barcoding phylogeography and species boundaries in clownfishes of the indian ocean

DNA Barcodes 2015; Volume 3 5–16 1 Introduction DNA barcoding is designed to provide with accurate species identifications through the use of molecular species tags based on short, standardised gene r[.]

 DNA Barcodes 2015; Volume 3: 5–16 Research Article Open Access Kottila Veettil Dhaneesh*, Thipramalai Thankappan Ajith Kumar, Appukuttannair Biju Kumar Barcoding, phylogeography and species boundaries in clownfishes of the Indian Ocean Abstract: In this study, barcoding of 13 clownfish species of the Indian Ocean was carried out to infer the phylogenetic relationships among them by analyzing cytochrome oxidase (CO1) and cytochrome b mitochondrial gene sequences The study also scrutinized species boundaries between four closely related species of the subgenus Phalerebus (Amphiprion akallopisos, A perideraion, A sandaracinos and A nigripes), three species of the subgenus Amphiprion (A frenatus, A melanopus and A ephippium) and two species of the subgenus Paramphiprion (A sebae and A polymnus) In addition, phylogeographic structure of A clarkii was calculated in terms of geographic isolation by phylogenetic analysis of mitochondrial control region and cytochrome b sequences The genetic distances between the subgenus Phalerebus species were 0.165-0.233 in control region and 0.021-0.065 in cytochrome b; and the genetic distance between the subgenus Amphiprion species was 0.122-0.171 in control region and 0.038-2.308 in cytochrome b Species of the subgenus Paramphiprion had a genetic distance of 0.016 in control region and 2.185 in cytochrome b A clarkii collected from four regions have genetic distance of 0.0190.06 (control region) and 0-0.025 (cytochrome b) Keywords: Anemonefish, species identification, molecular phylogeny, Amphiprion, Premnas Doi: 10.1515/dna-2015-0002 received January 31, 2014 accepted January 20, 2015 *Corresponding author: Kottila Veettil Dhaneesh: Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India, E-mail:dhanee121@gmail.com Thipramalai Thankappan Ajith Kumar: Centre of Advanced Study in Marine Biology, Faculty of Marine Sciences, Annamalai University, Parangipettai 608 502, Tamil Nadu, India Kottila Veettil Dhaneesh, Appukuttannair Biju Kumar: Department of Aquatic Biology and Fisheries, University of Kerala, Kariavattom, Thiruvananthapuram 695581, Kerala, India Thipramalai Thankappan Ajith Kumar: National Bureau of Fish Genetic Resources, Indian Council of Agricultural Research, Canal Ring Road, Dilkusha PO, Lucknow 226002, Uttar Pradesh, India Introduction DNA barcoding is designed to provide with accurate species identifications through the use of molecular species tags based on short, standardised gene regions [1] DNA barcode libraries are fully available as they are deposited in a major sequence database, and attached to a voucher specimen whose origin and current location are recorded [1] Once libraries are available, recent studies illustrate the vast array of applications that can be applied to forensic engineering [2], ecology of cryptic communities [3], tracking of invasive species [4], and identification of prey from predator stomach samples [5] The members of the sub-family Amphiprioninae (family Pomacentridae) are unique because all the species have an obligate symbiotic relationship with sea anemones 30 species of this subfamily are united by a number of morphological [6] and molecular [7, 8] characters However molecular studies on anemonefishes are scarce all over the world Studies available are mainly focused on taxonomy and evolution of the anemonefishes based on phylogenetic analysis of nuclear and mtDNA [9, 10, 11] Mitochondrial DNA (mtDNA) possesses several favourable characteristics, including large quantity in the cell, small genome size, maternal inheritance and extremely low probability of paternal leakage [12], higher mutation rate than nuclear DNA, and change mainly through mutation rather than recombination [13] For these reasons, mtDNA has been widely employed to address questions of genetic diversity, population structure, phylogeography and population evolution of animals [14] The 648-bp segment of the 5′ cytochrome c oxidase (CO1) gene sequences can act as a universal DNA marker for identification of animals (15) Besides, cytochrome b (Cyt b) has been considered as the most useful gene for phylogenetic work, and is probably the best known mitochondrial gene with respect to structure and function of its protein product [16] DNA barcoding works under the principle that inter-species variations are greater than the intra-species variations, allowing one to © 2015 Kottila Veettil Dhaneesh, et al., licensee De Gruyter Open This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivs 3.0 License Unauthenticated Download Date | 2/20/17 8:19 AM 6   K.V Dhaneesh et al distinguish the species using nucleotide sequences For the proper evaluation of the biodiversity, it is necessary to explain species boundaries, integrities and phylogenetic relationships [17] Many species of coral reef fishes are distinguished by their colour patterns, but genetic studies have shown that these are not always sufficient indicators of genetic isolation and species boundaries [18] Closely related species only show slight differences in their colour pattern in the subgenus Phalerebus, such as Amphiprion akallopisos and A sandaracinos; A perideraion and A nigripes Likewise, A frenatus and A melanopus of the sub genus Amphiprion also showed high similarity in colour pattern The sibling species A ocellaris and A percula showed more or less the same colour pattern, although A percula is described to have larger black bands in its colouration [19] Molecular techniques have become a major tool for systematic ichthyologists and may also be useful to fishery biologists for ratification of taxonomic problems at species and population levels [20] Due to advances in molecular biology techniques, large numbers of highly informative DNA markers have been developed for the identification of genetic polymorphism Hence, in the present study, sequence data from a portion of the CO1 and Cyt b gene were used to infer the phylogenetic relationships among representative species of clownfishes, to investigate the species boundaries and phylogenetic relationships of the clownfishes, and the mtDNA diversity among A clarkii in four different regions of the Indian Ocean This analysis may resolve the phylogenetic position of 13 species of clownfishes, providing with a more robust estimate of pomacentrid phylogeny and to find out the reason behind the high morphological similarity among the clownfishes and whether these species form distinct genetic clades within the subgenera Materials and methods 2.1 Sample collection Clownfishes (Fig 1) were collected by hand-nets while snorkeling and SCUBA diving from four different regions (Agatti island of Lakshadweep, Van island of Gulf of Mannar, Port Blair of Andaman and Bali island of Indonesia) located in the Indian Ocean (Fig 2) After collection, the live fishes were transported to the marine ornamental fish hatchery at Centre of Advanced Study in Marine Biology, Annamalai University, Tamil Nadu (India) A few specimens were also shifted after preserving in 95% ethanol Fishes from Bali Island of Indonesia were collected from the authorized marine ornamental fish trader A fin clip from the caudal and dorsal fin was taken and the live fishes were released to their host anemones It was therefore possible to obtain tissue samples without sacrificing the animals The samples were preserved in 95% ethanol and stored at °C Single specimens of yellowtail clownfish A clarkii (Fig 3) collected from four different regions in Indian Ocean were used to analyze the geographic variation of the species The meristic characters (D.X, 15-16; A.II, 13-14; P.19-20; V.I, 5) of all the four specimens were similar but showed slight colour variation and appearance 2.2 DNA isolation Total DNA was extracted from 50-100 mg of caudal fin tissue using standardised salting out protocol [21] The extracted DNA acted as a template for Polymerase Chain Reaction (PCR) The quality and concentration of DNA was checked on % agarose gel (dissolve agarose in X TAE) 2.3 Polymerase Chain Reaction For the barcoding study, two types of primers for CO1 and cytochrome b were used for PCR amplification CO1 primers such as FishF1 (5′-TCA ACC AAC CAC AAA GAC ATT GGC AC-3′) and FishR1 (5′-TAG ACT TCT GGG TGG CCA AAG AAT CA-3′) [22] were used for the amplification of CO1 gene The fragment of CO1 at maximum length of 648 bp was amplified by using Genie TL – 9700 Mastercycler with the following thermo-profile: initial denaturation at 95 °C for min, 35 cycles at 94 °C for 30 sec, annealing at 54 °C for 30 sec and extension at 72 °C for min, with a final extension at 72 °C for 10 min, followed by indefinite hold at °C For the amplification of the cytochrome b fragment of around 400 bp length, the primers tRNAgluF (AAA ACC ACC GTT GTT ATT CAA CTA CA; [23]) and H15149 (AAA CTG CAG CCC CTC AGA ATG ATA TTT GTC CTC A; [24]) were used with following programme of 95 °C for min, 35 cycles of 95 °C for 45 s, 58 °C for 45 s and 72 °C for 60 s The final elongation was 72 °C for A fragment with a maximum length of 420 bp of the mitochondrial control region (CR) was amplified with the primers CR-A (TTC CAC CTC TAA CTC CCA AAG CTA G) and CR-E (CCT GAA GTA GGA ACC AGA TG) [25] Target regions of mtDNA were amplified using the following thermal cycling profile at 95 °C initial denaturing for min, followed by 35 cycles of 95 °C denaturing for 30 s, 50 °C annealing for 30 s, 72 °C extension for 60 s The terminal elongation was at 72 °C for Unauthenticated Download Date | 2/20/17 8:19 AM  Barcoding and phylogeography of clownfishes  Figure 1: Clownfishes of Indian Ocean [(a) A polymnus, (b) A sebae, (c) A ocellaris, (d) A percula, (e) A frenatus, (f) A melanopus, (g) A sandaracinos, (h) A clarkii, (i) A nigripes, (j) A perideraion, (k) A akallopisos, (l) A ephippium and (m) P biaculeatus] Unauthenticated Download Date | 2/20/17 8:19 AM  7 8   K.V Dhaneesh et al Figure 2: Sampling areas in Indian Ocean Figure 3: A clarkii collected from (a) Van Island (Gulf of Mannar), (b) Agatti Island (Lakshadweep), (c) Bali Island (Indonesia) and (d) Port Blair (Andaman) All PCR products were checked on % agarose gel and samples with good results were purified with the QIA-quick PCR Purification Kit (Qiagen) Sequencing was conducted with the PCR primers by Dideoxy Sanger standard method in an automated ABI sequencer at Ramachandra Innovis, Chennai (India) The obtained sequences were edited based on the chromatogram peak clarities To obtain the similar sequence from NCBI database, BLAST program was performed Sequences were submitted to the NCBI GenBank through sequin 2.4 Sequence alignment and phylogenetic analyses Sequence alignment was done using ClustalW of MEGA version (Molecular Evolutionary Genetics Analysis) Phylogenetic analysis was conducted using MEGA version [26] Sequence divergences were calculated using the Maximum Composite Likelihood The statistical confidences were evaluated by 1000 non-parametric bootstrap replicates for ML analysis Unauthenticated Download Date | 2/20/17 8:19 AM  Barcoding and phylogeography of clownfishes  Results 3.1 Sequence features Amplified cytochrome oxidase subunit-1 (CO1) and cytochrome b (Cyt b) genes of 13 species of clownfishes were sequenced and the accession numbers are given in Table Sequence length of CO1 averaged 681 bp (range = 465 - 761 bp), and 92% of the amplified sequences were larger than 600 bp In the case of Cyt b, the sequence length ranged from 294 to 430 bp with an average of 406 bp, and 85% of the amplified sequences were larger than 350 bp For the study of phylogeography and species boundary, control region (CR) and Cyt b genes were amplified and sequenced CR fragment of average 373 bp (range = 355 398 bp) and Cyt b of average 400 bp (range = 288 - 430 bp) sequence length containing 13 sequences from ten species were obtained (Table 2) 100% of the amplified sequences of CR and 69% of the Cyt b sequences were larger than 350 bp The transition/transversion ratio (Ts/Tv ratio) of the sequences was calculated by the MEGA software (Version 6) The Ts/Tv ratio of data set of CO1, Cyt b and CR was 2.89, 2.69 and 2.05 respectively GC content in the sequences of CO1, Cyt b and CR genes was calculated by BioEdit software In CO1 region, maximum GC content was found in P biaculeatus (48.52%) and minimum was observed in A clarkii (43.76%) Regarding Cyt b region, the GC content ranged between  9 41.45 (A perideraion) and 47.03% (A frenatus) In CR region, it ranged between 27.86 (A clarkii of Lakshadweep) and 32.64% (A perideraion of Andaman) The average GC content in CO1, Cyt b and CR region was 45.74 ± 0.01%, 45.67 ± 0.01% and 30.17 ± 0.01% respectively 3.2 Phylogenetic analysis The evolutionary history of clownfishes was inferred using the Maximum Likelihood method (Figs and 5) All related specimens formed cohesive units and were separated from each other in the ML tree Similar species such as A melanopus, A frenatus and A ephippium were grouped in neighboring clades along with their identical species of GenBank Similarly, A sebae, A clarkii and A polymnus were also claded with similar species taken from database Premnas biaculeatus was clustered with similar one with highest bootstrap value (100%) Identical species A ocellaris and A percula were also grouped with similar species in adjoining clades with higher bootstrap values (100 and 99% respectively in CO1 but 78% in Cyt b) Phylogenetic analysis of the CR dataset is presented as a Maximum Likelihood cladogram with bootstrap values (Fig 6), showing a grouping of all species in four main clades One clade contained A frenatus, A melanopus and A ephippium and the other had A polymnus and A sebae Third clade contained A nigripes, A perideraion, A sandaracinos and A akallopisos with comparatively weaker bootstrap values but claded together Three Table 1: Sampled individuals of clownfishes of Indian Ocean, abbreviations used, accession numbers and sampled locations Species Location A polymnus Port Blair (Andaman) A sebae Port Blair (Andaman) A ocellaris A percula Abbreviations Accession number of CO1 Accession number of cytochrome b Apo JX975292 KP151502 ASe JX987298 JX163863 Port Blair (Andaman) AO JX548323 JQ314452 Port Blair (Andaman) APl JX573170 JQ314454 A frenatus Port Blair (Andaman) AF JX901062 KP151503 A melanopus Port Blair (Andaman) AM JX548321 JX233779 A sandaracinos Port Blair (Andaman) AS JX548320 JX035793 A clarkii Van Island (Gulf of Mannar) AC JX573169 JX233781 A nigripes Agatti Island (Lakshadweep) AN JX573171 JN637372 A perideraion Port Blair (Andaman) AP JX548324 JX233778 A akallopisos Port Blair (Andaman) AAk JX975291 JQ314453 A ephippium Port Blair (Andaman) AE JX987299 JQ314455 P biaculeatus Port Blair (Andaman) PB JX548322 JX233780 Unauthenticated Download Date | 2/20/17 8:19 AM 10   K.V Dhaneesh et al Table 2: Clownfishes sampled from four regions of Indian Ocean [abbreviations and their accession numbers] Species Location Abbreviations Accession number of CR Accession number of cytochrome b Saddleback complex A polymnus Port Blair (Andaman) Apo JX417170 KP151502 A sebae Port Blair (Andaman) ASe JX417172 JX163863 A akallopisos Port Blair (Andaman) AAk JX417178 JQ314453 A nigripes Agatti Island (Lakshadweep) AN JX417175 JN637372 A perideraion Port Blair (Andaman) AP JX417173 JX233778 A sandaracinos Port Blair (Andaman) AS JX417171 JX035793 A frenatus Port Blair (Andaman) AF JX901059 KP151503 A melanopus Port Blair (Andaman) AM JX417174 JX233779 A ephippium Port Blair (Andaman) AE JX417179 JQ314455 A clarkii Van Island (Gulf of Mannar) AC-I JX417176 JX233781 A clarkii Agatti Island (Lakshadweep) AC-AGT JX901061 JX901064 A clarkii Port Blair (Andaman) AC-ADM JX901060 JX901063 A clarkii Bali Island (Indonesia) AC-B JX417177 JX233777 Skunk complex Tomato complex Clarkii Figure 4: Maximum Likelihood (ML) tree of cytochrome oxidase subunit (CO1) sequences of clownfishes from the Indian waters along with the similar sequences Red colour indicates the sequences of the present study Unauthenticated Download Date | 2/20/17 8:19 AM  Barcoding and phylogeography of clownfishes   11 Figure 5: Maximum Likelihood (ML) tree of cytochrome b (Cyt b) sequences of clownfishes from the Indian waters along with the similar sequences Red colour indicates the sequences of the present study Figure 6: Maximum Likelihood tree of control region (CR) sequences of clownfishes from the genus Amphiprion in the Indian Ocean Unauthenticated Download Date | 2/20/17 8:19 AM 12   K.V Dhaneesh et al specimens of A clarkii were grouped in the fourth clade and to this, a fourth specimen of A clarkii was joined with high boot strap value (100%) Phylogenetic tree of Cyt b sequences was constructed according to the method of Maximum Likelihood (Fig 7) The species from sub genus Phalerebus were claded together and A clarkii from four regions were also joined together 3.3 Genetic distance Pair-wise genetic distance of each species was done based on pair-wise distance analysis using Maximum Composite Likelihood method A frenatus had genetically lesser distance with A melanopus (0.0074) followed by A ephippium (0.0099), in which all three belong to subgenus Amphiprion A percula had lesser distance with its counterpart A ocellaris (0.0379) A clarkii had lesser distance with A sebae (0.0251) while maximum distance with P biaculeatus (0.1328) A nigripes had lowest genetic distance with all species of subgenus Amphiprion (0.04) followed by A sandaracinos (0.0564), A perideraion (0.0785) and A akallopisos (0.0895) Genetic distance of P biaculeatus was less with A ephippium (0.1125) and maximum with A sebae (0.1361) Average evolutionary divergence over all sequence pairs (CO1) is 0.084±0.01 According to the Cyt b sequences, A clarkii showed minimum genetic distance with A sebae (0.078) while A perideraion had minimum genetic distance with A akallopisos (0.0310), A nigripes (0.04), A sandaracinos (0.017) respectively A melanopus was less distant with A ephippium (0.031) A ocellaris and A percula have not shown any genetic difference The base differences per sequence averaging from over all sequence pairs (overall mean distance) of Cyt b was 0.924±0.41 Based on CR region, among the A clarkii, specimen collected from Gulf of Mannar had maximum distance with the specimen of Lakshadweep Islands (0.046) and less distance with that of Andaman Islands (0.019) and Bali Island (0.023) A frenatus has less distance with A melanopus (0.122) and maximum with A perideraion (0.223) Among subgenus Phalerebus, A perideraion has lowest distance with A akallopisos (0.165) followed by A sandaracinos (0.171) A polymnus has lowest genetic distance with its associate of subgenus Paramphiprion A sebae (0.016) Based on cytochrome b sequences, among A clarkii, specimen of Gulf of Mannar had least genetic distance with that of Andaman and Bali Island compared to Lakshadweep Island In the case of members of Phalerebus, A sandaracinos has least genetic distance with A perideraion (0.021) followed by A akallopisos (0.051) and A nigripes (0.060) In subgenus Amphiprion, A ephippium has least distance with A melanopus (0.038) The overall mean distance among the CR sequences was 0.224 ± 0.034 and among Cyt b sequences, it was 0.665 ± 0.354 Discussion DNA barcoding is an efficient method for species-level identifications using an array of species specific molecular tags derived from the 5’ region of the mitochondrial cytochrome oxidase (CO1) gene In this context, identification of fish species with similar appearance is challenging and DNA barcoding provides with new perspectives in ecology and systematics of fishes Present study has strongly validated the efficacy of CO1 barcodes for identifying the fish species and also for phylogeny Figure 7: Maximum Likelihood tree of cytochrome b (Cyt b) sequences of clownfishes from the genus Amphiprion in the Indian Ocean Unauthenticated Download Date | 2/20/17 8:19 AM  It has been reported that lineages diversify more quickly within species than between species [5] This is due to the fact that diversification within species is driven by mutation at a rate higher than speciation within lineages Hence, the branch length between species tends to be much deeper than that of conspecific individuals leading to a gap in the distribution of the pairwise distance between conspecific individuals and between species that have been referred to the barcoding gap [27] The CO1 locus harbours a high mutational rate even for mtDNA [28] Present study confirms that, in the vast majority of taxa examined, barcoding gap exists and the mean genetic distance was generally much smaller than the average distance between the individual species In this study, 13 species of clownfishes were sequenced for the barcode region of CO1 and cytochrome b (Cyt b), with no exceptions all the sequenced species could be discriminated and all the species were amplified with specific primer sets GC content of the mitochondrial CO1 region was higher in P biaculeatus (48.52%) and those of Cyt b region were higher in A frenatus (47.03%) Average GC content in CO1 region was 45.74 ± 0.01% and in Cyt b region, it was 45.67 ± 0.01% Saccone et al [28] reviewed the complete mitochondrial genomes of nine Osteichthyes and three Chondrichthyes species, deriving GC contents of 43.2% and 38.4%, respectively These values correspond reasonably well with the present study, especially with respect to the higher GC content of the teleosts CO1 barcoding for species identification is far more powerful than protein fingerprinting Reliable discrimination of Thunnus species using conventional protein electrophoresis is hard [29], but Ward et al [22] found that the same samples were readily identified by CO1 sequencing However, methodologies for phylogeny reconstruction from molecular data remain somewhat controversial [30] It may not be possible to trace the true phylogeny of fishes from a 648 bp fragment of mitochondrial DNA through K2P distance Rather, more gene regions (including nuclear genes) and additional analytic methods should be used Mutation rate variation among genus and species has been recognized as a major problem for sequence divergence estimation and phylogenetic reconstructions [31], and may sometimes hinder the use of the DNA barcoding for cataloguing species diversity [32] This appears to be the reason for low levels of sequence divergence obtained in the present study among the sequences of clownfishes (CO1, 0.08% and Cyt b, 0.10%) Present results reveal that CO1 barcoding will permit the unambiguous identification of the vast majority of fish species With increasing application of DNA barcoding, Barcoding and phylogeography of clownfishes   13 many previously unrecognized fish species will be revealed through the discovery of deep divergence of CO1 sequences within currently recognised species There might also be instances of supposedly distinct species that have identical CO1 sequences, suggesting the possibility of species fusion Resolution of cases of this nature will require careful morphological analysis from expert taxonomists before any final recommendations can be made One way to evaluate the phylogenetic efficiency of a gene tree is by assessing congruence with a well-supported phylogeny based on independently derived data sets Frequently, this has involved comparisons among molecular phylogenies based on morphological data [33] However, tests of similarity have been increasingly used to compare several molecular phylogenetic estimates with one another [34] Therefore, main goal in the present study is to explore the utility of mtCR and Cyt b and to infer phylogenetic relationships at various taxonomic levels in the family Pomacentridae by assessing congruence among data sets Molecular phylogenetic analyses of closely related species give insights into their relationships and allow verifying their morphological taxonomic classification Sometimes, such studies indicate that the previously assumed classification is wrong or not sufficient This was the case in a study on the Dascyllus trimaculatus species complex [35], in which the authors reported inconsistence between morphological and colouration traits, as well as their molecular phylogenetic relationships Additionally, such studies can reveal the divergence and speciation processes, as well as geographic locations of these events 4.1 Amphiprion clarkii Phylogeographic structure of A clarkii is varied in terms of geographic isolation The genetic similarity within a population is expected to increase under certain conditions, such as inbreeding and non-random mating [23] Genetic variation is also thought to increase with effective population size Based on the phylogenetic tree (CR), the A clarkii specimen of the Gulf of Mannar was genetically closer to the specimen of Andaman; A clarkii of the Lakshadweep Islands showed more genetic similarity with the specimens collected from the Gulf of Mannar and Indonesia; and specimen from the Andaman waters showed more similarity with those of Gulf of Mannar Based on the phylogenetic tree of Cyt b sequences, A clarkii from the Gulf of Mannar got joined to the similar species of Bali Phylogeographic structure of A clarkii cannot be readily explained in terms of larval or adult Unauthenticated Download Date | 2/20/17 8:19 AM 14   K.V Dhaneesh et al dispersal Besides, the seasonal changes in the directions and speed of the circulating ocean currents would also have important bearing on plankton dynamics [23] Support for widespread, long-range dispersal of Pacific reef fishes is derived from genetic studies based on protein or isoenzyme polymorphisms Ehrlich [36] found that pomacentrids consistently showed no allelic variation even in populations separated by as much as 3,000 km Shaklee [37] found significant differentiation in the damselfish Stegastes fasciolatus throughout the 2,500 km range of the Hawaiian Islands Bell et al [38] carried out a pioneering study regarding the genetic structuring in damselfish with a pelagic phase, in which they reported significant allelic differentiation in A clarkii, over a 1,500 km range in Japan In this study, analogous to this, A clarkii throughout the 5,000 km range in the Indian Ocean (Lakshadweep to Indonesia) showed substantial genetic variation within the species Genetic distance of A clarkii from Andaman was more compared with other specimens of the same species collected from other geographical areas While genetic distance was low among specimens of Lakshadweep, Gulf of Mannar and Bali This was parallel to the result of phylogenetic trees of both primers and therefore it can be concluded that the variations could be present within the same species with respect to the features of their geographical areas It is very clear that, A clarkii dwelling in the Andaman waters was genetically more distant than the other three Gene flow among A clarkii populations is possible if the reef systems are strongly connected by ocean currents, although A clarkii is not considered to be a high dispersal species because of its relatively short larval period 4.2 Skunk complex (Amphiprion akallopisos/ perideraion/ sandaracinos/ nigripes) In this study, morphological characters and colouration pattern of A akallopisos, A perideraion, A sandaracinos and A nigripes were supported by the molecular phylogenetic analysis Comparably low sequence divergences in this group confirmed the close relatedness of these species Based on the colouration pattern, it is inferred that A akallopisos and A sandaracinos are sister species and A perideraion and A nigripes are other group of sister species Phylogenetic trees revealed that A akallopisos, A sandaracinos, A perideraion and A nigripes are more closely related This is in agreement to the findings of previous studies on the molecular phylogeny of anemonefishes [10, 39] CR divergence times were within the ranges calculated for Cyt b It showed a consistency for the divergence times between the two markers A akallopisos, A sandaracinos, A nigripes and A perideraion were commonly found in the Indian Ocean and the Pacific Ocean (except A nigripes) The sea level low stands between the Pacific and Indian Ocean might have triggered the divergence and speciation in the anemonefishes [39] 4.3 Tomato complex (Amphiprion frenatus/ melanopus/ephippium) All the three species were collected from the shallow waters of Andaman close to the coastline Based on the phylogenetic analysis (CR), specimens of A frenatus was claded with A melanopus which was accurate to their morphological similarities and A ephippium was joined to this According to sequence identity matrix (CR), A frenatus and A ephippium were more identical to A melanopus But according to Cyt b sequences, the specimens of A ephippium and A melanopus were more similar to A nigripes Probably, there is a tendency of mutation saturation present for the strong diverged clades for the Cyt b gene [40] and in the highly variable CR sequences as well, indicated by the comparably small Ts/Tv ratios in both markers (3.9 in labroid fishes [41]; in lionfishes [42]; 3.93 in cichlid fishes [40]) On the other hand, the genetic distances between the Amphiprion frenatus/ melanopus/ ephippium species complex were smaller than between the species in the subgenus Phalerebus Sequence divergences of 0.122– 0.17 (CR) present between the species of tomato complex are quite at the genus level This finding is supported by comparison with the lower genetic distances among the clearly distinct species in the subgenus Phalerebus (CR 0.165 – 0.233) 4.4 Saddleback complex (Amphiprion sebae/ polymnus) These two species from the Andaman waters were clearly claded together with highest bootstrap values (99%) by CR maximum likelihood tree Morphological resemblance and colouration pattern of A sebae and A polymnus were supported by the molecular phylogenetic analysis Additionally, the comparably low sequence divergences in this group confirmed the close relatedness of these species (0.016) A sebae was distributed throughout the entire Indian Ocean (Southern coast of Arabia, India, Sri Lanka, Maldives and Indian Ocean coasts of Sumatra and Java), however A polymnus was restricted to the Andaman Unauthenticated Download Date | 2/20/17 8:19 AM  waters and scattered from nearly located China to the Solomon Islands Within the A akallopisos/ perideraion/ sandaracinos/ nigripes species complex, A frenatus/ melanopus/ ephippium species complex and A sebae/ polymnus complex, the genetic clades follow with species boundaries, defined by the morphology and colour pattern Results of the present study revealed that the morphologically similar species of each subgenus of clownfishes are closely related which was supported by the molecular phylogeny The species of the two subgenera (Amphiprion and Phalerebus) are biologically very similar to each other They share the same food sources and behave and reproduce in a similar way [39] Conclusion Based on the present study, it can be stated that the CO1 barcoding will permit the unambiguous identification of vast majority of fish species With increasing application of DNA barcoding, many previously unrecognized fish species will be revealed through the discovery of deep divergence of CO1 sequences within currently recognised species Barcoding and morphological analysis should go hand-in-hand for species identification Phylogeography of A clarkii revealed that the variations were present within the species with respect to the features of their geographical areas It is very clear that, A clarkii dwelling in the Andaman waters was genetically more distant than the specimens of the Lakshadweep, the Gulf of Mannar and Bali Islands Within the Phalerebus species complex, Amphiprion species complex and Paramphiprion complex, genetic clades trail specific species limitations revealed by the morphological characters Results of this study disclose that the morphologically similar species of each subgenus of clownfishes are closely related, as supported by the molecular phylogeny data Acknowledgements: The authors are thankful to the authorities of Annamalai University for the facilities We are also thankful to Prof L Kannan for critically going through the manuscript and special thanks go to Dr M Gopi, Mr D Thiyagarajan, Dr V Karuppiah, Dr C Prasanna Kumar, Dr S Guru Prasad, Dr S Kumaran, Dr Subburaj, Dr Visruth Prem, Dr Muthazhakan and Dr Ramana Devi for their timely help The first author is grateful to University Grants Commission, Government of India, New Delhi for “UGC - Dr D S Kothari Post-Doctoral Fellowship” (Grant No BL/12-13/0365) Barcoding and phylogeography of clownfishes   15 References [1] Hebert P.D.N., Gregory 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Dendrochirus and Pterois (Scorpaenidae: Pteroidae) based on mitochondrial DNA sequences Mol Phyl Evol., 2003, 28, 396-403 Unauthenticated Download Date | 2/20/17 8:19 AM ... reveal that CO1 barcoding will permit the unambiguous identification of the vast majority of fish species With increasing application of DNA barcoding, Barcoding and phylogeography of clownfishes? ??... found in the Indian Ocean and the Pacific Ocean (except A nigripes) The sea level low stands between the Pacific and Indian Ocean might have triggered the divergence and speciation in the anemonefishes... regions (Agatti island of Lakshadweep, Van island of Gulf of Mannar, Port Blair of Andaman and Bali island of Indonesia) located in the Indian Ocean (Fig 2) After collection, the live fishes were