ZOOTAXA

ISSN 1175-5326 (print edition)

ISSN 1175-5334 (online edition)Copyright © 2014 Magnolia Press

Zootaxa 3861 (3): 249–264

www.mapress.com/zootaxa/Article

http://dx.doi.org/10.11646/zootaxa.3861.3.3

http://zoobank.org/urn:lsid:zoobank.org:pub:FFCFBCF2-F2BF-44ED-ACD7-652643DBE362

A new species of Pseudopaludicola Miranda-Ribeiro, 1926 (Anura:

Leptodactylidae: Leiuperinae) from northwestern state of São Paulo, Brazil

ANDRÉ PANSONATO1,7, JESSICA RHAIZA MUDREK2, ANA CRISTINA PRADO VEIGA-MENONCELLO3,

DENISE DE CERQUEIRA ROSSA-FERES4, ITAMAR ALVES MARTINS1,5 & CHRISTINE STRÜSSMANN2,6

1Pós-Graduação em Biologia Animal, Universidade Estadual Paulista (UNESP), Rua Cristóvão Colombo, 2265, Jardim Nazareth,

15054–000, São José do Rio Preto, São Paulo, Brazil2Pós-Graduação em Ecologia e Conservação da Biodiversidade, Instituto de Biociências, Universidade Federal de Mato Grosso

(UFMT), Instituto de Biociências, Universidade Federal de Mato Grosso (UFMT), Av. Fernando Correa da Costa, 2367, Boa Esper-

ança, 78060–900, Cuiabá, Mato Grosso, Brazil3Departamento de Biologia Estrutural e Funcional, Instituto de Biologia, Universidade Estadual de Campinas (UNICAMP),

13083–863, Campinas, São Paulo, Brazil4Departamento de Zoologia e Botânica, Universidade Estadual Paulista (UNESP), Rua Cristóvão Colombo, 2265, Jardim Nazareth,

15054–000, São José do Rio Preto, São Paulo, Brazil5Laboratório de Zoologia, Instituto Básico de Biociências, Universidade de Taubaté (UNITAU), Av. Tiradentes, 500, 12030-180, Tau-

baté, São Paulo, Brazil6Departamento de Ciências Básicas e Produção Animal, Faculdade de Agronomia, Medicina Veterinária e Zootecnia, Universidade

Federal de Mato Grosso (UFMT), Av. Fernando Correa da Costa, 2367, Boa Esperança, 78060–900, Cuiabá, Mato Grosso, Brazil7Corresponding author. E-mail: andreufmt@gmail.com

Abstract

A new species of Pseudopaludicola is described from human-altered areas originally covered by Semideciduous Forest

in northwestern state of São Paulo, southeastern Brazil. Morphologically, the new species differs from four species be-

longing to the P. pusilla group by the absence of either T-shaped terminal phalanges or toe tips expanded, and from all

other congeners except P. canga and P. facureae by possessing an areolate vocal sac, with dark reticulation. The higher

duration (300–700 ms) of each single, pulsed note (9–36 nonconcatenated pulses) that compose the call in the new species

distinguishes it from all other 14 species of Pseudopaludicola with calls already described (10–290 ms). Absence of har-

monics also differ the advertisement call of the new species from the call of its sister species P. facureae, even though

these two species presented unexpected low genetic distances. Although we could not identify any single morphological

character distinguishing the new species from P. facureae, a PCA and DFA performed using 12 morphometric variables

evidenced significant size differences between these two species.

Key words: Amphibia, taxonomy, advertisement call, genetic distance

Introduction

Species from the genus Pseudopaludicola Miranda-Ribeiro, 1926 are distributed in open grasslands (seasonally flooded or not), open environments in the vicinities or amidst tropical rainforests, and dry forests that occur throughout South America (Lynch 1989; Pansonato et al. 2012a; Frost 2014). The genus currently comprises 17 valid species (Frost 2014; Pansonato et al. 2014).

According to former phylogenetic analyses by Lobo (1995) and by Cardozo and Suárez (2012), only the Pseudopaludicola pusilla group, characterized by T-shaped terminal phalanges was recognized as monophyletic.Four species of Pseudopaludicola were assigned to this group: P. boliviana Parker, 1927; P. ceratophyes Rivero & Serna, 1984; P. llanera Lynch, 1989; and P. pusilla (Ruthven, 1916). Until recently, other 14 species remained unassigned to this or to any other monophyletic group (Lynch 1989; Cardozo & Suárez 2012; Andrade & Carvalho, 2013; Roberto et al. 2013; Magalhães et al. 2014): P. ameghini (Cope, 1887); P. canga Giaretta & Kokubum, 2003;

Accepted by M. Vences: 22 Aug. 2014; published: 15 Sept. 2014 249

P. facureae Andrade & Carvalho, 2013; P. falcipes (Hensel, 1867); P. giarettai Carvalho, 2012; P. hyleaustralis

Pansonato, Morais, Ávila, Kawashita-Ribeiro, Strüssmann & Martins, 2012; P. mineira Lobo, 1994; P. mystacalis

(Cope, 1887); P. murundu Toledo, Siqueira, Duarte, Veiga-Menoncello, Recco-Pimentel & Haddad, 2010; P.

parnaiba Roberto, Cardozo & Ávila 2013; P. pocoto Magalhães, Loebmann, Kokubum, Haddad, Garda, 2014; P.saltica (Cope, 1887); P. serrana Toledo, 2010 (recently synonymized to P. murundu; Pansonato et al. 2014) and P.ternetzi Miranda-Ribeiro, 1937.

A more embracing phylogenetic analysis, including 11 species of Pseudopaludicola, reinforced the monophyly of the genus, and revealed that contained species can be organized into four major clades (Veiga-Menoncello et al.

2014). The study by Veiga-Menoncello et al. (2014) corroborated the formerly recognized P. pusilla group. It also confirmed, in consonance with previous morphological analysis (Lynch 1989), that the assemblage of species that lack T-shaped terminal phalanges is paraphyletic with respect to the P. pusilla group, thus providing strong evidences that taxonomic changes and a full generic revision are needed in Pseudopaludicola.

During an ongoing taxonomic review of the genus Pseudopaludicola, we identified a new, small, short-hindlimbed species of this genus, which occurs syntopically with two other congeners in agricultural areas in northwestern state of São Paulo, southeastern Brazil. Herein we provide a formal description of this new taxon, based in morphological and bioacoustical data. Additionally, we re-analyzed topotypic advertisement calls of Pseudopaludicola facureae and identified the presence of harmonics in their structure, a character previously unnoticed in this species.

Material and methods

Specimens of the new species (Appendix 1, 2), collected with permission of the “Instituto Chico Mendes de Conservação da Biodiversidade” (ICMBIO 27231-1), are deposited in the “Coleção Zoológica de Vertebrados, Universidade Federal de Mato Grosso” (UFMT; Cuiabá, Mato Grosso, Brazil) and in the “Coleção de Amphibia, Universidade Estadual Paulista Júlio de Mesquita Filho” (DZJSRP-Amphibia, UNESP; São José do Rio Preto, São Paulo, Brazil).

The measurements of 29 adult specimens (22 males and seven females) were taken with digital calipers to the nearest 0.1 mm. Measurements for hand length (HAL), tibia length (TL), tarsus length (TAL), and foot length (FL) followed Heyer et al. (1990). Measurements for snout-vent length (SVL), head length (HL), head width (HW), inter-orbital distance (IOD), eye diameter (ED), eye-nostril distance (END), internarial distance (IND), and thigh length (THL) followed Duellman (2001). Color was described using field notes and photographs of live specimens. Principal Component Analyses (PCAs) and Discriminant Function Analyses (DFA) were performed on correlations matrices from log-transformed morphometric parameters using R platform version 3.0.1 (R Development Core Team 2012).

Nine paratypes of Pseudopaludicola sp. nov. (UFMT 16198–205; UFMT 18131) were cleared and stained using the technique of Taylor and Van Dyke (1985), for morphological comparisons with the osteology of terminal phalanges of the toe tips of congeneric species, presented by Lynch (1989) and Cardozo and Suárez (2012).

Vocalizations of topotypic specimens of Pseudopaludicola sp. nov. were recorded at the type-locality by André Pansonato, with a professional digital recorder Marantz PMD 660 equipped with a Yoga EM-9600 external directional microphone. Digital recordings were sampled at 44.1 kHz sampling rate, resolution of 16 bit, saved in uncompressed wave files. Spectrograms were edited using Raven Pro 1.3 software with the following configuration for brightness 69%, contrast 60%, DFT size 256 points samples, and time grid overlap 50%. The following temporal parameters were measured from the waveform: number of notes per series and pulses per note; note, pulse, and series duration (s); inter-note, inter-pulse, and inter-series interval (s). Note repetition rate pulse and repetition rate were calculated per minute. The range frequency (portrayed in spectrogram; Hz), dominant frequency (peak of frequency of note; Hz) and harmonics were obtained from spectrograms. Terminology for acoustic parameters follows Heyer et al. (1990), Pansonato et al. (2013), and Magalhães et al. (2014). Vocalizations are archived in the “Banco de Registros Bioacústicos”, housed at the “Laboratório de Herpetologia do Instituto de Biociências da Universidade Federal de Mato Grosso” (LH; Cuiabá, Mato Grosso). Original morphometric and bioacoustic measurements were submitted to the Dryad data repository (www.datadryad.org) under DOI 10.5061/dryad.d1g94.

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Considering the morphological and cytogenetic similarity, and the geographical proximity (Duarte et al. 2010, Andrade & Carvalho 2013) between the new species and Pseudopaludicola facureae, the advertisement calls of topotypic specimens, described by Andrade and Carvalho (2013), were re-evaluated. Original sound files are archived in the “Collection of frogs of the Universidade Federal de Uberlândia” (voucher record number Pseudop_facureaeUberlândiaMG9aAAGm671 and Pseudop_facureaeUberlândiaMG11aAAGm671). Additional topotypic recordings of P. facureae were obtained by André Pansonato and Jessica R. Mudrek (record number LH 721–728).

To estimate genetic distances between Pseudopaludicola sp. nov. and related species, sequences of a fragment of the 16S mitochondrial rRNA gene, corresponding to a standard DNA barcoding marker in amphibians (Vences

et al. 2005; Vieites et al. 2009), were taken from Genbank® (access numbers KJ146972; KJ146975; KJ146978; KJ146983; KJ146988; KJ146992; KJ147002; KJ147025; KJ147031; KJ147049; KJ147054; KJ146996 – KJ14998). Uncorrected genetic distances (p-distances) were calculated using MEGA, version 5 (Tamura et al.

2011).

Results

Pseudopaludicola atragula sp. nov.

(Figures 1–3)

Holotype. Adult male (UFMT 16576; Figure 1) collected on 10 January 2013 by A. Pansonato and J. R. Mudrek, on a permanent swamp (20º20’31”S; 49º11’42”W) in the smallholding named “sítio Boa Esperança”, municipality of Icém, northwest region of the state of São Paulo, Brazil.

FIGURE 1. Holotype of Pseudopaludicola atragula sp. nov.. Adult male (UFMT 16576; SVL = 14.07 mm). Dorsal (A) and ventral (B) views.

Paratypes. Three adult males (DZJSRP 8727–8; 8747) collected on 23 February 2005 by C. P. Candeira, R. A. Silva and F. R. Silva; two adult males (UFMT 16198–9) and one adult female (UFMT 16550) collected on 02 March 2012 by A. Pansonato and R. A. Silva; nine adult males (UFMT 16200–1; 16551–5; 16557; 16571), three adult females (UFMT 16202; 16572; 16582), and one juvenile (UFMT 16556) collected on 10 January 2013 by A. Pansonato and J. R. Mudrek, from the same locality of the holotype. Nine adult males (UFMT 16203–6; 16576–80), three adult females (UFMT 16573–5), and one juvenile (UFMT 16581) collected on 10 January 2013 by A. Pansonato and J. R. Mudrek at district of Nova Itapirema (21º04’54”S; 49º31’11”W), municipality of Nova Aliança, São Paulo, Brazil.

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FIGURE 2. Holotype of Pseudopaludicola atragula sp. nov. (UFMT 16576): (A) ventral view of vocal sac; (B) profile of head; (C) ventral view of right foot; (D) ventral view of right hand. Scale bar = 1 mm.

Referred specimens. We here provide a list of all name usages that we have found in the literature, i.e., names once applied to material that we recognize as strictly corresponding to the species being described here: Pseudopaludicola aff. canga (from Icém, SP)—Duarte et al. (2010: 4) and Andrade & Carvalho (2013: 396); Pseudopaludicola aff. falcipes – Silva et al. (2008: 126–130) and Silva et al. (2011: 613–614); Pseudopaludicola

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falcipes—Silva et al. (2012: 1416–1422); Pseudopaludicola sp. 2 (aff. canga)—Veiga-Menoncello et al. (2014: 263–270).

Diagnosis. The new species is assigned to the genus Pseudopaludicola based on the presence of hypertrophied antebrachial tubercles (Lynch 1989). Pseudopaludicola atragula sp. nov. is diagnosed by the following combination of characters: (1) small size (SVL 12.2–15 mm in males and 15.5–16.7 mm in females); (2) absence of either T-shaped terminal phalanges or expanded toe tips (disks or pads); (3) short hindlimbs, with tibiotarsal articulation only reaching the eye; (4) areolate vocal sac, with dark reticulation; (5) karyotype with 2n=18 chromosomes; (6) advertisement call composed of a single note with 9–36 nonconcatenated pulses (resembling the sound of clockwork motors used in wind-up toys); (7) note duration of 0.3–0.7 s; (8) note repetition rate of 42–98 notes/min; (9) absence of harmonics; (10) dominant frequency of advertisement call 3617.6–4263.6 Hz.

FIGURE 3. Live specimens of Pseudopaludicola atragula sp. nov. from the municipality of Icém, São Paulo, Brazil. Dorsal and lateral views of an adult male, UFMT 16199 (A–C), and of an adult female, UFMT 16550 (B–D).

Description of holotype. Small size (SVL 14.07 mm). Canthus rostralis indistinct, rounded, smooth; vocal sac single (subgular), with areolate, whitish dark reticulations (Figure 2A); vocal slits present; choanae well-separated from each other. Surfaces of dorsum, venter, upper eyelids, belly and throat smooth; H-shaped glandular ridge on shoulder region. Snout rounded to subovoid in dorsal view, and acuminate in profile; head wider than long; sides of head (upper lips, under the eyes, and tympanum area) and flanks with white granules (Figure 2B). Nostrils slightly protuberant, directed anterolaterally, closer to tip of snout than to eyes; pupil rounded; upper eyelids; tympanum

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indistinct; vomerine teeth absent; tongue ovoid, posteriorly free. Cloaca smooth. Inner metatarsal tubercle oval; outer metatarsal tubercle conical; toe tips slightly pointed, not webbed and extensively fringed to almost their tips (Figure 2C); relative length of toes I<II<III~V<IV; subarticular tubercles large, rounded; supernumerary tubercles absent. Hand with fingers free (Figure 2D), with tips rounded, not expanded; relative length of fingers I<II~IV<III; thumb thickened, with keratinized gray nuptial pad on internal surface; two subconical antebrachial tubercles, the distal one scarcely perceptible; outer metacarpal tubercle round; inner metacarpal tubercle elongated; subarticular finger tubercles rounded; supernumerary finger tubercles indistinct.

Color. In life (Figure 3A–B), dorsum dark gray; flanks pale gray; no vertebral line from the snout to vent; belly white; upper lips with white spots which extend to the insertion of the forelimbs; vocal sac with large white areolations (nearly circular protuberances), interspersed among dark reticulation. Dark reticulation is particularly evident in the anterior half of the vocal sac. In preservative, dorsum grayish brown, with darker brown blotches; belly whitish; ventral surface of hands and feet dark gray, mottled; white spots on upper lips, extending to the dorsal insertion of the forelimbs and to the flanks; vocal sac with large white areolations over a grey, nearly translucent skin, peppered with black melanophores. Dark transverse stripes in thigh and shank, tarsus and foot, and forearm.

Variation. Dorsal coloration is highly variable, with nearly 25% of the examined specimens presenting a light, thin vertebral stripe, from the tip of the snout to vent. In preserved specimens, the gular region may vary from light to darker grey, always peppered with black melanophores. Females have a more robust body and their gular region, although areolate, is always unpigmented, clear white (Table 1).

TABLE 1. Measurements (mm) of the type series adult specimens of Pseudopaludicola atragula sp. nov.. Values are

presented as mean ± standard deviation (minimum–maximum). N = number of specimens analyzed. “Males” include the

holotype.

Description of the advertisement call. We analyzed 41 calls of four specimens of Pseudopaludicola atragula

(Table 2). A typical advertisement call of the new species (Figure 4) consists of a single pulsed note, with 9–36 (mean 22.8±5.4) pulses emitted in a very short period of time, during a single airflow (McLister et al. 1995). The call (= one note) resembles the sound of clockwork motors used in wind-up toys. Mean duration of each note is 0.45±0.07 s (0.3–0.7 s). Inter-notes interval ranges from 0.5±0.2 s (0.04–0.9 s) and average rate of notes per minute is 65.3±15.1 notes/min (42–98). Mean frequency ranged from 2500.01 (±341.1) Hz to 5088.5 (±249.2) Hz, and mean dominant frequency from 3981.5±156.9 Hz (3617.6–4263.6 Hz). Mean duration of each pulse is 0.003±0.001 s (0.001–0.007 s). Inter-pulses interval ranges from 0.01±0.008 s (0.002–0.064 s). Average rate of pulses per minute is 2935.5±549.8 pulses/min (1436–3957).

Comparison with other species. A combination of external and/or internal morphological characters, bioacoustic properties of the call, and/or karyotype allows to distinguish the new species from all other 17 Pseudopaludicola species (from now on, characters of compared species in parentheses). From eight out of the 13

Holotype Males Females

(N=22) (N=7)

SVL 14.07 13.6±0.9 (12.2–15.1) 15.9±0.5 (15.5–16.7)

HL 5.47 5.3±0.3 (4.7–5.9) 6.1±0.3 (5.7–6.5)

HW 4.69 4.6±0.2 (4.1–5.1) 5.2±0.5 (4.6–5.7)

IOD 1.18 1.1±0.08 (1–1.3) 1.2±0.1 (1–1.4)

ED 1.35 1.4±0.1 (1.2–1.7) 1.6±0.1 (1.5–1.9)

END 1.2 1.3±0.1 (1–1.6) 1.5±0.2 (1.3–1.8)

IND 1.1 1.1±0.1 (0.9–1.5) 1.3±0.1 (1.1–1.4)

HAL 3.93 3.9±0.3 (3.3–4.4) 4.5±0.3 (4–4.8)

THL 6.43 6.2±0.3 (5.7–6.8) 7.2±0.4 (6.7–7.9)

TL 7.09 6.7±0.5 (5.9–7.7) 7.9±0.3 (7.4–8.3)

TAL 4.02 3.8±0.3 (3.3–4.2) 4.6±0.2 (4.1–4.9)

FL 8.3 7.7±0.6 (6.5–8.7) 9.2±0.5 (8.5–9.6)

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Pseudopaludicola species not belonging to P. pusilla group (P. ameghini, P. canga, P. facureae, P. falcipes, P.

giarettai, P. hyleaustralis, P. mineira, P. mystacalis, P. murundu, P. parnaiba, P. pocoto, P. saltica, and P. ternetzi) the new species differs: from P. falcipes, by the presence of an abdominal fold (abdominal fold interrupted or absent; Lobo 1996; Lavilla & Cei 2001); from P. murundu, and P. saltica, by the shorter hindlimbs, with tibio-tarsal articulation reaching the nostrils (presence of very long hindlimbs, with tibio-tarsal articulation reaching beyond the end of the snout; Toledo et al. 2010; Pansonato et al. 2013); from P. giarettai, by the smaller body size (16.2–18.0 mm SVL in males) and by the relative length of fingers (finger I shorter than finger IV; Carvalho 2012); from P. ameghini, by the smaller body size (14.1–19.3 mm SVL in males and 17.3–22.7 mm SVL in females; Haddad & Cardoso 1987; Pansonato et al. 2013); from P. mineira, by head longer than wide (head as long as wide; Pereira & Nascimento 2004); from P. ternetzi, by the smaller body size (SVL of males 16.0–18.6 mm; SVL of females 16.0–22.2 mm; Lobo 1996).

TABLE 2. Measurements of acoustic parameters of specimens of Pseudopaludicola atragula sp. nov. from the type

locality (Icém, São Paulo, Brazil). Values are: mean ± standard deviation (minimum–maximum). N: number of calls (=

notes) analyzed (number of specimens recorded).

FIGURE 4. Oscillograms (A) and audiospectrograms (B) of a single pulsed note of Pseudopaludicola atragula sp. nov., with 26 nonconcatenated pulses (voucher record LH 703, from specimen UFMT 16206, recorded on 02 March 2012, 17:30 h, air temperature 23.8ºC, municipality of Icém, São Paulo, Brazil) and of non-pulsed notes of Pseudopaludicola facureae (voucher record: Pseudop_facureae UberlândiaMG11aAAGm671, recorded on 29 October 2011, 17:30 h, air temperature 21ºC, municipality of Uberlândia, Minas Gerais, Brazil). Note absence of harmonics in P. atragula sp. nov., and presence of three harmonics (H1–H3; FD = dominant frequency) on each note in the call of P. facureae. Time interval is the same for both species.

The appearance of the vocal sac of male Pseudopaludicola atragula—areolate, whitish, with dark reticulation—distinguishes the new species from P. ameghini, P. falcipes, P. giarettai, P. hyleaustralis, and P.

Advertisement call parameters Pseudopaludicola atragula sp. nov.

N=41 (4)

Number of pulses per note 22.8±5.4 (9–36)

Note duration (s) 0.45±0.07 (0.3–0.7)

Inter-notes interval (s) 0.5±0.2 (0.04–0.9)

Pulse duration (s) 0.003±0.001 (0.001–0.007)

Inter-pulses interval (s) 0.01±0.008 (0.002–0.064)

Minimum frequency (Hz) 2500.01±341.1 (1932.7–3331.3)

Maximum frequency (Hz) 5088.5±249.2 (4322.8–5578.4)

Dominant frequency (Hz) 3981.5±156.9 (3617.6–4263.6)

Average rate of notes/min 65.3±15.1 (42–98)

Average rate of pulses/min 2935.5±549.8 (1436–3957)

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pocoto, which present vocal sac yellowish or light cream (Haddad & Cardoso 1987; Carvalho 2012; Pansonato et

al. 2012a; Pansonato et al. 2013; Magalhães et al. 2014), from P. boliviana, P. mineira, P. mystacalis, P. saltica, and from topotypic specimens of P. ternetzi, which present vocal sac not areolate and uniformly whitish (Miranda-Ribeiro 1837; Lobo 1994; De la Riva et al 2000; Pansonato et al. 2013), from P. murundu, by the vocal sac not areolate and uniformly dark (Toledo et al. 2010).

Presently, only four species cannot be distinguished from Pseudopaludicola atragula based solely on external morphology: P. canga, P. facureae, P. parnaiba, and P. pusilla. Internally, however, P. atragula is distinguished from congeners belonging to the P. pusilla group (P. boliviana, P. ceratophyes, P. llanera and P. pusilla) by the absence of either T-shaped terminal phalanges or expanded toe tips (Lynch 1989). Advertisement call structure distinguishes Pseudopaludicola atragula from all 14 congeners (Figure 5–6) for which call descriptions are available (P. ameghini, P. boliviana, P. canga, P. facureae, P. falcipes, P. giarettai, P. hyleaustralis, P. mineira, P.

mystacalis, P. murundu, P. parnaiba, P. pocoto, P. saltica and P. ternetzi). Call consisting of a single pulsed note and lower dominant frequency (3617.6–4263.6 Hz) differ Pseudopaludicola atragula from two congeners having notes with concatenated pulses (Figure 5A): P. mystacalis (Figure 6A; call consisting of sequences of notes, each note composed of 12–20 pulses, with dominant frequency 4478.9–5340.2 Hz; Pansonato et al. 2013; 2014), and P.boliviana (Figure 6B; call consisting of sequences of notes, each note composed of 3–6 pulses, with dominant frequency 4942–5224 Hz; Duré et al. 2004).

FIGURE 5. Comparative spectrograms (DFT size =1024 samples) of sequences of the advertisement calls of 15 species of Pseudopaludicola, divided in three distinct groups according to type of notes: (A) species having notes with concatenated pulses; (B) species having notes with nonconcatenated pulses (three notes for each species); (C) species having non-pulsed notes.

Among species for which calls composed of pulsed notes with nonconcatenated pulses were reported (Figure 5B; see also Table 3 in Magalhães et al. 2014), Pseudopaludicola atragula (Figure 6C) differs from P. ameghini, P.

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falcipes, P. mineira, P. murundu, P. saltica and P. ternetzi by a higher number of pulses per note (9–36 pulses),higher note duration (0.3–0.7 s), and/or lower dominant frequency (3617.6–4263.6 Hz). In P. ameghini (Figure 6D) there are 3–6 pulses per note, and note duration is 0.04–0.11 s (Pansonato et al. 2013); in P. ternetzi (Figure 6E) there are 3–6 pulses per note, and note duration is 0.01–0.09 s in populations from Minas Gerais and São Paulo (Cardozo & Toledo 2013); in P. murundu (Figure 6F), there are 2–6 pulses per note, note duration is 0.03–0.14 s, and dominant frequency is 4875–6370 Hz (Toledo et al. 2010; Pansonato et al. 2014); in P. saltica (Figure 6G), there are 1–4 pulses per note, and note duration is 0.03–0.1 s (Pansonato et al. 2013); in P. falcipes (Figure 6H), there are 2 pulses per note and dominant frequency is 4200–5800 Hz (Haddad & Cardoso 1987); in P. mineira

(Figure 6I), there are 2 pulses per note, note duration is 0.04 s, and dominant frequency is 4306–4823 Hz (Pereira & Nascimento 2004); in P. pocoto (Figure 6J), there are 2–3 pulses per note, note duration is 0.13–0.29 s, and dominant frequency is 5168–6374 Hz (Magalhães et al. 2014).

FIGURE 6. Comparative oscillogram of a single note of the advertisement calls of 15 species of Pseudopaludicola: (A–B) species having notes with concatenated pulses; (C–J) species having notes with nonconcatenated pulses; (L–P) species having non-pulsed notes. Total time of each oscillogram corresponds to 0.5 s.

The new species differs from P. canga, P. facureae, P. giarettai, P. hyleaustralis, and P. parnaiba (Figure 5C) by having pulsed (9–36 pulses) notes, with higher note duration (0.3–0.7 s) and lower note repetition rate (42–98 notes/min). All five above-mentioned species present non-pulsed notes (Giaretta & Kokubum 2003; Carvalho

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2012; Pansonato et al. 2012; Andrade & Carvalho 2013; Pansonato et al. 2013; Roberto et al. 2013). Advertisement calls correspond to long series of non-pulsed notes emitted at a relatively fast rate in P. parnaiba

(Figure 6L; 6–46 non-pulsed notes, note duration 0.01–0.04 s and note repetition rate 16–162 notes/min; Roberto et

al. 2013), in P. hyleaustralis (Figure 6M; 11–74 non-pulsed notes, note duration 0.02–0.05 s and note repetition rate 503.8–623.2 notes/min; Pansonato et al. 2012), and in P. facureae (Figure 6N; 3–53 non-pulsed notes, note duration 0.02–0.035 s and note repetition rate 480–1860 notes/min; Andrade & Carvalho 2013). In P. canga, the advertisement call consists of short series of up to 19 non-pulsed notes (Figure 6O; note duration 0.01–0.03 s and note repetition rate 689–944 notes/min; Giaretta & Kokubum 2003; Pansonato et al. 2012). In P. giarettai (Figure 6P; Carvalho 2012) the advertisement call consists of a non-pulsed note resembling a whistle.

TABLE 3. Uncorrected p-distances of the 16S mitochondrial rRNA gene between Pseudopaludicola atragula sp. nov.

and 12 congeners.

A lower dominant frequency also differ the advertisement call of Pseudopaludicola atragula from the calls of P.

facureae (4076–5108 Hz; Andrade & Carvalho 2013) and P. parnaiba (4220.5–5168 Hz; Roberto et al. 2013). Absence

of harmonics differ the call of the new species from that of P. giarettai (with four harmonics; Carvalho 2012) and from P.

facureae (with three harmonics; present study).

Finally, the number of chromosomes (2n=18) distinguishes Pseudopaludicola atragula (Duarte et al. 2010; treated therein as “Pseudopaludicola aff. canga”) from P. falcipes, P. mineira, P. murundu, and P. saltica (2n=22), P. ameghini and P. ternetzi (2n=20), and P. mystacalis (2n=16) (Duarte et al. 2010; Fávero et al. 2011).

Geographic distribution. Pseudopaludicola atragula is a Brazilian endemic species, presently known from two localities in the northwest region of the state of São Paulo, in southeastern Brazil: the type locality (municipality of Icém, 20º21’33”S; 49º11’55”W), and the district of Nova Itapirema, municipality of Nova Aliança (21º04’54”S; 49º31’11”W), approximately 100 km in a straight line southwards from Icém.

Genetic distance. The intraspecific genetic distances (uncorrected p-distances) between Pseudopaludicola

atragula and species in the P. pusilla group were greater (> 17%) than distances between P. atragula and closely related species that lack T-shaped terminal phalanges and share the same diploid number 2n=18 chromosomes (3.2 to 7.9%; Table 3).

Habitat and Conservation. In the northwestern region of the state of São Paulo, Pseudopaludicola atragula is always found in sympatry with P. mystacalis, which also occurs together with P. ternetzi in some localities (R.M. Pelinson pers. comm.). These three Pseudopaludicola species occur syntopically at the type-locality of P. atragula

Species; Genbank access

number

1 2 3 4 5 6 7 8 9 10 11 12

1. P. atragula;

KJ146996–KJ14998

2. P. falcipes; KJ146972 0.166

3. P. ameghini; KJ146975 0.101 0.122

4. P. facureae; KJ146978 0.032 0.161 0.103

5. P. mystacalis;

KJ146983

0.094 0.148 0.098 0.094

6. P. canga; KJ146988 0.079 0.137 0.088 0.083 0.085

7. Pseudopaludicola

sp.—P. pusilla group;

KJ146992

0.173 0.126 0.115 0.163 0.133 0.147

8. P. saltica; KJ147002 0.147 0.090 0.123 0.149 0.144 0.146 0.124

9. P. mineira; KJ147025 0.170 0.075 0.122 0.174 0.146 0.154 0.122 0.048

10. P. murundu;

KJ147031

0.149 0.073 0.114 0.151 0.139 0.133 0.110 0.025 0.043

11. P. boliviana—P.

pusilla group; KJ147049

0.179 0.121 0.134 0.174 0.152 0.162 0.071 0.121 0.115 0.110

12. P. ternetzi; KJ147054 0.106 0.126 0.016 0.108 0.099 0.083 0.119 0.125 0.128 0.119 0.134

13. Pseudopaludicola sp.

3 (aff. canga); KJ147013

0.072 0.135 0.080 0.076 0.080 0.018 0.145 0.140 0.152 0.128 0.158 0.078

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which, although being the least frequent of the three, can be locally abundant, their representatives being found along the whole rainy season, from September to March (Santos et al. 2007; Silva et al. 2008; 2011).

Pseudopaludicola atragula occurs in groundwater upwelling sites where the water runs slowly, forming temporary and permanent ponds and swamps with a large amount of marginal herbaceous vegetation (see Fig. 1f in Silva et al. 2008, who treated the species as Pseudopaludicola aff. falcipes and listed other anuran species found syntopically; see also Silva et al. 2012). Males call from both wet sites and sites with shallow water, generally from the base of small individual grasses, in areas sparsely covered by vegetation and interspersed with areas without vegetation (Santos et al. 2007; Silva et al. 2011).

In the municipalities of northwestern region of the state of São Paulo, Brazil, populations of Pseudopaludicola

and of other anuran species are under strong pressure due to habitat destruction. The region is considered the most deforested and fragmented in that state, with less than 9% of the original vegetation of seasonal Semideciduous Forests and Cerrado savannahs. Main causes of habitat loss are due to agribusiness (mainly sugarcane crops) and urbanization. Unsustainable farming practices are leading to high levels of soil erosion, sedimentation of streams, and degradation of aquatic ecosystems. Additionally, conservation units are scarce in the northwestern region of São Paulo (Martinelli & Filoso 2008; Necchi Jr. 2011).

Etymology. The specific epithet is derived from the Latin words “atra”, meaning dark, black, and “gula”, meaning throat or gullet. It is used in reference to the dark gular region of most males (in life) – unusual within the genus Pseudopaludicola – with white areolations interspersed among dark reticulation.

Concluding remarks. Morphologically and cytogenetically, Pseudopaludicola atragula most closely resembles P. facureae. Indeed, based on similarities in the chromosome numbers of the two species (2n=18), Duarte et al. (2010) suggested that the populations from Icém (São Paulo, Brazil) and Uberlândia (Minas Gerais, Brazil), both treated by those authors as “Pseudopaludicola aff. canga”, could belong to the same taxon.

Visual examination of qualitative morphological characters of males and females in our sample of Pseudopaludicola atragula did not evidenced any diagnostic character to distinguish this taxon from P. facureae. Comparative analyses of morphometric data through a PCA, however, clearly allowed distinguishing the two taxa (Figure 7). The first two axes of the PCA explained 87% of the total variability in males (Figure 7A) and higher loadings corresponded to snout-vent length, head length, and thigh length. The PCA explained 85% of the total variability in females (Figure 7B) and higher loadings corresponded to snout-vent length, head length, and foot length. The DFA with morphometric measurements, using all specimens, assigned 100% of the individuals of Pseudopaludicola atragula and 100% of the individuals of P. facureae to the correct species (Table 4).

TABLE 4. Summary of the number of specimens correctly classified as Pseudopaludicola atragula sp. nov. and P.

facureae using Discriminant Function Analysis of 12 morphometric measurements.

Bioacoustic parameters and spectrograms presented herein also evidenced the taxonomic validity of the species from northwestern state of São Paulo. As here shown, with a single contraction of the trunk muscles, males of P. atragula emit an advertisement call composed of a single pulsed note with 9–36 nonconcatenated pulses, without harmonics, resembling the sound of clockwork motors used in wind-up toys. Calls are also composed of single notes and present harmonics only in P. giarettai, but in this species notes are non-pulsed, there are four harmonics, and the call resembles a whistle. In the remaining species of Pseudopaludicola where bioacoustic parameters were already analyzed, the call consists in a succession of series of notes. In particular, we call attention to the fact that calls already described within the genus Pseudopaludicola might be divided in three distinct groups according to type of notes: non-pulsed notes; notes with concatenated pulses, and notes with nonconcatenated

Males Females

True group True group

Assigned to P. atragula P. facureae P. atragula P. facureae

P. atragula 22 0 7 0

P. facureae 0 13 0 9

Number correct 22 13 7 9

% 100 100 100 100

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pulses (Figure 5–6). Bioacoustic data, therefore, are here confirmed to represent a reliable tool in the recognition of cryptic species within the genus Pseudopaludicola.

FIGURE 7. Principal component analysis (PCA) of 12 morphometric characters of males (A) and females (B) of Pseudopaludicola atragula sp. nov. (open circles) and P. facureae (closed circles), along the first two axes.

Acoustic parameters of calls and the spectrogram of topotypes of Pseudopaludicola atragula are readily distinguished from topotypical advertisement calls of P. facureae, composed of series of 2–55 (mean 15.8±10.1) non-pulsed notes, with series duration of 0.1–4.3 s, emitted at intervals of 0.2–1.7 s (Table 5; Figure 4). Mean note duration in this species is 0.02±0.005 (0.01–0.04), emitted on average at intervals of 0.03±0.03 (0.01–0.4) with average rate of notes per minute of 1249.1±388.5 (646–2000). Mean frequency ranges from 2688.5 (±573.9) Hz to 5947.9 (±336.8) Hz, and mean dominant frequency is 4559.4±212.2 (4306.6–4995.7), usually concentrated in the first harmonic (Figure 4B). Therefore, the call of P. atragula differs from P. facureae by having pulsed notes, higher note duration, lower note repetition rate and dominant frequency, and especially in the absence of harmonics (Figure 8).

Evaluation of uncorrected pairwise distances revealed high genetic divergences between Pseudopaludicola

atragula and its congeners (9.4–17.9%), except for species that share with P. atragula the diploid number of 2n=18 chromosomes. These findings seem to be in agreement to its phylogenetic relationships: P. atragula + P. facureae

(genetic divergence of 3.2%) and P. canga + Pseudopaludicola sp. (aff. canga), showing genetic divergences of 7.2 and 7.9%, respectively, to P. atragula, were recovered as sister groups (Veiga-Menoncello et al. 2014). Although our results revealed an overall high genetic distance among species of Pseudopaludicola, a lower genetic divergence was revealed when closely related species are compared (e.g., 1.6% between P. ameghini and P.

ternetzi, and 2.5% between P. saltica and P. murundu). This scenario is similar to that reported for certain species in the dendrobatid genus Ameerega (Lötters et al. 2009) and in the hylid genus Osteocephalus (Jungfer et al. 2013). The uncorrected p-distance of 3.2% found between P. atragula and P. facureae is very similar to values regarded by some authors as a “strong molecular” and “substantial genetic” differentiation between related taxa, even though they could represent conspecific lineages, or cryptic species instead (e.g., Vences et al. 2011). By integrating morphological, bioacoustic and molecular data, evidence presented herein support the recognition of P. atragula as a new, valid species of the genus Pseudopaludicola.

As here reported, Pseudopaludicola atragula occurs syntopically with other two congeners at the type-locality: P. mystacalis and P. ternetzi. Although not yet studied in detail, co-occurrence is a rather frequent situation throughout the range of the genus (e.g., Silva et al. 2008; Pansonato et al. 2012b; Pansonato et al. 2013). In general, individuals of Pseudopaludicola spp. seem to occupy habitats with vegetation consisting only in sparse, tall grasses, in seasonally flooded shallow areas or in permanent wet areas with a thin water film flow (Santos et al. 2007; Silva et al. 2008; Giaretta & Facure 2009).

PANSONATO ET AL.260 · Zootaxa 3861 (3) © 2014 Magnolia Press

FIGURE 8. Comparative power spectrum of advertisement calls of the two species of Pseudopaludicola (A–B) having harmonics (H1–H4), and of Pseudopaludicola atragula sp. nov., without harmonics (C). FD = dominant frequency. Voucher records: Pseudop_giarettaiMG8TRC_AAGmt, from P. giarettai (see Carvalho 2012); Pseudop_facureaeUberlândiaMG11aAAGm671, from P. facureae, and LH 727, from P. atragula sp. nov.

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Geographical, morphological, ecological, bioacoustic, and genetic patterns of co-occurring species of Pseudopaludicola should be evaluated using integrative approaches, for a better understanding of habitat use, geographic distribution, and evolution in the genus.

TABLE 5. Measurements of acoustic parameters of specimens of Pseudopaludicola facureae from the type locality

(Uberlândia, Minas, Brazil). Values are: mean ± standard deviation (minimum–maximum). N: number of calls (= notes)

analyzed (number of specimens recorded).

Acknowledgements

Special thanks go to the owner of Boa Esperança smallholding, Toninho, for allowing our field work there; to Miguel Vences, for comments on earlier versions of the manuscript; to Felipe Curcio, for allowing access to material under his care at “Coleção Zoológica de Vertebrados da Universidade Federal de Mato Grosso” (UFMT); to Ariovaldo Giaretta and Felipe de Andrade for allowing access to topotypic calls and measurements of P. facureae; to CNPq (project “Rede de pesquisa em anfíbios e répteis de ecossistemas não florestais brasileiros”; process # 563352/2010-8) and to Evandson Silva (FAPEMAT process # 285060/2010), for financial and logistical support, respectively. To Rodrigo Silva for help during field sampling; to Jéssica Cantarini for clearing and staining specimens; to Jeffrey Himmelstein for language review. Ecological information about habitat use was obtained during several research projects accomplished by students from the Theoretical Ecology Laboratory at UNESP to whom we are grateful: Rodolfo Pelinson, Rodrigo Silva, Michel Garey, Thiago Pereira, Veronica Sousa and Tales Cordeiro. AP thanks “Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior” (CAPES) for a PhD scholarship; CS and DCRF thank to CNPq for research fellowships (processes # 309541/2012-3 and # 303522/ 2013-5).

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APPENDIX 1. Voucher specimens examined.

Pseudopaludicola facureae: BRAZIL: MINAS GERAIS: Uberlândia: Males: AAG-UFU 853–855; 2277–8; 2528; 2622; 2681; 3586; 3588; UFMT 17745–5; 17748; and ZUEC 13650–2. Females: AAG-UFU 2279; 2282; 3585; 3587; 3589; 3591; 4731 and ZUEC 13653–4.

APPENDIX 2. Analyzed sound files.

Pseudopaludicola atragula sp. nov.: BRAZIL: SÃO PAULO: Icém: LH 702 (voucher specimen UFMT 16198); LH 703 (voucher specimen UFMT 16206); LH 439–440 (no voucher specimens).

Pseudopaludicola facureae: BRAZIL: MINAS GERAIS: Uberlândia: LH 721–723 (no voucher specimens); LH 724 (voucher specimen UFMT 17745); LH 725 (voucher specimen UFMT 17746); LH 726 (voucher specimen UFMT 17748).

PANSONATO ET AL.264 · Zootaxa 3861 (3) © 2014 Magnolia Press