Fifty years after: A taxonomic revision of the amphibian species from the Ecuadorian biodiversity hotspot Abra de Zamora, with description of two new Pristimantis species

call characterized by a call series composed by clicking calls repeated for long periods of time, similar to the call of P. tiktik (Fig 9). The calls are composed only by two short, single-pulsed notes (tonal sounds). Because the males can call continuously for long periods of time, the call series duration is unknown. The calls are characterized by a mean duration of 0.022 s (SD = 0.004), a mean inter-call interval of 0.196 s (SD = 0.028) and a mean call rate of 291.3 calls/min (SD = 36.817). The mean dominant frequency of the call was 3302.2 Hz (SD = 124.359), with a mean 90% bandwidth of 3109.6–3605.6 Hz. The fundamental frequency is not recognizable, but 2 to 4 harmonics are usually visible. Compared to the call of P. tiktik, which is single-noted, the call of P.vidua is composed of two notes: a first short one, and a second, slightly longer. Also, its call is very similar to the advertisement call of P. samaniegoi sp. nov. (the differences between the two calls are pre- sented further below, in the description of the P. samaniegoi sp. nov. call). Distribution. Pristimantis vidua presence is confirmed (with DNA samples) from Parque Nacional Podocarpus (Cajanuma entrance), Reserva Madrigal del Podocarpus, Abra de Zamora and Bosque Protector Washapamba, in Loja and Zamora Chinchipe provinces (Fig 6). The presence of this species in the Azuay province [9] is yet to be confirmed. We consider this unlikely, since we do not have any valid records of this species in any inventoried similar eco-

systems north of Saraguro (close to the interprovincial border). The species was encountered at an altitudinal range between 2750 and 3000 m a.s.l. in montane cloud forest and subpa´ramo ecosystems. Natural history. Lynch (1979) described the species based on 15 females, and hypothesized that maybe this species might be “all-female”, or at the least that P. vidua exhibits a “prepon- derance of females” (female-biased sex ratio). We found males although most of the Table 2. Quantitative description of the advertisement calls (mean ± SD, range and n) of the Pristimantis tiktik, P. vidua, P. samaniegoi sp. nov., P. balionotus, and P. versicolor. Pristimantis tiktik (11) Pristimantis vidua (3) P. samaniegoi sp. nov. (5) Pristimantis balionotus (3) Pristimantis versicolor (4) Notes per call 1 2 usually 2 1–8 (usually 1 or 2) 1–5 (usually 1) Call duration (s) 0.013 ± 0.004 (0.007–0.024) n = 330 0.022 ± 0.004 (0.013–0.029) n = 117 0.027 ± 0.008 (0.017–0.040) n = 116 0.069 ± 0.005 (0.061– 0.078) n = 31 0.084 ± 0.006 (0.070– 0.099) n = 109 Inter-call interval (s) 0.287 ± 0.029 (0.225–0.354) n = 319 0.196 ± 0.028 (0.157–0.259) n = 114 0.272 ± 0.027 (0.234–0.362) n = 112 6.075 ± 2.371 (3.499– 15.677) n = 67 1.804 ± 0.315 (1.364– 3.337) n = 136 Call rate (calls/ min) 200.6 ± 18.905 (173.6– 237.0) n = 11 291.3 ± 36.817 (249.0– 316.4) n = 3 185.9 ± 25.398 (160.8– 212.6) n = 5 10.0 ± 1.610 (8.6–11.8) n = 3 30.3 ± 3.138 (27.0–33.8) n = 4 Short notes duration (s) N/A 0.004 ± 0.001 (0.002–0.006) n = 97 0.004 ± 0.001 (0.002–0.006) n = 116 N/A N/A Long notes duration (s)

N/A 0.009 ± 0.004 (0.003–0.017) n = 97 0.007 ± 0.001 (0.003–0.010) n = 116 N/A N/A Inter-note interval (s) N/A 0.009 ± 0.005 (0.002–0.017) n = 97 0.015 ± 0.007 (0.006–0.028) n = 115 0.215 ± 0.011 (0.196– 0.249) n = 39 0.208 ± 0.027 (0.168– 0.325) n = 94 Dominant frequency (Hz) 3190.8 ± 99.873 (3014.6– 3359.2) n = 330 3302.2 ± 124.359 (3100.8– 3531.5) n = 273 3140.3 ± 144.886 (2842.4– 3359.2) n = 294 2431.7 ± 59.371 (2325.6– 2584.0) n = 69 1753.6 ± 80.323 (1636.5– 2067.2) n = 139 Frequency 5% (Hz) 3059.5 ± 112.207 (2842.4– 3273.1) n = 330 3109.6 ± 93.588 (2842.4– 3273.1) n = 273 2994.4 ± 156.623 (2670.1– 3186.9) n = 294 2331.8 ± 39.947 (2239.5– 2411.7) n = 69 1542.3 ± 60.568 (1378.1– 1636.5) n = 138 Frequency 95% (Hz) 3390.5 ± 86.571 (3186.9– 3617.6) n = 330 3605.6 ± 170.730 (3273.1– 3876.0) n = 273 3496.2 ± 284.2 (3186.9– 4306.6) n = 294 2542.8 ± 45.772 (2497.9– 2670.1) n = 69 3269.2 ± 283.50 (2842.4– 4478.9) n = 138 The call of P. tiktik is presented with comparative purposes as it is very similar to the P. vidua and P. samaniegoi sp. nov. calls. In the case of P. balionotus and P. versicolor the call duration is presented for the one-noted calls and the Inter-note interval is for the multi-noted calls. The number of recorded individuals is given in brackets after species name. https://doi.org/10.1371/journal.pone.0238306.t002 PLOS ONE Taxonomic revision of the amphibian species from Abra de Zamora PLOS ONE | https://doi.org/10.1371/journal.pone.0238306 September 10, 2020 19 / 59