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The current study was focused on documentation of amphibian assemblage in North Punjab and Islamabad Capital Territory, Pakistan, by using mitochondrial gene sequences of 16S rRNA. Our study entailed 37% of the known amphibian species of the country. We provided a phylogenetic analysis based on 74 newly generated mitochondrial 16S rRNAs from nine species of genus Microlyla, Duttaphrynus, Allopaa, Nanorana, Sphaerotheca, Minervarya, Hoplobatrachus, and Euphlyctis. We employed the maximum‐likelihood inference and Bayesian analysis to assess the taxonomic status of the samples obtained from Pakistan, with respect to other congeneric species from surrounding regions. Our findings confirmed the taxonomic status of South Asian anuran species Duttaphrynus stomaticus, Duttaphrynus melanostictus, Microhyla nilphamariensis, Allopaa hazarensis, Nanorana vicina, Sphaerotheca maskeyi (synonym: S. pashchima), Minervarya pierrei, Hoplobatrachus tigerinus, and Euphlyctis kalasgramensis in Pakistan. We have reported new country records of genus Minervarya ( M. pierrei). Minervarya pierrei was previously misidentified as Fejervarya limnocharis, due to dearth of genetic information. We provided the first genetic records of our endemic species N. vicina. The results revealed the taxonomic placement of N. vicina with respect to its congeners and validated the taxonomic status of N. vicina from its type locality (Murree) for the first time. The findings of the present study also indicated the paraphyletic relationship of A.‐ hazarensis with Nanorana species. So, based on our phylogenetic inferences, morphological characters, and habitat preferences, validity of generic status of A. hazarensis is undecided. As our data were not enough to resolve this issue, we suggest sequencing of additional mitochondrial and nuclear genes in the future studies to get a better resolution. We recommend carrying out extensive surveys throughout the country for proper scientific documentation of amphibians of Pakistan. Many new species, some of them might be endemic to Pakistan, are expected to be discovered, and taxonomic status of other species would be resolved., The current study was focused to document amphibian assemblage of North Punjab and Islamabad Capital Territory, Pakistan, by using 16S rRNA. Our findings on the taxonomic status of South Asian anuran species confirmed the presence of Duttaphrynus stomaticus, Duttaphrynus melanostictus, Microhyla nilphamariensis, Sphaerotheca maskeyi (synonym: S. pashchima) Hoplobatrachus tigerinus, Euphlyctis kalasgramensis and Allopaa hazarensis. We have reported new country records of Minervarya pierrei and reported the first genetic records of south asian endemic species Nanorana vicina from its type locality.

Endemic anurans are particularly vulnerable to environmental changes, and are susceptible to population declines because of their restricted distribution ranges. The Murree Hills Frog Nanorana vicina and Hazara Torrent Frog Allopaa hazarensis are associated with the torrential streams and nearby clear water pools in subtropical chir pine forest and other forest types, at elevations higher than 1000 m in Pakistan. In this study, we have provided data on the extent of movement of these frog species for the first time. We installed radio transmitters on a total of 13 Murree Hills Frogs and 13 Hazara Torrent Frogs during eight consecutive days in September 2017 and 2018. Our results showed that these frogs did not move long distances along the stream or away from the stream into the forest. All the radio-tracked frogs showed movement of < 3 m. We found a significant differences only in the distance moved by Murree Hills Frogs between the two years studied. Based on our findings, we propose a movement paradigm that focuses on conservation implications for these endemic frogs.

The genus Nanorana, also called Yunnan Slow Frogs (Frank & Ramus 1995), belongs to the family Dicroglossidae. Murree Hills Frog N. vicina, is an endemic species of Southeast Asian uplands in Pakistan and India and was first reported by Stoliczka (1872) from Murree, Punjab Province, Pakistan. Later Rais et al. (2014) recaptured it from its type locality and described morphology of one juvenile female and one adult male. The species is listed as Least Concern in IUCN Red List of Threatened Species while the EDGE (Evolutionarily Distinct and Globally Endangered) score is 2.64. In Pakistan, it is reported from brook water springs and streams of Murree, Galiat (North Punjab), Ayubia (Khyber Pakhtunkhwa), and parts of Azad Jammu and Kashmir (Rais et al. 2014). Although the adult form has been characterized (Khan 2006; Rais et al. 2014), the morphology of the tadpoles have not yet been described. The current study aimed to describe external morphology and morphometry of tadpoles of N. vicina.

Murree Hills Frog N. vicina , is an endemic species of Southeast Asian uplands in Pakistan and India and was first reported by Stoliczka (1872) from Murree, Punjab Province, Pakistan. Later Rais et al. (2014) recaptured it from its type locality and described morphology of one juvenile female and one adult male. The species is listed as Least Concern in IUCN Red List of Threatened Species while the EDGE (Evolutionarily Distinct and Globally Endangered) score is 2.64. In Pakistan, it is reported from brook water springs and streams of Murree, Galiat (North Punjab), Ayubia (Khyber Pakhtunkhwa), and parts of Azad Jammu and Kashmir (Rais et al. 2014). Although the adult form has been characterized (Khan 2006; Rais et al. 2014), the morphology of the tadpoles have not yet been described. The current study aimed to describe external morphology and morphometry of tadpoles of N. vicina. We collected 154 tadpoles during March 2018 from the streams of Village Perhana (UTMX 358388.4, UTMY 3745826.6, zone 43), Tehsil Murree, District Rawalpindi, Province Punjab, Pakistan. All were studied alive and later one tadpole of each Gosner (1960) stage (26 to 41) was euthanized and preserved in 20% Bouin���s solution (Khan 1982). The samples were housed at the lab of Department of Wildlife Management at PMAS Arid Agriculture University Rawalpindi, with voucher numbers SG-19926 to SG-19941. The morphological terminology followed Altig (2007) and McDiarmid and Altig (1999). Twenty tadpoles of stage 36 (SG-19936) were used to describe the external morphology. The photographs were taken with the help of Canon EOS D60 DSLR with EF-S 18���55 mm lens. Eighteen variables were measured with INSIZE Electronic Calipers series 1112 (0.01 mm). One specimen at stage 36 was studied using scanning electron microscope (2380 N Hitachi) for studying the oral apparatus at Central Hi-Tech Lab, University of Agriculture Faisalabad, Pakistan. Description of tadpoles. The morphological measurements are given in mm as mean �� standard error: total length (TL) = 50.96 �� 4.14, snout-vent distance (SVL) = 22.37 �� 1.45, body length (BL) = 18.9 �� 1.48, maximum body width (MBW) = 10.76 �� 0.95, body height (BH) = 6.52 �� 0.87, eye diameter (ED) = 1.8 �� 0.23, internarial distance (IND) = 1.66 �� 0.26, interorbital distance (IOD) = 3.93 �� 0.52, snout-narial distance (SND) = 2.33 �� 0.44, orbito-narial distance (OND) = 1.33 �� 0.28, snout-spiracle distance (SSD) = 12.14 �� 0.87, tail length (TLL) = 36.55 �� 3.46, maximum tail height (MTH) = 7.17 �� 0.85, tail muscle height (TMH) = 5.16 �� 0.7, tail muscle width (TMW) = 4.58 �� 0.56, dorsal fin height (DFH) = 2.44 �� 0.33, ventral fin height (VFH) = 2.36 �� 0.37 and oral disc width (ODW) = 5.84 �� 0.49. The tadpoles were medium sized. Body depressed in lateral view (Fig. 1A) and oval in dorsal view (Fig. 1B); longer than broad (BL/MBW = 1.76) and broader than deep (MDW/BH = 1.67). Snout broad and round in dorsal view, slightly sloped and pointed in lateral view. Interorbital distance more than twice the internarial distance (IOD/IND = 2.4). Nares small, rounded, slightly rimmed and closer to eyes than snout (SND/END = 1.79). Eyes round, small, positioned dorsally and directed dorsolaterally (Fig. 1B). Spiracle single, sinistral, distinct, oval opening, posterodorsally directed, spiracular tube attached to the body, visible in lateral, dorsal and ventral views. Tail robust, muscular and well-developed, more than 1.5 times longer than body length (TLL/BL = 1.93), tail length five times the tail height (TLL/MTH = 5.13), tail height similar to body height (MTH/BH = 1.11); tail tip widely rounded. Tail muscle height slightly greater than tail muscle width (TMH/TMW = 1.13), dorsal and ventral fin origin nonparallel, fins of same height (DFH/VFH = 1.04), tail muscle height more than twice dorsal fin height (TMH/DFH = 2.14) and ventral fin height (TMH/VFH = 2.22). Dorsal fin originates posteriorly on the body; ventral fin originates attached to the vent tube. Vent tube medial, attached to the left side of the ventral tail fin with dextral opening (Fig. 1C). Oral disc medium sized (MBW/ODW = 1.85), anteroventral, not emarginated. Marginal papillae continuous on posterior labium with a wide dorsal gap on anterior labium; submarginal papillae larger, arranged in a single row in posterior labium and some scattered at the lateral margins of the anterior labium and posterior labium. Labial tooth row formula (LTRF) 5(3-5)/3(1). The jaw sheaths fully keratinized, dark, serrated (22 serrations/mm), upper sheath widely arched and lower sheath wide ���V���-shaped (Fig. 1D). Labial teeth (35/ 0.5 mm) with narrow base and head with multiple cusps. Color in life. The body dorsum was dark, greenish-brown or greenish-gray, while the ventral side was transparent with visible digestive tract. Tail was greenish-gray in dorsal view having dark greenish-brown speckles. In lateral view tail muscle was darker dorsally and gradually became lighter creamish-green color ventrally. The speckles also decreased gradually from dorsal to ventral side and anterior to posterior side of the tail. Color in preservative: The body dorsum dark, green in appearance; ventral side yellow with visible intestines. Yellow staining due to picric acid in Bouin���s solution (Fig. 1 A, B and C). The comparison of the tadpoles of Nanorana vicina with other studied tadpoles of the genus, N. aenea, N. unculuanus, N. chayuensis, N. conaensis, N. maculosa, N. medogensis, N. taihangnica, N. parkeri, N. pleskei and N. ventripunc- tata, showed that the posterior labium had three labial tooth rows with one medially interrupted 3(1) in all species while five anterior tooth rows of N. vicina was a shared character with N. conaensis. Two rows of papillae on posterior labium were also common in all except N. parkeri and N. pleskei tadpoles. Tail end was also round in most with the exception of N. chayuensis and N. maculosa (Chuaynkern et al. 2018; Fei et al. 2012). The tadpole of Murree Hills Frog (Nanorana vicina) were type IV Orton (1953) and type 5 Inthara et al. (2005) larvae. Some features, such as anteroventral oral disc, dorsoventrally flattened body and low tail fins, are frequent in tadpoles of shallow, permanent lotic water bodies (Ampai et al. 2015). The anteroventral placement of the oral disc, and the structure of keratinized mouthparts suggest a detritivorous and suspension-rasper feeding ecology (Khan & Mufti 1994). The description of Nanorana vicina provides an essential morphological basis for the identification and classification. Further study on buccopharyngeal morphology, diet analysis and population dynamics may help understand natural history of the species., Published as part of Gill, Sumbul, Rais, Muhammad, Saeed, Muhammad, Ahmed, Waseem & Akram, Ayesha, 2020, The tadpoles of Murree Hills Frog Nanorana vicina (Anura: Dicroglossidae), pp. 440-442 in Zootaxa 4759 (3) on pages 440-442, DOI: 10.11646/zootaxa.4759.3.11, http://zenodo.org/record/3742127, {"references":["Stoliczka, F. (1872) Notes on some new species of Reptilia and Amphibia, collected by Dr. W. Waagen in North-Western Punjab. Proceedings of the Asiatic Society of Bengal, 1872, 124 - 131.","Rais, M., Abbassi, S., Batool, T., Jilani, M. J., Assadi, M. A., Mubarak, H. & Baloch, S. (2014) A note on recapture of Nanorana vicina (Anura: Amphibia) from Murree, Pakistan. Journal of Animal and Plant Sciences, 24, 455 - 458.","Khan, M. S. (2006) Amphibians and reptiles of Pakistan. Krieger Publishing Company, Malabar, Florida, 311 pp.","Gosner, K. L. (1960) A simplified table for staging anuran embryos and larvae with notes on identification. Herpetologica, 16, 183 - 190.","Khan, M. S. (1982) Key for the identification of amphibian tadpoles from the plains of Pakistan. Pakistan Journal of Zoology, 14, 133 - 145.","Altig, R. (2007) A primer for the morphology of anuran tadpoles. Herpetological Conservation Biology, 2, 71 - 74.","McDiarmid, R. W. & Altig, R. (1999) Tadpoles: The biology of anuran larvae. The University of Chicago Press, Chicago, Illinois, 444 pp.","Chuaynkern, C., Kaewtongkum, N., Ohler, A., Duengkae, P., Duangjai, S., Makchai, S. & Chuaynkern, Y. (2018) First description of the Nanorana (Chaparana) aenea (Smith, 1922) tadpole from northern Thailand and additional information on the Quasipaa (Eripaa) fasciculispina (Inger, 1970) tadpole buccopharyngeal anatomy. Alytes, 36, 93 - 108.","Fei, L., Ye, C. & Jiang, J. (2012) Colored atlas of Chinese amphibians and their distributions. Sichuan Publishing Group, Chengdu, 620 pp.","Orton, G. L. (1953) The systematics of vertebrate larvae. Systematic Zoology, 2, 63 - 75. https: // doi. org / 10.2307 / 2411661","Inthara, C., Lauhachinda, V., Nabhitabhata, J., Chuaynkern, Y. & Kumtong, P. (2005) Mouth part structures and distribution of some tadpoles from Thailand. The Thailand Natural History Museum Journal, 1, 55 - 78.","Ampai, N., Rujirawan, A., Arkajag, J., McLeod, D. S. & Aowphol, A. (2015) Description of the tadpoles of two endemic frogs: the Phu Luang cascade frog Odorrana aureola (Anura: Ranidae) and the Isan big-headed frog Limnonectes isanensis (Anura: Dicroglossidae) from northeastern Thailand. Zootaxa, 3981 (4), 508 - 520. https: // doi. org / 10.11646 / zootaxa. 3981.4.3","Khan, M. S. & Mufti, S. A. (1994) Oral disc morphology of amphibian tadpole and its functional correlates. Pakistan Journal of Zoology, 26, 25 - 30."]}

The larvae of some amphibian species grow and develop more slowly at lower temperature and attain larger body size at the end of metamorphosis, but this is not true for all amphibians. We have for the first time provided empirical data by using a combination of field and lab-based studies with a robust sample size (360 adults, 1000 tadpoles) for Hazara Torrent Frog (Allopaa hazarensis) and Murree Hills Frog (Nanorana vicina). In this study, we examined how tadpoles responded to different temperatures through metamorphosis, body size, developmental complications or deformities, fitness and survival. We found that the tadpoles of A. hazarensis and N. vicina showed the same response to elevated temperatures under laboratory conditions including faster metamorphosis, reduction in the body size, more frequent developmental complications or deformities such as edema and tail kinks, lower fitness and higher mortality at elevated temperatures (>26 °C). The comparison of thermal sensitivity between the two species showed that N. vicina was more sensitive to higher temperatures. Pakistan has been experiencing changes in climatic patterns, and the ecosystems in northern Pakistan are losing biodiversity due to increasing temperature, frequency of drought and intensity of floods. It is feared that these frogs may experience local extinction in future. Our findings contribute to filling the information gap regarding impacts of temperature increase on biodiversity of high altitude forested montane ecosystems and is an important contribution to future studies associating biodiversity and climate change.