Bioacoustics of Pithecopus gonzagai

Differential diagnosis Pithecopus gonzagai sp. nov. is promptly distinguished from P. centralis , P. megacephalus , P. ayeaye , P. oreades and P. rusticus by the absence of the reticulate pattern of colouration on flanks ( Bokermann 1965 ; Lutz 1966 ; Brandṳo 2002; Caramaschi 2006 ; Bruschi et al. 2014). Pithecopus gonzagai sp. nov. is distinguished from P.centralis and P. megacephalus by its smaller head width ( 8.9–11.2 mm in P. gonzagai sp. nov. vs 12.2–14.5 mm , combined values for other species). Also, P. gonzagai sp. nov. differs from P. ayeaye , P. megacephalus , P. oreades and P. rusticus ( 9.9–12.7 mm , combined values) by its smaller head length ( 6.2–8.9 mm ). The new species is distinguished from P. centralis and P. megacephalus (3.0– 3.5 mm , combined values) by its smaller eye-nostril distance ( 1.8–2.8 mm ) ( Bokermann 1965 ; Lutz 1966 ; Brandṳo 2002; Caramaschi 2006 ; Bruschi et al. 2014). Pithecopus gonzagai sp. nov. is also distinguished from P. centralis (40.0–42.0 mm) by its smaller SVL ( 28.5–37.8 mm ) ( Bokermann 1965 ). The new species is distinguished from P. rusticus by the absence of the slightly reticulated pattern on the palpebral membrane and the throat region (pattern unique to P. rusticus ; Bruschi et al. 2014). P. gonzagai sp. nov. P. nordestinus Type series Additional specimens Traits Males n = 24 Females n = 4 Males n = 135 Females n = 15 Males n = 52 Snout-vent length 33.1 ± 1.4 (29.5–35.3) 38.0 ± 0.8 (36.3–38.1) 33.3 ± 2.0 (28.5–37.8) 37.9 ± 1.6 (35.3–40.7) 36.3 ± 1.9 (32.9–42.5) Head length 7.6 ± 0.7 (6.2–8.9) 9.1 ± 0.5 (7.8–9.1) 7.3 ± 0.5 (6.3–8.6) 7.9 ± 0.4 (7.3–8.6) 7.5 ± 0.7 (6.3–9.7) Head width 9.9 ± 0.3 (9.1–10.4) 11.9 ± 0.4 (10.9–11.9) 10.2 ± 0.5 (8.9–11.2) 11.4 ± 0.5 (10.8–12.4) 11.1 ± 0.6 (9.9–12.7) Axilla-groin length 16.3 ± 1.7 (12.6–18.8) 19.1 ± 1.3 (16.4–19.4) 15.9 ± 1.8 (11.8–19.5) 18.5 ± 1.9 (15.4–22.8) 17.3 ± 1.5 (14.5–20.8) Eye diameter 4.1 ± 0.3 (3.6–4.6) 4.8 ± 0.4 (3.9–4.8) 4.0 ± 0.3 (3.0–4.9) 4.4 ± 0.3 (3.9–5.0) 4.2 ± 0.4 (3.3–4.9) Tympanum diameter 1.5 ± 0.2 (1.2–1.8) 2.2 ± 0.3 (1.6–2.2) 1.7 ± 0.3 (1.1–2.3) 1.9 ± 0.2 (1.5–2.3) 1.8 ± 0.3 (1.2–2.3) Eye-nostril distance 2.1 ± 0.2 (1.8–2.6) 2.6 ± 0.2 (2.0–2.5) 2.3 ± 0.2 (1.8–2.8) 2.6 ± 0.2 (2.3–3.0) 2.5 ± 0.2 (2.0–3.1) Internarial distance 3.1 ± 0.3 (2.6–3.5) 3.7 ± 0.3 (3.1–3.7) 3.0 ± 0.3 (2.3–3.7) 3.4 ± 0.3 (2.9–3.8) 3.4 ± 0.3 (2.9–4.0) Upper arm length 6.2 ± 0.4 (5.4–6.9) 7.8 ± 0.4 (7.0–7.9) 6.7 ± 0.6 (5.3–8.2) 8.0 ± 0.7 (6.6–9.0) 7.3 ± 0.5 (6.5–8.5) Forearm length 7.2 ± 0.5 (6.3–8.0) 9.8 ± 0.6 (8.3–9.6) 7.5 ± 0.5 (6.3–8.8) 9.3 ± 0.6 (8.4–10.2) 8.2 ± 0.6 (6.9–9.3) Hand length 8.5 ± 0.6 (7.7–9.9) 11.0 ± 0.9 (8.8–11.0) 8.5 ± 0.6 (7.0–9.7) 9.8 ± 0.6 (8.5–10.4) 9.4 ± 0.7 (8.0–11.2) Thigh length 13.5 ± 0.7 (12.1–14.6) 17.0 ± 0.6 (15.8–17.2) 14.1 ± 0.8 (12.0–15.9) 16.2 ± 0.9 (14.6–18.0) 15.1 ± 0.8 (13.6–16.6) Tibia length 13.5 ± 0.7 (11.9–14.8) 16.5 ± 0.6 (15.2–16.6) 14.0 ± 0.7 (12.4–16.0) 16.0 ± 0.8 (14.4–17.3) 14.8 ± 0.7 (13.4–16.2) Tarsus length 9.0 ± 0.5 (8.1–9.9) 11.0 ± 0.4 (10.2–11.1) 9.3 ± 0.5 (7.9–10.5) 10.4 ± 0.6 (9.5–11.4) 9.8 ± 0.6 (8.8–11.1) Foot length 11.0 ± 0.7 (9.6–12.6) 13.4 ± 0.7 (11.8–13.2) 11.1 ± 0.7 (9.5–12.9) 12.8 ± 0.8 (11.6–14.1) 11.7 ± 0.6 (10.4–13.1) Call traits P. gonzagai sp . nov . n = 6 males /73 calls P. nordestinus n = 6 males / 105 calls Call duration (ms) 28.5 ± 6.7 (17–49) 51.0 ± 8.5 (19–85) Calls per minute 34.9 ± 17.1 (16.8–54.4) 42.7 ± 21.0 (11–82) Pulses / call 3.3 ± 0.4 (3–4) 4.0 ± 1.0 (3–6) Pulse duration (ms) 7.3 ± 1.3 (5–12) 7.5 ± 0.9 (3–16) Inter-pulse interval within core (ms) 0.9 ± 0.7 (0–4) 0.6 ± 0.4 (0–6) Core duration (ms) 22.0 ± 4.1 (17–35) 24.2 ± 2.2 (17–33) Pulses / core 3.0 ± 0.0 (3–3) 3.0 ± 0.0 (3–4) Duration of isolated pulses (ms) 7.7 ± 2.5 (4–12) 8.3 ± 1.7 (2–17)* Number of isolated pulses 1.0 (1–1) 1.2 ± 0.1 (1–2) Interval between core and isolated pulse (ms) 14.2 ± 3.8 (8–22) 17.2 ± 4.7 (6–48)** Pulses per second 141.5 ± 22 (86–177) 127.0 ± 11.7 (91–177) Min. of dominant frequency (Hz) 1,563 ± 124 (1,188–1,927) 1,508 ± 126 (1,133–1,897) Max. of dominant frequency (Hz) 2,856 ± 215 (2,446–3,592) 2,539 ± 260 (2,174–3,124) Peak of dominant frequency (Hz) 2,118 ± 63.2 (1,969–2,391) 2,074 ± 47 (1,969–2,250) Air temperature (°C) 28.6 ± 0.5 (28–29) 24.6 ± 3.0 (21–27) Time of recording (hour:minutes) 19:11–22:07 21:04–00:00 From its closer relatives (the lowland species), Pithecopus gonzagai sp. nov. is distinguished by being smaller than P. nordestinus , P. azureus and P. hypochondrialis in SVL , head width and length of axillagroin, internarial distance, and upper arm, hand, thigh, tibia and foot lengths (Exact Wilcoxon-Mann-Whitney Test: P <0.01; see Table 3 ). In addition, based on P values obtained from the Exact Wilcoxon-Mann-Whitney Test ( Table 3 ), P. gonzagai sp. nov. is smaller than P. nordestinus in eye-nostril distance ( 1.8–2.8 mm in the new species vs 2.0– 3.1 mm in P. nordestinus ), forearm length ( 6.3–8.8 mm in the new species vs 6.9–9.3 mm in P. nordestinus ) and tarsus length ( 7.9–10.5 mm in the new species vs 8.8–11.1 mm in P. nordestinus ). The new species is larger than P . araguaius in SVL , head length, head width, axilla-groin and eye-nostril lengths, and upper arm, hand, thigh and foot lengths (see Table 3 ). The randomForest model on morphometric traits classified 90% of the males of the new species correctly ( Table 4 ). It was the species with the highest percentage of males classified correctly in comparison with the other four taxa. DAPC based on morphological traits yielded no noticeable discrimination among the new species and P. azureus , P. nordestinus and P. araguaius ( Fig. 4A ). However, it is possible to notice slight discrimination between the new species and P. hypochondrialis ( Fig. 4A ), with a greater separation along axis 1 (LD1 = 58%; axis x), but the axis 2 (LD2 = 25%; axis y) also contributes to the separation. Tarsus length (17%), head width (12%), tibia (12%) and foot (11%) lengths mainly accounted for species separation along LD1 ( Fig. 4A ); while tarsus (25%) and hand (16%) lengths, eyenostril distance (11%) and internarial distance (10%) accounted for the separation along LD2 ( Fig. 4A ). Morphometric measurements of all examined specimens and the eigenvectors of the DAPC based on morphometric traits are in Supplementary files 3 and 4, respectively. Traits vs P. azureus (n = 25) vs P. nordestinus (n = 52) vs P. hypochondrialis (n = 93) vs P. araguaius (n = 36) Snout-vent length 1 . 09 × 10 -8 8 . 80 × 10 -16 8 . 80 × 10 -16 2 . 44 × 10 -5 Head length 3.79 × 10-1 3.79 × 10-1 1 . 13 × 10 -11 1 . 40 × 10 -3 Head width 5 . 32 × 10 -5 8 . 80 × 10 -16 8 . 80 × 10 -16 4 . 50 × 10 -9 Axilla-groin length 7 . 56 × 10 -4 3 . 65 × 10 -5 8 . 47 × 10 -4 7 . 56 × 10 -4 Eye diameter 5.89 × 10-1 2.29 × 10-1 1 . 25 × 10 -15 2.42 × 10-1 Tympanum diameter 9.43 × 10-1 3.58 × 10-2 1 . 06 × 10 -3 8.56 × 10-1 Eye-nostril distance 2.84 × 10-1 3 . 26 × 10 -11 1 . 57 × 10 -8 1 . 51 × 10 -12 Internarial distance 2 . 14 × 10 -7 1 . 66 × 10 -15 8 . 80 × 10 -16 2.59 × 10-1 Upper arm length 1 . 08 × 10 -4 6 . 26 × 10 -13 8 . 80 × 10 -16 2 . 98 × 10 -4 Forearm length 2.20 × 10-2 5 . 87 × 10 -14 8 . 80 × 10 -16 9.33 × 10-1 Hand length 8 . 29 × 10 -5 1 . 29 × 10 -12 8 . 80 × 10 -16 3 . 21 × 10 -6 Thigh length 6 . 75 × 10 -4 5 . 54 × 10 -14 8 . 80 × 10 -16 9 . 29 × 10 -3 Tibia length 2 . 82 × 10 -3 7 . 06 × 10 -11 8 . 80 × 10 -16 2.50 × 10-2 Tarsus length 2.19 × 10-1 1 . 10 × 10 -8 8 . 80 × 10 -16 7.90 × 10-1 Foot length 8 . 41 × 10 -5 3 . 18 × 10 -6 8 . 80 × 10 -16 1 . 15 × 10 -5 In comparison with the lowland species, the new species differs from P. palliatus by having the advertisement call with a higher dominant frequency (above 1900 Hz in P. gonzagai sp. nov. vs 1580 Hz in P. palliatus ) and with a single note (= core portion) (double notes in P. palliatus ; K̂hler & L̂tters 1999 ). Based on the Exact Wilcoxon-Mann-Whitney Test results, the new species can be distinguished from P. hypochondrialis and P. araguaius by having a lower number of pulses per advertisement call (new species: 3.3 ± 0.4 [3–4], P. araguaius : 6.0 ± 0.6 [5–8; P = 3.50 × 10 -3 ], P. hypochondrialis : 4.2 ± 0.4 [3–6; P = 7.42 × 10 -4 ]; present study; Haga et al. 2017a ) and pulses per core (new species: 3.0 ± 0.0 [3–3], P. araguaius : 6.0 ± 0.5 [5–8; P = 3.50 × 10 -3 ], P. hypochondrialis : 3.9 ± 0.3 [3–5; P = 2.61 × 10 -6 ]; present study; Haga et al. 2017a ), and shorter core duration (new species: 22.0 ± 4.1 [17–35 ms], P. araguaius : 39.3 ± 5.4 [28–48 ms; P = 3.50 × 10 -3 ], P. hypochondrialis : 32.0 ± 4.0 [19–61 ms; P = 1.77 × 10 -4 ]; present study; Haga et al. 2017a ). In addition, the new species can also be differentiated from P. araguaius by the lower peak of the dominant frequency of its advertisement call (new species: 2118 ± 63.2 [1969–2391 Hz], P. araguaius : 2540 ± 308 [2240–3316 Hz; P = 4.66 × 10 -3 ]; present study; Haga et al. 2017a ), and from P. hypochondrialis by its shorter inter-pulse interval within the core (new species: 1.8 ± 0.8 [0–4 ms], P. hypochondrialis : 2.0 ± 1.0 [0–7 ms; P = 1.06 × 10 -2 ]; present study; Haga et al. 2017a ). We were unable to find qualitative or quantitative diagnostic acoustic characters (absence of overlaps) between the new species, P. nordestinus and P. azureus . The randomForest model on acoustic traits was unable to distinguish one male of the new species from those of P. hypochondrialis ( Table 4 ); the other five males were correctly classified. All six males of P. nordestinus were correctly classified. The univariate analyses did not recover differences between calls of the new species, P. azureus and P. nordestinus . In addition, there are overlaps in the acoustic traits of these three species. Among the lowland species, P. araguaius is the more acoustically distinct species ( Fig. 4B ). The greater separation among lowland species was along axis 1 (LD1 = 91%), while axis 2 contributed much less to the separation (LD2 = 7%). Pulses per core (30%), core duration (22%), isolated pulse (21%) and number of pulses per call (18%) mainly accounted for species separation along axis 1 ( Fig. 4B ). Eigenvectors of the DAPC based on acoustic traits are in Supplementary file 5.

Phylogenetic inferences and species delimitation test Bayesian inference was congruent with the phylogenetic intrageneric relations recovered by Faivovich et al. (2010) , Duellman et al. (2016) and Haga et al. (2017a) . In our topologies, we recovered two wellsupported divergent clades (North and South) traditionally assigned to P. nordestinus , coincident with the phylogeographic breaks reported by Bruschi et al. (2019) . The paratopotypes of P. gonzagai sp. nov. were recovered nested all with populations from north of SFR while the specimens of P. nordestinus were assembled with populations from south of SFR ( Fig. 5 , Supplementary file 6). The P. gonzagai sp. nov. + P. nordestinus are sister-group of the P. azureus ( Fig. 5 , Supplementary file 6). The single-locus species discovery strategies by GMYC and bPTP approaches were performed in a dataset composed of 148 sequences consisting of 1049 nucleotides from mitochondrial DNA 16S gene, 134 sequences consisting of 892 nucleotides from ND2 and 73 sequences consisting of 422 nucleotides from Siah . Both the GMYC and bPTP identified within the stringent threshold two taxonomic entities, congruent with two main lineages recovered by Bayesian inference (see Fig. 5 , Supplementary files 6, 7, 8, 9). Our results recovered same species clusters as shown by Bruschi et al. (2019) in a multi-locus study. GMYC analysis under an estimated ultrametric tree showed the threshold time (16S: -0.0001845922; ND2 : -0.01166751; Siah : -0.0002401328) indicating the time all nodes reflect coalescent events; the likelihood of the null model was 1631.647 (16S), 1289.623 ( ND2 ) and 644.3782 ( Siah ) and the maximum likelihood of the GMYC model was 1635.787 (16S), 1291.181 ( ND2 ), 651.7409 ( Siah ). Because the differentiation among samples of P. nordestinus and P. gonzagai sp. nov. based on mitochondrial markers revealed high genetic distances level, the GMYC model suggested 52 entities composed of 32 distinct clusters for 16S, three entities composed of three clusters for ND2 and 11 entities composed of ten clusters for Siah , however, with the exception of two individuals from Areia Branca, no mixture between samples of P. nordestinus and P. gonzagai sp. nov. was recovered in our inferences. The tree resulting from the bPTP analysis recovered two species from the best ML search, completely separating individuals of the south ( P. nordestinus ) from individuals of the north ( Pithecopus gonzagai sp. nov. ). The highest Bayesian solution was similar to the ML tree; however, a total of 118 (16S), 85 ( ND2 ) and 54 ( Siah ) putative entities were recovered by a simple heuristic search. In contrast, a mass sampling can contain individuals from real isolated clades and individuals in different states of structured populations, which may be the cause of the high number of entities found in the results from GMYC and bPTP analyses. Advertisement call Six males and 73 advertisement calls of the new species were recorded and analysed. Quantitative call traits are summarized in Table 2 . The advertisement call of P. gonzagai sp. nov. consists of a single pulsed note emitted sporadically ( Fig. 6 A–B). The calls are generally composed of a main group of pulses (= core portion) ( Fig. 6A ), which may be followed by one low-amplitude final pulse ( Fig. 6B ). When present (37 % of analysed calls), the isolated pulses were limited to one and lasted 4–12 ms, separated from the core portion by a long interval of 8–22 ms. The pattern of three-pulsed core without the isolated pulse was more common (63% of analysed calls; Fig. 6A ), and lasted 17–35 ms. Taking into account the isolated pulses, the call lasted 17–49 ms and has 3–4 pulses. The pulse duration varied from 5–12 ms, emitted at rates of 86–177 pulses / second. The inter-pulse interval (or no interval) within the core lasted 0–4 ms. The peak of dominant frequency varied from 1969 to 2391 Hz ( Fig. 6 A–B). Six males and 105 advertisement calls of P. nordestinus (sister species) were recorded and analysed. The advertisement call of the sister species of the new taxon also contains a single pulsed note, with sporadic emission (quantitative call traits are summarized in Table 2 ; Fig. 6 C–D). Core duration varied from 17 to 33 ms and the intervals between the core and the isolated pulses varied from 6 to 48 ms. The number of pulses per core varied from 3 to 4 pulses, with pulse intervals (or no interval) within the core from 0 to 6 ms. Pulses were arranged in: (1) a three-pulsed core with one isolated pulse (62.9%); (2) a threepulsed core with no isolated pulse (16.2%); (3) a three-pulsed core followed by two isolated pulses, with long inter-pulse interval between them (17.1%; Fig. 6D ); (4) a four-pulsed core followed by an isolated pulse (2.9%); and (5) a four-pulsed core followed by two isolated pulses, with long inter-pulse interval between them (1.0%). The entire call duration lasted 19–85 ms and has 3–6 pulses, the duration of which varied from 3 to 16 ms, emitted at rates of 91–177 pulses / second. The peak of dominant frequency varied from 1969–2250 Hz ( Fig. 6D ).

Natural history Adult males of P. gonzagai sp. nov. call in the open areas, by the margins of lentic environments (mostly ponds) during the rainy season of the year (which could vary across its distribution). Females lay eggs on leaves over the water bodies ( Fig. 3D ), from where exotrophic tadpoles hatch and drop into the water. Therefore, the reproductive mode is the number 24 ( sensu Haddad & Prado 2005 ). Different call types (advertisement, distress, warning and fighting calls) were described and previously attributed to P. nordestinus (see Toledo et al. 2015 ). However, part of those calls must now be attributed to P. gonzagai sp. nov. : the distress and warning calls described by Toledo et al. (2015) were recorded from individuals sampled in the state of Rio Grande do Norte . Therefore, this population falls within the distribution of P. gonzagai sp. nov. , not P. nordestinus . Thus, it is clear that both species ( P. nordestinus and P. gonzagai sp. nov. ) have a complex vocal repertoire that should be further explored.