Qiucheng LIU, Tongliang WANG, Xiaofei ZHAI and Jichao WANG

Ministry of Education Key Laboratory for Tropical Island Ecology, College of Life Sciences, Hainan Normal University,Haikou 571158, China

Abstract Anuran calls are usually species-specific and therefore valued as a tool for species identification. Call characteristics are a potential honest signal in sexual selection because they often re flect male body size. Polypedates megacephalus and P. mutus are two sympatric and morphologically similar tree frogs, but it remains unknown whether their calls are associated with body size. In this study, we compared call characteristics of these two species and investigated any potential relationships with body size. We found that P. megacephalus, males produced six call types which consisting of three distinct notes, while P. mutus males produced five types consisting of two types of notes.Dominant frequency, note duration, pulse duration, and call duration exhibited signi ficant interspeci fic differences. In P. megacephalus, one note exhibited a dominant frequency that was negatively correlated with body mass, snout-vent length, head length, and head width. In P. mutus, the duration of one note type was positively correlated with body mass and head width. These differences in call characteristics may play an important role in interspeci fic recognition.Additionally, because interspeci fic acoustic variation re flects body size, calls may be relevant for sexual selection. Taken together, our results con firmed that calls are a valid tool for distinguishing between the two tree-frog species in the field.

Keywords body size, call characteristics, Polypedates, sexual selection

1. Introduction

Amphibian communication is primarily dependent on vocalizations (Gerhardt and Huber, 2002; Cui et al.,2011). In most anurans, acoustic interactions are key to male-male competition and female choice (Narins et al., 2006; Xu et al., 2011). The spectral and temporal parameters of acoustic calls are species-specific, and hence can be used for species identification and for determining phylogenetic relationships (Jiang et al.,2002; Heyer and Barrio-Amorós, 2009). Thus, acoustic studies are an important tool for clarifying the taxonomy of sympatric and morphologically similar species.

Body size may affect the outcomes of sexual selection through male-male competition and female choice.Larger males usually defeat smaller males in aggressive encounters, and females often preferentially choose larger males (Wells, 1978; Given, 1988; Richardson et al., 2010;Liao and Lu, 2011; Rausch et al., 2014). Previous studies have shown that the spectral and temporal parameters of calls could re flect frog body size (Martin, 1972; Gerhardt,1994; Wang et al., 2012; Wei et al., 2012; Zhu et al.,2016a). However, other studies suggest that acoustic properties and morphological characteristics are unrelated in some frog species (Penna, 2004; Márquez et al., 2005).

Polypedates megacephalus and P. mutus (family Rhacophoridae) are tree frogs common throughout southern China and Southeast Asia (Fei et al., 2012).The two species have controversial taxonomic status. In China, Hong Kong populations previously considered to be P. leucomystax were named as P. megacephalus,a new species (Hallowell, 1861), a classification that subsequent taxonomists conferred to all P. leucomystax populations in continental China (Matsui et al., 1986;Zhao and Adler, 1993; Fei, 1999; Fei et al., 2005, 2009).Polypedates mutus was identified from Hekou Yunnan,China, based on samples with a “striped” morphology and the lack of a vocal sac (Liu et al., 1960); this species was later found throughout southern China (Inger et al.,1999). Recent phylogenetic and taxonomic evidence supports the presence of P. megacephalus and P. mutus in southern China, including Hainan (Kuraishi et al.,2012; Pan et al., 2013). Acoustic call characteristics of both species have been previously described (Xu,2005), but limited samples allowed only a few simple analyses of acoustic parameters and structure. A later study identi fied three note types in the complex call of P.megacephalus from Hong Kong (type locality) (Kuraishi et al., 2011). However, no comprehensive comparison of call characteristics has been made between the two species thus far. It also remains unknown whether their calls reflect body size. This missing information complicates field identification of these two sympatric and morphologically similar species.

The objective of the present study was to compare the spectral and temporal parameters of vocalizations from sympatric P. megacephalus and P. mutus. We investigated the relationship between call characteristics and male body size. Our study contributes to existing knowledge of vocal repertoires across tree frog species and provides comprehensive data that will help to distinguish interspeci fic call types.

2. Materials and Methods

2.1. Study species Snout-vent lengths of adult P.megacephalus males and females are 41–48 and 57–65 mm, respectively (Table 1). No black or brown spots are present on the sides of the body, but spots are arranged in reticulate patterns along the posterior (Figure 1A).Numerous small reticulate structures are present on the back (Figure 1C) (Liu et al., 2018). Hind legs are stout and tibial-tarsal joints stretch between the eyes and nostrils. Male frogs have a pair of pharyngeal subcapsular structures. Polypedates megacephalus inhabits altitudes ranging between 80 and 2200 m (Fei et al., 2012; Wang et al., 2014).

Figure 1 Morphological characteristics of Polypedates megacephalus (left) and P. mutus (right).

Snout-vent lengths of adult males and females of P.mutus are 52–63 and 53–77 mm, respectively (Table 1).Large and dark brown spots are arranged continuously on the sides of the body, and the posterior lacks any reticulate patterns (Figure 1B). A few large reticulate structures are present on the back (Figure 1D) (Liu et al., 2018).Hind legs are slender, with tibial-tarsal joints stretching to the snout or nostrils, and male frogs lack a vocal sac.Polypedates mutus inhabits altitudes ranging between 340 and 1100 m (Fei et al., 2012; Wang et al., 2014).

2.2. Vocalization recording and analyses From June 10 to July 9, 2015, we recorded male P. megacephalus and P. mutus vocalizations in their original habitat of Mt. Diaoluo National Nature Reserve (18°43'22.02" N,109°52'6.19" E, 933 m a.s.l.), Hainan, China. Recorded calls were always of isolated individuals and never from a mixed chorus. Vocalizations were recorded from 19:30 to 23:30 h (air temperature: 20.7 ± 1.4 °C, relative humidity:94.5% ± 5.3%). Each recording lasted 10 min per male,using a directional microphone (Sennheiser ME-66 with K6 power module; Sennheiser, Wedemark, Germany)connected to a digital recorder (PMD-660, 16 bit, 44.1 kHz; Marantz, Kanagawa, Japan), placed approximately 1 m away from the subject. Calls of individuals from both species were recorded on the same night as far as possible. Actual species recorded was decided based on whichever individuals were found in one night. Data were saved as wav files.

Call parameters were defined and illustrated based on K?hler et al. (2017). Dominant frequency was sound energy concentrated within the whole power spectrum,while fundamental frequency was the base or lowest frequency band in each note. Acoustic parameters measured included the following temporal and spectral properties: note duration (ND), call duration (CD), note number (NN), pulse number (PN), pulse duration (PD),fundamental frequency (FF), and dominant frequency(DF). According to the recording quality, we selected 6–41 and 1–10 calls used for each individual of P.megacephalus and P. mutus, respectively. Oscillograms and spectrograms were prepared in PRAAT (version 5.1.11; Boersma and Weenink, 2017). Body mass, snouttovent length (SVL), head length (HL), and head width(HW) were measured after the calls were recorded,respectively. Correlation analyses between morphological indexes and call parameters were conducted.

Table 1 Body size and call properties of male Polypedates megacephalus and P. mutus.

The ratio of CVb(between-male coefficients of variation) to CVw(within-male coef ficients of variability)was calculated to find relative variability of between and within individuals’ variation (Pettitt et al., 2013; Fang et al., 2018). If CVb/CVw＞ 1.0 for a call trait, there is more variability among males and this may have behavioral consequences for individual recognition (Bee et al., 2001;Robisson et al., 2010; Pettitt et al., 2013). In the present study, when the number of calls ＞ 1 from recorded individual, this subject was used to calculate CVw.

2.3. Statistical analysis Data were statistically analyzed using SPSS 19.0 (IBM Corp., Chicago, IL, USA). To compare the differences of DF, FF or ND among the three note types in P. megacephalus, Friedman test was used. If the main effect was significant, Wilcoxon test was used to determine the difference for each parameter.Wilcoxon test was also conducted for each parameter in P. mutus. Mann-Whitney U tests were used to compare the differences in DF, FF, ND, CD, PN, and PD between species. Spearman’s correlation analysis was used to detect possible relationships between vocalization characters and body size. Prior to statistical analyses,we calculated the average value for each call trait of each male frog, and then used those average values to calculate mean ± SD which represented the trait value of one species frog. Data are expressed as the mean ± SD,and P ＜ 0.05 and P ＜ 0.001 were considered statistically signi ficant and highly statistically signi ficant.

3. Results

3.1. Call characteristics Calls consisting of a single note type are simple calls, and calls consisting of different note types are complex calls (K?hler et al., 2017). We recorded and analyzed spontaneous vocalizations from 18 P. megacephalus and 13 P. mutus. Male P. megacephalus produced six call types: I (note A), II (note B), III (note C), IV (note A + B), V (note B + A), and VI (note C + B),where notes A–C are three distinct note types. Male P.mutus produced five call types: VII (note D), VIII (note E), IX (note D + E), X (note E + D), and XI (note D + E+ D), with notes D and E being distinct. Call waveforms and spectrograms are presented in Figures 2 and 3. The seven acoustic characteristics (DF, FF, ND, NN, PN, PD,and CD) are summarized in Table 1.

Polypedates megacephalus Call type I (Figure 2A) is a simple call, consisting of 1–23 (average 6.01 ± 1.80)note-A renditions, CD ranging from 21.00–1684.00 ms (n= 18). The ND, DF, and FF of note A were 26.08 ± 3.78 ms (n = 18), 1281.23 ± 315.23 Hz (n = 18), and 227.86 ±21.03 Hz (n = 18), respectively.

Call type II (Figure 2B) is a simple call, consisting of 1–9 (average 2.77 ± 1.14) note-B renditions, CD ranging from 63.80–2670.60 ms (n = 18). The ND, DF, and FF of note B were 124.16 ± 26.06 ms (n = 18), 1134.94 ±353.94 Hz (n = 18), and 213.81 ± 18.94 Hz (n = 18),respectively. Calls exhibited 6.54 ± 1.07 pulses, with a PD of 13.11 ± 1.00 ms.

Call type III (Figure 2C) is a simple call, consisting of 2–4 (average 3.58 ± 0.58) note-C renditions, CD ranging from 111.00 – 416.00 ms (n = 12). The ND, DF, and FF of note C were 30.15 ± 4.23 ms (n = 12), 1963.64 ± 110.35 Hz (n = 12), and 236.65 ± 34.14 Hz (n = 12) respectively,with clear harmonics.

Call type IV (Figure 2D) is a complex call, with 2.00± 0.71 renditions of note A, followed by 2.00 ± 1.41 renditions of note B (n = 4).

Call type V (Figure 2E) is a complex call, with 2.75± 0.96 renditions of note B, followed by 8.75 ± 7.89 renditions of note A (n = 4).

Call type VI (Figure 2F) has 3.27 ± 0.70 renditions of note C, followed by 2.96 ± 1.20 renditions of note B (n =14).

Type II calls were dominant across 18 individuals(57.43%), followed by type I (25.69%), type VI (7.50%),type III (6.60%), type IV (1.50%), and type V (1.30%).Friedman test results showed that DF and ND differed significantly among notes A, B, and C (P ＜ 0.001), but FF (P = 0.083) did not. Wilcoxon tests revealed that DF differed signi ficantly between notes A and C (P ＜ 0.002),as well as note B and C (P ＜ 0.002), but not between notes A and B (P = 0.170). Signi ficant differences in ND were observed between notes A and B (P ＜ 0.001), A and C (P ＜ 0.05), as well as B and C (P = 0.002).

Polypedates mutus Call type VII (Figure 3A) is a simple call, consisting of 1–33 (average 14.14 ± 5.84) note-D renditions, CD ranging from 18.00–7731.00 ms (n = 13).The ND, DF, and FF of note D were 20.32 ± 1.93 ms (n= 13), 1155.32 ± 123.10 Hz (n = 13), and 209.44 ± 19.36 Hz (n = 13), respectively, with clear harmonics.

Call type VIII (Figure 3B) consisted of 2–4 (average 3.00 ± 0.71) note-E renditions, CD ranging from 421.00–971.00 ms (n = 5). The ND of note E was 133.96 ± 29.95 ms (n = 5), and its DF and FF were 1088.22 ± 111.69 Hz(n = 5) and 217.65 ± 18.14 Hz (n = 5), respectively, with 6.98 ± 1.11 pulses and PD was 16.60 ± 1.35 ms.

Figure 2 Amplitude-modulated waveforms and spectrograms of Polypedates megacephalus calls: (A) call type I, (B) call type II, (C) call type III, (D) call type IV, (E) call type V, and (F) call type VI.

Call type IX (Figure 3C) is complex, with 7 ± 8.7 renditions of note D, followed by 1.33 ± 0.58 renditions of note E (n = 3).

Call type X (Figure 3D) included 2.90 ± 0.74 renditions of note E, followed by 15.47 ± 8.98 renditions of note D (n = 5).

Call type XI (Figure 3E) was 12.33 ± 18.77 renditions of note D, followed by 1.67 ± 0.58 renditions of note E and 14.33 ± 3.51 renditions of note D (n = 4).

Call type VII was dominant across 13 individuals(66.67%), followed by type X (15.50%), type XI (9.50%),type IX (4.80%), and type VIII (3.60%).Wilcoxon tests revealed that note D and E differed signi ficantly in ND (P= 0.043), but not in DF (P = 0.345) or FF (P = 0.225) .

3.2. Comparisons of acoustic characteristics for simple calls in the two species of tree frogs The results of the Mann-Whitney U-test revealed no signi ficant differences in DF, except between notes C and note D (P ＜ 0.001),as well as notes C and note E (P ＜ 0.001). Significant ND differences were observed between different notes of the two species, except between notes B and note E (P= 0.371). Excluding the comparisons between CD I and CD VIII (P = 0.201), CD I and CD IX (P = 0.100), CD II and CD VIII (P = 1.000), CD II and CD IX (P = 0.100),CD III and CD VIII (P = 0.109), CD III and CD IX (P =0.109), CD IV and CD VIII (P = 1.000), CD IV and CD IX (P = 0.480), CD V and CD VIII (P = 0.157), CD V and CD IX (P = 1.000), CD VI and CD VIII (P = 0.105),and CD VI and CD IX (P = 0.487), CD was signi ficantly different between all other comparisons. Notes B and E did not differ signi ficantly in PN (P = 0.455), but did in PD (P = 0.001). In P. megacephalus and P. mutus, CVb/CVw＞1.0 was 11 and 7 call traits, respectively (Table 1).

3.3. Relationship between body size and call structures Correlation analysis was used to determine whether DF and ND were associated with body size. Mean male BM,SVL, HL, and HW values of P. megacephalus were 6.78± 1.25 g, 53.38 ± 3.29 mm, 17.79 ± 1.3 mm, and 15.66 ±1.31 mm, respectively (n = 11) (Table 1). Only the DF of note B was negatively correlated with BM (r = –0.665, P= 0.026, n = 11; Figure 4A), SVL (r = –0.763, P = 0.006,n = 11; Figure 4B), HL (r = –0.759, P = 0.007, n = 11;Figure 4C), and HW (r = –0.623, P = 0.040, n = 11; Figure 4D). However, no signi ficant correlation existed between ND and body size in P. megacephalus (Figure 4E–F).

Figure 3 Amplitude-modulated waveforms and spectrograms of Polypedates mutus calls: (A) call type VII, (B) call type VIII, (C) call type IX, (D) call type X, and (E) call type XI.

Figure 4 Relationship between dominant frequency of note B and body mass (A), snout–vent length (B), head length (C), and head width (D).Relationship between duration of note B and body mass (E) and head width (F) for Polypedates megacephalus.

Figure 5 Relationship between dominant frequency of note D and body mass (A), snout–vent length (B), head length (C), and head width (D).Relationship between duration of note D and body mass (E) and head width (F) for Polypedates mutus.

Mean BM, SVL, HL, and HW of P. mutus males were 12.2 ± 1.83 g, 63.19 ± 3.89 mm, 20.35 ± 1.04 mm, and 18.1 ± 1.1 mm, respectively (n = 10) (Table 1). Only the ND of note D was positively correlated with BM(r = 0.716, P = 0.020, n = 10; Figure 5A) and HW (r =0.643, P = 0.045, n = 10; Figure 5B), whereas DF was not signi ficantly correlated with body size (Figure 5A–D).

4. Discussion

In the present study, calls from 18 P. megacephalus and 13 P. mutus were recorded. Calls of both species comprised multiple note types: three in P. megacephalus and two in P. mutus. In P. megacephalus, note C had harmonics, while notes A and B did not. In P. mutus,note D had harmonics, while note E did not. Because the two species differed in the proportion of each call type per unit time, and P. mutus was less abundant in the field, statistical between-species comparisons were only performed on simple call structure and their parameters.The investigation of between-species acoustic parameters revealed significant differences in DF, ND, PD, and CD. Similar results were also reported in two sympatric species of Chiasmocleis, suggesting that the observed differences could be due to variation in social behavior(Forlani et al., 2013). In general, the difference in call characteristics reinforces existing species distinctions and indicates that calls are important as a mechanism to avoid interspecific mating (Haddad et al., 1994; Bastos et al.,2011). Based on the described acoustic differences, the two species of tree frogs could be easily distinguished in the field.

Frogs and toads generally produce relatively simple calls consisting of a single note or a series of identical repeated notes (Wang et al., 2016). Some anuran males, however, can produce dozens of complex calls.A previous study reported that the endemic Boophis madagascariensis possesses the largest known anuran vocal repertoire, with 28 distinct call types that differ in temporal pattern and spectral bandwidth (Narins et al., 2000). Polypedates leucomystax in Southeast Asia produces nine different call notes and at least 12 different call types (Christensen-Dalsgaard et al., 2002).In the present study, P. megacephalus produced more complex calls than did P. mutus. Previous research has suggested that in some anuran species, different note types have distinct functions (Kelley, 2004; Zhu et al.,2017). Although we did not test the meaning of each call property via playback experiments, CVb/CVwvalues suggested that dominant frequency, note duration, note number, and call duration are important features useful for individual identi fication in both species.

Xu (2005) reported similar DF in a Guangxi population of P. megacephalus. However, calls from the two populations differed in FF and ND. Previously reports of Hong Kong P. megacephalus call structure also had three note types, similar to our findings (Kuraishi et al., 2011).The DF of Hainan P. mutus and a Guangxi population was similar, but FF differed between them (Xu, 2005). Thus,these findings suggest that geographic variation may exist in P. megacephalus and P. mutus calls. This hypothesis is being thoroughly tested in our ongoing research.

Call characteristics are correlated with body size in some species and are potentially important signals related to male-male competition and mate choice (Given, 1987;Morris and Yoon, 1989; Bee et al., 1999; Gerhardt and Huber, 2002; Narins et al., 2006; Zhu et al., 2016b). In the present study, we found that the DF of note B was negatively correlated with body size in P. megacephalus,thus corroborating findings from many other anuran species and providing evidence that DF is a reliable indicator of male body size (Wang et al., 2012, 2016; Zhu et al., 2016a). However, note duration and body size were not signi ficantly correlated in P. megacephalus, nor were dominant frequency and body size correlated in P. mutus.The ND of note D was positively correlated with BM and HW in P. mutus. This pattern probably occurs because longer ND results in longer call duration, which might be metabolically costly to produce and requires a larger body size (Wang et al., 2012; Zhu et al., 2016a). In conclusion,although the sample size was small for each species in the present study, the current results still suggest that interspeci fic acoustic differences could re flect body size and facilitate intraspeci fic identi fication, thereby playing an important role in sexual selection.

Limitations In the present study, calls in 18 individuals of P. megacephalus and 13 individuals of P. mutus were recorded to analyze the call characteristics. Because the proportion of each call type per unit time in both species was different, and the density of P. mutus was also lower in the field, only some call parameters were statistically compared between the two species of tree frogs.

AcknowledgementsWe thank Hao ZHANG for the help in field experiment. We are grateful to Zhixin SUN,Bicheng ZHU, and Longhui ZHAO for helpful and advice on analyses. This work was supported by the National Natural Science Foundation of China (31260518 to JW) and the Education Department of Hainan Province(00501023523).References

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