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Geographic variation in the advertisement calls of the giant spiny frog (Paa spinosa)
Acta Ecologica Sinica 35 (2015) 39–45
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Acta Ecologica Sinica j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c h n a e s
Geographic variation in the advertisement calls of the giant spiny frog (Paa spinosa) Bing Shen, Ping Chen, Rong Quan Zheng *, Jin Jin Min, Bao Juan Dong, Meng Meng Zhao Institute of Ecology, Zhejiang Normal University, Jinhua 321004, China
A R T I C L E
I N F O
Article history: Received 17 June 2014 Revised 29 December 2014 Accepted 29 January 2015 Available online Keywords: Paa spinosa Advertisement calls Geographic variation
A B S T R A C T
We compared the advertisement calls of the giant spiny frog Paa spinosa from six localities with Paa yunnanensis, which to examine the geographical variation in the call of P. spinosa and prove it exist cryptic species. The method of discriminate analysis was used to check the variation of the calls with seven acoustic parameters. Call analysis showed very significant geographic variation of the call among the frog P. spinosa, and the variations were close to those between P. spinosa and P. yunnanensis. Furthermore, the six localities P. spinosa were roughly divided into two areas, based on the discriminate analysis. Through the significant differences in call properties, we can distinguish between the localities and confirm the P. spinosa exist cryptic species. In addition, 1-note, 2-note, and 8-note calls were presented in this paper as they have not been described before. The analysis of acoustical signals can be used as a basis for the taxonomy of P. spinosa and other complex frog species. © 2015 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
1. Introduction Acoustic signaling is used by many species of anuran to broadcast information on species identity, mate fitness, sexual state, and location [1]. Based on their function and environmental contexts, these signals are categorized into four groups, namely, reciprocation calls, release calls, distress calls, and advertisement calls [2,3]. Of the four groups, advertisement calls in anurans play an important role in female mate choice, and are regarded as one of the key characters responsible for reproductive isolation and speciation events in this animal group [3–5]. Based on its species specificity, the advertisement calls are frequently used to delimit species in taxonomically complex groups of frogs [6–9], and studies on intraspecific variation of advertisement calls prove valuable in understanding the evolution of mating signal [10–13]. For example, acoustic features continue to be the most reliable cue in differentiating Eleutherodactylus glamyrus from the other four cryptic species of the E. auriculatus species group [14]. The advertisement calls are highly variable, and the differences in the calls across species should be correlated with some factors including (1) morphological and physiological characteristics, such as the body size and the weight of male [15–17]; (2) geographic habitat, such as elevation [18,19]; and (3) environmental conditions, such as air temperature [20,21]. In general, the advertisement call characters mainly include temporal characters and spectral
* Corresponding author. Institute of Ecology, Zhejiang Normal University, Jinhua 321004, China. Tel.: +86 579 82282269; fax: +86 579 82282269. E-mail address: [email protected] (R.Q. Zheng).
characters. Previous studies suggested that the spectral characters were determined by the morphological and physiological characters of the frogs, there was a negative correlation between the laryngeal apparatus or body size and the call spectral properties [22,23]. However, the temporal characters often varied largely with many environment conditions. In much anuran species, the temperature, daily period and density of population can really affect the temporal characters of the advertisement calls [24,25]. Geographic variation of phenotypes such as mating calls is a subject of considerable interest because it may lead to assortative mating, reproductive isolation, and reduced gene flow between populations [26,27]. Many studies on birds and frogs show that the acoustic properties of advertisement calls differ substantially among individuals or populations living in different geographic areas [24,28–30]. Geographic variation in the advertisement call of frogs and toads is commonly observed among conspecific populations with a wide distribution [31], and it functions in mating attraction and assessment [32,33]. The documentation of geographical variation in mating calls had not only provided insights into the mechanisms of diversification, but also led to the discovery of cryptic species [34–37]. P. spinosa a giant spiny-frog most distinctly characterized with keratinized skin spines on chest is currently distributed in 10 Chinese provinces and in northern Vietnam [38,39]. The taxonomy of frogs in this genus is subject to controversy, and according to nuclear and mitochondrial genes analyses P. spinosa is likely a species complex rather than single species [40,41]. According to these authors, three divergent lineages can be identified within P. spinosa probably representing three cryptic species. The current study aims to present more description of the calls of the P. spinosa, examine the geographical variation in the advertisement calls of the giant spiny frog,
http://dx.doi.org/10.1016/j.chnaes.2015.01.001 1872-2032/© 2015 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
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and prove the P. spinosa exist cryptic species through bioacoustics. Considering their greater separation by geographical distance and geographic variation in body size and sexual dimorphism [42], we expected to observe high divergence in call traits among the different geographic populations to clearly distinguish P. spinosa species groups. According to field surveys and habitat selection studies, P. yunnanensis and P. spinosa exists distribution overlap. To verify whether the variation among P. spinosa was approximate to the variation between P. yunnanensis and P. spinosa, we also compared the variation of calls between P. yunnanensis and P. spinosa. 2. Research methods 2.1. Research locations During April to July 2011 and May to June 2013, we collected 102 P. spinosa from six different locations, including Lanxi (LX) (n = 27), Jixi (JX) (n = 31), Taining (TN) (n = 12), Liuyang (LY) (n = 6), Cangnan (CN) (n = 7) and Yongxiu (YX) (n = 19), and collected 5 P. yunnanensis from Qujing (QJ) (Fig. 1), placed them in the Lanxi frog farm. All the frogs were released into separate artificial pond (3 m × 2 m × 0.8 m) which located to a slow flowing stream respectively, and kept in artificial imitation of the ecological environment which was characterized by rocks, branches, and roots. The frogs were fed with mealworms (larvae of Tenebrio molitor) and water enriched with vitamins and minerals.
night, we recorded the calls at 19:00–21:00 in the consecutive nights. For each male, vocalizations were recorded using Newsmy XD20 Recording pen and Sony PCM-D50 recorder with Sony ECM-77B Condenser Lavalier microphone held at an approximate distance of 1 m from the calling frog which was in separate ponds [43]. Air temperature was 24–24.5 °C during the recording of advertisement calls.
2.3. Statistical analyses A total of 1089 calls were recorded, including LX (N = 270), JX (N = 317), YX (N = 197), TN (N = 118), LY (N = 62), CN (N = 70) and QJ (N = 55). Calls were analyzed using Praat version 4.2.23 [44] at a sampling frequency resolution of 8 kHz and 16 bit resolution. Seven call properties were measured and terminology of calls was followed [24]: call duration(s), note number (n), pulse rate (pulses/s), dominant frequency (Hz), and the formant (F1, F2, and F3) (Hz). Statistical analyses were performed with the SPSS18.0. All data of call parameters were tested for normality using the Shapiro– Wilks test [45]. We employed discriminant analysis to compare the variation of the advertisement calls of the frogs with seven acoustic parameters. All values were presented as mean ± SD, and the significance level was set at 0.05.
3. Results
2.2. Recording
3.1. Advertisement call of P. spinosa and P. yunnanensis
From 24 to 26 July 2011 and 5 to 7 July 2013, we recorded the advertisement calls emitted by the frogs which were at separate artificial ponds. Because the calling mainly took place during the
Oscillograms and sound spectrogram of the advertisement calls of the three localities of P. spinosa are shown in Fig. 2. By analyzing the advertisement calls of P. spinosa, they emit call consisting
Fig. 1. Phylogeographical lineages (Ye et al. 2013) from which sampled localities are outlined on the map. The P. spinosa were collected in 6 allopatric localities: Lanxi (LX: 119°28′N: 29°12′E), Yongxiu (YX: 115°35′N: 28°59′E), Jixi (JX: 118°58′N: 30°04′E), Taining (TN: 117°12′N: 26°45′E), Liuyang (LY:113°37′N: 28°09′E), Cangnan (CN: 120°07′N: 27°06′E). P. yunnanensis were collected in Qujin (QJ: 103°47′N: 25°29′E).
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Fig. 2. Oscillogram and sound spectrogram of the advertisement call of P. spinosa from three localities (A: 5-note calls from JX; B: 5-note calls from YX; C: 5-note calls from LX).
of one to eight notes. However, the advertisement calls of P. yunnanensis were composed of single note (Fig. 3). 3.2. Geographic variation in the advertisement calls of P. spinosa and comparison of P. spinosa and P. yunnanensis The average values of the call characteristics produced by males of the six localities P. spinosa and P. yunnanensis were summarized in Table 1. Discriminant analysis showed very significant geographic variation of the advertisement call of frogs with seven acoustic parameters (Table 2a). A clear variation pattern comprising longer call duration and note number in the P. spinosa relative to those of the P. yunnanensis was observed. But the P. yunnanensis had a longer dominant frequency and F1. Among the six localities P. spinosa, the seven call parameters also showed significant variation. In general, frogs
from the LX, TN, CN, JX had longer call duration and note number relative to frogs from LY and YX. Dominant frequency from sites JX and YX was longer than the other sites, and F1 from sites TN and LY was smaller. A discriminant function plot of functions 1 and 2, as shown in Fig. 4, illustrates these variations. The advertisement calls of P. yunnanensis and P. spinosa were correctly predicted by the variables selected. Within the six localities P. spinosa the clustering was close, but the cluster formed by any two localities was different. Percentage classification success of the discriminant functions for discrimination of the variation of the frogs was summarized in Table 2b. Classification success of the discriminant functions for all seven localities, measured by correct assignment, gave more than 50% correct classification. Correct assignment was 100% for P. yunnanensis from QJ, but just 51.1% for P. spinosa from LX.
F1 (Hz)
2103.67 ± 196.17 2089.68 ± 156.10 2080.05 ± 185.03 1618.63 ± 250.34 2529.62 ± 404.87 2238.14 ± 349.92 2254.68 ± 83.38 1258.24 ± 97.99 1201.35 ± 87.00 1361.40 ± 109.77 1050.91 ± 99.99 1342.32 ± 223.46 1105.19 ± 99.41 1311.99 ± 63.01
F2 (Hz)
F3 (Hz)
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816.20 ± 121.21 841.03 ± 123.92 900.68 ± 146.91 684.13 ± 90.95 750.77 ± 164.24 828.90 ± 117.36 1041.11 ± 52.70
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Abbreviations: LX (Lanxi), YX (Yongxiu), JX (Jixi), TN (Taining), LY (Liuyang), CN (Cangnan), QJ (Qujin).
D F (Hz)
950.87 ± 307.54 1047.30 ± 186.50 1227.16 ± 182.11 884.75 ± 241.21 812.90 ± 412.60 945.71 ± 217.65 1147.82 ± 87.36 94.87 ± 61.76 138.33 ± 75.71 94.84 ± 49.09 231.57 ± 80.69 208.94 ± 130.54 314.27 ± 101.09 90.90 ± 71.58 4.62 ± 1.16 3.36 ± 0.66 4.25 ± 1.16 4.40 ± 0.85 3.95 ± 1.21 4.74 ± 1.00 1.000
Pulse rate (n/s) Note number (n)
1.50 ± 0.49 0.95 ± 0.20 1.27 ± 0.32 1.51 ± 0.29 1.23 ± 0.27 1.57 ± 0.31 0.31 ± 0.06 270 197 317 118 62 70 55
Call duration (s) Sample size (calls)
The current study shows that P. spinosa emit the calls with one to eight notes, whereas Yu et al. [43] pointed only three to seven note calls during the breeding period. Compared with their results, 1-note, 2-note, and 8-note calls are first discovered (Fig. 6). Based on our observation, frogs often release 1-note call when some people approach the frog pool or open the passive defend net. Once one of the frogs releases this call, the others stop calling. The frogs will not call until the environment is quiet. The function of 1-note calls was similar to that of distress call. But whether it belongs to distress call is needed more research to prove. In addition, we cannot determine the call function of the 2-note and 8-note calls which are only found in JX. To determine their function, more field observation and a series of behavioral experiment should be made in the future. Our results indicated that the call characteristics including call duration, note number, F1, and F2 are distinct different in six geographic populations of P. spinosa. Further, the rate of multi-note call which the three geographic population of P. spinosa released is quite different. It is more common to release the multi-note advertisement call in frogs [46,47]. The Booroolong frog (Litoria booroolongensis) from different locations release different numbers of notes [25]. The rate of multi-note call may be an indicator of geographic variation, and used to distinguish among P. spinosa species groups. But in this paper, because of the number of call we recorded from some sites was small, we only examined the rate of multi-note call only in three geographic sites of P. spinosa. To test the hypothesis, we need to record and analyze more advertisement calls from different geographic population. Cryptic species are too similar to be separated morphologically by traditional taxonomy, but show behavioral, physiological, or other differences resulting to reproductive isolation [48]. Given the weak of migration and strong philopatric, frogs usually have a strong genetic differentiation, and do not show morphological changes. Frogs have species recognition and mate choice systems that rely on nonmorphological characteristics (e.g. the advertisement call), which
P. spinosa LX P. spinosa YX P. spinosa JX P. spinosa TN P. spinosa LY P. spinosa CN P. yunnanensis QJ
4. Discussion
Species
Except for these call parameters, the rate of multi-note call in the localities LX, YX and JX was quite different (Fig. 5). Calls from LX and JX had 3–7 notes, but YX only had 3–5 note calls.
Table 1 Locality and number of calls, mean and standard deviation of seven call parameters of advertisement calls of the P. spinosa and P. yunnanensis.
Fig. 3. Oscillogram and sound spectrogram of the advertisement call of P. yunnanensis (A).
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Table 2 Differentiation of seven call parameters of advertisement calls of the P. spinosa and P. yunnanensis by discriminant analyses. (a) Significance of discriminant functions for the seven call parameters Call parameters
Wilk’s lambda
Call duration Note number Pulse rate DF F1 F2 F3
F
0.562 0.598 0.546 0.756 0.717 0.535 0.558
df1
140.38 121.06 149.96 58.262 71.261 156.82 142.75
6 6 6 6 6 6 6
df2
P
1082 1082 1082 1082 1082 1082 1082
<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
(b) Classification success of the P. spinosa and P. yunnanensis Species P. spinosa LX P. spinosa YX P. spinosa JX P. spinosa TN P. spinosa LY P. spinosa CN P. yunnanensis QJ
P. spinosa LX
P. spinosa YX
P. spinosa JX
P. spinosa TN
P. spinosa LY
P. spinosa CN
P. yunnanensis QJ
Classification success (%)
138 11 69 3 1 5 0
59 155 18 0 3 2 0
50 17 218 0 10 0 0
6 1 3 105 4 3 0
10 3 8 1 38 2 0
6 9 1 9 6 58 0
1 1 0 0 0 0 55
51.1 78.7 68.8 89.0 61.3 82.9 100.0
Fig. 5. The rate of different note calls in three geographic populations of P. spinosa (LX, JX and YX).
Fig. 4. Discriminant function plots depicting call parameter differentiation within frog group from the seven sites (LX, Lanxi; YX, Yongxiu; JX, Jixi; TN, Taining; LY, Liuyang; CN, Cangnan; QJ, Qujin). Different shapes inside graph indicate different localities.
can be storehouses of tremendous cryptic diversity [49]. The acoustic mating signals are used to discriminate closely related cryptic species in a variety of frogs [7,50]. The P. spinosa is a widespread native species of amphibians in the southern and southeast parts of China. In the current study, our results reveal significant geographic variation in male advertisement calls of the six localities P. spinosa and also between P. spinosa and P. yunnanensis according to the discriminant analysis. It is expected that the differences in call characteristics among the P. spinosa populations do not reach the level of difference between P. spinosa and P. yunnanensis. But in this study, we found that the variation among the six P. spinosa was close to those between P. spinosa and P. yunnanensis, such as the variation between CN and YX. It indicated that the variation among the P. spinosa was approximate to the species level. By contrast, our results supported the idea that the variation of advertisement call characteristics such as call duration, note number, F1, and F2 among the localities P. spinosa was found to be congruent with mitochondrial and nuclear data. The six populations which we recorded were
from two lineages, and the population of LX, CN and JX came from the same lineages [41]. There was much overlap among the P. spinosa and the seven acoustic parameters showed close clustering within the four localities (LX, JX, LY and YX) in Fig. 4. The cluster formed by the sites CN and TN was roughly equidistant from those of the four localities. Discriminant function plots showed that the six localities were roughly divided into two areas. Thus, we provided prediction that there may exist rather than single species in the lineage or the variation in the lineage was approximate to the species level. According to these bioacoustics analyses, we can hold that there was distinct geographic variation among the P. spinosa and proved that there exist cryptic species. And we can distinguish between the frogs from six localities through their significant difference in call characteristics. More researchers had used bioacoustics to assess the taxonomic status of the frog, distinguish among complex frog groups. In anurans as well as in many other species, including birds, fish and insects, male call traits can provide much information. But the advertisement call was highly variable, especially the temporal characters varied largely with the environment conditions. For instance, air temperature and density of population showed a conspicuous influence on the temporal characters of the calls. So in the present study, we feed the frogs in separate ponds and provided the frogs with similar living environment. And all the advertisement calls were recorded at 19:00–21:00 on July, and there was an approximate environment condition on the days we do the experiment. Despite there
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Fig. 6. Oscillogram and sound spectrogram of the advertisement call of P. spinosa of 1-note call (A), 2-note call (B) and 8-note call (C).
were still a modicum of limitations, our analysis was unlikely to be significantly affected by this condition, and our study reveals substantial differences in advertisement calls. Above all, our study reveals range-wide geographical variation in male advertisement calls of the giant spiny frog, and proves that the P. spinosa exist cryptic species. What is more, through acoustic and statistical analyses of advertisement calls, we can distinguish between cryptic species in P. spinosa and other complex frog species. In the future studies, more sampling from other regions and more accurate statistical procedures are needed to fully understand the taxonomic status of P. spinosa and enrich the bioacoustics data of Amphibian. Acknowledgements This research is supported by the National Natural Science Foundation of China (No. 31172116), the Major Science and Technology Specific Projects of Zhejiang Province, China (Nos. 2010C12008 and 2012C12907-9), Project of Science and Technology Commission of
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Contents lists available at ScienceDirect
Acta Ecologica Sinica j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / c h n a e s
Geographic variation in the advertisement calls of the giant spiny frog (Paa spinosa) Bing Shen, Ping Chen, Rong Quan Zheng *, Jin Jin Min, Bao Juan Dong, Meng Meng Zhao Institute of Ecology, Zhejiang Normal University, Jinhua 321004, China
A R T I C L E
I N F O
Article history: Received 17 June 2014 Revised 29 December 2014 Accepted 29 January 2015 Available online Keywords: Paa spinosa Advertisement calls Geographic variation
A B S T R A C T
We compared the advertisement calls of the giant spiny frog Paa spinosa from six localities with Paa yunnanensis, which to examine the geographical variation in the call of P. spinosa and prove it exist cryptic species. The method of discriminate analysis was used to check the variation of the calls with seven acoustic parameters. Call analysis showed very significant geographic variation of the call among the frog P. spinosa, and the variations were close to those between P. spinosa and P. yunnanensis. Furthermore, the six localities P. spinosa were roughly divided into two areas, based on the discriminate analysis. Through the significant differences in call properties, we can distinguish between the localities and confirm the P. spinosa exist cryptic species. In addition, 1-note, 2-note, and 8-note calls were presented in this paper as they have not been described before. The analysis of acoustical signals can be used as a basis for the taxonomy of P. spinosa and other complex frog species. © 2015 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
1. Introduction Acoustic signaling is used by many species of anuran to broadcast information on species identity, mate fitness, sexual state, and location [1]. Based on their function and environmental contexts, these signals are categorized into four groups, namely, reciprocation calls, release calls, distress calls, and advertisement calls [2,3]. Of the four groups, advertisement calls in anurans play an important role in female mate choice, and are regarded as one of the key characters responsible for reproductive isolation and speciation events in this animal group [3–5]. Based on its species specificity, the advertisement calls are frequently used to delimit species in taxonomically complex groups of frogs [6–9], and studies on intraspecific variation of advertisement calls prove valuable in understanding the evolution of mating signal [10–13]. For example, acoustic features continue to be the most reliable cue in differentiating Eleutherodactylus glamyrus from the other four cryptic species of the E. auriculatus species group [14]. The advertisement calls are highly variable, and the differences in the calls across species should be correlated with some factors including (1) morphological and physiological characteristics, such as the body size and the weight of male [15–17]; (2) geographic habitat, such as elevation [18,19]; and (3) environmental conditions, such as air temperature [20,21]. In general, the advertisement call characters mainly include temporal characters and spectral
* Corresponding author. Institute of Ecology, Zhejiang Normal University, Jinhua 321004, China. Tel.: +86 579 82282269; fax: +86 579 82282269. E-mail address: [email protected] (R.Q. Zheng).
characters. Previous studies suggested that the spectral characters were determined by the morphological and physiological characters of the frogs, there was a negative correlation between the laryngeal apparatus or body size and the call spectral properties [22,23]. However, the temporal characters often varied largely with many environment conditions. In much anuran species, the temperature, daily period and density of population can really affect the temporal characters of the advertisement calls [24,25]. Geographic variation of phenotypes such as mating calls is a subject of considerable interest because it may lead to assortative mating, reproductive isolation, and reduced gene flow between populations [26,27]. Many studies on birds and frogs show that the acoustic properties of advertisement calls differ substantially among individuals or populations living in different geographic areas [24,28–30]. Geographic variation in the advertisement call of frogs and toads is commonly observed among conspecific populations with a wide distribution [31], and it functions in mating attraction and assessment [32,33]. The documentation of geographical variation in mating calls had not only provided insights into the mechanisms of diversification, but also led to the discovery of cryptic species [34–37]. P. spinosa a giant spiny-frog most distinctly characterized with keratinized skin spines on chest is currently distributed in 10 Chinese provinces and in northern Vietnam [38,39]. The taxonomy of frogs in this genus is subject to controversy, and according to nuclear and mitochondrial genes analyses P. spinosa is likely a species complex rather than single species [40,41]. According to these authors, three divergent lineages can be identified within P. spinosa probably representing three cryptic species. The current study aims to present more description of the calls of the P. spinosa, examine the geographical variation in the advertisement calls of the giant spiny frog,
http://dx.doi.org/10.1016/j.chnaes.2015.01.001 1872-2032/© 2015 Ecological Society of China. Published by Elsevier B.V. All rights reserved.
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and prove the P. spinosa exist cryptic species through bioacoustics. Considering their greater separation by geographical distance and geographic variation in body size and sexual dimorphism [42], we expected to observe high divergence in call traits among the different geographic populations to clearly distinguish P. spinosa species groups. According to field surveys and habitat selection studies, P. yunnanensis and P. spinosa exists distribution overlap. To verify whether the variation among P. spinosa was approximate to the variation between P. yunnanensis and P. spinosa, we also compared the variation of calls between P. yunnanensis and P. spinosa. 2. Research methods 2.1. Research locations During April to July 2011 and May to June 2013, we collected 102 P. spinosa from six different locations, including Lanxi (LX) (n = 27), Jixi (JX) (n = 31), Taining (TN) (n = 12), Liuyang (LY) (n = 6), Cangnan (CN) (n = 7) and Yongxiu (YX) (n = 19), and collected 5 P. yunnanensis from Qujing (QJ) (Fig. 1), placed them in the Lanxi frog farm. All the frogs were released into separate artificial pond (3 m × 2 m × 0.8 m) which located to a slow flowing stream respectively, and kept in artificial imitation of the ecological environment which was characterized by rocks, branches, and roots. The frogs were fed with mealworms (larvae of Tenebrio molitor) and water enriched with vitamins and minerals.
night, we recorded the calls at 19:00–21:00 in the consecutive nights. For each male, vocalizations were recorded using Newsmy XD20 Recording pen and Sony PCM-D50 recorder with Sony ECM-77B Condenser Lavalier microphone held at an approximate distance of 1 m from the calling frog which was in separate ponds [43]. Air temperature was 24–24.5 °C during the recording of advertisement calls.
2.3. Statistical analyses A total of 1089 calls were recorded, including LX (N = 270), JX (N = 317), YX (N = 197), TN (N = 118), LY (N = 62), CN (N = 70) and QJ (N = 55). Calls were analyzed using Praat version 4.2.23 [44] at a sampling frequency resolution of 8 kHz and 16 bit resolution. Seven call properties were measured and terminology of calls was followed [24]: call duration(s), note number (n), pulse rate (pulses/s), dominant frequency (Hz), and the formant (F1, F2, and F3) (Hz). Statistical analyses were performed with the SPSS18.0. All data of call parameters were tested for normality using the Shapiro– Wilks test [45]. We employed discriminant analysis to compare the variation of the advertisement calls of the frogs with seven acoustic parameters. All values were presented as mean ± SD, and the significance level was set at 0.05.
3. Results
2.2. Recording
3.1. Advertisement call of P. spinosa and P. yunnanensis
From 24 to 26 July 2011 and 5 to 7 July 2013, we recorded the advertisement calls emitted by the frogs which were at separate artificial ponds. Because the calling mainly took place during the
Oscillograms and sound spectrogram of the advertisement calls of the three localities of P. spinosa are shown in Fig. 2. By analyzing the advertisement calls of P. spinosa, they emit call consisting
Fig. 1. Phylogeographical lineages (Ye et al. 2013) from which sampled localities are outlined on the map. The P. spinosa were collected in 6 allopatric localities: Lanxi (LX: 119°28′N: 29°12′E), Yongxiu (YX: 115°35′N: 28°59′E), Jixi (JX: 118°58′N: 30°04′E), Taining (TN: 117°12′N: 26°45′E), Liuyang (LY:113°37′N: 28°09′E), Cangnan (CN: 120°07′N: 27°06′E). P. yunnanensis were collected in Qujin (QJ: 103°47′N: 25°29′E).
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Fig. 2. Oscillogram and sound spectrogram of the advertisement call of P. spinosa from three localities (A: 5-note calls from JX; B: 5-note calls from YX; C: 5-note calls from LX).
of one to eight notes. However, the advertisement calls of P. yunnanensis were composed of single note (Fig. 3). 3.2. Geographic variation in the advertisement calls of P. spinosa and comparison of P. spinosa and P. yunnanensis The average values of the call characteristics produced by males of the six localities P. spinosa and P. yunnanensis were summarized in Table 1. Discriminant analysis showed very significant geographic variation of the advertisement call of frogs with seven acoustic parameters (Table 2a). A clear variation pattern comprising longer call duration and note number in the P. spinosa relative to those of the P. yunnanensis was observed. But the P. yunnanensis had a longer dominant frequency and F1. Among the six localities P. spinosa, the seven call parameters also showed significant variation. In general, frogs
from the LX, TN, CN, JX had longer call duration and note number relative to frogs from LY and YX. Dominant frequency from sites JX and YX was longer than the other sites, and F1 from sites TN and LY was smaller. A discriminant function plot of functions 1 and 2, as shown in Fig. 4, illustrates these variations. The advertisement calls of P. yunnanensis and P. spinosa were correctly predicted by the variables selected. Within the six localities P. spinosa the clustering was close, but the cluster formed by any two localities was different. Percentage classification success of the discriminant functions for discrimination of the variation of the frogs was summarized in Table 2b. Classification success of the discriminant functions for all seven localities, measured by correct assignment, gave more than 50% correct classification. Correct assignment was 100% for P. yunnanensis from QJ, but just 51.1% for P. spinosa from LX.
F1 (Hz)
2103.67 ± 196.17 2089.68 ± 156.10 2080.05 ± 185.03 1618.63 ± 250.34 2529.62 ± 404.87 2238.14 ± 349.92 2254.68 ± 83.38 1258.24 ± 97.99 1201.35 ± 87.00 1361.40 ± 109.77 1050.91 ± 99.99 1342.32 ± 223.46 1105.19 ± 99.41 1311.99 ± 63.01
F2 (Hz)
F3 (Hz)
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816.20 ± 121.21 841.03 ± 123.92 900.68 ± 146.91 684.13 ± 90.95 750.77 ± 164.24 828.90 ± 117.36 1041.11 ± 52.70
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Abbreviations: LX (Lanxi), YX (Yongxiu), JX (Jixi), TN (Taining), LY (Liuyang), CN (Cangnan), QJ (Qujin).
D F (Hz)
950.87 ± 307.54 1047.30 ± 186.50 1227.16 ± 182.11 884.75 ± 241.21 812.90 ± 412.60 945.71 ± 217.65 1147.82 ± 87.36 94.87 ± 61.76 138.33 ± 75.71 94.84 ± 49.09 231.57 ± 80.69 208.94 ± 130.54 314.27 ± 101.09 90.90 ± 71.58 4.62 ± 1.16 3.36 ± 0.66 4.25 ± 1.16 4.40 ± 0.85 3.95 ± 1.21 4.74 ± 1.00 1.000
Pulse rate (n/s) Note number (n)
1.50 ± 0.49 0.95 ± 0.20 1.27 ± 0.32 1.51 ± 0.29 1.23 ± 0.27 1.57 ± 0.31 0.31 ± 0.06 270 197 317 118 62 70 55
Call duration (s) Sample size (calls)
The current study shows that P. spinosa emit the calls with one to eight notes, whereas Yu et al. [43] pointed only three to seven note calls during the breeding period. Compared with their results, 1-note, 2-note, and 8-note calls are first discovered (Fig. 6). Based on our observation, frogs often release 1-note call when some people approach the frog pool or open the passive defend net. Once one of the frogs releases this call, the others stop calling. The frogs will not call until the environment is quiet. The function of 1-note calls was similar to that of distress call. But whether it belongs to distress call is needed more research to prove. In addition, we cannot determine the call function of the 2-note and 8-note calls which are only found in JX. To determine their function, more field observation and a series of behavioral experiment should be made in the future. Our results indicated that the call characteristics including call duration, note number, F1, and F2 are distinct different in six geographic populations of P. spinosa. Further, the rate of multi-note call which the three geographic population of P. spinosa released is quite different. It is more common to release the multi-note advertisement call in frogs [46,47]. The Booroolong frog (Litoria booroolongensis) from different locations release different numbers of notes [25]. The rate of multi-note call may be an indicator of geographic variation, and used to distinguish among P. spinosa species groups. But in this paper, because of the number of call we recorded from some sites was small, we only examined the rate of multi-note call only in three geographic sites of P. spinosa. To test the hypothesis, we need to record and analyze more advertisement calls from different geographic population. Cryptic species are too similar to be separated morphologically by traditional taxonomy, but show behavioral, physiological, or other differences resulting to reproductive isolation [48]. Given the weak of migration and strong philopatric, frogs usually have a strong genetic differentiation, and do not show morphological changes. Frogs have species recognition and mate choice systems that rely on nonmorphological characteristics (e.g. the advertisement call), which
P. spinosa LX P. spinosa YX P. spinosa JX P. spinosa TN P. spinosa LY P. spinosa CN P. yunnanensis QJ
4. Discussion
Species
Except for these call parameters, the rate of multi-note call in the localities LX, YX and JX was quite different (Fig. 5). Calls from LX and JX had 3–7 notes, but YX only had 3–5 note calls.
Table 1 Locality and number of calls, mean and standard deviation of seven call parameters of advertisement calls of the P. spinosa and P. yunnanensis.
Fig. 3. Oscillogram and sound spectrogram of the advertisement call of P. yunnanensis (A).
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Table 2 Differentiation of seven call parameters of advertisement calls of the P. spinosa and P. yunnanensis by discriminant analyses. (a) Significance of discriminant functions for the seven call parameters Call parameters
Wilk’s lambda
Call duration Note number Pulse rate DF F1 F2 F3
F
0.562 0.598 0.546 0.756 0.717 0.535 0.558
df1
140.38 121.06 149.96 58.262 71.261 156.82 142.75
6 6 6 6 6 6 6
df2
P
1082 1082 1082 1082 1082 1082 1082
<0.01 <0.01 <0.01 <0.01 <0.01 <0.01 <0.01
(b) Classification success of the P. spinosa and P. yunnanensis Species P. spinosa LX P. spinosa YX P. spinosa JX P. spinosa TN P. spinosa LY P. spinosa CN P. yunnanensis QJ
P. spinosa LX
P. spinosa YX
P. spinosa JX
P. spinosa TN
P. spinosa LY
P. spinosa CN
P. yunnanensis QJ
Classification success (%)
138 11 69 3 1 5 0
59 155 18 0 3 2 0
50 17 218 0 10 0 0
6 1 3 105 4 3 0
10 3 8 1 38 2 0
6 9 1 9 6 58 0
1 1 0 0 0 0 55
51.1 78.7 68.8 89.0 61.3 82.9 100.0
Fig. 5. The rate of different note calls in three geographic populations of P. spinosa (LX, JX and YX).
Fig. 4. Discriminant function plots depicting call parameter differentiation within frog group from the seven sites (LX, Lanxi; YX, Yongxiu; JX, Jixi; TN, Taining; LY, Liuyang; CN, Cangnan; QJ, Qujin). Different shapes inside graph indicate different localities.
can be storehouses of tremendous cryptic diversity [49]. The acoustic mating signals are used to discriminate closely related cryptic species in a variety of frogs [7,50]. The P. spinosa is a widespread native species of amphibians in the southern and southeast parts of China. In the current study, our results reveal significant geographic variation in male advertisement calls of the six localities P. spinosa and also between P. spinosa and P. yunnanensis according to the discriminant analysis. It is expected that the differences in call characteristics among the P. spinosa populations do not reach the level of difference between P. spinosa and P. yunnanensis. But in this study, we found that the variation among the six P. spinosa was close to those between P. spinosa and P. yunnanensis, such as the variation between CN and YX. It indicated that the variation among the P. spinosa was approximate to the species level. By contrast, our results supported the idea that the variation of advertisement call characteristics such as call duration, note number, F1, and F2 among the localities P. spinosa was found to be congruent with mitochondrial and nuclear data. The six populations which we recorded were
from two lineages, and the population of LX, CN and JX came from the same lineages [41]. There was much overlap among the P. spinosa and the seven acoustic parameters showed close clustering within the four localities (LX, JX, LY and YX) in Fig. 4. The cluster formed by the sites CN and TN was roughly equidistant from those of the four localities. Discriminant function plots showed that the six localities were roughly divided into two areas. Thus, we provided prediction that there may exist rather than single species in the lineage or the variation in the lineage was approximate to the species level. According to these bioacoustics analyses, we can hold that there was distinct geographic variation among the P. spinosa and proved that there exist cryptic species. And we can distinguish between the frogs from six localities through their significant difference in call characteristics. More researchers had used bioacoustics to assess the taxonomic status of the frog, distinguish among complex frog groups. In anurans as well as in many other species, including birds, fish and insects, male call traits can provide much information. But the advertisement call was highly variable, especially the temporal characters varied largely with the environment conditions. For instance, air temperature and density of population showed a conspicuous influence on the temporal characters of the calls. So in the present study, we feed the frogs in separate ponds and provided the frogs with similar living environment. And all the advertisement calls were recorded at 19:00–21:00 on July, and there was an approximate environment condition on the days we do the experiment. Despite there
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Fig. 6. Oscillogram and sound spectrogram of the advertisement call of P. spinosa of 1-note call (A), 2-note call (B) and 8-note call (C).
were still a modicum of limitations, our analysis was unlikely to be significantly affected by this condition, and our study reveals substantial differences in advertisement calls. Above all, our study reveals range-wide geographical variation in male advertisement calls of the giant spiny frog, and proves that the P. spinosa exist cryptic species. What is more, through acoustic and statistical analyses of advertisement calls, we can distinguish between cryptic species in P. spinosa and other complex frog species. In the future studies, more sampling from other regions and more accurate statistical procedures are needed to fully understand the taxonomic status of P. spinosa and enrich the bioacoustics data of Amphibian. Acknowledgements This research is supported by the National Natural Science Foundation of China (No. 31172116), the Major Science and Technology Specific Projects of Zhejiang Province, China (Nos. 2010C12008 and 2012C12907-9), Project of Science and Technology Commission of
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