# Geographical Variation in Morphological and Bioacoustic Traits ofPseudopaludicola mystacalis (Cope, 1887) and a Reassessment of the Taxonomic Status of Pseudopaludicola serrana Toledo, 2010 (Anura: Leptodactylidae: Leiuperinae)

Authors: André Pansonato; Jessica Rhaiza Mudrek; Fernanda Simioni; Itamar Alves Martins; Christine Strüssmann
Journal: Advances in Zoology
 (2014)

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## Abstract

Comparisons of advertisement calls of anurans can be used to determine intra- and interspecific differences or affinities. Described from midwestern Brazil,Pseudopaludicola mystacalis is widely distributed and abundant in major open Brazilian ecosystems. However, researchers frequently fail to determine the true taxonomic status of some of these populations and attribute them to unidentified or misidentified species. Herein, we employ morphological and bioacoustic data to reassess the distribution range and to evaluate intraspecific variation in P. mystacalis based on specimens from fifteen localities and seven Brazilian states. We also reassess the distribution and taxonomic status of Pseudopaludicola serrana, herein considered as a junior synonym of P. murundu based on morphology, bioacoustics, and molecular data.


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## Body

## 1. Introduction

Advertisement calls are important for mate recognition in anurans and hence contribute to premating isolation among sympatric species [1]. Comparison of advertisement calls can be used to determine differences and affinities among different anuran species and so to determine taxonomic identity and phylogenetic relationships [2, 3]. Such comparisons may be especially useful for situations in which the taxonomic identity of a species is uncertain or contested involving synonymies, redescriptions, and species resurrections. Indeed, in the absence of consistent morphological characters for interspecific distinctiveness, advertisement calls have proven to be of great help in revealing and supporting hypotheses of putative new species ofPseudopaludicola [4–9].Described from midwestern Brazil (Chapada dos Guimarães, Mato Grosso),Pseudopaludicola mystacalis is widely distributed and abundant in open environments along South American “diagonal of open formations” [8]. However, some populations along the species’ range are not recognized as belonging to this taxon, being rather attributed to other taxonomic entities, such asPseudopaludicola aff.falcipes,Pseudopaludicola aff.mystacalis, andPseudopaludicola sp. [10–14]. Therefore, an integrative taxonomic revision is needed to clarify the distribution range and variation in this species [9].In spite of great morphological similarity among most of the 18 species ofPseudopaludicolacurrently recognized, three of them are readily distinguished from the others by having longer hindlimbs [4–6, 8, 9]:Pseudopaludicola saltica (Cope, 1887),Pseudopaludicola murundu Toledo, Siqueira, Duarte, Veiga-Menoncello, Recco-Pimentel, and Haddad, 2010, andPseudopaludicola serrana Toledo, 2010. Advertisement calls of all three species are similarly composed of series of notes with nonconcatenated pulses [8, 15–17] and they all share 2
n
=
22 chromosomes [16, 18] comprising a monophyletic clade [19]. However, the results of a recent molecular phylogenetic analysis suggested thatP. serrana is a junior synonym ofP. murundu [19].Objectives of the present article are (1) to provide and to discuss the intraspecific variation in morphological and bioacoustic traits ofP. mystacalisalong its distribution range in Brazil; (2) to reassess the taxonomic status ofP. serrana, based on the reinterpretation of the literature data and on the examination of new materials (specimens and calls), freshly obtained at the type-locality and/or its vicinities ofP. serranaand related species.


## 2. Material and Methods

Examined specimens ofP. mystacalis,P. murundu, P. saltica, andP. serrana(Appendices A and B) are deposited in the following collections: “Coleção Zoológica de Vertebrados, Universidade Federal de Mato Grosso” (UFMT, Cuiabá, Mato Grosso, Brazil), “Coleção Herpetológica da Universidade Federal do Ceará” (UFC, Fortaleza, Ceará), “Fonoteca Neotropical Jacques Vielliard” (FNJV, Campinas, São Paulo), and “Museu de Ciências Naturais” (MCNAM, Belo Horizonte, Minas Gerais). Material deposited at UFMT was collected with permission of the “Instituto Chico Mendes de Conservação da Biodiversidade” (ICMBIO 27231-1).Since the available diagnoses forP. serrana andP. murundu do not allow clearly distinguishing specimens from these two species, new material from additional localities was putatively associated with each of them based solely on the positioning of collecting and/or recording sites: populations from easternmost localities in Minas Gerais were considered to representP. serrana and populations from westernmost localities in Minas Gerais (and those from the state of São Paulo) were considered to representP. murundu.The measurements of 218 adult specimens (Tables1 and 2) ofP. murundu (6 males; 1 female),P. mystacalis (101 males; 37 females),P. saltica (39 males; 3 females; original data in Pansonato et al. [8]), andP. serrana (22 males; 6 females) were taken with a digital caliper 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. [20]. Measurements for snout-vent length (SVL), head length (HL), head width (HW), interorbital distance (IOD), eye diameter (ED), eye-nostril distance (END), internarial distance (IND), and thigh length (THL) followed Duellman [21].Table 1
Measurements (mm) of specimens ofPseudopaludicola mystacalis from localities in four Brazilian states. Values are presented as mean ± standard deviation (minimum–maximum). N = number of specimens analyzed.
Mato Grosso
São Paulo
Goiás
Pará
Males
Females
Males
Females
Males
Females
Males
(N
=
53)
(N
=
20)
(N
=
14)
(N
=
7)
(N
=
20)
(N
=
6)
(N
=
14)
SVL
13.9
±
1.3
16.3
±
0.9
13.9
±
0.5
15.3
±
0.7
14.1
±
0.1
15.9
±
0.9
15.4
±
0.8
(10.9–16.3)
(14.3–18.1)
(13–14.9)
(14.3–16)
(11.7–16.5)
(14.6–17)
(14.6–16.6)
HL
5.4
±
0.4
6.2
±
0.5
5.4
±
0.3
5.5
±
0.3
5.3
±
0.4
5.8
±
0.5
5.6
±
0.2
(4.4–6.5)
(5.2–7.5)
(4.6–5.9)
(5.1–6.0)
(4.7–6.1)
(5.0–6.3)
(5.3–5.9)
HW
4.8
±
0.5
5.9
±
0.6
4.8
±
0.2
5.1
±
0.4
4.9
±
0.5
5.5
±
0.4
4.8
±
0.3
(3.7–6.7)
(5.0–6.8)
(4.3–5.1)
(4.6–5.8)
(4.3–6.4)
(4.9–5.9)
(4.5–5.3)
IOD
1.1
±
0.2
1.4
±
0.2
1.2
±
0.1
1.3
±
0.1
1.2
±
0.2
1.4
±
0.2
1.4
±
0.2
(0.7–1.6)
(0.8–1.7)
(1.0–1.4)
(1.1–1.4)
(1.0–1.7)
(1.2–1.8)
(1.1–1.5)
ED
1.6
±
0.2
1.7
±
0.2
1.4
±
0.1
1.5
±
0.2
1.5
±
0.1
1.6
±
0.1
1.7
±
0.08
(1.0–1.9)
(1.3–2.3)
(1.1–1.7)
(1.3–1.9)
(1.2–1.7)
(1.4–1.8)
(1.6–1.8)
END
1.2
±
0.2
1.4
±
0.2
1.1
±
0.07
1.3
±
0.2
1.2
±
0.1
1.3
±
0.1
1.2
±
0.1
(0.7–1.8)
(1.1–2.1)
(1.0–1.2)
(1.1–1.8)
(0.9–1.5)
(1.1–1.4)
(1.1–1.4)
IND
1.2
±
0.2
1.5
±
0.2
1.3
±
0.1
1.4
±
0.09
1.4
±
0.1
1.5
±
0.2
1.3
±
0.09
(0.8–1.7)
(1.0–1.9)
(1.1–1.5)
(1.3–1.5)
(1.1–1.7)
(1.2–1.7)
(1.2–1.4)
HAL
3.7
±
0.3
4.4
±
0.7
3.9
±
0.2
4.2
±
0.4
3.8
±
0.2
4.1
±
0.3
3.9
±
0.2
(3.1–4.4)
(3.2–6.3)
(3.5–4.3)
(3.8–4.8)
(3.4–4.4)
(3.8–4.5)
(3.6–4.1)
THL
6.3
±
0.5
7.7
±
0.9
6.2
±
0.3
6.6
±
0.6
6.5
±
0.5
7.4
±
0.2
6.8
±
0.2
(5.1–7.3)
(6.4–9.4)
(5.6–6.9)
(5.3–7.1)
(5.6–7.3)
(7.1–7.7)
(6.6–7.2)
TL
6.9
±
0.5
8.3
±
0.9
7
±
0.3
7.5
±
0.3
7.1
±
0.4
7.8
±
0.2
7.4
±
0.2
(5.9–8.2)
(7.2–9.8)
(6.7–7.6)
(7.1–7.8)
(6.4–8.0)
(7.5–8.1)
(7.2–7.6)
TAL
3.9
±
0.4
4.5
±
0.6
3.9
±
0.2
4.3
±
0.3
3.9
±
0.3
4.3
±
0.3
3.9
±
0.3
(3.1–4.6)
(3.1–5.2)
(3.5–4.5)
(3.9–4.6)
(3.2–4.5)
(4.0–4.8)
(3.4–4.2)
FL
7.7
±
0.5
9.2
±
0.8
7.9
±
0.3
8.4
±
0.5
7.7
±
0.4
8.5
±
0.6
8.1
±
0.5
(6.3–9.2)
(7.9–10.6)
(7.3–8.4)
(7.6–8.9)
(6.9–8.2)
(7.8–9.2)
(7.6–8.8)Table 2
Measurements (mm) of the topotypes ofPseudopaludicola serranaand P. murundu. Values are presented as mean ± standard deviation (minimum–maximum). Additional localities (state of Minas Gerais, municipalities of Itabirito, Lavras Novas, Mariana, Ouro Branco, São João Del Rei, and Botumirim). N = number of specimens analyzed.
Pseudopaludicola  serrana
Pseudopaludicola  murundu
Topotypes
Additional localities
Topotypes
Males
Females
Males
Females
Males
Females
(N
=
5)
(N
=
1)
(N
=
16)
(N
=
5)
(N
=
5)
(N
=
1)
SVL
16.3
±
1.2
17.7
15.2
±
0.9
17.1
±
1.2
15.9
±
0.8
17.3
(14.6–17.5)
(12.6–16.7)
(15.9–18.5)
(15.2–16.9)
HL
6.5
±
0.5
6.8
6.0
±
0.4
6.7
±
0.3
6.1
±
0.4
6.8
(6.2–7.3)
(4.8–6.9)
(6.3–7.1)
(5.6–6.7)
HW
6.0
±
0.2
6.6
5.5
±
0.4
6.3
±
0.3
5.5
±
0.2
5.9
(5.7–6.3)
(4.7–6.1)
(5.9–6.7)
(5.3–5.8)
IOD
1.4
±
0.1
1.8
1.4
±
0.1
1.6
±
0.07
1.4
±
0.0
.
8
1.5
(1.3–1.6)
(1.2–1.6)
(1.5–1.7)
(1.3–1.5)
ED
1.6
±
0.07
1.7
1.5
±
0.1
1.6
±
0.09
1.6
±
0.2
1.9
(1.5–1.7)
(1.3–1.7)
(1.5–1.7)
(1.4–1.8)
END
1.6
±
0.09
1.6
1.5
±
0.1
1.5
±
0.08
1.5
±
0.08
1.6
(1.5–1.7)
(1.3–1.7)
(1.4–1.6)
(1.4–1.6)
IND
1.5
±
0.1
1.8
1.4
±
0.1
1.6
±
0.08
1.5
±
0.09
1.6
(1.3–1.7)
(1.2–1.7)
(1.5–1.7)
(1.4–1.7)
HAL
4.4
±
0.1
4.9
4.1
±
0.4
4.5
±
0.1
4.3
±
0.2
4.9
(4.3–4.6)
(3.4–4.6)
(4.3–4.7)
(4.2–4.6)
THL
9.0
±
0.3
10.2
8.5
±
0.7
9.4
±
0.5
8.9
±
0.3
9.5
(8.5–9.3)
(6.8–9.4)
(8.7–10.1)
(8.6–9.3)
TL
10.9
±
0.3
12.7
10.7
±
0.7
11.8
±
0.5
11.2
±
0.3
12.3
(10.6–11.3)
(8.9–11.5)
(10.9–12.2)
(10.8–11.6)
TAL
5.3
±
0.2
5.8
5.1
±
0.4
5.7
±
0.4
5.4
±
0.07
6.3
(5.1–5.7)
(4.2–5.5)
(5.0–6.1)
(5.4–5.6)
FL
9.8
±
0.3
11.1
9.3
±
0.8
10.2
±
0.5
10.6
±
0.4
11.4
(9.5–10.3)
(7.4–10.4)
(9.5–10.6)
(9.9–10.9)Vocalizations of specimens ofP. murundu,P. mystacalis,P. saltica, andP. serrana were recorded 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 and 16 bit resolution and saved in uncompressed wave files. Spectrograms were edited using Raven Pro 1.3 software with the following configuration: forP. mystacalis, brightness 75%; contrast 80%; DFT size 512 samples; 3 dB filter bandwidth 112 Hz; time grid overlap 50%; forP. murundu,P. saltica, andP. serrana, brightness 70%; contrast 80%; DFT size 512 samples; 3 dB filter bandwidth 124 Hz; time grid overlap 50%.We analyzed 1894 notes (Tables3 and 4): 503 fromP. murundu, 444 notes fromP. mystacalis, 577 fromP. saltica, and 370 fromP. serrana. The following temporal variables were measured from the waveform: number of pulses per note; note and pulse duration (ms); internote and interpulse interval (ms). Note that repetition rate was calculated in notes per minute. Dominant frequency (note peak frequency; Hz) was obtained from the spectrogram. Terminology for bioacoustic variables follows Magalhães et al. [7], Pansonato et al. [8], and Heyer et al. [20]. Vocalizations are archived in the “Banco de Registros Bioacústicos” and housed at the “Laboratório de Herpetologia do Instituto de Biociências da Universidade Federal de Mato Grosso” (LH, Cuiabá, Mato Grosso).Table 3
Measurements of bioacoustic variables of the advertisement calls of specimens ofPseudopaludicola mystacalis from 12 localities in seven Brazilian states (CE: Ceará; GO: Goiás; MA: Maranhão; MT: Mato Grosso; PA: Pará; PI: Piauí; SP: São Paulo). Values are presented as mean ± standard deviation (minimum–maximum). N: number of notes analyzed.
Locality—State
Air temp
Note duration (ms)
Internote interval (ms)
Pulses per note
Pulse duration (ms)
Dominant frequency (Hz)
Notes/min(mean)
Poconé—MT (N
=
30)
25.6°C
40
±
5 (30–50)
80
±
10 (60–100)
16
±
1.2 (14–18)
2
±
0.3  (1-2)
4684.2
±
115.7 (4522–4909.6)
511.1
N.S. Livramento—MT (N
=
25)
25.2°C
40
±
4 (40–50)
70
±
30 (50–200)
17
±
1.0 (14–19)
2
±
0.3  (1-2)
4811.4
±
56.3 (4737.3–4909.6)
506.4
C. Guimarães—MT (N
=
110)
26.3°C
40
±
4 (30–50)
60
±
10 (40–100)
16
±
0.8 (14–17)
2
±
0.4  (1-2)
4962.4
±
181.5 (4651.2–5254.1)
576.9
Cuiabá—MT (N
=
25)
26°C
50
±
4 (30–50)
90
±
10 (60–100)
14
±
0.8 (12–16)
2
±
0.4  (1–3)
4775.2
±
82.8 (4651.2–4909.6)
443.3
Cuiabá—MT (N
=
25)
26°C
45
±
4 (30–50)
80
±
30 (50–200)
16
±
1.0 (13–18)
2
±
0.3  (1-2)
4837.2
±
150 (4478.9–4995.7)
524.3
Cáceres—MT (N
=
25)
26°C
50
±
3 (40–50)
60
±
50 (50–70)
16
±
1.0 (14–18)
2
±
0.3  (1-2)
4901
±
51.2 (4737.3–4995.7)
538.9
Balsas—MA (N
=
30)
26.5°C
40
±
2  (37–45)
80
±
5 (70–90)
16
±
1.0 (14–18)
2
±
0.4  (1-2)
5076.09
±
205.68 (4780.4–5340.2)
513.7
Icém—SP (N
=
28)
23°C
50
±
5 (50–60)
110
±
7 (100–150)
17
±
1.7 (14–20)
2
±
0.4  (1-2)
4872.2
±
79.9 (4737.3–4974.2)
363.3
Itapipoca—CE (N
=
30)
—
50
±
3  (40–50)
90
±
10  (70–130)
16
±
1.2  (14–18)
2
±
0.4   (1-2)
5128.2
±
98.1  (4780.4–5211)
420.7
Taíba—CE (N
=
30)
—
40
±
2  (40–50)
70
±
2  (60–70)
16±1.1  (13–18)
2±0.5(1–3)
4795.2±85.2  (4694.2–4952.6)
535.3
Brejo do Buriti—PI (N
=
30)
27°C
50
±
1  (45–50)
90
±
20  (60–140)
19
±
0.6  (18–20)
2
4860.75
±
31.48  (4823.4–4909.6)
429.6
Primavera—PA (N
=
30)
25.5°C
50
±
2  (40–50)
80
±
5  (70–100)
18±1.2  (16–20)
2±0.5(1-2)
5040.6±45.6  (4921.9–5109.4)
463.7
Uruaçu—GO (N
=
26)
24.5°C
50±2(40–50)
60±5 (50–80)
16±1.2  (14–18)
2
±
0.5   (1-2)
4633.4
±
86.6  (4500–4968.8)
543.5
Mean ± SD
50
±
4
(30–60)
80
±
10
(40–200)
16
±
1.7
(12–20)
2
±
0.4
(1–3)
4887.4
±
149.5
(4478.9–5340.2)
490.1±61.1 (363.3–576.9)Table 4
Measurements of bioacoustic variables of the advertisement calls of specimens ofPseudopaludicola murundu, P. saltica, andP. serrana. Values are presented as mean ± standard deviation (minimum–maximum).
Species(voucher record)
Note duration (ms)
Internote interval (ms)
Pulse duration (ms)
Interpulses interval (ms)
Dominant frequency (Hz)
Pulses/note
Notes/min
Municipality/state
P. murundu   (LH 676)
80
±
12 (58–105)
100
±
15 (69–141)
8
±
2 (4–12)
26
±
7 (7–40)
5600.7
±
204.3 (5168–5857)
3.3
±
0.7 (2–5)
348.9
±
21.9 (314.5–370.4)
Rio Claro/SP
P. murundu  (LH 677)
94
±
11 (49–114)
98
±
16 (70–140)
13
±
2 (9–17)
23
±
9 (6–39)
5579.9
±
150.2 (5254.1–5857)
2.9
±
0.2 (2-3)
317.2
±
17.6 (295.6–341.9)
Rio Claro/SP
P. murundu   (LH 678)
89
±
13 (33–104)
107
±
12 (89–137)
9
±
2 (5–17)
20
±
8 (8–46)
5725.9
±
129.7 (5340.2–5900.1)
3.7
±
0.5 (2–4)
308.7
±
13.9 (292.6–326.4)
Rio Claro/SP
P. murundu   (FNJV 12976)
81
±
15 (56–103)
152
±
50 (79–247)
9
±
2 (5–14)
18
±
5 (10–29)
5755.7
±
322.6 (5168–6201.6)
3.7
±
0.4 (3-4)
280.3
Rio Claro/SP
P. murundu   (FNJV 4575)
81
±
13 (54–105)
132
±
20 (97–193)
10
±
2 (5–16)
23
±
9 (5–50)
5575.5
±
149.3 (4875–5812.5)
3.4
±
0.7 (2–5)
284.3
±
31.3 (218.2–369.1)
São Roque de Minas/MG
P. murundu   (FNJV 4576)
118
±
18 (77–143)
159
±
14 (129–194)
13
±
2 (8–18)
27
±
9 (10–54)
5460.3
±
76.6 (5250–5625)
3.6
±
0.5 (2–4)
218.3
±
19.8 (189.3–266.6)
Poços de Caldas/MG
P. serrana   (LH 673)
98
±
12 (65–131)
184
±
18 (131–224)
12
±
2 (8–18)
16
±
5 (6–32)
5655.6
±
120.2 (5168–5814)
4.1
±
0.3 (3–5)
218.3
±
7.4 (206.1–225.8)
Brumadinho/MG
P. serrana  (LH 674)
91
±
14 (49–117)
236
±
25 (184–288)
12
±
2 (6–17)
25
±
8 (5–41)
5340.2
±
29.3 (5168–5512.5)
2.9
±
0.4 (2–4)
179.5
±
8.4 (170.9–187.7)
Brumadinho/MG
P. serrana  (LH 675)
83
±
15 (49–99)
161
±
51 (81–197)
10
±
2 (7–16)
16
±
7 (5–33)
5857
3.7
±
0.4 (3-4)
249.4
±
22.3 (228.2–272.7)
São João Del Rei/MG
P. serrana    (FNJV 12879)
60
±
6 (47–72)
86
±
8 (63–101)
8
±
2 (4–14)
8
±
2 (4–16)
5707.0
±
115.5 (5512.5–5857)
3.9
±
0.3 (3-4)
411.9
±
23.7 (361.4–476.2)
São João Del Rei/MG
P. serrana   (FNJV 12880)
63
±
13 (26–97)
112
±
20 (75–166)
5
±
1 (1–9)
13
±
5 (4–33)
5773.7
±
169.3 (5512.5–6029.3)
3.8
±
0.5 (2–5)
348.7
±
47.3 (251.1–517.2)
São João Del Rei/MG
P. saltica    (13A-01)
65
±
9 (48–86)
120
±
19 (75–170)
9
±
2 (5–13)
35
±
14 (5–55)
4751.0
±
104.2 (4608.1–4952.6)
2.5
±
0.5 (2-3)
383.9
±
36 (342.4–406.2)
Chapada dos Guimarães/MT
P. saltica   (42A-06)
58
±
6 (46–70)
109
±
14 (90–137)
8
±
1 (4–11)
15
±
7 (5–30)
4914.3
±
35.3 (4823.4–4952.6)
3.2
±
0
.
3
(3-4)
365.4
±
41.9 (298.5–441.2)
Pontes e Lacerda/MT
P. saltica    (42A-07)
66
±
13 (48–87)
186
±
23 (144–224)
11
±
4 (4–19)
36
±
5 (30–46)
4500.4
±
339.8 (4005.2–4952.6)
2.2
±
0.4 (2-3)
239.3
±
18.2 (213.5–283)
Pontes e Lacerda/MT
P. saltica   (LH 12)
77
±
8 (63–95)
113
±
18 (80–172)
6
±
1 (4–9)
21
±
18 (3–49)
5023.0
±
135.7 (4866.5–5512.5)
3.9
±
0.4 (3–5)
318.3
±
32.8 (229.9–394.7)
Porto Estrela/MT
P. saltica   (LH 13)
97
±
6 (85–106)
127
±
10 (99–158)
5
±
1 (3–7)
18
±
17 (2–56)
5236.8
±
61.5 (5081.8–5297.2)
5
±
0.1 (5–6)
268.7
±
14.7 (230.8–314.1)
Porto Estrela/MT
P. saltica   (LH 14)
78
±
5 (70–92)
134
±
17 (107–210)
17
±
13 (4–44)
6
±
2 (4–10)
4806.2
±
67.7 (4651.2–4995.7)
2.1
±
0.3 (2-3)
283.7
±
25.3 (206.2–338.9)
Porto Estrela/MT
P. saltica   (LH 16)
97
±
10 (64–118)
127
±
18 (93–162)
10
±
3 (6–18)
23
±
17 (3–56)
5035.9
±
299.8 (4306.6–5340.2)
4.5
±
0.6 (3–5)
271.2
±
26.5 (226.4–335.2)
Porto Estrela/MT
P. saltica   (LH 17)
73
±
26 (33–122)
106
±
24 (79–228)
7
±
1 (4–8)
19
±
13 (5–54)
4883.7
±
209.9 (4478.9–5168)
3.4
±
0.5 (3–5)
352.4
±
77.6 (211.3–483.8)
Porto Estrela/MT
P. saltica   (LH 18)
83
±
4 (75–100)
143
±
17 (100–212)
5
±
2 (3–16)
18
±
14 (3–47)
5248.3
±
44.3 (5081.8–5297.2)
4.1
±
0.3 (4-5)
267.0
±
21.4 (198.7–338.9)
Porto Estrela/MT
P. saltica   (LH 709)
86
±
14 (51–117)
122
±
55 (58–383)
6
±
1 (3–10)
11
±
8 (3–33)
5113.9
±
116.9 (4996.7–5340.2)
4.4
±
0.5 (4-5)
306.7
±
72.5 (126.6–483.8)
Uberlândia/MGDiscriminant function analyses (DFA) were conducted in order to evidence the set of morphometric and bioacoustic variables that mostly distinguish between the species ofPseudopaludicolacompared herein. DFA were performed on correlation matrices from 12 log-transformed morphometric and six bioacoustic variables using R platform version 3.0.1 [22]. Bioacoustic comparisons through the DFA were performed using mean individual values.


## 3. Results

### 3.1. Geographical Variation inPseudopaludicola mystacalis(Cope, 1887)

Based on morphology and bioacoustics,Pseudopaludicola mystacalis was positively identified in fifteen localities (Figure 1) from seven Brazilian states: Ceará, Goiás, Maranhão, Mato Grosso, Pará, Piauí, and São Paulo. The advertisement calls ofPseudopaludicola mystacalisalong its distribution range in Brazil (Figures 1 and 2) consisted of series composed of 9–229 notes with 16
±
1.7 (12–20) concatenated pulses (i.e., no interpulse intervals). Mean note repetition rate was 490.1
±
61.1 notes/min (363.3–576.9); mean duration of each note was 50
±
4 ms (30–60); average internote interval varied from 80
±
10 ms (40–200). Mean pulse duration was 2
±
0.4 ms (1–3). Frequency ranged from 2686.2 Hz to 7343.3 Hz and dominant frequency ranged from 4478.9 to 5340.2 Hz (Table 3).Figure 1
Geographical variation in the advertisement calls (oscillograms) ofPseudopaludicola mystacalis from twelve localities in seven Brazilian states: Pará (PA): Primavera (1); Ceará (CE): Itapipoca (2); Taíba (3); Maranhão (MA): Balsas (4); Piauí (PI): Brejo do Piauí (5); Goiás (GO): Uruaçu (6); Mato Grosso (MT): Cuiabá (7); Chapada dos Guimarães (8); Nossa Senhora do Livramento (9); Poconé (10); Cáceres (11); São Paulo (SP): Icém (12). Total time of each oscillogram corresponds to 0.2 s.Figure 2
Spectrograms (DFT size = 512 samples) of the advertisement calls ofPseudopaludicola mystacalisfrom twelve localities (two notes for each locality) in seven Brazilian states (CE: Ceará; GO: Goiás; MA: Maranhão; MT: Mato Grosso; PA: Pará; PI: Piauí; SP: São Paulo).Discriminant function analysis (DFA) with our data onP. mystacalis did not group localities, neither regarding morphometric characters (males and females) nor regarding bioacoustic variables. The first function (DF1) of DFA with morphometric data explained 63% of the variation among males of different populations (Figure 3(a)) and higher loadings corresponded to thigh length (10.5), hand length (9.8), and foot length (8.4). The DF1 explained 85% of the variation among females (Figure 3(b)) and higher loadings corresponded to hand length (12.3), foot length (10.7), and snout-vent length (8.8). The DF1 of DFA with bioacoustic variables explained 79% of the total variation in our sample (Figure 3(c)) and higher loadings corresponded to note rate (38.5), internote interval (29.5), and note duration (22.1). No distinct groups were formed along the first function axis of the DFA.Scatterplots, on the two discriminant axes (DF1 and DF2), of scores of 12 morphometric characters of 101 males (a), 37 females (b), and six bioacoustic variables (c) ofPseudopaludicola mystacalisfrom 15 different localities in Brazil.
(a)
(b)
(c)


### 3.2. Taxonomic Status ofPseudopaludicola serranaToledo, 2010

Advertisement calls ofPseudopaludicola serrana recorded at the type-locality (Brumadinho, Minas Gerais) and nearly 120 km southwestwards at Serra do Lenheiro, Minas Gerais were similar to those of topotypes ofP. murundu and distinct from topotypes ofP. saltica, both in spectral and temporal parameters (Figures 4 and 5). The intraspecific variation in bioacoustic parameters ofP. murundu andP. serrana, presented below, is based on calls from topotypical specimens and on calls recorded in additional localities listed in Appendix B.Figure 4
Spectrograms (DFT size = 512 samples; two notes for each locality) of advertisement calls ofPseudopaludicola serrana (A–C) andP. murundu (D–F) recorded at their respective type-localities andP. saltica recorded in state of Minas Gerais-Uberlândia (G); state of Mato Grosso-Chapada dos Guimarães (type-locality; H) and Porto Estrela (I).Oscillograms of advertisement calls ofPseudopaludicola serrana (a)-(b) andP. murundu (c)–(e) recorded at their respective type-localities andP. saltica recorded in state of Minas Gerais: Uberlândia (f); state of Mato Grosso: Chapada dos Guimarães (type-locality (g)) and Porto Estrela (h). Total time of each oscillogram corresponds to 0.5 s.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)Advertisement calls ofP. serrana were composed of series of pulsed notes with nonconcatenated pulses. Each note consisted of 3.6
±
0.6 (range 2–5) pulses. Mean note duration was 82
±
58 ms (26–131) emitted, on average, at intervals of 167
±
58 ms (63–288). Mean duration of each pulse was 9
±
3 ms (2–18). Interpulse intervals were 16
±
7 ms long (4–41). Mean frequency ranged from 4019.6
±
453 Hz to 7098.6
±
312 Hz and mean dominant frequency was 5625.1
±
212 Hz (5168–6029.3). Advertisement calls ofP. murundu were composed of series of pulsed notes with nonconcatenated pulses. Each note consisted of 3.4
±
0.6 (range 2–5) pulses. Mean note duration was 92
±
18 ms (33–143) emitted, on average, at intervals of 121
±
30 ms (69–247). Mean duration of each pulse was 10
±
3 ms (4–18). Interpulse intervals were 24
±
8 ms (5–54). Mean frequency ranged from 3628.5
±
513 Hz to 7330.1
±
355 Hz and mean dominant frequency was 5590.1
±
176 Hz (4875–6201.6 Hz).A discriminant function analysis (DFA) with morphometric data revealed thatP. serrana andP. murunduoverlapped along the first function (DF1) of DFA, which explained 98% of the total variation (Figure 6(a)). While the DFA did not separateP. serrana fromP. murundu, both were separated from a third species (Pseudopaludicola saltica) whose individuals also have long hindlimbs. Higher loadings corresponded to eye diameter (12.1), tarsus length (7.5), and tibia length (4.6). DFA from bioacoustic data also separatedPseudopaludicola saltica fromP. murundu andP. serrana (Figure 6(b)). The first function (DF1) of DFA explained 99% of the total variation and higher loadings corresponded to dominant frequency (64.1) and number of pulses per note (6.1).Scatterplots, on the two discriminant axes (DF1 and DF2), of scores of 12 morphometric characters of males (a) and six bioacoustic variables (b) ofPseudopaludicola murundu(open circles),P. saltica (triangles), andP. serrana(closed circles).
(a)
(b)DFA of morphometric measurements using all specimens assigned 100% of the individuals ofPseudopaludicola saltica, 86% of the individuals ofP. serrana, and only 16% of the individuals ofP. murundu to the correct species (Table 5). DFA of bioacoustic variables using all recorded males correctly assigned 89% of the specimens ofP. saltica, 83% of the specimens ofP. murundu, and 40% ofP. serrana (Table 5). In contrast, DFA with both morphometric and bioacoustic data using specimens ofP. saltica and a mixed sample of specimens attributable to eitherP. murunduorP. serranaassigned 100% of these latter to a single taxon.Table 5
Summary of the number of male specimens correctly classified asPseudopaludicola murundu, P. saltica, or P. serrana using discriminant function analysis of 12 morphometric measurements and seven bioacoustic variables.
Morphometric measurements
True group
P. murundu
P. saltica
P. serrana
P. murundu
1
0
5
P. saltica
0
39
0
P. serrana
3
0
19
Total number
6
39
22
%
16
100
86
Bioacoustic variables
True group
P. murundu
P. saltica
P. serrana
P. murundu
5
0
1
P. saltica
0
8
1
P. serrana
3
0
2
Total number
6
9
5
%
83
88.9
40The overall similarity in morphology and general structure of the call ofP. murunduandP. serrana, evidenced by the results of DFA with 12 morphometric and six bioacoustic variables, strongly support the rejection of the specific status ofPseudopaludicola serrana, here considered to be a junior synonym ofPseudopaludicola murundu. Geographical distribution ofPseudopaludicola murundu, as presently recognized, is not anymore restricted to its type-locality [16] but includes instead twelve distinct localities in southeastern Brazil in the states of Minas Gerais and São Paulo (Figure 7).Figure 7
Geographical distribution ofPseudopaludicola murundu (circles) andP. saltica (triangles) in southeastern Brazil: state of São Paulo—Rio Claro (type locality (1)) and Campinas (2); state of Minas Gerais—Poços de Caldas (3), Alpinópolis (4) (see [17]), São Roque de Minas (5), São João Del Rei (6), Mariana (7), Ouro Branco (8), Itabirito (9), Brumadinho (10), Lavras Novas (11), Botumirim (12), and Uberlândia (13).


## 3.1. Geographical Variation inPseudopaludicola mystacalis(Cope, 1887)

Based on morphology and bioacoustics,Pseudopaludicola mystacalis was positively identified in fifteen localities (Figure 1) from seven Brazilian states: Ceará, Goiás, Maranhão, Mato Grosso, Pará, Piauí, and São Paulo. The advertisement calls ofPseudopaludicola mystacalisalong its distribution range in Brazil (Figures 1 and 2) consisted of series composed of 9–229 notes with 16
±
1.7 (12–20) concatenated pulses (i.e., no interpulse intervals). Mean note repetition rate was 490.1
±
61.1 notes/min (363.3–576.9); mean duration of each note was 50
±
4 ms (30–60); average internote interval varied from 80
±
10 ms (40–200). Mean pulse duration was 2
±
0.4 ms (1–3). Frequency ranged from 2686.2 Hz to 7343.3 Hz and dominant frequency ranged from 4478.9 to 5340.2 Hz (Table 3).Figure 1
Geographical variation in the advertisement calls (oscillograms) ofPseudopaludicola mystacalis from twelve localities in seven Brazilian states: Pará (PA): Primavera (1); Ceará (CE): Itapipoca (2); Taíba (3); Maranhão (MA): Balsas (4); Piauí (PI): Brejo do Piauí (5); Goiás (GO): Uruaçu (6); Mato Grosso (MT): Cuiabá (7); Chapada dos Guimarães (8); Nossa Senhora do Livramento (9); Poconé (10); Cáceres (11); São Paulo (SP): Icém (12). Total time of each oscillogram corresponds to 0.2 s.Figure 2
Spectrograms (DFT size = 512 samples) of the advertisement calls ofPseudopaludicola mystacalisfrom twelve localities (two notes for each locality) in seven Brazilian states (CE: Ceará; GO: Goiás; MA: Maranhão; MT: Mato Grosso; PA: Pará; PI: Piauí; SP: São Paulo).Discriminant function analysis (DFA) with our data onP. mystacalis did not group localities, neither regarding morphometric characters (males and females) nor regarding bioacoustic variables. The first function (DF1) of DFA with morphometric data explained 63% of the variation among males of different populations (Figure 3(a)) and higher loadings corresponded to thigh length (10.5), hand length (9.8), and foot length (8.4). The DF1 explained 85% of the variation among females (Figure 3(b)) and higher loadings corresponded to hand length (12.3), foot length (10.7), and snout-vent length (8.8). The DF1 of DFA with bioacoustic variables explained 79% of the total variation in our sample (Figure 3(c)) and higher loadings corresponded to note rate (38.5), internote interval (29.5), and note duration (22.1). No distinct groups were formed along the first function axis of the DFA.Scatterplots, on the two discriminant axes (DF1 and DF2), of scores of 12 morphometric characters of 101 males (a), 37 females (b), and six bioacoustic variables (c) ofPseudopaludicola mystacalisfrom 15 different localities in Brazil.
(a)
(b)
(c)


## 3.2. Taxonomic Status ofPseudopaludicola serranaToledo, 2010

Advertisement calls ofPseudopaludicola serrana recorded at the type-locality (Brumadinho, Minas Gerais) and nearly 120 km southwestwards at Serra do Lenheiro, Minas Gerais were similar to those of topotypes ofP. murundu and distinct from topotypes ofP. saltica, both in spectral and temporal parameters (Figures 4 and 5). The intraspecific variation in bioacoustic parameters ofP. murundu andP. serrana, presented below, is based on calls from topotypical specimens and on calls recorded in additional localities listed in Appendix B.Figure 4
Spectrograms (DFT size = 512 samples; two notes for each locality) of advertisement calls ofPseudopaludicola serrana (A–C) andP. murundu (D–F) recorded at their respective type-localities andP. saltica recorded in state of Minas Gerais-Uberlândia (G); state of Mato Grosso-Chapada dos Guimarães (type-locality; H) and Porto Estrela (I).Oscillograms of advertisement calls ofPseudopaludicola serrana (a)-(b) andP. murundu (c)–(e) recorded at their respective type-localities andP. saltica recorded in state of Minas Gerais: Uberlândia (f); state of Mato Grosso: Chapada dos Guimarães (type-locality (g)) and Porto Estrela (h). Total time of each oscillogram corresponds to 0.5 s.
(a)
(b)
(c)
(d)
(e)
(f)
(g)
(h)Advertisement calls ofP. serrana were composed of series of pulsed notes with nonconcatenated pulses. Each note consisted of 3.6
±
0.6 (range 2–5) pulses. Mean note duration was 82
±
58 ms (26–131) emitted, on average, at intervals of 167
±
58 ms (63–288). Mean duration of each pulse was 9
±
3 ms (2–18). Interpulse intervals were 16
±
7 ms long (4–41). Mean frequency ranged from 4019.6
±
453 Hz to 7098.6
±
312 Hz and mean dominant frequency was 5625.1
±
212 Hz (5168–6029.3). Advertisement calls ofP. murundu were composed of series of pulsed notes with nonconcatenated pulses. Each note consisted of 3.4
±
0.6 (range 2–5) pulses. Mean note duration was 92
±
18 ms (33–143) emitted, on average, at intervals of 121
±
30 ms (69–247). Mean duration of each pulse was 10
±
3 ms (4–18). Interpulse intervals were 24
±
8 ms (5–54). Mean frequency ranged from 3628.5
±
513 Hz to 7330.1
±
355 Hz and mean dominant frequency was 5590.1
±
176 Hz (4875–6201.6 Hz).A discriminant function analysis (DFA) with morphometric data revealed thatP. serrana andP. murunduoverlapped along the first function (DF1) of DFA, which explained 98% of the total variation (Figure 6(a)). While the DFA did not separateP. serrana fromP. murundu, both were separated from a third species (Pseudopaludicola saltica) whose individuals also have long hindlimbs. Higher loadings corresponded to eye diameter (12.1), tarsus length (7.5), and tibia length (4.6). DFA from bioacoustic data also separatedPseudopaludicola saltica fromP. murundu andP. serrana (Figure 6(b)). The first function (DF1) of DFA explained 99% of the total variation and higher loadings corresponded to dominant frequency (64.1) and number of pulses per note (6.1).Scatterplots, on the two discriminant axes (DF1 and DF2), of scores of 12 morphometric characters of males (a) and six bioacoustic variables (b) ofPseudopaludicola murundu(open circles),P. saltica (triangles), andP. serrana(closed circles).
(a)
(b)DFA of morphometric measurements using all specimens assigned 100% of the individuals ofPseudopaludicola saltica, 86% of the individuals ofP. serrana, and only 16% of the individuals ofP. murundu to the correct species (Table 5). DFA of bioacoustic variables using all recorded males correctly assigned 89% of the specimens ofP. saltica, 83% of the specimens ofP. murundu, and 40% ofP. serrana (Table 5). In contrast, DFA with both morphometric and bioacoustic data using specimens ofP. saltica and a mixed sample of specimens attributable to eitherP. murunduorP. serranaassigned 100% of these latter to a single taxon.Table 5
Summary of the number of male specimens correctly classified asPseudopaludicola murundu, P. saltica, or P. serrana using discriminant function analysis of 12 morphometric measurements and seven bioacoustic variables.
Morphometric measurements
True group
P. murundu
P. saltica
P. serrana
P. murundu
1
0
5
P. saltica
0
39
0
P. serrana
3
0
19
Total number
6
39
22
%
16
100
86
Bioacoustic variables
True group
P. murundu
P. saltica
P. serrana
P. murundu
5
0
1
P. saltica
0
8
1
P. serrana
3
0
2
Total number
6
9
5
%
83
88.9
40The overall similarity in morphology and general structure of the call ofP. murunduandP. serrana, evidenced by the results of DFA with 12 morphometric and six bioacoustic variables, strongly support the rejection of the specific status ofPseudopaludicola serrana, here considered to be a junior synonym ofPseudopaludicola murundu. Geographical distribution ofPseudopaludicola murundu, as presently recognized, is not anymore restricted to its type-locality [16] but includes instead twelve distinct localities in southeastern Brazil in the states of Minas Gerais and São Paulo (Figure 7).Figure 7
Geographical distribution ofPseudopaludicola murundu (circles) andP. saltica (triangles) in southeastern Brazil: state of São Paulo—Rio Claro (type locality (1)) and Campinas (2); state of Minas Gerais—Poços de Caldas (3), Alpinópolis (4) (see [17]), São Roque de Minas (5), São João Del Rei (6), Mariana (7), Ouro Branco (8), Itabirito (9), Brumadinho (10), Lavras Novas (11), Botumirim (12), and Uberlândia (13).


## 4. Discussion

### 4.1. Geographical Variation inPseudopaludicola mystacalis(Cope, 1887)

Among the 18 valid species ofPseudopaludicola,P. mystacalisis currently the one with the most widespread distribution range. Extent of occurrence in Brazil is nearly two million square kilometers encompassing areas in three different ecoregions along all the “diagonal of open formations” from South America [23]: Caatinga, Cerrado, and Pantanal. It is also found in areas of Chaco in Argentina [24] and Paraguay [25]. Although plausible, the occurrence in Bolivian Chaco was based on material that morphologically and bioacoustically would correspond toP. ameghini [12, 26]. Habitats where individuals of this species were found include “restinga” [10, present study], “campo sujo” in elevated plateaus of the Cerrado Domain, and seasonally flooded fields in the Pantanal lowlands [8].Advertisement calls ofPseudopaludicola mystacalis from different Brazilian localities are composed of series of notes with concatenated pulses. Maximum variation in dominant frequency of the calls of all specimens analyzed is 861.3 Hz (from 4478.9 Hz, in Cuiabá, Mato Grosso, to 5340.2 Hz, in Balsas, Maranhão; Table 3) without any evident latitudinal or longitudinal trend. Dominant frequency in the most distant known locations (Primavera, state of Pará, and Icém, state of São Paulo) varied by 372.1 Hz. Considering subsets of populations from Brazilian Northeast (states of Ceará, Maranhão, and Piauí) and from Brazilian Midwest (states of Mato Grosso and Goiás), maximum interpopulational variation in dominant frequency is 646 Hz and 775.2 Hz, respectively. Maximum intrapopulational variation in dominant frequency, evaluated for four localities in the lower portion of the species’ range (Cáceres, Cuiabá, Nossa Senhora do Livramento, and Poconé, state of Mato Grosso), is 516.8 Hz in Cuiabá (see Table 3 and [8]).Maximum variation in temporal variables (notes per minute and number of pulses) of the calls of all specimens analyzed is 213.6 notes/min and 8 pulses/note. In the most distant known locations, the difference in the number of notes per minute and in the number of pulses is 100.3 notes/min and 6 pulses/note, respectively. Considering subsets of populations from Brazilian Northeast and Brazilian Midwest, maximum interpopulational variation is 114.6 notes/min and 133.5 notes/min, respectively. There are 7 pulses/note in calls from populations of both areas. Maximum intrapopulational variation in the number of notes per minute and in the number of pulses in four localities from the state of Mato Grosso is 81 notes/min in Cuiabá and 5 pulses/note in Cuiabá and Nossa Senhora do Livramento (see Table3 and [8]). Mean note duration does not differ significantly in all the examined calls (Table 3; F
=
4.9; df
=
2; P
=
0.03).Acoustic analysis of geographical variation in species with an extent of occurrence of the magnitude seen inP. mystacalis sometimes reveals cryptic taxa being treated under a single name. However, this was not the case inP. mystacalis, a taxon that remained erroneously characterized and poorly recognized in the literature until recently [8]. Our results reveal a relatively small variation in morphological and acoustical traits ofP. mystacalis and allow confirming the presence of the species in a wide geographical area along the Brazilian open ecosystems.


### 4.2. Taxonomic Status ofPseudopaludicola serranaToledo, 2010

When describingP. serrana, Toledo [15] used as morphological characteristics separating the three species belonging to theP. salticagroup the aspect (relative size and coloration) of nuptial pads in the external part of finger I and aspect of the vocal sac. Such characteristics allow differingP. serrana andP. murundu fromP. saltica but do not allow differingP. serrana fromP. murundu[15]. Previously, Haddad and Cardoso [17] had called attention to the fact that nuptial pads were more developed in specimens ofP. saltica from the type-locality (Chapada do Guimarães, Mato Grosso) than in specimens collected and recorded in localities in the states of Minas Gerais and São Paulo. In the same article, Haddad and Cardoso [17] also presented a spectrogram of the call of a specimen from Campinas, São Paulo. Based on such reported morphological evidence and acoustic parameters, we here argue that specimens from Minas Gerais and São Paulo attributed toP. saltica in Haddad and Cardoso [17] in fact correspond toP. murundu.The only external morphological characteristic used by Toledo [15] to differP. serrana fromP. murundu was a shorter head length/head width ratio (HL/HW = 0.97
±
0.06 in the five individuals ofP. serrana evaluated; 1.11
±
0.07 in 11 individuals ofP. murundu). After having analyzed a higher number of specimens attributed toP. serrana(N
=
21), we found that HL/HW (1.10
±
0.06; see Table 6) in this sample does not differ from the values presented in the original description, and confirmed herein, forP. murundu.Table 6
Ratios of selected measurements (in mm) of males ofPseudopaludicola murundu, P. saltica,andP. serrana. Values are presented as mean ± standard deviation. N = number of specimens analyzed.
HL/SVL
HW/SVL
HL/HW
HAL/SVL
FL/SVL
THL/SVL
TL/SVL
P. serrana (N
=
21)
0.40
±
0.03
0.36
±
0.02
1.10
±
0.06
0.27
±
0.02
0.61
±
0.04
0.56
±
0.03
0.70
±
0.03
P. murundu(N
=
5)
0.39
±
0.04
0.35
±
0.03
1.11
±
0.09
0.27
±
0.01
0.66
±
0.02
0.56
±
0.02
0.70
±
0.03
P. saltica (N
=
44)
0.41
±
0.04
0.38
±
0.03
1.09
±
0.11
0.26
±
0.03
0.68
±
0.06
0.55
±
0.05
0.73
±
0.05
HAL: hand length; HL: head length; HW: head width; FL: foot length; SVL: snout-vent length; THL: thigh length; TL: tibia length.The description ofP. serrana was also based on physical characteristics of its advertisement calls [15]. However, the original dataset is relatively low: 12 notes of a single male ofP. saltica, 14 notes of 2 males ofP. murundu, and 15 notes of 3 males ofP. serranawere analyzed and compared. Only one out of four acoustic variables (pulse duration) was considered diagnostic for the new species. Toledo [15] also showed that dominant frequency range ofP. serrana was completely nested within the dominant frequency range ofP. murundu. However, when later proposing the synonymization ofPseudopaludicola riopiedadensis Mercadal de Barrio and Barrio, 1994 toPseudopaludicola ternetziMiranda-Ribeiro, 1937, Cardozo and Toledo [5] based their decision on “several important overlaps”, including dominant frequency ranges, between the two species. Indeed, our data showed that dominant frequency contributed to higher loadings to separate species in theP. saltica group. Nonoverlapping dominant frequencies would therefore be expected between related but distinct species ofPseudopaludicola.Bioacoustic data have proven to be useful for uncovering morphologically cryptic species in the genusPseudopaludicola [4–9, 15, 16]. Advertisement calls of species in this genus might be divided into three distinct groups according to type of notes: nonpulsed notes, notes with concatenated pulses, and notes with nonconcatenated pulses [7]. Each group might be diagnosed by temporal (pulses per note, note and pulse duration, internotes and interpulses intervals, note rate, and pulse rate) and spectral variables (dominant frequency and harmonics), which in combination allow characterizing all the species for which calls were already analyzed, except forP. serrana andP. murundu. As evidenced by the recent synonymization ofPseudopaludicola riopiedadensis toP. ternetzi [15], and reinforced herein by the proposed synonymization ofP. serrana toP. murundu, morphologically similar species ofPseudopaludicola presenting the same type of note and an overlap in their spectral acoustic variables are expected to represent the same taxon. Therefore, no arguments remain to considerP. serrana as a distinct species fromP. murundu and the former must be considered as a junior synonym ofP. murundu.


## 4.1. Geographical Variation inPseudopaludicola mystacalis(Cope, 1887)

Among the 18 valid species ofPseudopaludicola,P. mystacalisis currently the one with the most widespread distribution range. Extent of occurrence in Brazil is nearly two million square kilometers encompassing areas in three different ecoregions along all the “diagonal of open formations” from South America [23]: Caatinga, Cerrado, and Pantanal. It is also found in areas of Chaco in Argentina [24] and Paraguay [25]. Although plausible, the occurrence in Bolivian Chaco was based on material that morphologically and bioacoustically would correspond toP. ameghini [12, 26]. Habitats where individuals of this species were found include “restinga” [10, present study], “campo sujo” in elevated plateaus of the Cerrado Domain, and seasonally flooded fields in the Pantanal lowlands [8].Advertisement calls ofPseudopaludicola mystacalis from different Brazilian localities are composed of series of notes with concatenated pulses. Maximum variation in dominant frequency of the calls of all specimens analyzed is 861.3 Hz (from 4478.9 Hz, in Cuiabá, Mato Grosso, to 5340.2 Hz, in Balsas, Maranhão; Table 3) without any evident latitudinal or longitudinal trend. Dominant frequency in the most distant known locations (Primavera, state of Pará, and Icém, state of São Paulo) varied by 372.1 Hz. Considering subsets of populations from Brazilian Northeast (states of Ceará, Maranhão, and Piauí) and from Brazilian Midwest (states of Mato Grosso and Goiás), maximum interpopulational variation in dominant frequency is 646 Hz and 775.2 Hz, respectively. Maximum intrapopulational variation in dominant frequency, evaluated for four localities in the lower portion of the species’ range (Cáceres, Cuiabá, Nossa Senhora do Livramento, and Poconé, state of Mato Grosso), is 516.8 Hz in Cuiabá (see Table 3 and [8]).Maximum variation in temporal variables (notes per minute and number of pulses) of the calls of all specimens analyzed is 213.6 notes/min and 8 pulses/note. In the most distant known locations, the difference in the number of notes per minute and in the number of pulses is 100.3 notes/min and 6 pulses/note, respectively. Considering subsets of populations from Brazilian Northeast and Brazilian Midwest, maximum interpopulational variation is 114.6 notes/min and 133.5 notes/min, respectively. There are 7 pulses/note in calls from populations of both areas. Maximum intrapopulational variation in the number of notes per minute and in the number of pulses in four localities from the state of Mato Grosso is 81 notes/min in Cuiabá and 5 pulses/note in Cuiabá and Nossa Senhora do Livramento (see Table3 and [8]). Mean note duration does not differ significantly in all the examined calls (Table 3; F
=
4.9; df
=
2; P
=
0.03).Acoustic analysis of geographical variation in species with an extent of occurrence of the magnitude seen inP. mystacalis sometimes reveals cryptic taxa being treated under a single name. However, this was not the case inP. mystacalis, a taxon that remained erroneously characterized and poorly recognized in the literature until recently [8]. Our results reveal a relatively small variation in morphological and acoustical traits ofP. mystacalis and allow confirming the presence of the species in a wide geographical area along the Brazilian open ecosystems.


## 4.2. Taxonomic Status ofPseudopaludicola serranaToledo, 2010

When describingP. serrana, Toledo [15] used as morphological characteristics separating the three species belonging to theP. salticagroup the aspect (relative size and coloration) of nuptial pads in the external part of finger I and aspect of the vocal sac. Such characteristics allow differingP. serrana andP. murundu fromP. saltica but do not allow differingP. serrana fromP. murundu[15]. Previously, Haddad and Cardoso [17] had called attention to the fact that nuptial pads were more developed in specimens ofP. saltica from the type-locality (Chapada do Guimarães, Mato Grosso) than in specimens collected and recorded in localities in the states of Minas Gerais and São Paulo. In the same article, Haddad and Cardoso [17] also presented a spectrogram of the call of a specimen from Campinas, São Paulo. Based on such reported morphological evidence and acoustic parameters, we here argue that specimens from Minas Gerais and São Paulo attributed toP. saltica in Haddad and Cardoso [17] in fact correspond toP. murundu.The only external morphological characteristic used by Toledo [15] to differP. serrana fromP. murundu was a shorter head length/head width ratio (HL/HW = 0.97
±
0.06 in the five individuals ofP. serrana evaluated; 1.11
±
0.07 in 11 individuals ofP. murundu). After having analyzed a higher number of specimens attributed toP. serrana(N
=
21), we found that HL/HW (1.10
±
0.06; see Table 6) in this sample does not differ from the values presented in the original description, and confirmed herein, forP. murundu.Table 6
Ratios of selected measurements (in mm) of males ofPseudopaludicola murundu, P. saltica,andP. serrana. Values are presented as mean ± standard deviation. N = number of specimens analyzed.
HL/SVL
HW/SVL
HL/HW
HAL/SVL
FL/SVL
THL/SVL
TL/SVL
P. serrana (N
=
21)
0.40
±
0.03
0.36
±
0.02
1.10
±
0.06
0.27
±
0.02
0.61
±
0.04
0.56
±
0.03
0.70
±
0.03
P. murundu(N
=
5)
0.39
±
0.04
0.35
±
0.03
1.11
±
0.09
0.27
±
0.01
0.66
±
0.02
0.56
±
0.02
0.70
±
0.03
P. saltica (N
=
44)
0.41
±
0.04
0.38
±
0.03
1.09
±
0.11
0.26
±
0.03
0.68
±
0.06
0.55
±
0.05
0.73
±
0.05
HAL: hand length; HL: head length; HW: head width; FL: foot length; SVL: snout-vent length; THL: thigh length; TL: tibia length.The description ofP. serrana was also based on physical characteristics of its advertisement calls [15]. However, the original dataset is relatively low: 12 notes of a single male ofP. saltica, 14 notes of 2 males ofP. murundu, and 15 notes of 3 males ofP. serranawere analyzed and compared. Only one out of four acoustic variables (pulse duration) was considered diagnostic for the new species. Toledo [15] also showed that dominant frequency range ofP. serrana was completely nested within the dominant frequency range ofP. murundu. However, when later proposing the synonymization ofPseudopaludicola riopiedadensis Mercadal de Barrio and Barrio, 1994 toPseudopaludicola ternetziMiranda-Ribeiro, 1937, Cardozo and Toledo [5] based their decision on “several important overlaps”, including dominant frequency ranges, between the two species. Indeed, our data showed that dominant frequency contributed to higher loadings to separate species in theP. saltica group. Nonoverlapping dominant frequencies would therefore be expected between related but distinct species ofPseudopaludicola.Bioacoustic data have proven to be useful for uncovering morphologically cryptic species in the genusPseudopaludicola [4–9, 15, 16]. Advertisement calls of species in this genus might be divided into three distinct groups according to type of notes: nonpulsed notes, notes with concatenated pulses, and notes with nonconcatenated pulses [7]. Each group might be diagnosed by temporal (pulses per note, note and pulse duration, internotes and interpulses intervals, note rate, and pulse rate) and spectral variables (dominant frequency and harmonics), which in combination allow characterizing all the species for which calls were already analyzed, except forP. serrana andP. murundu. As evidenced by the recent synonymization ofPseudopaludicola riopiedadensis toP. ternetzi [15], and reinforced herein by the proposed synonymization ofP. serrana toP. murundu, morphologically similar species ofPseudopaludicola presenting the same type of note and an overlap in their spectral acoustic variables are expected to represent the same taxon. Therefore, no arguments remain to considerP. serrana as a distinct species fromP. murundu and the former must be considered as a junior synonym ofP. murundu.


## 5. Conclusions

We here report on the variation in morphometric characters and variation in bioacoustic variables of the advertisement calls in fifteen Brazilian populations ofPseudopaludicola mystacalis. Although limited, our sample evidenced no statistical differences in any of the evaluated attributes and allowed us to confirm that this species is widely distributed in Brazil. Morphometric and bioacoustic data did not differ, also, in populations currently attributed toPseudopaludicola serrana andPseudopaludicola murundu, and the former taxon is here considered as a junior synonym ofP. murundu. The data in the present work aims at contributing to a better understanding of the diversity and distribution patterns in the genusPseudopaludicola.



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*Source: 563165-2014-08-12.xml*