Badis kaladanensis, a new fish species (Teleostei: Badidae) from Mizoram, northeast India

Badis kaladanensis, a new percoid fish is described from the Kaladan basin of Mizoram, northeast India. It belongs to the Badis badis species group but can be easily distinguished from its congeners, except from B. kanabos and B. tuivaiei, in having a dark blotch on the dorsal fin between the base of 3rd to 5th spines. It is further distinguished from B. kanabos in having more scales in lateral row (27–30 vs. 25–26), more circumpeduncular scale rows (18–20 vs. 16–17) and smaller eye (7.5–8.9% SL vs. 9.5–12.7); and from B. tuivaiei in having fewer vertebrae (28–29 vs. 30–31) and more rakers on the first gill arch (9 vs. 6–8). The analysis of the mitochondrial DNA (coi and cytb) revealed the distinctness of B. kaladanensis from all other Badis species with the interspecific distance ranges from 5.4–20.4%. (coi) and 5.1–26.3% (cytb).


Introduction
Fishes of the genus Badis Bleeker 1854 are small freshwater fish normally inhabiting small streams or hill streams with slow to moderate flow, coastal drainages or ditches with stagnant waters. They are widely distributed over the Indian subcontinent, Pakistan, Nepal, Bangladesh, Myanmar, Peninsular Thailand, the Mae Khlong drainage, and part of the Mekong basin in South East Asia as well as the Upper Irrawaddy in southern Yunnan, China [1]. They belong to the family Badidae which consist of two genera viz., Badis and Dario. The genus Badis is distinguished from the genus Dario in having tubed lateral-line scales (vs. absent); 2-4 dentary foramina (vs. none); 3-toothed hypobranchial (vs. edentulous); short pelvic fin in males, not reaching the first anal-fin spine (vs. reaching beyond origin of anal fin); short dorsal fin lappets and rounded caudal fin (vs. prolonged and truncated respectively) [2].
Species belonging to the genus Badis, particularly the B. badis species group, exhibit very similar morphological traits including colour pattern [3]. Due to poor morphological diagnostic characters among some Badis species, accurate identification and/or delimitation of species remains a challenge. For instance, among the 25 currently recognized species of the genus Badis [4] [3]. The molecular approach of species delineation has been proved efficient and helpful when integrated with the conventional taxonomic approach [5][6][7]. It is therefore necessary to integrate morphological and the molecular approach for species delimitation, particularly among the Badis species. An ichthyological survey conducted along the Palak River and Sala River, tributaries of the Kaladan River, resulted in the collection of a Badis species which differs from the already reported congeners. A comparison of the morphology and mitochondrial DNA (coi and cytb) sequences of the species with congeners revealed it to be an unnamed species, which is described herein as Badis kaladanensis, new species.

Material and methods
The live fishes were collected specifically for this study after permission from Institutional Animal Ethics Committee of Pachhunga University College, Aizawl-Mizoram, India (approval no. PUC-IAEC-2011-A02). The study did not involve any endangered species or protected areas or private water bodies. The fishes were handled according to the guidelines laid down by the Committee for the Purpose of Control/ Supervision of Experiments on Animals (CPCSEA), and Animal Dissection Monitoring Committee of Pachhunga University College, Aizawl-Mizoram, India. In brief the live specimens of Badis kaladanensis sp.nov., were anesthetized using buffered solution of MS222 (TMS, Tricaine methanesulfonate) at the concentration of 70 mg/L in water, in a tank well oxygenated with external oxygen supply. The concentration of anesthesia was increased to 280 mg/L for euthanizing the fishes for preservation of voucher specimens. The fishes were kept for additional 20 minutes at this concentration after the opercular movement stopped in all fishes. In India no additional permission is required for collection and use of biodiversity within country.
A small amount of muscle was collected from the right side of each fish specimen and kept in 95% ethanol for DNA extraction. The fish specimens were fixed in 10% formalin and later transferred to 70% ethanol preservative. Counts and measurements were made on the left side of specimens, whenever possible, following Kullander & Britz [2]. Measurements were made point to point with digital calipers to the nearest 0.1 mm. Measurements of body parts are given as proportions of standard length (SL). For vertebral counts, four specimens were cleared and stained in alizarin. Vertebral count includes each of the first four vertebrae of the Weberian apparatus. Fin rays were counted using a stereomicroscope and were confirmed through cleared and stained specimens. The small posterior-most rays of the dorsal and anal fins, articulating with the same pterygiophore were counted together with the preceding ray as 1. Description and numbering of the laterosensory system on the head follows Fig 1 in Kullander & Britz [2]. Numbers in parentheses after a meristic value indicate the frequency of that value. All morphological measurements and meristic counts of Badis kaladanensis sp. nov. are given in Table 1.
Type specimens were deposited at the Zoological Survey of India, Kolkata (ZSI) and Pachhunga University College Museum of Fishes, Mizoram (PUCMF). Other collection code used herein is MUMF for Manipur University Museum of Fishes. 96 fast thermal cycler (Applied Biosystems, Inc., USA). A total of 25 μl PCR 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 were prepared. The PCR conditions (for both coi and cytb) 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 (49˚C for cytb) for 30 sec., extension at 72˚C for 80 sec. with final extension of 10 min at 72˚C. Sequencing was performed in forward and reverse direction using an automated ABI 3500 Genetic Analyzer (Applied Biosystems, Inc, USA).

DNA sequence analysis
The four sequences (2 each for B. kaladanensis and B. ferrarisi) obtained were analysed with the available GenBank sequences of Badis species (27 for coi and 31 for cytb) along with Dario dario as outgroup (KC774648 for coi and AY330958 for cytb) ( Table 2).
Sequences were aligned using CLUSTAL_W module integrated in MEGA 7 (Molecular Evolutionary Genetics Analysis) software [11]. The sequences were blasted in NCBI (http:// www.ncbi.nlm.nih.gov) for the nearest matches and submitted to NCBI GenBank (Accn. Nos. MT997154-MT997157 for cytb and MW000668-MW000671 for coi). The genetic distance was calculated by averaging pairwise comparisons of sequences across close relatives of Badis in MEGA7. The maximum likelihood (ML) tree was constructed using coi (Fig 1) and cytb dataset (Fig 2). Based on the lowest BIC (Bayesian Information Criterion), the best fit nucleotide substitution model for both the coi and cytb dataset was HKY+G+I given by Hasegawa et al. [12].

Nomenclature acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available under that Code from the electronic edition of this article. This published work and the nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix

PLOS ONE
"http://zoobank.org/". The LSID for this publication is: urn: lsid: zoobank.org: pub:9A23E022-F6F5-4226-BCD3-9F9D512AD4A8. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital repositories: PubMed Central, LOCKSS.

Molecular analysis
The coi and cytb gene sequences of Badis kaladanensis generated in this study are distinct compared to available GenBank sequences of other Badis species (5.4-20.4% for coi and 5.1 to 26.3 for cyt b), with the exception of one coi sequence of an unconfirmed Badis species, submitted earlier by first author (KF318338), with only 0.3% K2P distance, indicating that they are conspecific. Badis kaladanensis clustered in the same clade with species of the Badis badis species group [2], indicating that B. kaladanensis belongs to the B. badis species group.

PLOS ONE
Diagnosis. Badis kaladanensis is distinguished from its congeners in having the following combination of characters: a post nuchal hump, a conspicuous dark blotch covering superficial part of cleithrum above pectoral-fin base, a dark anterior dorsal-fin blotch between 3 rd to 5 th spines (consistently present in live and preserved specimens), 27-30 scales in lateral row, 18-20 circumpeduncular scale rows, 28-29 vertebrae, 9 rakers on the first gill arch and lacking a dark blotch on the dorsolateral aspect of caudal peduncle.
Description. See Table 1 for morphometric data and Fig 3 for appearance. Body elongate, compressed laterally. Predorsal profile rising approximately evenly from tip of snout to first dorsal-fin spine (a post-nuchal hump present). Snout triangular or slightly pointed in lateral aspect. Orbit situated in anterior half of head at level of mid-lateral axis of body or slightly below it,

PLOS ONE
diameter smaller than ⅓ of head length. Mouth oblique, protrusible, lower jaw projecting slightly beyond upper jaw, maxilla reaching to ⅓ of orbit, lower jaw articulation below middle of orbit. Opercular spine slender, with a sharp tip. Palatine, vomer and parasphenoid toothed.
Coloration in preservative. Body pale brownish with brown or black markings. Preorbital stripe dark brown, continued across chin. Postorbital stripe distinct with a black blotch close to orbit, and a faint blotch one scale posterior to that blotch. No supraorbital stripe. Suborbital stripe indistinct below middle of orbit. No dark blotch on opercle. Cleithral blotch present, prominent and black.
Body with five pairs of dark brown vertical bars (usually composed of dark spots at bases of scales). Three pairs between head and anal fin-origin, and two pairs posterior to anal-fin origin. Bar above cleithral blotch absent or indistinct. Dorsal bar terminations form darker blotches, not observed in some specimens. Dorsal fin dark brown or blackish with contrasting narrow white margin. A dark blotch at base between 3 rd to 5 th dorsal-fin spines. Caudal-fin base with a dark or brownish blotch covering the last lateral-line scale on body; a vertical black bar across caudal peduncle, absorbing the caudal base blotch. Anal and pelvic fins dark brown with distal margin usually hyaline.
In life (Fig 4).Similar to preserved specimens except that a dark band along middle of dorsal fin immediately distal to scaly basal cover, and sub-marginal part of dorsal fin orange.
Distribution and habitat. Known from the Palak River ( Fig 5) and Sala River, a tributary of the Kaladan basin, in the vicinity of Phurra village and Lungpuk village respectively in Siaha District of Mizoram, India (Fig 6). It is found associated with Olyra saginata, Pethia expletiforis, Rasbora rasbora and R. daniconius.
Etymology. The species is named after the River drainage, the Kaladan River. An adjective.   [2]. The previously included member of the group, B. ferrarisi [2], however, based on the genetic analysis, does not belong to this group and forms another group with B. kyar [3].
Badis kaladanensis markedly differs from members of the B. badis species group, apart from B. kanabos and B. tuivaiei, in having a dark blotch at base between 3 rd to 5 th dorsal fin spines (vs. absent in all other members). It differs from B. kanabos in having more scales in lateral row (27-30 vs. 25-26), more circumpeduncular scale rows (18-20 vs. 16-17), more vertebrae (28-29 vs. 26-28) and smaller eye diameter (7.5-8.9% SL vs. 9.5-12.7); from B. tuivaiei in having fewer vertebrae (28-29 vs. 30-31) and more rakers on the first gill arch (9 vs. [6][7][8]. The so far reported species of Badis from the Kaladan basin is B. badis [13] and the same species was reported form the Barak River drainage [14] inside Mizoram, India. Our previous collections from Tuirial and Tlawng Rivers (a tributaries of Barak River) and Tuichawng River (a tributary of Karnaphuli River) include B. badis and B. rhabdotus, but not a single specimen of B. badis from Kaladan basin. It is likely that the specimens previously reported as Badis badis from the Kaladan basin were in fact B. kaladanensis.
The morphological distinction of Badis kaladanensis from B. badis, B. chittagongis, B. pallidus and B. rhabdotus is less distinctive due to their overlapping characters. Moreover, B.  (Table 3).
Badis kaladanensis is also compared with members of other species groups. It differs from B. kyar (B. kyar group) in having more scales in lateral row (27-30 vs. 26-27) and the pattern of caudal-fin marking (vertical black bar across caudal peduncle absorbing the caudal base blotch vs. caudal fin marking with an entire curved band in B. kyar). Schindler & Linke [15]  The mitochondrial gene (coi and cytb) analysis of Badis kaladanensis and comparison with other Badis sequences available in GenBank revealed that it is significantly different from all of them, except the coi sequence of Badis sp. Kolodyne (0.3% only, indicating that they are conspecific). Badis sp Kolodyne is a specimen from the Lungbun river of Mizoram which is draining into the lower stretch of Kaladan drainage. This suggested that B. kaladanensis may have a wider distribution along the Kaladan River and its tributaries. Furthermore, the maximum likelihood tree inferred from coi and cytb confirmed that B. kaladanensis belongs to the Badis badis group. Badis kaladanensis is more closely related to B. rhabdotus, which has adjacent geographical distribution (type locality of B. rhabdotus being Karnafuli Drainage), compared to other species of Badis badis group. However as mentioned in the result section B. kaladanensis and B. rhabdotus belong to two distinct species (interspecies distance ranges from 4.9-5.1%). Moreover, the position in the tree (Figs 1 and 2) and the wide genetic distance from the closely related species confirms that B. kaladanensis is distinct from all other Badis species.
Despite the less pronounced morphological diagnostic characters among species of the Badis, many species have been described using morphological analysis leading to confusion. It is suggested to integrate morphological approach with molecular analysis. The integration of morphological and molecular approach of species delimitation will strengthen and speed up the taxonomy of Badis in future.