Herpetology Notes, volume 12: 113-204 (2019) (published online on 1 January 2019)

Filling knowledge gaps about the ‘Data Deficient’ species carvalhoi Izecksohn, 1983 (Anura, ): an endemic from the Atlantic Forest of southeastern Brazil

Jhonny J. M. Guedes1,*, Clodoaldo L. de Assis1, Camila M. Novaes2, Jorge A. Dergam3, and Renato N. Feio1

Abstract. Zachaenus carvalhoi is an endemic frog from the Atlantic Forest of southeastern Brazil. It is considered a rare species, being currently classified as ‘Data Deficient’ by the IUCN due to the shortage of information about its natural history, ecological requirements, distribution and population trends. In the present study, we aim to fill gaps in the current knowledge of this species by: updating its altitudinal distribution range; describing its release call; providing new natural history data; and describing its karyotype. During fieldwork in the Municipality of , State, we recorded individuals of Z. carvalhoi in three small forest fragments, one of them at only 230 m a.s.l., which now represents the lowest known altitudinal record for this species. In all occasions, we only recorded this frog in calling activity during rainy days, suggesting an opportunistic breeding strategy. Moreover, the karyotype of Z. carvalhoi (2n = 26) is similar to those of other species of the family Cycloramphidae, but it differs remarkably in morphology and fundamental number. The advertisement call presented differences in the spectral structure in comparison to descriptions from other populations. In conclusion, Z. carvalhoi seems to be an explosive breeder occurring along a wide altitudinal range (low- and highland Atlantic Forest), and physical barriers such as mountain chains may not be a constraint to its dispersal. Furthermore, this species represents a promising subject for investigations over both bioacoustics and cytogenetic studies.

Keywords. Altitudinal distribution, bioacoustics, karyotype, natural history

Introduction human-related impacts we now face a global decline of species (Blaustein et al., 1994; Collins Every year, dozens of anuran species are described and Storfer, 2003; Grant et al., 2016), the lack of such worldwide (Glaw and Köhler, 1998; Pimm et al., data may imply in severe consequences to biodiversity 2010; Frost, 2018), and more than 7,800 species have conservation and species management because it may been formally described so far (Frost, 2018). However, hamper a broader application of these data in basic and for many of these taxa, basic information on natural applied research, leading to inefficient conservation history and ecology are still missing (see Oliveira et planning (Bury, 2006). Therefore, providing such kind al., 2017). In a rapidly changing world, where due to of information is fundamental in decreasing current shortcomings, besides contributing to solve taxonomic, phylogenetic, biogeographic and conservation issues. The genus Zachaenus Cope, 1866 is composed of small leaf-litter endemic to the Brazilian Atlantic 1 Departamento de Biologia , Museu de Zoologia João Forest (Verdade et al., 2009), and currently comprises Moojen, Universidade Federal de Viçosa, Viçosa, Minas two valid species: (Girard, 1853) Gerais 36570-900, Brazil. and Z. carvalhoi Izecksohn, 1983 (Frost, 2018). The 2 Departamento de Biologia Geral, Laboratório de Biologia former occurs throughout Rio de Janeiro State, the Molecular de Insetos, Universidade Federal de Viçosa, northern region of São Paulo State, and the south- Viçosa, Minas Gerais 36570-900, Brazil. central region of Espírito Santo State (Verdade et al., 3 Departamento de Biologia Animal, Laboratório de Sistemática Molecular-Beagle, Universidade Federal de Viçosa, Viçosa, 2009; Oliveira et al., 2012). The latter is known only Minas Gerais 36570-900, Brazil. from eight localities, occurring along highland areas in * Corresponding author. E-mail: [email protected] the Mantiqueira mountain range in the State of Minas 114 Jhonny J. M. Guedes et al.

Gerais, and Cariacica and Santa Teresa (type-locality) classified as type Aw in Köppen’s system, with mean in the State of Espírito Santo (Mollo-Neto et al., 2016). annual temperature varying between 22 and 24° C, The available information regarding Zachaenus mean annual precipitation around 1,300 and 1,600 mm carvalhoi relies basically on anecdotal reports about its (Alvares et al., 2013), with a rainy season from October distribution, defensive behaviour, diet and advertisement to April and a dry season from May to September (Sá call (Dayrell et al., 2006; Verdade et al., 2009; Motta et Júnior et al., 2012). The region is part of the Atlantic al., 2010; Moura et al., 2010; Salles and Maciel, 2010; Forest biome (sensu IBGE and MMA 2004), and was Coelho-Augusto et al., 2013; Guimarães et al., 2013; originally covered by montane and submontane seasonal Zocca et al., 2014; Mollo-Neto et al., 2015). Only semideciduous forests (Veloso et al., 1991). Nowadays, recently a more comprehensive study was published it has less than 12% of forest cover remaining, mostly (Mollo-Neto et al., 2016), dealing with its reproduction distributed in small fragments of secondary forest (SOS and geographic variation. Furthermore, based on the Mata Atlântica and INPE, 2018). low densities at which individuals are known to occur, Fieldwork.—We performed monthly surveys from threats imposed by habitat loss and fragmentation, and December 2012 to November 2013 in a locality called fast decline of habitat quality along the Atlantic Forest biome, Mollo-Neto et al. (2016) also suggested the Serra da Neblina/Sapecado (21.2091°S, 42.7558°W; reallocation of Z. carvalhoi to the ‘Near Threatened’ Datum WGS84; 912 m a.s.l.). Individuals of Z. category of the IUCN Red List, instead of the current carvalhoi were surveyed using five linear pitfall trap ‘Data Deficient’ status. In fact, threats such as habitat arrays (Ribeiro-Júnior et al., 2011), where each trap loss and fragmentation can severely reduce amphibian line was 50 m long, made of six 65 l buckets buried survival rates and result in population declines into the ground, set at 10 m intervals, and connected (Eterovick et al., 2005; Cushman, 2006; Tarvin et al., by a 50 cm tall drift fence of plastic canvas. The traps 2014), especially in forest dependent species. Therefore, remained active during five days in each month, and we the effects of such threats to amphibian conservation visited each trap line daily, during daytime. Moreover, should be carefully investigated. between 2013 and 2017, we randomly searched for So far, we are aware that Z. carvalhoi was not yet individuals by active search (Fitzgerald, 2012) in the studied cytogenetically, still lacks description of some surroundings of the area in which the traps were set, as parameters of its advertisement call, and its release well as in different forest fragments at the study site. call is unknown. Besides that, the lack of data about We conducted the searches during afternoon and night, distribution and/or abundance imposes especial issues through transects in the interior and border of such when determining in which category a species should fragments. be placed regarding its conservation framework (IUCN, Collected specimens (see Appendix I) were killed 2017). In this sense, herein we: (1) describe the release by intraperitoneal injection of Xylocaine® (lidocaine call of Z. carvalhoi; (2) update its altitudinal distribution hydrochloridae), tagged, and fixed in 10% formalin range; (3) provide new data about its natural history; before being transferred to a solution of 70% ethanol and (4) describe its karyotype. for storage. We identified the specimens as Z. carvalhoi based on the absence of dorsal folds and axillary Material and Methods patagium, as well as by the dark-brown coloration of its venter, with white dots of variable sizes (Izecksohn, Study site.—All data presented herein are based on 1983 “1982”). We deposited vouchers in the Museu specimens collected and/or observed during the course de Zoologia João Moojen (MZUFV), Universidade of a herpetological inventory in the Municipality of Federal de Viçosa, Viçosa Municipality, Minas Gerais Cataguases (21.3752ºS, 42.6855°W; Datum WGS84; State, Brazil. 179 m a.s.l.), Minas Gerais State, southeastern Brazil. For detailed information on the methodology used, as Acoustic analysis.—In the field, we recorded the well as other taxa recorded in the study site, see Guedes advertisement call of Z. carvalhoi using a Sony ICD et al. (2017) for lizards and amphisbaenians, and Assis PX721 digital recorder with an internal microphone and Feio (2017) for . (only the temporal parameters of these recordings were The study site varies in elevation from 170 to 1,200 used in further call analysis). To convert the calls from m, being drained by the Pomba river sub-basin, within MP3 to WAV format we used the software Adobe the Paraíba do Sul river basin. Climate is tropical, Audition CC 2018. In the laboratory, we simulated a Filling knowledge gaps about Zachaenus carvalhoi in Brazil 115

Figure 1. Individual Zachaenus carvalhoi collected in Cataguases Municipality, Minas Gerais State, southeastern Brazil (A) and giemsa-stained karyotype of the specimen (B).

false axillary amplexus by gently pushing a collected spectral parameters we used: “Peak Frequency (Hz)”, specimen (MZUFV 18261; Fig. 1A), using thumb “Frequency 5% (Hz)”, and “Frequency 95% (Hz)”. and forefinger until a release call was emitted. In this Acoustic terminology follows Köhler et al. (2017). circumstance we used a Tascan DR-40 digital recorder We deposited all recordings at the Fonoteca Neotropical with an internal microphone, sampling rate of 48 kHz, Jacques Vielliard (FNJV) of the Universidade Estadual and resolution of 16 bits for recording. de Campinas, São Paulo, Brazil. We compared We measured spectral and temporal parameters using our recordings with other calls available in the Raven Pro 1.5 (Bioacoustics Research Program, 2014) literature, and their acronyms at FNJV are as follow: directly and respectively from the spectrogram and ZUFABC = Laboratório de Evolução e Diversidade oscillogram. Spectrogram was generated with window of the Universidade Federal do ABC; and ASUFRN = type Hann; window size = 512 samples; overlap = 70%; Laboratório de Anfíbios e Répteis of the Universidade hop size = 3.21 ms; DFT size = 1024 samples and; Federal do Rio Grande do Norte. grid spacing = 46.9 Hz. We observed and measured Cytogenetic analyses.—We treated one male specimen the following parameters: (1) Call structure (simple (MZUFV 18261; Fig. 1A) with 0.1% colchicine solution or compound); (2) spectral structure (harmonic or/and in the ratio of 1 ml/100 g body weight for a period of multipulsed); (3) call duration (between the beginning four hours before being processed. After its death, we and ending of a call within a series of calls); (4) call obtained the mitotic chromosomes from cell suspensions interval; (5) call rate per minute; (6) call series duration of intestinal epithelium through the crushing technique (between the beginning of the first call and the ending (Bogart, 1973). Chromosomes were stained with 3% of the last one within a series of calls); (7) call series Giemsa, and examined under a photomicroscope intervals; (8) calls per series; (9) notes per call; (10) OLYMPUS BX-60 coupled to an image capture and note duration; (11) notes interval; (12) note rate per analysis system. Finally, we organized the metaphases second; (13) pulses per note; (14) pulse duration; (Fig. 1B) using an image-editing program, established (15) pulses interval; (16) pulse rate per second; (17) the diploid number, and characterized the morphology dominant frequency; (18) minimum frequency; (19) of the chromosomes according to the ratio of arms maximum frequency. For measurements of temporal (Levan et al., 1964). parameters, we calculated the difference between begin and end time for a selected call/note/pulse, and for 116 Jhonny J. M. Guedes et al.

Results three visible harmonics without frequency modulation (Fig. 2). The series of calls have a duration of 0.81–1.83 Between the months of September and February s (n = 8 series of calls) and an interval from 14.7–43.1 (rainy season), we collected 10 specimens of Zachaenus s (n = 7 intervals). The calls had 3–4 pulsed notes (Fig. carvalhoi using pitfall traps. Through active search, we 2A), with duration of 0.12–0.29 s (n = 28 calls) and heard males in calling activity in five occasions: three intervals of 0.25–0.34 s (n = 20 intervals). The notes of them during 7–10 September 2013 and 2 October had 2–11 pulses (Fig. 2B), with duration of 0.009–0.13 2013, at the same site where the pitfall trap system s (n = 93 notes) and intervals of 0.01–0.05 s (n = 65 was installed. In all occasions specimens were calling intervals). Lastly, the duration of pulses varied between hidden under the leaf litter in the interior of the forest. 3–12 ms (n = 210 pulses) and the intervals between In September it had not rained yet, and in two occasions pulses ranged from 0–14 ms (n = 160 intervals). The we observed only a few specimens vocalizing, with repetition rates of calls, notes and pulses were 7.5/min, longer intervals between series of calls, allowing to 13.3–33.6/s and 74.7–250/s, respectively (Table 1). The individualize each of them. Vocalizations started in the specimen kept calling at this frequency until 17:30 h, afternoon around 17:30 and ceased after 20:30 h, when time in which the rain ceased, and completely stopped the air temperature rose from 16 to 19º C. calling activity at nightfall. In October, several males started to vocalize intensely The other two recordings were made in different at 14:00 h, during a heavy rain. Calls overlapped and forest fragments. One of them at a locality called it was not possible to precisely estimate the number “Santa Maria”, and the other in a small forest fragment of calling males. Between 15:00 and 15:30 h, with the at “Mata da EMPA” (Table 2). In the first locality, air temperature close to 19º C, we recorded within a on 16 October 2015 (18:00 h; air temperature 20º C) distance of 50 cm, a specimen of Z. carvalhoi (MZUFV we observed two specimens vocalizing discreetly and 16081). The advertisement call is composed by series of scarcely until approximately 19:00 h. In the second 2–4 calls with pulsatile-harmonic notes, including up to place, on 21 October 2016, we collected a specimen

Figure 2. Waveform (above) and spectogram (below) of the advertisement calls of Zachaenus carvalhoi (MZUFV 16081) from the Municipality of Cataguases, Minas Gerais State, southeastern Brazil: (A) sequence of two calls, and (B) sequence of six notes. Filling knowledge gaps about Zachaenus carvalhoi in Brazil 117

Table 1. Parameters of the release and advertisement call of Zachaenus carvalhoi. Data type: CS = call structure; SS = spectral structure; CD = call duration (s); CI = call interval (s); CR = call rate/min.; CSD = call series duration (s); CSI = call series interval (s); CPS = call per series; NC = notes per call; ND = notes duration (s); NR = note rate/sec.; NI = notes interval (s); PN = pulses per notes; PD = pulses duration (ms); PR = pulse rate/sec.; PI = pulses interval (ms); DF = dominant frequency (Hz); MinF = minimum frequency (Hz); MaxF = maximum frequency (Hz).

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Table 2. Recorded localities for Zachaenus carvalhoi arranged in increasing elevation. Coordinates are given in decimal degrees (Datum WGS84). References: 1, 2 and 11 = This study; 3 and 12 = Verdade et al. (2009); 4 = Dayrell et al. (2006); 5 = Mollo Neto et al. (2016); 6 = Salles and Maciel (2010); 7 = Izeckson (1983); 8 and 10 = Mollo Neto et al. (2015); 9 = Zocca et al. (2014); 13 Table 2. Recorded localities for Zachaenus carvalhoi arranged in increasing elevation. Coordinates are given in decimal degrees (Datum WGS84). References: 1, 2 = Guimarães et al. (2013); 14 = Motta et al. (2010); 15 = Moura et al. (2010). Legend: REBIO = Reserva Biológica; PARNA = and 11 = This study; 3 and 12 = Verdade et al. (2009); 4 = Dayrell et al. (2006); 5 = Mollo Neto et al. (2016); 6 = Salles and Maciel (2010); 7 = Izeckson (1983); 8 and 10 = Mollo Neto Parqueet al. (2015); Nacional; 9 = EMPAZocca et= al.EMPA (2014); S/A 13 Serviços= Guimarães de Engenharia. et al. (2013); 14 = Motta et al. (2010); 15 = Moura et al. (2010). Legend: REBIO = Reserva Biológica; PARNA = Parque Nacional; EMPA = EMPA S/A Serviços de Engenharia.

Nº on map Locality Latitude Longitude Elevation 1 Cataguases - Mata da EMPA 21.4290ºS 42.7033ºW 230 m 2 Cataguases - Santa Maria 21.2336ºS 42.6917ºW 415 m 3 Ca riacica - REBIO Duas Bocas 20.2852ºS 40.4738ºW 560 m 4 Pe dra Dourada 20.7830ºS 42.1666ºW ca. 630 m 5 Vi çosa - Mata do Seu Nico 20.7977ºS 42.8463ºW 770 m 6 Ju iz de Fora - Park 21.7925ºS 43.3677ºW 770–850 m 7 Sa nta Teresa* 19.9330ºS 40.6000ºW ca. 800 m 8 Ju iz de Fora - Floresta farm 21.7425ºS 43.2919ºW 809 m 9 Sa nta Teresa - REBIO Augusto Ruschi 19.8888ºS 40.5458ºW 834–880 m 10 - REBIO Poço D'Anta 21.7538ºS 43.3105ºW 859 m 11 Cataguases - Serra da Neblina 21 .2090ºS 42.7559ºW 912 m 12 Ibitirama - PARNA Caparaó 20 .4672ºS 41.7338ºW 1135 m 13 Ervália - District of Careço 20 .8875ºS 42.5227ºW 1236 m 14 Muriaé - Serra do Brigadeiro 20 .8835ºS 42.5170ºW 1236 m 15 Araponga - Serra do Brigadeiro 20.7216ºS 42.4783ºW 1380 m

*Type - locality

(MZUFV 18261; Fig. 1A). Males started vocalizing observed four frequency modulation types: sinusoidal around 20:30 h (air temperature 26º C), after a heavy rain (n = 3; Fig. 3A); descendant in the initial portion (n = that lasted ca. 15 minutes. Individuals were frequently 1, Fig. 3B); descendant in the beginning followed by calling, however, in this case the calls did not overlap, ascendant modulation in the final part (n = 3, Fig. 3B); being possible to individualize each one. When the rain and ascendant in the initial portion and descendant in stopped, vocalization ceased as well. We also recorded the final portion (n = 1, Fig. 3C). The peak of dominant other anuran species, such as Ischnocnema gr. parva , frequency coincided with the second harmonic in I. verrucosa , Haddadus binotatus and Physalaemus eight calls, and with the eighth harmonic in one call. signifer , vocalizing at the same time and close to The minimum and maximum frequency and the peak specimens of Z. carvalhoi . of dominant frequency was respectively, 937.5–2250 Regarding the release call, during stimulation, the Hz, 2156–12375 Hz and 1500–8671 Hz (n = 9 calls). male emitted 12 isolated calls or in series of up to Calls “A” have four types of amplitude modulation: three calls, which were recorded within a distance of ascending gradually, with a peak in the final portion 10 cm (air temperature not measured). The calls are of the call, followed by an acute decay (Fig. 3A); composed of one pulsatile-harmonic note, with up ascending gradually until the meddle portion, followed to 29 visible harmonics, where most of them present by a gradual decay (Fig. 3B); ascending sharply, with a frequency modulation. We identified to different peak in the initial part, followed by a gradual decay (Fig. call types: one with higher amplitude e duration time 3C); and ascending sharply, with a peak in the middle (release call “A”); and other with smaller amplitude portion, followed by a sharp decay (last call in Fig. 3F). and duration type, consisting of a click-like call (release The release call “B” has duration of 0.007–0.01 s, and call “B”). The release call type “A” has duration of the minimum and maximum frequency and the peak of 0.03–0.21 s (n = 9 calls) and intervals of 0.008–0.02 dominant frequency was respectively, 1453.1–2296.9 s (n = 3 intervals) (Fig. 3A–C, 3F). In this case, we Hz, 4171.9–8390.6 Hz and 1546.9–4078.1 Hz (n = 3 Filling knowledge gaps about Zachaenus carvalhoi in Brazil 119

Figure 3. Waveform (above) and spectogram (below) of the release calls of Zachaenus carvalhoi (MZUFV 18261) from the Municipality of Cataguases, Minas Gerais State, southeastern Brazil: (A–C) release call type “A”, (D–E) release call type “B”, and (F) sequence of three release calls, being the first type “A” and the other two type “B”.

calls). We did not observe frequency modulation for Discussion call type “B”, and the amplitude modulation was the The presence of Zachaenus carvalhoi at the study same for all three calls, with a peak in the initial part, site is somewhat expected due to its current geographic followed by a gradual descending (Fig. 3D–E and first call in 3F). distribution (see Mollo-Neto et al., 2016). However, We analysed the karyotype of a collected male this species is mainly associated to mountainous areas specimen, which presented 2n = 26 chromosomes, with (Verdade et al., 2009) and was previously known to four metacentric pairs (1, 2, 5, 10), three submetacentric occur at elevations above 560 m (lowest record at pairs (3, 4, 6) and six telocentric pairs (7, 8, 9, 11, 12, 13) Cariacica Municipality, Espírito Santo State), whereas with a fundamental number equal to 40 (Fig. 1B). We specimens collected in this study were found at 415 and did not detect any heteromorphic sexual chromosomes 230 m a.s.l. (new lowest altitudinal record). Therefore, or supernumerary elements in this species. since this species can be found from 230 to over 1300 200 Jhonny J. M. Guedes et al.

Figure 4. Updated geographic distribution of Zachaenus carvalhoi in southeastern Brazil. Inset map shows the Brazilian political divisions. Rectangle points out the general location of the distribution of Z. carvalhoi. Circles: literature records; Triangles: new records. States abbreviations: MG = Minas Gerais; RJ = Rio de Janeiro; ES = Espírito Santo. For specific localities (numbers on map) see Table 2.

m a.s.l. (Table 2; Fig. 4), elevation might not represent few specimens currently housed in scientific collections a barrier to dispersal for this particular species, which (Zocca et al., 2014). Data on its calling activity are based may present a high physiological plasticity to be able to on nocturnal records and mostly associated with rainy tolerate, for example, distinct gradients of moisture and days (Verdade et al., 2009; Motta et al., 2010; Guimarães temperature along its altitudinal range. et al., 2013; Mollo-Neto et al., 2016). In this study, we About Z. carvalhoi being commonly associated to recorded, at the same site, few specimens calling in days highland areas, it may be only an artefact derived from with no rain, as well as a great number of males calling the fact that most herpetological studies have been during a heavy rainy day. It suggests that this species conducted in such regions (e.g., Cruz and Feio, 2007; may present an opportunistic reproductive behaviour Lacerda et al., 2009; Moura et al., 2010; Santana et al., (sensu Abrunhosa et al., 2006), which is reinforced 2010; Zocca et al., 2014; Neves et al., 2017). This is by the seasonality of the region and its terrestrial quite understandable since most of the native remaining reproductive mode as well (sensu Haddad and Prado, vegetation, as well as protected areas, are concentrated in 2005), restricting them to moist environments for the such regions (Feio and Ferreira, 2005). Hence, lowland survival of their eggs (Crump, 2015). Moreover, the fact areas, which are highly fragmented and modified by we did not collect too many specimens, both in the pitfall human activities (Tabarelli et al., 2010), are generally traps and during active searches, may be related to the neglected and undersampled. However, such areas may semifossorial habit of this species, as it has been found house other populations of this species, aside from other from 5 to 20 cm deep into the leaf litter (Mollo-Neto et anurans considered so far as exclusive inhabitants of al., 2016). Therefore, the rarity commonly attributed to mid-elevation/highland areas. this species may be allied to a set of factors related to its Zachaenus carvalhoi is considered a rare species, biology, which makes it difficult to record. occurring at low population density and with only a In relation to its advertisement call, we found Filling knowledge gaps about Zachaenus carvalhoi in Brazil 201 discrepancies from the literature in relation to the be used in taxonomic and phylogenetic studies (Köhler interval between series of calls and the spectral et al., 2017). structure. Mollo-Neto et al. (2016) recorded, in early The diploid number (2n = 26) observed in Zachaenus November, a time interval of 20 minutes between series carvalhoi is identical to the diploid number in Z. parvulus of calls, or even more, for a population from Juiz de (Campos, 2010), as well as in other taxa that are part of Fora Municipality, Minas Gerais State. In this case, the family Cycloramphidae (Campos, 2010; Noleto et specimens were vocalizing at night, with air temperature al., 2011), corroborating the numerical conservatism of 20º C, under heavy rain, at a site between 700 and 900 the karyotypes of species within this family. However, m a.s.l. Despite some similarities between this record the morphology of the chromosomes of Z. carvalhoi and the one we provide herein (heavy rain, altitude of presents several differences when compared to other 912 m, air temperature 19º C), it is known that anurans species from its family, being noteworthy the presence of belonging to the same species may present distinct six pairs of telocentric chromosomes (7, 8, 9, 11, 12 and temporal parameters, or even reproductive strategies, 13; Fig. 1B), resulting in a fundamental number (n = 40) in different geographic areas or time periods (Pombal quite different from the others. This feature is unusual Jr. and Haddad, 2005; Morais et al., 2012). Therefore, in the karyotype of Cycloramphidae, since Zachaenus we expect that Z. carvalhoi may also display some parvulus (2n = 26, NF = 52) and miliaris (2n plasticity regarding its temporal parameters as well as in = 26, NF = 52) do not present chromosomes with such its reproductive strategy throughout its distribution. morphology (Campos, 2010), and some species within About the spectral structure of the advertisement call (2n = 26, NF = 50 and 52) have only one of Z. carvalhoi, we detected harmonics and therefore pair of telocentric chromosomes (Noleto et al., 2011). classified it as pulsatile-harmonic, which is a feature not This variation might be very relevant, considering the mentioned in the literature so far (see Guimarães et al., current taxonomic and phylogenetic confusion within 2013; Molo-Neto et al., 2016). However, the presence the family Cycloramphidae (Faivovich et al., 2014; or absence of this characteristic may be related to the Sabbag et al., 2018). In order to clarify such karyotypic way the recording was made, in which recordings at differences, additional studies such as chromosome a shorter distance are more prone to detect harmonics banding and sequence markings may be valuable. than recordings at longer distances (Köhler et al., Our results reveal novel and peculiar features of 2017). Since available recordings of Z. carvalhoi in the Zachaenus carvalhoi, such as: a karyotype quite different literature do not specify the distance in which specimens morphologically from those in other species of the were recorded, we cannot confirm whether the presence family Cycloramphidae; an advertisement call differing of harmonics represents variation among populations or in its spectral structure from the advertisement call of just methodological differences during recordings. other populations; and a wider altitudinal distribution The temporal and spectral parameters of the release than previously known. Moreover, we highlight that call of Z. carvalhoi overlapped in all values (except by this species is currently classified as ‘Data Deficient’ call duration in type “B” call) when compared to its by the IUCN Red List (Peixoto and Silvano, 2004), and advertisement call (Table 1; Fig. 3). Such similarities it has been proposed the reassignment of Z. carvalhoi are commonly observed for many anuran species (e.g., to the ‘Near Threatened’ category (Mollo-Neto et al., Garda et al., 2010; Dias et al., 2014; Nali et al., 2015; 2016). However, we suggest that such a change should Stănescu et al., 2018), what reinforces the hypothesis be based on sound biological information about this that the advertisement call originated from simple release species. Finally, we stress that Z. carvalhoi is a forest calls (see Schmidt, 1988, 1990). Also, we highlight inhabitant, and threats imposed by anthropic activities, the presence of two distinct release calls (type “A” especially habitat destruction and fragmentation, and “B”) in Z. carvalhoi, a feature described for other may severely impact this species causing population anuran species, where even differences in the spectral declines or even local extinctions. In this sense, the structure among call types were reported (Batista data we provide herein help to fill gaps on the current et al., 2017; Yakar et al., 2017). Moreover, release knowledge of Z. carvalhoi, and will certainly contribute calls are unknown for other species belonging to the to solve still existing ecological, biogeographic and family Cycloramphidae, which hampers comparisons phylogenetic questions, as well as to determine its due in the present study, but our data coupled with future conservation framework. descriptions may provide relevant information that can 202 Jhonny J. M. Guedes et al.

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Accepted by Fabrício Oda