Keywords

1 Introduction

Since the Convention of Biological Diversity summit at Rio-de-Janeiro in 1992, all over the world, there has been lot of interest in identification of biological resources. Identification of species in the classic way, using morphological characters takes longer time and manpower, hence scientists developed an alternative method in conjunction with the genetic make up of the species. DNA barcoding is a molecular method for species identification and classification of biological organisms based on the analysis of short, standardized gene sequences (Hebert et al. 2003). In most animals, the fragment of mitochondrial gene cytochrome c oxidase subunit I (COI) has been used as the target sequence. This novel system is designed to provide rapid, accurate, and automatable species identifications by using short, standardized gene regions as internal species tags. DNA barcoding is now gaining more attention in the field of assessment of cryptic species, taxon diagnosis, authentication and safety assessment of seafood, wildlife forensics, conservation genetics and detection of invasive alien species from whole fish, fillets, fins, fragments, juveniles, larvae, eggs, any properly preserved tissue or environment sample (Trivedi et al. 2016). The concerted global research FISH-BOL (Fish Barcode of Life) on DNA barcoding was launched in 2005, with the goal of collection and assembling specific DNA barcode sequences and associated voucher provenance data in accurate reference sequence library to aid the molecular identification of all fish species.

India has a rich natural heritage and nurtures a unique bio-diversity, placing it among the 12 biodiversity rich countries. Globally the number of valid fish species recorded so far is more than 31000, with the addition, at an average, of 100–150/year (Eschmeyer et al. 2010). Among these 2,508 are indigenous to Indian subcontinent (877 freshwater, 113 brackish water and 1,518 marine species) (NBFGR, 2013). Out of which about 500 marine species has been barcoded (Tables 1, 2 and Fig. 1), covering carangids, scombrids, serranids, scianids, polynemids, nemipterids, pomacanthids, gobiids, clupeids, mugilids, sharks, rays, skates and other taxonomically important groups, under national programme on DNA barcoding which was launched in 2005 with initiative of NBFGR and other research programmes. In view of the growing importance of fish DNA barcoding, Nagpure et al. (2012) developed Fish Barcode Information System (FBIS) database on Indian fishes. FBIS represent barcode sequences for Indian fishes, reported in India as well as from other countries. Presently, it comprises barcode sequence of 1083 marine, 349 Freshwater and 49 brackish water with 23429 specimen sequences (http://mail.nbfgr.res.in/fbis/) belonging to 213 families and 48 orders.

Table 1 List of Indian marine fish species barcoded (Bony fishes)
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Table 2 List of Indian marine fish species barcoded (Cartilaginous fishes)
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Fig. 1
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Different groups of fishes barcoded in India

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1.1 Carangids

The family Carangidae comprises 30 genera with 146 species distributed throughout Atlantic, Indian and Pacific Oceans (http://www.fishbase.org). In India approximately 58 species are recorded. Carangids are considered as a most delicious and highly valued fish in India. This group of fishes is categorized into five sub groups i.e., black pomfrets, queen fishes, trevallies, scads, and pompanos. The group has emerged as one of the important resources along Indian coast. A total of 51 fish species, covering 18 genera were barcoded in India from Indian water. NBFGR has completed barcoding of 45 carangid species belonging to 16 genera. Analaysis show that longfin trevally, Carangoides armatus is a species complex, suggesting cryptic species within the complex. Persis et al. (2009) has carried out the phylogenetic study using COI for 28 carangids from Kakinada coast in India and revealed that all these fishes fall into three distinct groups which are genetically distant from each other and exhibited identical phylogenetic reservation. In addition to that 17 fish species from 13 genera were analysed by Lakra et al. (2011) who observed that the average genetic distance within species was 0.32 % whereas between species it was 16.1 %. The NJ tree also revealed distinct clusters for species of same genera with 94–100 % bootstrap values.

1.2 Sciaenids

The family Sciaenidae, known as croakers or drums, is distributed worldwide with approximately 70 genera and 300 species including about 40 species from Indian waters. This group contributes approximately 4.6 % to the total Indian marine fish production. Out of these 14 species were barcoded from Indian waters. Lakra et al. (2009) studied the phylogenetic relationships of six species of Indian sciaenids  (Otolithes cuvieri, O. ruber, Johnius borneensis, J. dussumieri, Dendrophysa russelii and Nibea maculata) based on 16S rRNA and cytochrome c oxidase subunit I which revealed three genetic distinct groups. The average genetic distance within species was 0.28 % whereas the overall mean distance among the species was 18.20 %. The NJ tree clearly distinguished the species having same genus under one cluster with a bootstrap value of 96–100 %.

1.3 Scombrids

The family Scombridae consist of the mackerels, tunas, and bonitos, distributed worldwide in tropical and subtropical seas. The family consists of 15 genera and 54 species including 21 species from India. Out of which 12 fish comprising seven genera were barcodes from Indian waters. Based on the COI (655 bp) sequences genetic relationship of Rastrelliger kanagurta, R. faugni and R. brachysoma were carried out by Basheer et al. (2015) and it was observed that the mean genetic divergence between three mackerel species was 5 %. The pair-wise divergence between R. kanagurta and R. faugni was 0.08-0.09 and with R. brachysoma it was 0.03-0.04. R. kanagurta samples between Indian mainland and Andaman waters showed a divergence level of 1.2 %. Similar kind of work has been carried out by Lakra et al. (2011) covering five genera (Auxis thazard, A. rochei, R. kanagurta, Thunus albacares, T. tonggol, Euthynnus affinis, Katsuwonus pelamis) and revealed the 0.3 % genetic distance within species and 9.20 between the species. All the species were clearly separated into different clusters in the NJ tree with a bootstrap value ranging from 96 to 100 %.

1.4 Serranids

The family serranidae consist of 537 species, under 75 genera including 70 known species from India. Serranids are distributed throughout tropical and temperate oceans and it is represents one of the most important resources targeted by coastal fisheries in country. A total of 47 species of Serenade family were barcoded from Indian water by NBFGR and other agencies. Traditionally serranid fishes are identified based on visible morphological, meristic, and anatomical characters (Roy and Gopalakrishnan 2011). Sachithanandam et al. (2011) has shown the utility of COI divergences in identifying all the Plectropomus maculatus fishes in Andaman Islands with minimum base pair (133 bp). A molecular phylogeny study using COI sequences has shown that the overall mean distance among the species is 12.60 % and within species 0.24 %. In the NJ tree all the species were under one clad with bootstrap values of 94–98 % (Lakra et al. 2011).

At NBFGR, we barcoded 36 grouper species, including seven species listed in the IUCN red list under threatened category. Barcodes from six genera Aetheloperca, Cephalopholis, Epinephelus, Hyporthodus, Plectropomus and Variola were developed out of which E. polylepis and E. miliaris were developed for the first time. A comparison of the sequences generated were done with sequences available in NCBI to authenticate the species identification and resolve taxonomic ambiguity. Upon the sequence analysis, taxonomic ambiguity exists in E. polylepis and E. cholorostigma. E. polylepis has a distribution in northwest Indian Ocean (Craig et al. 2011) and there is no further report of the species after the description by Randall and Heemstra (1991) from India. It may be misidentification by earlier workers and reported as E. chlorostigma. Phylogenetic analysis using neighbour joining tree is given in Fig. 2.

Fig. 2
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NJ tree of COI gene sequences computed by using K2P distance of the groupers barcoded

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1.5 Nemipterids

Species belonging to the family nemipteridae are known as threadfin, whiptail breams and false snappers, widely distributed in tropical waters of the Indian and Western Pacific Oceans. The family consists of 67 species, under 5 genera. So far 30 species are reported from India, out of which 13 species were barcoded. Under this family most species are benthic carnivores in nature. Ravichandirane et al. (2012), distinguish nine Nemipterus species, using COI gene and revealed all the nine species are genetically distant from each other and exhibited identical phylogenetic reservation. 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).

1.6 Polynemids

Polynemids are known as threadfins, they are abundantly distributed in tropical to subtropical waters throughout the world. The polynemidae consist of 8 genera and 43 species, a total of 14 species are reported from the East and West coast of India, of which a total of 10 species belonging to five genera (Polydactylus, Polynemus, Eleutheronema, Leptomelanosoma and Filimanus) were barcoded. The molecular study of polynemids, by Lakra et al. (2011) has revealed that the average K2P distance within species was 0.35 % and 16.30 % for interspecies distance. In NJ tree three clusters were formed. The genus Polydactylus and Filimanus shared the first and second cluster respectively, whereas the third cluster was formed by Leptomelanosoma and Eleutheronema, with a bootstrap value ranging from 92–100 %.

1.7 Leiognathids

Leiognathids, the silverbellies are common fishes of coastal and estuarine waters in the tropical and subtropical Indo-Pacific Ocean. The leiognathidae consist of 9 genera and 48 species. A total of 23 species are reported from Indian waters, out of which total of 9 species from 5 genera were barcoded. Molecular study using COI showed the average genetic distance within species was 0.20 %.

1.8 Mullids

The family Mullidae are commonly called goatfish. The family consist of 85 species, under 6 genera. So far 23 species reported from India, out of which 8 species comprising three genera have been barcoded. Molecular phylogeny study by Lakra et al. (2011) of six fish species showed the average genetic distance within species was 0.38 % whereas the overall mean distance among the species was 13.90 %. The NJ tree revealed that the genera Parupeneus, Mulloidichthys and Upeneus formed three separate clades with a boostrap value of 95–99 %.

1.9 Lutjanids

Snappers are of mainly marine but some are found in estuaries and fresh water. The family includes about 110 species in 17 genera, some of which are considered as important food fish. A total of 47 fishes are reported from Indian waters of which barcoding has been completed for only 10 species. By using COI Victor et al. (2009) has successfully identified the larvae and newly-settled juveniles of the Cubera Snapper (Lutjanus cyanopterus) and observed that the nearest neighbor species, L. analis, was more than 11 % divergent. Recently Lutjanus johni, L.lutjanus, L. russelli, and L. malabaricus has been identified using COI by Krishna et al. (2012) and Lakra et al. (2011).

1.10 Pomacentrids

The family pomacentridae comprises damselfishes and clownfishes. They are primarily marine, while a few species inhabit freshwater and brackish environments. About 385 species are classified in this family with 29 genera, and a total of 52 species have been reported from Indian waters. The barcodes for 18 species under 4 genera have been generated in India by Lakra et al. (2011), Dhaneesh et al. (2015) and Bamaniya et al. (2015). The cytochrome oxidase 1 (COI) sequence of 13 clownfish species was used for construction of phylogenetic relationship, and scrutinized species boundaries between four closely related species of the sub-genera Phalerebus, Amphiprion and Paramphiprion (Dhaneesh et al. 2015).

1.11 Gobiids

Gobiids constitute a major proportion of fish population in both tropical and temperate freshwater as well as marine ecosystem and is one of the largest families of marine fishes containing 1718 species in 251 genera. Out of these a total of 100 species are reported from India. 14 species have been barcoded by Lakra et al. (2011), and Viswambharan et al. (2015). Due to their small size, cryptic ecology and ambiguous morphological characters, gobiid diversity was not documented completely. Viswambharan et al. (2015) generated COI barcode for 11 species of gobies for accurate species identification. The COI barcodes clearly distinguished all these species with higher interspecific genetic distance values than intraspecific values based on K2P (Kimura 2 Parameter) model. The average genetic distance (K2P model) within species, genus and family was 1.2 %, 22.2 % and 25.3 %, respectively.

1.12 Clupeids

Clupeidae include many of the most important food fishes in the world that  includes herrings, shads, sardines, hilsa, and menhadens. The family comprises 198 species under 54 genera. A total of 27 species have been reported from India, of which 10 species have been barcoded by several workers (Lakra et al. 2011). The phylogenetic study of clupeids has been carried out by Lakra et al. (2011) using COI gene of seven species and observed that overall mean distance among the species was very high (20.30 %). The average genetic distance within species was 0.41 %.

1.13 Engraulids

Engraulids are mainly known as a Anchovies, they are schooling fishes, mostly of shallow coastal waters and estuaries in tropical and temperate regions. Some are estuarine in nature. They are widely distributed across Atlantic, Indian and Pacific Oceans. The family consists of 198 species in 54 genera of which 34 species are recorded from Indian waters. Anchovies are considered as a delicious and preferred fish in India. Despite their importance as one of the important fishery resources, only 8 species under this group have been barcoded from India covering three genera. Two species, Thryssa purava and Thryssa setirostris were barcoded from estuaries of River Krishna by Krishna et al. (2012). The molecular phylogenetic study has been carried out in selected species using COI sequence, and revealed the average genetic distance between Stolephorus indicus and Stolephorus commersonnii was 9.11 % (Khan et al. 2010) and the average genetic distance within species was 0.41 % (Lakra et al. 2011). In the neighbor-joining tree both the species fall into same clade with a bootstrap value of 98 % (Khan et al. 2010; Lakra et al. 2011).

1.14 Mugilids

Mugilidae, the mullets or grey mullets are distributed worldwide in coastal temperate and tropical waters, with some species in fresh water. Mullets serve as an important source of food in coastal areas. The family includes about 76 species in 20 genera, although half of the species are in just two genera (Liza and Mugil). A total of 17 species have been reported from Indian waters, of which 14 species were barcoded. Rahman et al. (2013) reported that Canonical Analysis of Principal Coordinates (CAP) failed to separate the 10 species collected from Southeast Coast of India, which were clearly identified and differentiated using COI gene.

1.15 Ariids

 Ariidae is a family of  catfish, which are marine and esturine in nature, widely distributed in tropical to warm temperate zones. The family comprise about 143 species which includes 24 species from India, of which barcoding of 10 species was completed. Lakra et al. (2011) carried out the COI based molecular study of the catfishes of three genera namely Osteogeneiosus, Netuma and Arius under the family and observed the 0.23 % average K2P distance within species and 8.10 % within family. The NJ tree revealed two clusters, where first cluster is of Arius subrostratus and A. arius. The second cluster was shared by Netuma thalassinus and Osteogeneiosus militaris with a bootstrap value of 90 to 99 %.

1.16 Sphyraenids

The barracuda are marine ray-finned pelagic predatory fish of the genus Sphyraena, the only genus in the family Sphyraenidae. Globally there are more than 22 species of barracuda, So far seven species have been reported from Indian waters viz., S. barracuda, S. jello, S. putnamiae, S. qenie, S. forsteri, S. obtusata and S. novahollandae. All the species from Arabian Sea were characterized by NBFGR using DNA barcodes. Among the species examined, one was confirmed as new species and named Sphyraena arabiansis sp. nov. (Abdussamad et al. 2015). The intraspecies  genetic distance ranged from 0.000 to 0.007, while interspecies varied from 0.111 to 0.273. COI sequences of Sphyraena barracuda and Sphyraena arabiansis showed a clear barcode split (11.4 % divergence) congruent with morphological differences. The NJ tree revealed very distinct species clusters. The average interspecies distance among the seven species in the family sphyraenidae was 15.2 % (Jena et al. 2014).

1.17 Sharks and Rays

India is one of the leading Chondrichthyan fishing nations with an estimated landing of 46,471 tonnes (sharks 45.5 %, rays 49.5 % and skates 5 %) in 2013 that accounted for 1.23 % of the total marine fish landings in the country (CMFRI, 2013). Despite rich elasmobranch  diversity, only a few detailed studies have been undertaken that makes their management and conservation very difficult. Ward et al. (2005) validated the efficacy of COI barcodes for identifying chondrichthyans by sequencing 61 species of sharks and rays from Australian waters. Recent taxonomic studies that use both morphological and molecular markers resulted in taxonomic resolution of many species complexes and discovery of many new species. A total barcode record of 109 species are available of which 104 species were barcoded by NBFGR, India, representing 10 orders and 33 families of chondrichthyans from Indian waters. This is the largest study using genetic barcodes approach to identify sharks and rays found in the Indian waters. The average Kimura 2 parameter (K2P) distance separating individuals within species observed was 0.32 %, and the average distance separating species within genera 6.73 %. There are 37 and 29 species sequences generated in the present study representing new sequences for GenBank and BOLD respectively. During this study seven species were suggested as putative new species requiring formal descriptions and eleven elasmobranch species were confirmed first records for Indian waters. The present study uncovered the presence of eight undescribed or unrecognized batoid species including three in the genus Himantura. Barcode analysis result shows the presence of unrecognized species and highlights the need for further detailed taxonomic examinations of several families of elasmobranchs from Indian waters. Six species of Carcharinus were barcoded and phylogentic tree is given in Fig. 3. Barcode analysis shows very clear cut differentiation between these 6 species.

Fig. 3
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K2P distance neighbor-joining tree of COI sequence from six species of Carcharhinus

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1.18 Other Important Groups

Lizard fishes (Synodontidae), Puffers (Tetradontidae), Half beaks (Hemirahmphidae), Big eyes (Priacanthidae), Tongue fishes (Cynoglossidae), Butterfly fishes (Chaetodontidae), Mojarras (Gerreidae), Cutlass fishes (Trichuridae), Wrasses (Labridae), snake mackerels or escolars (Gempylidae), and Moray eels (Muraenidae) are some of the important groups of marine fishes, distributed along the coast of India. These groups of fishes contribute a substantial amount to total marine landing. The barcoding and species identification of this entire group using COI has already been taken up by several researchers, but in limited scale. Hence there is pressing need of DNA barcoding of all these groups along with other groups available in the country for effective conservation and management purpose.

2 Discussion

DNA barcoding has multiple implications in marine ecosystem; identification of species, cryptic species, larvae, new species, invasive species, illegal trade, biodiversity assessments, stock management, ecosystem monitoring, resolving taxonomic ambiguity and revisions of certain taxa, inference of phylogenetic relationships and speciation patterns (Hebert et al. 2003, 2004; Hogg and Hebert 2004; Jaafar et al. 2012; Trivedi et al. 2014; Trivedi et al. 2016). Molecular studies of selected species in the country using barcode sequences clearly discriminated taxonomic status of all the species examined and the NJ tree revealed identical phylogenetic relationship among the species. The phylogenetic relationship among the species was clearly established, and similar species were clustered under same clade while dissimilar species were clustered under separate clades with bootstrap values ranging from 90–100 %. Although barcode analysis seeks only to delineate species boundaries, there is clearly some phylogenetic signal in COI sequence data. Recent taxonomic research coupled with COI divergence analysis revealed discovery of many new marine fish species (Akhilesh et al. 2012; Bineesh et al. 2013; Greenfield et al. 2012). Sequence analysis of COI was used for resolution of taxonomic identity of many marine species in India (Bineesh et al. 2014). Based on COI sequence divergences a second species of Asian sea bass, Lates calcarifer is revealed (Ward et al. 2008; Vij et al. 2014). In addition to the species identification, DNA barcoding has been used for identification of protected whale shark, Rhyncodon typus (Sajeela et al. 2010). Barcoding is also being used for identifying processed seafood products (Nagalakshmi et al. 2016).

Despite rich natural biodiversity, comprising over 1518 native marine species, at present barcodes of just about 500 marine fish species are available, which is approximately 33 % of total Indian marine fish diversity. Whereas major portion of described marine fishes remain untouched. Hence more emphasis should be given to DNA barcoding, with mandate of barcoding all the species to establish global comprehensive reference libraries. The traditional taxonomists will play a vital role in completing such a global database; hence there is a pressing need to make a integration of DNA barcoding with traditional taxonomy. In nutshell it can be said that DNA barcoding can be taken up as pragmatic approach for resolving unambiguous identification of the fish fauna which can play a crucial role in biodiversity assessment and conservation of marine ecosystem of country.