Zootaxa 4521 (3): 404–416 ISSN 1175-5326 (print edition)

http://www.mapress.com/j/zt/
Copyright © 2018 Magnolia Press
Article ZOOTAXA
ISSN 1175-5334 (online edition)
https://doi.org/10.11646/zootaxa.4521.3.6
http://zoobank.org/urn:lsid:zoobank.org:pub:723C8CEC-D150-4B5D-8582-D482A1350AC4

Geographic variation in the advertisement call of Trachycephalus typhonius

(Anura: Hylidae) based on South American samples

VÍCTOR HUGO ZARACHO1, 3, LEONARDO DIONEL AGUIAR1 & ARIOVALDO ANTONIO GIARETTA2
1
Laboratorio de Herpetología. Facultad de Ciencias Exactas y Naturales y Agrimensura. Universidad Nacional del Nordeste. Av. Lib-
ertad 5470. Corrientes (CP 3400). Argentina. E-mail: victorzaracho@yahoo.com.ar, aguiardionel@gmail.com.
2
Laboratório de Taxonomia e Sistemática de Anuros Neotropicais. Faculdades Integradas do Pontal. Universidade Federal de Uber-
lândia. Rua 20 Nº 1600 - Bairro Tupã 38304-402. Ituiutaba (CEP: 38302-000). Minas Gerais, Brazil.
E-mail: aagiaretta@gmail.com
3
Corresponding author.

Abstract

Previously described calls of Trachycephalus typhonius (Linnaeus, 1758) correspond to several populations from Central
and South America, but in general, these descriptions were brief and often based on a single recorded individual. Here,
based on an expressive sample, we re-describe the advertisement calls of T. typhonius using recordings from populations
in Brazil and Argentina. Additionally, we discuss geographical variation of calls, comment on their frequency band struc-
ture and compare calls with those described for other species of Trachycephalus. Calls of 32 males were recorded and
temporal and spectral features of 269 calls were measured. To search for discrimination among three populations sampled
we used the Random Forest (RF) model, Multidimensional Scaling Analysis (MDS) and Wilcoxon-Mann-Whitney Rank
Sum Test. The advertisement calls of T. typhonius consist of a multipulsed note of 343–540 ms of duration, emitted at
regular intervals, with up to three emphasized frequency bands. Dominant frequency ranged between 1705–2750 Hz.
Calls from Rondônia (Brazil) were significantly different from those recorded in Argentina and Minas Gerais (Brazil) in
relation to pulse rate and dominant frequency. Populations from Minas Gerais and Argentina differed in dominant frequen-
cy of calls. Such population differences can be partly attributed to differences in prevalence of calling sites (immersed in
water vs. perches on vegetation), but can also hint at the existence of cryptic species diversity under this taxon.

Key words: Argentina, Amazon Milk Frog, Brazil, differentiation, call structure, vocalizations

Introduction

As presently defined, Trachycephalus (Tschudi, 1838) contains 17 species distributed in the Neotropical region,
from the lowlands of Mexico, Central and South America (east of the Andes) to northern Argentina and eastern
Brazil (Frost 2017). Trachycephalus typhonius (Linnaeus, 1758) is an explosive breeder, usually reproducing after
heavy rains (Savage 2002), and generally associated to lentic water bodies as permanent or temporary ponds near
forested areas. Males call while floating in the water or perched on marginal vegetation, inflating their back-turned
paired vocal sacs (De la Riva 1995). Trachycephalus typhonius occurs along almost all of the distribution of genus
Trachycephalus (La Marca et al. 2010; Ron et al. 2016), and due to its wide distribution, variation in external
morphology, and genetic evidence it has been suggested that it may represent a species complex (Lavilla et al.
2010; Murphy et al. 2017). In fact, two species previously considered as junior synonyms of T. typhonius have
been recently resurrected (Ron et al. 2016).
Advertisement calls represent a complementary tool in taxonomic studies of anurans (Köhler et al. 2017).
Previously described calls of T. typhonius correspond to several individuals recorded throughout Central and South
America, including records from Panama (Zweifel 1964; Duellman 1970), Costa Rica (Duellman 1970), French
Guiana, Peru (Zimmerman & Hödl 1983), Bolivia (De la Riva et al. 1995), Brazil (Guimarães et al. 2001), and
Venezuela (Rivero & Esteves 1969; Tárano 2010). In general, these descriptions were brief and based on a single
recorded individual.

404 Accepted by P. Simoes: 26 Sept. 2018; published: 15 Nov. 2018

 In this study, we compile all previous information about advertisement calls of T. typhonius and add detailed
descriptions of calls from populations distributed in Brazil and Argentina. The Argentinean populations represent
the southernmost records for this species, extending the known acoustic sampling for the species in over 1500 km.
In Brazil, a single male from the Cerrado Biome (Guimarães et al. 2001) had its calls analyzed to date. Herein, we
expressively increase the samples from the Cerrado and for the first time, present data on a population from the
Brazilian Amazonia region. Additionally, we evaluate the existence and discuss the geographical variation in call
features and comment on their frequency band structure. Finally, we compare calls of T. typhonius with those
described for other species of Trachycephalus: T. dibernardoi Kwet & Solé, 2008 (Kwet & Solé 2008), T. atlas
Bokermann, 1966 (Dos Santos-Silva et al. 2012), T. resinifictrix (Goeldi, 1907) (Zimmerman & Hödl 1983), T.
mesophaeus (Hensel, 1867) (Prado et al. 2003), T. nigromaculatus Tschudi, 1838 (Abrunhosa et al. 2001), T.
cunauaru Gordo, Toledo, Suárez, Kawashita-Ribeiro, Ávila, Morais & Nunes, 2013 (Hödl 1991; Gordo et al.
2013), and T. coriaceus (Peters, 1867) (De la Riva et al. 1995).

Material and methods

Advertisement calls of thirty-two males from three regions in South America were recorded and analyzed. Samples
include three sites in the region of Triângulo Mineiro (State of Minas Gerais, Southeastern Brazil), one site in
Southwestern Brazilian Amazonia (State of Rondônia) and three from Northeastern Argentina (Chaco, Corrientes
and Misiones Provinces) (Table 1, Fig. 1). Neighbor sampling sites in Southeastern Brazil and Argentina were
pooled for description of call features due to the lack of evidence of micro-regional differences (data not shown).
The three sampling regions are separated by 900–1200 km. Brazilian samples correspond to specimens living in the
Cerrado (Oliveira & Marquis 2002) and Amazonian Forest biomes, whereas Argentinean specimens were recorded
in areas within the Chaco (Oriental Region) and Paranaense phytogeographic provinces (Cabrera 1976).

TABLE 1. Localities, geographic coordinates, altitude, number of recorded males and number of calls of
Trachycephalus typhonius analyzed in this study. Approximate coordinates and altitudes were obtained in Google Earth
Pro (2017, Google Inc.; WGS84 datum).
Localities Geographic coordinates Altitude (m a.s.l) n males n calls
Brazil
Minas Gerais State, Ituiutaba 18°58’25” S, 49°24’38” W ca. 580 5 44
Minas Gerais State, Uberlândia 19°11’17” S, 48°24’18” W ca. 810 1 14
Minas Gerais State, Araguari 18°27’57” S, 48°31’46” W ca. 857 5 50
Rondônia State, Costa Marques 12°26’08” S, 64°13’38” W ca. 140 11 65
Argentina
Chaco Province, La Leonesa 27º05’24” S, 58º44’54” W ca. 66 3 52
Corrientes Province, Corrientes 27º25’50” S, 58º42’30” W ca .52 6 32
Misiones Province, Puerto Iguazú 25º37’00” S, 54º34’00” W ca. 162 1 12

In Argentina, calls of T. typhonius were recorded with an M-audio Microtrack II digital recorder coupled to a
Sennheiser ME66/K6 microphone. In Brazil, calls were recorded using Marantz PMD 670 or 671 digital recorders
coupled to Sennheiser ME67/K6 microphones. Calls were recorded in .wav format and all recorders were set at a
sampling rate of 44 or 48 kHz and 16-bit resolution. Air or water temperature at time of recordings varied between
21–29°C (Table 2) and considering that temperatures did not differ significantly among the three localities
(Approx. K-Sample Fisher-Pitman Permutation Test, chi-squared = 3.83, p-value = 0.15) we did not try to
incorporate this variable in the discrimination analysis (see below). Temporal and spectral properties of 269 calls
were measured: 96 calls of 10 males from Argentina (2–17 calls per male), 65 calls of 11 males from Minas Gerais
(5–16 calls per male) and 108 calls of 11 males from Rondônia (316 calls per male). Sounds were analyzed using
Raven Pro 1.4, 32-bit version (Bioacoustics Research Program 2011). Oscillogram and spectrogram illustrations
were generated using the Seewave v.1.6 package (Sueur et al. 2008) in R v.2.13.0 platform (R Development Core
Team 2016). Seewave settings were: Hanning window, 90% overlap and 512 points resolution (FFT). Temporal
acoustic variables were analyzed on oscillograms and spectral variables on spectrograms with the following
settings: window size 512 samples, Hann window, contrast 50 %, brightness 50 %, and time grid overlap 50 %. The

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dominant frequency was measured using Peak Frequency option. Additionally, we analyzed some spectral views
using a windows size of 1024 points in order to compare values with published data. Measurements of the
following acoustic variables, according to Köhler et al. (2017), were taken: call rate (ratio between number of calls
and the record duration in which these calls were emitted), call duration, inter-call interval, pulse rate (ratio of the
number of pulses and the duration in which these pulses were emitted), pulse duration, inter-pulse interval and
dominant frequency. Additionally, we measured the number of pulses per call and the frequency peaks in other
bands with noticeable energy. All analyzed sound files are listed in Appendix 1.

FIGURE 1. Geographic distribution (white shade) and localities from where advertisement calls of Trachycephalus typhonius
are known. 1) Costa Rica (Duellman, 1970); 2) Panama (Zweifel 1964; Duellman 1970); 3) Venezuela (Zimmerman & Hödl
1983, Tárano 2010); 4) French Guiana (Zimmerman & Hödl 1983); 5) Loreto, Peru (Zimmerman & Hödl 1983); 6) Costa
Marques, Rondônia, Brazil (this study); 7) Puerto Almacén, Bolivia (De la Riva et al. 1995); 8) Pontalina, Goiás, Brazil
(Guimarães et al. 2001); 9) Araguari /Uberlândia/Ituiutaba, Minas Gerais, Brazil (this study); 10) Puerto Iguazú, Misiones
Province, Argentina (this study); 11) La Leonesa, Chaco Province, Argentina; and 12) Corrientes, Corrientes Province,
Argentina (this study). Distribution map was based on estimates provided by The IUCN Red List of Threatened Species (La
Marca et al. 2010).

406 · Zootaxa 4521 (3) © 2018 Magnolia Press ZARACHO ET AL.

 We used the Random Forest (RF) model (Liaw & Wiener 2002) to search for discrimination among the three
populations (‘randomForest’ R package; R Development Core Team 2016). Random Forest constructs many
classification trees using bootstrap (each split on the best predictors chosen at each node). Then, it generates
classifiers and aggregates results to classes by voting (Liaw & Wiener 2002). Distance estimates among objects
resulting from the Randon Forest model were subject to a Multidimensional Scaling Analysis (MDS) and plotted
with the "proximity.plot" function of the Package ‘rfPermute’ version 2.0.1 (Archer 2016). We tested for
populational differences in call features through the Wilcoxon-Mann-Whitney Rank Sum Test in the ‘coin’ R
Package (Hothorn et al. 2008). As tests were carried on population pairs, the significance levels (“P”) were
adjusted considering the number of comparisons through the method of Holm (p.adjust function in R).
One voucher specimen from each sampling region is deposited in the collection of amphibians of Museu de
Biodiversidade do Cerrado, at Universidade Federal de Uberlândia, Minas Gerais, Brazil (AAG-UFU 4098,
Araguari, MG, Brazil; AAG-UFU 5339, Costa Marques, Rondônia, Brazil) and in the herpetological collection of
the Universidad Nacional del Nordeste, Corrientes, Argentina (UNNEC 13080, La Leonesa, Chaco, Argentina).
For comparative purposes, we analyzed unpublished calls of two males of T. mesophaeus recorded on 24
January 2018, in São Lourenco da Serra, São Paulo, Brazil (23°52’31” S, 46°56’00” W).

FIGURE 2. Audiospectrograms (upper graph, FFT = 512) and corresponding oscillograms (lower graph) of middle portions of
the advertisement calls of males from the three studied populations of Trachycephalus typhonius depicting seven pulses. From
left to right: La Leonesa (Chaco province, Argentina), Uberlândia (Minas Gerais, Brazil), and Costa Marques (Rondônia,
Brazil).

Results

Values of temporal and spectral features of the advertisement calls of Trachycephalus typhonius from Brazil and
Argentina are summarized in Table 2, as well as comparative data from the literature. In Minas Gerais, males were
found alongside temporary pools, at night and after heavy rain showers. They were calling in chorus perched on
trees up to 3–8 m high or immersed in the water. Water or air temperature at time of recording in this region ranged
between 21–27 ºC. In Rondônia, males were calling in chorus and were found at night around temporary pools,
perched on bushes, 1–2 m high. Air temperature at time of recording in this region ranged between 26–29 ºC. In
Argentina, males were found in a temporary pond, next to a small forest fragment after copious rain (70 mm in the

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previous 24 h), from dusk until midnight. They were calling in chorus, floating on the water surface. Water
temperature at time of recording in this region ranged between 22–29 ºC.
The advertisement calls of all males recorded consist of a multipulsed note emitted at regular intervals, with up
to three emphasized frequency bands: an inferior and a central (= dominant) always well-defined; and a superior
that could be present or not (Fig. 2). In all cases, pulses gradually reach maximum amplitude towards the end of the
call. In Argentinean populations, advertisement calls lasted 411–598 ms, spaced by intervals between 452 and 1180
ms and emitted at a rate between 35 and 61 calls/min. Calls are composed of 59–77 pulses emitted at a rate of 129–
144 pulses/s. The three frequency bands were always present, the dominant frequency ranged between 1705 and
2060 Hz. The frequency peak in the lower band ranged between 258 and 294 Hz, and in the upper band, between
2239 and 2727 Hz (Figs. 2, 3).
Calls of specimens recorded in Minas Gerais had a duration of 343–540 ms, were spaced by intervals of 565–
1095 ms and were emitted at a rate between 38 and 64 calls/min. Calls were composed of 52–77 pulses emitted at a
rate of 123–154 pulses/s. Three frequency bands were generally observed. The dominant frequency was between
1881 and 2359 Hz, while the lower band was between 258 and 656 Hz and the upper band between 2858 and 3194
Hz (Fig. 3).

FIGURE 3. Audiospectrogram (upper graph, FFT = 512) and corresponding oscillogram (lower graph) of one call of
Trachycephalus typhonius from La Leonesa, Chaco province, Argentina (UNNEC 13080). Note the low frequency band around
300 Hz and the dominant around 1800 Hz. Record file: Trachycephalus_typhonius_VHZ-
MT2_209_voucherUNNEC13080_LaLeonesa_file0053.wav. November 19, 2013; 20:58h; water = 22.2ºC.

Calls from Rondônia had a duration of 350–465 ms, were spaced by intervals of 637–1891 ms and were
emitted at a rate of 26–71 calls/min. Calls were composed by 59–87 pulses emitted at a rate of 130–204 pulses/s. In
most individuals only two frequency bands were observed: the dominant ranging between 2156 and 2750 Hz and a
lower between 281 and 786 Hz (Fig. 3).
The random forest model on call features resulted in an error rate of 22 %, with most individuals recovered
within their own population. Samples from Minas Gerais and Rondônia broadly overlapped along the first axis of
the MDS analysis. Likewise, the Minas Gerais sample could not be completely discriminated from the Argentina
sample (Fig. 4). Samples from Argentina and Rondônia were distantly separated along the first MDS axis (no
classification error between them). Acoustic features indicated as important in discriminating geographic samples
were pulse rate and dominant frequency, with calls from Rondônia being significantly different (Wilcox-Mann-

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Whitney test) from calls recorded in Argentina and Minas Gerais (pulse rate: vs. Argentina population, Z = -3.24, p
< 0.01; vs. Minas Gerais population, Z = -3.25, p < 0.01. Dominant frequency: vs. Argentina population, diagnostic
—no overlap—; vs. Minas Gerais population, Z = -3.19, p < 0.01) (Fig. 5). The difference in average dominant
frequency was significant between Minas Gerais and Argentina as well (Z = -3.66, p < 0.01).

FIGURE 4. Plot of the two first axes of a Multidimensional Scaling Analysis on the Random Forest result for the acoustic data
of Trachycephalus typhonius from Minas Gerais, Rondônia and Argentina. Note the complete discrimination between
Argentina and Rondônia populations. Discrimination was mostly due to differences in dominant frequency and pulse rate.

Calls of T. mesophaeus from São Paulo lasted between 148 and 178 ms, were spaced by intervals of 309–947
ms and were emitted at a rate of 95–108 calls/min (n = 22; air temp.: 22 ºC). Calls were composed of 45–55 pulses
emitted at a rate of 270–317 pulses/s. Five harmonics were observed. The third harmonic corresponded to the
dominant frequency and was located between 1292 and 1378 Hz. The fundamental frequency ranged between 602
and 689 (Fig. 6; Table 3).
Call comparisons with congeneric species. Acoustic features of the advertisement calls of eight
Trachycephalus species are summarized in Table 3. A multipulsed advertisement call is shared by all species of
Trachycephalus for which calls are known (Zimmerman & Hödl 1983; Hödl 1991; De la Riva et al. 1995;
Abrunhosa et al. 2001; Kwet & Solé 2008; Gordo et al. 2013; Dos Santos-Silva et al. 2012; present study).
Although not mentioned in a previous description of the calls of T. mesophaeus (Prado et al. 2003) we detected a
pulsed structure in call samples from São Paulo. Calls assigned to T. resinifrictix in Zimmerman & Hödl (1983) and
Hödl (1991) correspond to calls of T. cunauaru (Gordo et al. 2013).

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 TABLE 2. Temporal and spectral features of the advertisement calls of Trachycephalus typhonius populations evaluated in this study and reviewed in literature. For populations recorded in Argentina
and Brazil in this study, values of summary statistics are presented as mean ± 1 s.d. (range; n). For the remaining populations, values were transcribed as provided in the original publications.

Sites Argentina (Chaco, Brazil (Minas Brazil (Rondônia) Bolivia Brazil French Panama Panama and Peru Venezuela Venezuela
Corrientes, and Gerais) (n = 11 males) (Puerto (Goiás) Guiana (Nueva Costa Rica (Loreto) (n = not (Guárico)
Misiones) (n = 11 males) Almacén) (n (n = 1 male) (n = not Gorgona) (n = (n = not informed) (n = 7)
(n = 10 males) = not informed) (n = 1 male) 7males) informed)
Call features informed)
Call rate (calls/min) 46.8 ± 10.6 49.2 ± 10.0 44.9 ± 14.1 54.8 15.2 ± 6.8 26.75 67 47 33.3
(35.0–61.2; 10) (37.7–64.5; 11) (26.0–70.7; 11) (46–61) (8–26) (42– 52)

Call duration (ms) 509.0 ± 62.5 444.9 ± 71.2 413.1 ± 39.4 342.9 468.8 ± 470 400 300 400 400 456.04 ±
(410.7–598.2; 96) (343.1540.3; 108) (350.0–465.4; 65) (291–384) 60.3 (400–570) (230– 360) (380–410) 50.76
(422–573)
Inter-call interval (ms) 838.7 ± 243.0 824.7 ± 211.4 1062.± 358.7 1130 400–500 770
(452.0–1180.5; 88) (565.5–1094.9; 94) (637.0–1891.0; 48) (950–1300) (470–950)

Pulse per call 69.7 ± 6.7 63.9 ± 8.8 69.3 ± 8.2 52.2 65 ± 7.0 75.97 ±

410 · Zootaxa 4521 (3) © 2018 Magnolia Press

(59.0–77.3; 96) (51.8–76.9; 108) (58.5–87.0; 65) (45–60) (58–76) 8.95

Pulse rate (pulse/s) 137.1 ± 6.0 142.7 ± 8.3 167.2 ± 21.4 152.1 131.4 161 168.2
(129.5–144.5; 96) (123.1–153.7; 108) (129.6–204.4; 70) (140.1– (123.4– (150–175) (167.7–
157.3) 138.9) 172.8)

Pulse duration (ms) 3.4 ± 0.5 3.4 ± 0.4 3.1 ± 0.3 6.24 ± 0.6 8.33 ± 4.73
(3.0–4.2; 160) (2.7–4.0; 195) (2.9–3.8; 165) (5–8)

Inter-pulse interval (ms) 4.0 ± 0.5 3.5 ± 0.4 2.9 ± 1.0

(3.0–4.9; 160) (3.0–4.1; 195) (1.4–4.7; 165)
Dominant frequency 1824 ± 105 2146 ± 130 2430 ± 187 2143 2126 ± 84.2 1700–2420 1700– 2700 1622 not clear 2000–4000 2110 ± 220
(medial band)(Hz) (1705–2060; 98) (1881–2359; 108) (2156–2750; 65) (1878– (2074– (1392–
2546) 2276) 1946)
Lower band frequency 268 ± 14 565 ± 137 520 ± 184.0 652 270 159 250
peak (Hz) (258–294; 98) (258–656; 108) (281–785; 65) (626–666) (110–440) (139–183) (110–510)

Upper band frequency 2442 ± 181 2987 ± 132 3000 3530

peak (Hz) (2239–2727; 92) (2858–3194; 44) (2770– (2790–
3220) 3880)
Temperature (°C) 22–29 21–27 26–29 26 27.6

Reference Present study Present study This study De la Riva Guimarães Zimmerman Zweifel Duellman Zimmerman Rivero & Tárano
et al. (1995) et al. (2001) & Hödl (1964) (1970) & Hödl Esteves (2010)
(1983) (1983) (1969)

ZARACHO ET AL.
 TABLE 3. Temporal and spectral acoustic features of the advertisement calls of Trachycephalus species. When reported in more than one reference, means and ranges of acoustic features among
publications are provided.

T. atlas T. coriaceus T. cunauaru T. dibernardoi T. mesophaeus T. mesophaeus T. nigromaculatus T. resinifictrix T. typhonius

Call features

Call rate (calls/min) 71 29 101 8.6 47

59–80.2 27–34 24–36 95–108 3.8–15.2 26–70
Call duration (ms) 170 608 470 604 250 164 160 490 454
140 –220 399–970 260–550 470–760 148–178 150–170 350–630 343–598
Inter–call interval 350 1360 470 430–580 425 460 510 910
(ms) 220–540 310-960 309–947 290–1190 390–690 452–1891
Pulse per call 12 42 38 48 10 68
10–15 34–52 31–49 45–55 8–11 52–87
Pulse rate (pulse/s) 82 127–146 25–80 294 183 149
77–85 270–317 150–250 123–204

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Dominant frequency 1840 2090 830 ca. 1550 Hz 874–1153 1358 1290–1990 varies 2143
(kHz) 1690–1880 1959–2302 390–1400 (1100–1800) 1292–1378 1705–2750
Lower band frequency 780 300 600–900 646 370 457
peak (kHz) or 560–1500 210–380 602–689 230–460 258–785
fundamental
frequency
Upper band frequency ca. 3000 1980 2800–3400 1850 2637
peak (kHz) 1520–2690 1420–2200 2239–3194

Spectral structure 3 bands of 1 band of 6–13 harmonics 3 bands of 9 harmonics 5 harmonics 3 bands of 7–12 harmonics 3 bands of
frequency frequency frequency frequency frequency
Temperature (°C) 21-29 23 27 19 21 22 24 22–27 21–29

Locality Jeremoabo (Bahia, Puerto Almacén Paranaíta (Mato São Francisco de Reserva Biológica São Lourenco da Saquarema (Rio de INPA-WWF and NE Argentina,
Brazil) (Santa Cruz, Grosso) and Paula (Rio Grande de Duas Bocas, Serra (São Paulo, Janeiro, Brazil) Ducke Reserves NW and SE Brazil
Bolivia) Altamira do Sul, Brazil) Cariacica (Espírito Brazil) (Manaus) and
(Pará)(Brazil) Santo, Brazil) Juruti (Para)
(Brazil)
Reference Dos Santos–Silva De la Riva et al. Gordo et al. Kwet & Solé Prado et al. (2003) Present study Abrunhosa et al. Zimmerman & Present study
et al. (2012) (1995). (2013); (2008) (2001) Hödl (1983);
Zimmerman & Gordo et al.(2013)
Hödl (1983)

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411
FIGURE 5. Call features pointed as important in the discrimination of the three studied Trachycephalus typhonius populations
(Argentina, Minas Gerais and Rondônia).

Advertisement calls of Trachycephalus atlas and T. nigromaculatus can be distinguished from calls of all other
congeners by their lower number of pulses (10–15 and 8–11 pulses/call, respectively) and shorter duration (140–
220 and 150–170 ms, respectively) (Abrunhosa et al. 2001; Dos Santos-Silva et al. 2012). The call duration is also
short in T. mesophaeus (148–250 ms) (Prado et al. 2003; this study). Trachycephalus cunauaru, T. mesophaeus, T.
resinifrictix and T. typhonius (pulse rates above 100 pulses/s) have a higher pulse rate than T. dibernardoi (31–49
pulses/s, Kwet & Solé 2008), T. atlas (50–80 pulses/s, Dos Santos-Silva et al. 2012) and T. coriaceus (34–52
pulses/s, De la Riva et al. 1995). In turn, T. mesophaeus (270–317 pulses/s, this study) has the higher pulse rate
among Trachycephalus species. Moreover, calls of T. typhonius (2133 Hz) can be distinguished from T. cunauaru
(830 Hz) by its higher dominant frequency (Gordo et al. 2013), while from T. resinifrictix (4–33/min) (Zimmerman
& Hödl 1983) it can be distinguished by its higher call rate (35–71/min). Also, T. resinifrictix often emits calls in
series of 2–6 (Zimmerman & Hödl 1983). Additionally, the harmonic structure in the advertisement calls is shared
only by Trachycephalus mesophaeus (Prado et al. 2003; this study), T. cunauaru and T. resinifrictix (Zimmerman
& Hödl 1983, Gordo et al. 2013).

Discussion

In this study, we described calls of the geographically widespread treefrog Trachycephalus typhonius proceeding
from ecologically and climatically very disparate regions of South America. Call samples from Argentine Chaco

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correspond to the southernmost sound records for this species. Calls recorded in Rondônia represent the first calls
described for this species proceeding from the Brazilian Amazon forest. Along with those recorded in Minas
Gerais, in general, these call samples are similar to those of other previously described for T. typhonius populations
from Nueva Gorgona, Panamá (Zweifel, 1964), Venezuela (Rivero & Esteves 1969), other locations in Panamá and
Costa Rica (Duellman 1970), Peru, French Guiana (Zimmerman & Hödl 1983), Puerto Almacén, Bolivia (De la
Riva et al. 1995), and Goiás, Brazil (Guimarães et al. 2001) (Table 2).
We found significant statistical differences in pulse rate and dominant frequency among calls from Rondônia
and those from Minas Gerais and Argentina. Based on these acoustic differences, other characters with taxonomic
relevance, such as morphology and DNA sequences, should be considered in order to evaluate the existence of
cryptic species under this taxon. Recently, a study based in molecular data and external morphology assessed the
status of Ecuadorian and Peruvian populations of T. typhonius and, as result, two species were revalidated:
Trachycephalus quadrangulum (Boulenger, 1882) and Trachycephalus macrotis (Andersson, 1945) (Ron et al.
2016). Unfortunately, advertisement calls of these two species are unknown.
Even though not evaluated here, it is possible that different calling sites (perched vs. floating on water) may
influence call features of T. typhonius, as the relative frequency of perched and floating males varied greatly among
sampling regions. However, in order to evaluate this potential effect, an experimental approach is necessary,
comparing call recordings of the same specimens vocalizing while perched and while immersed in water.

FIGURE 6. Audiospectrogram (upper graph, FFT = 512) and corresponding oscillogram (lower graph) of one call of
Trachycephalus mesophaeus from São Lourenco da Serra (São Paulo, Brazil). Note the harmonics and pulsed structure of the
call. Record file: Trachycep_mesophaeusSLourencoSerraSP1bAAGm661MK2. January 24, 2018; 23:05h; air = 22 ºC.

A major taxonomic impediment to the recognition of the species diversity under the name T. typhonius is that
the original description of T. typhonius did not designate a precise type locality, stated simply as “América” by
Linnaeus (1758). Recently, the specimen UUZM 134 was identified as the holotype and the type locality was
restricted to Paramaribo, Suriname (Lavilla et al. 2010). Thus, the description of T. typhonius from calls from
French Guiana by Zimmerman & Hödl (1983) corresponds to the nearest acoustic record to the species type
locality (Fig. 1). Temporally and spectrally, calls described therein are similar to those provided in other
descriptions (including that of Zimmerman & Hödl 1983), except by the presence of multiple harmonics.
The presence of multiple harmonics (up to 23) was previously mentioned by Zweifel (1964), Duellman (1970)
and Zimmerman & Hödl (1983) based on populations from French Guiana, Mexico, Panama, Peru and Venezuela.

ADVERTISEMENT CALL OF TRACHYCEPHALUS TYPHONIUS Zootaxa 4521 (3) © 2018 Magnolia Press · 413
The populations analyzed in the present study exhibit up to three frequency bands, what has not been previously
reported in T. typhonius, but is a feature of the calls of T. dibernardoi (Kwet & Solé 2008) and T. atlas (Dos Santos-
Silva et al. 2012). Among these three bands, the central is the dominant and the upper band (a true harmonic) is
often barely discernible. However, the lower band is not harmonically related to the other two bands and can be
attributed to the pulsatile nature of the call (Hepp et al. 2017). We only observed additional frequency bands when
calls were viewed with a window size of 1024 points, what in reality represent sidebands (Frommolt 1999), as they
are not multiples of the fundamental frequency. Hence, it is possible that sidebands were previously inaccurately
taken as harmonics in some of the aforementioned studies. Sidebands appear also to be mistaken for true harmonics
in descriptions of calls of T. resinifictrix, T. mesophaeus and T. cunauaru (Zimmerman & Hödl 1983; Prado et al.
2003; Gordo et al. 2013), but this requires further investigation. For a better evaluation of the spectral features of
advertisement calls in Trachycephalus, a reevaluation of existent and new records is necessary, employing a unified
analysis and standardized terminology.

Acknowledgments

Diego Baldo kindly provided the record of a specimen from Puerto Iguazú (Argentina). Two anonymous reviewers
improved the manuscript. This research was supported by grants PICTO 2011-0244 and SGCYT-UNNE 16F012
(Argentina). A grant was provided to AAG by CNPq; financial support to AAG by CNPq and FAPEMIG. Davi L.
Bang helped AAG in fieldwork in Brazil.

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APPENDIX 1. Audio files analyzed

BRAZIL
Minas Gerais State
Uberlândia:
Trachyc_venulUberlMG1bAAGm . wav
Araguari:
Trachyc_venulAraguariMG1aAAGm1.wav
Trachyc_venulAraguariMG2aAAGm1.wav
Trachyc_venulAraguariMG3bAAGm.wav
Trachyc_venulAraguariMG4bAAGm.wav
Trachyc_venulAraguariMG5aAAGm.wav
Ituiutaba:
Trachyceph_venulosusItuiutabaMG13bAAGm671.wav
Trachyceph_venulosusItuiutabaMG13cAAGm671.wav
Trachyceph_venulosusItuiutabaMG15aAAGm671.wav
Trachyceph_venulosusItuiutabaMG17aAAGm671.wav

ADVERTISEMENT CALL OF TRACHYCEPHALUS TYPHONIUS Zootaxa 4521 (3) © 2018 Magnolia Press · 415
Trachyceph_venulosusItuiutabaMG18aAAGm671.wav

Rondônia State
Costa Marques:
Trachycep_typhoniusCostaMarquesRO1aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO2aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO3aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO4aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO6aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO7aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO8aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO9aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO10aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO11aAAGm671.wav
Trachycep_typhoniusCostaMarquesRO12aAAGm671.wav

ARGENTINA

Chaco Province:
Trachycephalus_typhonius_VHZ-MT2_208c_novoucher_LaLeonesa_file0051.wav
Trachycephalus_typhonius_VHZ-MT2_209_voucherUNNEC13080_LaLeonesa_file0053.wav
Trachycephalus_typhonius_VHZ-MT2_210a_novoucher_LaLeonesa_file0055.wav
Trachycephalus_typhonius_VHZ-MT2_210b_novoucher_LaLeonesa_file0056.wav

Corrientes Province:
Trachycephalus_typhonius_VHZ-MT2_347b_novoucher_Corrientes_file0719.wav
Trachycephalus_typhonius_VHZ-MT2_348_novoucher_Corrientes_file0721.wav
Trachycephalus_typhonius_VHZ-MT2_349b_novoucher_Corrientes_file0723.wav
Trachycephalus_typhonius_VHZ-MT2_350_novoucher_ Corrientes_file0724.wav
Trachycephalus_typhonius_VHZ-MT2_352_novoucher_ Corrientes_file0727.wav
Trachycephalus_typhonius_VHZ-MT2_366c_novoucher_ Corrientes_file0777.wav

416 · Zootaxa 4521 (3) © 2018 Magnolia Press ZARACHO ET AL.