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

“moderate support” for 50–70 and 0.90–9.95 and “weak support” or non-resolved for lower values of 50 or 0.90, respectively. Uncorrected p-genetic distances for gene 16S were estimated with software MEGA6 [36] (S3 Table). Call recordings and analysis We analyzed 16 calls of 15 individuals belonging to five species: release call of L. flavomacula- tus and advertisement calls of P. vidua, P. samaniegoi sp. nov., P. balionotus and P. versicolor. The calls were recorded in the field or in laboratory (L. flavomaculatus) using an Olympus LS- 11 Linear PCM Recorder and a RØDE NTG2 condenser shotgun microphone at 44.1 kHz sampling frequency and 16-bit resolution, in WAV file format. Air temperature and humidity were measured with a data logger (Lascar Electronics, model EL-USB-2-LCD, accuracy: ± 0.5˚C; ± 5%). All analyzed call recordings are deposited in original form, full length at Fono- teca UTPL (record IDs are provided in S4 Table). Acoustic analysis was conducted using Raven Pro 1.6 (Center for Conservation Bioacoustics 2019). We measured the temporal parameters from the oscillograms and the spectral parameters from spectrograms obtained with the Hanning window function, DFT: 512 samples, 3 dB filter bandwidth: 124 Hz, and a 50% overlap. The terminology and procedures for measuring call parameters follow Cocroft and Ryan [37], Toledo et al. [38] and Ko¨hler et al. [39] and a call-centered approach was used to dis- tinguish between a call and a note [sensu 39]. The following temporal and spectral parame-

ters were measured and analyzed: (1) call duration: time from the beginning to the end of a call; (2) inter-call interval: the interval between two consecutive calls, measured from the end of one call to the beginning of the consecutive call; (3) call rate: number of calls per min- ute, measured as the time between the beginning of the first call and the beginning of the last call; (4) note duration: the duration of a single note within a call, measured from begin- ning to the end of the note; (5) inter-note interval: the interval between two consecutive notes within the same call, measured from the end of one note to the beginning of the conse- cutive note; (6) note rate: number of notes per second, measured as the time between the beginning of the first note and the beginning of the last note; (7) number of pulses: the num- ber of pulses per note; (8) pulse rate: number of pulses per second, measured as the time between the beginning of the first pulse and the beginning of the last pulse; (9) dominant frequency: the frequency containing the highest sound energy, measured along the entire call; and (10) the 90% bandwidth, reported as frequency 5% and frequency 95%, or the mini- mum and maximum frequencies, excluding the 5% below and above the total energy in the selected call. Nomenclatural acts The electronic edition of this article conforms to the requirements of the amended Interna- tional Code of Zoological Nomenclature, and hence the new names contained herein are avail- able under that Code from the electronic edition of this article. This published work and the

nomenclatural acts it contains have been registered in ZooBank, the online registration system for the ICZN. The ZooBank LSIDs (Life Science Identifiers) can be resolved and the associated information viewed through any standard web browser by appending the LSID to the prefix "http://zoobank.org/". The LSID for this publication is: urn:lsid:zoobank.org:pub:32448A- CE-C45F-4D84-9C2A-670E0C29B389. The electronic edition of this work was published in a journal with an ISSN, and has been archived and is available from the following digital reposi- tories: PubMed Central and LOCKSS. 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 7 / 59