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Journal of Animal Diversity Online ISSN 2676-685X Volume 3, Issue 2 (2021)

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Journal of Animal Diversity Online ISSN 2676-685X Volume 3, Issue 2 (2021) Journal of Animal Diversity Online ISSN 2676-685X Volume 3, Issue 2 (2021) Research Article http://dx.doi.org/10.52547/JAD.2021.3.2.2 The first record of Kurixalus yangi Yu, Hui, Rao and Yang, 2018 (Anura: Rhacophoridae) from Mizoram State, India, with a reassessment of previous records of its congeners from Mizoram Lal Muansanga, Malnica Vanlal Malsawmtluangi, Gospel Zothanmawia Hmar, Lal Biakzuala, Vanlal Siammawii, Vabeiryureilai Mathipi and Hmar Tlawmte Lalremsanga* Developmental Biology and Herpetology Laboratory, Department of Zoology, Mizoram University, Aizawl, 796004, Mizoram, India *Corresponding author : [email protected] Abstract Based on a collection of Kurixalus specimens collected from different localities in Mizoram State, we report the occurrence of Kurixalus yangi from the State. Species identification was confirmed based on morphology and molecular data Received: 14 December 2020 (mitochondrial 16S rRNA marker gene sequence). In addition, we confirm the Accepted: 28 January 2021 previous record of Kurixalus naso from the same region to be erroneous as it is Published online: 12 August 2021 in fact K. yangi. Herein, we reassess the systematic status of this montane population of Kurixalus from Northeast India and provide evidence for its distinction from Kurixalus naso sensu stricto, thereby reporting K. yangi as an addition to the amphibian fauna of Mizoram. This record represents the southernmost distribution of the species. Key words: First record, Northeast India, range extension, re-identification, 16S rRNA gene, taxonomy Introduction ranges from the vicinity of the type locality into adjacent northern Myanmar on the border region and The family Rhacophoridae (Amphibia: Anura) Nagaland of northeastern India (Frost, 2021). comprises 442 species belonging to 22 genera and Previously, two species in the same genus, Kurixalus the genus Kurixalus Ye, Fei and Dubois, 1999 appendiculatus (Günther, 1858) and Kurixalus naso consists of 20 species of tree frogs, distributed in (Annandale, 1912) were reported from Arunachal Myanmar, India, China, southern Cambodia, Pradesh State of Northeast India (Dinesh et al., 2020). Vietnam, Thailand, Peninsular Malaysia, Sumatra, Recently, Kurixalus yangi was reported from Borneo and the Philippines (Frost, 2021). However, Nagaland, northeastern India by Humtsoe et al. (2020). cryptic species diagnoses on the basis of their morphology is challenging in Kurixalus (Yu et al., In this paper, we report the occurrence of Kurixalus 2018). Studies indicate that this genus originated on yangi from Mizoram of northeastern India, confirmed mainland Asia and subsequently dispersed to the on the basis of morphological and molecular data and continental islands of East Asia and Southeast Asia also re-assess the validity of early records of Kurixalus by multiple colonization events (Yu et al., 2020). naso from the same region. Contiguous land connection between Borneo and Material and Methods mainland Southeast Asia existed throughout much of the Neogene Period (during times of lowered sea Surveys were conducted in Mizoram State to explore levels), and might have facilitated migration of the local herpetofauna between 2010 and 2020, terrestrial biotas (Moss and Wilson, 1998). Kurixalus employing Visual Encounter Survey (VES) and yangi was first described by Yu et al. (2018) from Acoustic Encounter Survey (AES) techniques. We Nabang, Yingjiang County, Dehong Autonomous observed and collected several specimens of Kurixalus Prefecture, western Yunnan in China. Its distribution individuals from Mizoram. Specimens were identified This article is published with open access on www.jad.lu.ac.ir | © Lorestan University Press 9 Muansanga et al. 10 using Yu et al. (2018) at the Laboratory of deposited in the Department Museum of Zoology, Developmental Biology and Herpetology, Department Mizoram University (MZMU) for further studies. of Zoology, Mizoram University. Specimens were Specimens were collected from seven localities (Table preserved in 70% ethanol, and liver tissues extracted 1). The distribution map was created using an open for genetic analysis; voucher specimens were source QGIS version 3.10.8 (Fig. 1). Figure 1: Map showing the distribution of Kurixalus yangi (type locality in red star, published records in red circles, and new records in red diamonds) and previous records of Kurixalus naso (in blue triangles): 1. Reiek Community Reserved Forest, 2. Tamdil National Wetland, 3. Hmuifang Community Reserved Forest, 4. Bualpui V, 5. District Park (Lunglei), 6. Sialhawk, 7. Phawngpui National Park, 8. Nabang, 9. Dengga, 10. Nanjingli, 11. Mohynin, 12. Wokha, 13. Kohima, 14. Egar, and 15. Muotuo. Mensural data were taken by using a digital calliper proximal end of the outer palmar tubercle to the tip (MitutoyoTM 505-507) to the nearest 0.01 mm. of the longest finger); finger length (F, distance from Abbreviations used in this study for morphometric wrist-metacarpal joint to tip of finger); femur length measurements are: snout-vent length (SVL); head (FL); tibia length (TL); length of tarsus and foot width (HW); head length (HL); distance from the together (TFOL); foot length (FOL); fourth toe length back of the mandible to the nostril (MN); distance (FTL); toe length (T, distance from tarsus-metatarsal from the back of the mandible to the front of the eye joint to tip of finger); VA/ERS/ZSI (Vouchered (MFE); distance from the back of the mandible to the Amphibians/Eastern Regional Station/Zoological back of the eye (MBE); distance between the front of Survey of India). Detailed morphological data of the the eye (IFE); distance between the back of the eye specimens are provided in Table 2. (IBE); inter-narial distance (IN); distance from the Genomic DNA was extracted from the liver tissues eye to nostril (EN); eye length (EL); snout to nostril of adult amphibians according to manufacturer’s distance (SN); snout length (SL, distance from the protocol using a QIAamp DNA Mini Kit (Cat No. nostril to the tip of the snout); greatest tympanum ID: 51306). PCR reaction was prepared for 20 μL diameter (TYD); distance from tympanum to the reaction mixture contained 1X amplification buffer, back of the eye (TYE); inter-orbital distance (IO); 2.5 mM MgCl , 0.25 mM dNTPs, 0.2 pM each upper eyelid width (UEW); forelimb length (FLL, 2 forward and reverse primer, 1 μL genomic DNA, and distance from upper arm insertion to the tip of the 1U Taq DNA polymerase with a pair of partial 16S longest finger); hand length (HAL, distance from the Journal of Animal Diversity (2021) | © Lorestan University The first record of Kurixalus yangi Yu, Hui, Rao and Yang, 2018 … rRNA primers: forward (L02510- CGC CTG TTT 7 (Kumar et al., 2016), the Kimura 2 (K2P) and ATC AAA AAC AT) (Palumbi, 1996) and reverse genetic distances (Kimura, 1980) were calculated (H03063- CTC CGG TTT GAA CTC AGA TC) using MEGA 7 (Kumar et al., 2016). (Rassmann, 1997). The PCR thermal regime for The Bayesian Inference (BI) phylogenetic tree was amplification was 5 min at 95 °C for initial constructed in MrBayes 3.2.5 using GTR+I+G denaturation, followed by 35 cycles of 1 min at 95 °C model. The MCMC (one cold and three hot chains) for denaturation, 30s for annealing at 50.3 °C, was run for 100,000 generations by sampling every elongation for 1 min at 72 °C, and a final elongation 100 generations and set the burn-in to 25%. The for 5 min at 72 °C. PCR products were checked by analysis was terminated when the standard deviation gel electrophoresis on a 1.5% agarose gel containing of split frequencies was less than 0.01. The ethidium bromide. Samples were sequenced using percentage of trees in which the associated taxa Sanger’s dideoxy method and sequencing reactions clustered together is shown next to the branches were carried out in both directions on a sequencer (Ronquist and Huelsenbeck, 2003). The generated (Agrigenome Labs Pvt Ltd., Kochin, India). The phylogenetic tree was further modified using web- generated partial 16S rRNA sequence was deposited based iTOL software (Letunic and Bork, 2019). in GenBank (595 base pairs; accession number MT808303.1). Results and Discussion For the analysis of our dataset of 16S rRNA, we included 15 congener sequences obtained from NCBI The present study confirmed the first documentation of database and one sequence of Odorrana chloronata Kurixalus yangi from the Mizoram State of India (Fig. 2). (MZMU1842) was used as an outgroup. All sequences All specimens were collected during the monsoon were aligned by using MUSCLE algorithm in MEGA season from undisturbed riparian forests (Fig. 3). Table 1: Collection data of the present specimens and Mathew and Sen (2008; 2010) of Kurixalus yangi based on seven different localities in Mizoram state, India. 1- Reiek Community Reserved Forest, 2- Tamdil National Wetland, 3- Hmuifang Community Reserved Forest, 4- Bualpui V, 5- District Park, Lunglei, 6- Sialhawk, 7- Phawngpui National Park. Elevations are above the sea level (a.s.l.). District Park, Locality Lunglei 1 2 3 4 5 6 7 No. (Mathew and Sen 2008; 2010) Museum No. 243 243 615 618 621 617 (MZMU) 1014 1022 1016 1018 1815 1816 1924 2048 1013 1017 VA/ERS/ZSI/769 VA/ERS/ZSI/769 VA/ERS/ZSI/728 Collection date 1/6/2014 1/6/2014 2/9/2016 3/7/2016 5/6/2018 7/8/2020 4/8/2014 4/6/2006 30/7/2010 30/7/2010 29/5/2016 25/5/2016 14/5/2017 27/4/2019 23/6/2015 19/6/2016 12/6/2016 21/7/2014 22/9/2005 Coordinate 92°49' E 92°49' 22°50' N 22°50' N 92°57'2.23"E 92°45'7.71"E 92°49'3.54"E 93°5'30.89"E 93°1'56.03"E 23°7'12.42"N 23°7'12.42"N 92°36'21.93"E 92°47'39.32"E 23°41'36.48"N 23°41'36.48"N 23°44'30.35"N 23°27'15.03"N 22°50'52.60"N 23°17'43.39"N 22°37'19.79"N Elevation 1232 m 764 m 1484 m 925 m 1128 m 1292 m 1452 m 1128 m Journal of Animal Diversity (2021), 3 (2): 9–17 | www.jad.lu.ac.ir 11 Muansanga et al.
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