Variation And Evolution Of Acoustic Signals In Asian Horned Frogs(Megophryinae)

As a behavioral trait,the evolution of animal sounds tends to be more unpredictable than morphological traits.In recent years,research on the morphology and phylogenetic evolution of the amphibian subfamily Megophryinae has shed light on its unique evolutionary characteristics and diversity history.However,the study of its acoustic features has received relatively less attention.To investigate the variation and evolution of acoustic signals within this group,a comprehensive study was conducted from 2021 to 2022.The study involved field recordings,behavior observations,mark-recapture studies,acoustic analysis,playback experiments,collection of habitat features,and phylogenetic comparisons.The calls of six species of Megophryinae frogs found in the Mangshan National Nature Reserve,were observed and studied on-site.Additionally,call recordings from a total of 52 horned toads were collected between 2012 and 2022 from various provinces in southern China.This research represents the first systematic exploration of the diversity and variation in the calls of Megophryinae species,the relationship between behavioral characteristics and acoustic signals,the coexistence mechanisms of sympatric species,and the phylogenetic evolution of acoustic signals.Here are some key findings from the study:(1)Among the three Boulenophrys species distributed in the eastern region of Mangshan,Hunan,there were significant negative correlations between note duration and note interval of Bo.nanlingensis with air temperature.Conversely,note rate and note number showed significant positive correlations with air temperature in the same species.Additionally,note interval and pulse number of Bo.shimentaina exhibited significant positive correlations with air temperature,whereas the note rate showed a significant negative correlation.Temperature-corrected call parameters revealed that the dominant frequency was significantly negatively correlated with body mass in Bo.nanlingensis and with snout-vent length in Bo.shimentaina.Coefficient of variation analysis indicated that the dominant frequency was a statistical parameter with the lowest CVw value in each species.Moreover,analyses of repeated recordings of Bo.shimentaina and Bo.ombrophila demonstrated that the dominant frequency had the lowest mean percentage change and a moderate repeatability(R)value.(2)Based on behavioral observations of three Boulenophrys species and one Brachytarsophrys species in Mangshan,Hunan,it was observed that male Megophryinae species can emit both advertisement calls and territorial calls.The primary purpose of the advertisement call was to attract female mates,while territorial calls served to maintain spacing between males.Although the structure of territorial calls and advertisement calls differed,no significant difference in dominant frequency was detected.Br.popei’s territorial call consisted of an elongated single note,whereas the territorial calls of the three Boulenophrys species consisted of a combination of"introductive" and "explosive" portions.Similar call structures were also observed in other species within the Megophryinae subfamily,such as Bo.cheni,B.sanmingensis,Bo.tuberogranulata,Br.orientalis and Pelobatrachus kalimantanensis.The activity rhythm of different call types was analyzed using passive recording data during the breeding peak of the three Boulenophrys species.It was discovered that there was a peak of territorial call activity between 18:00 and 20:00 for each species.(3)Significant call differences were observed among species that shared habitats and called simultaneously.Principal component analysis revealed that note interval and dominant frequency were variables with high scores in each species assemblage.Distinct calling site preferences were observed among the six Megophyinae species in Mangshan.Bo.shimentaina and Xenophrys mangshanensis were more inclined to call from high positions,while the remaining four species tended to call from lower positions.Most of Bo.shimentaina’s calling sites were categorized as "open," whereas the calling sites of Bo.nanlingensis and Br.popei were classified as "hidden," and the calling sites of Bo.ombrophila and Bo.jiulianensis were predominantly categorized as"semi-open." Results from playback experiments conducted on male Bo.nanlingensis indicated a higher response rate to calls with similar dominant frequencies.The response percentage was 56%for calls from Bo.nanlingensis,54%for calls from Bo.ombrophila,and 63%for calls from "Bo.lishuiensis".No response was detected from the calls of Bo.shimentaina and "Bo.sanmingensis" with higher dominant frequencies.These findings suggest that reproductive interference between species with similar dominant frequencies may contribute to the distinct song combinations observed in sympatric species of Megophryinae.(4)Based on a phylogenetic comparative analysis of acoustic data,calling site data,and morphological data from 73 species of Megophryinae,several findings emerged.Pagel’s λ model indicated that the dominant frequency exhibited the highest phylogenetic signal,followed by note duration,while the pulse number demonstrated a moderate phylogenetic signal.Blomberg’s K model revealed significant phylogenetic signals for dominant frequency,note duration,note interval,pulse number,and note number.However,no significant phylogenetic signal was detected for calling site types.Phylogenetic generalized least squares analysis indicated a significant negative correlation between snout-vent length and dominant frequency in Megophryinae species.Moreover,the calling site type was also significantly correlated with the dominant frequency.Analysis of covariance,after controlling for the effect of snoutvent length on dominant frequency,revealed that the relationship between different calling site types(arboreal and terrestrial)and the dominant frequency of the call was not significant.Additionally,a significant negative correlation was found between snout-vent length and note interval,while a significant positive correlation was observed between snout-vent length and note duration.Head width ratio exhibited a significant negative correlation with dominant frequency,whereas tibial length ratio showed a significant positive correlation with note number.The results demonstrate a relationship between call parameters of Megophryinae species and temperature as well as body size,aligning with the general patterns observed in most anurans.Notably,the dominant frequency and note rate exhibited the lowest coefficient of variation.Territorial calls serve as the primary warning mechanism for maintaining spatial distance among male Megophryinae frogs and typically occur at the onset of male calling activities each night.These territorial calls commonly consist of an introduction portion and an explosive portion.Similar structures of territorial calls were observed in the ancient clades of Pelobatrachus and Brachytarsophrys,differing from the more advanced Boulenophrys.Megophryinae frogs inhabiting the same breeding habitat and calling simultaneously exhibit a mechanism of bioacoustic niche partitioning,primarily characterized by significant differences in dominant frequency.Reproductive interference occurs between species with similar dominant frequencies,leading to syntopic species with similar dominant frequencies avoiding breeding in the same season.Phylogenetic relationships were significantly correlated with the evolution of acoustic signals in Megophryinae species,but not significantly associated with the speciation of calling site preferences.Furthermore,in line with the general rule in anurans,species within Megophryinae demonstrated a negative correlation between body size and dominant frequency.Additionally,species preferring arboreal calling sites exhibited significantly higher dominant frequencies,primarily attributable to the influence of body size.