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Short Communication
The advertisement call of Leptobrachella dushanensis (Anura, Megophryidae)
expand article infoJing Liu, Yanlin Cheng, Shize Li, Gang Cheng§, Gang Wei§
‡ Moutai Institute, Renhuai, China
§ Guiyang College, Guiyang, China
Open Access

Abstract

We describe the advertisement call of the Dushan Leaf Litter Toad, Leptobrachella dushanensis, based on recordings obtained from three individuals from its type locality. The advertisement call of L. dushanensis consists of a single note with a dominant frequency of 4.84–4.91 kHz. Furthermore, we compare the differences in advertisement calls between L. dushanensis and its sister species, Leptobrachella dong. The advertisement calls of L. dushanensis and L. dong can be clearly distinguished. Our findings provide bioacoustic evidence supporting the taxonomic status of L. dushanensis and provide basic data for future behavioral and ecological studies on Leptobrachella.

Key Words

acoustic differences, bioacoustics, frogs, southern China

Introduction

The Asian leaf litter toads of the genus Leptobrachella Smith, 1925, are the largest clade of the family Megophryidae Bonaparte, 1950, currently comprising 105 species (Frost 2024). Species of the genus Leptobrachella are widely distributed, ranging from southern China, northeastern India, and Myanmar, through Thailand and Vietnam, and extending to the Malay Peninsula, Borneo, and Natuna Island (Frost 2024). During the breeding season, Leptobrachella species often inhabit rocks on the stream banks (Fei et al. 2005). Molecular phylogenetic frameworks proposed many cryptic species in the genus (Chen et al. 2018). In the past five years, more than 30% of new species in Leptobrachella have been described (Frost 2024). High levels of morphological similarity and rampant homoplasy appear to have misled estimates of diversity and evolutionary relationships (Shen et al. 2021). Addressing the aforementioned issues requires the application of integrative taxonomy, combining methods from morphology, genetics, and bioacoustics to reveal the species diversity within the genus Leptobrachella.

Vocalizations of anuran amphibians have received much attention in studies of behavioral ecology and species identification (Cui et al. 2012; Zhu et al. 2021; Shen et al. 2024). In Anura, nearly all species are capable of vocalization (Köhler et al. 2017). To further understand the relationship between frog behaviors and vocal communication, researchers have divided frog calls into four types: feeding calls, aggressive calls, reproductive calls, and defensive calls. Advertisement calls, courtship calls, amplectant calls, release calls, post-oviposition male release calls, and rain calls are examples of reproductive calls (Toledo et al. 2015; Köhler et al. 2017). Furthermore, advertisement calls exhibit substantial variation across species, making them a viable foundation for the systematic classification and identification of cryptic species (Sullivan et al. 1996).

Leptobrachella dushanensis (Anura, Megophryidae) is a newly described species in Southern China (Li et al. 2024). Information about their acoustic signals is not available in the literature. Here, we describe the advertisement call of L. dushanensis for the first time to understand its characteristics. Furthermore, we focus on comparing the advertisement call of L. dushanensis with that of its sister species, Leptobrachella dong. Our findings provide acoustic evidence that supports the taxonomic differentiation of L. dushanensis and contribute basic data for future behavioral and ecological studies on Leptobrachella species.

Materials and methods

Call recordings

A total of 197 calls were recorded from three individuals of L. dushanensis, collected from its type locality Dushan County, Guizhou Province, China (107.63°E, 25.94°N, ca. 1200 m elev., 17.0–19.0 ℃ air temperature, 98% ambient humidity) on April 1, 2024, between 19:00–23:00 h. Additionally, to compare the differences in advertisement calls between L. dushanensis and its sister species, Leptobrachella dong, we also collected advertisement calls from L. dong. A total of 117 calls were recorded from four individuals of L. dong, collected from Congjiang County, Guizhou Province, China (108.28°E, 25.57°N, ca. 1190 m elev., 12.0–13.0 ℃ air temperature, 90% ambient humidity) on March 4, 2022, between 19:00–23:00 h. Calls from each individual were obtained using a digital recorder, a SONY ICD-PX470 (sampling rate 44.1 kHz, 16-bit resolution). Each call was recorded within a 0.5 m distance from the calling individual. All recordings were obtained from isolated individuals, without overlapping calls or mixed choruses, and saved as WAV files. The recordings are publicly available in Figshare at https://doi.org/10.6084/m9.figshare.26969512.

Snout-vent lengths (SVLs) of all recorded males were measured in situ using a precision digital caliper to the nearest 0.1 mm. One L. dushanensis (specimen number: DS20240401001) and one L. dong (specimen number: CJ20220304001) were collected for species identification, and the others were released to their original habitat after measurement. After taking photos of the two specimens, we euthanized them using isoflurane and then fixed the specimens in 10% buffered formalin. Tissue samples were taken and preserved separately in 95% ethanol before fixation. Specimens were deposited in the Moutai Institute, China. Mitochondrial COI genes were extracted and amplified from muscle samples and sequenced, and the obtained sequence was verified and uploaded to GenBank (https://www.ncbi.nlm.nih.gov/). The sequencing results were compared to finally determine the species collected.

Acoustic analyses

The advertisement call characteristics were analyzed with the software Raven Pro 1.6 (K. Lisa Yang Center for Conservation Bioacoustics [at the Cornell Lab of Ornithology] 2023). Temporal properties were measured using Raven’s waveform display. Spectral properties were measured by averaging the spectrum over the entire duration of a call (Hann window, DFT = 512 samples, overlap = 50%, hop size = 256 samples). Only calls that had a high signal-to-noise ratio and were free from overlapping calls of nearby males were used for the analysis. We used “call-centred” terminology as summarised by Köhler et al. (2017), in which the fundamental unit was defined as a “call”; each “call” contains a single “note,” and each “note” contains several pulses. We measured all parameters and characteristics following the procedure described by Köhler et al. (2017) and Qian et al. (2023), including (1) call duration (ms), CD (2) inter-call interval, CI (3) call rate (calls/s), CR (4) note per call, NPC (5) no. of pulses per call, NP (6) pulse rate (pulses/s), PR (7) dominant frequency (kHz), DF. Oscillograms and spectrograms were generated using Seewave v.2.2.0 (Sueur et al. 2008) and TuneR 1.4.2 (Ligges et al. 2013) packages in R programme 4.2.2 (R Core Team 2021) with a “Hanning” window size of 256 samples and an overlap of 50%. Descriptive statistics of call characteristics (mean, standard deviation (SD), and range) were computed using SPSS 23.0. Furthermore, principal component analysis (PCA) was conducted to compare the differences in advertisement calls between L. dushanensis and its sister species, L. dong.

Results

Call characteristics

Males were observed calling on rocks in streams surrounded by shrubs. After molecular identification, we determined that the species captured in Dushan County is L. dushanensis (Fig. 1A). The GenBank accession number for the COI sequence is PQ288957 (specimen number: DS20240401001). The SVL of L. dushanensis ranged from 31.9 mm to 32.6 mm (Table 1). We analyzed the spontaneous vocalizations (197 calls) from three L. dushanensis males. The advertisement calls of L. dushanensis are pulse-repetition calls (Fig. 2A, B). All calls of L. dushanensis exhibited a consistent structure, with the amplitude reaching its peak at the beginning of the call, then gradually decreasing. About one-third into the call, the amplitude increased again but did not exceed the initial peak before gradually decreasing until the end of the call (Fig. 2B). The advertisement call of L. dushanensis comprised a single note with a call duration ranging from 78.19–123.78 ms (Table 1). The inter-call interval ranged from 394.88–499.59 ms. The call rate ranged from 1.78–2.31 calls/s. The pulse number ranged from 14.00–21.00, and the pulse rate ranged from 164.26–174.06 pulses/second. The dominant frequency ranged from 4.84–4.91 kHz, and no obvious harmonics were found (Fig. 2B, Table 1).

Figure 1. 

Photograph of the specimen. A. Adult male of Leptobrachella dushanensis (DS20240401001) from Dushan County, Guizhou Province, China; B. Adult male of Leptobrachella dong (CJ20220304001) from Congjiang County, Guizhou Province, China.

Table 1.

Descriptive statistics for call parameters of advertisement calls of Leptobrachella dushanensis.

Individuals Calls analyzed Air temperature (°C) SVL (mm) Call duration (ms) Inter-call interval (ms) Call rate Note per call Note rate No. of pulses per call Pulse rate Dominant frequency (kHz)
DS20220401001 75 17.0 31.9 123.78 ± 16.61 94.00–167.10 499.59 ± 49.57 410.80–594.31 1.78 ± 0.14 1.65–1.93 1 NA 21.00 ± 4.50 14.00–33.00 164.26 ± 21.72 134.30–223.40 4.91 ± 0.15 4.82–5.17
DS20240401002 77 19.0 32.3 86.76 ± 9.73 68.40–115.40 469.77 ± 77.65 303.27–669.00 2.09 ± 0.18 1.92–2.39 1 NA 15.00 ± 1.50 12.00–24.00 174.06 ± 12.40 152.01–205.72 4.87 ± 0.17 4.82–5.51
DS20240401003 45 19.0 32.6 78.19 ± 6.52 64.60–97.10 394.88 ± 68.53 288.00–588.02 2.31 ± 0.15 2.19–2.48 1 NA 14.00 ± 0.50 10.00–20.00 164.56 ± 12.13 139.32–195.67 4.84 ± 0.10 4.82–5.51
All Individuals 3/197 17.0–19.0 31.9–32.6 78.19–123.78 394.88–499.59 1.78–2.31 1 NA 14.00–21.00 164.26–174.06 4.84–4.91

After molecular identification, we confirmed that the species collected from Congjiang County is L. dong (Fig. 1B). The GenBank accession number for the COI sequence of L. dong is PQ438741 (specimen number: CJ20220304001). The SVL of L. dong ranged from 29.4 mm to 29.8 mm (Table 2). We analyzed the spontaneous vocalizations of 117 calls from four L. dong males. The advertisement calls of L. dong are also pulse-repetition calls (Fig. 2C, D). The advertisement call of L. dong reaches its peak amplitude at the beginning and then gradually decreases until reaching approximately halfway through the call. Subsequently, at around two-thirds of the call duration, the amplitude suddenly increases again but does not exceed the initial peak, and then gradually decreases until the end of the call (Fig. 2D). The advertisement call of L. dong comprised a single note with a call duration ranging from 63.54–90.82 ms (Table 2). The inter-call interval ranged from 299.20–380.01 ms. The call rate ranged from 2.23–2.72 calls/s. The pulse number ranged from 9.00–11.00, with a pulse rate of 102.13–133.77 pulses/second. The dominant frequency was 4.82 kHz, and no obvious harmonics were detected (Fig. 2D, Table 2).

Figure 2. 

Advertisement calls of the two Leptobrachella species. A. 5 s oscillograms showing nine calls of Leptobrachella dushanensis; B. 0.2 s oscillograms and corresponding spectrograms showing one call of L. dushanensis; C. 5 s oscillograms showing fourteen calls of Leptobrachella dong; D. 0.2 s oscillograms and corresponding spectrograms showing one call of L. dong.

Table 2.

Descriptive statistics for call parameters of advertisement calls of Leptobrachella dong.

Individuals Calls analyzed Air temperature (°C) SVL (mm) Call duration (ms) Inter-call interval (ms) Call rate Note per call Note rate No. of pulses per call Pulse rate Dominant frequency (kHz)
CJ20220304001 27 13.0 29.6 82.55 ± 5.08 65.60–94.00 380.01 ± 42.06 312.95–478.13 2.23 1 NA 11.00 ± 0.00 8.00–13.00 121.87 ± 5.43 106.71–133.63 4.82
CJ20220304002 30 13.0 29.8 63.54 ± 4.84 48.40–72.80 336.30 ± 47.67 274.14–441.65 2.57 1 NA 9.00 ± 1.13 5.00–13.00 133.77 ± 21.62 70.80–165.41 4.82
CJ20220304003 30 12.0 29.5 90.82 ± 8.50 63.10–105.60 311.65 ± 37.97 270.60–435.54 2.55 1 NA 10.50 ± 0.50 6.00–12.00 102.13 ± 7.24 79.24–119.18 4.82
CJ20220304004 30 12.5 29.4 66.56 ± 6.15 54.40–81.60 299.20 ± 27.75 242.72–360.42 2.72 1 NA 9.00 ± 0.50 5.00–10.00 112.59 ± 16.92 66.89–147.06 4.82
All Individuals 4/117 12.0–13.0 29.4–29.8 63.54–90.82 299.20–380.01 2.23–2.72 1 NA 9.00–11.00 102.13–133.77 4.82

Comparison of advertisement calls

The principal component analysis (PCA) of call parameters between L. dushanensis and L. dong resulted in two PCs, explaining a cumulated variance of 79.77% (Fig. 3). The PC1 axis accounted for 62.09% of the total variation and was primarily associated with temporal parameters, as it comprised the number of pulses, call duration, and pulse rate (Table 3). The PC2 axis explains 17.69% of the variation, primarily driven by the spectral parameter of dominant frequency (Table 3). The advertisement calls of the two species showed no overlap in PC space, indicating acoustic separation between the species.

Figure 3. 

Plots of the first principal component (PC1) versus the second (PC2) for advertisement calls of Leptobrachella dushanensis and Leptobrachella dong from a principal component analysis.

Table 3.

Factor loadings of the principal component analysis (PCA) on the advertisement call parameters of the Leptobrachella dushanensis and Leptobrachella dong.

Call parameters Principal components
1 2 3 4 5
Call duration 0.830 0.074 -0.551 0.030 0.043
Inter-call interval 0.765 -0.395 0.210 0.463 -0.003
Number of pulses 0.967 -0.004 -0.178 -0.175 -0.060
Pulse rate 0.814 -0.192 0.428 -0.341 0.031
Dominant frequency 0.483 0.829 0.247 0.138 0.001
Eigenvalue 3.104 0.884 0.624 0.381 0.006
Variance (%) 62.085 17.686 12.473 7.628 0.128
Cumulative variance (%) 62.085 79.771 92.244 99.872 100.000

Discussion

From the perspective of phylogenetic relationships, L. dushanensis was assigned to the L. oshanensis species group (Li et al. 2024). Previous studies have shown that species within the L. oshanensis species group typically produce more complex advertisement calls (Wang et al. 2019; Shi et al. 2021; Shen et al. 2023). However, unlike other species within the species group, we only recorded simple calls of L. dushanensis and did not notice any complex calls. Compared to the published calls of the other species in the L. oshanensis species group, the advertisement call of L. dushanensis differs considerably, reinforcing the specific identity of this species. For example, the species of Leptobrachella yeae, Leptobrachella jinyunensis, Leptobrachella chishuiensis, Leptobrachella bijie, Leptobrachella jinshaensis, Leptobrachella suiyangensis, Leptobrachella purpuraventra, Leptobrachella wulingensis, and Leptobrachella oshanensis exhibit multiple types of advertisement calls (Wang et al. 2019; Li et al. 2020; Cheng et al. 2021; Shi et al. 2021; Shen et al. 2023; Shi et al. 2023; Qian et al. 2023). The dominant frequency of L. dushanensis (4.88 kHz) is lower than that of Leptobrachella alpian (6.70 kHz; Xu et al. 2005). As the sister species of L. dushanensis, L. dong exhibits a similar advertisement call structure to that of L. dushanensis. Their advertisement calls can be distinguished by call parameters such as the number of pulses, call duration, pulse rate, and dominant frequency (Table 3). In taxonomic studies, an increasing number of researchers are utilizing acoustic diagnostic traits to identify cryptic species (Cui et al. 2023; Chen et al. 2024). Species within the genus Leptobrachella are highly morphologically conserved, but L. dushanensis exhibits distinct advertisement call characteristics in its calls compared to other congeneric species, supporting the species-specific nature of its acoustic signals. The differentiation of advertisement calls in Leptobrachella can serve as evidence for their differentiation in taxonomic status. Our study will support further studies on the behavior, physiology, and ecology of the species.

Acknowledgements

This work was supported by the projects from the National Natural Science Foundation of China (Nos. 32270498, 31960099, 32260136, and 32070426), Guizhou Provincial Science and Technology Projects (Nos. ZK[2022]540 and [2023] 099), and high-level personnel research start-up funding projects of Moutai Institute (Nos. mygccrc[2022]055, mygccrc[2022]067, and mygccrc[2022]083).

References

  • Chen JM, Poyarkov NAJ, Suwannapoom C, Lathrop A, Wu YH, Zhou WW, Yuan ZY, Jin JQ, Chen HM, Liu HQ, Nguyen TQ, Nguyen SN, Duong TV, Eto K, Nishikawa K, Matsui M, Orlov NL, Stuart BL, Brown RM, Rowley JJL, Murphy RW, Wang YY, Che J (2018) Large-scale phylogenetic analyses provide insights into unrecognized diversity and historical biogeography of Asian leaf-litter frogs, genus Leptolalax (Anura: Megophryidae). Molecular Phylogenetics and Evolution 124: 162–171. https://doi.org/10.1016/j.ympev.2018.02.020
  • Chen WC, Li P, Peng WX, Liu YJ, Huang Y (2024) The fourth species of Leptobrachella (Anura, Megophryidae) found at Shiwandashan National Nature Reserve, Guangxi, China. ZooKeys 1192: 257–279. https://doi.org/10.3897/zookeys.1192.98352
  • Cheng YL, Shi SC, Li JQ, Liu J, Li SZ, Wang B (2021) A new species of the Asian leaf litter toad genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from northwest Guizhou Province, China. ZooKeys 1021: 81–107. https://doi.org/10.3897/zookeys.1021.60729
  • Cui JG, Tang YZ, Narins PM (2012) Real estate ads in Emei music frog vocalizations: Female preference for calls emanating from burrows. Biology Letters 8: 337–340. https://doi.org/10.1098/rsbl.2011.1091
  • Fei L, Ye CY, Jiang JP, Xie F, Huang YZ (2005) An illustrated key to Chinese amphibians. Sichuan Publishing House of Science and Technology: 71–73.
  • K. Lisa Yang Center for Conservation Bioacoustics (2023) Raven Pro: interactive sound analysis software (Version 1.6.4). The Cornell Lab of Ornithology, Ithaca. https://ravensoundsoftware.com/
  • Köhler J, Jansen M, Rodríguez A, Kok PJR, Toledo LF, Emmrich M, Glaw F, Haddad CFB, Rödel MO, Vences M (2017) The use of bioacoustics in anuran taxonomy: theory, terminology, methods and recommendations for best practice. Zootaxa 4251(1): 1. https://doi.org/10.11646/zootaxa.4251.1.1
  • Li SZ, Li W, Cheng YL, Liu J, Wei G, Wang B (2024) Description of a new Asian Leaf Litter Toad of the genus Leptobrachella Smith, 1925 (Anura, Megophryidae) from southern Guizhou Province, China. Biodiversity Data Journal 12: e113427. https://doi.org/10.3897/BDJ.12.e113427
  • Li SZ, Liu J, Wei G, Wang B (2020) A new species of the Asian leaf litter toad genus Leptobrachella (Amphibia, Anura, Megophryidae) from southwest China. ZooKeys 943: 91–118. https://doi.org/10.3897/zookeys.943.51572
  • R Core Team (2021) R: a language and environment for statistical computing. R Foundation for Statistical Computing, Vienna. https://www.R-project.org/
  • Shen T, Li SZ, Liu J, Li GR, Xiao X, Su HJ (2023) Advertisement calls of Leptobrachella suiyangensis and Leptobrachella bashaensis (Anura, Megophryidae). Herpetozoa 36: 273–280. https://doi.org/10.3897/herpetozoa.36.e108125
  • Shen T, Li SZ, Liu J, Wei G, Mu L, Su HJ (2022) Analysis on morphological characteristics of six species of Leptobrachella in Guizhou Plateau. Chinese Journal of Zoology 57(1): 49–68. https://doi.org/10.13859/j.cjz.202201006
  • Shen T, Liu J, Tang XJ, Peng CC, Li SZ, Feng CB, Mu L, Su HJ (2024) Acoustic difference in advertisement calls among two sympatric Boulenophrys species: A confirmatory case to acoustic niche hypothesis and morphological constraint hypothesis. Ecology and Evolution 14: e11318. https://doi.org/10.1002/ece3.11318
  • Shi SC, Hou YM, Song ZB, Jiang JP, Wang B (2021) A new Leaf Litter Toad of Leptobrachella Smith, 1925 (Anura, Megophryidae) from Sichuan Province, China with supplementary description of L. oshanensis. Asian Herpetological Research 12(2): 143–166.
  • Shi SC, Shen T, Wang X, Jiang JP, Wang B (2023) Multiple data sources reveal a new Asian Leaf Litter Toad of Leptobrachella Smith, 1925 (Anura, Megophryidae) from Southwestern China. Asian Herpetological Research 14(1): 62–94.
  • Sullivan BK, Malmos KB, Given MF (1996) Systematics of the Bufo woodhousii complex (Anura: Bufonidae): advertisement call variation. Copeia 1996(2): 274–280. https://doi.org/10.2307/1446843
  • Toledo LF, Martins IA, Bruschi DP, Passos MA, Alexandre C, Haddad CFB (2015) The anuran calling repertoire in the light of social context. Acta Ethologica 18: 87–99. https://doi.org/10.1007/s10211-014-0194-4
  • Wang J, Li YL, Li Y, Chen HH, Zeng YJ, Shen JM, Wang YY (2019) Morphology, molecular genetics, and acoustics reveal two new species of the genus Leptobrachella from northwestern Guizhou Province, China (Anura, Megophryidae). ZooKeys 848: 119–154. https://doi.org/10.3897/zookeys.848.29181
  • Xu JX, Xie F, Jiang JP, Mo YM, Zheng ZH (2005) The Acoustic Features of the Mating Call of 12 Anuran Species. Chinese Journal of Zoology 40(3): 12–19.
  • Zhu BC, Zhou Y, Yang Y, Deng K, Wang TL, Wang JC, Tang YZ, Ryan MJ, Cui JG (2021) Multisensory modalities increase working memory for mating signals in a treefrog. Journal of Animal Ecology 90: 1455–1465. https://doi.org/10.1111/1365-2656.13465

1 Jing Liu and Yanlin Cheng contributed equally to this work.
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