by a final extension at 72 C for 10 min. PCR products were checked on 2% agarose E-GelÒ 96-well system (Invitrogen). 2.3. PCR sequencing Amplicons were diluted 4 times and prepared as the template for PCR sequencing. The PCR sequencing protocol used M13 primers with cycling condition as below: an initial step of 2 min at 96 C and 35 cycles of 30 s at 96 C, 15 s at 55 C, and 4 min at 60 C. Sequencing reactions were performed in an ABI 3730 capillary sequencer with BigDyeÒ Terminator Kit Version 3.1 (Applied Biosystems). 2.4. Data analyses Sequence alignments were implemented with SeqScape v.2.1.1 software (Applied Biosystems). Measuring Genetic differentiation was based on Kimura two parameter (K2P) distance, a suitable metric model when genetic distances are low (Nei and Kumar, 2000). The unrooted neighbor-joining (NJ) tree was established in MEGA 3 (Kumar et al., 2004). The correlation between geographical distances and genetic distances was analyzed by the Mantel test in R 2.9.2 software (R Development Core Team, 2008). 3. Results 3.1. Comparisons of genetic variations One COI pseudogene was excluded (pseudogenes derived from polypeptide sequences generally are punctuated with stop codons, effectively rendering them incapable of producing a functional protein), and 229 bidirectional sequences were
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Fig. 2. a–c Neighbor-joining (NJ) tree of COI sequences. Scale: 5.0% K2P distance. The first number behind species name is sequence ID, and the latter is sample ID.
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Fig. 2. (continued).
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
Fig. 2. (continued).
39
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obtained (sequence information in BOLD was presented in Table 1). There were many haplotypes in some species. The average of intraspecific K2P distance was only 0.3%, while the average genetic distance rose sharply to 17.6% among congeners. Overall, the average of genetic distances among congeners was over 60-fold higher than among conspecifics. However, two exceptions were observed. For Kyphosus vaigiensis and Pterocaesio digramma, the intraspecific genetic variation came to 2.7% and 4.0%, respectively. Within the higher taxonomic ranks (family, order and class), interspecific K2P genetic distance increased gently with the average value of 18.5%, 23.2% and 24.6%, respectively (Table 2 and Fig. 1a–e). As showed in the NJ tree (Fig. 2a–c), all COI sequences formed species units were clustered in monophyletic groups at the genus level, whereas at the family level, there were paraphyletic clusters for five families (Carangidae, Gobiidae Serranidae, Soleidae and Cynoglossidae). It indicates that K2P genetic divergences tend to saturate at high taxonomic levels. 3.2. The association between geographic and genetic distance To determine if intraspecific genetic variations depend upon the species and global scale to which they are applied, we submitted all 229 sequences in BOLD and obtained intraspecific K2P distance matrix for these 158 species. The association between geographical distance and intraspecific genetic divergence was assessed using the Mantel test, and the result implied that there was no significant correlation between geographical distances and intraspecific genetic distances (r ¼ 0.016, P ¼ 0.451). 4. Discussion Fish are the largest group of vertebrates, which exhibit a remarkable diversity of morphological attributes and biological adaptations (Eschmeyer et al., 1998; Nelson, 2006). Species are typically circumscribed based on the presence of fixed diagnostic morphological characters which distinguish them from other species (Wiens and Servedio, 2000). But for fishes, there are a large number of intraspecific invariants or interspecific overlappings, so fish identification is challenging for taxonomists when facing rich biotas. The limitations inherent in morphology-based identification systems and the dwindling pool of taxonomists call for the molecular approach to species recognition (Steinke et al., 2009a). The efficiency of species identification by molecular methods is judged by the levels of intraspecific homogeneity and interspecific heterogenenity displayed by the intended method (Hallden et al., 1994; Lievens et al., 2001). Mitochondrial COI gene, as an attractive “species barcode”, its high efficiency in species identification has been reported in Australia marine fishes (Ward et al., 2005), Canadian freshwater fishes (Hubert et al., 2008) and ornamental fishes in the market of North America (Steinke et al., 2009b). In all projects of Fish-BoL (near 50,000 sequences of over 7000 species have been uploaded so far), the average of intraspecific K2P distance is about 0.3%, and the average genetic distance among congeners is at least 30fold higher than among conspecifics. COI gene is a reliable species tag and DNA barcoding can deliver species-level identifications (reviewed by Hebert et al., 2003a), particularly in some cases where the morphological taxonomy is little to the purpose of species identification. Apogon are known to be a number of different species groups, based on variations of color patterns, slight differences in body and caudal-fin shape, gill-raker and pectoral fin-ray counts for which the limits are gradually becoming defined, but some of them are often difficult to identify (Fraser and Randall, 2003). Two species Apogon lineatus and Apogon semilineatus, which have similar characteristics with 3 or more head and body stripes (blackish, brownish or yellowish in life), are often misidentified. In this study, the pairwise genetic variation between Apogon lineatus and Apogon semilineatus reached 16.0%, which can provide definite species delimitation for the two species. Japan is a major fishing nation with a focus on the marine conservation, and studies about the early life history (ELH) of marine fishes are essential for the management of its marine resources. Nevertheless, species identification of eggs and larvae seems to be very difficult only based on morphological characteristics. If so, a lot of misidentifications may introduce extreme biases in surveys of biodiversity and community structure. Most fishes go through metamorphism in the ontogeny, and during the larval stage, many morphometric characteristics such as pigmentations and the number of myomeres, would change during the ontogenetic development. Therefore, species identification of eggs and larvae is bewildered by overlapping meristic characteristics (Graves et al., 1989; Chow et al., 2003). In this study, each species formed monophyletic units in the NJ tree and could be clearly separated from others (Fig. 2a–c). Mantel test also showed that there was no significant correlation between geographical distances and intraspecific genetic distances (r ¼ 0.016，P ¼ 0.451). These results demonstrate that no deep genetic divergence of COI among conspecifics and DNA barcodes are effective species tags. The application of DNA barcoding to identify species at all life-history stages is beneficial and helpful for better understanding the ecological mechanisms (Katoh, 2001; Pegg et al., 2006; Rocha-Olivares and Chávez-González, 2008; Victor et al., 2009). In Japan, warm ‘Black Current’ runs parallel with and south of the main islands, bringing with it huge range of migratory fish, including many varieties of tropic fish (Saiki, 1982). Migration is a strategy to exploit seasonal environments, and migratory species occur disproportionately at higher latitudes and their habitats display marked seasonality, thus their availability fluctuates greatly (Robinson et al., 2008). In fact, few congeneric species were captured in this study, and high interspecific genetic distances were observed due to the scarcity of pairwise comparisons between species of close phylogenetic relationships (Table 2). We tested assigned identifications through pairwise comparisons with all sequences in BOLD, and two morphological identifications against taxonomic records in FAO (Food and Agriculture Organization) and the project FTW (fish of Taiwan, voucher specimens preserved in Research Center for Biodiversity, Academia Sinica) were detected. K2P genetic distance
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
41
between the specimen ABFJ021-06 (Kyphosus vaigiensis) and FOAJ442-09 (Kyphosus cinerascens) was 0.2%, and K2P genetic distance between the specimen ABFJ007-06 (Pterocaesio digramma) and ASIZP0801062 (Pterocaesio marri) was zero. This is in stark contrast with the intraspecific genetic values in Kyphosus vaigiensis (2.7%) and Pterocaesio digramma (4.0%) in this study. We checked the preserved specimens, and some intermediate morphological characteristics were found in them. The factors responsible for deviations from taxonomic monophyly may be varied and complex (including taxonomic criteria and methodology) (Funk and Omland, 2003); the main cause of species-level paryphyly is the occasional mating between species of close phylogenetic relationship, resulting in hybrid offspring carrying a mix of genes from both parent species. For mitochondrial genes, which are maternally inherited, the introgressive hybridization can lead to the phylogentic paraphyly (Arnold, 1993; Barraclough and Nee, 2001; Funk and Omland, 2003). In BOLD, 1.0% K2P genetic distance is settled for the threshold of species delimitation, in other words, if the intraspecific genetic variation is above 1.0% or interspecific distance is below 1.0%, testing uncertain identification in BOLD should be performed for verification of the assigned identification. Indeed, intraspecific divergences are rarely greater than 2.0% and most are less than 1.0%, and higher genetic divergences generally involve taxonomic uncertainty and imply recognition of new species (Avise, 2000). For some taxonomic groups, there are few trained taxonomists to describe them, and even for species that have been adequately described, relative information of systematic classification is often incomplete or unreliable (Romano and Palumbi, 1996; Balakrishnan, 2005; Neigel et al., 2007; Swartz et al., 2008). Due to the high efficiency in species identification, some ichthyologists advocate the inclusion of a DNA barcode in the formal description of species (e.g. Victor, 2007; Astarloa et al., 2008). Somehow, it deserves attention to recent speciation, introgressive hybridization, and taxonomic splitting, which may cause the inability of COI barcodes. In such cases, a secondary independent molecular marker is required to solidify or confirm identification if applicable (Smith et al., 2007). Acknowledgements We are very grateful to Dr. Seinen Chow, National Research Institute of Fisheries Science, Japan for providing samples and specimen data. This research was supported through funding to the Canadian Barcode of Life Network from Genome Canada, NSERC and other sponsors listed at www.BOLNET.ca, and Chinese National Funding 40776089, “Dongfang” and “Shuguang” funds from Shanghai Education Committee (B-9500-09-0003, 09SG48). References Arnold, J., 1993. Cytonuclear disequilibria in hybrid zones. Annu. Rev. Ecol. Evol. Syst. 24, 521–554. Astarloa, J.M.D., Mabragaña, E., Hanner, R., 2008. Morphological and molecular evidence for a new species of longnose skate (Rajiformes: Rajidae: Dipturus) from Argentinean waters based on DNA barcoding. Zootaxa 1921, 35–46. Avise, J.C., 2000. Phylogeography: The history and formation of species. Harvard University Press, Cambridge, MA. Balakrishnan, R., 2005. Species concepts, species boundaries and species identification: a view from the Tropics. Syst. Bio. 54, 689–693. Barraclough, T.G., Nee, S., 2001. Phylogenetics and speciation. Trends Ecol. Evol. 16, 391–399. Chow, S., Nohara, K., Tanabe, T., Itoh, T., Tsuji, S., Nishikawa, Y., Uyeyanagi, S., Uchikawa, K., 2003. Genetic and morphological identification of larval and small juvenile tunas (Pisces: Scombridae) caught by a mid-water trawl in the western Pacific. Bul. Fish. Res. Agency 8, 1–14. Eschmeyer, W.N., Ferraris, J.C.J., Hoang, M.D., Long, D.J., 1998. Part 1. Species of fishes. In: Eschmeyer, W.N. (Ed.), Catalog of Fishes. California Academy of Sciences, San Franscisco. Fraser, T.H., Randall, J.E., 2003. Two new species of deeper dwelling Apogon (Perciformes: apogonidae) from Micronesia and south Pacific Ocean. Zootaxa 171, 1–11. Funk, D.J., Omland, K.E., 2003. Species-level paraphyly and polyphyly: frequency, causes, and consequences, with insights from animal mitochondrial DNA. Annu. Rev. Ecol. Evol. Syst. 34, 397–423. Graves, J.E., Simovich, M.A., Schaefer, K.M., 1989. Electrophoretic identification of early juvenile yellowfin tuna, Thunnus albacares. Fish. Bul 86, 835–838. Hallden, C., Nilsson, N.O., Rading, I.N., Sall, T., 1994. Evaluation of RFLP and RAPD markers in a comparison of Brassica napus breeding lines. Theor. Appl. Genet. 88, 123–128. Hebert, P.D.N., Cywinska, A., Ball, S.L., deWaard, J.R., 2003a. Biological identifications through DNA barcodes. P. Roy. Soc. Lond. B. Bio. 270, 313–322. Hebert, P.D.N., Ratnasingham, S., DeWaard, J.R., 2003b. Barcoding animal life: cytochrome c oxidase subunit 1 divergences among closely related species. P. Roy. Soc. Lond. B. Bio. 270 (Suppl.), S96–S99. Hubert, N., Hanner, R., Holm, E., Mandrak, N.E., Taylor, E., Burridge, M., Watkinson, D., Dumont, P., Curry, A., Bentzen, P., Zhang, J., April, J., Bernatchez, L., 2008. Identifying Canadian freshwater fishes through DNA barcodes. PLos ONE 3, e2490–2490. Ivanova, N.V., deWaard, J.R., Hebert, P.D.N., 2006. An inexpensive, automation-friendly protocol for recovering high-quality DNA. Mol. Ecol. Notes 6, 998–1002. Ivanova, N.V., Zemlak, T.S., Hanner, R.H., Hebert, P.D.N., 2007. Universal primer cocktails for fish DNA barcoding. Mol. Ecol. Notes 7, 544–548. Katoh, M., 2001. Genetic and morphological identification of Sebastiscus tertius in the East China Sea (Scorpaeniformes: Scorpaenidae). Ichthyol. Res. 48, 247–255. Kumar, S., Tamura, K., Nei, M., 2004. MEGA3: integrated software for molecular evolutionary genetics analysis and sequence alignment. Brief. Bioinf 5, 150–216. Lievens, S., Goormachtig, S., Holsters, M., 2001. A critical evaluation of differential display as a tool to identify genes involved in legume nodulation: looking back and looking forward. Nucl. Acids Res. 29, 3459–3468. Marshall, E., 2005. Taxonomy: will DNA bar codes breathe life into classification? Science 307, 1037. Nei, M., Kumar, S., 2000. Molecular Evolution and Phylogenetics. Oxford University Press, New York. Neigel, J., Domingo, A., Stake, J., 2007. DNA barcoding as a tool for coral reef conservation. Coral Reefs 26, 487–499. Nelson, J.S., 2006. Fishes of the World, fourth ed. John Wiley & Sons, Hoboken, NJ. Pegg, G.G., Sinclair, B., Briskey, L., Aspden, W.J., 2006. MtDNA barcode identification of fish larvae in the southern Great Barrier Reef, Australia. Sci. Mar. 70S2, 7–12. R Development Core Team, 2008. R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. http://www.R-project.org. Ratnasingham, S., Hebert, P.D.N., 2007. BOLD: the barcode of life data system. Mol. Ecol. Notes 7, 355–364. Robinson, R.A., Crick, H.Q.P., Learmonth, J.A., Maclean, I.M.D., Thomas, C.D., Bairlein, F., Forchhammer, M.C., Francis, C.M., Gill, J.A., Godley, B.J., Harwood, J., Hays, G.C., Huntley, B., Hutson, A.M., Pierce, G.J., Rehfisch, M.M., Sims, D.W., Santos, M.B., Sparks, T.H., Stroud, D.A., Visser, M.A., 2008. Travelling through a warming world: climate change and migratory species. Endanger. Species Res.. doi:10.3354/esr00095.
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Rocha-Olivares, A., Chávez-González, J.P., 2008. Molecular identification of dolphinfish species (genus Coryphaena) using multiplex haplotype-specific PCR of mitochondrial DNA. Ichthyol.Res. 55, 389–393. Romano, S.L., Palumbi, S.R., 1996. Evolution of scleractinian corals inferred from molecular systematics. Science 271, 640–642. Saiki, M., 1982. Relation between the geostrophic flux of the Kuroshio in the Eastern China Sea and its large meander in the south of Japan. Oceanogra. Mag. 32, 11–18. Smith, M.A., Wood, D.M., Janzen, D.H., Hallwachs, W., Hebert, P.D.N., 2007. DNA barcodes affirm that 16 species of apparently generalist tropical parasitoid flies (Diptera, Tachinidae) are not all generalists. Proc. Natl. Acad.Sci. USA 104, 4967–4972. Steinke, D., Zemlak, T.S., Boutillier, J.A., Hebert, P.D.N., 2009a. DNA barcoding of Pacific Canada’s fishes. Mar. Bio 156, 2641–2647. Steinke, D., Zemlak, T.S., Hebert, P.D.N., 2009b. Barcoding Nemo: DNA-Based identifications for the ornamental fish Trade. PLos ONE 4, e6300. Swartz, E.R., Mwale, M., Hanner, R., 2008. A role for barcoding in the study of African fish diversity and conservation. S. Afr. J. Sci. 104, 293–298. Victor, B.C., 2007. Coryphopterus kuna, a new goby (Perciformes: Gobiidae: Gobiinae) from the western Caribbean, with the identification of the late larval stage and an estimate of the pelagic larval duration. Zootaxa 1526, 51–61. Victor, B.C., Hanner, R., Shivji, M., Hyde, J., Caldow, C., 2009. Identification of the larval and juvenile stages of the Cubera Snapper, Lutjanus cyanopterus, using DNA barcoding. Zootaxa 2215, 24–36. Ward, R.D., Zemlak, T.S., Innes, B.H., Last, P.R., Hebert, P.D.N., 2005. DNA barcoding Australia’s fish species. Philos. Trans. R. Soc. Lond. B. 360, 1847–1857. Wiens, J.J., Servedio, M.R., 2000. Species delimitation in systematics: inferring diagnostic differences between species. P. Roy. Soc. Lond. B. Bio 267, 631–636.
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Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
DNA barcoding is a useful tool for the identification of marine fishes from Japan Jun-Bin Zhang a, b, *, Robert Hanner b a
Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China b Department of Integrative Biology, University of Guelph, Guelph ON, Canada
a r t i c l e i n f o
a b s t r a c t
Article history: Received 1 April 2010 Accepted 21 December 2010 Available online 22 January 2011
In this study, 229 DNA sequences of cytochrome oxidase subunit I gene (COI) from 158 marine fishes of Japan were employed to test the efficacy of species identification by DNA barcoding. The average genetic distance was 60-fold higher between species than within species, as Kimura two parameter (K2P) genetic distances averaged 17.6% among congeners and only 0.3% among conspecifics. There were no overlaps between intraspecific and interspecific K2P distances, and all sequences formed species units in the neighbor-joining dendrogram. Hybridization phenomena in two species (Kyphosus vaigiensis and Pterocaesio digramma) were also detected through searches in Barcode of Life Data Systems (BOLD). DNA barcoding provides a new way for fish identification. Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved.
Keywords: DNA barcoding Marine fishes Japan
1. Introduction DNA barcoding, which was advocated by Hebert et al. (2003a,b), seeks to facilitate identifying the increasing number of unfamiliar taxa in biological conservation and biodiversity surveys, based on sequence diversity within a short and standardized gene region (Hebert et al., 2003a; Marshall, 2005). So far, scientists from over 30 different countries have participated in this international plan. For coordinating the collection data of specimens and performing data analysis with barcode data, the website Barcode of Life Data Systems (BOLD) (http://www.boldsystems.org) has been established (Ratnasingham and Hebert, 2007). BOLD is an accessible database that aids in management, analysis, dissemination, and searching of DNA barcodes. Fish Barcode of Life Initiative (FISH-BOL), as a campaign of the International Barcode of Life Project (iBOL), is to build up a standardized database of reference sequences for all fishes. With regard to fishes, the target DNA segment of 652 base-pairs near 50 end of the mitochondrial cytochrome oxidase subunit I gene is strongly proposed (Hebert et al., 2003a). These sequences are derived from voucher specimens preserved in the museums all around the world. Storage information (taxonomic identification, catalogue number and institution storing) and collection data (collector, collection date and location with longitude-latitude coordinates) are required to inject specimen records when creating projects for them. In addition, DNA barcodes (sequences of at least 500 bps and trace files) need to be uploaded to projects in FISH-BOL after finishing PCR amplification and sequencing. Thus, any problem concerning morphological identification can be solved by searching the relative data in BOLD or sending enquiries for checking voucher specimens preserved in natural history collections. Assignment of specimens to known species can be achieved through the comparison of genetic similarity with the
* Corresponding author. Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China. Tel.: þ86 21 61900421. E-mail address: [email protected] (J.-B. Zhang). 0305-1978/$ – see front matter Crown Copyright Ó 2011 Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bse.2010.12.017
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Table 1 Voucher and sequence information for these 229 specimens. Process IDs are sequence numbers of voucher specimens in Barcode of Life Database (www. barcodinglife.org), and Voucher IDs are voucher numbers in National Research Institute of Fisheries Science, Japan. Order
Family
Genus/Species
Voucher ID
Process ID
Anguilliformes
Congridae
Ariosoma meeki
Atheriniformes
Ophichthidae Synaphobranchidae Atherinidae
Conger japonicus Pisodonophis zophistius Synaphobranchus kaupii Hypoatherina valenciennei
Aulopiformes
Alepisauridae Synodontidae
Alepisaurus ferox Saurida elongata
Beloniformes
Exocoetidae
Hirundichthys oxycephalus
Belonidae
Ablennes hians
Exocoetidae Hemiramphidae
Cheilopogon pinnatibarbatus Hyporhamphus sajori
Berycidae Holocentridae Chimaeridae Clupeidae
Beryx splendens Sargocentron spiniferum Hydrolagus barbouri Engraulis japonicus Etrumeus teres
NRIFS:fish:HAA6 NRIFS:fish:HAA7 NRIFS:fish:MANG1 NRIFS:fish:BUM9 NRIFS:fish:IRAN1 NRIFS:fish:TGI1 NRIFS:fish:TGI2 NRIFS:fish:MIZU1 NRIFS:fish:TKE6 NRIFS:fish:TKE7 NRIFS:fish:TKE8 NRIFS:fish:HTB1 NRIFS:fish:HTB2 NRIFS:fish:DAT6 NRIFS:fish:DAT7 NRIFS:fish:HTBU1 NRIFS:fish:SDLG1 NRIFS:fish:SUJL1 NRIFS:fish:BERX1 NRIFS:fish:ISI6 NRIFS:fish:KHGZ1 NRIFS:fish:KTK1 NRIFS:fish:AMDK1 NRIFS:fish:AGM1 NRIFS:fish:KNOS1 NRIFS:fish:KNOS2 NRIFS:fish:SAPP2 NRIFS:fish:SAPP1 NRIFS:fish:MIW5 NRIFS:fish:MIW4 NRIFS:fish:MIW2 NRIFS:fish:MIW1 NRIFS:fish:KBG2 NRIFS:fish:KBG1 NRIFS:fish:KTK2 NRIFS:fish:95 KL NRIFS:fish:ISG35 NRIFS:fish:KNDR1 NRIFS:fish:HIMD1 NRIFS:fish:HANS1 NRIFS:fish:MADR1 NRIFS:fish:HONT1 NRIFS:fish:HGDR1 NRIFS:fish:EZIA1 NRIFS:fish:WAPL1 NRIFS:fish:NEZZ1 NRIFS:fish:KIAK1 NRIFS:fish:NUT6 NRIFS:fish:NUT7 NRIFS:fish:NUT8 NRIFS:fish:ISS1 NRIFS:fish:BBR1 NRIFS:fish:NIZ5 NRIFS:fish:NIZ4 NRIFS:fish:SK8 NRIFS:fish:SK9 NRIFS:fish:SK10 NRIFS:fish:KSI1 NRIFS:fish:TGD7 NRIFS:fish:TGD8 NRIFS:fish:TGD9 NRIFS:fish:NBD2 NRIFS:fish:NBD1 NRIFS:fish:NENB6 NRIFS:fish:NENB7 NRIFS:fish:NZGP2 NRIFS:fish:BJAP1 NRIFS:fish:TGE1
ABFJ241-07 ABFJ242-07 ABFJ138-06 ABFJ240-07 ABFJ130-06 ABFJ032-06 ABFJ172-06 ABFJ132-06 ABFJ234-07 ABFJ235-07 ABFJ236-07 ABFJ063-06 ABFJ064-06 ABFJ238-07 ABFJ239-07 ABFJ141-06 ABFJ154-06 ABFJ153-06 ABFJ182-06 ABFJ006-06 ABFJ147-06 ABFJ098-06 ABFJ156-06 ABFJ155-06 ABFJ202-07 ABFJ203-07 ABFJ177-06 ABFJ163-06 ABFJ171-06 ABFJ170-06 ABFJ081-06 ABFJ080-06 ABFJ087-06 ABFJ086-06 ABFJ099-06 ABFJ214-07 ABFJ245-07 ABFJ144-06 ABFJ109-06 ABFJ108-06 ABFJ131-06 ABFJ157-06 ABFJ143-06 ABFJ118-06 ABFJ129-06 ABFJ148-06 ABFJ128-06 ABFJ228-07 ABFJ229-07 ABFJ230-07 ABFJ037-06 ABFJ103-06 ABFJ039-06 ABFJ038-06 ABFJ247-07 ABFJ248-07 ABFJ249-07 ABFJ079-06 ABFJ257-07 ABFJ258-07 ABFJ259-07 ABFJ060-06 ABFJ059-06 ABFJ217-07 ABFJ218-07 ABFJ197-07 ABFJ113-06 ABFJ111-06
Beryciformes Chimaeriformes Clupeiformes
Konosirus punctatus Sardinella zunasi Sardinops melanostictus
Spratelloides gracilis
Gadidae
Engraulis japonicus Elops hawaiensis Megalops cyprinoides Antimora microlepis Coryphaenoides longifilis Coryphaenoides nasutus Gadus macrocephalus
Lamniformes Lophiiformes Myxiniformes
Macrouridae Moridae Gadidae Lamnidae Lophiidae Myxinidae
Nezumia proxima Physiculus maximowiczi Theragra chalcogramma Lamna ditropis Lophius litulon Eptatretus burgeri
Osmeriformes Osmeriformes Perciformes
Osmeridae Microstomatidae Acanthuridae
Salangichthys ishikawae Microstoma ashne Prionurus scalprum
Acropomatidae
Synagrops japonicus
Apogonidae
Apogon endekataenia Apogon lineatus
Elopiformes Gadiformes
Engraulidae Elopidae Megalopidae Moridae Macrouridae
Apogon semilineatus
Blenniidae Bramidae
Petroscirtes breviceps Brama japonica Taractes rubescens
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
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Table 1 (continued ) Order
Family
Genus/Species
Voucher ID
Process ID
Caesionidae
Pterocaesio digramma
Callionymidae
Repomucenus valenciennei
Carangidae
Alectis ciliaris
NRIFS:fish:ISI7 NRIFS:fish:ISI8 NRIFS:fish:HTTN1 NRIFS:fish:HTTN2 NRIFS:fish:ITHJ1 NRIFS:fish:ITHJ2 NRIFS:fish:GGAJ1 NRIFS:fish:GGAJ2 NRIFS:fish:MARJ1 NRIFS:fish:ON8 NRIFS:fish:MAJ1 NRIFS:fish:MAJ2 NRIFS:fish:OKAJ1 NRIFS:fish:IBD7 NRIFS:fish:MEDA1 NRIFS:fish:KUMD1 NRIFS:fish:TAK1 NRIFS:fish:TAK10 NRIFS:fish:TAK11 NRIFS:fish:SIR2 NRIFS:fish:SIR3 NRIFS:fish:HSKB1 NRIFS:fish:UM1J5 NRIFS:fish:OKT2 NRIFS:fish:OKT1 NRIFS:fish:MHZ1 NRIFS:fish:KMJ7 NRIFS:fish:KMJ8 NRIFS:fish:BIG1 NRIFS:fish:ITHZ1 NRIFS:fish:DIYG1 NRIFS:fish:DIYG2 NRIFS:fish:ISK2 NRIFS:fish:ISK1 NRIFS:fish:ISK5 NRIFS:fish:ISK3 NRIFS:fish:KOSH2 NRIFS:fish:ISI33 NRIFS:fish:MEJ10 NRIFS:fish:ISZ9 NRIFS:fish:ISI31 NRIFS:fish:KYS1 NRIFS:fish:TUMK1 NRIFS:fish:SSH2 NRIFS:fish:SSH1 NRIFS:fish:SZK1 NRIFS:fish:HIG1 NRIFS:fish:HIRG7 NRIFS:fish:HIRG8 NRIFS:fish:OKH1 NRIFS:fish:OKH2 NRIFS:fish:OKHG6 NRIFS:fish:ISI10 NRIFS:fish:ISI14 NRIFS:fish:ISI2 NRIFS:fish:ISI13 NRIFS:fish:ISI32 NRIFS:fish:ISI12 NRIFS:fish:ISI15 NRIFS:fish:ISI3 NRIFS:fish:ISI11 NRIFS:fish:ITFD1 NRIFS:fish:ISI37 NRIFS:fish:AKAM1 NRIFS:fish:BOR2 NRIFS:fish:BOR1.1 NRIFS:fish:HMJ6 NRIFS:fish:HOM9 NRIFS:fish:HBRU1 NRIFS:fish:TG1J5
ABFJ007-06 ABFJ008-06 ABFJ160-06 ABFJ178-06 ABFJ200-07 ABFJ201-07 ABFJ207-07 ABFJ208-07 ABFJ204-07 ABFJ246-07 ABFJ061-06 ABFJ062-06 ABFJ181-06 ABFJ268-07 ABFJ127-06 ABFJ107-06 ABFJ040-06 ABFJ194-07 ABFJ195-07 ABFJ075-06 ABFJ076-06 ABFJ114-06 ABFJ050-06 ABFJ072-06 ABFJ071-06 ABFJ035-06 ABFJ270-07 ABFJ271-07 ABFJ033-06 ABFJ165-06 ABFJ198-07 ABFJ199-07 ABFJ074-06 ABFJ073-06 ABFJ224-07 ABFJ169-06 ABFJ159-06 ABFJ023-06 ABFJ168-06 ABFJ232-07 ABFJ021-06 ABFJ093-06 ABFJ158-06 ABFJ097-06 ABFJ096-06 ABFJ090-06 ABFJ077-06 ABFJ219-07 ABFJ220-07 ABFJ082-06 ABFJ083-06 ABFJ221-07 ABFJ009-06 ABFJ013-06 ABFJ002-06 ABFJ012-06 ABFJ022-06 ABFJ011-06 ABFJ014-06 ABFJ003-06 ABFJ010-06 ABFJ205-07 ABFJ026-06 ABFJ187-07 ABFJ101-06 ABFJ100-06 ABFJ227-07 ABFJ231-07 ABFJ140-06 ABFJ048-06
Caranx sexfasciatus Decapterus akaadsi Megalaspis cordyla Trachurus japonicus
Cheilodactylidae
Uraspis helvola Psenopsis anomala Hyperoglyphe japonica Icichthys lockingtoni Goniistius zonatus
Coryphaenidae
Coryphaena hippurus
Echeneidae Embiotocidae
Remora osteochir Ditrema temminckii Neoditrema ransonnetii
Gerreidae
Acanthogobius flavimanus Amblychaeturichthys sciistius
Centrolophidae
Chaenogobius castaneus Cryptocentrus filifer Gerres japonicus Haemulidae
Kyphosidae
Parapristipoma trilineatum
Plectorhinchus cinctus Plectorhinchus orientalis Girella punctata Kyphosus vaigiensis
Labridae
Halichoeres poecilopterus Choerodon azurio Pteragogus flagellifer
Lateolabracidae Leiognathidae
Lateolabrax japonicus Leiognathus nuchalis
Leiognathus rivulatus
Lethrinidae
Lutjanidae
Malacanthidae Mugilidae
Nomeidae Pentacerotidae
Gnathodentex aureolineatus Gymnocranius elongatus Lethrinus harak Lethrinus semicinctus Lethrinus mahsena Lethrinus nebulosus Monotaxis grandoculis Etelis carbunculus Lutjanus bohar Lutjanus monostigma Lutjanus quinquelineatus Branchiostegus japonicus Mugil cephalus Upeneus japonicus Cubiceps baxteri Psenes pellucidus Evistias acutirostris
(continued on next page)
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Table 1 (continued ) Order
Family
Genus/Species
Voucher ID
Process ID
Polynemidae Pomacanthidae
Polydactylus plebeius Amblyglyphidodon leucogaster Chaetodontoplus septentrionalis Chromis notatus notatus Cookeolus boops Argyrosomus argentatus
NRIFS:fish:TUB10 NRIFS:fish:ISI28 NRIFS:fish:PM1 NRIFS:fish:SM1J5 NRIFS:fish:TKK1 NRIFS:fish:SIRG1 NRIFS:fish:GIRG2 NRIFS:fish:TSG2 NRIFS:fish:TSG1 NRIFS:fish:MSB2 NRIFS:fish:MSB1 NRIFS:fish:WAHO1 NRIFS:fish:KURM7 NRIFS:fish:KURM8 NRIFS:fish:ISI40 NRIFS:fish:ISI43 NRIFS:fish:ISI16 NRIFS:fish:ISI5 NRIFS:fish:HMD7 NRIFS:fish:ISI39 NRIFS:fish:KISS1 NRIFS:fish:KISS2 NRIFS:fish:MTG1 NRIFS:fish:CDI1 NRIFS:fish:MADA1 NRIFS:fish:SHAK1 NRIFS:fish:HED6 NRIFS:fish:ISI1 NRIFS:fish:KAM1 NRIFS:fish:KAM2 NRIFS:fish:GNP2 NRIFS:fish:DUB1 NRIFS:fish:TACU3 NRIFS:fish:TACU2 NRIFS:fish:SWT1 NRIFS:fish:KTGG1 NRIFS:fish:GEK7 NRIFS:fish:GEK8 NRIFS:fish:HME1 NRIFS:fish:HME2 NRIFS:fish:TGB7 NRIFS:fish:TGB8 NRIFS:fish:TGB9 NRIFS:fish:SOHH1 NRIFS:fish:MIGG1 NRIFS:fish:HIRG1 NRIFS:fish:AKGR1 NRIFS:fish:IGR1 NRIFS:fish:ISGR1 NRIFS:fish:MGR2 NRIFS:fish:MGR1 NRIFS:fish:SSU7 NRIFS:fish:SSU8 NRIFS:fish:SSU9 NRIFS:fish:AKE1 NRIFS:fish:TOKB1 NRIFS:fish:HACH5 NRIFS:fish:HACH6 NRIFS:fish:AIKK1 NRIFS:fish:HOTU1 NRIFS:fish:KMKK1 NRIFS:fish:HOKE1 NRIFS:fish:SKBN1 NRIFS:fish:KUSA1 NRIFS:fish:YGO1 NRIFS:fish:KOC2 NRIFS:fish:WANG1 NRIFS:fish:AKDK1 NRIFS:fish:ISI20 NRIFS:fish:ONKS5
ABFJ244-07 ABFJ020-06 ABFJ078-06 ABFJ047-06 ABFJ110-06 ABFJ166-06 ABFJ180-06 ABFJ058-06 ABFJ057-06 ABFJ066-06 ABFJ065-06 ABFJ134-06 ABFJ222-07 ABFJ223-07 ABFJ028-06 ABFJ029-06 ABFJ015-06 ABFJ005-06 ABFJ233-07 ABFJ027-06 ABFJ161-06 ABFJ179-06 ABFJ152-06 ABFJ186-06 ABFJ211-07 ABFJ164-06 ABFJ243-07 ABFJ001-06 ABFJ184-06 ABFJ185-06 ABFJ030-06 ABFJ116-06 ABFJ206-07 ABFJ183-06 ABFJ174-06 ABFJ119-06 ABFJ264-07 ABFJ265-07 ABFJ092-06 ABFJ176-06 ABFJ260-07 ABFJ261-07 ABFJ262-07 ABFJ135-06 ABFJ121-06 ABFJ136-06 ABFJ106-06 ABFJ036-06 ABFJ175-06 ABFJ085-06 ABFJ084-06 ABFJ254-07 ABFJ255-07 ABFJ256-07 ABFJ146-06 ABFJ117-06 ABFJ215-07 ABFJ216-07 ABFJ125-06 ABFJ145-06 ABFJ124-06 ABFJ112-06 ABFJ120-06 ABFJ123-06 ABFJ067-06 ABFJ068-06 ABFJ212-07 ABFJ122-06 ABFJ017-06 ABFJ190-07
Priacanthidae Sciaenidae
Argyrosomus macrocephalus Scombridae
Scomber australasicus
Scombropidae
Acanthocybium solandri Scombrops gilberti
Serranidae
Epinephelus coioides Epinephelus macrospilos
Siganidae Sillaginidae
Plectropomus leopardus Tosana niwae Siganus guttatus Sillago japonica
Sparidae
Evynnis japonica Pagrus major
Pleuronectiformes
Sphyraenidae
Rhabdosargus sarba Sphyraena forsteri Sphyraena pinguis
Stichaeidae Tetragonuridae Trichiuridae
Dictyosoma burgeri Tetragonurus cuvieri Trichiurus lepturus
Xiphiidae Zoarcidae Cynoglossidae
Xiphias gladius Bothrocarina microcephala Cynoglossus interruptus
Paralichthyidae
Paralichthys olivaceus Pseudorhombus pentophthalmus
Pleuronectidae
Cleisthenes pinetorum Dexistes rikuzenius Glyptocephalus stelleri Hippoglossoides dubius Kareius bicoloratus Pleuronectes herzensteini
Rajiformes Scorpaeniformes
Soleidae
Heteromycteris japonicus
Dasyatidae Agonidae
Dasyatis akajei Podothecus sachi Apistus carinatus
Cottidae Cyclopteridae Hemitripteridae Hexagrammidae Liparidae
Gymnocanthus intermedius Aptocyclus ventricosus Hemitripterus villosus Pleurogrammus azonus Careproctus rastrinus Liparis tanakai Cociella crocodila Platycephalus indicus Inegocia guttata Ebinania vermiculata Dendrochirus zebra Scorpaenopsis cirrosa
Platycephalidae
Psychrolutidae Scorpaenidae
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
35
Table 1 (continued ) Order
Siluriformes Squaliformes Tetraodontiformes
Family
Genus/Species
Sebastidae
Sebastes inermis
Plotosidae Somniosidae Squalidae Molidae Monacanthidae
Sebastolobus macrochir Plotosus lineatus Somniosus pacificus Squalus suckleyi Mola mola Stephanolepis cirrhifer
Ostraciidae Tetraodontidae
Zeiformes
Thamnaconus modestus Ostracion immaculatus Canthigaster rivulata Lagocephalus wheeleri Takifugu poecilonotus Canthigaster rivulata Arothron hispidus Triacanthus biaculeatus Zenopsis nebulosa
Triacanthidae Zeidae
Voucher ID
Process ID
NRIFS:fish:ONKS4 NRIFS:fish:Nee1 NRIFS:fish:MB1J5 NRIFS:fish:KINK1 NRIFS:fish:GOZ2 NRIFS:fish:ONDZ1 NRIFS:fish:ABRZ1 NRIFS:fish:MNB1 NRIFS:fish:KW2J5 NRIFS:fish:KAW4 NRIFS:fish:KW1J5 NRIFS:fish:UMZH1 NRIFS:fish:HF1J5 NRIFS:fish:KTM1 NRIFS:fish:SIFU1 NRIFS:fish:KMFU1 NRIFS:fish:KITM6 NRIFS:fish:KITM7 NRIFS:fish:ISI17 NRIFS:fish:GIMA1 NRIFS:fish:KGMD1
ABFJ189-07 ABFJ054-06 ABFJ053-06 ABFJ102-06 ABFJ031-06 ABFJ149-06 ABFJ150-06 ABFJ213-07 ABFJ052-06 ABFJ188-07 ABFJ051-06 ABFJ133-06 ABFJ055-06 ABFJ088-06 ABFJ191-07 ABFJ209-07 ABFJ225-07 ABFJ226-07 ABFJ016-06 ABFJ162-06 ABFJ126-06
sequence database. FISH-BOL provides a platform for ichthyologists to collaborate more closely, but also greatly accelerate species identification and discovery (Swartz et al., 2008). In this study, a data set composed of 229 sequences of mitochondrial cytochrome oxidase subunit I gene (COI) from 158 species, some of which are represented by multiple sequences, was employed to elucidate the rationale of DNA barcoding and test its accuracy in species identification. 2. Material and methods 2.1. Fish specimens and DNA extraction Fish specimens were captured with a trawl at 5 localities in South Japan (35 000 N 139 300 E; 24 000 N 124 000 E; 35 250 N 139 500 E; 34 400 N 138 200 E; 23 000 N 124 000 E) in 2005 and 2006. Vouchers were deposited in National Research Institute of Fisheries Sciences, Japan. Genomic DNA was extracted according to the protocol for Barcode of Life (Ivanova et al., 2006). 2.2. PCR amplification Fragments of the 50 region of mitochondrial COI gene were initially amplified with VF2_t1/FR1d_t1 primers (Ivanova et al., 2007), and the failed ones were tried again with primers FishF2_t1/FR1d_t1. These primers are described as follows: FishF2_t1: *50 TGTAAAACGACGGCCAGTCGACTAATCATAAAGATATCGGCAC 30 . VF2_t1: 50 TGTAAAACGACGGCCAGTCAACCAACCACAAAGACATTGGCAC 30 . FishR2_t1: *50 CAGGAAACAGCTATGACACTTCAGGGTGACCGAAGAATCAGAA 30 . FR1d_t1: 50 CAGGAAACAGCTATGACACCTCAGGGTGTCCGAARAAYCARAA 30 . *M13 tails were underlined. PCRs were performed in 96-well plates using MastercyclerÒ Eppendorf gradient thermal cyclers. The reaction mixture, which consists of 825 ml water, 125 ml 10buffer, 62.5 ml MgCl2 (25 mM), 6.25 ml dNTP (10 mM), 6.25 ml each primer (0.01 mM) and 6.25 ml Taq DNA polymerase (5 U/ml), was prepared for each plate, and 10.5 ml mixture was added to each well with 2 ml genomic DNA. PCR procedures were an initial step of 2 min at 95 C and 35 cycles of 30 s at 94 C, 40 s at 52 C, and 1 min at Table 2 Genetic divergences (percentage, K2P distance) within various taxonomic levels. Comparisons within
Taxa
Number of comparisons
Mean (%)
Minimum (%)
Maximum (%)
S.E.a
Species Genus Family Order Class
158 144 93 21 2
91 48 220 7588 16372
0.3 17.6 18.5 23.2 24.6
0 8.4 3.8 12.1 11.5
4.0b 22.9 27.7 31.7 34.8
0.05 0.47 0.37 0.03 0.02
a b
Standard error. Pterocaesio digramma.
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J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
Fig. 1. The distribution of pairwise K2P genetic distance a. within species; b. within genus; c. within family; d. within order; e. within class.
72 C, followed by a final extension at 72 C for 10 min. PCR products were checked on 2% agarose E-GelÒ 96-well system (Invitrogen). 2.3. PCR sequencing Amplicons were diluted 4 times and prepared as the template for PCR sequencing. The PCR sequencing protocol used M13 primers with cycling condition as below: an initial step of 2 min at 96 C and 35 cycles of 30 s at 96 C, 15 s at 55 C, and 4 min at 60 C. Sequencing reactions were performed in an ABI 3730 capillary sequencer with BigDyeÒ Terminator Kit Version 3.1 (Applied Biosystems). 2.4. Data analyses Sequence alignments were implemented with SeqScape v.2.1.1 software (Applied Biosystems). Measuring Genetic differentiation was based on Kimura two parameter (K2P) distance, a suitable metric model when genetic distances are low (Nei and Kumar, 2000). The unrooted neighbor-joining (NJ) tree was established in MEGA 3 (Kumar et al., 2004). The correlation between geographical distances and genetic distances was analyzed by the Mantel test in R 2.9.2 software (R Development Core Team, 2008). 3. Results 3.1. Comparisons of genetic variations One COI pseudogene was excluded (pseudogenes derived from polypeptide sequences generally are punctuated with stop codons, effectively rendering them incapable of producing a functional protein), and 229 bidirectional sequences were
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Fig. 2. a–c Neighbor-joining (NJ) tree of COI sequences. Scale: 5.0% K2P distance. The first number behind species name is sequence ID, and the latter is sample ID.
38
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
Fig. 2. (continued).
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
Fig. 2. (continued).
39
40
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
obtained (sequence information in BOLD was presented in Table 1). There were many haplotypes in some species. The average of intraspecific K2P distance was only 0.3%, while the average genetic distance rose sharply to 17.6% among congeners. Overall, the average of genetic distances among congeners was over 60-fold higher than among conspecifics. However, two exceptions were observed. For Kyphosus vaigiensis and Pterocaesio digramma, the intraspecific genetic variation came to 2.7% and 4.0%, respectively. Within the higher taxonomic ranks (family, order and class), interspecific K2P genetic distance increased gently with the average value of 18.5%, 23.2% and 24.6%, respectively (Table 2 and Fig. 1a–e). As showed in the NJ tree (Fig. 2a–c), all COI sequences formed species units were clustered in monophyletic groups at the genus level, whereas at the family level, there were paraphyletic clusters for five families (Carangidae, Gobiidae Serranidae, Soleidae and Cynoglossidae). It indicates that K2P genetic divergences tend to saturate at high taxonomic levels. 3.2. The association between geographic and genetic distance To determine if intraspecific genetic variations depend upon the species and global scale to which they are applied, we submitted all 229 sequences in BOLD and obtained intraspecific K2P distance matrix for these 158 species. The association between geographical distance and intraspecific genetic divergence was assessed using the Mantel test, and the result implied that there was no significant correlation between geographical distances and intraspecific genetic distances (r ¼ 0.016, P ¼ 0.451). 4. Discussion Fish are the largest group of vertebrates, which exhibit a remarkable diversity of morphological attributes and biological adaptations (Eschmeyer et al., 1998; Nelson, 2006). Species are typically circumscribed based on the presence of fixed diagnostic morphological characters which distinguish them from other species (Wiens and Servedio, 2000). But for fishes, there are a large number of intraspecific invariants or interspecific overlappings, so fish identification is challenging for taxonomists when facing rich biotas. The limitations inherent in morphology-based identification systems and the dwindling pool of taxonomists call for the molecular approach to species recognition (Steinke et al., 2009a). The efficiency of species identification by molecular methods is judged by the levels of intraspecific homogeneity and interspecific heterogenenity displayed by the intended method (Hallden et al., 1994; Lievens et al., 2001). Mitochondrial COI gene, as an attractive “species barcode”, its high efficiency in species identification has been reported in Australia marine fishes (Ward et al., 2005), Canadian freshwater fishes (Hubert et al., 2008) and ornamental fishes in the market of North America (Steinke et al., 2009b). In all projects of Fish-BoL (near 50,000 sequences of over 7000 species have been uploaded so far), the average of intraspecific K2P distance is about 0.3%, and the average genetic distance among congeners is at least 30fold higher than among conspecifics. COI gene is a reliable species tag and DNA barcoding can deliver species-level identifications (reviewed by Hebert et al., 2003a), particularly in some cases where the morphological taxonomy is little to the purpose of species identification. Apogon are known to be a number of different species groups, based on variations of color patterns, slight differences in body and caudal-fin shape, gill-raker and pectoral fin-ray counts for which the limits are gradually becoming defined, but some of them are often difficult to identify (Fraser and Randall, 2003). Two species Apogon lineatus and Apogon semilineatus, which have similar characteristics with 3 or more head and body stripes (blackish, brownish or yellowish in life), are often misidentified. In this study, the pairwise genetic variation between Apogon lineatus and Apogon semilineatus reached 16.0%, which can provide definite species delimitation for the two species. Japan is a major fishing nation with a focus on the marine conservation, and studies about the early life history (ELH) of marine fishes are essential for the management of its marine resources. Nevertheless, species identification of eggs and larvae seems to be very difficult only based on morphological characteristics. If so, a lot of misidentifications may introduce extreme biases in surveys of biodiversity and community structure. Most fishes go through metamorphism in the ontogeny, and during the larval stage, many morphometric characteristics such as pigmentations and the number of myomeres, would change during the ontogenetic development. Therefore, species identification of eggs and larvae is bewildered by overlapping meristic characteristics (Graves et al., 1989; Chow et al., 2003). In this study, each species formed monophyletic units in the NJ tree and could be clearly separated from others (Fig. 2a–c). Mantel test also showed that there was no significant correlation between geographical distances and intraspecific genetic distances (r ¼ 0.016，P ¼ 0.451). These results demonstrate that no deep genetic divergence of COI among conspecifics and DNA barcodes are effective species tags. The application of DNA barcoding to identify species at all life-history stages is beneficial and helpful for better understanding the ecological mechanisms (Katoh, 2001; Pegg et al., 2006; Rocha-Olivares and Chávez-González, 2008; Victor et al., 2009). In Japan, warm ‘Black Current’ runs parallel with and south of the main islands, bringing with it huge range of migratory fish, including many varieties of tropic fish (Saiki, 1982). Migration is a strategy to exploit seasonal environments, and migratory species occur disproportionately at higher latitudes and their habitats display marked seasonality, thus their availability fluctuates greatly (Robinson et al., 2008). In fact, few congeneric species were captured in this study, and high interspecific genetic distances were observed due to the scarcity of pairwise comparisons between species of close phylogenetic relationships (Table 2). We tested assigned identifications through pairwise comparisons with all sequences in BOLD, and two morphological identifications against taxonomic records in FAO (Food and Agriculture Organization) and the project FTW (fish of Taiwan, voucher specimens preserved in Research Center for Biodiversity, Academia Sinica) were detected. K2P genetic distance
J.-B. Zhang, R. Hanner / Biochemical Systematics and Ecology 39 (2011) 31–42
41
between the specimen ABFJ021-06 (Kyphosus vaigiensis) and FOAJ442-09 (Kyphosus cinerascens) was 0.2%, and K2P genetic distance between the specimen ABFJ007-06 (Pterocaesio digramma) and ASIZP0801062 (Pterocaesio marri) was zero. This is in stark contrast with the intraspecific genetic values in Kyphosus vaigiensis (2.7%) and Pterocaesio digramma (4.0%) in this study. We checked the preserved specimens, and some intermediate morphological characteristics were found in them. The factors responsible for deviations from taxonomic monophyly may be varied and complex (including taxonomic criteria and methodology) (Funk and Omland, 2003); the main cause of species-level paryphyly is the occasional mating between species of close phylogenetic relationship, resulting in hybrid offspring carrying a mix of genes from both parent species. For mitochondrial genes, which are maternally inherited, the introgressive hybridization can lead to the phylogentic paraphyly (Arnold, 1993; Barraclough and Nee, 2001; Funk and Omland, 2003). In BOLD, 1.0% K2P genetic distance is settled for the threshold of species delimitation, in other words, if the intraspecific genetic variation is above 1.0% or interspecific distance is below 1.0%, testing uncertain identification in BOLD should be performed for verification of the assigned identification. Indeed, intraspecific divergences are rarely greater than 2.0% and most are less than 1.0%, and higher genetic divergences generally involve taxonomic uncertainty and imply recognition of new species (Avise, 2000). For some taxonomic groups, there are few trained taxonomists to describe them, and even for species that have been adequately described, relative information of systematic classification is often incomplete or unreliable (Romano and Palumbi, 1996; Balakrishnan, 2005; Neigel et al., 2007; Swartz et al., 2008). Due to the high efficiency in species identification, some ichthyologists advocate the inclusion of a DNA barcode in the formal description of species (e.g. Victor, 2007; Astarloa et al., 2008). Somehow, it deserves attention to recent speciation, introgressive hybridization, and taxonomic splitting, which may cause the inability of COI barcodes. In such cases, a secondary independent molecular marker is required to solidify or confirm identification if applicable (Smith et al., 2007). Acknowledgements We are very grateful to Dr. Seinen Chow, National Research Institute of Fisheries Science, Japan for providing samples and specimen data. This research was supported through funding to the Canadian Barcode of Life Network from Genome Canada, NSERC and other sponsors listed at www.BOLNET.ca, and Chinese National Funding 40776089, “Dongfang” and “Shuguang” funds from Shanghai Education Committee (B-9500-09-0003, 09SG48). References Arnold, J., 1993. Cytonuclear disequilibria in hybrid zones. Annu. Rev. Ecol. Evol. Syst. 24, 521–554. Astarloa, J.M.D., Mabragaña, E., Hanner, R., 2008. Morphological and molecular evidence for a new species of longnose skate (Rajiformes: Rajidae: Dipturus) from Argentinean waters based on DNA barcoding. Zootaxa 1921, 35–46. Avise, J.C., 2000. Phylogeography: The history and formation of species. Harvard University Press, Cambridge, MA. Balakrishnan, R., 2005. Species concepts, species boundaries and species identification: a view from the Tropics. Syst. Bio. 54, 689–693. Barraclough, T.G., Nee, S., 2001. Phylogenetics and speciation. Trends Ecol. Evol. 16, 391–399. 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