SpeciesIdentificationofMarineFishesinChina...
Transcript of SpeciesIdentificationofMarineFishesinChina...
Hindawi Publishing CorporationEvidence-Based Complementary and Alternative MedicineVolume 2011, Article ID 978253, 10 pagesdoi:10.1155/2011/978253
Research Article
Species Identification of Marine Fishes in Chinawith DNA Barcoding
Junbin Zhang
College of Fisheries and Life Science, Shanghai Ocean University, Shanghai 201306, China
Correspondence should be addressed to Junbin Zhang, [email protected]
Received 10 December 2010; Accepted 27 February 2011
Copyright © 2011 Junbin Zhang. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
DNA barcoding is a molecular method that uses a short standardized DNA sequence as a species identification tool. In this study,the standard 652 base-pair region of the mitochondrial cytochrome oxidase subunit I gene (COI) was sequenced in marine fishspecimens captured in China. The average genetic distance was 50-fold higher between species than within species, as Kimura twoparameter (K2P) genetic distances averaged 15.742% among congeners and only 0.319% for intraspecific individuals. There are nooverlaps of pairwise genetic variations between conspecific and interspecific comparisons apart from the genera Pampus in whichthe introgressive hybridization was detected. High efficiency of species identification was demonstrated in the present study byDNA barcoding. Due to the incidence of cryptic species, an assumed threshold is suggested to expedite discovering of new speciesand biodiversity, especially involving biotas of few studies.
1. Introduction
Fishes are important animal protein sources for humanbeings, and they are frequently used in complementary andalternative medicine/traditional medicine (CAM/TM). Thedelimitation and recognition of fish species is not only ofinterest for taxonomy and systematics, but also a requirementin management of fisheries, authentication of food products,and identification of CAM/TM materials [1–3].
Due to the complexity and limitations of morphologicalcharacters used in traditional taxonomy, several PCR-basedmethods of genotype analysis have been developed for theidentification of fish species, particularly for eggs, larvae, andcommercial products. Sequence analysis of species-specificDNA fragments (often mitochondrial or ribosomal genes)and multiplex PCR of species-conserved DNA fragmentsare efficient for fish species identification [4–10]. However,these molecular methods are limited to particular knownspecies and are not easily applicable to a wide range oftaxa. Therefore, Hebert et al. advocated using a standardDNA sequence that is DNA barcoding to identify speciesand uncover biological diversity [11, 12]. For many animaltaxa, sequence divergences within the 5′ region of themitochondrial cytochrome oxidase subunit I (COI) genewere much greater between species than within them, andthis in turn suggests that the approach is widely applicable
across phylogenetically distant animal groups [12, 13]. Todate, some published papers explicitly address that COI bar-codes effectively discriminate different species for a varietyof organisms [14–23]. However, several scientists expressconcerns that species identification based on variations ofsingle mitochondrial gene fragment may remain incorrector ambiguous assignments, particularly in cases of possiblemitochondrial polyphyly or paraphyly [24, 25]. In thecurrent study, we test the efficacy of DNA barcoding inmarine fishes of China. The sea area of China is part of theIndo-West Pacific Ocean, which is regarded as the centerof the world’s marine biodiversity [26]. Highly species-richbiotas are particularly attractive to test the reliability andefficiency of DNA barcoding.
2. Material and Methods
The majority of fish specimens were captured with the drawlnet at 20 localities along the coast of China (collectioninformation available at http://www.barcodinglife.org/). Atotal of 329 specimens from one hundred species of fishwere collected. Vouchers were deposited in the South ChinaSea Institute of Oceanography, Chinese Academy of Sciences,and all specimens were preserved in 70% ethanol. Tissuesamples were dissected from the dorsal muscle, and genomicDNA was extracted according to the standard Barcode of Life
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protocol [27]. Firstly, fragments of the 5′ region of the mito-chondrial COI gene were PCR-amplified using C FishF1t1/C FishR1t1 primer cocktails [28]. The cocktail C FishF1t1contained two primers (FishF2 t1/VF2 t1), and C FishR1t1also contained two primers (FishR2 t1/ FR1d t1). All PCRprimers were tailed with M13 sequences to facilitate sequenc-ing of products. The nucleotide sequences of the primerswere
FishF2 t1: ∗5′-TGTAAAACGACGGCCAGTCGACT-AATCATAAAGATATCGGCAC-3′.
VF2 t1: ∗5′-TGTAAAACGACGGCCAGTCAACCA-ACCACAAAGACATTGGCAC-3′.
FishR2 t1: ∗∗5′-CAGGAAACAGCTATGACACTTC-AGGGTGACCGAAGAATCAGAA-3′.
FR1d t1: ∗∗5′-CAGGAAACAGCTATGACACCTCA-GGGTGTCCGAARAAYCARAA-3′.∗The M13F primer sequence is underlined; ∗∗theM13R primer sequence is underlined.
PCR reactions were carried out in 96-well plates usingMastercycler Eppendorf gradient thermal cyclers (Brink-mann Instruments, Inc.). The reaction mixture of 825 µlwater, 125 µl 10× buffer, 62.5 µl MgCl2 (25 mM), 6.25 µldNTP (10 mM), 6.25 µl each primer (0.01 mM), and 6.25 µlTaq DNA polymerase (5 U/µl) was prepared for 96 wells ofeach plate, in which each well contained 10.5 µl mixture and2 µl genomic DNA. Thermocycling comprised an initial stepof 2 min at 95◦C and 35 cycles of 30 sec at 94◦C, 40 sec at52◦C, and 1 min at 72◦C, with a final extension at 72◦C for10 min. Amplicons were visualized on 2% agarose E-Gel 96-well system (Invitrogen). PCR products were amplified againwith the primers M13F (5′-TGTAAAACGACGGCCAGT-3′)and M13R (5′-CAGGAAACAGCTATGAC-3′), respectively,using the BigDye Terminator v.3.1 Cycle Sequencing Kit(Applied Biosystems, Inc.). Thermocycling conditions wereas follows: an initial step of 2 min at 96◦C and 35 cycles of30 sec at 96◦C, 15 sec at 55◦C, and 4 min at 60◦C. Sequencingwas performed on an ABI 3730 capillary sequencer accordingto manufacturer’s instructions.
For specimens that failed to yield sequences using theprimer combinations above, a second round of PCR usingthe alternative C VF1LFt1/ C VR1LRt1 primer combinationwas carried out. C VF1LFt1 consisted of four primers (VF1t1/VF1d t1/LepF1 t1/VFli t1), and C VR1LRt1 also compri-sed four primers (VR1 t1/VR1d t1/LepR1 t1/VRli t1) [28].
VF1 t1: ∗5′-TGTAAAACGACGGCCAGTTCTCAA-CCAACCACAAAGACATTGG-3′.
VF1d t1: ∗5′-TGTAAAACGACGGCCAGTTCTCA-ACCAACCACAARGAYATYGG-3′.
LepF1 t1: ∗5′-TGTAAAACGACGGCCAGTATTCA-ACCAATCATAAAGATATTGG-3′.
VFli t1: ∗5′-TGTAAAACGACGGCCAGTTCTCAA-CCAACCAIAAIGAIATIGG-3′.
VR1 t1: ∗∗5′-CAGGAAACAGCTATGACTAGACT-
TCTGGGTGGCCRAARAAYCA-3′.
VR1d t1: ∗∗5′-CAGGAAACAGCTATGACTAGACT-TCTGGGTGGCCAAAGAATCA-3′.
LepR1 t1: ∗∗5′-CAGGAAACAGCTATGACTAAAC-TTCTGGATGTCCAAAAAATCA-3′.
VRli t1: ∗∗5′-CAGGAAACAGCTATGACTAGACT-TCTGGGTGICCIAAIAAICA-3′.∗The M13F primer sequence is underlined; ∗∗theM13R primer sequence is underlined.
The thermocycling protocol used was 1 min at 95◦C and35 cycles of 30 sec at 94◦C, 40 sec at 50◦C, and 1 min at 72◦C,with a final extension at 72◦C for 10 min. Sequecing PCR andsequencing followed above procedure.
DNA sequences were aligned with SEQSCAPE v.2.5software (Applied Biosystems, Inc.). Sequence divergenceswere calculated using the Kimura two parameter (K2P)distance model [29], and unrooted NJ trees based on K2Pdistances were created in MEGA software [30]. In the chosentaxonomic group, phylogenetic analysis was carried out inPAUP 4.010b using the maximum parsimony (MP) method,with 1,000 replications of the full heuristic search.
The following categories of K2P distances were calcu-lated: intraspecific distances (S), interspecies within the con-gener (G), and interspecies from different genus but withinintrafamily (F). These values were plotted using the boxplotrepresentation of R. Boxplots [31] in SPSS 11.5 software(SPSS Inc., Chicago, IL, USA). Only for families containing2 or more genera, separate boxplot was constructed for thesake of comparisons among taxonomic categories. Boxplotsdescribe median (central bar), interquartile range (IQR:between upper (Q3) and low (Q1) quartile), values lyingwithin 1.5× IQR beneath Q1 or 1.5× above Q3 (“whiskers”),and extreme values (outliers). Mann-Whitney tests wereperformed between S, G, and F distributions to estimate theoverlap among taxonomic ranks.
3. Results
A total of 329 specimens were analyzed, from which 321sequences (all >500 bp) belonging to 121 species (anotherspecies was identified to the genus level) were ultimately obt-ained (GenBank accession numbers: EF607296-EF607616).These species cover the majority of fishes living in the coast-line of the South China Sea. All sequences were aligned witha consensus length of 652 bp, and no insertions, deletions,or stop codons were observed in any sequence. However,multiple haplotypes were detected for some species.
Except for Acentrogobius caninus, Scomber japonicus,Terapon jarbua, Upeneus sulphureus, Elops hawaiensis, Gym-nothorax pseudothyrsoideus, Dendrophysa russelii, and Pen-nahia anea (which reached the maximum value of 2.02%),intraspecific genetic distances were generally below 1%, andsome decreased to zero (between some intraspecific individ-uals of Thryssa setirostris, Parapercis ommatura, Scatophagusargus, etc.).
The mean intraspecies K2P (Kimura two-parameter)distance was 0.319%; the distance increased sharply to15.742% among individuals of congeneric species. Overall,
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Table 1: Genetic divergences (percentage, K2P distance) within various taxonomic levels. Data are based on 321 sequences (>500 bp) from122 species.
Comparisons within Taxa Number of comparisons Mean Median Minimum Maximum s.e.#
Species 121 453 0.319 0.150 0 2.021∗ 0.018
Genus 85 397 15.742 16.490 0.154∗∗ 25.189 0.292
Family 55 848 20.199 19.850 11.532 34.333 0.134
Order 15 17881 24.656 — 12.923 39.627 0.024
Class 2 29262 25.225 — 15.730 40.800 0.016∗
Pennahia anea; ∗∗ Pampus argenteus versus Pampus cinereus.#Standard error.
p.aneaP.argenteus versus P.cinereus
S G F−10
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%)
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Figure 1: Box plots of K2P distances. IQR: interval into whichthe “central” 50% of the data fall. Black bar in the box indicatesthe median. Circle: “mild outlier” and asterisks: “extreme outliers”.Extreme outliers are discussed in the text.
the average genetic distance among congeneric species isnearly 50-fold higher than that among individuals withinspecies. For the higher taxonomic ranks (family, order, andclass), mean pairwise genetic distances increased graduallyand reached 20.199%, 24.656%, and 25.225%, respectively(Table 1). Standard errors for K2P genetic distances weresmall, and values of the mean and median were closewithin different taxonomic ranks (Table 1). This indicatesfluctuations of K2P genetic distances tend to be convergent(Figures 1 and 2).
In the unrooted NJ (neighbour-joining) tree (Figure 3),three specimens of Pampus argentenus were grouped togetherand contained within the cluster of Pampus cinereus. ThesePampus argentenus specimens were collected in the same siteoff the west coast of the South China Sea, and were difficultto identify because of their complex morphological char-acteristics (available at http://www.barcodinglife.org/). Theypossessed combined characteristics of Pampus cinereus andPampus argentenus: the asymmetrical tail of Pampus cinereusand silver color of Pampus argentenus. If the suspiciouscongeneric K2P distances in the genera Pampus are excluded(the extreme outliers in Figure 1), the pairwise genetic
S G F S G F S G F S G F S G F S G F S G F S G F S G F S G F−10
k2P
gen
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%)
0
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40 Mu
raenesocidae
Mon
acanth
idae
Cyn
oglossidae
Synodon
tidae
Mu
llidae
Leiognath
idae
Scombrbidae
Caran
gidae
En
graulidae
Clu
peidae
Figure 2: Boxplot distributions of S, G, and F. Intra-species (S),interspecies among congeneric species (G), and intergenera butintrafamily (F) K2P distances for different families.
divergences among congeneric species are above 10%. Thereare no overlaps between intraspecific and congeneric K2Pdistances within the same family (Figure 3).
At the species level, all COI sequences clustered inmonophyletic species units. At the family level, there wereparaphyletic clusters for three families (Carangidae, Gobi-idae, and Ariidae) (Figure 3), though over 98% of specimensfell into the expected division of families. Intrafamily K2Pdistances (F) were generally higher than congeneric (G)distances, which were definitely higher than intraspecific(S) distances (Table 1, all Mann-Whitney tests were highlysignificant, P value <10−6). However, overlaps between Fand G distances were observed in Clupeidae, Carangidae,Mullidae, and Muraenesocidae.
4. Discussion
In morphological taxonomy, characters are delimited usuallywithout any explicit criteria for character selection or coding,and morphological data sets have the potential to be quitearbitrary. For example, morphologists do not generallyreport their criteria for including or excluding characters,
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Harpadon nehereus|FSCS326 06|FSCS 113 st 1|SynodontidaeFistularia commersonii|FSCS214-06|FSCS 81-l-1|Fistulariidae
Fistularia commersonii|FSCS215-06|FSCS 81-l-2|FistulariidaeArnoglossus polyspilus|FSCS116-06|FSCS 52-w-1|BothidaeArnoglossus polyspilus|FSCS117-06|FSCS 52-w-2|BothidaeArnoglossus polyspilus|FSCS118-06|FSCS 52-w-3|Bothidae
Chirocentrus nudus|FSCS175-06|FSCS 26-l-1|ChirocentridaeChirocentrus nudus|FSCS058-06|FSCS 26-W-3|ChirocentridaeChirocentrus nudus|FSCS057-06|FSCS 26-W-2|ChirocentridaeChirocentrus nudus|FSCS056-06|FSCS 26-W-1|ChirocentridaeChirocentrus dorab|FSCS176-06|FSCS 26-l-2|Chirocentridae
Thryssa setirostris|FSCS158-06|FSCS 8-l-1|EngraulidaeThryssa setirostris|FSCS014-06|FSCS 8-w-1|EngraulidaeThryssa setirostris|FSCS018-06|FSCS 9-w-4|EngraulidaeThryssa kammalensis|FSCS159-06|FSCS 9-l-1|EngraulidaeThryssa kammalensis|FSCS160-06|FSCS 9-l-2|EngraulidaeThryssa kammalensis|FSCS240-06|FSCS 9-yz-1|EngraulidaeThryssa kammalensis|FSCS017-06|FSCS 9-w-3|EngraulidaeThryssa kammalensis|FSCS015-06|FSCS 9-w-1|EngraulidaeThryssa kammalensis|FSCS241-06|FSCS 9-yz-2|EngraulidaeThryssa kammalensis|FSCS161-06|FSCS 9-l-3|Engraulidae
Thryssa hamiltonii|FSCS016-06|FSCS 9-w-2|EngraulidaeThryssa hamiltonii|FSCS019-06|FSCS 9-w-5|Engraulidae
Engraulis japonicus|FSCS263-06|FSCS 17-z-1|EngraulidaeEngraulis japonicus|FSCS266-06|FSCS 17-z-4|EngraulidaeEngraulis japonicus|FSCS267-06|FSCS 17-z-5|Engraulidae
Anchoviella sp.|FSCS265-06|FSCS 17-z-3|EngraulidaeDussumieria elopsoides|FSCS262-06|FSCS 16-z-1|ClupeidaeDussumieria elopsoides|FSCS035-06|FSCS 16-W-1|ClupeidaeDussumieria elopsoides|FSCS036-06|FSCS 16-W-2|Clupeidae
Sardinella sp.|FSCS287-06|FSCS 94-z-2|ClupeidaeSardinella melanura|FSCS168-06|FSCS 18-l-1|ClupeidaeSardinella melanura|FSCS268-06|FSCS 18-z-1|ClupeidaeSardinella melanura|FSCS269-06|FSCS 18-z-2|ClupeidaeSardinella melanura|FSCS169-06|FSCS 18-l-2|ClupeidaeSardinella melanura|FSCS235-06|FSCS 107-l-1|Clupeidae
Sardinella jussieu|FSCS271-06|FSCS 18-z-4|ClupeidaeSardinella jussieu|FSCS272-06|FSCS 18-z-5|ClupeidaeAmblygaster clupeoides|FSCS286-06|FSCS 94-z-1|Clupeidae
Sardinella aurita|FSCS327-06|FSCS 114-st-1|ClupeidaeSardinella aurita|FSCS328-06|FSCS 114-st-2|ClupeidaeSardinella aurita|FSCS329-06|FSCS 114-st-3|Clupeidae
Upeneus tragula|FSCS279-06|FSCS 78-z-1|MullidaeUpeneus tragula|FSCS207-06|FSCS 78-l-1|MullidaeUpeneus tragula|FSCS208-06|FSCS 78-l-2|MullidaeUpeneus tragula|FSCS209-06|FSCS 78-l-3|MullidaeUpeneus subvittatus|FSCS130-06|FSCS 56-w-5|Mullidae
Upeneus japonicus|FSCS126-06|FSCS 56-w-1|MullidaeUpeneus japonicus|FSCS128-06|FSCS 56-w-3|MullidaeUpeneus japonicus|FSCS127-06|FSCS 56-w-2|MullidaeUpeneus japonicus|FSCS129-06|FSCS 56-w-4|MullidaeUpeneus japonicus|FSCS245-06|FSCS 56-yz-1|Mullidae
Upeneus sulphureus|FSCS171-06|FSCS 22-l-1|MullidaeUpeneus sulphureus|FSCS045-06|FSCS 22-W-1|MullidaeUpeneus sulphureus|FSCS046-06|FSCS 22-W-2|Mullidae
Parupeneus ciliatus|FSCS210-06|FSCS 79-l-1|MullidaeParupeneus ciliatus|FSCS211-06|FSCS 79-l-2|MullidaeTrichiurus lepturus|FSCS155-06|FSCS 3-l-1|TrichiuridaeTrichiurus lepturus|FSCS156-06|FSCS 3-l-2|Trichiuridae
Lepturacanthus savala|FSCS290-06|FSCS 3-b-1|TrichiuridaeLepturacanthus savala|FSCS007-06|FSCS 3-w-1|Trichiuridae
Scomber sp.|FSCS093-06|FSCS 38-W-5|ScombridaeScomber japonicus|FSCS092-06|FSCS 38-W-4|ScombridaeScomber japonicus|FSCS181-06|FSCS 38-l-2|ScombridaeScomber japonicus|FSCS089-06|FSCS 38-W-1|ScombridaeScomber japonicus|FSCS309-06|FSCS 38-sz-1|ScombridaeScomber japonicus|FSCS091-06|FSCS 38-W-3|ScombridaeScomber japonicus|FSCS180-06|FSCS 38-l-1|ScombridaeScomber japonicus|FSCS090-06|FSCS 38-W-2|Scombridae
Scomberomorus guttatus|FSCS249-06|FSCS 86-yz-2|ScombridaeScomberomorus guttatus|FSCS298-06|FSCS 86-b-1|ScombridaeScomberomorus guttatus|FSCS228-06|FSCS 86-l-2|ScombridaeScomberomorus commerson|FSCS248-06|FSCS 86-yz-1|Scombridae
Johnius belangerii|FSCS200-06|FSCS 76-l-1|SciaenidaeJohnius belangerii|FSCS201-06|FSCS 76-l-2|SciaenidaeJohnius belangerii|FSCS297-06|FSCS 76-b-1|Sciaenidae
Dendrophysa russelii|FSCS068-06|FSCS 29-W-1|SciaenidaeDendrophysa russelii|FSCS072-06|FSCS 29-W-5|SciaenidaeDendrophysa russelii|FSCS069-06|FSCS 29-W-2|SciaenidaeDendrophysa russelii|FSCS070-06|FSCS 29-W-3|SciaenidaeDendrophysa russelii|FSCS071-06|FSCS 29-W-4|Sciaenidae
5%
(a)
Figure 3: Continued.
Evidence-Based Complementary and Alternative Medicine 5
Terapon jarbua|FSCS121-06|FSCS 54-w-2|TerapontidaeTerapon jarbua|FSCS123-06|FSCS 54-w-4|Terapontidae
Terapon jarbua|FSCS244-06|FSCS 54-yz-1|TerapontidaeGerres oblongus|FSCS223-06|FSCS 84-l-2|Gerreidae
Gerres limbatus|FSCS232-06|FSCS 92-l-2|GerreidaeGerres limbatus|FSCS231-06|FSCS 92-l-1|GerreidaeGerres limbatus|FSCS282-06|FSCS 92-z-1|GerreidaeGerres limbatus|FSCS283-06|FSCS 92-z-2|Gerreidae
Gerres limbatus|FSCS250-06|FSCS 92-yz-1|GerreidaeThamnaconus tessellatus|FSCS185-06|FSCS 46-l-1|Monacanthidae
Thamnaconus tessellatus|FSCS278-06|FSCS 46-z-1|MonacanthidaeParamonacanthus sulcatus|FSCS101-06|FSCS 45-w-1|MonacanthidaeParamonacanthus sulcatus|FSCS102-06|FSCS 45-w-2|MonacanthidaeParamonacanthus sulcatus|FSCS183-06|FSCS 45-l-1|Monacanthidae
Paramonacanthus sulcatus|FSCS103-06|FSCS 45-w-3|MonacanthidaeParamonacanthus sulcatus|FSCS105-06|FSCS 46-w-2|MonacanthidaeParamonacanthus sulcatus|FSCS106-06|FSCS 46-w-3|MonacanthidaeParamonacanthus sulcatus|FSCS243-06|FSCS 45-yz-1|MonacanthidaeParamonacanthus sulcatus|FSCS184-06|FSCS 45-l-2|MonacanthidaeParamonacanthus sulcatus|FSCS104-06|FSCS 46-w-1|MonacanthidaeThamnaconus septentrionalis|FSCS107-06|FSCS 47-w-1|MonacanthidaeThamnaconus septentrionalis|FSCS108-06|FSCS 47-w-2|MonacanthidaeThamnaconus septentrionalis|FSCS186-06|FSCS 47-l-1|Monacanthidae
Thalassoma lunare|FSCS198-06|FSCS 72-l-1|LabridaeScomberoides tol|FSCS063-06|FSCS 28-W-1|Carangidae
Scomberoides tol|FSCS064-06|FSCS 28-W-2|CarangidaeScomberoides tol|FSCS065-06|FSCS 28-W-3|CarangidaeScomberoides tol|FSCS067-06|FSCS 28-W-5|CarangidaeScomberoides tol|FSCS066-06|FSCS 28-W-4|CarangidaeDrepane punctata|FSCS239-06|FSCS 116-l-1|Drepaneidae
Sillago sihama|FSCS196-06|FSCS 70-l-1|SillaginidaeSillago sihama|FSCS197-06|FSCS 70-l-2|SillaginidaeSillago sihama|FSCS246-06|FSCS 70-yz-1|SillaginidaeSillago sihama|FSCS247-06|FSCS 70-yz-2|Sillaginidae
Sillago maculata|FSCS081-06|FSCS 34-W-1|SillaginidaeSelaroides leptolepis|FSCS087-06|FSCS 36-W-5|CarangidaeSelaroides leptolepis|FSCS083-06|FSCS 36-W-1|CarangidaeSelaroides leptolepis|FSCS084-06|FSCS 36-W-2|CarangidaeSelaroides leptolepis|FSCS085-06|FSCS 36-W-3|CarangidaeSelaroides leptolepis|FSCS086-06|FSCS 36-W-4|CarangidaeSelaroides leptolepis|FSCS179-06|FSCS 36-l-1|CarangidaeCarangoides malabaricus|FSCS012-06|FSCS 7-w-1|CarangidaeCarangoides malabaricus|FSCS013-06|FSCS 7-w-2|CarangidaeCarangoides hedlandensis|FSCS096-06|FSCS 41-w-1|CarangidaeCarangoides hedlandensis|FSCS097-06|FSCS 41-w-2|Carangidae
Atule mate|FSCS194-06|FSCS 69-l-1|CarangidaeAtule mate|FSCS195-06|FSCS 69-l-2|CarangidaeAtule mate|FSCS296-06|FSCS 69-b-1|Carangidae
Atule sp.|FSCS257-06|FSCS 111-yz-1|CarangidaeAlepes djedaba|FSCS088-06|FSCS 37-W-1|Carangidae
Alepes djedaba|FSCS261-06|FSCS 15-z-2|CarangidaeAlepes djedaba|FSCS292-06|FSCS 15-b-1|CarangidaeAlepes djedaba|FSCS167-06|FSCS 15-l-2|CarangidaeAlepes djedaba|FSCS166-06|FSCS 15-l-1|CarangidaeAlepes djedaba|FSCS260-06|FSCS 15-z-1|Carangidae
Rachycentron canadum|FSCS095-06|FSCS 40-W-1|RachycentridaeLactarius lactarius|FSCS157-06|FSCS 6-l-1|RussulaceaeLactarius lactarius|FSCS010-06|FSCS 6-w-1|RussulaceaeLactarius lactarius|FSCS011-06|FSCS 6-w-2|Russulaceae
Cynoglossus sp.|FSCS111-06|FSCS 49-w-2|CynoglossidaeCynoglossus puncticeps|FSCS110-06|FSCS 49-w-1|CynoglossidaeCynoglossus puncticeps|FSCS112-06|FSCS 49-w-3|Cynoglossidae
Cynoglossus bilineatus|FSCS114-06|FSCS 51-w-1|CynoglossidaeCynoglossus bilineatus|FSCS125-06|FSCS 55-w-2|CynoglossidaeCynoglossus bilineatus|FSCS115-06|FSCS 51-w-2|Cynoglossidae
Polydactylus sextarius|FSCS305-06|FSCS 100-b-1|PolynemidaeParaplagusia japonica|FSCS113-06|FSCS 50-w-1|Cynoglossidae
Brachirus orientalis|FSCS009-06|FSCS 5-w-1|SoleidaeMene maculata|FSCS001-06|FSCS 1-w-1|MenidaeMene maculata|FSCS002-06|FSCS 1-w-2|MenidaeMene maculata|FSCS003-06|FSCS 1-w-3|MenidaeMene maculata|FSCS004-06|FSCS 1-w-4|Menidae
Pardachirus pavoninus|FSCS253-06|FSCS 103-yz-1|SoleidaePardachirus pavoninus|FSCS255-06|FSCS 105-yz-1|Soleidae
Epinephelus sexfasciatus|FSCS098-06|FSCS 42-w-1|SerranidaeZebrias quagga|FSCS109-06|FSCS 48-w-1|Soleidae
Lagocephalus spadiceus|FSCS302-06|FSCS 98-b-1|TetraodontidaeSaurida sp.|FSCS277-06|FSCS 44-z-1|Synodontidae
Saurida elongata|FSCS100-06|FSCS 44-w-1|SynodontidaeHarpadon nehereus|FSCS307-06|FSCS 113-b-1|SynodontidaeHarpadon nehereus|FSCS326-06|FSCS 113-st-1|Synodontidae
Fistularia commersonii|FSCS214-06|FSCS 81-l-1|FistulariidaeFistularia commersonii|FSCS215-06|FSCS 81-l-2|Fistulariidae
(b)
Figure 3: Continued.
6 Evidence-Based Complementary and Alternative Medicine
Oxyconger leptognathus|FSCS145-06|FSCS 63-w-2|MuraenesocidaeOxyconger leptognathus|FSCS146-06|FSCS 63-w-3|Muraenesocidae
Muraenesox cinereus|FSCS234-06|FSCS 95-l-2|MuraenesocidaeMuraenesox cinereus|FSCS288-06|FSCS 95-z-1|Muraenesocidae
Elops hawaiensis|FSCS062-06|FSCS 27-W-4|ElopidaeElops hawaiensis|FSCS059-06|FSCS 27-W-1|ElopidaeElops hawaiensis|FSCS060-06|FSCS 27-W-2|ElopidaeElops hawaiensis|FSCS061-06|FSCS 27-W-3|Elopidae
Apogon taeniatus|FSCS191-06|FSCS 67-l-1|ApogonidaeApogon taeniatus|FSCS192-06|FSCS 67-l-2|Apogonidae
Apogon quadrifasciatus|FSCS149-06|FSCS 66-w-1|ApogonidaeApogon quadrifasciatus|FSCS150-06|FSCS 66-w-2|ApogonidaeApogon quadrifasciatus|FSCS295-06|FSCS 66-b-1|ApogonidaeApogon quadrifasciatus|FSCS256-06|FSCS 109-yz-1|ApogonidaeApogon fuscus|FSCS190-06|FSCS 66-l-1|ApogonidaeApogon erythrinus|FSCS193-06|FSCS 68-l-1|Apogonidae
Repomucenus richardsonii|FSCS136-06|FSCS 60-w-1|CallionymidaeRepomucenus richardsonii|FSCS137-06|FSCS 60-w-2|CallionymidaeRepomucenus richardsonii|FSCS139-06|FSCS 60-w-4|CallionymidaeRepomucenus richardsonii|FSCS138-06|FSCS 60-w-3|Callionymidae
Bathycallionymus kaianus|FSCS310-06|FSCS 60-sz-1|CallionymidaePriacanthus macracanthus|FSCS321-06|FSCS 110-st-1|PriacanthidaePriacanthus macracanthus|FSCS322-06|FSCS 110-st-2|Priacanthidae
Acentrogobius caninus|FSCS170-06|FSCS 19-l-1|GobiidaeAcentrogobius caninus|FSCS039-06|FSCS 19-W-3|GobiidaeAcentrogobius caninus|FSCS273-06|FSCS 19-z-1|GobiidaeAcentrogobius caninus|FSCS274-06|FSCS 19-z-2|GobiidaeAcentrogobius caninus|FSCS038-06|FSCS 19-W-2|Gobiidae
Acentrogobius caninus|FSCS037-06|FSCS 19-W-1|GobiidaeHypodytes indicus|FSCS131-06|FSCS 57-w-1|Congiopodidae
Parapercis ommatura|FSCS040-06|FSCS 20-W-1|PinguipedidaeParapercis ommatura|FSCS043-06|FSCS 20-W-4|PinguipedidaeParapercis ommatura|FSCS041-06|FSCS 20-W-2|PinguipedidaeParapercis ommatura|FSCS042-06|FSCS 20-W-3|Pinguipedidae
Glossogobius aureus|FSCS082-06|FSCS 35-W-1|GobiidaeSphyraena pinguis|FSCS099-06|FSCS 43-w-1|Sphyraenidae
Sphyraena helleri|FSCS182-06|FSCS 43-l-1|SphyraenidaeKumococius rodericensis|FSCS132-06|FSCS 58-w-1|PlatycephalidaeKumococius rodericensis|FSCS134-06|FSCS 58-w-3|Platycephalidae
Kumococius rodericensis|FSCS133-06|FSCS 58-w-2|PlatycephalidaeSeriola dumerili|FSCS218-06|FSCS 83-l-1|CarangidaeSeriola dumerili|FSCS221-06|FSCS 83-l-4|CarangidaeSeriola dumerili|FSCS219-06|FSCS 83-l-2|CarangidaeSeriola dumerili|FSCS220-06|FSCS 83-l-3|Carangidae
Lutjanus fulvus|FSCS229-06|FSCS 88-l-1|LutjanidaePomadasys hasta|FSCS174-06|FSCS 24-l-1|HaemulidaePomadasys hasta|FSCS051-06|FSCS 24-W-3|HaemulidaePomadasys hasta|FSCS049-06|FSCS 24-W-1|HaemulidaePomadasys hasta|FSCS050-06|FSCS 24-W-2|HaemulidaePomadasys hasta|FSCS052-06|FSCS 24-W-4|Haemulidae
Parapristipoma sp.|FSCS216-06|FSCS 82-l-1|HaemulidaeParapristipoma sp.|FSCS217-06|FSCS 82-l-2|Haemulidae
Hemiramphus far|FSCS177-06|FSCS 31-l-1|HemiramphidaeHemiramphus dussumieri|FSCS076-06|FSCS 31-W-1|HemiramphidaeHemiramphus dussumieri|FSCS077-06|FSCS 31-W-2|Hemiramphidae
Acanthopagrus schlegelii schlegelii|FSCS143-06|FSCS 62-w-1|SparidaeAcanthopagrus berda|FSCS078-06|FSCS 32-W-1|Sparidae
Acanthopagrus latus|FSCS178-06|FSCS 32-l-1|SparidaeAcanthopagrus latus|FSCS308-06|FSCS 32-sz-1|Sparidae
Hypoatherina valenciennei|FSCS151-06|FSCS 80-w-1|AtherinidaeHypoatherina valenciennei|FSCS152-06|FSCS 80-w-2|AtherinidaeHypoatherina valenciennei|FSCS212-06|FSCS 80-l-1|AtherinidaeHypoatherina valenciennei|FSCS213-06|FSCS 80-l-2|Atherinidae
Platycephalus indicus|FSCS135-06|FSCS 59-w-1|PlatycephalidaeGerres filamentosus|FSCS254-06|FSCS 104-yz-1|GerreidaeGerres filamentosus|FSCS312-06|FSCS 104-sz-1|GerreidaeGerres filamentosus|FSCS222-06|FSCS 84-l-1|Gerreidae
Valamugil cunnesius|FSCS251-06|FSCS 99-yz-1|MugilidaeMugil cephalus|FSCS303-06|FSCS 99-b-1|MugilidaeMugil cephalus|FSCS304-06|FSCS 99-b-2|Mugilidae
Siganus argenteus|FSCS311-06|FSCS 89-sz-1|SiganidaeSiganus argenteus|FSCS319-06|FSCS 89-st-1|SiganidaeSiganus argenteus|FSCS230-06|FSCS 89-l-1|SiganidaeSiganus argenteus|FSCS317-06|FSCS 13-st-1|Siganidae
Terapon theraps|FSCS318-06|FSCS 54-st-1|TerapontidaeTerapon jarbua|FSCS119-06|FSCS 53-w-1|TerapontidaeTerapon jarbua|FSCS122-06|FSCS 54-w-3|TerapontidaeTerapon jarbua|FSCS293-06|FSCS 54-b-1|TerapontidaeTerapon jarbua|FSCS294-06|FSCS 54-b-2|TerapontidaeTerapon jarbua|FSCS120-06|FSCS 54-w-1|TerapontidaeTerapon jarbua|FSCS121-06|FSCS 54-w-2|TerapontidaeTerapon jarbua|FSCS123-06|FSCS 54-w-4|Terapontidae
Terapon jarbua|FSCS244-06|FSCS 54-yz-1|Terapontidae
Oxyconger leptognathus|FSCS144-06|FSCS 62-w-1|Muraenesocidae
(c)
Figure 3: Continued.
Evidence-Based Complementary and Alternative Medicine 7
Pennahia anea|FSCS291-06|FSCS 11-b-1|SciaenidaePennahia anea|FSCS313-06|FSCS 11-st-1|Sciaenidae
Chrysochir aureus|FSCS148-06|FSCS 65-w-1|SciaenidaeOtolithes ruber|FSCS252-06|FSCS 101-yz-1|SciaenidaeOtolithes ruber|FSCS306-06|FSCS 101-b-1|Sciaenidae
Arius leiotetocephalus|FSCS284-06|FSCS 93-z-1|AriidaeArius leiotetocephalus|FSCS285-06|FSCS 93-z-2|Ariidae
Abudefduf septemfasciatus|FSCS199-06|FSCS 74-l-1|PomacentridaeTakifugu oblongus|FSCS094-06|FSCS 39-W-1|Tetradontidae
Arius thalassinus|FSCS202-06|FSCS 77-l-1|AriidaeArius thalassinus|FSCS205-06|FSCS 77-l-4|AriidaeArius thalassinus|FSCS206-06|FSCS 77-l-5|AriidaeArius thalassinus|FSCS204-06|FSCS 77-l-3|Ariidae
Scorpaenopsis vittapinna|FSCS224-06|FSCS 85-l-1|ScorpaenidaeScorpaenopsis vittapinna|FSCS226-06|FSCS 85-l-3|ScorpaenidaeScorpaenopsis vittapinna|FSCS225-06|FSCS 85-l-2|Scorpaenidae
Pampus cinereus|FSCS289-06|FSCS 112-z-1|StromateidaePampus cinereus|FSCS073-06|FSCS 30-W-1|StromateidaePampus argenteus|FSCS140-06|FSCS 61-w-1|StromateidaePampus argenteus|FSCS141-06|FSCS 61-w-2|StromateidaePampus argenteus|FSCS142-06|FSCS 61-w-3|Stromateidae
Pampus cinereus|FSCS075-06|FSCS 30-W-3|StromateidaePampus cinereus|FSCS325-06|FSCS 112-st-3|StromateidaePampus cinereus|FSCS324-06|FSCS 112-st-2|StromateidaePampus cinereus|FSCS323-06|FSCS 112-st-1|Stromateidae
Pampus argenteus|FSCS074-06|FSCS 30-W-2|StromateidaeGymnothorax pseudothyrsoideus|FSCS147-06|FSCS 64-w-1|Muraenidae
Gymnothorax pseudothyrsoideus|FSCS189-06|FSCS 64-l-2|MuraenidaeGymnothorax pseudothyrsoideus|FSCS188-06|FSCS 64-l-1|MuraenidaeFavonigobius gymnauchen|FSCS044-06|FSCS 21-W-1|Gobiidae
Etmopterus lucifer|FSCS162-06|FSCS 9-l-4|SqualidaeEtmopterus lucifer|FSCS238-06|FSCS 115-l-3|SqualidaeEtmopterus lucifer|FSCS237-06|FSCS 115-l-2|SqualidaeEtmopterus lucifer|FSCS236-06|FSCS 115-l-1|Squalidae
Dasyatis zugei|FSCS008-06|FSCS 4-w-1|DasyatididaeStrongylura strongylura|FSCS153-06|FSCS 2-l-1|BelonidaeStrongylura strongylura|FSCS154-06|FSCS 2-l-2|BelonidaeStrongylura strongylura|FSCS187-06|FSCS 59-l-1|BelonidaeStrongylura leiura|FSCS005-06|FSCS 2-w-1|BelonidaeStrongylura leiura|FSCS006-06|FSCS 2-w-2|Belonidae
Strongylura sp.|FSCS300-06|FSCS 96-b-2|BelonidaeScatophagus argus|FSCS299-06|FSCS 96-b-1|ScatophagidaeScatophagus argus|FSCS053-06|FSCS 25-W-1|ScatophagidaeScatophagus argus|FSCS054-06|FSCS 25-W-2|ScatophagidaeScatophagus argus|FSCS055-06|FSCS 25-W-3|Scatophagidae
Escualosa thoracata|FSCS258-06|FSCS 10-z-1|ClupeidaeEscualosa thoracata|FSCS259-06|FSCS 10-z-2|ClupeidaeEscualosa thoracata|FSCS020-06|FSCS 10-w-1|Clupeidae
Escualosa thoracata|FSCS024-06|FSCS 10-w-5|ClupeidaeEscualosa thoracata|FSCS021-06|FSCS 10-w-2|ClupeidaeEscualosa thoracata|FSCS022-06|FSCS 10-w-3|Clupeidae
Secutor ruconius|FSCS314-06|FSCS 12-st-1|LeiognathidaeSecutor ruconius|FSCS026-06|FSCS 12-W-1|LeiognathidaeSecutor ruconius|FSCS027-06|FSCS 12-W-2|Leiognathidae
Secutor insidiator|FSCS315-06|FSCS 12-st-2|LeiognathidaeSecutor insidiator|FSCS316-06|FSCS 12-st-3|Leiognathidae
Leiognathus nuchalis|FSCS275-06|FSCS 23-z-1|LeiognathidaeLeiognathus nuchalis|FSCS276-06|FSCS 23-z-2|Leiognathidae
Leiognathus bindus|FSCS172-06|FSCS 23-l-1|LeiognathidaeLeiognathus bindus|FSCS173-06|FSCS 23-l-2|LeiognathidaeLeiognathus bindus|FSCS242-06|FSCS 23-yz-1|Leiognathidae
Leiognathus bindus|FSCS031-06|FSCS 14-W-1|LeiognathidaeLeiognathus bindus|FSCS034-06|FSCS 14-W-4|Leiognathidae
Leiognathus bindus|FSCS033-06|FSCS 14-W-3|LeiognathidaeLeiognathus bindus|FSCS032-06|FSCS 14-W-2|Leiognathidae
Leiognathus leuciscus|FSCS163-06|FSCS 13-l-1|LeiognathidaeLeiognathus leuciscus|FSCS164-06|FSCS 13-l-2|LeiognathidaeLeiognathus leuciscus|FSCS165-06|FSCS 13-l-3|LeiognathidaeLeiognathus leuciscus|FSCS029-06|FSCS 13-W-2|LeiognathidaeLeiognathus leuciscus|FSCS030-06|FSCS 13-W-3|Leiognathidae
Leiognathus leuciscus|FSCS028-06|FSCS 13-W-1|LeiognathidaeLeiognathus brevirostris|FSCS047-06|FSCS 23-W-1|LeiognathidaeLeiognathus brevirostris|FSCS048-06|FSCS 23-W-2|Leiognathidae
Lethrinus lentjan|FSCS280-06|FSCS 91-z-1|LethrinidaeLethrinus lentjan|FSCS281-06|FSCS 91-z-2|Lethrinidae
Hirundichthys rondeletii|FSCS079-06|FSCS 33-W-1|ExocoetidaeHirundichthys rondeletii|FSCS080-06|FSCS 33-W-2|Exocoetidae
Oxyconger sp.|FSCS233-06|FSCS 95-l-1|MuraenesocidaeOxyconger sp.|FSCS320-06|FSCS 95-st-1|Muraenesocidae
Oxyconger leptognathus|FSCS144-06|FSCS 63-w-1|MuraenesocidaeOxyconger leptognathus|FSCS145-06|FSCS 63-w-2|MuraenesocidaeOxyconger leptognathus|FSCS146-06|FSCS 63-w-3|Muraenesocidae
(d)
Figure 3: Neighbor-joining (NJ) tree of COI sequences. Scale: 5% K2P distance. The first numbers following species names are the processIDs, and the latter are the sample IDs.
8 Evidence-Based Complementary and Alternative Medicine
and when criteria are given, they vary considerably amongstudies [32]. Thus, it is not surprising that there are so manysynonyms for organisms [33], and an objective, rigorousspecies delimitation according to explicit criteria is thereforenecessary for many taxonomic studies [34]. While DNAbarcoding provides taxonomic identification for a specimen,the accuracy of such an assignment depends on whetherspecies are monophyletic with respect to sequence variationsof the COI gene. That is, individuals of a given speciesare more closely related to all other conspecifics than toany member of other species. Except for the hybridizedspecimens in the genus Pampus, there are no overlapsbetween genetic variations of S and G (Figure 1).
The factors responsible for deviations from taxonomicmonophyly may be varied and complex [35]; one potentialcause of species-level polyphyly is the occasional mat-ing between distinct species, resulting in hybrid offspringcarrying a mixture of genes from both parent species.Furthermore, mitochondrial genes are generally subjectedto introgression more frequently than nuclear ones, andintrogression also leads to phylogenetic paraphyly [35–38], like the hybridization between Pampus argentenus andPampus cinereus in this study. In such cases, combinationsof morphological and genotypic data are needed for speciesassignment of hybrids.
Biological mechanisms, water dynamics, or historicalevents may cause deep genetic structuring of populationsin marine species [26, 39]. Many explanations for geneticpopulation structuring on local and regional scales involvebehaviors such as the adoption of pelagic early life stagesand movement over broad geographic ranges, and thesefactors are theoretically associated with gene flow [40–42].For many marine fishes, there is a lack of phylogeographicstructure among populations [43, 44]; in this study, forindividuals from long distance localities, some intraspecificgenetic variations reduced to zero within families Carangi-dae, Sciaenidae, and Mullidae. However, some pairwiseK2P distances exceeded 1.00% within the coastal speciessuch as Acentrogobius caninus, Scomber japonicus, Teraponjarbua, Upeneus sulphureus, Elops hawaiensis, Gymnothoraxpseudothyrsoideus, and Dendrophysa russelii. It implied thatbiological mechanisms were responsible for the fluctuationof intraspecific genetic divergences in marine fishes.
The neighbor-joining method was originally employed inthis study for species identification, but some phylogeneticinformation was also revealed by the dendrogram, and over98% of specimens were allocated into different families with-out polyphyly/paraphyly in the NJ tree (Figure 3). However,DNA barcoding is independent of the way the taxonomy hasbeen built, and it cannot be regarded as the “taxonomic” tag[45]. DNA barcoding is no substitute for taxonomy Ebachand Holdrege [46], and a great deal of work is neededto bring about the reconciliation between traditional andmolecular taxonomy. It is unfeasible to build the phylogenyof fishes only based on mitochondrial DNA fragments.Polyphyly/paraphyly in the NJ tree probably results from“bad taxonomy” when named species fail to identify thegenetic limits of separate evolutionary entities, particularlyfor perplexing taxa involving cryptic species [47]. If we
cannot set a threshold of the genetic variation in speciesdelimitation, we find ourselves sunk in the dilemma facingnew or cryptic species. On the one hand, the morphologicaltaxonomy cannot give a definite identification. On theother hand, we cannot claim that it may be a new speciesbased on molecular analysis without the species delimitation.An assumed threshold is helpful to expedite discovery ofnew species and biodiversity, especially in dealing withlittle-studied biotas, although a single, uniform thresholdfor species delimitation seems arbitrary because rates ofmolecular evolution vary widely within and among lineages[24, 25, 48].
Acknowledgments
Thanks are due to Chinese National Funding U0633007 and40776089 and CAS Founding KZCX2-YW-213.
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