Scaling new heights: first record of Boulenger’s Lazy Toad Scutiger boulengeri (Amphibia: Anura: Megophryidae) from high altitude lake in Sikkim Himalaya, India

hotspots of the World (Mittermeier et al. 2004). This region has a high altitudinal range from 300–8598 m and harbors several endemic taxa. The high altitude flora and fauna of this region are important for global biological diversity because there are very few places on Earth with such a unique ecosystem (Mani 1994; Shrestha et al. 2012). In Sikkim Himalaya, 23 species of amphibians are known (Chettri et al. 2011); some species have narrow distribution range, and for many, there is still a lack of knowledge about their distribution, life history and ecology (Acharya & Vijayan 2011; Chettri et. al. 2011). These areas, due to their inaccessibility, have not been surveyed extensively, especially for amphibians. Scaling new heights: first record of Boulenger’s Lazy Toad Scutiger boulengeri (Amphibia: Anura: Megophryidae) from high altitude lake in Sikkim Himalaya, India

Among the several restricted taxa in the Himalayan region is the genus Scutiger, belonging to the family Megophryidae.It is one of the most diverse families of non-neobatrachian frogs.Twenty species of Scutiger occur in the high altitudes of the Tibetan plateau in southwestern China, Myanmar, Nepal and northern India (Frost 2015).One interesting species in this genus Scutiger boulengeri, commonly known as Boulenger's Lazy Toad or Xizang Alpine Toad, holds the record for being the highest altitude frog in the world (Hock 1964), found at altitudes up to 5100m.Scutiger boulengeri is known to occur in northwestern Nepal, and southeastern Qinghai, eastern & southern part of Xizang, southern Gansu and northwestern Sichuan provinces in western China (Ohler et al. 2014).
Another species of Scutiger found in Sikkim is Scutiger sikimmensis, commonly known as the Sikkim Snow Toad.Scutiger boulengeri can be easily mistaken for S. sikimmensis, as they are morphologically similar.Scutiger sikimmensis is very common in the high altitudes of Sikkim Himalaya.In this paper, using an integrative taxonomic approach, we delineate S. boulengeri from S. sikimmensis and hence confirm the presence of two species of Scutiger in Sikkim.

Sampling
A total of four individuals of S. boulengeri and four individuals of S. sikimmensis collected from five locations in Sikkim were included in this study (Fig. 1).The specimens are presently deposited in ATREE museum, Bangalore, India and will be deposited in the Natural History Museum of Sikkim as per the agreement with the forest department (15/DIR(REE)/FEWMD/GOS/2011) once the museum becomes functional.Latitude and longitude were recorded using Garmin eTrex Vista H GPS with the datum set to WGS 84.
Specimens were fixed in 5% formalin for 12 to 18 hours and were subsequently transferred to 70% ethanol.Samples for molecular analysis were taken from hind limb muscle tissue, and were preserved in 70% ethanol prior to fixing in formalin.

Morphometry
Morphometric data were recorded using digital caliper (+/-0.1mm).All measurements follow Ohler et al. (2011).The measurements were used to compute the Principle Component Analysis (PCA) between the two species.For PCA, data were transformed into their base 10 logarithm to correct for size and analysis was performed in PAST 3 (Hammer et al. 2001).
Body: SVL snout-vent length.Head: EL eye length; EN distance from anterior corner of eye to posterior edge of nostril; HL head length from posterior corner of mandible to tip of snout; HW head width, at the angle of jaws; IBE distance between posterior corners of eyes; IFE distance between anterior corners of eyes; IN internarial distance; IUE minimum distance between upper eyelids; MBE distance from posterior corner of mandible to posterior corner of eye; MFE distance from posterior corner of mandible to anterior corner of eye; MN distance from posterior corner of mandible to posterior edge of nostril; NS distance from anterior edge of nostril to tip of snout; SL distance from anterior corner of eye to tip of snout; UEW maximum width of upper eyelid.Forelimb: FLL forearm length, from elbow to base of outer palmar tubercle; HAL hand length, from base of outer palmar tubercle to tip of third finger; TFL third finger length, from articulation of proximal and intermediate phalange.Hindlimb: FFTF distance from maximum incurvation of web between fourth and fifth toe to tip of fourth toe, toes being spread; FL thigh length, from vent to knee; FOL foot length, from base of inner metatarsal tubercle to tip of fourth toe; FTL fourth toe length, from articulation of proximal and intermediate phalange; ITL inner toe length; MTFF distance from distal edge of metatarsal tubercle to maximum incurvation of web between fourth and fifth toe, toes being spread; MTTF distance from distal edge of metatarsal tubercle to maximum incurvation of web between third and fourth toe, toes being spread; TFOL length of tarsus and foot from base of tarsus to tip of fourth toe; TFTF distance from maximum incurvation of web between third and fourth toe to tip of fourth toe, toes being spread; TL shank length.

DNA extraction, amplification and sequencing
Genomic DNA extraction was carried out using thigh tissue samples preserved in 70% alcohol.The extraction was carried out following the protocol described in Vences et al. (2012).We used the primers 16Sar-L 5'-CGCCTGTTTATCAAAAACAT-3' and 16Sbr-H 5'-GAGGGTGACGGGCGGTGTGT-3' to amplify the fragment of the mitochondrial 16S ribosomal RNA gene following Hebbar et al. (2015).The PCR cycling procedure was as follows; initial denaturation at 94 0 C for 3 mins, 36 cycles of 94 0 C for 45 sec, 55 0 C for 1 min and 72 0 C for 45 sec and a final extension step at 72 0 C for 10 mins.Amplified PCR products were sent for purification and sequencing to AMNION Sequencing Service, Bangalore, India.

Sequence Data Analysis
The sequence chromatogram obtained were edited using Chromas (Version 2.4, Copyright 1998-2012; Technelysium Pvt. Ltd., South Brisbane, Australia) and the BLAST (Altschul et al. 1990) analysis was performed with the sequences as queries to identify similar nucleotide sequences in the NCBI sequence database.Along with the sequences with highest homology, maximum query coverage and maximum score, the available DNA sequences of Scutiger species were downloaded as FASTA format from the GenBank.We obtained the sequences of S. glandulatus and S. boulengeri from the GenBank and these were included in the analyses (Appendix 1).These sequences along with the ones obtained from the current study were aligned using MUSCLE (Edgar 2004) nested in MEGA 5 (Tamura et al. 2011).All sequences were deposited in GenBank (Accession no.KR781480 -KR781486).The best fit model of sequence evolution/ DNA substitution was determined using J-Model Test Version 2.1.7(Darriba et al. 2012).The DNA substitution models chosen using the Akaike information criterion (AIC) in jModelTest ver.2.1.7 was GTR+I+G for 16S rRNA dataset (AIC value = 2881.698,-lnL= 1396.84880).Phylogenetic tree were constructed using Bayesian and Likelihood methods implemented in MrBayes Version 3.2 (Ronquist et al. 2011) and RaxML Version 1.3.1 (Silvestro & Michalak 2012) respectively.Ten million generations were executed in MrBayes out of which 10% (10,000 trees) were discarded as 'burn in'.The bootstrap support was determined for 1000 pseudoreplicates for the ML tree using rapid bootstrapping in RAxML Version 1.3.1.The convergence of MCMC chains was determined visually by the software TRACER Version 1.6 and by analyzing Potential Side Reduction Factor (PSRF).The final tree was visualized using FIGTREE (Rambaut 2009).

Results and Discussion
Four males and four females of the Scutiger species were used for both the morphometric as well as genetic analysis.Of these, two males and two females belonged to each of the two species, S. boulengeri and S. sikimmensis.Scutiger sikimmensis was collected from its type locality, Sikkim (Theobald 1868) and this is the first sequence submission of this species to GenBank.The morphometeric data analysis conducted confirmed that the individuals of the genus Scutiger found in Sikkim are two distinct species namely S. boulengeri and S. sikimmensis (Table 1; Image 1).The sequence analysis confirmed that these are two distinct species (Fig. 2).The PCA analysis based on morphometric data also supported the genetic analysis (Fig. 3).Thus, both morphological characters as well as molecular tools clearly established that these are two distinct species of Scutiger.
The obtained sequences of individual species of Scutiger comprised of 484bp out of which there were 54 parsimony informative sites.The phylogenetic tree based on Maximum Likelihood analysis showed distinct clusters corresponding to S. sikimmensis, S. glandulatus and S. boulengeri.The nodes with poor support (<50%) are not shown (Fig. 2).The sequences, which were attributed to S. boulengeri based on morphological characters, namely KR781481, KR781482 and KR781483, formed a distinct cluster with S. boulengeri reported from China, thus conforming the identity of the species.The pairwise genetic distance (K2P) between S. boulengeri from the current study and Fu et al. (2007) study were equal to or less than 3% (Table 2).Genus Scutiger is restricted to the high altitudes of the Himalaya.Here we report the occurrence of S. boulengeri for the first time in India, from a high altitude lake in Sikkim.This species was found in the glacier Lake Gurudongmar Complex 1 and 2 (28 0 01'34.79"N& 88 0 42'51.72"E;28 0 00'34.17"N& 88 0 42'56.69"E)at an elevation of 5160m and 5270m respectively, which is the highest altitude ever recorded for an amphibian species.

Characters
At present, 20 species of Scutiger have been reported out of which 15 are endemic to China (Frost 2015).Only two species (S. sikimmensis, S. nyingchiensis) have been reported so far from India (Molur & Walker 1998).In this paper, we report S. boulengeri for the first time from the Indian region taking the number of amphibian species known from India to 385 (Dinesh et al. 2015).The IUCN lists S. boulengeri as Least Concern (Ohler et al. 2004) and its population trend as unknown.In our study area in Sikkim, both species were quite abundant.
During our field survey we found S. sikimmensis between 2619m to 4395m.It has been reported to occur at an altitude of 2500m and has been found upto 4600m in Sikkim (Chettri et al. 2011).We recorded S. boulengeri at 4005m and upto 5270m.
Compared to other terrestrial vertebrates, amphibians are relatively more vulnerable to the effects of rapid climate change as temperature can have a pervasive effect on amphibian biology, including reproduction because of their limited mobility (Donnelly & Crump 1998).A recent study by Shrestha et al. (2012) has shown that the Himalaya are among the most vulnerable regions to future climate change.According to IPCC Himalayan glaciers are receding faster than in any other part of the world (IPCC 2007).For example, Zemu Glacier in Sikkim has retracted by 194m between 1977 and 1984 with an average retreat of 27.7m/year (IPCC 2007).Increasing mean temperature in the Himalaya has led to rapid melting of snow covers and glaciers bringing irreversible changes to the Himalayan cryosphere and rivers that are enriched by snowmelt (Bolch et al. 2012).This rapid climate change could have a serious impact on high altitude amphibians.Over 50% of amphibians are reported to be particularly susceptible to climate change (Bellard et al. 2012).Climate change also has implications on species differentiation of amphibians.A species restricted to a narrow altitudinal zone could migrate to higher altitudes due to global warming (Moritz & Agudo 2013).The future of this extremely high altitude frog remains uncertain in the face of impending climate variation and global warming.Further studies on its physiology, temperature and moisture tolerance can give an insight on a batrachian strategy to adapt to climate change.
Head flat, wider than long.Snout rounded.Snout tip protrudes beyond lower jaw.Supratympanal fold present.Tympanum indistinct.Eyes protruding.Pupil vertical.Fingers free.Subpalmar tubercles below I and IV fingers.Toes moderately webbed.One large and oblong metatarsal tubercle present.Canthus rostralis banded.Males have nuptial spines on fingers I,II and III and a pair of small axillary and a pair of large oval pectoral shields.The belly in reproductive males is dotted with numerous small warts with black tips.Similar granules also present near cloaca.The black tip wears off in preserved specimen if not fixed in formalin.
Coloration: Color in life olive or greenish-grey with numerous warts arranged somewhat in a longitudinal row, on the dorsal side.Ventral side yellowish in color.Dorsal pattern with slightly darker color than the body color.Dorsal pattern forms a bar between the eyes.Pupil golden.

Habit and Habitat
High altitude lakes, ponds and streams.As winter sets in towards the end of September, they come out of water and burrow into lose soil to hibernate.The hibernating individuals come out of the burrows and from under the rocks with the first rains (March/April).

Distribution
Nepal, China (Qinghai, Xizang, Gansu Sichuan).This is the first report of this species from Sikkim, India.

Morphological characters
SVL: Male 42-62 mm; Female 45-67 mm Head wider than long.Snout rounded, slightly protrudes over the lower jaw.Tympanum hidden.Pupil vertical.Parotid glands present.Supratympanal fold in contact with the parotoids and does not reach the upper arm insertion.Fingers free.Toe webbing rudimentary.Metatarsal tubercle absent.Males have four-rounded sub-quadrangular or oval pectoral plate-like shields, two larger mid pectoral and two smaller axillary ones.Nuptual spines present on the upper side of fingers I and II and laterally on the third finger.During reproductive phase males develop tiny warts with black tips on the sides of the head, and on the anterior side of the lower jaw, dorsal region of the legs and on the ventral region of toes IV and V.
Coloration: Dorsal color in life olive green, brown or greyish-brown with numerous warts.Dorsal pattern is variable.Ventral region yellowish, uniform and smooth.Pupil golden.

Habit and Habitat
Found near mountain streams and rivers, grasslands, ox-bow lakes at higher elevation.They remain hidden under the stones.During breeding season males call from under the rocks at night. Altitudinal

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Image 1. A-E -Scutiger sikimmensis.A -Live specimen, (B-E -Preserved specimen) B -ventral view, C -dorsal view, D -ventral view of left foot, E -dorsal view of right hand; F-J -Scutiger boulengeri.F -live specimen, (G-J -preserved specimen) G -ventral view, H -dorsal view, I -dorsal view of left foot, J -dorsal view of right hand.

Figure 2 .
Figure 2. Molecular phylogenetic tree of S. boulengeri, S. glandulatus and S. sikimmensis using 16SrRNA dataset with Oreolalax spp. as outgroup.The values above the branches are Maximum Likelihood Bootstrap and below the branches are Bayesian Posterior Probability (BPP) generated using MrBayes.The nodes having value below 50% bootstrap and BPP 0.75 are not shown