Male Advertisement Call of the Endangered Leptobrachella tengchongensis (Anura:Megophryidae) from Mount Gaoligongshan,Yunnan Province,China

Ho Yuen YEUNG,Xiangyuan HUANG,Shenpin YANG and Jianhuan YANG＊

1 Kadoorie Conservation China Department,Kadoorie Farm and Botanic Garden,Lam Kam Road,Tai Po,N.T.,Hong Kong,China

2 Gaoligongshan National Nature Reserve (Tengchong Bureau),Tengchong 679100,Yunnan,China

Abstract We present the first description of the acoustic characters of the endangered Tengchong leaf litter toad (Leptobrachella tengchongensis),aiming to inform future ecological studies of the species and taxonomic studies in the genus Leptobrachella.A total of 1179 calls,belonging to 6 adults male individuals,were recorded using TASCAM DR-40 digital sound recorder from its type locality in May 2015 and then analyzed using Raven Pro v.1.6.1 software.The advertisement call of male L.tengchongensis consists of primary advertisement call and secondary advertisement call.The primary advertisement call is invariably composed of two notes and has a call duration of 61±5 ms (23-87 ms,n = 1142).The secondary advertisement call is composed of 3-10 notes (mean 5.25±1.41 notes/call,n = 37) with a call duration of 90±6 ms (37-127 ms,n = 37).The mean inter-call interval is 212±121 ms (88-1121 ms,n = 1092).An introductory note is absent in advertisement calls and the dominant of calls is 4.1-4.8 kHz (at 20 ℃).We discuss the interspecific and intraspecific variations on advertisement calls in L.tengchongensis and among Leptobrachella species,and provide a summary on the major acoustic characters of all Leptobrachella species occurring north of the Isthmus of Kra.

Keywords acoustic analysis,amphibians,bioacoustics,breeding ecology,Leptolalax,taxonomy,Tengchong

1.Introduction

Vocalizations is the main communication mechanism in anuran amphibians,at both interspecific and intraspecific levels (K?hler

et al

.,2017).Almost all anurans depend on acoustics signals to locate each other during breeding season (Wells and Schwartz,2007).Advertisement calls are the main type of vocalization in anurans (K?hler

et al

.,2017).Advertisement calls are species specific and,therefore,considered a prezygotic mechanism of reproductive isolation (Littlejohn,1998).They also provide informative characters for identifying and delimiting species and,as such,are widely used in taxonomic research (Goicoechea

et al

.,2010).The Asian leaf litter toads of the genus

Leptobrachella

Dubois,1983 is widely distributed from southern China to northeastern India and Myanmar,through Indochina mainland to peninsular Malaysia and the island of Borneo (Frost,2020).Frogs in the genus inhabit the forest floor and rocky streams in hilly evergreen forest (Fei

et al

.,2012).Due to their small size and cryptic coloration,

Leptobrachella

are often difficult to detect and identify in the field;advertisement calls produced by adult males during the breeding seasons are a major clue to trace these elusive anurans (Matsui

et al

.,2009).Moreover,acoustic data can assist in rapidly surveying anurans,and further offer a new insight into species diversity,species boundaries and evolutionary relationships within this morphologically conserved group (Rowley

et al

.,2011;Rowley

et al

.,2016).Acoustic data has become increasingly important in the taxonomic study of the genus

Leptobrachella

in recent years (see Rowley

et al

.,2016;Yang

et al

.,2018 as examples),and the use of an integrative approach incorporating acoustic,morphological,and molecular data has resulted in the continued discovery of new anuran species in the region (Yang

et al

.,2018;Wang

et al

.,2019;Li

et al

.,2020).The Tengchong leaf litter toad (

Leptobrachella tengchongensis

) is a recently described species discovered on Mount Gaoligongshan in Yunnan Province in China (Yang

et al

.,2016).The species is currently categorized as Endangered by the IUCN Red List of Threatened Species due to its restricted and small distribution range as well as ongoing habitat degradation (IUCN SSC Amphibian Specialist Group,2020).However,the male advertisement call of this endangered species remains undocumented.Herein,we describe the acoustic characteristics and calling behavior of

L.tengchongensis

based on recordings of male advertisement calls from the type locality of the species.We aim to provide informative characters for future ecological studies of the species and for taxonomic research in the genus

Leptobrachella

.

2.Material and Methods

2.1.Study site

The advertisement calls of male

Leptobrachella tengchongensis

were recorded at the type locality of the species,at the Linjiapu substation of the Tengchong Section of Gaoligongshan National Nature Reserve,Tengchong County,Yunnan Province,China (25°17'51.26'' N,98°42'03.93'' E,2100 m above sea level).See Yang

et al

.(2016) for detailed information and photos regarding the macro- and micro-habitats of the locality.The call recordings were made between 21:00 to 23:40 h on 16 May 2015 by J.Yang,and the ambient air temperature was approximately 20 ℃.The ambient air temperature of the calling site was taken with a digital thermometer (Peakmeter MS6508).

2.2.Call recording

Advertisement calls were recorded using a TASCAM DR-40 digital sound recorder held within ca.30-50 cm of the calling individuals.All recordings consist of a minimum duration of 30 seconds call.The sound files in wave format were resampled at 48 kHz with sampling depth 24 bits.

2.3.Acoustic analyses

The sonograms and waveforms were generated by Raven Pro 1.6.1 software (The Cornell Lab of Ornithology,available from http://www.birds.cornell.edu/raven) with Fast Fourier samples 512 points and overlap 50%,from which all parameters and characters were measured as following Rowley

et al

.(2012,2016) and Yang

et al

.(2018):call duration (ms),call repetition rate (calls/s),Inter-call interval (ms),number of notes per call and dominant frequency (kHz).The recordings of the advertisement call of the male holotype was deposited at Amphibiaweb and is available at the url https://amphibiaweb.org/sounds/Leptobrachella_tengchongensis56.mp3.

2.4.Data comparison

Comparative advertisement call characters for

Leptobrachella

species occurring north of the Isthmus of Kra were taken from references (Jiang

et al

.,2002;Xu

et al

.,2005;Matsui,2006;Rowley and Cao,2009;Rowley

et al

.,2010a,2010b,2010c,2011,2012,2013,2015a,2015b,2016,2017a,2017b;Poyarkov

et al

.,2015;Nguyen

et al

.,2018;Yang

et al

.,2018;Chen

et al

.,2019;Ding

et al

.,2019;Wang

et al

.,2019;Li

et al

.,2020).The minimum and maximum values are given as min-max,all numeric parameters are given as mean±SE.

3.Results

3.1.Calling behavior

During the survey,males of

Leptobrachella tengchongensis

were mostly found calling on the forest floor with dense understory,close to the main stream and near a seepage.The majority of calling males were found perched on or hidden under leaf litter.Some were found calling under rocks by the side of the main stream.Sympatric amphibians recorded in the locality during the survey in May 2015 included the Spiny stream toad (

Bufo tuberospinius

),Glandular horned toad (

Megophrys glandulosa

),Yunnan spiny frog (

Nanorana yunnanensis

),Anderson's frog (

Odorrana andersonii

),Chaochiao brown frog (

Rana chaochiaoensis

),Green-spotted torrent frog (

Amolops viridimaculatus

),Burmese treefrog (

Rhacophorus burmanus

),and an unidentified small-sized

Megophrys

.Among them,advertisement calls of

M.glandulosa

,

R.burmanus

and the unknown

Megophrys

sp.were also heard during the survey.

3.2.Acoustic characteristics analyses

Recordings of six calling males of

L.tengchongensis

were obtained during the survey.A total of 271 seconds of recordings contained 1179 clear advertisement calls and were used for acoustic analyses.Within the recordings sampled,all individuals emitted a continuous call series with durations of minimum 15 seconds to maximum 120 seconds.We recognized two different types of advertisement calls among the recordings sampled (Table 1 and Figure 1),namely the primary advertisement call (PAC) and secondary advertisement call (SAC),following Rowley

et al

.(2013;2017a,b).While PACs were the dominant call of a continuous call series,SACs were rarely and irregularly emitted within a call series and sometimes absent.PACs invariably contained two notes per call and were repeated rapidly with a regular call duration and inter-call interval (Figures 1A and B).SACs consisted of 3-10 notes per call with an irregular call duration (Figures 1A and C),but shared the same dominant frequency with PACs in a call series.The call duration was longer in SACs (Table 1 and Figure 1).To the human ear,the PAC is a very rapid,highpitched ticking,similar to calling songs of orthopteran species,while the SAC sounds like an irregular,prolonged PAC.

Figure 1 Advertisement call of male Leptobrachella tengchongensis. A:waveform (MU) and spectrogram (kHz) showing a six-call portion of five PACs and a SAC (the 5th call) in a continue call series.B:waveform (MU) and spectrogram (kHz) showing a 2-notes PAC.C:waveform (MU) and spectrogram (kHz) showing a 10-notes SAC.

The PACs of six individuals were uniform in call structure,which invariably consisted of two pulsed notes with amplitudepeaking at the start of each note.The call durations of PACs were similar among individuals,with the average duration ranging between 54-67 ms (Table 1).

Table 1 Measurements of male advertisement call parameters in Leptobrachella tengchongensis.

SACs were only recorded in two individuals (individual A and C,see Table 1).Unlike the PACs,the call durations and notes/call of SACs showed great variations (see Table 1).The 37 SACs measured were in highly variable in both call structure and call duration (90±6 ms,37-127 ms,see Table 1).

The advertisement calls of

L.tengchongensis

were typically emitted in continuous call series.The average inter-call interval of continuous call series sampled was 212±121 ms (88-1121 ms,

n

= 1092).Within call series,advertisement calls were repeated at a rate of approximately 4.28±1.76 calls per second (1.32-6.49 calls/s,

n

= 8).

The dominant frequency of advertisement calls presented a certain degree of variation both within and among individuals (see Table 1).The dominant frequency ranged from 4.12 to 4.82 kHz in individual C,and the mean dominant frequency ranged between 4.39-4.73 kHz among six individuals.We found two individuals (individual A and B) calling simultaneously nearby (ca.3 meters apart) during the survey.While call durations and inter-call intervals of these two individuals were relatively similar,the mean dominant frequency was slightly different,4.52 kHz and 4.73 kHz in individual A and individual B respectively.

4.Discussion

The advertisement calls of the three other

Leptobrachella

species from northern Vietnam and northern Laos,namely

L.botsfordi

,

L.petrops

and

L.puhoatensis

,were also reported containing both PACs and SACs (Rowley

et al

.,2013;Rowley

et al

.,2017a,b).Furthermore,based on our field observations,most species of

Leptrobrachell

a from China (i.e.,

L.laui,L.liui

,

L.mangshanensis

,

L.oshanensis

,and

L.ventripunctatus

) can also emit SACs as part of long call series of PACs (J.Yang and H.Y.Yeung,unpublished data).Thus,we assume that the SAC may actually commonly occur in advertisement calls of

Leptobrachella

species from China and northern Indochina (=lineage A of the genus in Chen

et al

.,2018),and the lack of descriptions of SACs in some previous studies in the region may be due to the insufficient sample size of recordings and/or the fact they have been overlooked by other author(s).In general,SACs are similar to the dominant PACs in note structure,but have more notes per call and longer call durations,which sounds to the human ear like prolonged PACs (Rowley

et al

.,2013;Rowley

et al

.,2017a,b;this study).However,SACs are rarely and irregularly emitted in a continuous call series.Taking this study as an example,only recordings of two out of six individuals of

L.tengchongensis

sampled in the wild contained SACs.Therefore,more calling individuals sampled and longer recordings are desirable in order to determine whether SACs are present in the male advertisement call of a species.For most species of the

Leptobrachella applebyi

species group (= lineage B of the genus in Chen

et al

.,2018) from the central highlands of southern Indochina,the advertisement call typically contains a distinct introductory note,which is a longer note occurring at the beginning of each call (Rowley

et al

.,2010b,2016).However,this introductory note was not observed in

L.tengchongensis

in this study,and has never been documented for other

Leptobrachella

species from China,northern Indochina and south of Isthmus of Kra (see Matsui,1997;Matsui,2006;Matsui

et al

.,2009;Rowley

et al

.,2013;Yang

et al

.,2018 as examples).Therefore,we assume that the introductory note may only occur in advertisement calls of

L.applebyi

species group,and may be considered as a unique acoustic character of this species group,although not present in all species in the group.Our results indicate that while the SACs of

L.tengchongensis

had great intraspecific variations in both call structure (no.of notes per call) and call duration,PACs had a stable call structure and had very small intraspecific variations in call duration among all individuals (Table 1),which can be considered as a key diagnostic character of the species.However,PACs do show a certain degree of intraspecific variations in call structure in some

Leptobrachella

species (Table 2).The dominant frequency of advertisement calls is commonly used as an important diagnostic acoustic character in the species comparison of the genus

Leptobrachella

(see Rowley

et al

.,2013;Rowley

et al

.,2017a,b;Yang

et al

.,2018;Chen

et al

.,2019 as examples).It is worth noting that,in this study,we observed a certain degree of intraspecific variation on the dominant frequency in

L.tengchongensis

,ranging in between 4.1-4.8 kHz.Such variation had also been documented in some congeneric species,for examples

L.aerea

(6.2-7.9 kHz,Rowley

et al

.,2010b) and

L.firthi

(5.4-6.6 kHz,Rowley

et al

.,2012).Similar great intraspecific variation in inter-call interval and call repetition rate were also found in

L.tengchongensis

and in other

Leptobrachella

species (see Tables 1 and 2).As a result,we recommend that researchers pay more attention to the intraspecific variation in these parameters when using acoustic data in taxonomic work,and try to obtain advertisement call recordings that are longer in duration,from more individuals,and from a greater number of locations.

Species Secondary call Notes/Call Call duration (ms)Inter-call interval (ms)Individuals analyzed No.of calls analyzed Introductory note Dominant frequency (kHz) Calling season Elevation (m)Temperature (°C)Source aerea not documented 2-16 13-130 4-582 613734 120 absent 6.2-7.9 Jun, Nov 284-511 22.4-25.7 Rowley et al.,2010b alpina not documented 270 unknown 11 absent 6.7 May 1150-2400 16 Fei et al.,2012;Xu et al.,2005 applebyi not documented 93-6 213-404 unknown unknown present 3.9-4.3 Mar, Jul 1200-1500 21.5 Rowley and Cao, 2009;Rowley et al.,2016 ardens not documented 3-10 114-373 unknown unknown present (not all calls)3.1-3.4 Aug 1041-1450 21.4-24.7 Rowley et al.,2016 bidoupensis not documented 5-82 308-400 unknown 48 absent 1.9-2.3 May, Jul 1620-1730 19-20.4 Rowley et al.,2011;Rowley et al.,2016 bijie not documented 98.8-264 101.9-245.2 200 absent 4.8-5.1 Jul 1670-1750 18.6 Wang et al.,2019 botsfordi present 3 (PAC) 1 (SAC)239-303 (PAC) 194-283 (SAC)406-1028 1 15 absent 2.6-3.2 (PAC) 2.8-3.4 (SAC)June 3143 14 Rowley et al.,2013 chishuiensis not documented 1-4 75-353 8-98 166 325760 absent 6.1-6.3 May 270-604 20 Li et al.,2020 croceus not documented 4-6 164-249 400-14100 absent 2.6-3.0 Jul 1300 21.6-25.1 Rowley et al.,2010a firthi not documented 2-5 18-51 327-2285 absent 5.4-6.6 Mar, Apr 860-1720 18.3-21.2 Rowley et al.,2012 fuliginosus not documented 6-934-5 51-80 unknown unknown unknown absent 2.3-2.4 Dec unknown 19.3-19.6 Matsui, 2006 isos not documented 28-34 248-1060 4152213 40 absent 5.8-6.2 Jun, Aug,Oct 650-1100 22.4-22.8 Rowley et al.,2015a kalonensis not documented 201 unknown unknown present 2.8 Aug 200-791 26.4 Rowley et al.,2016 liui not documented 2-4 26-78 unknown 245 absent 4.8-5.5 Apr 730-1400 14.7-20.2 Ding et al.,2019;Fei et al.,2012 maculosa not documented 7-38 889-907 unknown unknown indistinct 2.7-2.8 Sep 900-1166 23.3-24.1 Rowley et al.,2016 melicus not documented 4-11 168-484 214-3821 80 present 2.6-4.0 Jun, Oct 600-850 26.1-26.2 Rowley et al.,2010b oshanensis not documented 29-67 unknown unknown absent 4.4-4.6 May 720-1800 14 Fei et al.,2012;Jiang et al.,2002 pallidus not documented 34-7 627-729 unknown unknown present (short)2.4-2.7 Jun, Jul 1644-1681 14-21.4 Rowley et al.,2016 petrops present 1-6 (PAC) 13-28 (SAC)5-62 (PAC) 196-379 (SAC)174-805 7 unknown absent 5.6-6.4 (PAC) 5.3-6.6 (SAC)May 234-1283 24.5-25.3 Rowley et al.,2017b puhoatensis present 1 (PAC) 41 (SAC)6-14 (PAC) 299 (SAC)74-128 5 100 absent 4.9-5.6 (PAC) 5.3 (SAC)Jun 1123-1352 22.3-25.8 24.3 Rowley et al.,2017a purpura not documented 12 86-117 430-1557 151414 20 absent 4.3-4.5 Apr 1615 15 Yang et al.,2018 purpuraventra not documented 90.3-192.2 90.8-217.8 19374030 absent 4.7-4.8 Jul 1600-1900 18.8-19.3 Wang et al.,2019 pyrrhops not documented 5-6 208-297 3260-5630 present 1.9-2.2 Apr 800-1100 25 Poyarkov et al.,2015 rowleyae not documented 4-61 276-517 68-576 absent 3.2-3.5 Aug to Feb 380-645 20.8-21.5 Nguyen et al.,2018 shangsiensis not documented 64-69 184-289 absent 5.5-6.5 Apr 450-550 21.5 Chen et al.,2019 tadungensis not documented 4-7 248-353 unknown unknown present 2.6-3.1 Jul 720-1932 12.9-22.3 Rowley et al.,2016 tengchongensis present 2 (PAC) 3-10 (SAC)23-87 (PAC) 37-127 (SAC)88-1121 6 1179 absent 4.2-4.8 (PAC) 4.1-4.7 (SAC)Apr, May 2000-2100 20This study tuberosus not documented 1 54-78 1800-13800 3 20 absent 2.6-2.8 Jul 836-1401 22.5-24.5 Rowley et al.,2010a ventripunctata present 4-6 (PAC) 9-17 (SAC)87-562 120-1369 1 73 absent 6.1-6.6 Apr, May,Jun 850-1360 15This study; Fei et al.,2012 yingjiangensis not documented 1 28-42 113-174 2 35 absent 5.7-5.9 May, Jun 1615 19 Yang et al.,2018 Table 2 Summary of male advertisement call parameters for species of Leptobrachella occuring north of the Isthmus of Kra with documented calls.Remarks: Notes/call,call duration and dominant frequency were given separately in two different type of advertisement calls, namely primary advertisement call (PAC) and secondary advertisement call (SAC),for some species for which these data were available.

According to Yang

et al

.(2019),three

Leptobrachella

species occur on Mount Gaoligongshan,namely

L.tengchongensis

,

L.ventripunctata

and an unknown

Leptobrachella

sp..Acoustic characteristics and breeding behaviors appear to different among the species.The peak frequency is 4.2-4.8 kHz in

L.tengchongensis

(20 ℃),6.1-6.6 kHz in

L.ventripunctata

(15 ℃),and 4.1-4.3 kHz in

Leptobrachella

sp.(3 ℃);the PACs are composed of 2 notes in

L.tengchongensis

,4-6 notes in

L.ventripunctata

and 3-11 notes in

Leptobrachella

sp.(J.Yang and H.Y.Yeung,unpublished data).Furthermore,while

L.tengchongensis

breeds in April and May and inhabits in 2000-2100 m elevations,calling males of

L.ventripunctata

can be heard from April to June and only recorded at relatively lower elevation at 1,350 m on Mount Gaoligongshan;while

Leptobrachella

sp.only breeds in March when night time air temperature was ca.3 ℃ and inhabits in 1 950-2 100 m elevations (Yang

et al

.2019;J.Yang and H.Y.Yeung,unpublished data).A summary of the major acoustic characters of the advertisement calls of

Leptobrachella

species occurring north of the Isthmus of Kra for which comparable acoustic data are available is presented in the Table 2,in order to provide a quick key for future taxonomic research on the genus.Compared with its congeners,the major acoustic characters of

L.tengchongensis

include the presence of two types of the advertisement calls,primary advertisement call and secondary advertisement call;the primary advertisement call is invariably composed of two notes and has a call duration of 61±5 ms (23-87 ms,

n

= 1142);the secondary advertisement call is composed of 3-10 notes (mean 5.25±1.41 notes/call,

n

= 37) with a call duration of 90±6 ms (37-127 ms,

n

= 37);the mean inter-call interval is 212±121 ms (88-1121 ms,

n

= 1092);and the dominant frequency of the calls is 4.1-4.8 kHz (at 20 ℃).Anurans can emit different call types that contains different social functions (Camurugi

et al

.,2015;K?hler

et al

.,2017).We speculate that the two different types of advertisement call in

L.tengchongensis

should also have distinct different signals.The dominant PACs with stable and regular call characters should serve to attract females and convey territorial information to competitors,which are commonly observed in anuran amphibians as breeding calls (Wells and Schwartz,2007;Morais

et al

.,2012).However,the social function of the SACs is unclear and needs to be clarified in further studies.Currently,advertisement calls for only nine out of the twenty-six

Leptobrachella

species occurring in China have been described.Moreover,most previous descriptions of

Leptobrachella

species in the region were based on only a few dozen advertisement calls (see Table 2),and mostly belonging to only a single individual,which cannot provide a better understanding on the intraspecific variation.Herein,we encourage field herpetologists should record and analysis the undocumented acoustic characteristics of anuran amphibians,and try to obtain more recording samples from more individuals if possible.

Acknowledgements

This study was supported by Kadoorie Farm and Botanic Garden,Hong Kong.We thank rangers of Linjiapu substation and colleagues of KFBG who helped in the fieldwork.