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. 2020 Oct 29;15(10):e0241316. doi: 10.1371/journal.pone.0241316

Use of bioacoustics in species identification: Piranhas from genus Pygocentrus (Teleostei: Serrasalmidae) as a case study

Xavier Raick 1,*, Alessia Huby 1, Gregório Kurchevski 2, Alexandre Lima Godinho 2, Éric Parmentier 1
Editor: Gianni Pavan3
PMCID: PMC7595327  PMID: 33119694

Abstract

The genus Pygocentrus contains three valid piranha species (P. cariba, P. nattereri and P. piraya) that are allopatric in tropical and subtropical freshwater environments of South America. This study uses acoustic features to differentiate the three species. Sounds were recorded in P. cariba, two populations of P. nattereri (red- and yellow-bellied) and P. piraya; providing sound description for the first time in P. cariba and P. piraya. Calls of P. cariba were distinct from all the other studied populations. Red- and yellow-bellied P. nattereri calls were different from each other but yellow-bellied P. nattereri calls were similar to those of P. piraya. These observations can be explained by considering that the studied specimens of yellow-bellied P. nattereri have been wrongly identified and are actually a sub-population of P. piraya. Morphological examinations and recent fish field recordings in the Araguari River strongly support our hypothesis. This study shows for the first time that sounds can be used to discover identification errors in the teleost taxa.

Introduction

Sounds for communication purposes are emitted by a high number of different zoological groups. Many species have evolved calls that are assumed to be species-specific [1–3] but that can sometimes show intraspecific variability since age, sex, and size can affect different call parameters such as frequency and amplitude [4–11]. The specific features of calls easily explain why sounds were successfully used to differentiate species of orthopterans [12], teleosts [13], birds [14,15], bats [16,17], and cetaceans [18–20]. Bioacoustics is also used as part of the diagnosis in bird species [14] where call features complement genetic and morphological data [21–27] simply because the use of acoustic signals is far easier to distinguish the species in the field. To the best of our knowledge, the consolidation of a fish species diagnosis with acoustic data has only been done in Encheliophis chardewalli (Carapidae) in which the comparison with other sympatric species of the same tribe supports a distinct repertoire [28].

“Cryptic” or “twin-species” are species that cannot be separated using characters of external morphology [29,30]. “Cryptic” species are mainly found in taxa that communicate reproductive signals via nonvisual cues [30]. Acoustic mating signals were thus effective in discriminating cryptic species in different invertebrates and vertebrates [30]. This kind of investigation is only suggested in teleosts [31]. For example, the cusk-eels Ophidion barbatum and O. rochei possess almost the same external phenotype [32] but show remarkable differences between their sound-producing apparatuses, supporting the production of different kinds of calls [32–34]. In electric fish, cryptic species were discriminated using features of their electric organ discharges [35].

Serrasalmidae includes pacus, silver dollars and piranhas [36,37]. Among piranha species, the Pygocentrus genus has three valid species: P. cariba, P. nattereri, and P. piraya. In addition, P. palometa, found in the Orinoco River basin, was described by Valenciennes in 1850 but is currently considered nomen dubium [38]. The three valid species are allopatric (Fig 1A). Pygocentrus cariba occurs in the Orinoco River basin (Northern South America) while P. piraya is restricted to the São Francisco River basin (Eastern South America). Pygocentrus nattereri has a wider distribution since it can be found in nine countries in South America between the areas inhabited by both previous species. It can be found in the watershed of the Essequibo, Amazon, and Paraná-Paraguay rivers, as well as in the north-eastern Brazilian coastal river basins [38]. Moreover, it has been introduced in many places, for example, the lakes of the Doce River [39–41]. Contrary to the two other species, Pygocentrus cariba has a black humeral spot. Pygocentrus piraya and southern populations of P. nattereri possess a yellow belly whereas P. nattereri from other locations all possess a red belly [42,43].

Fig 1.

Fig 1

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A. Distribution of the different species of genus Pygocentrus modified from [48] and adapted with data from non-native populations [39–41,49]. The range of P. cariba is represented in black, that of P. nattereri is approximately divided into red and yellow according to the colouration of the belly and the river basins, and that of P. piraya is in blue. B. Locations of sampling sites (black stars) in Minas Gerais state, Brazil.

In piranhas, Pygocentrus and Serrasalmus possess a well-developed sound-producing apparatus that is used in acoustic communication [10,44] and therefore plays a role in their ecology [45]. These sounds are warning calls [10] and are identical to those recorded during frontal displays [46]. Behavioural studies have shown that these calls are produced 80% of the time during frontal display between two fish and 20% of the time, these sounds could not be associated with a given behaviour [46]. They are species-specific, harmonic and generally made of continuous peaks with a low fundamental frequency below 150 Hz [10,46,47]. This study aimed to use acoustic features to differentiate the three current valid Pygocentrus species, providing sound description for the first time in P. cariba and P. piraya. In addition, this study also investigates the difference between yellow- and red-bellied populations of P. nattereri.

Materials and methods

Biological materials

Sounds were recorded from specimens maintained in aquaria and from specimens captured in the wild (Table 1). Three specimens of Pygocentrus cariba (standard length, SL: 90 ± 2 mm; mean ± standard deviation) and five specimens of P. piraya (SL: 98 ± 8 mm) were purchased in an exotic import aquarium store (Table 1). They were housed at the University of Liège (Belgium) in tanks (volume ≥ 100 L) with external filters, internal heaters (26 ± 1°C), bubblers and light (12 h light/dark cycle). They were fed three times a week with Mytilus edulis. Four additional specimens of P. piraya (SL: 409 ± 19 mm) from the aquarium Aquário da Bacia do Rio São Francisco of Belo Horizonte were used for sound recordings (Table 1). We did not use specimens of P. nattereri from aquarium stores because for this species there is a lot of mixing of specimens from different river basins and a lot of artificial selection.

Table 1. Specimens recorded in this study.

Species Variety Localisation N (fish) N (sounds)
Pygocentrus cariba University of Liège (origin: São Francisco River Basin) 3 26
Pygocentrus piraya University of Liège (origin: Orinoco River Basin) 5 50
Aquário da Bacia do Rio São Francisco (Belo Horizonte) 4 0
Paraopeba River 3 30
Pygocentrus nattereri Red-bellied Parque Estadual do Rio Doce 150 1500
Yellow-bellied Araguari River 11 107
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Additional specimens and species were fished in Brazil in July 2018 (Table 1). Three specimens of P. piraya (SL: 185 ± 86 mm) were captured with gillnets and by hook-and-line in the Paraopeba River (18°52′35″ S, 44°46′49″ W; São Francisco River Basin). Two populations of P. nattereri were studied: red-bellied and yellow-bellied. Red-bellied P. nattereri were from Dom Helvécio Lake (Parque Estadual do Rio Doce; 19°46′29″ S, 42°35′57″ W; Doce River Basin) where 150 specimens (SL: 178 ± 24 mm) were captured with gillnets. Specimens of yellow-bellied P. nattereri were from Araguari River (18°39′37″ S, 48°26′20″ W; Upper Paraná River Basin) where eleven specimens (SL: 328 ± 18 mm) were captured with gillnets and by hook-and-line. There was no effect of the fishing method on the sounds recorded. There is no geographical natural connection between red-bellied and yellow-bellied P. nattereri. Pictures of a specimen from each geographical group can be found in S1 Fig. Some specimens are available at the Aquarium-Museum of the University of Liège (vouchers R.E. 15271, 15272, 15273, 15274, 15275 and 15276).

Recordings & sound-analysis

The sounds were recorded in a glass-tank (108 L), with a hydrophone (HTI-96-Min, High Tech Inc., USA; sensitivity: –164.4 dB re 1V μPa-1) placed in the middle of the aquarium and connected to a TASCAM DR5 recorder (TEAC Corporation, USA). The fish were gently hand-held between the thumb and the index of the left hand without any pressure at approximatively five centimetres from the hydrophone in order to homogenize as much as possible the sounds recorded. During recordings in the University of Liège, the water temperature was 26 ± 1°C and the filters, heater, bubblers and the light of the aquarium were turned off to reduce non-biological noises. For the specimens recorded in Brazil, the water temperature in the aquarium was 26 ± 2°C. The resonance frequency was 3252 Hz (for details on recordings in tanks, see [50]).

In total, 1713 sounds were recorded and analysed. For each vocal specimen, ten sounds were analysed, except for P. cariba and yellow-bellied P. nattereri from the Araguari River for which 26 and 107 sounds have been analysed, respectively. The sounds were digitised on mono-channel at 44.1 kHz with a 16 bit-resolution and then they were sub-sampled at 4000 Hz and a high-pass filter at 50 Hz was applied. The analysis was manually carried out with Avisoft SAS-Lab Pro 5.2 (Avisoft Bioacoustics, Germany).

The temporal features were measured on oscillograms (Fig 2A) while the fundamental frequency (F0, in Hz) was obtained from the power-spectra of the sounds (Fig 2B). The temporal features were the sound duration (d, in ms); the number of peaks in a sound (N), and the period between consecutive peaks (p, in ms). The period of the first peaks has been shown to be longer than that of the rest of the sound in some piranha species [10]. In consequence, the same three features were measured in the most energetic zone (abbreviated “ez”), i.e., the continuous area with an intensity of maximum 3 dB less than the maximum intensity peak (as the dB scale is a logarithmic scale, it corresponds to a decrease of a factor 1.4, see Fig 2A) and named dez, Nez, and pez.

Fig 2.

Fig 2

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A. Oscillogram and B. power spectrum of a sound with the features measured. d = duration, dez = duration of the energetic zone, p = period, pez = period in the energetic zone, F0 = fundamental frequency.

Statistics

The description of the sounds was carried on the totality of the sounds. For each species, the different features were compared using Wilcoxon signed-rank tests and correlations between features were calculated using Bland-Altman repeated measures correlations.

The comparison between the different species required knowledge of the effect of the fish size because this could affect acoustic features [10]. For this reason, the effect of size was removed in order to conduct suitable comparisons. All the statistics relative to the effect of body size and the species comparison were realised on means per individuals, i.e., the ten sounds were averaged to obtain a single value per specimen. Correlation matrices were calculated using Spearman correlation coefficients (rS) and their associated P-value matrices corrected by the Holm-Bonferroni method. When a correlation between an acoustic feature and the SL was found, a linear regression was performed. Logarithmic and polynomial regressions were also tested.

Wilcoxon-Mann-Whitney tests (also named Mann-Whitney U tests) and Kruskal-Wallis tests, with the Dunn’s test as post-hoc with a Benjamini-Hochberg correction on the P, were used to compare acoustic features between species. The values of the statistics W and Z are presented with P and adjusted P when necessary. To reduce the multivariate aspect of the data, principal component analyses (PCAs) were conducted on the correlation matrices with all acoustic features except those whose R2 ≥ |0.60|, for which the features divided by the SL were used instead in order to remove the effect of size. In parallel, we carried out the same analyses on the residuals of the linear regressions. As the statistical significances and the biological conclusions with the two methods were identical, we present only the first method in this manuscript. We did not perform phylogenetic corrections because the number of species was small and it would have not been possible to take into account the difference between red- and yellow-belly P. nattereri. The principal components (PCs) are presented with their associated percentage of variance and their cumulative percentage of variance. PCs for each group were compared using Wilcoxon-Mann-Whitney and Kruskal-Wallis tests with the Dunn’s test as post-hoc. All the statistics were carried-out using R 3.3.0. (GNU General Public License) and the significance level was α = 0.05.

Authorisations

The experiment was approved by the ethical commission of the University of Liège (case 1532) and the capture of the Brazilian specimens was achieved under the license 10306–1 from the Brazilian Ministry of the Environment. In the Paraopeba River, the fish were captured near the Retiro Baixo Hydropower Dam with an authorisation n° PT-04/07/2018 issued by Retiro Baixo Energética. In the Parque Estadual do Rio Doce, the fish were sampled under the license 049/2018 from the Instituto Estadual de Florestas of the State of Minas Gerais. In the Araguari River, the fish were captured near the Capim Branco II Hydropower Dam with an authorisation from Consórcio Capim Branco. Six specimens have been euthanasied with an overdose of eugenol (CAS: 97-53-0) and imported to Belgium with the authorisation of the Federal Agency for the Safety of the Food Chain (COBT/IEC/CMY/1546595), the Federal University of Minas Gerais (MTA/TTM n° 02/2019) and the Conselho de Gestão do Patrimônio Genético of the Ministério do Meio Ambiente (n°R2B8FF7 et n°A57A7E9).

Results

Description of the sounds

Sounds of P. cariba, two populations of P. nattereri (red- and yellow-bellied) and P. piraya were recorded in this study. Sonic muscles were similar in the three species. All specimens produced sounds except four specimens of P. piraya coming from the Aquário da Bacia do Rio São Francisco of Belo Horizonte, for which body size was between 390 and 430 mm.

Description of Pygocentrus cariba sounds

Pygocentrus cariba SLs were between 87 and 92 mm. Their sounds (nsounds = 26) were low-frequency harmonic drumming sounds and consisted of 16 ± 7 (mean ± standard deviation) continuous peaks with a peak period of 11.5 ± 0.5 ms (Fig 3A). Sounds lasted from 54 to 388 ms (183 ± 74 ms) with a fundamental frequency of 89 ± 6 Hz. The most energetic zone contained between 2 and 18 peaks (7 ± 5 peaks) that corresponded to 15 to 80% (45 ± 16%) of the total number of peaks. The peak period in the most energetic zone was equivalent to the mean peak period of the sound (Wilcoxon signed rank test: V = 208, P = .42). The sound duration was positively correlated with the number of peaks (r = 0.99, P < .0001) but not with the period (r = –0.22, P = .30). In other words, the number of peaks determines the sound duration. NEZ and d were correlated with dEZ (r = 0.94 and 0.78, both P < .0001).

Fig 3.

Fig 3

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Waveform (top) and spectrogram (bottom) of a sound produced by A. Pygocentrus cariba, B. Pygocentrus piraya. C. red-bellied Pygocentrus nattereri and D. yellow-bellied Pygocentrus nattereri. FFT Length: 512, window: Hamming, frame: 100%.

Description of Pygocentrus piraya sounds

Pygocentrus piraya SLs were between 90 and 281 mm. Their sounds (nsounds = 80) were made of 9 ± 3 continuous peaks with a peak period of 15 ± 5 ms (Fig 3B). Sounds lasted from 71 to 266 ms (131 ± 50 ms) with a fundamental frequency of 79 ± 13 Hz. The most energetic zone of P. piraya sounds was much smaller than in P. cariba sounds (34 ± 30 ms vs. 94 ± 63 ms) with 4 ± 2 peaks and covered 38 ± 17% of the total number of peaks. The period in the energetic zone was 14 ± 5 ms. The number of peaks was independent of the duration of the energetic zone (r = 0.16, P = .18) and weakly correlated to the number of peaks in the energetic zone (r = 0.24, P = .04).

Description of Pygocentrus nattereri sounds

The specimens of yellow-bellied P. nattereri were much bigger than the specimens of red-bellied P. nattereri (Wilcoxon-Mann-Whitney: W = 0, P < .0001). Red-bellied P. nattereri sounds (Nsounds = 1500) were made of 11 ± 3 continuous peaks with a peak period of 14 ± 2 ms. Sounds lasted from 24 to 417 ms (147 ± 43 ms) with a fundamental frequency of 79 ± 12 Hz. The most energetic zone contained between 1 and 23 peaks (4 ± 2 peaks) that corresponded to 42 ± 16 of the total number of peaks (Fig 3C). Yellow-bellied P. nattereri sounds (Nsounds = 107) were made of 6 ± 3 continuous peaks with a peak period of 31 ± 8 ms. Sounds lasted from 40 to 466 ms (194 ± 90 ms) with a fundamental frequency of 70 ± 9 Hz. The most energetic zone contained between 1 and 7 peaks (2 ± 1 peaks) that corresponded to 36 ± 22% of the total number of peaks (Fig 3D).

Effect of the fish size

For all species, all the frequency features and the peak periods were correlated to the SL (all P < .001). The p and pez were highly positively correlated to the SL (rS = 0.64 & 0.73) while the fundamental frequency was highly negatively correlated to the SL (rS = –0.62). When p and pez were predicted, it was found that the SL was a significant predictor (both P < .001, both R2 = 0.68). To remove the effect of the size, the features divided by the SL (written “X SL-1” for the feature “X”) were used instead of the features themselves. In fish, some sound features, such as pulse duration or fundamental frequency, are known to be affected by their body size [8,9,51–54]. We combined our results with data also recorded in glass tanks from [55] and [10] to study the frequency as a function of size with 182 different specimens of red-bellied P. nattereri ranging from 44 to 270 mm (Fig 4). Besides linear regression, other models such as logarithmic models (Table 2) could explain the relation between the size and the fundamental frequency, with larger specimens having a smaller fundamental frequency.

Fig 4. Fundamental frequency as a function of the standard length for red-bellied Pygocentrus nattereri.

Fig 4

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The three boxes show the data from [55]. Each box corresponds to the mean ± SD of a given size class. The green “■” are from [10] and the blue “♦” from this study. Linear regressions are presented for separated datasets and a polynomial model of all the data.

Table 2. Adjustment models with their respective R2 for the fundamental frequency as a function of the standard length in red-bellied Pygocentrus nattereri.

Type of model Equation R2
Linear y = –0.50 x + 168.96 0.84
Logarithmic y = –59.79 ln (x) + 388.77 0.88
Polynomial y = 0.0018 x2–0.99 x + 198 0.88
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Species discrimination

The dez, N, and Nez of the sounds of Pygocentrus cariba were different from those of all the other species (Kruskal-Wallis: χ2 = 10, 29 & 30; df = 3; P = .02, < .001 & < .001; Dunn: Z = 2.64, 3.06 & 2.80; P = .02, .01 & .02 for dEZ, Z = 2.43, 3.00 & 4.30; P = .02, .005 & < .001 for N, Z = 2.22, 2.71 & 4.25; P = .04, .01 & < .001 for NEZ). The two features p SL-1 and pez SL-1 were the only ones that separated the red-bellied P. nattereri from all the others (Kruskal-Wallis: both χ2 = 38, df = 3, both P < .001; Dunn: Z = 3.08, –4.47 & –3.36; P = .004, < .001 & .002 for p SL-1 and Z = 3.12, –4.66 & –3.18; P = .004, < .001 & .004 for pez SL-1)and were equivalent for all the other comparisons (Dunn: Z = 0.26, 1.15 & 1.23; P = .80, .30 & .33 for p SL-1 and Z = .18, 1.27 & 1.51; P = .85, .25 & .20). The PCA performed on all the features (or on the features divided by the SL when applicable) showed that PC1 (45%) values were statistically different between all the groups (Kruskal-Wallis: χ2 = 19, df = 3, P < .001; Dunn: Z = –2.30, –3.12, –2.12, –3.49 & –2.98; P = .03, .005, .04, .003 & .006)) except between P. piraya and yellow-bellied P. nattereri (Dunn: Z = –0.35, P = .73). PC1 was highly negatively correlated with d, dez, N, Nez (rS = –0.66, –0.75, –0.90 & –0.86; all P < .001). PC2 (29%, 74% cumulative) separated red-bellied P. nattereri from all the others (Table 3 and Fig 5). The comparison between the studied specimens of red-bellied and yellow-bellied P. nattereri showed that their sounds differ on all the features (Wilcoxon-Mann-Whitney: W = 401, 1328, 1246, 1486, 1534, 0, 6; P = .004, < .001, .005, < .001, < .001, < .001 & < .001; Fig 6) except dez (Wilcoxon-Mann-Whitney: W = 962, P = .36). Similar results were obtained after dividing by the SL. No difference was found between the acoustic features of yellow-bellied P. nattereri and P. piraya.

Table 3. Scores of the Dunn post-hoc test on the principal components.

Significant P-values in red.

Species/variety comparison Z Non adjusted P Adjusted P
PC1 PC2 PC1 PC2 PC1 PC2
P. cariba vs. red-bellied P. nattereri –2.30 3.13 .02 .0017 .03 .003
P. cariba vs. P. piraya –3.12 0.34 .002 .73 .005 .73
Red-bellied P. nattereri vs. P. piraya –2.12 –4.40 .034 < .001 .04 < .001
P. cariba vs. yellow-bellied P. nattereri –3.49 1.10 < .001 .27 .003 .40
Red- vs. yellow-bellied P. nattereri –2.98 –3.55 < .001 < .001 .006 .001
P. piraya vs yellow-bellied P. nattereri –0.35 1.05 .73 .29 .73 .35
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Fig 5. Comparison of the scores from principal component analysis (PCA) in the genus Pygocentrus.

Fig 5

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Boxes represent the median ± interquartile range (IQR) and lines represent Q1–1.5 IQR and Q3 + 1.5 IQR. * = P < .05; ** = P < .01; *** = P < .001; Kruskal-Wallis + Dunn.

Fig 6. Comparison of acoustic features of red and yellow-bellied Pygocentrus nattereri.

Fig 6

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Boxes represent the median ± interquartile range (IQR) and lines represent Q1–1.5 IQR and Q3 + 1.5 IQR. ** = P < .01; *** P < .001; Wilcoxon-Mann-Whitney.

Discussion

This study described for the first time sounds of hand-held P. cariba and P. piraya. These results are important for future passive acoustics monitoring (PAM) of those species. However, caution need to be taken when compared fish sounds recorded in tanks (see [50] for details). Another important consideration is the fish size effect. Non-linear regression models has also been suggested in Balistidae [8,56] but with fewer specimens. In red-bellied P. nattereri, old adult specimens would be mute [55] because sonic muscles have been invaded by fat cells and there are several modifications in the ultrastructure of the myofibrils and sarcoplasmic reticulum [55]. In the present study, the four biggest specimens of P. piraya did not produce any sounds, supporting the hypothesis that older piranha specimens lose their sound-producing ability. Deep analysis of sonic muscles from these specimens did not take place because these fish were from a public aquarium and were returned after this experiment.

The challenge of this study was to discriminate three piranha species using their sound features. The easiest to discriminate was P. cariba which differs from all the others in dez, N, and Nez. The greater differences in acoustic features between P. cariba and congeners could be related to their higher genetic distance [57,58]. Pygocentrus cariba and the potential ancestor of both the other sister species (P. nattereri and P. piraya) would have separated around 8 Ma, whereas Pygocentrus nattereri separated from P. piraya only more recently [48,57], at 2.63 ± 0.2 Ma [57–59]. Interestingly, the studied specimens of yellow-bellied P. nattereri and P. piraya cannot be distinguished using acoustic features, whereas the studied specimens of yellow-bellied and red-bellied P. nattereri can. This observation does not correspond to the theory that sounds are species-specific [60–63]. This paradox could be solved with two complementary hypotheses: yellow-bellied and red-bellied P. nattereri are two different species and yellow-bellied P. nattereri have been wrongly identified, being in fact P. piraya.

The current yellow-bellied P. nattereri was first described as Serrasalmo ternetzi by Steindachner in 1908 [64] before being considered the holotype of the Gastropristis genus by Eigenmann 1915 [65,66]. The main distinctive feature was the morphology of the anal fin. Unfortunately, the holotype was mutilated and is lost to science [42]. This yellow-bellied population of piranha was then described as Serrasalmus ternetzi [67] based on three specimens that differ from P. nattereri at the level of the anterior profile, the length of the dorsal-fin base and the distance of dorsal fin from adipose fin [42,67]. Subsequent studies on a higher number of individuals confirmed the status of S. ternetzi. The diagnosis used the yellow coloration (on about 100 specimens), the convexity of the head and other meristic data in thirteen specimens [42,68]. However, using the body shape and head convexity of 60 specimens, Fink (1993) considered S. ternetzi as a junior synonym of P. nattereri [42]. More recently, a study observed differences in meristic data from north and south populations of P. nattereri: yellow-bellied P. nattereri possessed a deeper body relative to body length than red-bellied P. nattereri [43]. They considered this species as a nonlinear cline [43] and like in our study, their specimens of yellow-bellied P. nattereri were again bigger than those of red-bellied P. nattereri. Molecular studies show that specimens from different geographical areas (the red-bellied from Amazonas and yellow-bellied from Paraná-Paraguay) have exclusive haplotypes, supporting that a split happened at around 1.8 Ma [48,57,69]. Morphological, meristic, colour pattern, and molecular data all support deep differences between yellow-bellied and red-bellied P. nattereri. On the studied specimens, acoustic features clearly reinforce this assertion.

According to Fink (1993), “Pygocentrus piraya is diagnosed by the presence of adipose fin rays and is restricted to the Rio São Francisco of Brazil” whereas “P. nattereri is undiagnosed and highly variable in pigmentation” [42]. Fink (1993) reported that P. nattereri can be distinguished from P. cariba by the lack of a black humeral spot and from P. piraya by a lack of adipose fin rays [42]. We did not observe black humeral spots but we observed adipose fin rays in the so-called yellow-bellied P. nattereri (S1 Fig) sampled in the Araguari River (Paraná River basin). However, Pygocentrus is not native to this location [70,71] and was recorded for the first time in 2009 (S2 Fig; Godinho, personal data) [72–74] and identified as yellow-bellied P. nattereri by Langeani & Lacerda-Rêgo (2014) [71]. According to the characters they use in their study, it is however not possible to precisely identify the P. nattereri species but only the Pygocentrus genus. According to our results, having a population of yellow-bellied P. nattereri in Araguari River is deeply questionable and could result from misidentification. Based on historical morphological description and on the main argument that they both have the same acoustic features, the so-called yellow-bellied P. nattereri of the Araguari River should be P. piraya. Yearly records show a progressive migration from East to West (S2 Fig), suggesting fish were introduced to the east part of Araguari River [49] (S2 Fig; Godinho, personal data). This greatly supports our sonic-based assertion: P. piraya are now found in the Paraná River basin.

Our study supports that acoustic features can be important to reveal misidentified fish species. Up to now, this tool has been only used in other zoological groups. The distinction between species appears to be possible because acoustic signalling may be more flexible than morphology [17]. In birds, sounds were used for the distinction of three species of Phylloscopidae 30 years before their official description as different species [75]. Songs were also used to clarify systematic relationships in Passeriformes [76] and to contribute to the description of new species in Rhinocryptidae [77] and Tyrannidae [78]. Insects (Orthoptera, Hemiptera, and Neuroptera) [79–81] and anurans (Leptodactylidae, Ceratobatrachidae, Ranidae) [82–88] also possess many cryptic species that can be mainly differentiated acoustically. This role of bioacoustics in taxonomy supports the application of PAM in conservation programmes. However, more studies on frequency discrimination by fish are still necessary to know if acoustic differences are distinguishable by the fish.

Conclusions

Calls of P. cariba (Orinoco River basin) were distinct from all the other, red- and yellow-bellied P. nattereri (Parque Estadual do Rio Doce and Araguari River respectively) calls were different, and yellow-bellied P. nattereri calls were similar to those of P. piraya (São Francisco River basin). As the yellow-bellied P. nattereri found in the Araguari River basin possess adipose fin rays and the same acoustic features as Pygocentrus piraya, we have to consider that the population from Araguari River is most probably Pygocentrus piraya. This means that the geographical area of Pygocentrus piraya is larger than expected; and more likely, P. piraya has been introduced to the Paraná River basin. The use of acoustic features was made possible due to the removal of the effect of the fish size.

Supporting information

S1 Fig

Pictures of a studied specimen of Pygocentrus cariba (A), P. piraya (B), red-bellied P. nattereri (C) and yellow-bellied P. nattereri (D) and a magnification of its adipose fin showing adipose fin rays (E). Millimetre paper (10 x 10 mm) as scale.

(TIF)

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S2 Fig

A. Minas Gerais and B. São Francisco River basin (in blue) and Paraná River basin (in yellow) with the sites and dates of first observations of Pygocentrus in the upper Paraná River basin.

(TIF)

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S1 Table. Acoustic features of the sounds of the three valid species of Pygocentrus.

SD = standard deviation, SE = standard error, IQR = interquartile range, CV = coefficient of variation, Min = minimal value, Max = maximal value. Acoustic features symbols as defined in Fig 2. Body size (standard length) is given under each species name.

(DOCX)

Click here for additional data file. (19KB, docx)

Acknowledgments

The authors thank Thiago Carvalho and the team of the Aquário da Bacia do Rio Sao Francisco (Belo Horizonte) for letting us study some of their specimens. We also thank Janaína Aguiar, Rosinalva da Cunha Santos, and Vinicius de Assis Moreira from the Instituto Estadual de Florestas of the State of Minas Gerais for the authorisation to sample in Rio Doce State Park, and Samuel Bruno de Santana Vieira and Guilherme Coelho Melazo for authorisations in Retiro Baixo and Capim Branco II, respectively. In addition, we thank Sebastião Leandro Pinto and Antônio Bezerra da Silva for their help in Rio Doce State Park and Grande River, respectively.

Data Availability

All relevant data are within the manuscript and its Supporting Information files. Some specimens are available at the Aquarium-Museum of the University of Liège (vouchers R.E. 15271, 15272, 15273, 15274, 15275 and 17276).

Funding Statement

X.R.: Fonds de la Recherche Scientifique (2018/V 3/5/199 - JG/MS - 2718) A.H.: Fonds de la Recherche Scientifique (2018/V 3/5/200 - JG/MS - 2717) X.R. & A. H.: King Leopold III Fund for Nature Exploration and Conservation (2018/1.12.) E.P. & X.R.: Fonds de la Recherche Scientifique (PDR no. 23625340) https://www.frs-fnrs.be/fr/http://www.naturalsciences.be/LIII/ The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.Walters CL, Freeman R, Collen A, Dietz C, Brock Fenton M, Jones G, et al. 2012. A continental-scale tool for acoustic identification of European bats. J Appl Ecol. 49, 1064–74. ( 10.1111/j.1365-2664.2012.02182.x) [DOI] [Google Scholar]
  • 2.Xie J, Indraswari K, Schwarzkopf L, Towsey M, Zhang J, Roe P. 2018. Acoustic classification of frog within-species and species-specific calls. Appl Acoust. 131, 79–86. ( 10.1016/j.apacoust.2017.10.024) [DOI] [Google Scholar]
  • 3.Bernstein SK, Sheeran LK, Wagner RS, Li J-H, Koda H. 2016. The vocal repertoire of Tibetan macaques (Macaca thibetana): A quantitative classification. Am J Primatol. 78, 937–49. ( 10.1002/ajp.22564) [DOI] [PubMed] [Google Scholar]
  • 4.Kalko EV, Schnitzler H-U. 1993. Plasticity in echolocation signals of European pipistrelle bats in search flight: implications for habitat use and prey detection. Behav Ecol Sociobiol. 33, 415–28. ( 10.1007/BF00170257) [DOI] [Google Scholar]
  • 5.Obrist MK. 1995. Flexible bat echolocation: the influence of individual, habitat and conspecifics on sonar signal design. Behav Ecol Sociobiol. 36, 207–19. ( 10.1007/BF00177798) [DOI] [Google Scholar]
  • 6.Murray KL, Britzke ER, Robbins LW. 2001. Variation in search-phase calls of bats. J Mammal. 82, 728 () [DOI] [Google Scholar]
  • 7.Jones G, Siemers BM. 2011. The communicative potential of bat echolocation pulses. J Comp Physiol A. 197, 447–57. ( 10.1007/s00359-010-0565-x) [DOI] [PubMed] [Google Scholar]
  • 8.Parmentier E, Raick X, Lecchini D, Boyle K, Vanwassenbergh S, Bertucci F, et al. 2017. Unusual sound production mechanism in the triggerfish Rhinecanthus aculeatus (Balistidae). J Exp Biol. 220, 186–93. ( 10.1242/jeb.146514) [DOI] [PubMed] [Google Scholar]
  • 9.Colleye O, Frederich B, Vandewalle P, Casadevall M, Parmentier E. 2009. Agonistic sounds in the skunk clownfish Amphiprion akallopisos: size-related variation in acoustic features. J Fish Biol. 75, 908–16. ( 10.1111/j.1095-8649.2009.02316.x) [DOI] [PubMed] [Google Scholar]
  • 10.Mélotte G, Vigouroux R, Michel C, Parmentier E. 2016. Interspecific variation of warning calls in piranhas: a comparative analysis. Sci Rep. Nature Publishing Group; 6, 36127 ( 10.1038/srep36127) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Kéver L, Boyle KS, Dragičević B, Dulčić J, Casadevall M, Parmentier E. 2012. Sexual dimorphism of sonic apparatus and extreme intersexual variation of sounds in Ophidion rochei (Ophidiidae): first evidence of a tight relationship between morphology and sound characteristics in Ophidiidae. Front Zool. 9, 34 ( 10.1186/1742-9994-9-34) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Chesmore ED, Nellenbach C. 2001. Acoustic methods for the automated detection and identification of insects. Acta Hortic. 562, 223–31. ( 10.17660/ActaHortic.2001.562.26) [DOI] [Google Scholar]
  • 13.Parmentier E, Di Iorio L, Picciulin M, Malavasi S, Lagardère J-P, Bertucci F. 2018. Consistency of spatiotemporal sound features supports the use of passive acoustics for long-term monitoring. Anim Conserv. 21, 211–20. ( 10.1111/acv.12362) [DOI] [Google Scholar]
  • 14.Tubaro PL. 1999. Bioacústica aplicada a la sistemática, conservación y manejo de poblaciones naturales de aves. Etologia. 7, 19–32 [Google Scholar]
  • 15.Parkin DT. 2003. Birding and DNA: species for the new millennium. Bird Study. 50, 223–42. ( 10.1080/00063650309461316). [DOI] [Google Scholar]
  • 16.Ahléen I, BaagØe HJ. 1999. Use of ultrasound detectors for bat studies in Europe: experiences from field identification, surveys, and monitoring. Acta Chiropterologica. 1, 137–50. [Google Scholar]
  • 17.Jones G. 1997. Acoustic Signals and Speciation: The Roles of Natural and Sexual Selection in the Evolution of Cryptic Species. Adv study Behav. p. 317–54. ( 10.1016/S0065-3454(08)60383-6) [DOI] [Google Scholar]
  • 18.Oswald JN, Barlow J, Norris TF. 2003. Acoustic identification of nine delphinid species in the Eastern Tropical Pacific Ocean. Mar Mammal Sci. 19, 20–037. ( 10.1111/j.1748-7692.2003.tb01090.x) [DOI] [Google Scholar]
  • 19.Martin B, Kowarski K, Mouy X, Moors-Murphy H. 2014. Recording and identification of marine mammal vocalizations on the scotian shelf and slope 2014 Ocean—St John’s. St. John’s: IEEE; p. 1–6. ( 10.1109/OCEANS.2014.7003212) [DOI] [Google Scholar]
  • 20.Lin T-H, Yu H-Y, Chou L-S, Chen C-F. 2014. Passive acoustic monitoring on the seasonal species composition of cetaceans from the marine cable hosted observatory Ocean 2014—TAIPEI. Taipei: IEEE; p. 1–6. ( 10.1109/OCEANS-TAIPEI.2014.6964392) [DOI] [Google Scholar]
  • 21.Renner SC, Rappole JH, Kyaw M, Milensky CM, Päckert M. 2018. Genetic confirmation of the species status of Jabouilleia naungmungensis. J Ornithol. 159, 63–71. ( 10.1007/s10336-017-1493-0) [DOI] [Google Scholar]
  • 22.Van Doren BM, Freeman BG, Aristizabal N, Alvarez-R M, Pérez-Emán J, Cuervo AM, et al. 2018. Species limits in the Rusty-breasted Antpitta (Grallaricula ferrugineipectus) complex. Wilson J Ornithol. 130, 152 ( 10.1676/16-126.1) [DOI] [Google Scholar]
  • 23.Freeman BG, Montgomery GA. 2017. Using song playback experiments to measure species recognition between geographically isolated populations: A comparison with acoustic trait analyses. Auk. 134, 857–70. ( 10.1642/AUK-17-63.1) [DOI] [Google Scholar]
  • 24.Negret PJ, Laverde-R O. 2015. The enigmatic Black Tinamou: Do distribution, climate, and vocalizations reveal more than one species? Auk. 132, 132–9. ( 10.1642/AUK-14-183.1) [DOI] [Google Scholar]
  • 25.Feo TJ, Musser JM, Berv J, Clark CJ. 2015. Divergence in morphology, calls, song, mechanical sounds, and genetics supports species status for the Inaguan hummingbird (Trochilidae: Calliphlox “evelynae” lyrura). Auk. 132, 248–64. ( 10.1642/AUK-14-108.1) [DOI] [Google Scholar]
  • 26.Van Balen SB, Eaton JA, Rheindt FE. 2013. Biology, taxonomy and conservation status of the Short-tailed Green Magpie Cissa [t.] thalassina from Java. Bird Conserv Int. 23, 91–109. ( 10.1017/S0959270911000360) [DOI] [Google Scholar]
  • 27.Päckert M, Martens J, Eck S, Nazarenko AA, Valchuk OP, Petri B, et al. 2005. The great tit (Parus major)—a misclassified ring species. Biol J Linn Soc. 86, 153–74. ( 10.1111/j.1095-8312.2005.00529.x) [DOI] [Google Scholar]
  • 28.Parmentier E, Fine ML, Berthe C, Lecchini D. 2018. Taxonomic validation of Encheliophis chardewalli with description of calling abilities. J Morphol. 279, 864–70. ( 10.1002/jmor.20816) [DOI] [PubMed] [Google Scholar]
  • 29.Borkin LYA, Litvinchuk SN, Rozanov YUN, Skorinov D V. 2004. On cryptic species (exemplified by amphibians). Zool Zhurnal. 83, 936–60. [Google Scholar]
  • 30.Bickford D, Lohman DJ, Sodhi NS, Ng PKL, Meier R, Winker K, et al. 2007. Cryptic species as a window on diversity and conservation. Trends Ecol Evol. 22, 148–55. ( 10.1016/j.tree.2006.11.004) [DOI] [PubMed] [Google Scholar]
  • 31.Fish MP, Mowbray WH. 1959. The production of underwater sound by Opsanus sp., a new toadfish from Bimini, Bahamas. Zool Sci Contrib New York Zool Soc. 44, 71–6. [Google Scholar]
  • 32.Casadevall M, Matallanas J, Carrasson M, Muñoz M. 1996. Morphometric, meristic and anatomical differences between Ophidion barbatum L., 1758 and O. rochei Müller, 1845 (Pisces, Ophidiidae). Publicaciones Espec Inst Esp Oceanogr. 21, 45–61. [Google Scholar]
  • 33.Parmentier E, Bouillac G, Dragicevic B, Dulcic J, Fine M. 2010. Call properties and morphology of the sound-producing organ in Ophidion rochei (Ophidiidae). J Exp Biol. 213, 3230–6. ( 10.1242/jeb.044701) [DOI] [PubMed] [Google Scholar]
  • 34.Parmentier E, Fine M, Vandewalle P, Ducamp J-J, Lagardère J-P. 2006. Sound production in two carapids (Carapus acus and C. mourlani) and through the sea cucumber tegument. Acta Zool. 87, 113–9. ( 10.1111/j.1463-6395.2006.00221.x) [DOI] [Google Scholar]
  • 35.Feulner PGD, Kirschbaum F, Schugardt C, Ketmaier V, Tiedemann R. 2006. Electrophysiological and molecular genetic evidence for sympatrically occuring cryptic species in African weakly electric fishes (Teleostei: Mormyridae: Campylomormyrus). Mol Phylogenet Evol. 39, 198–208. ( 10.1016/j.ympev.2005.09.008) [DOI] [PubMed] [Google Scholar]
  • 36.Huby A, Lowie A, Herrel A, Vigouroux R, Frédérich B, Raick X, et al. 2019. Functional diversity in biters: the evolutionary morphology of the oral jaw system in pacus, piranhas and relatives (Teleostei: Serrasalmidae). Biol J Linn Soc. 127, 722–41. ( 10.1093/biolinnean/blz048) [DOI] [Google Scholar]
  • 37.Thompson AW, Betancur-R R, López-Fernández H, Ortí G. 2014. A time-calibrated, multi-locus phylogeny of piranhas and pacus (Characiformes: Serrasalmidae) and a comparison of species tree methods. Mol Phylogenet Evol. 81, 242–57. 10.1016/j.ympev.2014.06.018 [DOI] [PubMed] [Google Scholar]
  • 38.Jégu M. 2003. Subfamily Serrasalminae (pacus and piranhas)In: Reis RE, Kullander SO, Ferraris CJ, Ferraris J, editors. Check List Freshw Fishes South Cent Am. Edipucrs. Porto Alegre; p. 182–96. [Google Scholar]
  • 39.Sunaga T, Verani JR. 1985. Preliminary report of comparative study on fish community of the Rio Doce Valley lakesIn: Saijo Y, Tundisi JG, editors. Limnol Stud Rio Doce Val Lakes Pantanal Wetl Brazil Vol 1. Nagoya: Nagoya University Press; p. 167–174. [Google Scholar]
  • 40.Sunaga T, Verani JR. 1987. Second report of comparative study on fish community of the Rio Doce Valley lakesIn: Saijo Y, Tundisi JG, editors. Limnol Stud Rio Doce Val Lakes Pantanal Wetl Brazil Vol 2. Nagoya: Nagoya University Press; p. 129–35. [Google Scholar]
  • 41.Godinho AL, Formagio PS. 1992. Efeitos da introducão de Cichla ocellaris e Pygocentrus sp. sobre a comunidade de peixes da Lagoa Dom Helvecio. Encontro Anu Aquicultura Minas Gerais. 10, 93–102. [Google Scholar]
  • 42.Fink WL. 1993. Revision of the Piranha Genus Pygocentrus (Teleostei, Characiformes). Copeia. 1993, 665 ( 10.2307/1447228) [DOI] [Google Scholar]
  • 43.Fink WL, Zelditch ML. 1997. Shape Analysis and Taxonomic Status of Pygocentrus Piranhas (Ostariophysi, Characiformes) from the Paraguay and Paraná River Basins of South America. Copeia. 1997, 179 ( 10.2307/1447854) [DOI] [Google Scholar]
  • 44.Mélotte G, Raick X, Vigouroux R, Parmentier E. 2019. Origin and evolution of sound production in Serrasalmidae. Biol J Linn Soc. 20, 1–12. ( 10.1093/biolinnean/blz105) [DOI] [Google Scholar]
  • 45.Mélotte G, Parmentier E, Michel C, Herrel A, Boyle K. 2018. Hearing capacities and morphology of the auditory system in Serrasalmidae (Teleostei: Otophysi). Sci Rep. 8, 1281 ( 10.1038/s41598-018-19812-1) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Millot S, Vandewalle P, Parmentier E. 2011. Sound production in red-bellied piranhas (Pygocentrus nattereri, Kner): an acoustical, behavioural and morphofunctional study. J Exp Biol. 214, 3613–8. ( 10.1242/jeb.061218) [DOI] [PubMed] [Google Scholar]
  • 47.Raick X, Huby A, Kurchevski G, Godinho AL, Parmentier É. 2020. Yellow‐eyed piranhas produce louder sounds than red‐eyed piranhas in an invasive population of Serrasalmus marginatus. J Fish Biol. ( 10.1111/jfb.14529) [DOI] [PubMed] [Google Scholar]
  • 48.Hubert N, Duponchelle F, Nuñez J, Garcia-Davila C, Paugy D, Renno J-F. 2007. Phylogeography of the piranha genera Serrasalmus and Pygocentrus: implications for the diversification of the Neotropical ichthyofauna. Mol Ecol. 16, 2115–36. ( 10.1111/j.1365-294X.2007.03267.x) [DOI] [PubMed] [Google Scholar]
  • 49.Belei F, Sampaio WMS, Millani TJ, Trazzi A, Dergam JA. 2012. First record of Pygocentrus piraya (Cuvier 1819) (Characiformes: Serrasalmidae), a new predatory species introduction in the middle and lower Doce River basin. Check List. 8, 787 ( 10.15560/8.4.787) [DOI] [Google Scholar]
  • 50.Akamatsu T, Okumura T, Novarini N, Yan HY. 2002. Empirical refinements applicable to the recording of fish sounds in small tanks. J Acoust Soc Am. 112, 3073–82. ( 10.1121/1.1515799) [DOI] [PubMed] [Google Scholar]
  • 51.Ladich F, Bischof C, Schleinzer G, Fuchs A. 1992. Intra- and interspecific differences in agonistic vocalization in croaking gouramis (genus: Trichopsis, Anabantoidei, Teleostei). Bioacoustics. 4, 131–41. ( 10.1080/09524622.1992.9753212) [DOI] [Google Scholar]
  • 52.Myrberg AA, Ha SJ, Shamblott MJ. 1993. The sounds of bicolor damselfish (Pomacentrus partitus): Predictors of body size and a spectral basis for individual recognition and assessment. J Acoust Soc Am. 94, 3067–70. ( 10.1121/1.407267) [DOI] [Google Scholar]
  • 53.Henglmuller SM, Ladichm F. 1999. Development of agonistic behaviour and vocalization in croaking gouramis. J Fish Biol. 54, 380–95. ( 10.1111/j.1095-8649.1999.tb00837.x) [DOI] [Google Scholar]
  • 54.Amorim MCP, Hawkins AD. 2005. Ontogeny of Acoustic and Feeding Behaviour in the Grey Gurnard, Eutrigla gurnardus. Ethology. 111, 255–69. ( 10.1111/j.1439-0310.2004.01061.x) [DOI] [Google Scholar]
  • 55.Millot S, Parmentier E. 2014. Development of the ultrastructure of sonic muscles: a kind of neoteny? BMC Evol Biol. 14, 24 ( 10.1186/1471-2148-14-24) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Raick X, Lecchini D, Kéver L, Colleye O, Bertucci F, Parmentier É. 2018. Sound production mechanism in triggerfish (Balistidae): a synapomorphy. J Exp Biol. 221, jeb168948. ( 10.1242/jeb.168948) [DOI] [PubMed] [Google Scholar]
  • 57.Mateussi NTB, Melo BF, Foresti F, Oliveira C. 2019. Molecular data reveal multiple lineages in piranhas of the genus Pygocentrus (Teleostei, Characiformes). Genes (Basel). 10, 371 ( 10.3390/genes10050371) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Kolman MA, Hughes LC, Hernandez LP, Arcila D, Betancur R, Sabaj MH, et al. 2020. Phylogenomics of piranhas and pacus (Serrasalmidae) uncovers how convergent diets obfuscate traditional morphological taxonomy. bioRxiv. [preprint]. [DOI] [PubMed] [Google Scholar]
  • 59.Machado VN, Collins RA, Ota RP, Andrade MC, Farias IP, Hrbek T. 2018. One thousand DNA barcodes of piranhas and pacus reveal geographic structure and unrecognised diversity in the Amazon. Sci Rep. Springer US; 8, 8387 ( 10.1038/s41598-018-26550-x) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Myrberg AAJ. 1997. Sound Production by a Coral Reef Fish (Pomacentrus partitus): Evidence for a Vocal, Territorial “Keep-Out” Signal. Bull Mar Sci. 60, 1017–25. [Google Scholar]
  • 61.Wang Z-T, Nowacek DP, Akamatsu T, Wang K-X, Liu J-C, Duan G-Q, et al. 2017. Diversity of fish sound types in the Pearl River Estuary, China. PeerJ. 5, e3924 ( 10.7717/peerj.3924) [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 62.Rountree RA, Juanes F. 2020. Potential for use of passive acoustic monitoring of piranhas in the Pacaya–Samiria National Reserve in Peru. Freshw Biol. 65, 55–65. ( 10.1111/fwb.13185) [DOI] [Google Scholar]
  • 63.Di Iorio L, Raick X, Parmentier E, Boissery P, Valentini-Poirier C-A, Gervaise C. 2018. ‘ Posidonia meadows calling’: a ubiquitous fish sound with monitoring potential.Pettorelli N, Merchant N, editors. Remote Sens Ecol Conserv. 4, 248–63. ( 10.1002/rse2.72) [DOI] [Google Scholar]
  • 64.Steindachner F. 1908. Über sechs neue Serrasalmo- und Myletes-Arten aus Südamerika, von denen vier von ihm während der zoologischen Expedition der kaiserl. Akademie der Wissenschaften nach Brasilien im Jahre 1903 gesammelt wurden. Anzeiger der Kais Akad der Wissenschaften, Wien, Math Klasse. 45, 359–67. [Google Scholar]
  • 65.Eigenmann CH. 1915. The Serrasalminae and Mylinae. Ann Carnegie Museum. 9, 225–72. [Google Scholar]
  • 66.Machado-Allison A, Fink W. 1996. Los Peces Caribes de Venezuela: Diagnosis, Claves, Aspectos Ecologicos y Evolutivos. Universida.Caracas.
  • 67.Nokman JR. 1928. The South American Characid fishes of the subfamily Serrasalmoninae, with a revision of the genus Serrasalmus Lacepède. Proc Zool Soc London. 98, 781–829. ( 10.1111/j.1469-7998.1928.tb07169.x) [DOI] [Google Scholar]
  • 68.Géry J, Mahnert V, Dlouhy C. 1987. Poissons Characoïdes non Characidae du Paraguay (Pisces, Ostariophysi). Rev suisse Zool. 94, 357–464. [Google Scholar]
  • 69.Bignotto TS, Gomes VN, Maniglia TC, Boni TA, Agostinho CS, Prioli SMAP, et al. 2019. Molecular characterization and genetic relationships of seven piranha species of the genera Serrasalmus and Pygocentrus (Characiformes: Serrasalmidae) from Paraná-Paraguay, São Francisco and Tocantins River basins in Brazil. Brazilian J Biol. ( 10.1590/1519-6984.219020) [DOI] [PubMed] [Google Scholar]
  • 70.Martins I, Sanches B, Kaufmann PR, Hughes RM, Santos GB, Molozzi J, et al. 2015. Ecological assessment of a southeastern Brazil reservoir. Biota Neotrop. 15, e20140061 ( 10.1590/1676-06032015006114) [DOI] [Google Scholar]
  • 71.Langeani F, Lacerda-Rêgo AC. 2014. Guia ilustrado dos peixes da bacia do rio Araguari. Grupo de M.Uberlândia.
  • 72.Centro de Transposição de Peixes (Universidade Federal de Minas Gerais). 2007 UHE Capim Branco II—Programa de monitoramento e de conservação da ictiofauna.Belo Horizonte.
  • 73.Centro de Transposição de Peixes (Universidade Federal de Minas Gerais). 2008 UHE CAPIM BRANCO I—Programa de monitoramento e de conservação da ictiofauna.Belo Horizonte.
  • 74.Vono V. 2002. Efeitos da implantação de duas barragens sobre a estrutura da comunidade de peixes do rio Araguari (bacia do alto Paraná, MG).Universidade Federal de Minas Gerais.
  • 75.White G. 1813. The Natural History and Antiquities of Selborne in the county of Southampton.White B, editor. London.
  • 76.Gebauer A, Kaiser M, Wassmann C. 2006. Remarks on biology, vocalisations and systematics of Urocynchramus pylzowi Przewalski (Aves, Passeriformes). Zootaxa. 1325, 75 ( 10.11646/zootaxa.1325.1.6) [DOI] [Google Scholar]
  • 77.Cuervo AM, Cadena CD, Krabbe N, Renjifo LM. 2005. Scytalopus stilesi, a new species of tapaculo (Rhinocryptidae) from the Cordillera Central of Colombia. Auk. 122, 445–63. [Google Scholar]
  • 78.Johnson NK, Jones RE. 2001. A new species of Tody-Tyrant (Tyrannidae: Poecilotriccus) from Northern Peru. Auk. 118, 334–41. ( 10.2307/4089795) [DOI] [Google Scholar]
  • 79.Shaw KL. 2000. Further acoustic diversity in Hawaiian forests: two new species of Hawaiian cricket (Orthoptera: Gryllidae: Trigonidiinae Laupala). Zool J Linn Soc. 129, 73–91. ( 10.1006/zjls.1998.0201) [DOI] [Google Scholar]
  • 80.Henry CS. 1994. Singing and cryptic speciation insects. Trends Ecol Evol. 9, 388–92. ( 10.1016/0169-5347(94)90061-2) [DOI] [PubMed] [Google Scholar]
  • 81.Henry CS. 1985. Sibling species, call differences, and speciation in green lacewings (Neuroptera: Chrysopidae: Chrysoperla). Evolution (N Y). 39, 965–84. ( 10.1111/j.1558-5646.1985.tb00441.x) [DOI] [PubMed] [Google Scholar]
  • 82.Angulo A, Reichle S. 2008. Acoustic signals, species diagnosis, and species concepts: the case of a new cryptic species of Leptodactylus (Amphibia, Anura, Leptodactylidae) from the Chapare region, Bolivia. Zool J Linn Soc. 152, 59–77. ( 10.1111/j.1096-3642.2007.00338.x) [DOI] [Google Scholar]
  • 83.García-Lopez JM, Heyer WR, Cardoso AJ. 1996. Advertisement call variation in the Leptodactylus mystaceus species complex (Amphibia: Leptodactylidae) with a description of a new sibling species. Amphibia-Reptilia. 17, 7–31. ( 10.1163/156853896X00252) [DOI] [Google Scholar]
  • 84.Reichle S, Lötters S, de la Riva I, Lotters S. 2001. A new species of the discoidalis group of Eleutherodactylus (Anura, Leptodactylidae) from inner-Andean dry valleys of Bolivia. J Herpetol. 35, 21 ( 10.2307/1566018) [DOI] [Google Scholar]
  • 85.Kwet A, Steiner J, Zillikens A. 2009. A new species of Adenomera (Amphibia: Anura: Leptodactylidae) from the Atlantic rain forest in Santa Catarina, southern Brazil. Stud Neotrop Fauna Environ. 44, 93–107. ( 10.1080/01650520902901659) [DOI] [Google Scholar]
  • 86.Angulo A, Cocroft RB, Reichle S. 2003. Species identity in the genus Adenomera (Anura: Leptodactylidae) in Southeastern Peru. Herpetologica. 59, 490–504. ( 10.1655/20-104) [DOI] [Google Scholar]
  • 87.BROWN RM, RICHARDS SJ, SUKUMARAN J, FOUFOPOULOS J. 2006. A new morphologically cryptic species of forest frog (genus Platymantis) from New Britain Island, Bismarck Archipelago. Zootaxa. 1334, 45 ( 10.11646/zootaxa.1334.1.3) [DOI] [Google Scholar]
  • 88.Littlejohn MJ, Oldham RS. 1968. Rana pipiens complex: Mating call structure and taxonomy. Science (80-). 162, 1003–5. ( 10.1126/science.162.3857.1003) [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Fig

Pictures of a studied specimen of Pygocentrus cariba (A), P. piraya (B), red-bellied P. nattereri (C) and yellow-bellied P. nattereri (D) and a magnification of its adipose fin showing adipose fin rays (E). Millimetre paper (10 x 10 mm) as scale.

(TIF)

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S2 Fig

A. Minas Gerais and B. São Francisco River basin (in blue) and Paraná River basin (in yellow) with the sites and dates of first observations of Pygocentrus in the upper Paraná River basin.

(TIF)

Click here for additional data file. (1.3MB, tif)
S1 Table. Acoustic features of the sounds of the three valid species of Pygocentrus.

SD = standard deviation, SE = standard error, IQR = interquartile range, CV = coefficient of variation, Min = minimal value, Max = maximal value. Acoustic features symbols as defined in Fig 2. Body size (standard length) is given under each species name.

(DOCX)

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Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files. Some specimens are available at the Aquarium-Museum of the University of Liège (vouchers R.E. 15271, 15272, 15273, 15274, 15275 and 17276).

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