DNA barcoding indicates the range extension in an endemic frog Nyctibatrachus jog, from the Western Ghats, India

Abstract The frogs of genus Nyctibatrachus from the Western Ghats are endemic, with some taxa showing a narrow distribution range. Nyctibatrachus jog was known only from the type locality, Jog falls from Sharavathi river basin suggesting a restricted distribution. In this study, using DNA barcoding, we studied the distribution patterns of N. jog by sampling multiple river basins. 16S rRNA and Cytochrome b genes were used to distinguish N. jog from its congeners as well as to infer intra-species relationships. The results from the 16S rRNA gene showed 99% similarity of the collected individuals with the type specimen from Jog Falls confirming the identity of N. jog. The results indicate that N. jog has wide distribution extending its range in multiple river basins in the Western Ghats, India. This study also provides the Area of Occurrence and Extent of Occurrence of N. jog which could help in developing strategies for its conservation.


Introduction
DNA barcoding has been routinely used to identify species, contributing to biodiversity research (Hebert et al. 2003;Hajibabaei et al. 2007). It has also been useful to understand the geographical distributions of populations, providing insights into the patterns of geneflow within a species (Hajibabaei et al. 2007). Using DNA barcoding, many studies have identified the new extent of the species thus expanding their distribution range (Seshadri et al. 2016;Kundu et al. 2020;Lim et al. 2020).
The Western Ghats of India is known for the high diversity of amphibians with more than 90% of the species endemic to the region. There are nine families of anurans found here, of which three families, Micrixalidae Dubois et al. (2001), Ranixalidae Dubois (1987) and Nasikabatrachidae Biju and Bossuyt (2003) are endemic to the Western Ghats while family Nyctibatrachidae Blommers-Schl€ osser (1993) has genera Nyctibatrachus and Astrobatrachus distributed in the Western Ghats and genus Lankanectus endemic to Sri Lanka. Some of these taxa have narrow distribution ranges, making them of high conservation concern (Vijayakumar et al. 2019).
The genus Nyctibatrachus is endemic to the Western Ghats, India. There are 36 species at present (Biju et al. 2011;Gururaja et al. 2014;Garg et al. 2017;Krutha et al. 2017). The characteristics of Nyctibatrachus frogs include wrinkled skin, rhomboid pupils, pointed vomerine teeth, subocular glands and a notched tongue (Biju et al. 2011). The larger Nyctibatrachus species are associated with torrential streams whereas the smaller Nyctibatrachus species are associated with marshy pools. (Biju et al. 2011). A study by Van Bocxlaer et al. (2012) revealed that the species show mountain-associated clade level endemism with a narrow distribution range. The authors mentioned that torrentially adapted Nyctibatrachus species disperse along the river systems but may have restricted distribution range due to the east-west orientation of the river system, thus limiting their expansion from one river system to another. However, fine-scale studies understanding the distribution range for many Nyctibatrachus species is lacking. Knowledge of the distribution extent can be helpful for their conservation.
Nyctibatrachus jog Biju et al. (2011) is an endemic frog from central Western Ghats India. The species was discovered from Jog falls, Karnataka at an elevation of 600 m by Biju et al. (2011). The species is adapted to fast-flowing streams and torrents (Biju et al. 2011). The authors mentioned that N. jog was found only at the type locality, Jog Falls, central Western Ghats, India. However, the exact distribution range of N. jog is not known. In this study, we provide additional distribution records of N. jog, identified using DNA barcoding methods. We also provide the Extent of Occurrence (EOO) and Area of Occurrence (AOO) for N. jog.
Western Ghats and flow westwards to join the Arabian sea. The river basins are differentiated based on their catchment areas and delineated using Digital Elevation models (DEM). 43 streams were sampled from the three river basins. The streams were sampled for both adults and tadpoles. The tadpoles were sampled using dipnets (15 cm by 15 cm). To distinguish N. jog from its congeners, individuals were identified by their calls, size, sex, clutch size following Biju et al. (2011), Gururaja et al. (2014 and by DNA barcoding. A small tadpole tail tip was excised and preserved in 80% ethanol for molecular work and the tadpoles were released back into the streams.

Molecular methods
The DNA was extracted using the salt extraction method as mentioned in Vences et al. (2012). A 530 bp of 16S rRNA gene (Palumbi et al. 2002) was used for identification as it has been widely used for DNA barcoding of amphibians (Vences et al. 2005). We also used 427 bp Cytochrome b (Cytb) gene (mcb 398: TACCATGAGGACAAATATCATTCTG3 mcb 869: CCTCCTAGTTTGTTAGGGATTGATCG3, Verma and Singh 2002). 23 individuals were sequenced for 16S rRNA and 26 individuals were sequenced for Cyt-b. The sequences were checked for identity using nucleotide BLAST (https://blast. ncbi.nlm.nih.gov/) and deposited in GenBank (16S Accession numbers MW081889-MW081909, Cyt-b Accession numbers MW762640-MW762665). Two 16S sequences viz adult N. jog (Voucher number BNHS5457) with accession number JN644900 (Van Bocxlaer et al. 2012) and tadpole N. jog (Voucher number BNHS5900) with accession number KP317819 (Priti et al. 2015) were downloaded from GenBank to confirm the identity of genetic sequences generated in this study. The monophyly of N. jog individuals were analyzed by building phylogenetic tree for 16S rRNA gene using Bayesian inference in Mr Bayes (Ronquist and Huelsenbeck 2003) and Maximum Likelihood methods in RAxMLGUI (Silvestro and Michalak 2012). GTR þ I þ G was used as substitution model as selected by Partition Finder v 1.1.1 (Lanfear et al. 2012). Lankanectes corrugatus (Accession number AF215393) was used as an outgroup. The genetic distance was calculated using Kimura 2 parameter method in MEGA 7 (Kumar et al. 2016). To understand the distribution range of N. jog, the Area of Occurrence (AOO) and the Extent of Occurrence (EEO) were estimated using GeoCat (Bachman et al. 2011).

Discussion
The range extension of N. jog suggests either lack of finescale sampling by previous studies or the difficulty in sighting this species due to its specific microhabitat preferences. N. jog prefers fast flowing streams and waterfalls which may not be easily accessible for sampling leading to few observations. Even in this study, the sightings were only in 9 streams out of the 43 streams sampled. Seasonality could be another reason as N. jog calls are not heard post monsoon making it difficult to identify in the field. Also, Nyctibatrachus genus harbors cryptic species, hence previous studies based on morphological observations may have misidentified N. jog for another Nyctibatrachus species as it was earlier known to have restriction distribution. This DNA barcoding study now indicates the wide distribution of N. jog as against the previous notion that it is distributed only in the type locality of the Sharavathi river basin (Biju et al. 2011). The habitats of N. jog are perennial fast-flowing streams and torrents. The major threats to its habitats are the conversion of the streams into areca plantations and paddy fields. Since N. jog inhabits torrents, there is a threat to their habitat by tourists who use the torrents for bathing and defecating. According to the International Union of Conservation of Nature (IUCN) Global Redlist assessment, the status of N. jog is not evaluated (Dandekar et al. 2020). From this study, it is evident that the EOO of N. jog has increased, thus it is no longer restricted to its type locality, Jog Falls of the Sharavathi river basin. Since this species appears to have wide distribution, efforts are needed for extensive fieldwork locating new populations. To increase the conservation efforts, multiple stakeholders must be involved that includes educating local people and forest department officials. Besides that, it is also essential to study population dynamics and population genetic studies for developing conservation strategies.