Field surveys across the Pamir of Tajikistan and the mountain regions of northern Pakistan, supported by mitochondrial 16S ribosomal RNA sequencing, have substantially expanded the documented geographical distributions of three Eurasian green toad species within the genus Bufotes. Most prominently, the findings extended the known range of the hybridogenetic triploid Batura toad, Bufotes baturae, southward and southwestward by approximately seventy kilometers into northern Pakistan, placing confirmed populations within the Deosai Plateau, the Tarshing Valley near Nanga Parbat, and the Kumrat Valley. Concurrently, field sampling in the Shokhdara Valley of southwestern Tajikistan revealed populations of Bufotes baturae that closely approach the range of the tetraploid Pevzov toad, Bufotes pewzowi, significantly narrowing the previously observed geographical gap between these two lineages. The newly gathered molecular and geographic data also documented an unrecorded locality for Bufotes pewzowi in the northwestern Pamir and confirmed an occurrence of the diploid green toad Bufotes latastii in Baltistan.<\/p>\n
The Pamir and Karakoram mountain systems together constitute the western segment of the Pan-Tibetan Highlands, establishing an intricate, high-elevation crossroads that physically links the Tibetan Plateau, Central Asia, and the western Himalaya. Their complex topographical relief, extensive elevational gradients, and dynamic paleoclimatic history have fostered pronounced biogeographic differentiation and exceptionally high levels of regional endemism across native wildlife. However, steep slopes, rugged terrain, and limited accessibility have long impeded systematic biological exploration throughout the territory, leaving extensive portions of these high mountains poorly surveyed. Consequently, the actual distributions and spatial boundaries of numerous amphibian species across this mountainous zone remain incompletely documented, making targeted field surveys essential for clarifying baseline biodiversity across Central Asia.<\/p>\n
Eurasian green toads of the genus Bufotes Rafinesque, 1815 are among the most widespread Palearctic amphibians, occupying diverse ecological habitats ranging from Mediterranean coastal plains and temperate woodlands to arid lowlands and high mountain valleys of Central Asia. The genus also represents an exceptionally intricate amphibian radiation whose evolutionary history is defined by repeated episodes of secondary contact among divergent lineages, leading to extensive interspecific hybridization, recurrent allopolyploidization, and reticulate evolution. Modern systematic frameworks recognize seven diploid and five polyploid species within the genus. The diploid taxa belong to the Bufotes latastii and Bufotes viridis species groups, whereas all five polyploid taxa\u2014the triploid Bufotes baturae along with the tetraploids Bufotes oblongus, Bufotes pewzowi, Bufotes pseudoraddei, and Bufotes zugmayeri\u2014occur exclusively in Central Asia and arose through historic hybridization.<\/p>\n
The complex diversity and morphological similarities within the genus, particularly among members of the Bufotes viridis group, have historically made species identification difficult using external morphology alone, even though morphometric indices can reliably discriminate adult polyploids under specific conditions. Recent integrative and phylogenomic revisions have provided a robust molecular framework for species assignment. Among Central Asian taxa, the hybridogenetic triploid Batura toad, Bufotes baturae, and the tetraploid Pevzov toad, Bufotes pewzowi, are especially notable because they inhabit the highest elevations recorded for amphibians in northern Eurasia, reaching 4,000 to 4,200 meters above sea level. Their persistence in these harsh alpine steppes and deserts is thought to be facilitated by overwintering in permanent water bodies that do not freeze at the bottom, including geothermal springs, which also serve as vital breeding sites for larval development and metamorphosis.<\/p>\n
To clarify distributions and investigate potential contact areas, researchers carried out exploratory visual encounter surveys across northern Pakistan in September 2020 and in the Pamir of Tajikistan during July and August 2025. Surveys took place both day and night across open mountain valleys, targeting aquatic microhabitats such as temporary pools, riverbeds, and anthropogenic water bodies. Because expeditions were exploratory, teams did not utilize systematic transects or trapping grids, but photographed encountered toads in situ and recorded precise coordinates with a Garmin 60CSx GPS receiver before mapping distributions in ArcGIS Pro. Voucher specimens were subsequently deposited in the Museum Koenig, Leibniz Institute for the Analysis of Biodiversity Change in Bonn, the Institute of Zoology and Parasitology in Dushanbe, the Pakistan Museum of Natural History in Islamabad, and Comenius University in Bratislava, under formal institutional permits adhering to the Nagoya Protocol and national wildlife legislation.<\/p>\n
Following preliminary identification from external morphology using regional identification guides, species assignments were confirmed using approximately 580 base pairs of the mitochondrial 16S ribosomal RNA gene to facilitate comparison with published reference datasets. Genomic DNA was extracted from ethanol-preserved tissue samples using the Qiagen DNeasy Blood and Tissue Kit. Polymerase chain reaction amplification used primers 16sar-L and 16sbr-H with Standard PCR Master Mix under cycling conditions consisting of an initial denaturation at 95 degrees Celsius for five minutes, followed by 35 cycles of 94 degrees Celsius for 30 seconds, 54 degrees Celsius for 45 seconds, and 72 degrees Celsius for two minutes, concluding with a final extension at 72 degrees Celsius for ten minutes. The resulting PCR products were purified with ExoSAP-IT and sequenced by Macrogen to generate unambiguous sequence reads for phylogeographic analysis.<\/p>\n
Sequence alignment was performed based on RNA secondary structure using RNAsalsa, and phylogenetic relationships were inferred via Bayesian analysis in MrBayes version 3.2.6. Paired stem regions of the 16S alignment were analyzed under the specialized sixteen-by-sixteen doublet substitution model of Sch\u00f6niger and von Haeseler, whereas unpaired loop regions were evaluated under a general time reversible model using the standard four-by-four nucleotide substitution matrix. The analysis was initiated from a random starting tree and run for ten million generations, sampling every 500 generations across four independent Markov chain Monte Carlo simulations, each comprising four chains. After discarding the first 25 percent of sampled trees as burn-in, a majority-rule consensus tree was constructed, with convergence verified in Tracer by confirming that average standard deviations of split frequencies remained low and effective sample sizes exceeded 200.<\/p>\n
The Bayesian consensus tree recovered the principal Bufotes lineages recognized in recent systematic literature, assigning all newly generated sequences to their corresponding mitochondrial clades with posterior probability values greater than or equal to 0.70. Newly sampled toads from the Pamir belonged to Bufotes baturae and Bufotes pewzowi, whereas northern Pakistani specimens grouped with Bufotes latastii and Bufotes baturae. Across all 27 surveyed localities, the species exhibited pronounced elevational ranges within open, high-elevation mountain valleys characterized by streams, shallow pools, and marshy habitats in dry landscapes. Bufotes baturae occurred from 1,864 meters along the Gilgit River to 3,700 meters at Bulunkul, Bufotes pewzowi spanned 2,050 meters in the Bartang Valley to 3,140 meters in the Shokhdara Valley, and Bufotes latastii was documented at 2,360 meters along the Braldu River and at 3,400 meters at Lulusar Lake.<\/p>\n
The northern Pakistan records considerably expanded the documented geographical distribution of Bufotes baturae southward and southwestward by approximately 70 kilometers. Previous records showed this triploid species concentrated in extreme northern Pakistan, adjacent northeastern Afghanistan, and the Pamir of Tajikistan. The new surveys confirmed its presence on the western margin of the Deosai Plateau at 3,600 meters, in the Tarshing Valley near Nanga Parbat at 2,700 meters, and at three distinct stations in the Kumrat Valley between 2,310 and 2,370 meters, alongside additional localities at Shandur La, Phander Lake, Mastuj, Batura Glacier, and Hunza River. The Kumrat Valley records are particularly noteworthy because they document Bufotes baturae within the broader geographical area from which the tetraploid green toad Bufotes pseudoraddei has also been reported, identifying another potential contact zone between distinct polyploid toad lineages.<\/p>\n
In Tajikistan, surveys throughout the Shokhdara Valley extended the known range of Bufotes baturae into the southwestern Pamir, where its populations closely approach the distribution of Bufotes pewzowi. Recorded localities for Bufotes baturae between 3,010 and 3,370 meters in the valley approach within close proximity to Bufotes pewzowi sites found between 2,890 and 3,140 meters, demonstrating that the distributions of both species within common valley networks are more intricate than shown on previous range maps. The deeply dissected relief of the Pamir likely drives this pattern, as elevated mountain ridges act as physical barriers to toad dispersal while river valley corridors facilitate connectivity between otherwise isolated populations. Field encounters also documented Bufotes baturae larvae and juveniles in warm geothermal spring waters in the Shokhdara region at localities 16 and 17, providing direct evidence of localized breeding and developmental success under rigorous alpine conditions.<\/p>\n
The investigation also refined geographic records for the other two examined toad species while highlighting key limitations inherent to mitochondrial DNA datasets. A new occurrence of Bufotes pewzowi in the Viskharv Valley at 2,460 meters expanded documented localities in the northwestern Pamir, complementing records from the Bartang, Rivakdara, and Shokhdara valleys. For Bufotes latastii, genetic confirmation along the Braldu River at 2,360 meters provided an additional verified occurrence in Baltistan, which together with the Lulusar Lake population confirms that the species occupies a broad elevational range. However, because Eurasian green toads undergo recurrent interspecific hybridization that can lead to cytonuclear discordance, mitochondrial sequences reflect only maternal lineages. The researchers note that broader genomic analyses and fine-scale sampling across contact areas will be essential to establish whether approaching populations of Bufotes baturae and Bufotes pewzowi occur in true sympatry or remain ecologically separated across the high mountain valleys.<\/p>\n
Subject of Research: Distributional records and mitochondrial phylogenetics of Bufotes green toads in the Pamir and Karakoram<\/p>\n
Article Title: Green toads at the Roof of Asia: new distributional and mitochondrial DNA sequence data for the genus Bufotes ( Anura , Bufonidae ) from the Pamir and adjacent mountain regions<\/p>\n
Article References: Hofmann, S., Garibmamadov, G., Masroor, R., Dufresnes, C., Jablonski, D., & Saidov, K. (2026). Green toads at the Roof of Asia: new distributional and mitochondrial DNA sequence data for the genus Bufotes (Anura, Bufonidae) from the Pamir and adjacent mountain regions. Herpetozoa, 39, 447\u2013453. https:\/\/doi.org\/10.3897\/herpetozoa.39.e215296<\/a><\/p>\n Image Credits: AI Generated<\/p>\n