Chinese Journal of Oceanology and LimnologyVol. 29 No. 5, P. 967-972, 2011DOI: 10.1007/s00343-011-0040-8

Genetic identification of two species of Pleuronichthys by DNA barcoding*

ZHANG Hui (张辉)1, ZHANG Yan (张岩)2, GAO Tianxiang (高天翔)1, **, LI Pengfei (李鹏飞)3, XU Hanxiang (徐汉祥)3

1 Fisheries College, Ocean University of China, Qingdao 266003, China2 Yellow Sea Fisheries Research Institute, Chinese Academy of Fishery Sciences, Qingdao 266071, China3 Marine Fisheries Research Institute of Zhejiang Province, Zhoushan 316100, China

Received Sep. 24, 2010; revision accepted Nov. 1, 2010

© Chinese Society for Oceanology and Limnology, Science Press, and Springer-Verlag Berlin Heidelberg 2011

Abstract DNA barcoding is a new method for biological taxonomy, offering the ability to identify species from fragments in any life-history stage. Pleuronichthys cornutus and P. japonicus are two morphologically similar species. Pleuronichthys japonicus has never been found previously in China. However, in this study, we identified both species using DNA barcoding (cytochrome c oxidase subunit I (COI)), the mtDNA control region and cytochrome b. The results reveal that: 1) intraspecific variation in the DNA barcode is much less than interspecific variation; 2) the two morphologically similar species were placed into separate clades distinguishable by high bootstrap values; 3) COI barcodes are more powerful for identifying the two species than the other two mtDNA fragments.

Keyword: Pleuronichthys; DNA barcoding; COI; CR; Cyt b

1 INTRODUCTION

DNA barcoding, which is based on molecular biology techniques and bioinformatics, is a new method for biological taxonomy. As the barcodes on products are recognized, Hebert et al. (2003a) believed each living species can be identified using a short fragment of the mitochondrial gene (mtDNA) cytochrome c oxidase I (COI). Tautz et al. (2002) proposed that DNA sequences could be used as a tool to identify a species or taxon. Hebert et al. (2003a) were convinced that the prospect for sustainable identification lay in the construction of systems that employed DNA sequences as taxon “barcodes”, and established that the COI gene can serve as the core of a global bio-identification system for animals. Their later study indicated that sequence divergences at the COI gene regularly enabled the discrimination of closely allied species in all animal phyla except the Cnidaria (Hebert et al., 2003b). Consequently, many investigations have been carried out to check the feasibility of using the COI gene as the DNA barcodes to identify species in many different animal groups. The results indicated that using COI gene sequences as DNA barcodes was

generally effective, delivering more than 95% species-level resolution (Hebert et al., 2004a, 2004b; Barrett et al., 2005; Hajibabaei et al., 2005; Meyer et al., 2005; Ward et al., 2005; Hajibabaei et al., 2006; Cooper et al., 2007; Ratnasingham et al., 2007; Pyle et al., 2008; Ward et al., 2008).

Pleuronichthys cornutus and P. japonicus (Fig.1), which belong to the order Pleuronectiformes, are the only two members of the genus (Nakabo, 2000; Suzuki, 2009). Both species are commercially important fishery resources in the Northwestern Pacific Ocean (Li et al., 1995; Wang et al., 1999; Suzuki, 2009). Pleuronichthys japonicus is distributed from southern Hokkaido southward along the Sea of Japan and Pacific coasts of Japan to the southern East China Sea and Iyo Nada in the Seto Inland Sea (Suzuki, 2009). However, morphological differences between the two species appear to overlap. Nakabo (2000) and Suzuki (2009) identified P. japonicus from P. cornutus by the presence of an antrose

* Supported by the Special Fund for Agro-scientific Research in the

Public Interest (No. 201003068) and Special Key Program of Zhejiang

Provincial Department of Science and Technology (No. 2008C12011)

** Corresponding author: gaozhang@ouc.edu.cn

968 Vol.29CHIN. J. OCEANOL. LIMNOL., 29(5), 2011

branch of the supratemporal lateral line on the ocular and blind sides of P. japonicas, and by scale shape as well as other counts and proportional measurements. At present, only P. cornutus was recognized in Chinese waters (Li et al., 1995) and P. japonicus has never been reported. In this study, analyses of the mitochondrial DNA sequences of COI, the control region (CR), and cytochrome b (Cyt b) were employed to estimate the degree of genetic divergence between the two species. The main objective of this paper was to identify the two morphologically similar species based on DNA barcodes. Conservation programs and effective management of fishery resources will benefit from proper resource identification.

2 MATERIAL AND METHOD

2.1 Sample collection

Eighteen Pleuronichthys cornutus and four P. japonicus were obtained from the East China Sea in August, 2008. Muscle samples were preserved in

95% ethanol before DNA extraction. Sequences for Hippoglossus hippoglossus and H. stenolepis used as outgroups were obtained from GenBank and the accession numbers were AM749124 and AM749128 respectively.

2.2 DNA extraction, PCR and sequencing

Genomic DNA was isolated from muscle tissue by proteinase K digestion followed by a standard phenol-chloroform method (Sambrook et al., 1989). Each polymerase chain reaction (PCR) was performed in a volume of 50 μL containing 20–50 ng template DNA, 5 μL of 10 × PCR buffer, 5 μL of MgCl2 (25 mmol/L), 1 μL of dNTPs (10 mmol/L), 10 pmol/L of each primer and 2.5 units of Taq DNA polymerase (TaKaRa biotechnology (Dalian) Co, Ltd.) in an Eppendorf Mastercycler 5533 (Eppendorf, Hamburg, Germany).

CR was amplified with primers Dl-S: 5′-CCCACCACTAACTCCCAAAGC-3′ (forward) (Han et al., 2008) and Dielei-D: 5′-TAGGAACCAAATGCCAGGAA-3′ (reverse). Initial denaturation was for 5 min at 94°C, followed by 35 cycles of 45 s at 94°C for denaturation, 45 s at 52°C for annealing, 45 s at 72°C for extension, and a final extension at 72°C for 10 min. A Cyt b gene fragment was amplified with primers L14734-Glu: 5′-AACCACCGTTGTTATTCAACT-3′ (forward) and H15149-Cyb: 5′-CTCAGAATGACATTTGTCCTCA-3′ (reverse) (Gao et al., 2004). Initial denaturation was for 5 min at 95°C, followed by 35 cycles of 1 m at 95°C for denaturation, 1 m at 50°C for annealing, 1 m at 72°C for extension and a final extension at 72°C for 10 min. The COI gene fragment was amplified with primers FishF1: 5′-TCAACCAACCACAAAGACATTGGCAC-3′ (forward) and FishR1:5′-TAGACTTCTGGGTGGCCAAAGAATCA-3′ (reverse) following Ward et al. (2005).

All sets of PCR reactions included a negative control reaction tube in which all reagents were included, except template DNA. PCR products were separated on a 1.5% agarose gel and purified with the BioDev Gel Extraction System B (BioDev Technology (Beijing) Co., Ltd). Both strands of the purified products were bi-directionally sequenced using the BigDye Terminator cycle sequencing kit v2.0 (Applied Biosystems, Foster City, CA, USA), and sequencing was conducted on an ABI Prism 3730 automatic sequencer (Applied Biosystems).

All sequences have been deposited in the GenBank database under Accession Nos. COI: JN204300–JN204303 and JN204304–JN204310;

Fig.1 Pleuronichthys cornutus (upper) and Pleuronichthys japonicus (lower)

No.5 969ZHANG et al.: Genetic identification of two species of Pleuronichthys

CR: JN204328–JN204331 and JN204311–JN204327; Cyt b: JN204288–JN204291and JN204292–JN204299 for P. japonicus and P. cornutus.

2.3 Sequence analysis

Sequences were edited and aligned using DNASTAR software (DNASTAR, Inc., Madison, USA) and refined manually. Genetic diversity indices such as number of haplotypes, polymorphic sites, transitions, transversions, and indels were obtained using ARLEQUIN (Ver. 2.0) (Schneider et al., 2000). Haplotype diversity (h) and nucleotide diversity (π) were calculated with DNASP 4.0 (Rozas et al., 1995). The nucleotide compositions and the average distances between species and within species were calculated with MEGA 4.0 (Tamura et al., 2007). The gamma distribution shape parameters for the rates sites of heterogeneity were calculated using Modeltest 3.7 (Posada et al., 1998). COI and Cyt b gene sequences were translated into amino acid sequences in MEGA 4.0 (Tamura et al., 2007).

2.4 Phylogenetic analysis

Phylogenetic relationships between the two species of Pleuronichthys were reconstructed using neighbor joining (NJ), maximum parsimony (MP), maximum likelihood (ML) and Bayesian methods. The optimal base substitution model and the optimized parameters for NJ analysis were estimated by Modeltest 3.7 (Posada et al., 1998) using the hierarchical likelihood ratio tests (hLRTs). The best model for CR, Cyt b,

and COI fragments was HKY+G. NJ and MP trees were reconstructed by PAUP4.0b10 (Swofford, 2002). ML trees were reconstructed online at http://www.atgc-montpellier.fr/phyml/. Bayesian analysis was conducted by MrBayes 3.1.2 (Huelsenbeck et al., 2001; Ronquist et al., 2003).

3 RESULT

3.1 Nucleotide compositions and genetic diversity

The lengths of amplified fragments of COI, CR and Cyt b were 655 bp, 388 bp and 414 bp respectively. The first 3 bp from the 5′ of Cyt b belong to the tRNAGlu gene. The average nucleotide composition for each fragment is shown in Table 2. All the fragments showed an A+T bias (Table 1). There was bias against the G content at the 3rd codon position in the two protein coding genes (Fig.2).

Several genetic diversity indices of the three fragments are shown in Table 2. No haplotype was shared between the two species based on the three fragments. The nucleotide diversity and ratio of transitions/transversions of COI genes were higher in comparison with CR and Cyt b. Most substitutions were non-synonymous including 33 mutations at the 1st codon, four mutations at the 2nd codon, and no mutation at the 3rd codon in Cyt b. Most substitutions were synonymous, including 66 mutations at the 3rd codon and four mutations at 1st codon positions in COI gene sequences.

Table 1 The nucleotide composition and nucleotide diversity (π) based on COI, CR and Cyt b fragments of P. cornustus and P. japonicus

Fragment π A (%) T (%) G (%) C (%)

COI 0.046 23.6 30.0 18.4 28.0

CR 0.022 30.0 38.3 16.0 15.7

Cyt b 0.028 25.2 30.1 15.7 29.0

30

25

20

15

10

5

0A T G C A T G C

Frqu

ency

(%)

3035

2520151050

Frqu

ency

(%)

P. cornutus

Cyt b 3rd COI 3rd

P. japonicus

Fig.2 The nucleotide composition of the third codons in Cyt b and COI genes of P. cornutus and P. japonicus

970 Vol.29CHIN. J. OCEANOL. LIMNOL., 29(5), 2011

Translation of 654 bp of the COI gene resulted in 218 amino acids with only two non-synonymous amino acid substitutions (at the 1st codon). One hundred and thirty seven amino acids were coded by the 411 bp of the Cyt b gene. Thirty one amino acid substitutions were found at the 1st codon, caused by the non-synonymous substitutions.

Net average genetic distances (Kimura two parameter distances) between the two species were 0.094, 0.065 and 0.044 for COI, Cyt b and CR respectively.

The genetic distances within P. cornutus based on three fragments were 0.007, 0.010 and 0.005 for COI, CR and Cyt b respectively, while those in P. japonicus were 0.006, 0.007 and 0.004 for COI, CR and Cyt b respectively.

3.2 Phylogenetic relationships

The ratio of transitions/transversions for the three fragments is shown in Table 2. Transition and transversion substitutions of the three fragments increased linearly against the F84 distance (not given) (Felsenstein et al., 1996), indicating that base changes at these sites were not saturated.

The NJ, MP, ML and Bayesian trees were reconstructed based on the haplotype datasets of the three fragments. The trees reconstructed by different methods were mostly similar to each other using Hippoglossus hippoglossus and H. stenolepis as outgroups. All the trees indicated two clades belonging to P. cornutus and P. japonicus, respectively, with high bootstrap values for NJ, MP and ML trees and a high posterior probability for the Bayesian tree (Fig.3).

4 DISCUSSION

DNA barcoding is a new method for biological taxonomy. It permits the identification of species from fragments, and from any life-history stage, as well as the standardization of a universal master key in a format that reduces ambiguity and enables direct comparison of specimens to a global reference database. This has been shown to provide species

Table 2 Diversity indices for COI, CR and Cyt b of genus Pleuronichthys

Fragment Ns Nh L (bp) P Indels Pi h R

COI 13 12 655 70 0 63 0.987 2 5.55

CR 22 21 388 28 2 23 0.995 7 2.3

Cyt b 18 12 414 37 0 28 0.895 4 2.55

Note: Ns: the number of the sequences; Nh: the number of the haplotypes; L: the sequence length; P: polymorphic sites; Pi: Parsimony informative sites; R: ratio of transitions/transversions; h: haplotype diversity

68/59/100/100

100/100/100/100

100/97/100/100

62/75/100/99

100/100/100/100

87/51/100/98

99/94/99/99

100/100/100/100

100/99/100/100

P. cor 07P. cor 05P. cor 04P. cor 03P. cor 02P. cor 06P. cor 01P. jap 03P. jap 04P. jap 02P. jap 01H. steH. hip

H. steH. hip

P. cor 12P. cor 15P. cor 16P. cor 11P. cor 17P. cor 04P. cor 03P. cor 10P. cor 01P. cor 09P. cor 08P. cor 06P. cor 05P. cor 13P. cor 14P. cor 07P. cor 02P. jap 03P. jap 04P. jap 02P. jap 01

H. steH. hip

P. cor 04

P. cor 03

P. cor 01

P. cor 08

P. cor 06

P. cor 05

P. cor 07

P. cor 02

P. jap 03

P. jap 04P. jap 02

P. jap 01

a

b

c

Fig.3 Phylogenetic trees of P. cornutus and P. japonicus based on haplotype datasets of three fragments (from above to below: COI, CR and Cyt b)

Numbers on the branches from left to right were bootstrap values of

NJ, MP and ML trees and posterior probability of the Bayesian tree.

Values lower than 50% were not given. P. cor, P. jap, H. ste and H. hip

indicate P. cornutus, P. japonicus, H. stenolepis and H. hippoglossus,

respectively

No.5 971ZHANG et al.: Genetic identification of two species of Pleuronichthys

level resolution of the vast bulk of species in a wide range of animal taxa, including ants, bats, birds, butterflies, crustaceans, fish and spiders (Hebert et al., 2004a, 2004b; Barrett et al., 2005; Hajibabaei et al., 2005; Meyer et al., 2005; Ward et al., 2005; Hajibabaei et al., 2006; Cooper et al., 2007; Ratnasingham et al., 2007; Ward et al., 2008; Pyle et al., 2008.) The success in species diagnosis reflects both the high rates of sequence change at COI in most animal groups, and the low intraspecies rates. This study validates the efficiency of COI barcodes for identifying P. cornutus and P. japonicus.

As for other teleosts (Gao et al., 2004; Ward et al., 2005; Zhang et al., 2009), AT content was higher than GC content for all three mtDNA fragments. We observed more nucleotide changes at the 3rd codon positions (66 mutations) than the 1st (4 mutations), and more at the 1st than the 2nd in the COI gene. This reflected the fact that most synonymous mutations occurred at the 3rd codon, with a few at the 1st codon and none at the 2nd codon (Meyer, 1993; Gao et al., 2004; Ward et al., 2005; Zhang et al., 2009). In the Cyt b gene, most substitutions were non-synonymous with 33 mutations at the 1st codon, 4 mutations at the 2nd codon and none at the 3rd codon. The result was different from other fishes (Meyer, 1993; Gao et al., 2004; Zhang et al., 2009) in which most substitutions happened at the 3rd codon. This reflected an unusual character of the Cyt b gene in Pleuronichthys and further study should be done to find the reason. Thirty one amino acid substitutions were found in the 137 amino acids coded by the Cyt b gene between P. japonicus and P. cortunus. Among the 218 amino acids coded by the COI gene, only two amino acid substitutions were found. The amino acid substitution rate of the Cyt b gene was higher than that of the COI gene in the genus Pleuronichthys.

Despite its functional importance, the CR region was the most rapidly evolving part of the mtDNA, generating heterogeneity in both length and base composition (Sbisà et al., 1997). In this study, sequence comparison of the CR segment between P. cortunus and P. japonicus revealed 28 polymorphic sites (23 parsimony informative sites) with 21 transitions, 9 transversions and 2 indels, and the substitution rate was 0.072, while the substitution rates for the Cyt b and COI genes were 0.089 and 0.107 respectively. Comparatively, among the mtDNA fragments, the divergence level of the CR segment was the lowest between P. japonicus and P. cortunus. This result differs from other fish in the Pleuronectiformes (Gao et al., 2004; Zhang et al., 2009). The genetic distances between the two species

based on COI, CR and Cyt b fragments were 0.094, 0.044 and 0.065 respectively. Within species, the genetic distances in P. cornutus based on three fragments were 0.01 (CR) > 0.007 (COI) > 0.005 (Cyt b), while the genetic distances in P. japonicus were 0.007 (CR) > 0.006 (Cyt b) > 0.004 (COI). Applying the Cyt b gene divergence rate of 2%/MY, the divergence of the two species occurred about 3 250 000 years before present (BP), indicating that divergence between these two species occurred in the Pliocene.

Although any of the three fragments studied here could be used to identify and differentiate P. japonicus from P. cornutus, the COI gene showed the highest (0.094) genetic distance between two species. However, the genetic divergence based on COI within species was very low, 0.007 for P. cornutus and 0.004 for P. japonicus. As DNA barcoding requires that intraspecies DNA barcode variation should be substantially less than interspecific variation to allow accurate identification of individuals, this result indicated the effectiveness of COI in identifying the species. Two hundred and seven species of fish, mostly Australian marine fish, were sequenced (bar coded) for a 655 bp region of the COI gene by Ward et al. (2005). Our research based on COI is in agreement with Ward et al. (2005), who found average within-species, genus, family, order and class genetic distances as 0.003 9, 0.099 3, 0.154 6, 0.221 8 and 0.232 7, respectively. This study has proved the efficiency of COI barcodes for identifying P. cornutus and P. japonicus when compared with the other two mtDNA fragments. In conclusion, different segments in mtDNA should be used for different research purposes (Meyer, 1993; Gao et al., 2004). According to this study, the CR fragment can be chosen to study the genetic population structure within species of both P. cornutus and P. japonicus, while the COI and Cyt b genes appear more suitable to study the phylogenetic relationships in the genus Pleuronichthys. The COI was best in identifying P. cornutus and P. japonicas.

In conclusion, P. cornutus and P. japonicus are similar in morphology, but genetic divergences between them are extensive both in gene and amino acid sequences. CR, Cyt b and COI fragments, according to this study, could be used to identify the two species, but the COI gene proved more powerful for their identification. We suggest that COI gene sequences can be used as DNA barcodes to diagnose P. cornutus and P. japonicus even for the egg and larval stages. This study is therefore useful for the

972 Vol.29CHIN. J. OCEANOL. LIMNOL., 29(5), 2011

identification of Pleuronichthys species through DNA barcodes, especially for the identification of P. japonicus. As P. japonicus has never been reported in Chinese waters, this study will be useful for diagnosing this species, examining the phylogenetic relationships in the genus Pleuronichthys, and assisting fishery management.

5 ACKNOWLEDGMENT

We thank Dr. Nwafili SYLVANUS and Dr. Yongshuang XIAO for their contributions and proofreading.

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