First recording reveals the hidden voice of a newly discovered Chinese frog

Deep in the stream valleys of Dushan County in China’s Guizhou Province, a tiny frog is singing a song that no scientist had ever documented before. The Dushan Leaf Litter Toad, Leptobrachella dushanensis, was described as a new species only recently, and like many of its relatives, its biology remains almost entirely a mystery. Now, a team of researchers has captured and analyzed the male’s advertisement call for the first time, providing the missing acoustic fingerprint that helps confirm the species’ identity and separates it from its closest living relative.

The genus Leptobrachella, part of the family Megophryidae, is the largest clade within that family, currently encompassing 105 recognized species. These small amphibians are spread across a remarkable range, from southern China, northeastern India, and Myanmar, through Thailand and Vietnam, and extending to the Malay Peninsula, Borneo, and Natuna Island. During the breeding season, Leptobrachella species typically perch on rocks along stream banks, where males call to attract females. Despite their diversity, the genus is notorious for its morphological conservatism: species look so alike that past estimates of diversity and evolutionary relationships have been repeatedly misled by high levels of similarity and rampant homoplasy. Molecular phylogenetic frameworks have consequently proposed large numbers of cryptic species, and in the past five years alone, more than thirty percent of the species currently recognized in the genus have been formally described.

This explosion of new species descriptions has made integrative taxonomy, which combines morphology, genetics, and bioacoustics, essential for untangling the true diversity of the group. Vocalizations are particularly powerful tools in this effort. Nearly all species of anuran amphibians are capable of vocalization, and researchers have organized frog calls into four broad functional types: feeding calls, aggressive calls, reproductive calls, and defensive calls. Reproductive calls include advertisement calls, courtship calls, amplectant calls, release calls, post-oviposition male release calls, and rain calls. Among these, advertisement calls, the signals males produce to attract mates and signal their presence to rivals, exhibit substantial variation across species. This variation makes them a viable foundation for systematic classification and, crucially, for the identification of cryptic species that are nearly indistinguishable by eye.

Leptobrachella dushanensis was formally described as a new species from southern China in 2024, but no information about its acoustic signals existed in the literature. To fill this gap, researchers visited the species’ type locality in Dushan County on the night of April 1, 2024, between 19:00 and 23:00 hours. Conditions at the site were warm and extremely humid, with air temperatures of 17.0 to 19.0 degrees Celsius and ambient humidity of 98 percent. The team recorded a total of 197 calls from three adult males, using a Sony ICD-PX470 digital recorder at a sampling rate of 44.1 kHz and 16-bit resolution, with the microphone held within half a meter of each calling animal. All recordings came from isolated individuals, with no overlapping calls or mixed choruses, and were saved as WAV files for analysis. The researchers have made these recordings publicly available through the Figshare repository.

Species identity was verified with rigorous rigor. Snout-vent lengths of all recorded males were measured in situ with a precision digital caliper to the nearest 0.1 millimeter, ranging from 31.9 to 32.6 millimeters. One male was collected as a voucher specimen, euthanized with isoflurane, fixed in ten percent buffered formalin, and deposited at the Moutai Institute in China, with tissue samples preserved separately in 95 percent ethanol before fixation. The mitochondrial COI gene was extracted, amplified, sequenced, and uploaded to GenBank under accession number PQ288957, confirming that the calling frogs were indeed Leptobrachella dushanensis. The remaining recorded males were released back to their original habitat after measurement.

Acoustic analysis was performed with Raven Pro 1.6, following call-centred terminology in which the fundamental unit is a call, each call contains a single note, and each note contains several pulses. Only calls with high signal-to-noise ratios, free from overlapping calls of nearby males, were analyzed. The results revealed a remarkably consistent signal: the advertisement call of Leptobrachella dushanensis consists of a single note with a duration of 78.19 to 123.78 milliseconds, delivered at intervals of 394.88 to 499.59 milliseconds, yielding call rates of 1.78 to 2.31 calls per second. Each call contained 14 to 21 pulses produced at a rapid rate of 164.26 to 174.06 pulses per second. The dominant frequency, the frequency carrying the most acoustic energy, fell in a narrow band between 4.84 and 4.91 kilohertz, and no obvious harmonics were detected.

The amplitude structure of the call followed a distinctive pattern. In every recorded call, the amplitude peaked at the very beginning of the signal and then gradually decreased. About one-third of the way into the call, the amplitude rose again, though it never exceeded the initial peak, before declining steadily until the call ended. Males were observed calling from rocks in streams surrounded by shrubs, a typical setting for the genus during the breeding season.

To test whether these acoustic traits could distinguish the new species from its closest relative, the team also recorded Leptobrachella dong, the sister species of L. dushanensis. Working in Congjiang County, also in Guizhou Province, on March 4, 2022, at temperatures of 12.0 to 13.0 degrees Celsius and 90 percent humidity, they captured 117 calls from four males with snout-vent lengths of 29.4 to 29.8 millimeters. Molecular verification again confirmed the species identity via a COI sequence deposited in GenBank under accession number PQ438741. The calls of L. dong showed a broadly similar architecture: single-note, pulse-repetition calls with an initial amplitude peak, a mid-call dip, and a secondary rise around two-thirds of the way through that again failed to surpass the opening peak. Dominant frequency was a uniform 4.82 kilohertz, again without obvious harmonics.

Despite these structural similarities, the two species are acoustically separable on several parameters. L. dong calls are shorter, lasting 63.54 to 90.82 milliseconds, are delivered faster at rates of 2.23 to 2.72 calls per second, contain fewer pulses, only 9 to 11, at a markedly slower pulse rate of 102.13 to 133.77 pulses per second. A principal component analysis of the combined call dataset confirmed the separation quantitatively. The first two principal components together explained 79.77 percent of the total variance, with PC1 accounting for 62.09 percent and loading primarily on temporal parameters, including the number of pulses, call duration, and pulse rate, while PC2 explained 17.69 percent and was driven mainly by dominant frequency. In the resulting PC space, the calls of the two species showed no overlap at all, providing clear acoustic evidence supporting the taxonomic differentiation of L. dushanensis.

The findings also carry broader phylogenetic interest. L. dushanensis belongs to the L. oshanensis species group, whose members typically produce more complex advertisement calls, often with multiple call types. Unlike its relatives, L. dushanensis produced only simple calls in the recordings, and its call differs considerably from those of group members such as L. yeae, L. jinyunensis, L. chishuiensis, L. bijie, L. jinshaensis, L. suiyangensis, L. purpuraventra, L. wulingensis, and L. oshanensis, all of which exhibit multiple advertisement call types. Its dominant frequency of roughly 4.88 kilohertz is also lower than the 6.70 kilohertz reported for L. alpian. As more researchers adopt acoustic diagnostic traits to identify cryptic species in taxonomic studies, detailed descriptions like this one become indispensable. For a genus in which species are highly morphologically conserved, the species-specific nature of the Dushan Leaf Litter Toad’s call not only reinforces its status as a distinct species but also lays the basic groundwork for future behavioral, physiological, and ecological studies of Leptobrachella frogs across Asia.

Subject of Research: First description of the advertisement call of the frog Leptobrachella dushanensis and its bioacoustic distinction from its sister species Leptobrachella dong

Article Title: The advertisement call of Leptobrachella dushanensis ( Anura , Megophryidae )

Article References: Liu, J., Cheng, Y., Li, S., Cheng, G., & Wei, G. (2024). The advertisement call of Leptobrachella dushanensis (Anura, Megophryidae). Herpetozoa, 37, 373-378. https://doi.org/10.3897/herpetozoa.37.e136688

Image Credits: AI Generated

DOI: 10.3897/herpetozoa.37.e136688

Keywords: Leptobrachella dushanensis, advertisement call, bioacoustics, anuran vocalization, Megophryidae, cryptic species, integrative taxonomy, Guizhou Province, principal component analysis, amphibian conservation, dominant frequency, sister species