ADVERTISEMENT CALLS and REPRODUCTIVE ACTIVITY of Hylarana Guentheri (BOULENGER, 1882) from BACH MA NATIONAL PARK

Russian Journal of Herpetology Vol. 19, No. 3, 2012, pp. 239 – 250

ADVERTISEMENT CALLS AND REPRODUCTIVE ACTIVITY OF Hylarana guentheri (BOULENGER, 1882) FROM BACH MA NATIONAL PARK

Binh V. Ngo,1 Chung D. Ngo,2 Xuyen T. Nguyen,3 Ping-Chun L. Hou†,4 and Nhon T. Tran2

Submitted April 19, 2011.

We analyzed variations in advertisement calls from frogs of different localities and reproductive activity of Hylarana guentheri living in a tropical region. We estimated the variation of each call property among three localities of the calling male on acoustical features. The spectral characteristics of single-note and multi-note advertisement calls of the species were described using acoustic spectral parameters. Call duration and dominant frequency were the most stereotyped properties with the average dominant frequency of single-note advertisement call was 1.152 kHz; call rate exhibited an intermediate level of variation among populations; whereas rise time and frequency modulation were highly variable within individuals. Call duration, call rate, and rise time were significantly different between localities. Snout-vent length ranged between 50.2 and 80.2 mm among sexually mature males and between 58.9 and 91.5 mm among females. Histological analyses of testes revealed that sperm did not occur throughout the entire year, indicating discontinuous reproductive activity for males. Reproductive females were observed only during the auxiliary rainy season months (mainly April to July), indicating that females have a seasonal reproductive activity.

Keywords: Amphibia; Anura; Hylarana guentheri; Bach Ma; reproductive activity.

species in taxonomically complex groups of frogs based INTRODUCTION on species-specificity of advertisement calls (Angulo and Reichle, 2008; Channing et al., 2002; De la Riva et al., Sound communication plays a key role in the repro- 1996; Padial et al., 2008). In most of the studied cases, duction of most anurans. In the majority of species, fe- differentiation of advertisement calls corresponds to that males use the acoustic signals of males to discriminate of other characteristics (DNA sequences, karyotype, and between conspecifics and heterospecifics (reviewed in morphology) and the taxonomic conclusions that can Blair, 1964, 1968; Gerhardt and Huber, 2002; Wells, be drawn widely coincide (Schneider and Sinsch, 2007). 2007), and to locate mates (Arch et al., 2011). The adver- Although useful for delimiting species, call variations tisement calls also play a role in attracting females and between species do not necessarily have a strong phylo- repelling intruding males (Duellman and Trueb, 1986). In genetic signal and thus might not accurately predict evo- most studied species females have shown preferences lutionary relationships between species (Cannatella et al., for conspecific over heterospecific advertisement calls 1998; Wollenberg et al., 2007). (Gerhardt, 1994; Ryan, 2009). Various features of the ad- Most papers about the reproductive biology of vertisement calls have frequently been used to delimit amphibians treat fecundity, reproductive modes, vocal- 1 Department of Life Sciences, National Cheng Kung University, ization, and courtship behavior (Silva et al., 2005). How- No. 1 University Road, Tainan City 701, Taiwan; ever, the reproductive biology of many species, including e-mail: [email protected] Hylarana guentheri species (formerly Rana or Sylvira- 2 College of Pedagogy, Hue University, No. 34 Le Loi Road, Hue City, na), is unknown or poorly understood (Dubois, 1992, Vietnam. 3 2005). Much of our current knowledge on the reproduc- Huong Lam High School, Aluoi District, Thua Thien-Hue Province, tion within a community has been inferred from studies Vietnam. 4 of autecologies of single species or of small subsets of the Department of Life Sciences and Institute of Biodiversity, National Cheng Kung University, No.1, University Road, Tainan City 701, whole community in a given place (Huang et al., 1996, Taiwan. 1997). Anuran amphibians are good models for repro-

Fig. 1. Map of Thua Thien-Hue Province showing the geographic location of Bach Ma National Park and the three localities where recordings (red circles) of Hylarana guentheri were obtained: (1) Phu Loc District (PLD); (2) Do Quyen Stream (DQS); (3) Ta Trach River (TTR). ductive studies, because they are poikilothermic and are reproductive biology (reproductive activity, clutches, greatly affected by physiological constraints (proximate fecundity, and breeding season) and the advertisement factors) such as temperature, rainfall, and daylight length calls of populations of H. guentheri inhabiting the tropi- or by phylogenetic constraints (ultimate factors), or by a cal region in Central Vietnam. We attempted to document mixture of these factors (Zug, 1993). intraspecific variations in the species’ advertisement calls On the other hand, anuran amphibians exhibit a spec- at different localities in the Bach Ma National Park in trum of reproductive strategies ranging from species that greater detail, in order to address the influence of temper- abandon large clutches of small eggs in aquatic habitats ature and body size on acoustic features of the advertise- to species that provide extensive parental care to small ment calls, and to quantitatively describe the multinote clutches of large eggs in terrestrial habitats (Salthe and call of the species. Mecham, 1974; Duellman and Trueb, 1986). The greatest diversity of reproductive patterns occurs among tropical MATERIAL AND METHODS frogs (Crump, 1974; Duellman, 1978). However, most of the detailed studies of reproductive strategies have been Description of Study Site conducted on temperate, aquatic-breeding species (Em- len, 1976; Wells, 1977a; Howard, 1978a, b, 1980; Ryan, This study was conducted at the Bach Ma Natio- 1980; Berven, 1981; Robertson, 1986), while very few nal Park, Thua Thien Hue province, Central Vietnam. studies (for example Kluge, 1981; Ryan, 1985) have been A total core area of approximately 37,487 ha, with geo- performed on the reproductive strategies of tropical graphic coordinates (WGS 84) from 15°59¢ – 16°16¢ N anurans. to 107°37¢ – 107°54¢ E (Fig. 1). The area consists of In Vietnam, H. guentheri is widely distributed 32.18% primary forest, 53.98% ecological restoration throughout the entire country at different altitudes (Ngu- forest, and 13.84% administrative management area yen et al., 2009). In the present paper, we describe the (Oikawa, 2009, unpublished data). According to Nguyen Behavioral Ecology of Hylarana guentheri (Boulenger, 1882) 241 et al. (2004) the climate and weather are characterized by 30 1200 the tropical monsoon, dominated by a montane rain for- 28 1000 est (between 400 and 1400 m above sea level), and cloud 800 forests from 1450 m above sea level to the summits 26 (1712 m above sea level), subtropical climates, no dry 600 season, only a rainy season and a little rainy season 24

(Fig. 2). 400 Rainfall, mm 22 Temperature, °C 200 Data Collection 20 0 Recording was conducted in two trips in 2011. Little rain Auxiliary rain Mainly rain 18 Thirty-nine calls were recorded successfully in three lo- Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec calities with coordinates: (1) Phu Loc District Month (16°15¢18¢¢ N 107°46¢22¢¢ E), average elevation of 219 m Fig. 2. Monthly mean rainfall and temperature in the study area. Data correspond to the monthly mean precipitation (broken line, open cir- above sea level, twelve recordings; (2) Do Quyen Stream cles, in millimeters) and temperature (solid line, filled circles, in °C) (16°11¢15¢ N 107°50¢53¢¢ E), average elevation of over the last 20 years. Data recorded from statistic of the climatic- 1130 m above sea level, nine recordings; (3) Ta Trach hydrology characters of Thua Thien-Hue Province (Nguyen et al., 2004). River (16°04¢59¢¢ N 107°45¢21¢¢ E), average elevation of 455 m above sea level, eighteen recordings. We calculated the coefficients of variation (CV =[SD/mean] × × 100%) of the acoustic features measured in 10 calls Data Analyses from a single-note call bout per individual in order to quantify within-individual variations of the single-note The recording memory cards were digitized on Asus ® call. We employed the criteria of Gerhardt (1991) and computers with Intel Core™ i3. Sound analysis was classified as static those acoustic properties with average performed with the aid of Avisoft-SASLab Pro software within individual CV < 5% and dynamic those with CV > (Version 5.1.13) at a 44.1 kHz sampling frequency and 12%, and CV from 5-12% was intermediate level of 16-bit resolution. The software was set with the spectrogram parameters: Hamming window function, 1024 within-individual variation. points fast Fourier transformation (FFT), frame size Recording was performed by a Marantz Professional 100%, overlap 96.87%, color 5%, color table: colordar. Recorder (Solid State Recorder PMD671, Japan), with pal, and the power spectrum (logarithmic) of the call. a memory card, unidirectional Sony ECM-G7M micro- Using a random set of recordings, the appropriate call phone which was pointed at the calling male from a dis- recognition settings were adjusted so that all calls were tance of approximately 100 cm (Marshall and Gerhardt, accurately delineated in oscillograms and spectrograms. 2010). Calling individuals were located between The same settings were used for all analyses. Following 1900 – 0300 h using headlamps. Recording proceeded call detection, oscillograms and spectrograms were visu- for 5 – 10 min, registering both the silent periods and at ally inspected to reject incorrectly measured calls. least two entire call sequences of each male. Following In Bach Ma National Park, adult males of H. guen- the recordings, males were captured and their snout-vent theri emit two types of advertisement calls, a single-note lengths (SVL) were measured to the nearest 0.01 mm call and a multi-note call. Measurements of the single- with a digital caliper. Each individual was marked with a note advertisement calls were taken from 10 calls in the unique combination of toe clips to prevent duplicate re- middle of the longest call group and all single-note calls cordings. Air temperature at the recording site was mea- emitted by a given male were measured. Five acoustic sured to the nearest 0.1°C with a quick-reading thermom- features of advertisement calls were measured for each eter (Wisewind 0912, Taiwan). The individuals used in call: call duration (measured at zero amplitude on the the reproductive activity study were also captured by vi- oscillogram); rise time (time from call onset to peak am- sual encounters during two nights of surveys plitude in the oscillogram); call rate (reciprocal of the (1900 – 0200 h) each month between January and De- intercall interval); dominant frequency (frequency of ma- cember 2009, covering different areas of the forest. The ximum energy in the spectrogram); and frequency modu- sampling was standardized with two persons for seven lation (frequency range between onset and offset of the hours each night. The specimens were collected by hand dominant harmonic in the call spectrogram) of each adult and placed in individually labeled bags. male’s advertisement call. We also measured minimum 242 Binh V. Ngo et al.

Fig. 3. The spectral characteristics of lower quartile (25%), mean frequency (50%), and upper quartile (75%) of two-note calls in advertisement calls of Hylarana guentheri from Bach Ma National Park (Spectrogram Parameters: 1024 points FFT, Window: Hamming, Frame size 100%, Over- lap 96.87%, Color 5%, and Color table: colordar.pal). and maximum frequency such as the lower quartile in order to determine reproductive activity and document (25%), mean (50%), and upper quartile (75%) of fre- the SVL range for onset of sexual maturity. The stage of quency (the frequency that carries the most energy of 25, spermatogenesis was determined following Chan’s 50, and 75%, respectively; see Fig. 3); total bandwidth (2003) classification: stage 1: primary spermatocytes; (Hz) of the threshold at amplitude –10 dB of each adult stage 2: secondary spermatocytes; stage 3: spermatozoa. male’s advertisement call (Table 1). Female sexual maturity and reproductive activity Reproductive Activity. The individuals used in the was confirmed by the presence of yolked follicles, reproductive study were captured by visual encounters oviductal eggs, and enlarged convoluted oviducts. Ovar- during two nights of surveys (1900 – 0200 h) each month ian volume and diameter of the yolked follicles or between January and December 2009. The specimens oviductal eggs were recorded. Reproductive activity data were collected by hand and placed into individually for each animal were used to establish the percent age of labeled bags. In the laboratory, Gunther’s Amoy Frogs males and females in each reproductive stage each month (H. guentheri) were dissected and their reproductive and season throughout the year. Ovarian egg data were tracts were removed for analysis of sexual maturity and used to estimate mean clutch size. The number of ovarian reproductive activity. We recorded the longest and short- eggs was correlated with female body size (SVL). est diameters, calculated testicular and ovarian volume Chi-square tests or G-tests were employed to detect sig- using the formula for a prolate spheroid (Biavati et al., nificant intrasexual and intersexual variations in repro- 2004; Caldwell and Vitt, 1999; Magnusson et al., 2003). ductive stages over time. Linear regressions between go- The volumes of testes and ovaries were estimated using nad volume and SVL of each individual were calculated. the formula for a prolate spheroid: Reproductive data of each month were grouped into 4p length æ width ö2 two-month intervals (corresponding to the three rainfall V = ç ÷ . periods that occur during the year, see Fig. 2). 32è 2ø The spectral characteristics of advertisement calls The left and right testis were extracted, fixed in 70% (single-note, two-note, and three-note calls) and repro- ethanol, embedded in paraffin, sectioned into 6 ìm slices ductive activity between seasons were tested by one-way using a microtome, and stained with hematoxylin-eosin analysis of variance (ANOVA) with LSD Post Hoc Tests. Behavioral Ecology of Hylarana guentheri (Boulenger, 1882) 243

TABLE 1. The Spectral Characteristics of Call Structure and Calling Behavior of Hylarana guentheri from Bach Ma National Park, mean ± SD (range: minimum – maximum). Spectral Characteristics Call types DF, kHz LQ, kHz MF, kHz UQ, kHz MinF, kHz MaxF, kHz TBW, Hz Single-note call a1.152 ± 0.057 a0.984 ± 0.039 a1.281 ± 0.041 a1.766 ± 0.060 a1.101 ± 0.032 a1.236 ± 0.093 a67 ± 1.699 (n = 39) (1.025 – 1.241) (0.899 – 1.023) (1.189 – 1.327) (1.684 – 1.891) (1.008 – 1.155) (1.127 – 1.379) (65 – 70) Two-note calls a1.136 ± 0.024 a0.973 ± 0.051 b1.164 ± 0.006 b1.648 ± 0.054 b1.164 ± 0.052 a1.207 ± 0.048 b36 ± 2.761 (n = 39) (1.058 – 1.257) (0.892 – 1.105) (1.155 – 1.179) (1.548 – 1.726) (1.035 – 1.238) (1.109 – 1.256) (30 – 40) Three-note calls b0.988 ± 0.022 b0.775 ± 0.031 c1.069 ± 0.022 c1.365 ± 0.041 c0.984 ± 0.025 b1.017 ± 0.032 c26 ± 2.797 (n = 25) (0.929 – 1.047) (0.742 – 0.821) (1.036 – 1.102) (1.299 – 1.435) (0.912 – 1.012) (0.971 – 1.105) (20 – 30) Total (n = 103) 1.106 ± 0.078 0.929 ± 0.097 1.185 ± 0.088 1.624 ± 0.165 1.096 ± 0.080 1.173 ± 0.111 46 ± 17.724 (0.929 – 1.257) (0.742 – 1.105) (1.036 – 1.327) (1.299 – 1.891) (0.912 – 1.238) (0.971 – 1.379) (20 – 70) Notes. DF, Dominant frequency; LQ, lower quartile 25%; MF, mean frequency 50%; UQ, upper quartile 75%; MinF, minimum frequency; MaxF, maximum frequency; TBW, total bandwidth; the same a column, different letters indicating the mean difference is significant at the á = 0.05 level (P < 0.05).

Statistical differences in reproductive activity between typical advertisement call of H. guentheri from the Bach seasons were assessed using analysis of covariance Ma National Park. It can be described as a short pulse: (ANCOVA) on the gonad volumetric data with body size call duration 95.7 ± 17.42 msec with a rapid rise time and (SVL) as a covariate by SPSS 14.0 software (SPSS Inc., a smoother decay of rise time 4.07 ± 0.51 msec, pro- Chicago, Illinois). The possible effects of climatic factors duced in relatively long call groups of call group duration on reproductive activity were tested with multiple linear 309.9 ± 76.19 sec at call rate 31.4 ± 5.56 calls/min with regressions between the monthly scores of rainfall (in dominant frequency 1.152 ± 0.057 kHz and frequency millimeters) and temperature (°C) and gonadal volume. modulation 16.744 ± 2.653 Hz (Table 2). Data are presented as mean ± SD and P < 0.05 was con- Rise time (mean CV = 21.35%) and frequency modu- sidered to be statistically significant. lation (mean CV = 15.37%) were highly variable within individuals; dominant frequency (mean CV = 0.59%) and call duration (mean CV = 2.19%) were the most stereo- RESULTS typed properties; whereas call rate exhibited an intermediate level of within-individual variation (mean Advertisement calls CV = 8.75%). According to the criteria of Gerhardt Descriptive statistics of call properties of H. guenthe- (1991), dominant frequency and call duration can be con- ri showed that this species had two call properties, a sidered static properties, while rise time and frequency single-note call and a multinote call (Table 1). Average modulation can be considered dynamic properties. On spectral characteristics (mean ± SD) of advertisement the other hand, in the study area showed that temperature calls were dominant frequency: 1.106 ± 0.078 kHz; had an negative influence on advertisement calls, when lower quartile (25%): 0.929 ± 0.097 kHz; mean fre- air temperature in the recording area decreased below quency (50%): 1.185 ± 0.088 kHz; upper quartile (75%): 17°C, advertisement calls of adult males virtually ceased, 1.624 ± 0.165 kHz; minimum frequency: 1.096 ± 0.080 while air humidity had hardly any influence. Acoustic kHz; maximum frequency: 1.173 ± 0.111 kHz; and total features derived from active muscular contraction, such bandwidth: 46 ± 17.724 Hz (Table 1). Our results as calling rate, are highly temperature-dependent in anu- showed when not controlling for air temperature and rans because of their ectothermic condition. SVL, dominant frequency of single-note and two-note One-way analysis of variance (ANOVA) with LSD calls compared to three-note calls were significantly dif- Post Hoc Tests showed that the spectral characteristics of ferent (P > 0.05): single-note calls 1.152 ± 0.057 kHz call structure and call behavior of H. guentheri with the (Table 1; Figs. 4a,5a); two-note calls 1.136 ± 0.024 kHz mean difference was significant, P < 0.05 [exclusion of (Table 1; Figs. 4b,5b); three-note calls 0.988 ± dominant frequency, lower quartile (25%), and maxi- 0.022 kHz (Table 1; Figs. 4c,5c). mum frequency of single-note calls compared with two- Analysis call types of adult males showed the most of note calls was not significantly different, P > 0.05 (Table 1)]. single-note were created by a main structure (large An ANOVA showed that dominant frequency: F[2, 102] = subnote) and an auxiliary structure (small subnote) = 149.04, P < 0.0001; lower quartile (25%): F[2, 102] = (Fig. 4d ). On the other hand, a single-note call was emit- = 221.57, P < 0.0001; mean frequency (50%): F[2, 102] = ted by thirty-nine males recorded and is most likely the = 452.50, P < 0.0001; upper quartile (75%): F[2, 102] = 244 Binh V. Ngo et al.

ab c

0.055 0.06 0.065 0.07 0.06 0.07 0.08 0.09 0.1 0.11 0.14 0.16 0.18 0.2 0.22 0.24

0.006 0.007 0.008 0.009 0.01 0.011 0.012 0.013 0.014 0.015 0.016 0.017 0.018 0.019 0.02 Time, sec Fig. 4. The advertisement calls of Hylarana guentheri from Bach Ma National Park, recorded in 2011. Air temperature 24.5°C, water temperature 21°C, and air humidity 91%. Oscillograms of single-note call (a), two-note calls (b ), three-note calls (c), and spectrogram of single-note (d ) showing two peaks per note.

0 1.141 kHz ab1.130 kHz 0.096 kHz c

–10

–20

–30

Amplitude, dB –40

–50

–60 1 2 3 4 5 6 7 8 9 1 2 3 4 5 6 7 1 2 3 4 5 6 7 8 Frequency, kHz Fig. 5. Power spectrum that shows the energy distribution across the calls of Hylarana guentheri from Bach Ma National Park: a, single-note call; b, two-note calls; c, three-note calls, the power spectrum (logarithmic) of the call.

= 431.27, P < 0.0001; minimum frequency: F[2, 102] = = 156.70, P < 0.0001; total bandwidth: F[2, 102] =

= 156.70, P < 0.0001; maximum frequency: F[2, 102] = = 2678.89, P < 0.0001 (Table 1). Behavioral Ecology of Hylarana guentheri (Boulenger, 1882) 245

TABLE 2. Descriptive Statistics of Call Features Measured in Single-Note Advertisement Calls of Hylarana guentheri in the Three Localities Studied from Bach Ma National Park Call property PLD (n = 12) DQS (n = 9) TTR (n = 18) Total (n = 39) Call duration, msec 106.9 ± 19.47 103.0 ± 11.17 84.6 ± 11.27 95.7 ± 17.42 (75 – 135) (89 – 125) (56 – 99) (56 – 135) Call rate, calls/min 28.1 ± 6.82 31.8 ± 1.14 33.5 ± 5.08 31.4 ± 5.56 (15.7 – 40.5) (30.4 – 33.2) (26.1 – 47.3) (15.7 – 47.3) Rise time, msec 3.91 ± 0.31 4.77 ± 0.51 3.84 ± 0.25 4.07 ± 0.51 (3.5 – 4.5) (3.6 – 5.6) (3.3 – 4.2) (3.3 – 5.6) Dominant frequency, kHz 1.152 ± 0.051 1.161 ± 0.056 1.147 ± 0.063 1.152 ± 0.057 (1.088 – 1.234) (1.089 – 1.237) (1.025 – 1.241) (1.025 – 1.241) Frequency modulation, Hz 16.417 ± 2.193 16.222 ± 2.863 17.222 ± 2.881 16.744 ± 2.653 (13 – 20) (12 – 21) (12 – 25) (12 – 25) Call group duration, sec 304.4 ± 101.7 282.3 ± 2.455 327.4 ± 73.2 309.9 ± 76.19 (125 – 545) (254 – 316) (158 – 447) (125 – 545) Note. PLD, Phu Loc District; DQS, Do Quyen Stream; TTR, Ta Trach River (n = number of adult males recorded). The mean ± 1 SD and range (minimum-maximum) are shown without adjustments for variation in SVL size or air temperature.

100 35

30 80 25

60 20

SVL, mm 40 10

Number of individuals

20 5

Little rain Auxiliary rain Mainly rain 0 0 <30 30–39.9 40–49.9 50–59.9 60–69.9 70–79.9 >80 Jan – Mar – May – Jul – Sep – Nov – SVL range, mm – Feb – Apr – Jun – Aug – Oct – Dec Fig. 7. Distribution of snout-vent length (SVL) for the individuals in Fig. 6. Distribution of age (SVL, mm) and sex of the individuals col- the different categories considered in the population of Hylarana guen- lected from the population of Hylarana guentheri. Reproductive adult theri. Notice the restricted size distribution of adult males compared to males (filled circles), nonreproductive males (open circles), reproduc- the wide range of sizes and larger SVL attained by females. Juveniles tive adult females (filled triangles), nonreproductive adult females of undetermined sex (gray bars); juvenile males (blue bars); juvenile (open triangles), juvenile males (filled squares), juvenile females (open females (red bars); adult males (black bars); adult females (white bars). squares), juveniles of undetermined sex (rhombuses). The arrows indicate the minimum size at sexual maturity for each sex.

(mean ± SD = 70.77 ± 8.09 mm, n = 51); mature females ranged between 58.9 and 91.5 mm SVL (mean ± SD = Reproductive Activity 80.64 ± 9.29 mm, n = 39, Fig. 6). This population did not We collected 159 individuals: 39 adult females, exhibit strong sexual dimorphism in body size (SVL, 51 adult males, and 69 juveniles. The number of col- t[88] = 6.58, P = 0.0001, Fig. 7). lected males, females, and juveniles was not significant- Reproductive females were collected only in the 2 ly different (X [;]22 = 3.156, P > 0.5, n = 159), showing months of auxiliary rain (in April to August). There was equal capture for both sexes and juveniles during all a significant relationship between SVL and ovarian 2 seasons. Mature males did not occur throughout the year volume (r = 0.615, F[1 – 37] = 32.67, P = 0.0001, n = 39). 2 (X []2 = 4.67, P < 0.001, n = 51) and the occurrence of re- There were significant differences in ovarian volume productive mature females varied significantly between between seasons (ANCOVA, F[1–2]= 7.58, P = 0.001, 2 months (X []2 = 25.73, P < 0.001, n = 39, Fig. 6). Sex- Fig. 8a). Between March and August, adult females had ually mature males ranged from 50.2 – 80.2 mm SVL yolked follicles and the ovaries had their largest volumes 246 Binh V. Ngo et al.

22 10 1200 a a 20 Mean ± SD 1000 18 3 8

3 800 16 14 6 600 12

4 400 Rainfall, mm 10

8 Ovarian volume, cm 200

Ovarian volume, cm 2 6 0 4 0 2 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Jan – Mar – May – Jul – Sep – Nov – 30 1200 –Feb –Apr –Jun –Aug –Oct –Dec b 70 1000 b Mean ± SD 25 3 800 60 20 600 3 50 400 15 Rainfall, mm 40 200 Testis volume, mm 10 30 0

Testis volume, mm 5 20 Jan Feb Mar Apr May Jun Jul Aug Sep Oct Nov Dec Little rain Auxiliary rain Mainly rain Fig. 9. Monthly changes in mean gonadal volume in males and fe- 10 males of Hylarana guentheri and its relationship with mean rainfall Jan – Mar – May – Jul – Sep – Nov – –Feb –Apr –Jun –Aug –Oct –Dec values: a, ovarian volume (solid lines, filled squares), rainfall values (solid lines, open circles); b, testis volume (solid lines, filled circles), Fig. 8. Seasonal changes in gonadal volume of males and females of rainfall values (solid lines, open circles). Hylarana guentheri: a, ovarian volume varied significantly among seasons; b, testis volume varied significantly among seasons. 0.257, beta rainfall (mm) = –0.359, r2 = 0.359, F = 4.352, P = 0.316]. (mean ± SD = 7.138 ± 1.713 cm3, n = 25; follicle diame- [2, 48] Reproductive adult males did not occur throughout ter £1.7 mm, and between 1859 and 3895 yolked folli- the year. Spermatogenesis was observed polarized in cles, mean ± SD = 2873 ± 740). A positive correlation each testis: only primary spermatocytes were present was found between body size and fecundity (number of in the anterior portion of the testis and only spermato- yolked follicles, r2 = 0.723, P < 0.05). By June, maxi- zoa were found in the posterior portion. The occurrence mum ovarian size was attained. The first reproductive fe- of males with testes in stage 3 (with spermatozoa) males (with distended oviducts) were found in March, was significantly different over time (G[10] = 17.362, and from September to February females had ovaries of P < 0.001). There was a significant relationship between 3 2 small volumes (mean ± SD = 3.092 ± 0.986 cm and fol- SVL and testicular volume (r = 0.523, F[1, 49] = 18.35, licle diameter £0.7 mm). Therefore, ovulation and P = 0.0001, n = 51). Testicular volume of mature males oviposition occurs between the end of the little rainy sea- was significantly different between seasons (ANCOVA, F = 2.36, P = 0.001, Fig. 8b). When testes volumes son and the end of the auxiliary rainy season of the year [1, 2] are compared between months, a dramatic increase was (March to August; Fig. 9a). Multiple regressions indi- observed from April to July, when adult females have the cated that only ovarian volume is positively associated largest follicular sizes and are preovulatory. Testes vol- with rainfall; in males, variation in rainfall and tempera- umes fell from September to January (Fig. 9b), when ture seem not to significantly affect testicular volume most of the females had no ripe eggs in their ovaries. Al- [ovarian volume: beta temperature (°C) = 0.401, beta most all adult males (45 males, 88%) producing sperm 2 rainfall (mm) = –0.687, r = 0.635, F[2, 36] = 6.742, P = had visible secondary sexual characteristics such as cal- = 0.001. Testicular volume: beta temperature (°C) = losities on the foreleg, bright tympanum (usually reddish Behavioral Ecology of Hylarana guentheri (Boulenger, 1882) 247

a 12 a

–12

Amplitude

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8

0.01 0.015 0.02 0.025 0.03 0.035 0.04 0.045 0.05 0.055 0.06 0.065 0.07 0.075 0.08 0.085 0.09 Time, sec Fig. 10. The oscillogram of advertisement calls of Hylarana guentheri from Bach Ma National Park: a, three-note calls; b, spectrogram of a three-note call showing two peaks per note.

b brown tympanum), and the throat speckled with yellow- ish (usually the throat speckled with brown). Twelve percent of the adult males had sperm in their testes and no discernible callosities.

DISCUSSION

Temperature negatively influenced the advertisement calls of H. guentheri. When air temperature in the recording area decreased below 17°C, the advertisement calls of adult males virtually ceased, while air humidity had Photo by Binh V. Ngo hardly any effect. Acoustic features derived from active muscular contraction, such as calling rate, are highly Fig. 11. Photograph of male (top) and female (bottom) of Hylarana guentheri in amplexus during the breeding season (a) and a clutch of temperature-dependent in anurans because of their ecto- this species (b ) from Bach Ma National Park. thermic condition (Castellano et al., 2002; Gayou, 1984; Wells, 2007). In adult males of H. guentheri, our field ob- servations indicate that the production of each call in- which exhibited two call types, a single-note call (fre- volves the contraction of trunk muscles. Hence, meta- quently) and a multinote call (less frequently) and only bolic constraints could explain the positive correlation twenty-five of thirty-nine males emitted a three-note call between air temperature and call rate. Furthermore, it has interspersed in the middle of a single-note call group been shown that for many frogs the energetic cost of call- (Fig. 10). The advertisement calls of three-note calls are ing increases with call rate (Bevier, 1997; Wells, 2007). only exhibited when in the population had appeared 3 – 5 Males of this species probably maintain a relatively con- or more individuals made advertisement calls together stant energetic expenditure and are unable to increase calling rate and call group duration simultaneously. where a small space was enough for recognizing the calls However, our results indicate that call duration and domi- of other individuals of the same species. By contrast, nant frequency were the most stereotyped properties of when in the population had 1 or 2 male individuals made H. guentheri. Call duration and dominant frequency have advertisement calls at the same time, single-note and been traditionally employed to assess species limits and two-note calls were essential and coherent. Adult males divergence processes in anurans (Gergus et al., 2004; have three-note calls were commonly selected by females Márquez and Bosch, 1995) and our findings could be rel- for amplexus (Fig. 11a). A total of 39 adult males was re- evant for future studies of the genus Hylarana living in corded from Bach Ma National Park, with 25 individuals the tropical region. (mean ± SD = 78.0 ± 0.71 mm SVL) producing three- Analysis of advertisement calls showed the evolution note calls with a lower frequency than single-note and of advertisement calls in adult males of this species, two-note calls (Figs. 5c,12c; Table 1) and all were suc- 248 Binh V. Ngo et al.

20ab 20 vitellogenesis at the end of the little rainy season months 15 15 and reach their maximal diameter from April to July, the

10 10 month when the little rainy season abruptly ends. Oviposition takes place during the auxiliary rainy season, 55 and egg development occurs during the auxiliary rainy season and the first mainly rainy season of the year. Sea- 0.2 0.2 0.4 0.6 sonality in female reproductive activity seems to be com- 20 mon in anurans from Bach Ma National Park. In fact, it 15 c has been suggested that in tropical anuran species living

Frequency, kHz in areas of pronounced rainy season seasonality, ovi- 10 position is concentrated in the end of the little rainy season and hatching occurs during the auxiliary rainy season 5 (Ngo et al., 2009). Some authors suggested that continual spermatoge- 0.2 0.4 0.6 Time, sec nesis may represent an evolutionarily stable strategy for amphibians if the cost of maintaining and producing ma- Fig. 12. Spectrogram of the call presented in: a, single-note call; b, two-note calls; c, three-note calls of Hylarana guentheri (the spectro- ture sperm is low (Houck, 1977; Yang, 1998). However, gram parameters: 1024 points FFT, Window: Hamming, Frame size using detailed histological examination of the testes, 100%, Overlap 96.87%, Color 5%, and Color table: colordar. pal). Chan (2003) identified subtle levels of variation and cryptic temporal patterns in testis activity not detectable by external appearance or the presence of secondary cessful in amplexus during the breeding season sexual characteristics. Although testis volume did change (Fig. 11a). When the SVL of adult male individuals was significantly among seasons, it was possible to detect this smaller than 77.29 mm, only single-note and two-note same variation in testis activity of H. guentheri when calls were observed, indicating that advertisement calls observing the monthly behavior of testis volume. correspond to individual development. On the other Throughout the study process, maximal testicular vol- hand, the strong relationship of dominant frequency and umes were observed in April to July when the females are rise time with the size of callers suggests that females of periovulatory, and testes volumes fell during the follow- this species may use these features as acoustic cues for ing months, perhaps in response to the increased mating selecting bigger males for amplexus as has been demon- opportunities because matings occur around the time of strated in many species of anurans (Wells, 2007), and de- egg-laying. pending on the specific conditions such as air tempera- Acknowledgments. We wish to thank the heads of the ture, locality, and SVL, all calls to get high frequency and Biological Faculty, College of Pedagogy, Hue University, Viet- large change (Table 1; Fig. 12). nam and of the Department of Life Sciences and Institute Females of H. guentheri mature at larger body sizes of Biodiversity, National Cheng Kung University, Taiwan and have larger maximal sizes than males. Sexual dimor- for the support of this study. We would like to thank Prof. phism in body size is common in anurans and tends to be Y. Steinberger (Department of Life Sciences, Bar Ilan Univer- more pronounced in larger species (Salthe and Duellman, sity, Ramat Gan, Israel) for his English spelling review on the entire manuscript. We also thank two anonymous reviewers for 1973) and may be associated with reproduction: larger critically reading the manuscript and of the management board body size in females may allow greater clutch sizes of Bach Ma National Park, staff of A Luoi and A Pat Forest (Fig. 11b). In H. guentheri, as in some other species of Stations, Border Stations 629 and 633 for the help during the anurans, female fecundity increases with increasing body field work in Nam Dong, Phu Loc, and A Luoi district, Thua size (Wake and Dickie, 1998), and according to Turner Thien-Hue province from 2007 – 2010. Last and best, we thank (1960), larger female size is caused by faster growth rates Cheng A. Ho, Oang V. Ho, Phong V. Ho, Hau Ngo, Liem Q. in females and not by lower survival rates in males. Dinh, Bach V. Ho, Bang V. Ho, and Sinh V. Ho for their support Larger female size is consistent with other studies of anu- during the field work throughout 2007 – 2010. ran species that show that larger female body size is related to increased fecundity (Salthe and Duellman, 1973; REFERENCES Elmberg, 1991). The reproductive activity of H. guentheri is seasonal, Angulo A. and Reichle S. (2008), “Acoustic signals, species related to the rainfall regime. Ovarian follicles begin diagnosis, and species concepts: the case of a new cryptic Behavioral Ecology of Hylarana guentheri (Boulenger, 1882) 249

species of Leptodactylus (Amphibia, Anura, Leptodactyli- Elmberg J. (1991), “Factors affecting male yearly mating suc- dae) from the Chapare region, Bolivia,” Zool. J. Linnean cess in the Common Frog, Rana temporaria,” Behav. Ecol. Soc., 152(1), 59 – 77. Sociobiol., 28(2), 125 – 131. Arch V. S., Richards-Zawaki C. L., and Feng A. S. (2011), Emlen S. T. (1976), “Lek organization and mating strategies in “Acoustic communication in the Kihansi Spray Toad (Nec- the bullfrog,” Behav. Ecol. Sociobiol., 1(3), 283 – 313. tophrynoides asperginis): Insights from a captive popu- Gayou D. C. (1984), “Effects of temperature on the mating lation,” J. Herpetol., 45(1), 45 – 49. call of Hyla versicolor,” Copeia, 1984(3), 733 – 738. Berven K. A. (1981), “Mate choice in the wood frog, Rana Gergus E. W. A., Reeder T. W., and Sullivan B. K. (2004), sylvatica,” Evolution, 35(4), 707 – 722. “Geographic variation in Hyla wrightorum: advertisement Bevier C. R. (1997), “Utilization of energy substrates during calls, allozymes, mtDNA, and morphology,” Copeia, calling activity in tropical frogs,” Behav. Ecol. Sociobiol., 2004(4), 758 – 769. 41(5), 343 – 352. Gerhardt H. C. (1991), “Female mate choice in treefrogs: static and dynamic acoustic criteriav,” Animal Behav., 42, Biavati G. M., Wiederhecker H. C., and Colli G. R. (2004), 615 – 635. “Diet of Epipedobates flavopictus (Anura: Dendrobatidae) Gerhardt H. C. (1994), “The evolution of vocalization in in a neotropical savanna,” J. Herpetol., 38(4), 510 – 518. frogs and toads,” Ann. Rev. Ecol. Syst., 1994(25), 193 – Blair W. F. (1964), “Isolating mechanisms and interspecific 324. interactions in anuran amphibians,” Quart. Rev. Biol., Gerhardt C. H. and Huber F. (2002), Acoustic Communica- 39(4), 334 – 344. tion in Insect and Anurans, Univ. of Chicago Press, Blair W. F. (1968), “Amphibians and Reptiles,” in: T. A. Se- Chicago. boek (ed.), Animal Communication, Indiana Univ. Press, Houck L. D. (1977), “Life history patterns and reproductive Bloomington, pp. 289 – 310. biology of Neotropical salamanders,” in: D. H. Taylor and Cannatella D. C., Hillis D. M., Chippindale P. T., Weigt L., S. J. Guttman (eds.), The Reproductive Biology of Amphib- Rand A. S., and Ryan M. J. (1998), “Phylogeny of frogs ians, Plenum Publ. Corp., New York, pp. 43 – 72. of the Physalaemus pustulosus species group, with an ex- Howard R. D. (1978a), “The evolution of mating strategies in amination of data incongruence,” Syst. Biol., 47(2), 311 – bullfrogs, Rana catesbeiana,” Evolution, 32(4), 850 – 871. 335. Howard R. D. (1978b), “The influence of male-defended ovi- Castellano S., Cuatto B., Rinella R., Rosso A., and Giaco- position sites on early embryo mortality in bull-frogs,” ma C. (2002), “The advertisement call of the European Ecology, 59(4), 789 – 798. treefrogs (Hyla arborea): a multilevel study of variation,” Howard R. D. (1980), “Mating behaviour and mating success Ethology, 108(1), 75 – 89. in woodfrogs, Rana sylvatica,” Animal Behav., 28(3), Caldwell J. P. and Vitt L. J. (1999), “Dietary asymmetry in 705 – 716. leaf litter frogs and lizards in a transitional northern Amazo- Huang W. S., Lin J. Y., and Yu Y. L. (1996), “The male re- nian rain forest,” Oikos, 84(3), 383 – 397. productive cycle of the toad, Bufo bankorensis, in Taiwan,” Chan L. M. (2003), “Seasonality, microhabitat and cryptic Zool. Studies, 35(2), 128 – 137. variation in tropical salamander reproductive cycles,” Biol. Huang W. S., Lin J. Y., and Yu Y. L. (1997), “Male reproduc- J. Linnean Soc., 78(4), 489 – 496. tive cycle of the toad Bufo melanostictus in Taiwan,” Zool. Channing A., Moyer D., and Burger M. (2002), “Cryptic Sci., 14(3), 497 – 503. species of Sharp-Nosed Reed Frogs in the Hyperolius nasu- Kluge A. G. (1981), “The life history, social organization, and tus complex: advertisement call differences,” Afr. Zool., parental behavior of Hyla rosenbergi Boulenger, a nest- 37(1), 91 – 99. building gladiator frog,” Misc. Publ. Mus. Zool. Univ. Mi- Crump M. L. (1974), “Reproductive strategies in a tropical chigan, 160, 1 – 170. anuran community,” Univ. Kansas Mus. Nat. Hist. Misc. Magnusson W. E., Lima A. P., Alves da Silva W., and Car- Publ., 61,1–68. mozina de Araújo M. (2003), “Use geometric forms to estimate volume of invertebrates in ecological studies of De la Riva I., Márquez R., and Bosch J. (1996), “Advertise- dietary overlap,” Copeia, 2003(1), 13 – 19. ment calls of four microhylid frogs from Bolivia (Amphi- Márquez R. and Bosch J. (1995), “Advertisement calls of the bia, Anura),” Am. Midland Naturalist, 136(2), 418 – 422. midwife toads Alytes (Amphibia, Anura, Discoglossidae) in Dubois A. (1992), “Notes sur la classification des Ranidae continental Spain,” J. Zool. Syst. Evol. Res., 33(3 – 4), (Amphibiens: Anoures),” Bull. Mensuel Soc. Linn. Lyon, 185 – 192. 61, 305 – 352. Marshall V. T. and Gerhardt H. C. (2010), “A precedence Dubois A. (2005), “Amphibia Mundi. 1.1. An ergotaxonomy effect underlies preferences for calls with leading pulses in of recent Amphibians,” Alytes, 23,1–24. the grey treefrog, Hyla versicolor,” Animal Behav., 80(1), Duellman W. E. (1978), “The biology of an equatorial herpe- 139 – 145. tofauna in Amazonian Ecuador,” Univ. Kansas Mus. Nat. Ngo V. B., Tran T. T. N., and Tran C. T. (2009), “Nutritional Hist. Misc. Publ., 165, 1 – 352. and reproductive characters of three species (Quasipaa Duellman W. E. and Trueb L. (1986), Biology of Amphibians, verrucospinosa, Hylarana guentheri, and Fejervarya lim- McGraw-Hill, New York. nocharis) in Thua Thien Hue,” in: C. D. Ngo, T. H. Ta, 250 Binh V. Ngo et al.

N. N. Le, Q. X. Hoang, P. V. Vo, S. V. Nguyen, T. V. Ngu- Schneider H. and Sinsch U. (2007), “Contributions of yen, T. T. Le, and D. Q. Tran (eds.), Proc. of the 1st Natl. bioacoustics to the taxonomy of the Anura,” in: Amphibian Sci. Workshop Amphibia and Reptile, Hue University Pub- Biology, Surrey Beatty and Sons, Chipping Norton, New lishing House, Vietnam, pp. 179 – 187. South Wales, Australia, pp. 2893 – 2933. Nguyen V. S., Ho T. C., and Nguyen Q. T. (2009), Herpeto- Silva W. R., Giaretta A. A., and Facure K. G. (2005), “On fauna of Vietnam, Edition Chimaira, Frankfurt am Main. the natural history of the pepper frog Leptodactylus labyrin- Nguyen V., Truong D. H., Hoang T. L., Nguyen V. H., thicus (Anura, Leptodactylidae),” J. Nat. Hist., 39(7), Phung D. V., Ha H. K., Tran D. H., Phan V. H., Ngu- yen D. A., and Le Q. V. (2004), The Climatic-Hydrology 555 – 566. Characters of Thua Thien-Hue Province, Thuan Hoa Pub- Turner F. B. (1960), “Population structure and dynamics of lishing House, Vietnam. the Western Spotted Frog, Rana p. pretiosa Baird & Girard, Padial J. M., Köhler J., Munöz A., and De la Riva I. (2008), in Yellowstone Park, Wyoming,” Ecol. Monogr., 30(3), “Assessing the taxonomic status of tropical frogs through 251 – 278. bioacoustics: geographical variation in the advertisement Wake M. H. and Dickie R. (1998), “Oviduct structure and calls in the Eleutherodactylus discoidalis species group function and reproductive modes in amphibians,” J. Exp. (Anura),” Zool. J. Linnean Soc., 152(2), 353 – 365. Zool., 282(4 – 5), 477 – 506. Robertson J. G. M. (1986), “Female choice, male strategies Wells K. D. (1977a), “Territoriality and male mating success in and the role of vocalizations in the Australian frog Upero- leia rugosa,” Animal Behav., 34(3), 773 – 784. the green frog (Rana clamitans),” Ecology, 58(4), 750 – Ryan M. J. (2009), Communication in Frogs and Toads. En- 762. cyclopedia of Neuroscience, Acad. Press, Oxford, Wells K. D. (2007), The Ecology and Behavior of Amphibians, pp. 1159 – 1166. Univ. of Chicago Press, Chicago. Ryan M. J. (1980), “The reproductive behavior of the bullfrog Wollenberg K. C., Glaw F., Meyer A., and Vences M. (Rana catesbeiana),” Copeia, 1980(1), 108 – 114. (2007), “Molecular phylogeny of Malagasy Reed Frogs, Ryan M. J. (1985), The Tungara Frog, Univ. of Chicago Press, Heterixalus, and the relative performance of bioacoustics Chicago. and color-patterns for resolving their systematics,” Mol. Salthe S. N. and Duellman W. E. (1973), “Quantitative con- Phylogen. Evol., 45(1), 14 – 22. strains associated with reproductive mode in anurans,” in: J. L. Vial (ed.), Evolutionary Biology of the Anurans, Univ. Yang Y. J. (1998), A Field Guide to the Frogs and Toads of of Missouri Press, Columbia, pp. 229 – 249. Taiwan, Nature and Ecology Photo Association, Taipei. Salthe S. N. and Mecham J. S. (1974), “Reproductive and Zug G. R. (1993), “Dynamics of reproduction,” in: G. R. Zug courtship patterns,” in: B. Lofts (ed.), Physiology of the Am- (ed.), Herpetology: An Introductory Biology of Amphibians phibia. Vol. II, Acad. Press, New York, pp. 309 – 521. and Reptiles, Acad. Press, California, pp. 195 – 207.