17 4 NOTES ON GEOGRAPHIC DISTRIBUTION Check List 17 (4): 1021–1029 https://doi.org/10.15560/17.4.1021 Range extension of Ichthyophis multicolor Wilkinson et al., 2014 to India and first molecular identification ofIchthyophis moustakius Kamei et al., 2009 Hmar Tlawmte Lalremsanga1, Jayaditya Purkayastha2, Mathipi Vabeiryureilai1, Lal Muansanga1, Ht Decemson1, Lal Biakzuala1* 1 Developmental Biology and Herpetology Laboratory, Department of Zoology, Mizoram University, Aizawl, Mizoram, 796004, India • HTL: [email protected] https://orcid.org/0000-0002-3080-8647 • MV: [email protected] https://orcid.org/0000-0001-8708-3686 • LM: [email protected] https://orcid.org/0000-0001-8182-9029 • HD: [email protected] https://orcid.org/0000-0002- 7460-8233 • LB: [email protected] https://orcid.org/0000-0001-5142-3511 2 Help Earth, Guwahati, Assam, 781007, India • [email protected] https://orcid.org/0000-0002-3236-156X * Corresponding author Abstract We report a substantial range extension of Ichthyophis multicolor Wilkinson, Presswell, Sherratt, Papadopoulou & Gower, 2014, with new material from Mizoram State, Northeast India. The species was previously known only from its type locality more than 800 km away in Ayeyarwady Region, Myanmar. The species was identified by both its morphology and 16s rRNA gene sequence data. One of the studied individuals represents the largest known speci- men for the species (total length = 501 mm; mid-body width = 18.8 mm). Brief comparisons of I. multicolor with the sympatric as well as parapatric congeners in the region, and first barcode data for I. moustakius Kamei, Wilkinson, Gower & Biju, 2009 are also presented. Keywords Caecilian, Ichthyophiidae, Mizoram, new record, phylogeny, 16s rRNA Academic editor: Nikhil Modak | Received 30 November 2020 | Accepted 5 July 2021 | Published 12 July 2021 Citation: Lalremsanga HT, Purkayastha J, Vabeiryureilai M, Muansanga L, Decemson H, Biakzuala L (2021) Range extension of Ichthyophis multicolor Wilkinson et al., 2014 to India and first molecular identification ofIchthyophis moustakius Kamei et al., 2009. Check List 17 (4): 1021– 1029. https://doi.org/10.15560/17.4.1021 Introduction The genus Ichthyophis Fitzinger, 1826 is recognized as the Mathew & Sen, 2009; I. garoensis Pillai & Ravichan- most speciose and widely distributed genus of the caeci- dran, 1999; I. khumhzi Kamei, Wilkinson, Gower & Biju, lian amphibians (e.g., Wilkinson et al. 2014; Taylor 1968; 2009; I. moustakius Kamei, Wilkinson, Gower & Biju, Gower et al. 2002). Currently, the genus is represented 2009; I. nokrekensis Mathew & Sen, 2009; I. sendenyu by 50 species, of which 13 species occur in India. Eight Kamei, Wilkinson, Gower & Biju, 2009; and I. sikkimen- of these 13 species are reported from Northeast India, sis Taylor, 1960 (Frost 2021). namely I. alfredi Mathew & Sen, 2009; I. daribokensis Recently, Wilkinson et al. (2014) described a new © The authors. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 1022 Check List 17 (4) species of Ichthyophis, I. multicolor Wilkinson, Wilkin- ribosomal RNA gene (16s) using primers L02510 (For- son, Presswell, Sherratt, Papadopoulou & Gower, 2014, ward: 5′-CGCCTGTTTATCAAAAACAT-3′) (Palumbi based on 14 specimens from Ayeyarwady Region, Myan- 1996) and H3056 (Reverse: 5′-CTCCGGTTTGAACT- mar. This species is known only from its type locality CAGATC-3′) (Rassmann et al. 1997). The derived and is presently not included in the IUCN Red List. We sequences were compared with congeners using pub- provide the first record of I. multicolor from Mizoram lished 16s rRNA sequence data (Gower et al. 2002, 2007; State, India, and compare this species with the conge- Nishikawa et al. 2012; Wilkinson et al. 2014). Sequences nerics reported from Northeast India. We also provide (maximum of 691 base pairs) were aligned with MEGA genetic data of I. moustakius for the first time and com- v. 7 using the MUSCLE algorithm with default parame- pare this species’ molecular proximity to its congeners ter settings (Edgar 2004). Best fit nucleotide substitution through molecular phylogenetic analysis. model was selected using ModelFinder (Kalyaanamoor- thy et al. 2017) based on the Bayesian Information Cri- Methods terion. Phylogenetic relationships were reconstructed using maximum likelihood (ML) in IQ TREE (Nguyen The specimens reported here were collected after obtain- et al. 2015) with 10000 ultrafast bootstrap replicates ing permission for herpetofaunal collection in the State (Hoang et al. 2017). Uncorrected p-distance was calcu- of Mizoram from the Environment, Forests and Climate lated in MEGA v. 7 (Kumar et al. 2016). Change Department, Government of Mizoram (permit no. A.33011/2/99-CWLW/225). The specimens were deposited and voucher numbers were obtained from the Results Departmental Museum of Zoology, Mizoram Univer- Ichthyophis multicolor Wilkinson, Presswell, sity (MZMU). Measurements were taken with Mitutoyo Sherratt, Papadopoulou & Gower, 2014 dial vernier calipers (Model 505–671) to the nearest 0.1 Figures 1, 2; Tables 1–3 mm. The body circumference and total length (TL) were measured with thread and ruler. Annular grooves were New records. INDIA – Mizoram • Kolasib District, Ko- counted by using ImageJ2 software (Rueden et al. 2017) lasib Hmar Veng; 23.3682°N, 093.1420°E; 638 m a.s.l.; and cross-checked manually. The numbers of vertebrae 17.V. 2016; Hmar Tlawmte Lalremsanga leg.; from a were counted by using digital radiography at 56 kVp and roadside stream; MZMU 911, 1♂, TL 431 mm • Aizawl 24 mAs (Allengers MARS 30 X-Ray Machine). Fresh District, Mualpui; 24.2018°N, 093.2176°E; 833 m a.s.l.; liver tissue was collected and stored at −20°C in 95% 23.VI. 2016; Hmar Tlawmte Lalremsanga leg.; from ethanol in the facility of Developmental Biology and a roadside, near riparian buffers; MZMU 913, 1♀, TL Herpetology Laboratory, Mizoram University, India. A 335 mm • Aizawl District, College Veng; 24.2071°N, map was produced using QGIS v. 3.16.2 software (QGIS 093.2139°E; 874 m a.s.l.; 18.VIII. 2019; Hmar Tlawmte Development Team 2020). Lalremsanga leg.; dug out of soil during road construc- The abbreviations of Wilkinson et al. (2014) were tion; MZMU 1480, 1 ♀, TL 406 mm • Aizawl Dis- used: AG = annular groove; AM = anterior most mar- trict, Tlangnuam; 23.6996°N, 092.7172°E; 997 m a.s.l.; gin of mouth on upper jaw; C1 = first nuchal collar; C2 29.IX.2019; Samuel Lianzela leg.; from a roadside canal, = second nuchal collar; CM = corner of the mouth; IM = near secondary forest; GenBank: MZ098157; MZMU inner mandibular tooth; NG1 = first nuchal groove; NG3 1541, 1 ♂, TL 501 mm • Aizawl District, Melthum; = third nuchal groove; OM = outer mandibular tooth, 23.6894°N, 092.7208°E; 1030 m a.s.l.; 27.VI. 2020; Lal PM = premaxillary-maxillary tooth; ST = snout tip; TA Muansanga leg.; captured while crossing a tarmac road; = tentacular aperture; TG = dorsal transverse groove on GenBank: MZ098156; MZMU 1739, 1 ♀, TL 361 mm collar; TP = tentacular papillus; VP = vomeropalatine • Aizawl District, Mission Vengthlang; 23.7120°N, tooth; L/H = total length divided by head length (the 092.7071°E; 934 m a.s.l.; 1 VII. 2020; Ro Malsawma leg.; latter = ST–NG1 measured directly behind CM); L/T = GenBank: MZ098155; MZMU 1740, 1 ♀, TL 310 mm • total length divided by tail length (the latter = distance Aizawl District, Tuirini bridge; 23.6844°N, 092.8847°E; behind vent); L/W = total length divided by midbody 272 m a.s.l.; 1.X. 2020; Ht Decemson leg.; dead on width; W/S = width at midbody divided by maximum road; MZMU 1956, 1 ♀, TL 339 mm • Aizawl District, width of stripe at midbody; AV = anterior limit of vent; Zemabawk; 23.7331°N, 092.7622°E; 860 m a.s.l.; 1.X. PV = posterior limit of vent; TT = tail tip and in mea- 2020; Gospel Zothanmawia Hmar leg.; dead on road; sures N = naris; and E = eye. MZMU 1965, 1 ♂, TL 338 mm • Aizawl District, Mis- Genomic DNA was extracted from the liver sample sion Vengthlang; 23.7122°N, 092.7075°E; 943 m a.s.l.; of three I. multicolor specimens (MZMU 1541, MZMU 19.X. 2020; Ro Malsawma leg.; MZMU 2003, 1 ♀, TL 1739, and MZMU 1740), and two I. moustakius speci- 468 mm • Aizawl District, Mizoram University campus; mens (MZMU 1758 and MZMU 1847) using the DNeasy 23.7381°N, 092.6618°E; 774 m a.s.l.; 21.X. 2020; Ht De- Blood and Tissue Kit (Qiagen, Valencia, California, cemson leg.; found on surface near Mizoram University USA) following the manufacturer’s standard protocol. park, after rain showers; MZMU 2005, 1 ♂, TL 464 mm. We amplified and sequenced the mitochondrial 16S Identification. The specimens MZMU 1541, MZMU Lalremsanga et al. | Ichthyophis multicolor in India 1023 Figure 1. A. Map showing the type locality of Ichthyophis multicolor in Ayeyarwady, Myanmar (red diamond), and new localities from Mizoram, India (red triangles); and the new collection sites (yellow circles) and previous records (yellow squares) of Ichthyophis moustakius from India: (1) Ayeyarwady, (2) Kolasib Hmar veng, (3) Mizoram University campus, (4) Mualpui, (5) College veng, (6, 7) Mission vength- lang, (8) Tlangnuam, (9) Zemabawk, (10) Melthum, (11) Tuirini, (12) Tamdil, (13) Dampa Tiger reserve, (14) Thakthing, (15) Sawleng, (16) Bamgaizaeng, (17) Aziuram, (18) Nswanram, (19) Nriangluang, (20) Guwahati. B. Maximum likelihood 16s tree showing the inferred rela- tionships of Northeast Indian and Myanmar Ichthyophis multicolor and congeners with 10000 bootstrap replicates (values at internal branches are bootstrap support values). The rhinatrematid Epicrionops marmoratus was used as outgroup. Sequences generated in this study are shown in bold, and * indicates species recently allocated to the genus Epicrium (see Dubois et al. 2021). C. Ichthyophis moustakius (MZMU 1847) in life.