Herpetology Notes, volume 13: 645-648 (2020) (published online on 05 August 2020) First report on the fossorial tadpole of Micrixalus kottigeharensis (Rao, 1937) Madhushri Mudke1,2,3,*, Neelavara Ananthram Aravind1, Kotambylu Vasudev Gururaja4, Benjamin Tapley5, Pavankumar Thunga1, Jyoti Das3, Harshith Prince1, and Samyamee Sreevathsa1 The Kottigehar dancing frog, Micrixalus District, Karnataka, India (13.5762°N, 75.1045°E; kottigeharensis (Rao, 1937) is an evolutionarily distinct elevation 638 m). During this survey, we observed an and Critically Endangered frog (Biju et al., 2004; Gumbs amplectant pair of M. kottigeharenesis (Fig. 2A) and et al., 2018) endemic to the central Western Ghats of subsequent oviposition. We returned to the site the India. Its common name derives from characteristic following day to document the clutch and again 16 foot-flagging behaviour used in mating and territorial d later to observe tadpoles. The oviposition site had displays (Fig. 1). The primary habitat of this species approximately 70% canopy cover and was 0.2 m wide is fast to slow flowing stretches of rocky streams at an along a stream bed with small round pebbles in the elevation of 500–800 m (Biju et al., 2014). These frogs centre of a 3.9 m wide stream. breed in first and second order perennial freshwater Site 2.—On 9 November 2019 we surveyed a primary streams in evergreen to semi-evergreen forests as well as stream at Unchalli Falls, Uttara Kannada District, in the Myristica swamps of Karnataka (Chandran et al., Karnataka, India (14.4089°N, 74.7443°E; elevation 478 2010). As a part of a larger research project into the life m) and observed approximately 20 tadpoles at the site history and ecology of the species, a conservation action where we had also observed adult and amplecting M. that is highlighted in the IUCN Red List assessment for kottigeharenesis. The oviposition site had approximately this species, we found the tadpole of M. kottigeharensis 60% canopy cover and was 0.5 m wide along a shingle at two locations and we describe it herein. stream bed in the centre of a 4.3 m wide stream. Throughout these observations, mean atmospheric Materials and Methods temperature was measured by a Checktemp® digital Localities and observations Site 1.—On 21 September 2019 we surveyed a primary stream on the outskirts of Agumbe Nature Reserve, Dasanakodige Village, Tirthahalli Taluk, Shimoga 1 Suri Sehgal Center for Biodiversity and Conservation, Ashoka Trust for Research in Ecology and the Environment, Royal Enclave, Jakkur Post, Bangalore 560064, India 2 Manipal Academy of Higher Education, Manipal 576104, India. 3 EDGE of Existence Programme, Zoological Society of London, Regent’s Park, London, England NW1 4RY. 4 Srishti Institute of Art, Design and Technology, N4 Campus, Yelahanka New Town, Bangalore 560064, India. 5 Zoological Society of London, Regent’s Park, London, NW1 Figure 1. A foot-flagging male Micrixalus kottigeharensis 4RY England. from Shimoga District, Karnataka State, India. Photo by * Corresponding author. E-mail: [email protected] Benjamin Tapley. 646 Madhushri Mudke et al. thermometer (HI98501), and mean ambient humidity in MEGA 10.1.6, and alignments were created using was measured using a Portable Thermohygrometer ClustalW. A consensus sequence was used to query the (HI9564; both instruments from Hanna Instruments, NCBI BLAST database (Biju et al., 2014; Gururaja et Woonsocket, Rhode Island, USA). Stream length was al., 2014). We calculated the uncorrected p-distance for measured using tape measures and canopy cover was our sequences with a specimen of M. kottigeharensis estimated using photographs. collected 68 km away from Site 1 and 168 km away from Site 2 at Charmadi Ghats (13.0753°N, 75.4552°E, Species identification GenBank accession number KJ711304.1; Bombay Natural History Society voucher BNHS 5750; Biju et Species identity was confirmed based on DNA al., 2014). extraction from buccal swabs (Broquet et al., 2007). Two adult individuals from each site were swabbed Results and Discussion using sterile swabs. A clean pair of powder free, nitrile gloves was worn to handle each individual frog. Frogs Site 1.—On 21 September 2019 between 17:00 and were swabbed in-situ and the process took ca. 30 s. Frogs 18:40 h a pair of M. kottigeharensis in axillary amplexus were immediately released after the process and did was seen immersed in the stream water with only the not exhibit any signs of discomfort. We did not collect eyes above water level (Fig. 2A). The female was seen voucher specimens of tadpoles because of the species’ digging a cavity with her hind limbs for egg-laying conservation status and follow Rowley et al. (2017) in as described by Gururaja (2010). While remaining in describing the tadpole from live specimens in the field, amplexus, the female laid eggs into the cavity that she supported by molecular data. Swabs were stored in had dug at 18:05 h. She rapidly (within 2 s) covered the 100% ethanol at room temperature and transferred to the cavity with gravel (Fig. 2B) and the male simultaneously molecular genetics lab of the Ashoka Trust for Research separated from her and jumped on a rock within the in Ecology and the Environment. Total genomic DNA vicinity. was extracted using a Qiagen DNeasy Blood and After the male and the female had moved away, we Tissue kit following the manufacturer’s protocol. The marked the egg-laying area for future observation. At mitochondrial 16S rRNA gene was chosen for species noon on the following day, 22 September 2019, one of identification based on earlier studies (Biju et al., 2014). us (MM) carefully exposed the cavity by removing one The primers used for PCR amplification were 16Sf piece of gravel at a time to photograph the clutch and (5’-CGCCTGTTTATCAAAAACAT-3’) and 16Sr (5’- measure cavity depth using a metal ruler. During this CCGGTCTGAACTCAGATCACGT-3’). PCR was process care was taken to not let the eggs float away in performed in a 25-µl reaction using the Ampliqon flowing water by obstructing the flow for 3 min using Taq DNA Polymerase Master Mix RED containing flat rocks from within the stream and a light mesh. Once 1.5 µM MgCl2, 0.4 µM dNTP (including dATP, dCTP, the cavity was open, photographs of the eggs were taken dGTP, and dTTP), 0.2 µM of each primer, and 15 ng of (Fig. 2C). The cavity was then covered again and the DNA template. The optimised PCR amplification was area left as closely to its original state as possible. Eggs carried with a touchdown PCR annealing approach to were counted on the photographs by counting thrice minimise nonspecific primer amplifications (annealing from each of the five different photographs. This was temperature decreasing from 60–55°C for five cycles) done to increase the accuracy of visual counts and to and to improve primer specificity to bind the target reduce the possibility of duplicate counts or omissions. DNA. The thermocycling program was initiated with an A total of 34 eggs was counted and cavity depth was initial denaturation at 95°C for 5 min, a ‘touchdown’ 32 mm. PCR step consisting of 95°C for 30 s, 60–55°C for 45 s On 6 October 2019 the team revisited the marked for five cycles (decreasing by 1°C/cycle), and 72°C for oviposition site and exposed the clutch according to the 45 s, followed by 30 cycles of 95°C for 30 s, 55°C for methods described previously. At least three tadpoles 1 min, and 72°C for 90 s. The cycle was finished with were clearly seen in the cavity. When exposed, tadpoles a final extension of 72°C for 7 min, and products were exhibited quick, wriggling, wormlike movements, most stored at 4°C until further analysis. of which seemed to be caused by the tail. They quickly Amplified PCR products were purified and sequenced settled back into the cavity and lay flat on the gravel. at Barcode Biosciences (Bangalore, India) using an ABI By using a mesh net, we were able to catch two live 3730xl DNA sequencer. DNA sequences were edited tadpoles (Fig. 2D). These were at Gosner Stages 21– First report on the fossorial tadpole of Micrixalus kottigeharensis 647 Figure 2. Observations on the reproductive activity of Micrixalus kottigeharensis in Karnataka State, India. (A) Male and female in amplexus in Shimoga District. (B) Male and female M. kottigeharensis (A) after amplexus, with the female seen closing the cavity with eggs near her hind limbs. (C) Exposed egg cavity prepared. After removal of covering gravel, showing 34 eggs. (D, E) Photographs of live tadpoles from Shimoga District (D) and Uttara Kannada District (E). Photographs by Samyamee Sreevathsa, Madhushri Mudke, Kotambylu Vasudev Gururaja and Harshith Prince. 25 (Gosner, 1960), with yolk sacs, oral suckers, gills, 20 tadpoles at Gosner Stages 21–25. Using a fish net, a beating heart and discernible eyes, but without any we removed a single tadpole and photographed it (Fig. limbs or limb buds. 2E) before releasing. The ambient temperature and At Site 1, ambient temperature and humidity humidity were 26.7 °C and 92.4% respectively. The remained relatively constant (26.0 °C and 85.0% during water temperature was 24.7 °C. oviposition, 25.0 °C and 86.0% during our follow-up Species identification.—The species identity of the visit). Stream water temperature ranged from 22.3–23.6 adults at Sites 1 and 2 matched 100% with GenBank °C. Based on observations at this site, it takes 16 d for accession number KJ711304.1 (Biju et al., 2014) and the M. kottigeharensis eggs to develop from Gosner Stage p-distances for our samples were 0.000 and 0.001 (for 1 to Stage 25.