Indian Journal of Geo Marine Sciences Vol. 47 (11), November 2018, pp. 2237-2240

DNA barcoding of False stone fish diabolus (Cuvier, 1829), a new record for the Andaman Islands and India

Chatragadda Ramesh*, Raju Mohanraju, Perumal Karthick and Kada Narayana Murthy Department of Ocean Studies and Marine Biology, Pondicherry Central University, Brookshabad Campus, Port Blair-744102, Andaman & Nicobar Islands, India. *[E-mail address: [email protected]]

Received 05 April 2017: revised 03 May 2017

A specimen of the false stone fish Scorpaenopsis diabolus(Cuvier, 1829)was captured at the Burmanallah coast in the Andaman Islands, India. Identification was done combining traditional , based on the observation and measurement of morphology traits, with DNA barcoding in an integrative taxonomy approach. On this basis, S. diabolus constitutes a new record in the faunal diversity of the Andaman Islands and India.

[Keywords: Scorpaenopsis diabolus, DNA barcoding, cytochrome oxidase subunit I, Andaman and Nicobar Islands.]

Introduction distributed in the Indo-Pacific region, at depths of 1 m The world oceans occupy 70% of the earth space, to 70 m 5. In India 35 species belong to 15 genera of where a wide variety of commercial and non- have been recorded6. This family commercial fishes are found to inhabit various depths includes venomous false stone fish (spines are of neritic and pelagic zones. So far, 33200 extant fish venomous) also referred to as devil scorpionfish that species have been reported across the world and 2455 mimics venomous true reef stone fish species of the are known from Indian subcontinent1. Identification family Synanceiidae. S. diabolus are one of the most of these fishes are based on morphometric important venomous fish in coral reef biome. characteristics that sometimes have failed to Commonly they are found as solitary or in pairs in discriminate morphologically similar fishes and early weed bottoms, rubbles and coral reefs up to at depths larval stages. The efficacy of DNA barcoding has of 70 m. So far only 500 out of 15661 species of emerged as a reliable technique in tackling have been barcoded7. The analysis taxonomical identification. DNA barcoding technique of COI nucleotide sequence in the present study has has an important role in conserving the genetical data shown the application of DNA barcoding in of many organisms especially rare and endangered confirming the taxonomic identification of a specimen species. Moreover, it has become a reliable genetical of the venomous false stone fish Scorpaenopsis tool in identification of highly species rich biotas, diabolus from the Andaman Islands. morphologically similar organisms and other organisms that are difficult for identification2. Materials and Methods Although morphological taxonomy is reliable for Sample collection identification, yet, DNA Barcoding (genetical The specimen of false stone fish also referred to as taxonomy) is still more important and accurate devil scorpionfish was caught cautiously with a hand because of genetical sequence similarity studies that net from a rocky shore with corals at Burmanallah do not misidentify any organisms. The universal gene coast (Latitude 11⁰ 34’14.48’’ N; Longitude marker of is 655 base pair of mitochondrial 92⁰44’22.73’’E ) during the low tide time (Fig. 1). cytochrome c oxidase I (COI or cox1) gene fragment most widely used for barcoding various organisms3,4. Morphological identification Scorpaenidae are a well-known fish family Initially the specimen was identified by comprised of 215 species belong to 26 genera and are counting morphometric characteristics followed by 2238 INDIAN J. MAR. SCI., VOL. 47, NO. 11, NOVEMBER 2018

comparative identification with reference keys5, 8. The genomic DNA was assessed using a UV specimen was coded as CPR4355, gently washed with spectrophotometer, measuring the absorbance values sterile distilled water and scales from a part of the at 260 nm and 280 nm and also by running the DNA body were neatly removed. From this part 7 gm of sample in 1.2% agarose gel electrophoresis. The tissue was dissected, transferred into 95% ethanol and concentration of the DNA preparation was adjusted to stored in deep freezer until further use for DNA 20 ng/µl and employed for PCR reaction. Barcoding studies. PCR amplification of COI gene Diagnostic characteristics PCR amplification of the COI barcode region Body colour of S. diabolus varies from of sample CPR4355 of S. diabolus was carried reddish to orange, blue, green or purple, but colour out using the forward primer FishF1 5’- shade camouflages with surroundings. The head is TCAACCAACCACAAAGACATTGGCAC-3’ and large with pointed snout, mouth upward, body has the reverse primer FishR1 5’- pronounced hump on back with short dorsal fin TAGACTTCTGGGTGGCCAAAGAATCA-3’ 10. spines. Branched upper post temporal spine places The reaction mixture for 50 µl contained 5 µl of 10x this species distinct from other species of PCR buffer, 0.25µl of 10 μM dNTP mixture, 0.5 µl of Scorpaenopsis. The inner surface of the pectoral fins 1 U of Taq DNA polymerase, 0.5 µl of 10 µM of each are yellowish in colour with a large black spot. forward and reverse oligonucleotide primers, , 5 µl of Maximum body length grows up to 30 cm. genomic DNA preparation (100 ng) and the appropriate volume of ultrapure PCR grade water. DNA extraction PCR amplification was performed in a GeneAmp Genomic DNA was extracted from 0.3 gm of tissue PCR system 2720-Thermal Cycler, (Applied sample of S. diabolus specimen using the lysis buffer Biosystem, Foster City, CA, USA). The amplification and standard proteinase-K/phenol-chloroform- profile for COI gene consisted of an initial isoamyl method9. The concentration and quality of the denaturation at 95 ºC for 5 min, followed by 35 cycles of 95 ºC for 1 min, 58 ºC for 30 sec, 72 ºC for 1min and a final extension step at 72 ºC for 7 min. The samples were held at 4 ºC until further analysis. PCR products were resolved in electrophoresis 1.2% agarose gels, which were afterwards stained with ethidium bromide and photographed employing a gel doc capture system (BIOTOP FluorShot ver. 1.5).

Sequencing and phylogenetic analysis The PCR product of COI gene was gel-purified with GeNoRime PCR Purification kit (Shrimpex, Chennai) and submitted to Sanger dideoxy chain termination cycle sequencing. The resulting products were resolved in an automated Sequencer (Applied Biosystems, Foster City, USA). The Nucleotide sequence of this specimen has been deposited in GenBank under the assigned accession number of KT354503 and was compared and with sequences available in public gene databases BOLD Systems, GenBank and EMBL. Alignment of sequences and their comparison was carried out using MEGA 6 software11 and uncertain alignments found at the ends of the sequences were omitted for further analyses. The evolutionary relationships among the nucleotide

sequences were evaluated by means of the Neighbour- Fig. 1 — Study area: Burmanallah in South Andaman, India. Joining (NJ) method (reference), employing the RAMESH et al.: DNA BARCODING OF FALSE STONE FISH SCORPAENOPSIS DIABOLUS 2239

Kimura two-parameter (K2P) distance model and sequence comparison was also performed with the 1000 bootstrap replicates. sequences held in GenBank and EMBL and identified this specimen as S. diabolus. The NJ tree revealed the Results and Discussion formation of two distinct clusters by the congeneric Based on morphometric characteristics (Table 1) and conspecific individuals (Fig. 4). Average within- this specimen was presumptively identified as species and genus Kimura two parameter distances Scorpaenopsis diabolus (Fig. 2). Also, the present were 0.56% and 0.81%, respectively. study successfully amplified and sequenced the 655 bp barcode region of COI gene of this specimen and identified it as S. diabolus that belonged to family Scorpaenidae under the order Scorpaneiformes (Fig. 3 & 4). DNA barcoding sequence search in BOLD Systems revealed a 98% sequence identity with Scorpaenopsis diabolus JQ35035412. Using BLAST,

Table 1 — Morphometric and Meristic characteristics of Fig. 2 — Scorpaenopsis diabolus from Burmanallah coast. the S. diabolus specimen collected in the Andaman Island. Morphometric and Meristic characters: Total length 17.3 cm Standard length 14 cm Body depth 5.6 cm Caudal peduncle depth 1.5 cm Predorsal length 5.9 cm Length of dorsal fin base 8 cm Height of dorsal fin 2.9 cm Length of anal fin base 2 cm Height of anal fin 3.1 cm Length of pectoral fin 2.6 cm Length of pelvic fin 1.5 cm Fig. 3 — PCR product of COI gene visualized through 1.2% Length of longest dorsal spine 2 cm agarose gel electrophoresis. Lane M code for DNA ladder and 1 for specimen COI gene. Dorsal fin rays 10

Dorsal fin spines 12 Anal fin rays 5 Anal fin spines 3 Pectoral fin rays 18 Pelvic fin rays 5 Pelvic fin spines 1 Caudal fin rays 13 Total gill rakers on first arch 90 Head length 6.5 cm Head width 3.5 cm Snout length 3.4 cm Suborbital width 1.7 cm Eye diameter 1 cm Upper jaw length 3.4 cm Mouth position Superior Snout type Short Habitat Reef biome Scale type Cycloid Fig. 4 — K2P distance NJ tree of COI sequence of S. diabolus. Bootstrap values based on 1000 replicates are shown at tree Teeth Villiform nodes. The scale bar at bottom of the tree indicates K2P genetic Sex Male distance. 2240 INDIAN J. MAR. SCI., VOL. 47, NO. 11, NOVEMBER 2018

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