68 (2): 165 –175 © Senckenberg Gesellschaft für Naturforschung, 2018. 15.8.2018 Integrative taxonomy reveals a new species of snakehead fish, Channa stiktos (Teleostei: Channidae), from Mizoram, North Eastern India Lalramliana 1 *, John Daniel Marcus Knight 2, Denis Van Lalhlimpuia 1 & Mahender Singh 3 1 Department of Zoology, Pachhunga University College, Aizawl-Mizoram, India, 796001; [email protected]; [email protected] — 2 Flat ‘L’, Sri Balaji Apartments, 7th Main Road, Dhandeeswaram, Velachery, Chennai, Tamil Nadu 600042, India; [email protected] — 3 Molecular Biology & Biotechnology Division, ICAR-National Bureau of Fish Genetic Resources, Lucknow 226002, Uttar Pradesh, India; [email protected] — * Corresponding author: [email protected] Accepted July 09, 2018. Published online at www.senckenberg.de/vertebrate-zoology on July 27, 2018. Editor in charge: Uwe Fritz Abstract Channa stiktos, a new species of snakehead fish, is described from the River Kaladan and its tributaries, Mizoram, North Eastern India based on comparison of morphological and molecular features with closely related species. Channa stiktos is morphologically similar to C. ornatipinnis described from the Rakhine State of Myanmar, however, differs from it in having black spots on dorsal and ventral sides of the head (vs. no spots on dorsal and ventral sides of the head, but rather spots restricted to the post-orbital lateral region of the head), and lacking dark spot on the anal fin of juveniles (vs. presence of series of upto 10 dark spots). The molecular analysis, based on cytochrome c oxidase subunit I (COI) sequences, shows that C. stiktos is distinct from other close Channa species, except clade 3 of C. ornatipinnis, available in the GenBank (interspecies distance ranges from 8.24 – 25.33%). Channa stiktos clustered cohesively with clade 3 of C. ornati- pinnis (only 1.43% genetic distance) indicating that they are conspecific. The genetic distance betweenChanna stiktos and C. ornatipinnis (from the type locality and another locality in the Ayeyarwaddy basin) are 8.24 – 8.59%, and between C. stiktos and C. pulchra is 12.92%, supporting the conclusion that they are different species. Key words Channa ornatipinnis, C. pulchra, COI, Taxonomy, Freshwater fish, River Kaladan, Indo-Burma Biodiversity hotspot. Introduction Fishes of the family Channidae are widely distributed in cylindrical body, a large mouth with well-developed tropical Africa, parts of the Middle East, and Asia. They teeth on the jaws, vomer and palatines, long dorsal- and are known to inhabit a wide variety of freshwater habi- anal-fin bases with soft rays, and accessory air breath- tats ranging from hill streams to swamps (GEETAKUMARI ing organs (modified epibranchial 1) situated in the su- & VISHWANATH, 2011), and are able to survive out of wa- prabranchial cavity in the head (MUSIKASINTHORN, 1998; ter for short periods as they possess accessory air breath- COURTENAY & WILLIAMS, 2004). Currently there are 40 ing organs (BRITZ, 2013). Although Channidae is a small valid species under the genus Channa (ESCHMEYER et al., family of fishes, the knowledge about their species-level 2017), some of these were recently discovered from the diversity is far from complete, and the taxonomy of some Eastern Himalayan region (BRITZ, 2008 & 2013; GEETA- species group is still complicated (BRITZ, 2013). Species KUMARI & VISHWANATH, 2011; KNIGHT, 2016; LALHLIMPUIA of the genus Channa are characterized by an elongated et al., 2016). ISSN 1864-5755 165 Lalramliana et al.: New species of Channa from Mizoram, NE India. Many of the snakehead fish species currently recog- LAND (1842) for O. montanus; GÜNTHER, A. (1861) for nized were discovered before the beginning of the 20th O. kelaarti; MUSIKASINTHORN (2000) for Channa auran- century, and the last two decades have witnessed quite a timaculata; BRITZ, (2008) for C. ornatipinnis and C. pul- few discoveries (BRITZ, 2013). However, due to the exis- chra; and BRITZ, (2013) for C. andrao were used for tence of species complexes among some species group comparison. viz. C. gachua, C. marulius, C. punctata and C. striata (COURTENAY & WILLIAMS, 2004; RAINBOTH, 1996; ADAM- SON et al. 2010; SERRAO et al., 2014; CONTE-GRAND et al., DNA extraction, PCR amplification and 2017), accurate identification to the species level using DNA sequencing morphological approach alone is sometimes challenging. Therefore, a morphological approach when dealing with The extraction of DNA was performed from approxi- these species complexes likely benefits from supporting mately 25 mg of caudal muscle tissue following SAM- molecular barcoding analyses. Although the molecular BROOK & RUSSELL (2001). The partial COI gene was approach, also known as DNA barcoding, which employs amplified using the universal fish primers Fish-F1 and the conserved short stretch sequences of cytochrome c Fish-R1 (WARD et al. 2005). The PCR reaction was car- oxidase sub-unit I gene of the mitochondria (HEBERT et ried out in 25 µl volume containing 1X buffer, 100 µM al., 2003), has some limitations and pitfalls (MORITZ & dNTPs, 2 mM MgCl2, 5 pmol of each primer, 2U Taq CICERO, 2004; RUBINOFF et al., 2006; DUDU et al., 2016), it DNA polymerase and 100 ng template DNA. Amplifi- has proven to be useful in species identification of fishes cations were performed in Veriti 96 fast thermal cycler (WARD et al., 2005; HUBERT et al., 2008), particularly of (Applied Biosystems, Inc., USA) and the conditions are: Channa species (ZHU et al., 2013; SERRAO et al., 2014; initial denaturation of 3 min at 94 °C, followed by 35 CONTE-GRAND et al., 2017). cycles of denaturation at 94 °C for 50 sec, annealing at During field surveys in the River Kaladan and its 54 °C for 30 sec, extension at 72 °C for 80 sec with final tributaries, Mizoram, North Eastern India, specimens extension of 10 min at 72 °C. Sequencing was performed very similar to Channa ornatipinnis and C. pulchra were in forward direction only using an automated ABI 3500 collected. These specimens were previously identified as sequencer (Applied Biosystems, Inc, USA). Channa ornatipinnis due to lack of conspicuous mor- phological differences between the two species. How- ever, detailed morphological examination of the col- DNA sequence analysis lected specimens and comparisons with similar species, as well as molecular (COI) comparison with the results The cytochrome c oxidase subunit-I (COI) gene of sev- of recent studies (SERRAO et al., 2014; CONTE-GRAND et en individuals of Channa (5 C. aurantipectoralis and 2 al., 2017) revealed that these specimens were an unde- C. stiktos) were sequenced. Altogether, the 7 developed scribed species, which we herein describe as Channa sequences and another 44 sequences of species belong- stiktos sp. nov. ing to Channa gachua species group alongwith one out- group, Parachanna obscura (KJ937424), from GenBank were included to make a comparative analysis. The se- quences were aligned using CLUSTAL_W integrated in Materials and methods MEGA 7 (Molecular Evolutionary Genetics Analysis) software (KUMAR et al., 2016). The sequences were blasted in NCBI (http://www.ncbi.nlm.nih.gov) for the Collected specimens were fixed in 10% formalin and nearest matches and submitted to NCBI GenBank (NCBI subsequently preserved in 70% alcohol. Tissue samples Accession Nos. MH559824 & MH559825 for Channa were taken from the specimens prior to formalin fixation. stiktos and MH559819 – MH559823 for C. aurantipec- The specimens examined in this study are registered in toralis). The genetic distance was calculated by averag- Zoological Survey of India (ZSI), Kolkata, India and ing pairwise comparisons of 51 sequences across close the Pachhunga University College Museum of Fishes relatives of Channa stiktos by the Kimura 2 parameter in (PUCMF), Mizoram, India. Methods for taking counts MEGA 7 (Table 3). The maximum likelihood (ML) phy- and measurements follow MUSIKASINTHORN (1998). Mea- logenetic tree was constructed using COI dataset of 41 surements were taken with digital calipers to the near- sequences of Channa species, including seven sequences est 0.1 mm. Numbers in parentheses after a count denote generated in this study and one sequence of Parachanna the frequency of that count. Osteological preparations obscura (KJ937424) as outgroup. Based on the lowest were made following the method of TAYLOR & VAN DYKE BIC (Bayesian Information Criterion), the best fit nucle- (1985). Osteological nomenclature follows BRITZ (2008). otide substitution model (out of 24 models) for present Vertebrae were counted from two cleared and stained COI dataset was TN93+G+I given by TAMURA & NEI specimens (PUCMF 16010). Data from the original (1993) (Tamura-Nei + Gamma distribution with 5 rate descriptions of HAMILTON (1822) for Ophiocephalus au- categories + certain fraction of sites are evolutionarily rantiacus; CUVIER, (1829) for Ophicephalus marginatus, invariable). CUVIER & VALENCIENNES (1831) for O. fuscus; MCCLEL- 166 VERTEBRATE ZOOLOGY — 68 (2) 2018 Results Diagnosis. Channa stiktos is distinguished from all other species of Channa, except C. ornatipinnis, C. pulchra, C. pardalis, C. melanostigma and C. stewartii, by a Channa stiktos sp. nov. unique colour pattern consisting of numerous large, black Figs. 1 and 2 spots on the head and the body. Channa stiktos is dis- tinguished from C. ornatipinnis in having black spots on Holotype: ZSI FF 7727, 188.3 mm SL; India, Mizoram, Tiau Ri- dorsal and ventral sides of the head (vs. no spots on dorsal