www.nature.com/scientificreports OPEN DNA Barcoding of Freshwater Fishes of Indo-Myanmar Biodiversity Hotspot Received: 30 January 2018 Anindya Sundar Barman, Mamta Singh, Soibam Khogen Singh, Himadri Saha, Yumlembam Accepted: 22 May 2018 Jackie Singh, Martina Laishram & Pramod Kumar Pandey Published: xx xx xxxx To develop an effective conservation and management strategy, it is required to assess the biodiversity status of an ecosystem, especially when we deal with Indo-Myanmar biodiversity hotspot. Importance of this reaches to an entirely different level as the hotspot represents the area of high endemism which is under continuous threat. Therefore, the need of the present study was conceptualized, dealing with molecular assessment of the fish fauna of Indo-Myanmar region, which covers the Indian states namely, Manipur, Meghalaya, Mizoram, and Nagaland. A total of 363 specimens, representing 109 species were collected and barcoded from the different rivers and their tributaries of the region. The analyses performed in the present study, i.e. Kimura 2-Parameter genetic divergence, Neighbor- Joining, Automated Barcode Gap Discovery and Bayesian Poisson Tree Processes suggest that DNA barcoding is an efficient and reliable tool for species identification. Most of the species were clearly delineated. However, presence of intra-specific and inter-specific genetic distance overlap in few species, revealed the existence of putative cryptic species. A reliable DNA barcode reference library, established in our study provides an adequate knowledge base to the groups of non-taxonomists, researchers, biodiversity managers and policy makers in sketching effective conservation measures for this ecosystem. Spread over an approximate expanse of two million square kilometers of tropical Asia, the Indo-Myanmar region is a kaleidoscope of an amazing geographical diversity and is among the top 10 biodiversity hotspots of the world. Stunning variation in terms of landforms and climatic zones supports an enormous variety of habitats, areas of endemism and plethora of biodiversity of the region. Ecology and lives of the people of the region are shaped by sweeping expanses of Asia’s mighty rivers such as Mekong, Chao Phraya, Irrawaddy, Salween, Chindwin, Sittaung, Red and Pearl (Information adapted from Ecosystem Profile of Indo-Burma Biodiversity Hotspot 2011 update, prepared by the Critical Ecosystem Partnership Fund, CEPF). Freshwater fishes support rural livelihoods in the region but they are among the most threatened organism in Indo-Myanmar region , due to unsustainable fishing practices, invasive species, and habitat alteration and loss. Further, level of threat to the entire river ecosystem of the region could drastically increase in near future due to several anthropogenic activities. Therefore, sustainable management of the aquatic resources of the region is desperately needed to conserve the icthyofauna. A prereq- uisite for this is a careful and accurate assessment of fish species to devise appropriate conservation measures for the target species. Paramount works have been done by several ichthyologists on exploration, but available data on the fish fauna of the region is incomplete and inconclusive. Reason for this includes a lack of extensive survey due to difficult hilly terrain, inaccessibility and prevalence of many indigenous languages/dialects for communication with var- ious ethnic communities of the region. A total of 291 species has been reported from the entire northeast India 2 3–5 as per the records of Zoological Survey of India . Drainage-wise exploration studies are scanty in the region . Majority of the studies on ichthyofauna of the region are limited to the description of new species and s fi h diver - 2,6–14 sity, entirely based on morphometric & meristic characters and phylogenetics . Further, species identic fi ation through morphometric and meristic characters oen le ft ads to misidentification, taxon ambiguities and fluctuation in species number. The main culprits for this include phenotypic and genotypic plasticity, cryptic diversity or pos- sible hidden species and variation in color pattern at different stages of life history of the same species. College of Fisheries (Central Agricultural University, Imphal), Lembucherra, Tripura (West), 799210, India. Correspondence and requests for materials should be addressed to A.S.B. (email: firstname.lastname@example.org) Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 1 www.nature.com/scientificreports/ Order-wise (%) All species Sl. No. Nucleotide (%) Ost Ang Clu Cyp Slu Sym Per 1 G 18.0 17.4 18.2 19.2 17.9 17.9 17.9 18.4 2 C 27.6 26.2 26.9 27.7 27.1 27.5 28.3 29.4 3 A 25.7 27.7 26.2 23.3 26.2 25.9 26.0 23.4 4 T 28.7 28.7 28.7 29.8 28.8 28.7 27.8 28.7 5 GC 45.6 43.6 45.1 46.9 45.0 45.4 46.2 47.8 6 GC1 44.0 43.4 43.9 43.9 44.2 43.9 44.0 43.7 7 GC2 36.0 31.2 33.7 39.5 34.2 35.0 37.6 44.0 8 GC3 56.7 56.4 57.9 57.4 56.8 57.3 57.2 55.9 Table 1. Nucleotide composition, overall and order wise GC content and GC at codon position 1, 2 & 3. Ost: Osteoglossiformes, Ang: Anguilliformes, Clu: Clupeiformes, Cyp: Cypriniformes, Slu: Siluriformes, Sym: Symbranchiformes, Per: Perciformes. Pioneers of DNA barcoding proposed the method of molecular taxonomy or systematic, employing stand- ardized and authenticated DNA based approach to provide accurate and automated species identification, using nucleotide sequence of partial region of mitochondrial gene, i.e. cytochrome oxidase I (COI) . Like several other methods, DNA barcoding approach was also debated and divided by two schools of researchers, one ques- 16–19 tioned the claims made by barcoding and another supported the taxonomic resolution efficiency, accuracy 20–29 and rapidity of the method . Further, use of DNA barcoding is not restricted to species identification only. It 5,25,30 31–33 also helps in flagging new species , identification of raw fish material used in the value-added products , identification of larvae and juveniles, collected from natural ecosystems for identification of breeding grounds 34,35 and assessment of die ff rent life history stages , identification of invasive alien species and forensics, including 36–38 illegal wildlife harvesting . In view of public interest, and in an effort to solve species substitution problem in the United States, US Food and Drug Administration (FDA) agency emphasized on the use of DNA barcoding of 39,40 s fi hes for regulatory compliance in export, import and marketing of seafood . e L Th ingua Franca of traditional taxonomic methods with DNA barcode gap estimation not only strengthens the species identification, but also oer ff s a new perspective to fish diversity assessment. Various automatic, fast and straightforward methods for performing species delimitation process, using specific bioinformatics analyses 41–43 44 like Generalized Mixed Yule Coalescent (GMYC) , Bayesian Poisson Tree Processes (bPTP) and Automatic Barcode Gap Discovery (ABGD) are widely used. These approaches have been widely applied to distinguish or 5,25–27,46–48 resolve taxonomic status of the specimen by several researchers . There is no comprehensive molecular assessment of fishes of river systems, in particular, Brahmaputra, Barak and Chindwin, located in the states of Meghalaya, Manipur, Mizoram and Nagaland. In the background of the above facts, this study was carried out with a view to establish a DNA barcode library and genetic diversity analyses for fish-fauna of four NE states of India (part of Indo-Myanmar Biodiversity hotspot). This study substantially aids in our understanding of fish diversity of the river systems of north-east India. Further, information generated through this study will provide an adequate knowledge base to non taxonomists, researchers, biodiversity managers and policy makers to sketch out effective conservation measures for this ecosystem. Result A total of 363 individuals, representing 109 morphologically identified species, were barcoded by amplification and nucleotide sequencing of partial 5′ region of the COI mitochondrial gene. The entire dataset of 363 COI sequences was represented by 109 species, 57 genera, 22 family and 7 orders. Out of 109 species, 37 were repre- sented by single specimens. We were not able to generate the good quality sequence reads for some of the species even aer r ft epeated attempts. Therefore, these species were excluded from the final analyses. All other amplified sequences were of >600 bp with no deletion, insertion or stop codon, indicating that they represented func- tional mitochondrial COI sequences. Multiple sequences were analyzed, in the studied species, to infer per cent intra-specific divergence (mean = 3.33 specimens per species, range 1–22). Nucleotide diversity of the entire data- set was found to be 0.19327 and a total of 328 polymorphic sites were identified. Parsimony informative sites with two, three and four variants were found to be 314, 38 and 129, respectively. A total number of 184 haplotype with diversity of 0.9924 were also identified in complete dataset. Overall nucleotide composition and mean GC content at codon position 1–3 is given in Table 1. Overall GC% was calculated to be 45.6 and the highest GC% of 47.8 was recorded in the fishes, representing order perciformes. All the generated barcodes were deposited in GenBank and accession numbers were obtained. Details about the species collected, number of individuals, IUCN red list status and GenBank Accession Number are furnished in Supplementary Table 1. A total of 16 DNA barcodes, previously unreported from nine species, were also generated in the present study. This includes barcodes of Semiplotus manipurensis (MG736433), Paracanthocobitis adelaidae (KX576654), Schistura naganesis (KU681461), S. mani- purensis (MG736508), Lepidocephalicthys berdmorei (KX886802, MG778696), Mystus cineracius (KX886803, MG736520-21.), Akysis manipurensis (KX886801, MG736543), Pillaia indica (KJ936644-47) and Badis ferrarisi (KX886804). In addition to that, 11 new barcodes of seven species (un-described and identified up-to generic level only) were also generated, representing genus Garra (MG736488-92), Paracanthocobitis (MG736495), Schistura (MG736506-07), Ompok (MG736537), Glyptothorax (MG736554) and Badis (MG736580). Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 2 www.nature.com/scientificreports/ Taxonomic Level Taxa/n Min (%) Mean (%) Max (%) SE ± (%) Within species 72/327 0.00 0.42 3.28 0.2 Within genus 20/242 1.72 10.19 17.35 0.8 Within family 12/340 4.78 12.77 17.87 0.8 Within order 4/358 15.75 19.21 23.15 1.2 Table 2. Mean K2P distance value within various taxonomic levels (n = no. of individuals, SE = Standard Error). Figure 1. ABGD based partition of the complete data set of COI sequences of fishes of the Indo Myanmar Biodiversity hotspot. This represents the number of groups or species in each partition at different prior intra- specific divergence. The initial partition is shown as (o) and recursive partition as (∆). Mean genetic divergence (K2P) analysis and NJ tree. Most of the morphologically identified species of the present study were well differentiated by COI sequences. As expected, a hierarchical increase in the mean Kimura 2-Parameter (K2P) genetic divergence with increasing taxonomic levels from within a species (0.42%), to within genus (10.19%), to within families (12.77%), to within orders (19.21%) was observed (Table 2). The minimum mean K2P con-generic divergence (1.72%, SE ± 0.3) was found to be almost 4 fold higher than the average con-specific (0.42%, SE ± 0.1) divergence suggesting the presence of distinct species boundaries among the studied species. The lowest mean inter-specific divergence was observed among the Devario species (1.72%, SE ± 0.3) and the highest among the Channa species (17.35%, SE ± 1.0). NJ tree, obtained from complete COI sequence dataset, contained 117 species clusters, including the singleton species with a high level of resolution between the species, supported by high bootstrap value (Supplementary Fig. 1). Individuals of six species, namely Neolissocheilus hexastichus, Labeo bata, L. dyocheilus, S. khugae, B. assamensis and Channa orientalis formed distinct clusters, supported by high bootstrap values, indicating high intra-species divergence and possibly the presence of hidden diversity. ABGD Analyses for species delimitation. To delimit the species based on single locus sequence data, species delimitation was performed in ABGD tool, using K80 Kimura distance with relative gap width (X) of 0.75 and Nb bins (for distance distribution) of 20. Partition with prior maximal distance, i.e. P = 0.0215 and 0.0129 delimited the entire dataset into 108 and 114 putative species, respectively (Fig. 1). Out of 109 morphologically identified species, 100 (90.83%) were delimited nicely through ABGD tool at a prior maximal distance of 0.0215 and were found concordant with the observations of genetic distance and NJ analysis. Further, at a prior maximal distance of 0.0215, few species of genus Devario, Neolissocheilus, Garra and Mystus could not be delimited into different putative species. Although pair-wise genetic distance and NJ analysis separated them clearly. On the other hand, few specimens of morphologically identified species N. hexastichus, L. bata, L. dyocheilus, S. khugae, B. assamensis, and C. orienlatis were delimited into two or more putative species in ABGD tool which was also confirmed by pair wise K2P distance estimation and NJ analysis. bPTP analysis for species delimitation. All the species delimitation results i.e. K2P genetic divergence, NJ and ABGD analyses, presented above, are based on genetic distance method. In order to further validate the distance based observation, we performed tree based species delimitation method i.e. bPTP analysis. For de novo Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 3 www.nature.com/scientificreports/ No. of Intra-specific Con-specific Bootstrap Prior max. Species individuals distance % (±SE) distance % (±SE) support value % distance for ABGD Channa orientalis 10 3.19–6.36 (0.9) 0.0–0.98 (0.2) 86–100 0.0215 Labeo bata 07 4.39 (0.9) 0.00 81 0.0215 Neolissocheilus hexastichus 22 2.7–3.7 (1.0) 0.22–0.45 (0.2) 96–100 0.0129 Badis assamensis 04 3.52 (0.7) 0.88 (0.3) 97 0.0215 Labeo dyocheilus 04 3.29 (0.7) 0.00 100 0.0215 Schistura khugae 04 2.16 (0.6) 0.00 100 0.0215 Table 3. High Intra-specific divergence in some of the studied taxa. species delimitation, PTP model generally outperforms GYMC as well as OTU-picking methods when evolu- tionary distances between species are small . bPTP analysis was performed with 100,000 MCMC sampling. The bPTP analyses returned with an acceptance rate of 0.93, with merges of 49,900, splits of 50,100, and estimation of a number of species between 90 and 145 with mean value of 119.58, using single locus nucleotide sequence data. e m Th ean number of species delimited by bPTP analysis was found close to the number of species delineated by K2P (i.e. 117 putative species with >2.00% inter-specific divergence), NJ (i.e. 117 putative species with supported bootstrap value) and ABGD (114 putative species at prior maximal distance of 0.0129) analyses. Pair-wise intra-specific divergence. K2P pair-wise genetic divergences were determined among all the individuals of each species except species which was represented by a single individual. Pair-wise genetic diver- gence analyses of few individuals of C. orientalis, L. bata, N. hexastichus, B. assamensis, L. dyocheilus and S. khugae showed high intra-specific genetic divergence (> 2.0%) with no or very low con-specific variation in remaining individuals of the lineage. These observations were further confirmed by NJ (bootstrap support value) & ABGD analyses (prior maximal distance) as presented in Table 3. This possibly indicated the presence of a putative new species, species complex or geographically isolated population under the lineage. Notes on Specific Taxa. Garra species. Phenotypic plastic is always a hindrance in taxonomic identifica- tion of many Garra species and the same was experienced in the present study also. A total of 25 specimens of genus Garra were collected in the present survey. Out of this, 15 individuals were identified as G. annadalaei (07), G. lissorhynchus (01), G. litanensis (01), G. nasuta (05) and G. qiaojiensis (01). Remaining 10 individuals could be identified up-to generic level only and distributed into three morphologically distinct clusters. es Th e individuals were designated as Garra sp._01 (MG7364483-87), Garra sp._02 (MG736488-90) and Garra sp._03 (MG736491- 92). Homology search of COI sequence of Garra sp._01 showed 99% similarity with Garra sp. 8162 C NBFGRMU (KT896683) and Garra sp. Tuirivang (KF318331). The average pair-wise genetic distance, estimated between COI sequences of Garra sp.1, Garra sp. 8162 C NBFGRMU and Garra sp. Tuirivang, was found to be 0.56% (SE ± 0.2), revealing that they belonged to the same species which were also collected from the same geographical area and identified up-to generic level only. Specimens of Garra sp._02 were found to be morphologically close, but dis- tinct from G. naganensis. A high genetic divergence, i.e. 3.55% (SE ± 1.0) was estimated between the individuals of Garra sp._02 and sister species G. naganensis (KX951812) which was found to be higher than the maximum conspecific pair-wise divergence (3.28%, SE ± 1.0), estimated between all the species examined in the present study. Therefore, Garra sp. 2 represents a putative previously undescribed species. Same analyses were also con- ducted for Garra sp. 3 which was morphologically close, but distinct from G. mcclellandi (KX239495). The aver - age pair-wise genetic distance was estimated between Garra sp._03 and G. mcclellandi and was observed to be 11.31% (SE ± 2.0) whereas no inter-generic distance among Garra sp._03 individual was observed. e Th observed genetic divergence was found to be almost three and half times higher than the maximum conspecific pair-wise divergence, estimated between all the species examined. We further investigated to find out whether these are truly undescribed species. For this, we analyzed COI sequences of 22 Garra species, reported from the same or neighboring rivers and estimated pair-wise genetic divergence, followed by NJ and ABGD analyses. Out of these 22 species, COI sequence of five species, namely G. annandalaei, G. lissorhynchus, G. litanensis, G. nasuta and G. giaojiensis were generated in the present study whereas sequences of remaining 17 species were retrieved from NCBI database (species name & Accession no. is given in Fig. 2). NJ analyses also showed the clustering of Garra sp._01 with Garra sp. 8162 C NBFGRMU (KT896683) and Garra sp. Tuirivang (KF318331) with the highest supported bootstrap value (100%), suggesting that these individuals belonged to the same species. Individuals of Garra sp._02 were clustered separately from G. naganensis with the highest bootstrap value (100%). In same manner individuals of Garra sp._03 were also grouped separately from G. mccellandi with the supported bootstrap value. ABGD analyses at a prior maximal distance of 0.0215 also delineated Garra sp._02 and Garra sp._03 into a separate partition. These findings support that Garra sp._02 and Garra sp._03, collected in the present study, represents a putative and previously undescribed species. Paracanthocobitis species. One specimen of Paracanthocobitis species which was morphologically close, but distinct from Paracanthocobitis botia appeared to be unrecognized and designated as Paracanthocobitis sp._01 (MG736495). We further investigated to find out whether this is a truly undescribed species. For this, we analyzed the COI sequences of 10 species of Paracanthocobitis, available in the NCBI GenBank Database and reported from Indian and neighboring river (Species Name & Accession no. provided in Fig. 3). Paracanthocobitis sp._01 showed very high genetic divergence with the sister species Paracanthocobitis botia, i.e. 12.89%, SE ± 0.2 which was almost four times more than the maximum conspecific pair-wise divergence (3.28%, SE ± 1.0), estimated between all the species, Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 4 www.nature.com/scientificreports/ Figure 2. NJ analyses (based on K2P genetic distance of COI sequences) of Garra species (COI sequences of undescribed species are inside the box). Figure 3. NJ analyses (based on K2P genetic distance of COI sequences) of Paracanthocobitis species (COI sequences of undescribed species is inside the box). examined in the present study. Average distance with other studied species of Paracanthocobitis was recorded as 14.25%, SE ± 2.0. NJ analyses also showed separate clustering of Paracanthocobitis sp._01. ABGD analyses at a prior maximal distance of 0.0215 also delineated this species into a separate partition. These findings support that Paracanthocobitis sp._01, collected in the present study, represents a putative and previously undescribed species. Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 5 www.nature.com/scientificreports/ Figure 4. NJ analyses (based on K2P genetic distance of COI sequences) of Schistura species (COI sequences of undescribed species are inside the box). Schistura species. A total of 12 individuals of Schistura Species were collected and out of which 10 individuals were assigned as S. khugae (04), S. maculosa (03), S. manipurensis (01), S. naganesis (01), and S. prashadi (01). Remaining two individuals of Schistura species could be identified up-to genus level and designated as Schistura sp._01 (MG736506-07). We compared the sequence of Schistura sp._01 with 14 other species of Schistura, reported from same or neighboring rivers. Out of 14 species, COI sequences of six species namely S. khugae, S. maculosa, S. manipurensis, S. nagaensis, S. paucireticulata and S. prashadi were generated in the present study, whereas the sequences of remaining eight species were downloaded from the NCBI database (species name & Genbank Accession no. is given in Fig. 4). The closest genetic distance was observed with S. prashadi i.e. 9.55% (SE ± 2.0) which was found almost three times higher than the maximum conspecific pair-wise divergence (3.28%, SE ± 1.0), estimated between all the species examined. Average distance with other studied species of Schistura was recorded as 16.68% (SE ± 2.0). NJ analyses also showed separate clustering of Schistura sp._01 with high bootstrap support value i.e. 96.0%. ABGD analyses at a prior maximal distance of 0.0215 also delineated this species into a separate partition. These findings support that Schistura sp._01, collected in the present study, represents a putative and previously undescribed species. Ompok Species. One specimen of species Ompok, which was morphologically close, but distinct from Ompok pabda appeared to be unrecognized and designated as Ompok sp._01 (MG736537). We further investigated to find out whether this is a truly undescribed species. For this we analysed COI sequences of all the valid species of Ompok, reported from Indian water i.e. O. pabda, O. pabo, O. bimaculatus and O. malabaricus (restricted to Malabar Coast of India) except O. karunkodu (restricted to Amaravathi, a right-hand tributary of the River Kaveri in Southern India). Reason for excluding O. karunkodu from the analyses is unavailability of COI sequence in public databases. COI sequences of analysed species, namely O. pabda (KT762383), O. pabo (FJ230020), O. bimaculatus (FJ230051), and O. malabaricus (HQ009495) were retrieved from the NCBI GenBank database. Ompok sp. 1 showed very high genetic divergence with the sister species O. pabda i.e. 11.47%, SE ± 2.0 which was almost three and half times more than the maximum conspecific pair-wise divergence (3.28%, SE ± 1.0), estimated between all the species examined in the present study. Average distance with other studied species of Ompok was recorded as 13.39%, SE ± 2.0. NJ analyses also showed separate clustering of Ompok sp._01 with bootstrap value of 99.0%. ABGD analyses at a prior maximal distance of 0.0215 also delineated this species into a separate partition. These findings support that Ompok sp._01, collected in the present study, represents a putative and previously undescribed species. Glyptothorax species. One specimen of genus Glyptothorax was found morphologically close, but distinct from Glyptothorax verrucosus and appeared to be unrecognized and designated as Glyptothorax sp._01 (MG736554). We investigated to find out whether this is a truly undescribed species. For this, we analysed COI sequences of 16 Glyptothorax species, reported from the same or neighboring waters (species name & Genbank Accession no. is given in Fig. 5). Out of these 16 species, COI sequence of 05 species, namely G. ventrolineatus, G. ngapang, G. manipurensis, G. telchitta and G. trilineatus were generated in the present study. Glyptothorax sp. 1 showed high genetic divergence with the sister species G. verrucosus i.e. 3.16%, SE ± 1.0. Average distance with other studied species of Glyptothorax was recorded as 5.79%, SE ± 1.0. NJ analyses also showed separate clustering of Glyptothorax sp._01 and ABGD analyses at a prior maximal distance of 0.0215 also delineated this species into Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 6 www.nature.com/scientificreports/ Figure 5. NJ analyses (based on K2P genetic distance of COI sequences) of Glyptothorax species (COI sequences of undescribed species is inside the box). Figure 6. NJ analyses (based on K2P genetic distance of COI sequences) of Badis species (COI sequences of undescribed species is inside the box). a separate partition. These findings support that Glyptothorax sp._01, collected in the present study, represents a putative previously un-described species. Badis species. One specimen of genus Badis was found morphologically close, but distinct from Badis assamen- sis and appeared to be unrecognized and designated as Badis sp._01 (MG736580). We investigated to find out whether this is a truly un-described species. For this, we analyzed COI sequences of 08 species of Badis, reported from the same or neighboring waters (species name & Genbank Accession no. is given in Fig. 6). Sequence of B. assamensis, B. badis, B. ferrarisi and B. tuivaei were generated in the present study. Badis sp._01 showed high genetic divergence with the sister species B. assamensis i.e. 5.87%, SE ± 2.0 which was found higher than the max- imum conspecific pair-wise divergence (3.28%, SE ± 1.0), estimated between all the species examined. Average distance with other studied species of Badis was recorded as 16.04%, SE ± 1.0. NJ analyses also showed separate clustering of Badis sp._01 with the highest bootstrap support value of 100%. ABGD analyses at a prior maximal distance of 0.0215 also delineated this species into a separate partition. es Th e findings support that Badis sp._01, collected in the present study, represents a putative previously un-described species. Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 7 www.nature.com/scientificreports/ Note on distribution record. The collection sites of majority of the species, studied in the present report, were found concordant with their earlier distribution record. Although, some species, recorded from new locations (not reported earlier), are presented here. In the present study, a species namely, Devario derup- totalea, earlier reported only from the tributary of the Yu River (Chindwin drainage) in Manipur, India was also collected from river Tuphaleri of Nagaland. Another species Osteobrama feae, which was reported only from Myanmar & China, was also collected from a tributary of Chindwin in Manipur. One exotic species, Barbonymus gonionotus, with its natural distribution in the Mekong and Chao Phraya basins, Malay Peninsula, Sumatra and Java, was also reported from Mizoram and Meghalaya, which indicates the escape of this species from culture installations to natural water bodies. Another exotic species, Ctenopharyngodon idella (grass carp), was recorded from Meghalaya, indicating the escape from culture system to the river. Hypsibarbus myitkyinae, reported only from Myanmar, was also collected from the tributary of Chindwin from Manipur. Another species Schizothorax molesworthi, known from the Brahmaputra River drainage was also recorded from Chindwin drainage of Nagaland. A recently described species Schistura paucireticulata from Tuirial River, a major tributary of the Barak River of Mizoram, was also collected from the Tlwang River of Mizoram . Mystus cineraceus and B. ferrarisi described only from Irrawaddy drainage of Myanmar, was also recorded from Manipur. Few endemic species, described and reported from northeast India, were also collected from their type locality or nearby in the present study and are presented here. A miniature catfish Pseudolaguvia virgulata , described and reported from the Barak River drainage in Mizoram, was also collected from a nearby locality. A hill stream spineless eel Pillaia indica (reported from Garo Hills of Meghalaya) and freshwater glass perch Parambassis waikhomi (described and reported from Loktak Lake of Manipur) were also collected from their type locality in the present study. Discussion Species are considered as a basic unit of biodiversity and is the foundation of ecosystem services to which human well-being is closely linked. Therefore, the precise answer on biodiversity is needed to devise an effective meas- ure to conserve biodiversity (Source: Millennium Ecosystem Assessment: Ecosystems and Human Well-being: Biodiversity Synthesis, 2005). Indo-Myanmar Biodiversity hotspot represents the area of high endemism which is under continuous pressure . Therefore, the need of the present study was realized by the authors which deals with the first comprehensive molecular assessment of fish fauna of four states (i.e. Mizoram, Meghalaya, Manipur and Nagaland and comes under the biodiversity hotspot) of northeast India. This study includes the molecular identification of 109 species and development of the DNA barcode reference library which will provide efficient and easy identification system for fish fauna of the region. These 109 species includes 46.36% of the reported fish fauna of the region as per the Records of Zoological Survey of India . We observed hierarchical increase in mean K2P genetic divergence (%) with increasing taxonomic level (0.42, 10.19, 12.77 & 19.21) which is almost similar to the previous reports, documented on the freshwater fishes of 5,25,50,51 52 26 India , Canada and Salween river of southwestern China . Observations were also found similar for 53,54 Indian marine fishes . Earlier studies have attempted to use barcoding data alone for delineating the species 20,55–57 boundaries . Hebert and co-workers tried to establish a standard COI sequence threshold between con- specific and congeneric divergence i.e. also called as 10 X rule where 10 folds of mean intraspecific variation are adequate to draw the species boundary . In the present study, 10 X rule was not found to be fit for delineating the 55,59 species which was also in accordance with few previous reports . Further, frequent overlap between intraspe- cific and interspecific divergence has been reported in earlier studies, thus, generalizing the threshold for species level resolution has been considered difficult. er Th efore, combined use of mean K2P genetic divergence and vari - 5,25,26,47,60 ous species delimitation tools are more effective for delineating the species and defining the boundaries . The estimated number of species using ABGD model was found to be 108 and based on this value 90.83% of the studied species were delineated nicely. However, some taxa like N. hexastichus, L. bata, L. dyocheilus, B. assamensis, S. khugae, C. orientalis, Garra sp._01-03, Paracanthocobitis sp._01, Schistura sp._01, Ompok sp._01, Glyptothorax sp._01, and Badis sp._01 which did not show clear relationship and high divergence value, were also partitioned well with the help of NJ and ABGD species delineation analyses. The species which were not delin- eated properly through ABGD tool at a distance of 0.0215, were further separated at a low proximal distance of 0.0129, followed by confirmation using pair-wise genetic distance and NJ analysis. Tree based species delimitation i.e. bPTP, resulted into 119 number of species (mean value) which was found closed to the number of species delineated by K2P (i.e. 117 putative species with >2.00% inter-specific divergence), NJ (i.e. 117 putative species with supported bootstrap value) and ABGD (114 putative species at prior maximal distance of 0.0129) analyses. Therefore, the combination of genetic divergence along with NJ, ABGD & bPTP analyses may help in correct identification of the fish fauna of the region. A total of 291 species has been reported from the entire northeast India as per the Records of Zoological Survey of India . Out of these, 241 species were reported from Mizoram, Manipur, Meghalaya and Nagaland. Although, drainage-wise exploration studies are scanty in the region, but some workers like Vishwanath et al. reported 117 fish species in Chindwin drainage flowing in Manipur . Kar & Sen reported 151 fish fauna from Tripura, Barak drainage flows in parts of Assam, Manipur and Mizoram and Karnafuli and Koladyne/Kaladan drainage of Mizoram . Barman et al. also reported 51 fish species from the Kaladan River system, flowing in Mizoram . Four species, namely Tor putitora, P. manipurensis, P. indica and B. tuivaei, collected in present study, are included under IUCN Red list (2014) category of endangered species whereas seven species, namely, B. ngawa, P. atra, G. litanensis, S. khugae, S. nagaensis, S. prashadi and G. manipurensis under the category of vulnerable spe- cies and nine species, namely, A. bengalensis, N. hexagonolepis, N. hexastichus, O. belangeri, S. manipurensis, O. bimaculatus, O. pabda and W. attu under the category of near threatened species. Majority of the species which are included under the category of endangered, vulnerable and near threatened have sporadic distribution. The sporadic distribution of most of the above mentioned species indicated a decline in the population. Although, this Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 8 www.nature.com/scientificreports/ interpretation must be considered very cautiously because inadequate sampling cannot be ruled out in the present study. Variations in terms of number and type of species in comparison to previous studies can be explained by the cryptic diversity, phenotypic plasticity, misidentification, and taxonomic ambiguities. In conclusion, it is confirmed that the integrated use of DNA barcoding (genetic divergence, NJ, ABGD & bPTP species delineation) and morphological evidences are efficient and reliable tools for identification of fishes at the species level. Further, establishment of a reliable COI barcode database of fish fauna of the Indo-Myanmar region may serve as a reference library for accurate identification of fishes that will surely help ichthyologists, researchers, students, biodiversity managers and policy makers. Presence of high degree of pair-wise intra-specific divergence among the individuals of some species was revealed, suggesting the presence of putative sibling species or existence of hidden diversity among the fish species, advocating the immediate need for more comprehensive studies on fish fauna of the region. Materials and Methods Ethical statement. e fi Th shes, studied in the present study, are not protected under The Wildlife Protection Act, 1972 (Last amended in 2013), Government of India and caught with the help of local fisherman. No experi- mentation was conducted on live specimens in the laboratory and study was approved by the Institutional Animal Ethics Committee (IAEC) of College of Fisheries (Central Agricultural University, Imphal), India. Sample collection and DNA isolation. Fishes were collected from 40 distant locations of die ff rent rivers and tributaries, covering four states of northeast India (an essential part of Indo-Myanmar Biodiversity Hotspot). A total of 109 morphologically identified species were collected during August 2013 to February 2017 from differ - ent locations of Meghalaya, Manipur, Mizoram and Nagaland. Information about the sampling stations along with geographical coordinates is given in Supplementary Table 2. Present study includes fishes from Barak River and its tributaries, flowing in Mizoram and Manipur, tributaries of Brahmaputra River flowing in Nagaland and Meghalaya, tributaries of Chindwin river system flowing in Manipur and Nagaland and Karnafuli river of Mizoram. For species identification and nomenclature, information available at www.fishbase.org and taxonomic keys 7,49,61–74 prepared by various taxonomists were followed . The representative voucher specimen of all the collected species except Garra qiaojiensis, Lepidocephalichthys annandalei, Wallago attu and Paracanthocobitis zonalter- nans are maintained at the museum, established with partial financial assistance given by the Department of Biotechnology, Government of India under the center of excellence project at the College of Fisheries (Central Agricultural University), Tripura, India. Approximately 100 mg of white muscle tissue or 200-500 μl of whole blood from each specimen were preserved in 95% ethanol for genomic DNA isolation. Total genomic DNA was extracted from the muscle tissue or whole blood, using the standard phenol-chloroform-isoamyl-alcohol method described by Sambrook and Russell with some minor modifications . The integrity of the isolated samples was determined by running the samples in 0.8% agarose gel, stained with ethidium bromide. Concentration and purity of isolated samples were determined with the help of nano-volume spectrophotometer (mySpec Scientific GmbH) and stored at −20 °C for further use. COI Amplification and purification. For DNA barcoding, partial 5′ region of COI gene was ampli- fied in a final volume of 50 μl with final concentration of 1X reaction buffer (10 mM Tris-HCl , 50 mM pH 8.3 KCl,), 2.0 mM MgCl , 0.2 mM of dNTP mix, 10 pmol of forward and reverse primer, 2U of Taq DNA poly- merase and 100 ng of template DNA. The primers used for amplification of COI gene were 5′-TCAACCA ACCACAAAGACAT TGGCAC-3′ and 5′-TAGACT TCTGGGTGGCCAAAGAATCA-3′ . Each reaction included a negative control (no template DNA) and was carried out in an Veriti 96 well thermal cycler of Applied Biosystems under following thermal cycling conditions: initial denaturation at 95 °C for 4 min, followed by 35 cycles of denaturation at 94 °C for 35 sec, primer annealing at 52 °C for 30 sec and primer extension at 72 °C for 40 sec and final extension for 5 min at 72 °C. Amplified products of COI were separated on 1.5% agarose gel, stained with ethidium bromide, followed by gel elution using spin column based gel extraction kit of Thermo Fisher as per the manufacturer’s instructions. In the final step, purified products were eluted in 1X TE buffer. Nucleotide Sequencing of COI gene. e p Th urified products (free of salts and protein impurities) of COI were directly sequenced in Applied Biosystem 3500 genetic analyzer using BigDye Terminator Cycle Sequencing Kit (Applied Biosystems) as per manufacturer’s instructions. The sequencing (dye labeling) reaction was opti- mized to a final volume of 10 μl, using 50 ng template DNA, 33 μM primer, 1X BigDye Terminator reaction mix, followed by thermal cycling at initial denaturation at 96 °C for 4 min, 25 cycles of denaturation at 94 °C for 10 sec, annealing at 50 °C for 10 sec and extension at 60 °C for 4 min. The labeled products were precipitated with 0.1 vol- ume of 3 M sodium acetate (pH 4.6) and 2.5 volumes of ethanol, followed by centrifugation at room temperature for 30 min at 14,000 × g. The pelleted products were washed with 70% ethanol and air dried, followed by resus- pension in 15 μl of Hi-Di formamide solution. The samples were denatured at 95 °C for 5 min and chilled on ice for 5 min to prevent the renaturation. The denatured samples were transferred into 96 well sequencing plate with rubber closure and placed into ABI 3500 genetic analyzer for nucleotide sequencing at a DNA sequencing facility of central molecular biology laboratory of the College of Fisheries (CAU, Imphal), India. Genetic diversity analysis and phylogenetic tree construction. To rule out sequencing error, bidi- rectional 2X overage were performed (each base sequenced 2 times) for each studied sample. Chromatograms of each sequence were examined carefully to verify all base pair assignments, using ABI sequence viewing software. All the COI sequences of mtDNA region were assembled and aligned, using MEGA 7, followed by determination of nucleotide composition . DnaSp ver 5.10 was used to estimate the number of polymorphic Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 9 www.nature.com/scientificreports/ sites and nucleotide diversity (Pi) . Pair-wise and mean K2P genetic distance was calculated with the help of MEGA 7. Neighbor-joining (NJ) approach was employed to deduce phylogenetic relationships among species, using COI sequences with the help of MEGA 7 and support for monophyly was assessed with 10000 bootstrap pseudo-replicates . To delimit the species based on single locus sequence data, species delimitation was per- formed in ABGD tool, using JC69 Jukes-Cantor distance with relative gap width (X) of 0.75 and Nb bins (for distance distribution) of 20 . Tree based species delimition was also performed to validate the observations made by distance based delimitation method . For this, RAxML majority rule extended consensus (MRE) tree was obtained from unique haplotype sequences . The obtained MRE tree was imported to bPTP web server and analysis was performed with 100,000 MCMC generations. References 1. Allen, D. J., Molur, S., Daniel, B. 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Biol. 75(5), 758–771 (2008). Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 11 www.nature.com/scientificreports/ Acknowledgements Authors are thankful to the Vice-Chancellor, Central Agricultural University, Imphal for infrastructure support. Kind help of Tapas Sarkar, Tapan, Lalzamliana, Rebeck Lalrinpuii, George Lalnuntluanga, Samar Jamatia, Atom Arun Singh, Hijam, Chimwar, Amit, Apurva and Kim during the collection of different species from difficult geographical area are duly acknowledged. The authors are also thankful to the Department of Biotechnology (DBT), Ministry of Science and Technology, Government of India for research grants (DBT-NE/LIVE/05/2011). Author Contributions A.S.B. Investigator of the project, Envisaged and designed the study, collected sample from the state of Mizoram & Meghalaya, PCR amplification & purification, nucleotide Sequencing and Manuscript writing, M.S.: Associated Scientist of the project, conducted laboratory experiments like DNA Isolation, PCR amplification and nucleotide sequencing, data analyses & manuscript writing, S.K.S.: Associated faculty of the project and collected samples from the state of Manipur and manuscript editing, H.S.: Associated faculty of the project and collected samples from the state of Nagaland, Y.J.S.: Associated faculty of the project and collected sample from the state of Meghalaya, M.L.: Junior Research Fellow in the project and conducted laboratory experiments like DNA isolation, PCR amplification & Purification, P.K.P.: Team leader of the project and provided infrastructure support for study and guided the manuscript writing. Additional Information Supplementary information accompanies this paper at https://doi.org/10.1038/s41598-018-26976-3. Competing Interests: The authors declare no competing interests. Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Cre- ative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not per- mitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2018 Scientific REPO R TS | (2018) 8:8579 | DOI:10.1038/s41598-018-26976-3 12
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