Integrative taxonomy reveals a new species of snakehead fish , Channa stiktos ( Teleostei : Channidae ) , from Mizoram , North Eastern India

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. ornatipinnis (only 1.43% genetic distance) indicating that they are conspecific. The genetic distance between Channa 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.


Introduction
Fishes of the family Channidae are widely distributed in tropical Africa, parts of the Middle East, and Asia.They are known to inhabit a wide variety of freshwater habitats ranging from hill streams to swamps (Geetakumari & Vishwanath, 2011), and are able to survive out of water for short periods as they possess accessory air breathing organs (Britz, 2013).Although Channidae is a small family of fishes, the knowledge about their species-level diversity is far from complete, and the taxonomy of some species group is still complicated (Britz, 2013).Species of the genus Channa are characterized by an elongated cylindrical body, a large mouth with well-developed teeth on the jaws, vomer and palatines, long dorsal-and anal-fin bases with soft rays, and accessory air breathing organs (modified epibranchial 1) situated in the suprabranchial cavity in the head (musikasinthorn, 1998;Courtenay & williams, 2004).Currently there are 40 valid species under the genus Channa (esChmeyer et al., 2017), some of these were recently discovered from the Eastern Himalayan region (BRITZ, 2008(BRITZ, & 2013;;Geetakumari & Vishwanath, 2011;kniGht, 2016;lalhlimpuia et al., 2016).
Many of the snakehead fish species currently recognized were discovered before the beginning of the 20 th century, and the last two decades have witnessed quite a few discoveries (Britz, 2013).However, due to the existence of species complexes among some species group viz.C. gachua, C. marulius, C. punctata and C. striata (Courtenay & williams, 2004;rainBoth, 1996;adamson et al. 2010;serrao et al., 2014;Conte-Grand et al., 2017), accurate identification to the species level using morphological approach alone is sometimes challenging.Therefore, a morphological approach when dealing with these species complexes likely benefits from supporting molecular barcoding analyses.Although the molecular approach, also known as DNA barcoding, which employs the conserved short stretch sequences of cytochrome c oxidase sub-unit I gene of the mitochondria (heBert et al., 2003), has some limitations and pitfalls (moritz & CiCero, 2004;ruBinoff et al., 2006;dudu et al., 2016), it has proven to be useful in species identification of fishes (ward et al., 2005;huBert et al., 2008), particularly of Channa species (zhu et al., 2013;serrao et al., 2014;Conte-Grand et al., 2017).
During field surveys in the River Kaladan and its tributaries, Mizoram, North Eastern India, specimens very similar to Channa ornatipinnis and C. pulchra were collected.These specimens were previously identified as Channa ornatipinnis due to lack of conspicuous morphological differences between the two species.However, detailed morphological examination of the collected specimens and comparisons with similar species, as well as molecular (COI) comparison with the results of recent studies (serrao et al., 2014;Conte-Grand et al., 2017) revealed that these specimens were an undescribed species, which we herein describe as Channa stiktos sp.nov.

Materials and methods
Collected specimens were fixed in 10% formalin and subsequently preserved in 70% alcohol.Tissue samples were taken from the specimens prior to formalin fixation.The specimens examined in this study are registered in Zoological Survey of India (ZSI), Kolkata, India and the Pachhunga University College Museum of Fishes (PUCMF), Mizoram, India.Methods for taking counts and measurements follow musikasinthorn (1998).Measurements were taken with digital calipers to the nearest 0.1 mm.Numbers in parentheses after a count denote the frequency of that count.Osteological preparations were made following the method of taylor & Van dyke (1985).Osteological nomenclature follows Britz (2008) . kelaarti; musikasinthorn (2000) for Channa aurantimaculata ;Britz, (2008) for C. ornatipinnis and C. pulchra;and Britz, (2013) for C. andrao were used for comparison.

DNA extraction, PCR amplification and DNA sequencing
The extraction of DNA was performed from approximately 25 mg of caudal muscle tissue following SAM-BROOK & RUSSELL (2001).The partial COI gene was amplified using the universal fish primers Fish-F1 and Fish-R1 (WARD et al. 2005).The PCR reaction was carried out in 25 µl volume containing 1X buffer, 100 µM dNTPs, 2 mM MgCl 2 , 5 pmol of each primer, 2U Taq DNA polymerase and 100 ng template DNA.Amplifications were performed in Veriti 96 fast thermal cycler (Applied Biosystems, Inc., USA) and the conditions are: initial denaturation of 3 min at 94 °C, followed by 35 cycles of denaturation at 94 °C for 50 sec, annealing at 54 °C for 30 sec, extension at 72 °C for 80 sec with final extension of 10 min at 72 °C.Sequencing was performed in forward direction only using an automated ABI 3500 sequencer (Applied Biosystems, Inc, USA).

DNA sequence analysis
The cytochrome c oxidase subunit-I (COI) gene of seven individuals of Channa (5 C. aurantipectoralis and 2 C. stiktos) were sequenced.Altogether, the 7 developed sequences and another 44 sequences of species belonging to Channa gachua species group alongwith one outgroup, Parachanna obscura (KJ937424), from GenBank were included to make a comparative analysis.The sequences were aligned using CLUSTAL_W integrated in 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 nearest matches and submitted to NCBI GenBank (NCBI Accession Nos.MH559824 & MH559825 for Channa stiktos and MH559819 -MH559823 for C. aurantipectoralis).The genetic distance was calculated by averaging pairwise comparisons of 51 sequences across close relatives of Channa stiktos by the Kimura 2 parameter in MEGA 7 (Table 3).The maximum likelihood (ML) phylogenetic tree was constructed using COI dataset of 41 sequences of Channa species, including seven sequences generated in this study and one sequence of Parachanna obscura (KJ937424) as outgroup.Based on the lowest BIC (Bayesian Information Criterion), the best fit nucleotide substitution model (out of 24 models) for present COI dataset was TN93+G+I given by TAMURA & NEI (1993) (Tamura-Nei + Gamma distribution with 5 rate categories + certain fraction of sites are evolutionarily invariable).It can be distinguished from Channa pulchra in having black spots on the body well distributed above and below the lateral line (vs.sparsely distributed or absent on the body below the lateral line); from C. pardalis in having black spots all over the head (vs.spots restricted to post-orbital region of the head); and from both C. stewartii and C. melanostigma in having well defined black spots on the head (vs.spots on the head absent, and restricted to the body). 1 for morphological data and Figs.

Description. See Table
1 and 2 for general appearance.Body elongate, round in cross section anteriorly, its width a little less than its depth; gradually becoming compressed laterally towards caudal peduncle.Dorsal profile of head gently curved anteriorly; ventral profile almost straight.Caudal peduncle deep, its depth slightly greater than its length.Head large, dorsoventrally flattened, about one-third standard length, its widest portion between eye and opercle.Eyes small, located anteriorly on head, diameter less than snout length.
Mouth large, oblique; lips thick, angle of gape vertical through beyond posterior margin of eye.Lower jaw projecting slightly beyond upper jaw.Scales on cheeks in 5 horizontal rows.Both jaws with multiple rows of sharp teeth.Vomer with small sharp, pointed teeth.Premaxilla with 4 -5 rows of numerous minute inward curved teeth.Palatine with 2 or 3 stout inward curved canine teeth and a single row of minute sharp teeth.Fifth ceratobranchial slender, with numerous long sharp, inward curved, pointed teeth.Dentary with numerous minute, slender, inward curved, pointed teeth and an inner row of inward curved large teeth.
Colouration.In alcohol (Fig. 1), dorsum, side of head and body uniformly dark grey, gradually becoming lighter towards throat and abdomen.A series of 10 -12 cream-coloured chevron markings present on body along anal-fin base.Well-defined black spots, smaller than size of pupil present on head and body.Spots on head larger on post-orbital region, gradually becoming smaller on cranial region of head.Black spots on opercle and ventral surface of head.Black spots on flanks numerous, scattered above and below lateral line, smaller than spots on post-orbital region.Dorsal and anal fins dark grey with narrow white margin.Dorsal fin with 5 -6 black blotches on mid-dorsal fin region between rays 1 -22 in juveniles < 60 mm SL, either absent or persist as single blotch in specimens > 80 mm SL.Pectoral fin with 4 -7 (usually 4) alternating dark brown and white semi-circular bands.Pelvic fin creamy white.Caudal fin with series of black blotches irregularly arranged in two or three semicircular rows.
In life (Fig. 2), body uniformly greenish grey or brown with well-defined numerous black spots as in preserved specimens; black spots more conspicuous and set apart from body background colour.Lip light grey.Iris greyish with orange rim around pupil.Body with alternating dark and light chevron marks sometimes tinged with orange or red.Pectoral fin with 4 -7 (usually 4) alternating dark brown and white semi-circular bands as in preserved specimens.Dorsal fin greenish grey in adults with 1 -3 black blotches, absent sometime; anal fin pale bluish grey, both fins with faint white margin.Caudal fin colour pattern as in preserved specimens.
Juveniles (Fig. 2A) brown with large black spot above pectoral-fin origin and on caudal-fin base.A faint orange or yellow band running between these two spots, pigmentation below band being darker than above.Black spots on head and body smaller and inconspicuous.Anal fin hyaline without any marking or blotch.Dorsal fin with 5 -6 black blotches on mid-dorsal fin region in specimens < 60 mm SL, either disappear or reduce to 1 -3 blotches in adult.Other fins either hyaline or pale grey.
Etymology.The specific epithet 'stiktos' meaning 'spotted' in Greek, referring to the numerous conspicuous spots present on the body.It is use as an adjective.
Distribution and habitat.Channa stiktos was collected from the Ianava, Niawh and Tiau Rivers of the Kaladan River drainage, Mizoram, North Eastern India (Fig. 3).The streams were clear, slow flowing with cobble substrate and no aquatic vegetation (Fig. 4).

Molecular analysis
The two COI gene sequences of Channa stiktos generated in this study are distinct from all other Channa species available in GenBank (interspecies distance ranges from 8.24 -25.33%), with the exception of clade 3 of C. ornatipinnis, the samples of which originated from India (KU667357 -KU667361 & KJ847162 -KJ847166).Our samples of Channa stiktos clustered together with samples of clade 3 of C. ornatipinnis (only 1.43% genetic distance) indicating that both are conspecific.The genetic distance (K2P distance) between Channa stiktos and its morphologically closest species, clade 1 of C. ornatipinnis (C.ornatipinnis from the type locality   In addition to differences in the COI sequence and the distinguishing characters mentioned in the diagnosis section, adults of Channa stiktos can be further distinguished from C. ornatipinnis in having the lips and lachrymal region greyish green (vs.golden orange or reddish).Channa stiktos is distinguished from C. pulchra, another Rakhine species, in having more pored scales (47 -49 vs. 43 -46) in the lateral line, a shorter (26.5 -29.9 vs. 31.0-32.0%SL) and shallower (10.3 -13.3 vs. 13.8 -14.7% SL) head, and by the absence (vs.presence) of reddish blotches on the sides of the body.It can be distinguished from C. pardalis by the presence (vs.absence) of blotches on the mid dorsal fin region in juveniles and adults, by the pectoral    from C. harcourtbutleri in having more pored scales (47 -49 vs. 43 -46) in the lateral line and by the presence (vs.absence) of black spots on the body; and from C. aurantipectoralis in having striped (vs.plain) pectoral fins, fewer pored scales (47 -49 vs. 51 -64) in the lateral line and fewer scale rows (4½ vs. 5½ -6½) above the lateral line.
The taxonomic confusion between Channa gachua and C. limbata has been addressed by CONTE-GRAND et al. (2017).Using COI sequence data, they identified that a western lineage of gachua, the true Channa gachua (lineage 1 with specimens from west of the Indo-Burman ranges covering Sri Lanka, India, Nepal, Bangladesh, and the Rakhine area of Myanmar) differs from an eastern lineage of gachua, for which they applied the oldest available name C. limbata (lineage 2 with specimens from east of the Indo-Burman ranges from Myanmar reaching east to Vietnam and southern China and south to Indonesia and Malaysia).Channa stiktos markedly differs from C. limbata by the presence (vs.absence) of numerous large black spots on the head and the body.
There are some names still buried in the synonymy of Channa gachua.Of these, Ophiocephalus aurantiacus hamilton 1822 (type locality: Goyalpara on the North East frontier of Bengal), Ophicephalus marginatus Cu-Vier 1829 (type locality: Vizagapattam, see kottelat, 2000) and O. fuscus CuVier 1831 (type locality: Bengal) are species reported from India.Channa stiktos can be distinguished from O. aurantiacus, O. fuscus and O. marginatus (which are similar to C. gachua), by the presence (vs.absence) of well-defined black spots on the head and the body.Channa stiktos is further distinguished from C. fuscus and C. marginatus in having more than (vs.fewer than) 40 pored scales in the lateral line.Moreover, two other names, Ophicephalus montanus mCClelland, 1842 (type locality: Afghanistan?) and O. kelaarti Günther, 1861 (type locality: Sri Lanka) are also currently in the synonymy of C. gachua (kottelat, 2013).Channa stiktos can be distinguished from O. montanus by having more (34 -36 vs. 32) dorsal-fin rays and more (24 -25 vs. 17) anal-fin rays.Further, it can be distinguished from O. kelaarti by having more pored scales (47 -49 vs. 38) in the lateral line and more (24 -25 vs. 22) analfin rays.Another similar species, Ophiocephalus gachua var.basalis Günther, 1861 (type locality: East Indies), is most probably a synonym of C. gachua (kottelat, 2013) due to having a black ocellus with white edge on the hindmost part of the dorsal fin, a typical character of juvenile C. gachua.Channa stiktos can be distinguished from O. gachua var.basalis in having more (24 -25 vs. 21 -23) anal-fin rays, more scales (4½ vs. 3) above lateral line, and juveniles with blotches on the mid dorsal fin region, between rays (vs. a black ocellus with white edge on the hindmost part of the dorsal fin).
The discovery of Channa stiktos from the Kaladan River Basin of Mizoram, part of the Indo-Burma biodiversity hotspot region, highlights north-east India and north western Myanmar as hotspots of species level diversity for Channa as recognized previously (Britz,
bleheri and C. andrao by the presence (vs.absence) of pelvic fins.It can be distinguished from C. gachua by the presence (vs.absence) of numerous large black spots on the head and the body, pectoral fin with 4 -7 alternating dark brown and white bands (vs. 3 -4 grey bands fading towards the distal margin), presence (vs.absence) of blotches in the mid dorsal fin region in juveniles and adults, and more (28 vs. 24) circumpeduncular scales; from C. aurantimaculata and C. barca in having fewer (34 -36 vs. 45 -52) dorsal-fin rays and fewer pored scales (47 -49 vs. 51 -54 in C. aurantimaculata and 60 -64 in C. barca) in the lateral line;

Fig. 5 .
Fig. 5. Maximum Likelihood (ML) tree constructed by using TN93+G+I nucleotide substitution model with 1000 bootstraps for COI dataset of 41 sequences using MEGA7 software.
Channa stiktos is distinguished from all other species of Channa, except C. ornatipinnis, C. pulchra, C. pardalis, C. melanostigma and C. stewartii, by a unique colour pattern consisting of numerous large, black spots on the head and the body.Channa stiktos is distinguished from C. ornatipinnis 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 absence of dark spot on the anal fin of juveniles (vs.presence of series of up to 10 dark spots).
MUTHUKUMAR et al. (2017)oposed the 'Channa gachua species group' based on the diagnostic feature of having a varying number of dark and light semicircular bands on the pectoral fin.Channa stiktos shares this character indicating its inclusion into the C. gachua species group.Channa stiktos closely resembles C. ornatipinnis and C. pulchra in having prominent spots on the head and the body.Channa ornatipinnis was reported from the Tuivawl River (Barak River drainage) of Mizoram, India byMUTHUKUMAR et al. (2017).The snakeheadfigured in MUTHUKUMAR et al. (2017)is very similar to C. stiktos in morphological appearance and meristic counts, however, the numerous black spots on the body are sparsely distributed or absent below the lateral line (Fig.1of MUTHUKUMAR et al. 2017).Inaccessibility of the specimens reported in MUTHUKUMAR et al. (2017) for further study and absence of any DNAsequence data from these specimens do not allow us to comment further on the taxonomic identity and potential conspecificity of the C. ornatipinnis-like snakehead from Tuivawl River with C. stiktos.Recently, CONTE-GRAND et al. (2017) identified three significantly different clades when analyzing the COI sequences of Channa ornatipinnis.They observed high intraspecific divergence between C. ornatipinnis (of clade 1) from the type locality (Waloun Chaung, northern Rakhine State, Myanmar) and C. ornatipinnis (of clade 2) from another tributary of the Ayeyarwaddy (ca.70 km southeast of the type locality).A number of additional COI sequences deposited in GenBank as C. ornatipinnis from India, but without precise locality information, however, were even more divergent and considered to belong to a different species by CONTE-GRAND et al. (2017), who recommended a detailed taxonomic study of C. ornatipinnis specimens other than from the type locality.The COI analysis of our Channa stiktos and comparison with other Channa COI sequences available in GenBank revealed that it is significantly different from all of them, except CONTE-GRAND et al.'s (2017) clade 3 of C. ornatipinnis, the Indian samples.The sequence difference in the COI gene between specimens of CONTE-GRAND et al.'s (2017) clade 3 of C. ornatipinnis and C. stiktos is only 1.43% indicating that the two are conspecific.In contrast, the deep interspecies distance (8.24%) between C. stiktos with C. ornatipinnis from the type locality (clade 1 of C. ornatipinnis) and CONTE-GRAND et al.'s (2017) clade 2 of C. ornatipinnis (8.59%) supports the idea that C. stiktos and C. ornatipinnis are two different species.

Table 2 .
Detail of sequences (n=52) used in this study and NCBI GenBank accession numbers.

Table 3 .
The percent K2P genetic distance among Channa species, morphologically and genetically close to Channa stiktos sp.nov.
Furthermore, Channa stiktos is markedly different from other species of the C. gachua species group viz.C. orientalis, C. burmanica, C.