The genus Pelophylax (Amphibia, Ranidae) in Pakistan: museum collections and possible distribution

We provide the first comprehensive data on the questionable distribution of the genus Pelophylax and the family Ranidae from Pakistan. Based on a literature review and two specimens of the genus from Tasp, Panjgur District in Pakistani Balochistan (USNM 26194–95), stored in the Smithsonian National Museum of Natural History, Washington, DC, USA, we discuss the possible occurrence and affiliation of these frogs in the context of Central Asia. Our comparison shows that the nearest records of Pelophylax in relation to the Tasp specimens are reported from more than 280 km (air-line) away in Iran and Afghanistan, which are currently separated by hot and mostly desert environments. We suggest that possible surviving populations of this genus may still be present in Balochistan (Rakhshan River) or Khyber Pakhtunkhwa (Kabul River) Provinces of Pakistan. This would, however, need further field investigations.


Introduction
The genus Pelophylax Fitzinger, 1843 currently represents 20-21 species and 2-3 hemiclonals distributed from northern Africa to the Far East in Asia. They are morphologically very similar and often mentioned as particular species complexes (groups) with unresolved phylogenetic relationships (Akin et al. 2010). Populations from the Middle East and Central Asia are ranked under the P. ridibundus group and are geographically associated with the following species: P. ridibundus (Pallas, 1771), P. bedriagae (Camerano, 1882), P. caralitanus (Arikan, 1988) and P. terentievi (Mezhzherin, 1992). All these taxa have phenotypic, ecological and etiological similarities, yet the ongoing research shows that they will need comprehensive evolutionary-taxonomic re-evaluation (Mazepa et al., in prep.). The members of this group are paraphyletic, containing cryptic taxa (Plötner 2005). Populations of the ridibundus group have a continuous distribution and are most abundant in the temperate and subtropical zones of the Western Palearctic, with a scattered distribution in the southern dry regions in the Middle East where water bodies are available. Overall, they are distributed from eastern Spain to western China, through Central Europe, the Balkans, the Anatolian Peninsula, the Caspian Region and Central Asia (Akin et al. 2010). In the division between Central (Afghanistan, Iran, Turkmenistan, Uzbekistan) and Southern Asia, where the territory of Pakistan falls, three species are currently recognised: P. bedriagae, P. ridibundus and P. terentievi (Wagner et al. 2016;Pesarakloo et al. 2017;Showler 2018;Frost 2021).
Previous reports of the family Ranidae from Pakistan (Khan 2006) are for species currently recognized in the family Dicroglossidae. However, historical data mention the genus Pelophylax (formerly Rana Linnaeus, 1758), a true member of the family Ranidae, for Pakistan. The first Pelophylax record ("Rana esculenta, L.") was provided by Blanford in Hubrecht (1880: 621) from the locality Nál (Naal) of central Balochistan [coordinates given by Blanford as 29°30'N, 66°E, however, the correct position should be ~27.682°N, 66.183°E sensu St. John (1889)]. Another specimen was collected in Baghunna (~27.907°N, 66.298°E), "so badly preserved that doubts about its identity with this or any other species may be entertained" (Hubrecht 1880). These specimens were part of a small collection of amphibians and reptiles made by "Dr. C. Duke in April and May 1877" and deposited in the Leiden Museum (currently known as Naturalis Biodiversity Center, Leiden, The Netherlands; Hubrecht 1880). However, in the digital catalogue, we were able to find only a specimen RMNH. RENA.4024 of Trapelus agilis (Olivier, 1807) from that collection. We, moreover, think that both localities belong to Chrysopaa sternosignata (Murray, 1885), aquatic species that was described later than Blanford in Hubrecht published their observations. Authors thus could affiliate them erroneously with "Rana esculenta".
The adult frog vouchers came from the locality "near Sarawan, Baluchistan" (~28.67°N, 65.53°E; loc. 3) and were deposited by George E. Mason (Boulenger 1891: 380). The origin of some other amphibian specimens obtained from Mason was doubted by Boulenger (1920: 89), while no such doubt was raised for the Sarawan specimens.
All abovementioned records come from north-eastern Balochistan, near the border with Afghanistan (Fig. 1).

Materials and methods
During extensive research on the biogeography and taxonomy of the genus Pelophylax, we came across a forgotten collection comprising two specimens of Pelophylax originating from south-western Pakistan (Balochistan Province), deposited in the Smithsonian Institution National Museum of Natural History, Washington, DC, USA (USNM). We, therefore, provide important data and confirmation of (at least) the historical presence of these frogs for Pakistan (western Balochistan), supported by well available museum material. We took 14 morphometric characters (Table 1) and detailed photos of both specimens (Figs 2, 3). We also compiled, updated, or even corrected (see localities of vouchers CAS [California Academy of Sciences] 115915, FMNH [Field Museum of Natural History] 161114-16 in Suppl. material 1: Table  S1 and literature therein) the available field and museum records of the genus Pelophylax from eastern Iran and Afghanistan to present the possible geographical affinity to the closest populations and obtain a better view on the origin of Pakistani populations (see Suppl. material 1: Table S1). Moreover, we attempted to genotype the mitochondrial ND3 locus of specimens (USNM 26194-95) from Pakistan, as well as vouchers from one of the closest known localities of the genus from Iran (Zabol, MVZ [Museum of Vertebrate Zoology] 243552-63 and MVZ 243612-13). However, due to the age and preserved medium of specimens from Pakistan, we were able to extract DNA and sequences from only four of the Zabol vouchers, namely MVZ 243553-54 and 243612-13 (GenBank Accession Numbers: MW590982-MW590985).

Results
Two Pelophylax specimens ("Rana ridibunda"; Figs 2, 3) were collected from Tasp (26.99°N, 64.06°E; Fig. 1, loc. 1), south-western Balochistan, Pakistan, by George E. Mason (London, England) and deposited at the National Museum of Natural History, Smithsonian Institution, Washington, DC, USA, as an exchange for invertebrates (Fig. 4). The time of their collection is unknown, but before they were catalogued in July 1899 (Fig. 4). Both specimens were adult and have voucher numbers USNM 26194 (male; Fig. 2) and USNM 26195 (female, Fig. 3). Together with the material from Sarawan (BMNH 1884.11.20.6-7; see Introduction), these two specimens represent the only vouchered specimens of the genus from Pakistan. The specimens from Tasp are also the western-and southernmost known records of the genus for Pakistan. Morphological characters, together with the visible colour pattern, correspond to the P. ridibundus group. We compared the locality of Tasp with available locality records from Pakistan, Afghanistan and eastern Iran (overall 57 identified localities representing 147 vouchered specimens; Fig. 1 and Suppl. material 1: Table S1) to evaluate their geographic distances and closest locality. The nearest locality is "Hodar, Sarbaz River" (MMTT [Muzeye Melli-e Tarikh-e Tabiei, Tehran] 877; Fig. 1, loc. 57; Safaei-Mahroo and Ghaffari 2020) in Iran, approximately 280 km (air-line) from Tasp in Pakistan. Other Iranian and Afghan localities are more than 500 km from Tasp (Fig. 1). We also compared the distances to previously known historical records of the genus in Pakistan mentioned by Blanford in Humbrecht (1880) and    Boulenger (1889,1891,1920), namely the area of Quetta. These localities are ca. 370 km (air-line) from Tasp. Based on the distribution of the Tasp specimens, we expect that they should be identified as P. terentievi. The ND3 haplotypes of the sequenced specimens from the Zabol area, Iran (MVZ 243553-54 and 243612-13) are most similar to P. terentievi (GenBank Accession Number NC029199) from Ashgabat, Turkmenistan. Unfortunately, specimens from the Pakistani locality of Tasp (USNM 26194-95) were not successfully sequenced. However, we expect the same genetic affinity for the Pakistani specimens as is recorded for the Zabol specimens, but further confirmation is needed.

Discussion
As there exist no recent observations on Pelophylax from Pakistan and only four exact localities are historically known with only two corroborated by available voucher specimens, it is difficult to evaluate if Pelophylax is currently a member of the herpetofauna of the country. According to Masroor (2012), Sarwar et al. (2016), Frost (2021 and Jablonski et al. (2021), 20 to 24 species of amphibians are currently reported from the country, without any mention of the family Ranidae or the genus Pelophylax. Pakistani Balochistan is herpetologically the most diverse region of the country (Khan 2006), yet the predominantly arid conditions do not generally provide suitable and stable habitats for amphibians. Pelophylax frogs need permanent water sources around rivers or lakes, but they are also able to live in oases, for example, in northern Africa or the Middle East (e.g. Nicolas et al. 2015). Although such habitats are available in Balochistan, they are often ephemeral. This is also the case of the Rakhshan River flowing in the vicinity of Tasp. This river currently possesses little or no water; however, in Panjgur (near Tasp), it expands into a series of bright clear water pools (kor joh) connected by small water channels. The banks are bordered by numerous date palms and most of the water is used for irrigation. Speculatively, in the time of the collection (150 years ago), the situation with suitable habitats might have been different, providing conditions for a stable, broadly distributed population. However, we presume that some small populations of the genus Pelophylax might still occur there. This needs further investigations to determine not only the presence of Pelophylax, but also to investigate its origin with respect to the nearest localities in Iran and Afghanistan, which are situated more than 280 km from Tasp (Fig. 1). Due to the physiography of today's locality, the possible populations of Pelophylax may represent relicts, surviving in Balochistan after a more suitable past climate. Further field investigation is also needed from areas of historical records mentioned by Blanford in Hubrecht (1880) and Boulenger (1889Boulenger ( , 1891Boulenger ( , 1920 in the area of Quetta.
Moreover, potential localities of the genus in Pakistan may be found around the Kabul River in the Khyber Pakh- tunkhwa Province in the north-western part of the country (Fig. 1). The Kabul River is a biogeographic corridor for the Oriental fauna into the Palearctic area that probably allowed colonisation of valleys of Hindu Kush mountains around the river from Pakistan to eastern Afghanistan [e.g. Euphlyctis cyanophlyctis (Schneider, 1799); Calotes versicolor (Daudin, 1802); Ophisops jerdonii Blyth, 1853, Varanus bengalensis (Daudin, 1802); Fowlea piscator (Schneider, 1799); Wagner et al. 2016;Jablonski and Lesko 2018;Jablonski et al. 2019]. Given the records of Pelophylax in Afghanistan, for example, from Paghman near Kabul (FMNH: 161055; Anderson and Leviton 1969; Fig. 1 loc. 5) and especially from the locality "ca. 25 km SE (by air) Jalalabad" (MVZ: 236876; Fig. 1 loc. 4), only 60 km from the Pakistani border, such vice versa colonisation and presence of the genus Pelophylax in north-western Pakistan is highly possible. In this context, we checked the specimens stored in the Florida Museum of Natural History at the University of Florida (UF) and vouchered as UF 82322-24, collected in Yakh Tangi, Khyber Pakhtunkhwa Province, Pakistan (approximately 34.30°N, 71.65°E). These specimens were stored in the collection under the name "Rana "farooqi" nov. sp. Auffenberg", raising serious questions on the identification of these specimens as Pelophylax. However, available photos reveal that these specimens might be members of the genus Nanorana Günther, 1896, most likely N. vicina (Stoliczka, 1872.