Indian Journal of Biotechnology Vol 16, July 2017, pp 315-321

DNA barcoding of selected (Infra Class: Teleostei) from Indian coast

N Sadurudeen, A Pavan-Kumar*, P Gireesh-Babu, A K Jaiswar, A Chaudhari, Gopal Krishna and W S Lakra ICAR-Central Institute of Fisheries Education (CIFE), Versova, Mumbai-400061, India

Received 22 October 2014; revised 21 September 2015; accepted 6 October 2015

India has a rich aquatic biodiversity spreading across different ecosystems. Total 2358 endemic have been reported from India comprising marine (1368) , brackish water (113) and freshwater fishes (877) . Marine fish identification is seldom difficult because of their high diversity and profound changes in appearance during their development stages. DNA barcoding technique has been successfully used to discriminate all taxa. In this study, DNA barcodes were generated for 32 species representing 13 families of the order perciformes. The average genetic divergence values for within species, genus and family were 0.42, 13.91 and 18.05%, respectively. The genetic divergence values increased several folds (15-20 times) from lower to higher taxa. Barcode gap analysis showed the absence of overlapping between intra and interspecific divergence values. Intraspecific variation has increased several folds (15-20 times) after including conspecific sequences from different geographical locations. Presence of allopatric lineages / cryptic species was observed in several fishes that have Indo-pacific region distribution. The neighbour joining (NJ) tree constructed based on mitochondrial cytochrome oxidase submit 1 kimura two parameter (COI K2P) values showed distinct clusters shared by congeneric species. Around 100 nucleotide diagnostic characters exclusive to Perciformes fish species were also identified.

Keywords: Perciformes, DNA barcoding, cytochrome c oxidase subunit 1, allopatric divergence

Introduction difficult taxonomic questions and diagnose species in India is one of the mega biodiverse countries of 34 cases where morphology alone has proved biodiversity global hotspots. Around 2,649 fish inadequate4. Mitochondrial partial cytochrome species were reported from India including freshwater c oxidase subunit I (COI) gene has been successfully (877) , brackish water (113), marine water (1368) and used as a standard gene for discriminating all animal exotic (291), which together accounts for 7.8% of the life because of its faster rate of evolution at the global finfish diversity1. Accurate identification of synonymous codon third base position5. In India, fish species is essential for documentation of diversity DNA barcoding technique has been successfully used and assessing the stock structure thereby formulating to document major groups of Indian fish diversity6-9. management and conservation measures. Perciformes is the largest order in fishes and Traditionally, fish species identification is relied upon comprises nearly 40% of bony fishes10. Previous external morphological features, including body studies on this group from India have not included shape, pattern of colors, scale size and count, fin much diversity of these fishes and have not addressed number and its relative positions, numbers and types COI gene divergence pattern as geographical distance of fin rays and various relative measurements of the increases between conspecific individuals6. With this body parts2. However, these methods are constrained background, the present study was carried to document by phenotypic plasticity, life-stage specific the commercially important Perciformes fishes through identification cues and the occurrence of cryptic DNA barcoding and to investigate the pattern and level species3. These limitations of traditional led of divergence at COI locus between conspecific to the development of new methods such as DNA individuals collected from different locations. based taxonomy. DNA sequence data from mitochondrial genome have long been used to resolve Material and Methods Sample Collection —————— *Author for correspondence Around 86 specimens of order: Perciformes were Tel: 022-26361447 Ext. 452 collected from different landing centres of East and [email protected] West coast of India (Table 1). Wherever possible, 316 INDIAN J BIOTECHNOL, JULY 2017

more than two individuals per species were collected Genomic DNA isolation, PCR amplification and purification and the species were identified at the field by Total genomic DNA was isolated by salting out 12 observing morphological and meristic characters of method with slight modifications. Mitochondrial the specimens11. Voucher specimens were prepared by partial cytochrome c oxidase subunit I (COI) gene preserving the specimen in absolute alcohol with was amplified using reported universal primers, proper labelling. Fins and muscle tissues were collected Fish F1: 5’-TCAACCAACCACAAAGACATTGGC under aseptic conditions and preserved in absolute AC-3’; Fish R1: 5’-TAGACT TCTGGGTGGCCAA alcohol in the field, then deeply frozen at -80° C in the AGAATCA-3’; Fish F2: 5’-TCGACTAATCATAAA laboratory for further analysis. In addition, reported GATATCGGCAC-3’ and Fish R2: 5’-ACTTCA mitochondrial cytochrome c oxidase subunit I (COI) GGGTGACCGAAGAATCAGAA-3’. PCR was gene sequences for selected conspecific individuals performed in 25 μl reaction volume containing 100 ng were downloaded from GenBank National Centre for template DNA, 10 pmol of each specific primer, Biotechnology Information (NCBI), to calculate 200 μM of each dNTPs, 1.0 unit of Taq DNA divergence values between populations. polymerase and 1X Taq buffer containing 1.5 mM

Table 1 — List of species along with GenBank/BOLD accession numbers

S. No Family Species (number of specimen) GenBank Acc. Number / BOLD BIN 1. Carangidae Atropus atropus (1) KJ920133 2. Megalaspis cordyla (2) KJ920111, KM079290 3. Selaroides leptolepis (2) KJ920124, KM079293 4. Gnathanodon speciosus (2) KJ920129, KM079289 5. crocineus (3) KJ920118, KM079301-302 6. Lethrinus lentjan (2) KJ920116, KM079303 7. Lethrinus mahsena (3) KJ920117, KM079304-305 8. Lethrinus microdon (4) KJ920114, KM079306-308 9. Lethrinus nebulosus (3) KJ920115, KM079309-310 10. Lethrinus ornatus (4) KJ920113, KM079311-313 11. Epinephelus diacanthus (3) KJ920101, KM079296-97 12. Epinephelus longispinis (2) KJ920103, KM079298 13. Epinephelus undulosus (3) KJ920104, KM079299-300 14. Pogonoperca ocellata (1) KJ920102 15. Lutjanidae Lutjanus lutjanus (2) KJ920132, KM079315 16. Lutjanus russellii (3) KJ920119, KM079316-17 17. Lutjanus sebae (2) KJ920120, KM079314 18. Mullidae Mulloidichthys vanicolensis (4) KJ920106, KM079318-320 19. heptacanthus (4) KJ920105, KM079321-323 20. Parupeneus indicus (4) KJ920109, KM079324, KM079339-40 21. Upeneus moluccensis (4) KJ920110, KJ920112, KM079325-326 22. Upeneus sulphureus (4) KJ920107, KM079327-329. 23. Upeneus tragula (3) KJ920108, KM079330-331 24. jarbua (3) KJ920134, KM079294-295 25. Terapon puta (1) KJ920126 26. Stromatidae Pampus argenteus (2) KJ920121, KM079333 27. Pomacanthidae Pomacanthus semicirculatus (1) KJ920131 28. Latidae Psammoperca waigiensis (02) KJ920125, KM079334 29. Scatophagidae Scatophagus argus (3) KJ920130, KM079335-36 30. Nemipteridae Scolopsis bimaculatus (3) KJ920127, KM079337-38 31. Scombridae Scomberomorus commerson (4) KJ920122, KJ920128, KM079291-92 32. Drepaneidae punctata (2) KJ920123, KM079332 SADURUDEEN et al :DNA BARCODING OF PERCIFORMES FISHES 317

MgCl2. The thermocycler was programmed for initial position. The number of transitional pairs (Si = 69) denaturation at 95°C for 5 min, followed by 35 cycles were more than transversional pairs (Sv = 46) with an of 94°C for 30 sec, 54°C for 30 sec, 72°C for 1 min average ratio of 1.50. for denaturation, annealing and extension, with final The average distance values (K2P) of COI gene was extension at 72°C for 10 min. The PCR amplification increased from lower taxa towards the higher products were purified using gel extraction kit taxonomic rank (i.e. within species, genera, family). (Qiagen, Germany) following manufacturer’s The average interspecific distance value (D = 13.91%) protocols. The purified PCR products were sequenced was ~33 times higher than the average intraspecific directly using PCR primers. For all the genes, distance (D = 0.42%). whereas, the mean divergence sequencing was performed in both directions for value among genera within families was 18.05% better accuracy using ABI Big DYE terminator (Table 2). DNA barcode gap analysis showed that all method (Eurofins lab, Bangalore, India) . species were distinct from their nearest neighbour

Sequence Analysis (NN) species (Fig. 1). The COI partial gene sequences obtained for each The level and pattern of average barcode species were manually assembled using Gene Runner divergence for selected fish species (15) collected V 3.0 software. Assembled sequences were end- from different geographical locations showed less trimmed to a homologous region to avoid sequencing intraspecific divergence value (0.0 - 0.017) within errors. All the COI gene sequences were aligned using populations than between populations (0.007 - 0.192). ClustalW program14. Only those sequences with more Mantel test results also showed significant correlation than 550 bp in size were used for the analysis. between geographical and genetic distances (Table 3 Sequence divergence values within and between & Fig. 2). species were calculated using Kimura two parameter The nucleotide diagnostic (ND) approach was (K2P) distance model implemented in MEGA V.5.2 applied to COI gene to strengthen the utility of the software15. DNA barcode gap, the difference between DNA barcoding technique to identify species. In this maximum intraspecific distance value to minimum approach, species diagnosis is based on the presence interspecific distance was estimated by BOLD or absence of a diagnostic character (a DNA (Barcode of Life Database) engine as a test of the character), thereby preventing any ambiguity found in reliability for species discrimination. The neighbour- an analogue measurement of sequence similarity. joining (NJ) tree16 using COI K2P distance values was Around 100 diagnostic characters specific to created to provide a graphical representation of the respective fishes were identified (Table 4). divergence pattern between species and haplotypes Table 2 — Distance (K2P model) values (%) of COI gene with 1000 bootstrap replications. The relationship across Perciformes fishes. between geographical distance and intraspecific genetic divergence value was analysed using the Comparison N Taxa Comparisons Min Mean Max S.E Mantel test17 implemented in XLSTAT (version Within Species 79 27 85 0 0.421.81 0.01 2013.3.02; Addinsoft, Inc., NY, USA). between specimens

Results Within Genus 55 6 232 6.8613.9121.130.02 between species DNA barcodes were generated for 86 specimens representing 32 species and 13 families of the order Within family 38 3 178 7.6918.0525.010.02 between Genus Perciformes. The absence of insertions, deletions and stop codons in the COI gene sequences confirm that NUMTs (nuclear DNA sequences originating from mitochondrial DNA sequences) were not sequenced. All the sequences were submitted to NCBI GenBank and BOLD database (Table 1). The average GC content value (%) of COI gene across all collected fishes was 47.33 ± 0.22. Among species, GC content varies significantly for codon 3rd base position than for codon 1st and 2nd base position. This showed the nd absence of synonymous mutations at codon 2 base Fig. 1 — Scattered plot depicting COI gene barcode gap 318 INDIAN J BIOTECHNOL, JULY 2017

Table 3 — Maximum intraspecific distances for selected Perciformes fish species with barcode records from other nations. No. Species Maximum Intraspecific Countries with matches Mantel correlation distance values (K2P) statistics for geographical vs India Combined genetic distances (Specimen (Specimen (α=0.05) no.) no.) 1. Atropus atropos 0.005 (06) 0.025 (17) Malaysia (HQ560973, HQ560989, HQ560998, JX261490, r = 0.405; JX261352, JX261287, JX261411, JX261357, JX261368, P < 0.0001 JX261095, JX261172); India (EF609502-06) 2. Drepane 0.00 (01) 0.007 (13) China (JN242498-500; EU595100-102;FJ237983-84); r = 0.842; punctata Australia (DQ107748-51) P < 0.0001 3. Epinephelus 0.00 (02) 0.017 (15) Andaman, India (JX675001-05); Kerala, India (EF609521- r = 0.129; longispinis 22); South Africa (JF493444, GU805000, JF493442, p = 0.083 KF489584); Mozambique (JF493443, HQ945868) 4. Gnathanodon 0.002 (4) 0.027 (12) Malaysia (JX261186, JX261431, JX261245, JX261536); r = 0.670; speciosus Mozambique (JF493544); Iran (HQ149855-56, ); Kerala, P = < 0.0001 India (WLIND071-07, WLIND073-07, WLIND070-07) 5. Lethrinus 0.017 (3) 0.103 (9) South Africa (JF493743-748) r = 1.000; crocineus P = < 0.0001 6. Lethrinus 0 (2) 0.187 (15) China, Hainan (FJ237798-802; Mozambique (JF493749); r = 0.679; lentjan Australia (EF609386); China Guangdong (EF607439 -440); P = < 0.0001 Iran (HQ149867- 69); Madagascar (JQ350087) 7. Lethrinus 0.009 (2) 0.180 (9) Japan (JF952782); Mozambique (JF493750-52); Gulf of r = 0.728; mahsena Mannar (EF609387); Madagascar (JQ350088-89) P = < 0.0001 8. Lutjanus 0.003 (3) 0.046 (12) Philippines (N021228, JN021225-27) ; Australia r = 0.730; russellii (EF609400); China (EU595216-17, ); India (WLIND520-07, P = < 0.0001 WLIND521-07) 9. Megalaspis 0.00 (2) 0.020 (19) Malaysia Perak (HQ560983); Malaysia Pahang (JX261418, r = 0.567; cordyla JX261356); South Africa (HQ561501, JF493869, JF493868, P = < 0.0001 HQ945872, JF493867, GU804934); Iran (HQ149884, HQ149883, HQ149881, HQ149882, EF609552, ); Maharashtra, India (EF609551, EF609549, ); Japan (JF952790) 10. Mulloidichthys 0.0 (4) 0.192 (13) China, Hainan (, FJ237827-29); China, Guangdong r = 0.374; vanicolensis (EF607607); South Africa, Kwazulu natal (JF493909, P = < 0.0001 GU805044, KF489653); Mozambique (JF493910) ; French (JQ431926) . 11. Pampus 0.017 (2) 0.142 (24) Vietnam (DQ107599); Myanmar (DQ107597, -598, r = 0.449; argenteus DQ107600, ); China Guangdong (, EF607457- 458, P = < 0.0001 EF607460-66, EU595224); India (KF373005-12); Malaysia Sandakan (DQ107596) . 12. Parupeneus 0.01 (4) 0.184 (17) Mozambique (JF494099-100, GU805086-87, HQ561494, r = 0.234; heptacanthus HQ561525); Australia (DQ107802-03, DQ107788-89); Iran P = 0.026 (HQ149894-96) . 13. Parupeneus 0.01 (4) 0.012 (21) China Hainan (FJ237871-83); Australia (DQ107797, r = 0.203; indicus DQ107800); South Africa (DQ885036-37) . P = 0.009 14. Scatophagus 0.007 (3) 0.070 (23) Philippines (JN021246-50) ; Gujarat, India (JX983493); r = 0.746; argus Australia (DQ107757, ); China, Guangdong (EF607516-19); P = < 0.0001 Srilanka (FJ584086-90); Kerala, India (EF609604-07) 15. Selaroides 0.0 (02) 0.02 (21) Malaysia, Sabah (JX261308, JX261649, JX261054, r = 0.201; leptolepis JX261099, JX261176, JX261454, JX261390, JX261440, P = < 0.0001 JX261491, JX261367); China, Guangdong (EF607546-50); Malaysia, Johor (JX261137 ); Malaysia, Pahang (JX261038, JX261620, JX261163) SADURUDEEN et al :DNA BARCODING OF PERCIFORMES FISHES 319

Discussion In the present study, 86 fishes representing 32 species have been documented through DNA barcodes and 10 species are the first report from India in BOLD database. Although the proposed standard DNA barcode length was 600 bp, several researchers reported that short partially conserved sequences of 250 bp of COI region could be sufficient to delimit 19,20 species . In the present study, the mean sequence / barcode length was in the range of 500-600 bp. In all metazoans, nucleotide (especially GC) content of COI gene 5’ region has a strong correlation with overall nucleotide (GC) content of full mitochondrial genomes and it can be analysed as nucleotide diversity21,22. In the present study, the average GC content of the partial mitochondrial COI gene region was 47.77% and this value was similar to the previous studies13. The GC content variations can

affect different codon positions to a greater or lesser Fig.2 — Correlation between Genetic distance (K2P) and degree22. In the present study, significant variation of geographical distance for selected fishes rd GC content was observed at the codon 3 base position Table 4 — Nucleotide Diagnostics for each species are listed, followed by 1st base position. This variation is due to with sample sizes included in parentheses after the species name. the fact that codon 2nd base position is more constraint Character states (nucleotides) at selected positions of the COI gene region than other base positions and most synonymous rd S. No Species (Number) Diagnostic characters mutations occur at the codon 3 base position, a few at st nd 1. Epinephelus diacanthus (3) 42-G;290-C;337-C;507- the 1 base position and none in 2 position. A;511-T;602-T;614-T Nuclear mitochondrial DNAs (NUMTs) are non- 2. Epinephelus undulosus (3) 49-G;170-A; 290-T;351- functional copies of mitochondrial sequences that T;390-A;507-G have become incorporated into the nuclear genome23 3. Parupeneus heptacanthus (4) 272-A; 400-C;412-G;461-G 4. Mulloidichthys vanicolensis 46-C;302-G;363-C;378- and their presence may lead to overestimation of (4) G;554-T species. In the present study, NUMTs have not been 5. Upeneus sulphureus (4) 363-T;605-T observed and it was evident by the absence of stop 6. Parupeneus indicus (4) 178-C;204-T;496-T;527-A codons in open reading frame of COI gene. Several 7. Upeneus moluccensis (4) 281-C;311-A;375-G; 587-G previous studies have also reported the absence of 8. Lethrinus ornatus (4) 275-G;412-C NUMTs in 6, 13. 9. Lethrinus microdon (4) 192-T;238-T;293-G;305- The mutations (purines to pyrimidines and vice G;357-G;434-G;498-G; 10. Lethrinus nebulosus (3) 207-T versa) accumulated within COI gene of each species 11. Lethrinus mahsena (3) 63-G; 366-G since speciation forms the basis for estimating 12. Lethrinus crocineus (3) 213-C; 542-A; 605-G divergence value between two species. For higher 13. Lutjanus russellii (3) 158-G;245-A;257-A;330- vertebrates, several studies have reported that the A;452-C;513-G transitional frequencies were relatively more than the 14. Scomberomorus commerson (4) 55-T;112-C;520-A;521-T transversional frequencies24, 25. In the present study 15. Scolopsis bimaculatus (3) 100-T;165-T;178-G;191- also, the average number of transitions was more than G;258-T;266-G;267- A;324-T;351-G;354- transversions with the transition / transversion rate G;459-A;480-C;545- (Ti/Tv) ratio of R = 1.50. Generally, large number of G;578-T;587-A transversional pairs than transitions shows deep 16. Scatophagus argus (3) 94-A;137-G;139-A;142- divergence between species and often with sequence A;195-G;406-T;446-T;456- saturation26. G;457-C;474-T;492-A;504- T;514-A;517-A;578-G;585-T DNA barcodes can discriminate species based on 17. Terapon jarbua (3) 334-T;387-C;474-C;623-G the magnitude of difference between intraspecific and 320 INDIAN J BIOTECHNOL, JULY 2017

interspecific distance value. To assign a specimen to presence of allopatric divergence between haplotypes. species or to delimit the species, intraspecific distance Allopatric speciation happens when gene flow values (K2P) should be less than the interspecific between populations hampered for several generations distance values. In this study, the average within due to physical or chemical barriers. The fluctuations species, between species and within families distance of sea levels during the Pleistocene epoch resulted in values were 0.42, 13.91 and 18.05%, respectively. the exposure of Sunda shelf thereby prevented the These values were slightly higher than the values of gene flow between Indian and Pacific Oceans 32. 0.39, 9.93 and 15.46% that are previously reported for Leray et al 33 also concluded that the coral reef fish marine fishes13. This clearly showed the presence of populations between Indian and Pacific Ocean regions sufficient DNA barcoding gap among Perciformes have significant variation and supported the presence fishes. The Neighbor Joining (NJ) tree also revealed of cryptic or allopatric lineages. distinct clades that were shared by conspecific De Salle et al 34 have proposed character based individuals while dissimilar species were clustered DNA barcoding to accurately identify species in cases under separate nodes. of insufficient taxon sampling. In this method, species In DNA barcoding, several analytical methods / can be identified through the presence of diagnostic algorithms (distance, character and phylogeny) have characters or combination of characters within short been proposed and used to discriminate the species27. stretches of DNA sequences35. In the current study, In the distance based method, COI gene sequence nucleotide diagnostic characters of COI gene dissimilarity among species would be calculated and exclusive to each species were identified by then compared with within species sequence comparing the COI gene sequences of all other fishes. dissimilarity. Based on these values, threshold limit These diagnostic characters would complement distance can be proposed, upon which specimen will be based method for species identification. Several assigned to species. Hebert et al 5 proposed a standard previous studies have also developed nucleotide barcode sequence threshold of 10 × the mean diagnostic markers and successfully discriminated intraspecific divergence value for declaring different groups of aquatic vertebrates18, 36. genetically divergent specimens as provisional In conclusion, DNA barcodes were developed for species. However, distance based DNA barcoding commercially important Perciformes fishes. Few of method could be effective only if the genetic these barcodes were the first reference barcodes from variability of a species across its distribution range is 28 Indian coast in BOLD database. These barcodes can calculated . Further, limited sampling size or be efficiently used as complement markers in the restricted sampling distribution may lead to over conventional taxonomic studies, fish stock assessment estimation or under estimation of genetic variability. and bio-surveillance. A few previous studies have included samples from diverse geographical regions and investigated the Acknowledgement levels of COI sequence divergence. These studies The authors are thankful to Dr S. Ayyappan, showed that the DNA barcodes can delimit species 29-31 Director General, and Indian Council of Agricultural even the sampling coverage extended . Research for the guidance and support. In the present study, the intraspecific variation for fish species viz., Mulloidichthys vanicolensis, References Lutjanus russellii, Lethrinus mahsena and 1 Jena J K & Gopalakrishnan A, Aquatic biodiversity Gnathanodon speciosus that have distribution across management in India, Proc Nat Aca Sci, India Sec B: Bio Sci, 82 (2012) 363-379. Indo-Pacific region has been increased several folds 2 Strauss R E & Bond C E, Taxonomic methods: morphology, after including conspecific sequences from other in Methods for Fish Biology, edited by P Moyle & geographical locations. This increase (13-20 times) C. Schreck (American Fisheries Society, Bethesda) 1990, was more than the proposed threshold level (10X) , 109-140. which was reported for provisionally declaring a 3 Callejas C& Ochando M D, Molecular identification (RAPD) specimen as a species. The Mantel test results also of the eight species of the genus Barbus (Cyprinidae) in the Iberian Peninsula, J Fish Biol, 59 (2001) 1589-1599. showed significant positive correlation between 4 Teletchea F, Molecular identification methods of fish genetic distance and geographic distance and prove species: reassessment and possible applications, Rev Fish strong isolation by distance. This could be due to Biol Fisheries, 19 (2009) 265-293. SADURUDEEN et al :DNA BARCODING OF PERCIFORMES FISHES 321

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