Molecular Identification and Phylogenetic Relationships of Threadfin Breams ( Family : Nemipteridae ) Using mtDNA Marker

Cytochrome c oxidase-1 gene sequences of mitochondrial genome were analyzed for species identification and phylogenetic relationship among the commercially important Nemipterus species. Sequence analysis of COI gene clearly indicated that all the nine fish species fell into distinct clads, which are genetically distant from each other and exhibited identical phylogenetic reservation. All the COI gene sequences provide sufficient phylogenetic information and evolutionary relationship to distinguish the nine Nemipterus species unambiguously. As per the neighbour-joining (NJ) and maximum likelihood (ML) trees, all the nine species are genetically distant from each other and exhibited identical phylogenetic reservation. Based on the NJ and ML phylogenetic trees N. mesoprion, N. zysron, N. hexodon, N. nematophorus, N. virgatus and N. bipunctatus were closely related with high bootstrap value (97). The overall mean Kimura two parameter (K2P) distances between the nine species was 0.109. The intra species K2P distance was high in N. japonicus (0.069) followed by N. peronii (0.050) and N. mesoprion (0.002). This study proves the use of mtDNA COI gene sequence based approach is an alternative tool for identifying fish species at a faster pace.


Introduction
The threadfin breams, also called pink perch constitute an important demersal finfish resource in the Indian EEZ.These fishes are abundant beyond 50 m but show higher concentration at 100-200 m depth as revealed by the exploratory surveys and experimental fishing (Murty et al., 2001).Threadfin breams are one of the most dominant components among the demersal fisheries of India being exploited by commercial trawlers and available all the entire year (Russell, 1990).The classification of nemipterid fishes into different taxa is much confusing as, they are the members of one of the most confusing families (Russell, 1990).Threadfin bream's catches are usually mixed in India with representation of three or more species of genus Nemipterus (Murty et al., 2001).They are rarely reported as separate species because of problems in proper identification (Pawar et al., 2011).Morphometric characters and colour patterns are the most useful taxonomic tools which helps in segregation of fish species (Russell, 1990).But taxonomic ambiguity exists for juvenile and pre-adult fishes and it may lead to another synonym to a fish species.DNA-based approaches for taxon diagnosis exploiting DNA sequence diversity among species can be used to identify fishes and resolve taxonomic ambiguity including the discovery of new ⁄ cryptic species (Hebert et al., 2003).As there is no paternal contribution of mtDNA and no known recombination between mitochondrial genomes, the mtDNA genes are selected for species identification (Thangaraj and Lipton, 2011).Earlier studies have demonstrated that the COI gene is appropriate and accepted as a universal barcode for discriminating between closely related species across diverse animal phyla and this has been used for marine and freshwater fishes (Hebert et al., 2003;Hubert et al., 2008;Lakra et al., 2011;Ward et al., 2005).
Considering the importance of molecular identification and as there are no attempts to study genetic relation-ing a UV spectrophotometer.The DNA was diluted in TAE buffer to a final concentration of 100 ng ⁄ µL.

Amplification and sequencing
The COI gene was amplified in a 50 µL volume with 5 µL of 10X Taq polymerase buffer, 2 µL of MgCl 2 (50 mM), 0.25 µL of each dNTP (0.05 mM), 0.5 µL of each primer (0.01 mM), 0.6 U of Taq polymerase and 5 µl of genomic DNA.The primers used for the amplification of the COI gene were FishF1-5'TCAACCAACCACAAAGACATTGGCAC3' and FishR1-5'TAGACTTC TGGGTGGCCAAAGAAT-CA3' (Ward et al., 2005).The thermal regime consisted of an initial step of 2 min at 95°C followed by 35 cycles of 40 s at 94°C, 40 s at 54°C and 1 min 10 s at 72°C followed in turn by final extension of 10 min at 72°C.
The PCR products were visualized on 1.5% agarose gels, and the most intense products were selected for sequencing.The cleaned up PCR product was sequenced by a sequencing facility (Bioserv Biotechnologies Pvt Ltd, Hyderabad, India).

Sequence analysis
The CO1 gene partial sequences of the four Nemipterus species were unambiguously edited using BioEdit sequence alignment editor14 and aligned using CLUSTAL-W in BioEdit, and checked manually.Identical sequences were assigned in the same haplotype identity and only a single example of each species used in the phylogenetic divergences assuming that identical haplotypes shared the ship between various Nemipterus species, in spite of its economic importance and significant contribution in marine fishery, the present study was achieved.In this study, the genetic difference between four commonly available threadfin bream species in Bay of Bengal and other Nemipterus species were assessed using mitochondrial gene sequence.

Sample collection
Forty specimens from four species were collected from the Pondicherry coastal waters (11°46' and 12°03' N and 79°36' and 79°53' E).Immediately after the collection specimens were kept in the iceboxes for further studies.All the fishes were identified up to the species level using the FAO Fish Identification Sheets (Thomson, 1984) and further confirmation were carried out at Zoological Survey of India, Southern Regional Centre, Chennai.The voucher specimens are maintained in the Department of Zoology, Kanchi Mamunivar Centre for Post-Graduate Studies, Pondicherry.Approximately 100 mg of white muscle tissue and fin-clips from two to five individuals of each species were preserved in 95% ethanol and stored at 4°C until used.

DNA isolation
The DNA was isolated by standard Proteinase-K/Phenol-Chloroform-ethanol method (Sambrook et al., 1989) and the concentration of isolated DNA was estimated us- same evolutionary origin.Haplotype definitions have been submitted to the NCBI GenBank (Acc.No. HQ423413, JN992286, JN992287, JN992288).To support the present data, selective CO1 sequences in other five Nemipterus species and one outgroup (Lates calcarifer) were retrieved from Genbank (Tab.1).Nucleotide diversity, genetic variation and nucleotide composition and pairwise evolutionary distance among haplotypes was determined by the Kimura 2-Parameter method (Kimura, 1980) using the software program MEGA 3.1 (Molecular Evolutionary Genetics Analysis) (Kumar et al., 2004).The neighbourjoining (NJ) and maximum likelihood (ML) trees were constructed using MEGA 3.1 and to verify the robustness of the internal nodes of these trees, bootstrap analysis was carried out using 1000 pseudoreplications.

Results and discussion
A total of 21 sequences were analysed from nine Nemipterus species in this study.Simplicity and un-ambiguity were observed among all the sequences and no introns, deletions or stop codons were observed any of the sequences.The sequence analysis revealed the average nucleotide frequencies as A = 23.4 ± 0.75%, T = 31.6± 1.17%, G = 18.0 ± 0.60, C = 26.9 ± 0.76 (Tab.2).The average number of nucleotide difference (K) = 67.51 and nucleotide diversity (Pi) = 0.1342.Tajma's statistics (D) =-0.1109 and it was not significantly (p<0.01)different among sequences.Kimura 2 Parameter (K2P) genetic distance in thread-fin bream fish species is given in Tab. 3. The overall K2P distance between the nine species was 0.109.The K2P genetic distance was high (0.150) between N. perinii and N. hexodon.Very low K2P distance (0.009) was exhibited between N. mesoprion and N. zysron.
The intra-species K2P distance (Fig. 1) was high in N. japonicus (0.069) followed by N. peronii (0.050) and N. mesoprion (0.002).The codon based genetic distance in nine Nemipterus species is depicted in Tab. 4 (below diagonal).Based on the CO1 sequence data, the codon based genetic distance was high (0.372) between N. hexodon and N. nematophorus.The minimum level (0.007) of codon based genetic distance was displayed between N. mesopri-  All the twenty two sequences were subjected in the phylogenetic analysis.The neighbour joining tree (NJ) and maximum likelihood tree (ML) of K2P distance were   Ward et al. (2005) reported an overall higher GC content in fishes based on complete MtDNA genome ranging from 38.4-43.2% and in CO1 alone it was 42.2-47.1%,which reflects the 3 rd base variation.Peris et al. (2009) also reported considerable variation was exhibited in carangids in the 3 rd base position.In this study it has been observed the mean GC content was 36.8 (GC1)-42.6(GC3) among the nine Nemipterus species.The range of K2P intra-specific genetic distance in the present study was 0.000-0.069,which was slightly higher when compared with the previous studies of Indian carangids, 0.000-0.015(Peris et al., 2009), Australian fishes (Ward et al., 2005), North American Birds (Hebert et al., 2004), Moths (Hebert et al., 2003), and Primates (Hajibabaei et al., 2006).The mean nucleotide diversity (Pi) among all the species was estimated as 0.1342.In earlier studies it was estimated as 0.2029 in Indian marine fishes (Lakra et al., 2011).It has been shown that lineage diversity more quickly within species than between species (Pons et al., 2006).
An effective DNA-based identification system requires the satisfaction of three important conditions: it must be possible to recover the target DNA from all the species; the sequence information must be easily analysed; and the information content of the target sequence must be suf-with 97% bootstrap value.Another clad representing all the other three species such as N. japonicus, N. furcosus and N. peronii with only 44% bootstrap value.The outgroup, Lates calcarifer was highly divergent and deviated in to a separate clad forming the root to the phylogenetic tree.The ML tree also showed the same type of divergence and formed two sub-trees having the bootstrap value of 92% and 36% respectively.
Species identification and phylogenetic relationship based on traditional methods and molecular methods are mostly concordant (Ward et al., 2005).In this study nine Nemipterus species were found genetically distinct from each other based on CO1 gene sequence which demonstrates simplicity and unambiguity.Morphologically very similar species like N. mesoprion, N. zysron and N. virgatus form a sister clad.Whereas, N. japonicus, N. furcosus and N. peronii form an independent sister clad in both NJ and ML trees of CO1 gene sequence.Estimates of genetic divergence from CO1 gene were sufficient to differentiate individuals of different threadfin bream species.In this study the level of intra-species variation was low which may be due to low number of haplotype identified in the sample with limited numbers collected for this study.Similarly, Lakra et al. (2011) reported very low intra-specific genetic divergence for scombroid fished and Ward et al. (2005) in many marine teleost species.Peris et al. (2009) Fig. 3. Maximum likelihood (ML) phylogenetic tree of threadfin bream fish species from COI gene sequences ficient to enable species-level identification (Peris et al., 2009).All these three requirements were met in the present study, as all fish species examined could be recovered and targeted CO1 fragment aligned and analysed easily.Specimens of all species formed distinctive cluster and congruent with conventional morphological taxonomy.As per Ward et al. (2005), the CO1 analysis seeks only to delineate species boundaries yet there is some clear phylogenetic signal in CO1 sequence data, which is evident by the clustering of most of congeneric and confamilial species.The absolute clustering of conspecifics in the present study indicates the diagnostic ability of CO1 to correctly identify species.

Conclusions
The study has successfully proved the utility of COI divergences in identifying many of the Nemipterid fishes.The present analysis was not meant to be exhaustive, but to highlight the most important feature of COI based studies, that is the diagnostic ability of COI sequences in distinguishing closely related species and the intra-specific distances are lower than the inter-specific distances which was proved beyond doubt.The slight difference in the resolution factor in the present study and previous studies on Indian, Australian fishes and other groups of animals may be due to the low sample size.Further studies involving all the threadfin breams in the world and also by increasing the sample size in future studies will clarify the issue.
created to provide a graphical representation of the patterning of divergence of nine Nemipterus species (Fig.2 and 3).As per NJ tree two distinct clads as two sub-trees within the same genus were recognized with high bootstrap value.Among the two sub-trees the larger one has an independent assemblage of N. mesoprion, N. zysron, N. hexodon, N. nematophorus, N. virgatus and N. bipunctatus on and N. zysron.The disparity index in nine Nemipterus species is displayed in Tab. 4 (above diagonal).The maximum disparity index (0.249) was observed between N. nematophorus and N. hexodon.

Fig. 2 .
Fig. 2. Neighbour Joining (NJ) phylogenetic tree of threadfin bream fish species from COI gene sequences Tab. 1. Threadfin bream fish species and their COI sequence Genbank accession numbers Tab. 4. Codon based distance (below diagonal) and disparity index (above diagonal) in threadfin bream fish species