Our unknown neighbor: A new species of rain frog of the genus Pristimantis (Amphibia: Anura: Strabomantidae) from the city of Loja, southern Ecuador

We describe a new species of rain frog of the genus Pristimantis from the city of Loja, Southern Ecuador, based on an integrative taxonomy approach, combining molecular, morphological and bioacoustics data. Pristimantis lojanus sp. nov. is a medium sized species of the phylogenetically strongly supported P. phoxocephalus group, and its sister species is P. torresi. The new species can be easily distinguished from its closest congeners and morphologically similar species (that also have acuminate snout with a fleshy keel) by its characteristic advertisement call and morphological features (dorsum finely tuberculate with scattered larger tubercles, flanks without longitudinal lateral folds, no markings in axilla, groin or on concealed limb surfaces, and bronze iris). Additionally, we describe the advertisement call of its sister species, P. torresi. Finally, we detail the current situation of the amphibian species present in the city of Loja and its surroundings.


Introduction
Ecuador is one of the most biodiverse countries in the World [1,2], having the highest density of amphibian species-number of species per area unit [3], as well as a remarkable proportion of endemic species [4]. Despite its small size, Ecuador harbors several types of ecosystems in the Tropical Andes region, which have been recognized as hotspots of amphibian diversity [3]. Their distinctiveness is still far from completely known, numerous species being described during the recent years [5][6][7][8]. The city of Loja, the capital of Loja Province, is one of the largest cities from Southern Ecuador (with more than 250,000 inhabitants), and has two areas of high diversity in its vicinity: Parque Nacional Podocarpus and Abra de Zamora [7,9].
Loja itself has a fascinating history of amphibian records reported from the city and its surroundings. In 1932, the British zoologist Hampton Wildman Parker [10] described the frog project "Convive con los ríos" Campaña de comunicación ambiental, participación social y levantamiento de indicadores socioambientales de las cuencas manejadas por Foragua" (PROY_VIN_GAM_001_2016) JV received funding from the National Research, Development and Innovation Fund of Hungary (https://nkfih.gov.hu/ for-the-applicants), through the project no. KH130360, financed under the [KH_18]. Part of this study was funded by the Critical Ecosystem Partnership Fund (CEPF, https://www.cepf.net/ grants/grantee-projects/amphibian-conservationabra-de-zamora-key-biodiversity-area-ecuador), grant CEPF-108984, through the "Amphibian Conservation in the Abra de Zamora Key Biodiversity Area of Ecuador" project. PS received funding for fieldwork from Mohamed bin Zayed Species Conservation Fund project 172515484 "Conservation of the Ridge Marsupial Frog (Gastrotheca psychrophila) in southern Ecuador" (https://www.speciesconservation.org/casestudies-projects/ridge-marsupial-frog/15484). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Specimen collection and study site
Collected specimens were photographed alive, after which they were euthanized using 20% benzocaine, fixed in 10% formalin, and stored in 70% ethanol and the tissue samples for genetic analyses were preserved in 96% ethanol [7]. Examined specimens (listed in the typeseries and S1 Appendix) are housed in Museo de Zoología, Universidad Técnica Particular de Loja, Loja, Ecuador (MUTPL).

Morphology
The description of qualitative and quantitative morphological characters, as well as the format of the description largely follows Duellman & Lehr [22]. Sex was determined by the presence of vocal slits, nuptial pads and/or by gonadal inspection. Color data in life were based on field notes and digital photos. The specimens were weighted (body mass: BM) before euthanasia using a My Weigh Triton T3 portable scale with 0.01 g precision. Measurements were taken under a stereo microscope, with a Vernier caliper, and rounded to the nearest 0.1 mm. Specimens were measured for the following morphometric variables: (1) snout-vent length (SVL), distance from the tip of snout to posterior margin of vent; (2) head width (HW), widest portion of the head, measured at level of jaw articulation; (3) head length (HL), distance from the tip of snout to posterior angle of jaw articulation; (4) interorbital distance (IOD), minimum distance between the inner margins of the orbits; (5) internarial distance (IND), distance between the inner edges of the narial openings; (6) upper eyelid width (EW), the perpendicular distance to the outer edge of the eyelid; (7) eye diameter (ED), distance between anterior and posterior borders of eye; (8) eye-nostril distance (EN), distance from posterior margin of nostril to anterior margin of eye; (9) tympanum diameter (TD), horizontal distance between peripheral borders of tympanic annulus; (10) femur length (FL), length of femur from vent to knee; (11) tibia length (TL), length of flexed leg from knee to heel; (12) foot length (FoL), distance from proximal margin of inner metatarsal tubercle to tip of Toe IV; (13) hand length (HaL), distance from proximal edge of palmar tubercle to the tip of Finger III [7]. Measurements are given as mean ± SD.

DNA extraction, amplification and sequencing
DNA extraction was performed directly from 96% ethanol-preserved liver tissue, using the Extract-N-Amp™ Tissue PCR Kit (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany), followed by PCR reactions under the manufacturer's protocol [7]. Genomic extraction, amplification, and sequencing are as described in Székely et al. [7]. The newly generated DNA sequences (12S, 16S and RAG-1) were deposited in GenBank (S1 Table).
The sequences were edited, assembled, and aligned (MAFFT algorithm [24]) using the program Geneious Prime (Biomatters Ltd.). The edited alignments of 12S, 16S and RAG-1 sequences were manually inspected and concatenated into a single matrix, which was then used for the phylogenetic analyses [7]. The phylogenetic analyses were based on a 2339 bp dataset (909 bp 12S, 878 bp 16S and 612 bp RAG-1). The aligned matrix is available at https:// doi.org/10.5281/zenodo.5500445.
Molecular phylogenetic relationships were inferred using Maximum Likelihood (ML) and Bayesian Inference (BI). We used PartitionFinder v. 2.1.1 [25] to select the best-fitting models of sequence evolution and the best partition scheme with the AICc (for ML) and BIC (for BI) models of selection. ML analyses were conducted in GARLI v. 2.1 [26], performing four independent searches (two with the "streefname" set to random and two set to stepwise), with 250 replicates each, and with the "genthreshfortopoterm" set to 100,000 [7]. Node support was assessed with non-parametric bootstrapping [27] with 1,000 pseudoreplicates. The 50% majority rule consensus for the bootstrap trees was obtained with Geneious Prime (Biomatters Ltd.). BI analyses were conducted with MrBayes 3.2.6 [28], the Markov chain Monte Carlo runs being performed twice, independently, for 60 million generations, with a sampling frequency of 1000. Convergence of the runs was assessed from the average split frequency of standard deviations (p < 0.001) and by checking the potential scale reduction factors (PSRF~1.0) for all model parameters. The first 25% of the trees were discarded as burn-in and the remaining ones were used to generate a 50% majority rule consensus tree, as well as to estimate the Bayesian posterior probabilities [7]. Throughout the text, we considered that a node has "strong support" when its bootstrap value was > 70 and its Bayesian posterior probability was > 0.95, "moderate support" for 50-70 and 0.90-0.95 and "weak support" or non-resolved for lower values of 50 or 0.90, respectively [7]. Uncorrected p-genetic distances for gene 16S (S2 Table) were estimated with software MEGA6 [29].

Call recordings and analysis
The calls were recorded in the field using an Olympus LS-11 Linear PCM Recorder, a Tascam DR-100 MKIII 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 Fonoteca UTPL (record IDs are provided in S3 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 [7].
The terminology and procedures for measuring call parameters follow [30][31][32], with a call-centered approach to distinguish between a call and a note (sensu [32]). The following temporal and spectral parameters 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 minute, 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 beginning 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 consecutive 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) dominant frequency: the frequency containing the highest sound energy, measured along the entire call; and (8) the 90% bandwidth, reported as frequency 5% and frequency 95%, or the minimum and maximum frequencies, excluding the 5% below and above the total energy in the selected call [7].

Nomenclatural acts
The electronic edition of this article conforms to the requirements of the amended International Code of Zoological Nomenclature, and hence the new names contained herein are available 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:4-C8C6141-DC8A-4A0E-818E-B87B0F655278. 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 repositories: PubMed Central and LOCKSS. We recovered the Huicundomantis subgenus as monophyletic with strong support (bootstrap values = 99.5%; posterior probabilities = 1) in both ML and BI analyses similarly to the results of Páez & Ron [23]. The main difference between our phylogram and the one from [23] is in the position of the branch of P. hampatusami and an undescribed species (UCS3), as well as the position of several unresolved branches such as P. jimenezi, P. phoxocephalus, P. totoroi and P. tinguichaca, probably due to the different genes used in the analyses (Fig 1). Our tree has a very similar topology with the one presented in [7]. Pristimantis lojanus sp. nov. is part of the strongly supported P. phoxocephalus group (sensu [23]) and its sister species is P. torresi (Fig 1). Uncorrected p-genetic distances for the gene 16S between P. lojanus sp. nov. and all its relatives range from 5.0% to 10.9% (S2 Table). The intraspecific genetic distances of the revised specimens did not surpass 0.3%. Based on the large genetic distances to it congeners, advertisement call and morphological differences we describe this new species below.  ; asterisks indicate support values of 100% or 1 (posterior probabilities). Outgroup is not shown; the tree was rooted with Pristimantis galdi. With red is marked the new species and with green newly generated sequences for the present study. The collection number, species name, province and short locality name of the samples are shown next to each terminal; all samples are from Ecuador (associated data listed in S1 Table). Abbreviations: CCS = confirmed candidate species, UCS = unconfirmed candidate species (see [23]).

Taxonomic treatment
https://doi.org/10.1371/journal.pone.0258454.g001   Diagnosis. We assign this species to Pristimantis based on phylogenetic evidence (Fig 1) and on the general morphological similarity to other members of the genus. Pristimantis lojanus is a medium sized species, distinguished by the following combination of traits: (1) skin on  dorsum finely tuberculated with scattered larger tubercles (in life the skin tuberculated texture is more evident); skin on venter coarsely areolate to areolate; discoidal fold weak; dorsolateral folds absent; low middorsal fold present; (2) tympanic annulus prominent and tympanic membrane differentiated, its length about 45% of the length of eye; supratympanic fold present,   several small tubercles, similar in size and shape with the ones from the dorsum, about 80% IOD in females and 90% IOD in males; cranial crests absent; (5) dentigerous processes of vomers prominent, oblique, ovoid or triangular, separated medially by distance lower than the width of processes; each processes bearing 4 to 7 teeth; (6) males with a large subgular vocal sac and round vocal slits; nuptial pads present; (7) Finger I shorter than Finger II; discs on fingers broadly expanded, truncate; circumferential grooves present; (8) fingers bearing lateral fringes (trait more evident in life); subarticular tubercles prominent; supernumerary palmar tubercles present; palmar tubercle usually partially divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle elliptical, larger than the inner palmar tubercle; (9) ulnar tubercles present; (10) heel with several small, rounded tubercles; outer edge of tarsus with a row of small tubercles; inner tarsal fold present; (11) inner metatarsal tubercle broadly ovoid, about 4x or 5x the size of subconical (in profile) outer metatarsal tubercle; supernumerary plantar tubercles present; (12) toes bearing broad lateral fringes (trait more visible in life); webbing basal; Toe V much longer than Toe III; discs on toes broadly expanded, truncate, about same size as those on fingers; circumferential grooves present; (13) in life, dorsum and flanks of various shades of brown or reddish brown, with or without whitish spots or blotches, with or without whitish interorbital bars; venter cream, whitish or yellowish, with or without dark flecks and blotches; no markings in axilla, groin or on concealed limb surfaces; iris bronze with fine black reticulations and a median, horizontal read streak; (14)  Comparison with similar species. Pristimantis lojanus is morphologically similar to the species from the P. phoxocephalus group (sensu [23]), but it can be distinguished from all the resembling species. Pristimantis lojanus is most similar to its closest relative P. torresi (pgenetic distance between 5.0% and 8.4%; S2 Table), from which it differs by the following: slightly larger size (females SVL up to 44.3 mm vs. females SVL up to 39.5 mm; males SVL up to 30.4 mm vs. males SVL up to 30.0 mm), dorsum finely tuberculate with scattered larger tubercles (vs. shagreen dorsum in P. torresi), flanks without longitudinal lateral folds (vs. flanks with longitudinal lateral folds on anterior half), no markings in axilla, groin or on concealed limb surfaces (vs. many individuals with yellow, cream or orangey-yellow markings; Fig 6) and iris bronze (vs. iris much lighter, golden to beige; Figs 6, 8 and 9). However, the most evident difference between the two species is the advertisement call (see call descriptions below).
Somewhat similar are its northern, distant relatives that also have acuminate snout with a fleshy keel: P. atillo, P. jimenezi, P. phoxocephalus, P. teslai, P. totoroi, and P. verrucolatus. From these, P. teslai resembles most due to its tuberculate dorsum and darker iris, however P. lojanus is bigger (males SVL up to 30.4 mm vs. males SVL up to 27.3 mm; females are unknown in P. teslai), has finely tuberculate dorsum with scattered larger tubercles (vs. tuberculate dorsum, with some prominent tubercles) and bronze iris (vs. copper iris). Pristimantis lojanus can be distinguished from the others by the following characters (characters of P. lojanus in parenthesis): P. atillo has a shagreen dorsum with or without scattered small tubercles (vs. dorsum finely tuberculated with scattered larger tubercles) and usually has orange groins and black dots on the flanks (vs. no markings in axilla, groin or flanks); P. jimenezi has a shagreen dorsum with or without scattered small tubercles (vs. dorsum finely tuberculated with scattered larger tubercles), groins and posterior surfaces of thighs with small light brown to yellow spots (vs. no markings in axilla, groin or flanks) and iris reddish copper (vs. iris bronze); P. phoxocephalus has groins and concealed surfaces of thighs yellow with dark brown to black reticulations (vs. no markings in axilla, groin or on concealed limb surfaces); P. totoroi has a shagreen dorsum with or without scattered small tubercles (vs. dorsum finely tuberculated with scattered larger tubercles) and iris golden (vs. iris bronze); P. verrucolatus has a shagreen dorsum with or without scattered tubercles (vs. dorsum finely tuberculated with scattered larger tubercles) and with thick lateral folds, warts and larger tubercles on the flanks (vs. dorsolateral folds, warts and larger tubercles on the flanks absent).
Description of the holotype. Adult female (MUTPL 178; Figs 2, 3 and 5A), head narrower than body, wider than long, head length 81% of head width, head width 39% of SVL; head length 32% of SVL; snout moderately long (snout to eye distance 17% of SVL), acuminate with a vertical keel in dorsal view and subacuminate and inclined posteroventrally in profile (Figs 2 and 3); canthus rostralis weakly concave in dorsal view, rounded in profile; loreal region slightly convex; eye diameter smaller than eye-nostril distance; nostrils slightly protuberant, oriented posteriorly; lips not flared; cranial crests absent; upper eyelid bearing several small tubercles (three larger than the others), width of upper eyelid 74% of IOD; tympanic annulus prominent and tympanic membrane differentiated; thick supratympanic fold present, concealing the upper and posterior margin of the tympanum (Fig 2A); diameter of tympanum 46% of the length of eye; two larger, rounded postrictal tubercles surrounded by several smaller tubercles; choanae large, round, not concealed by palatal shelf of maxillary arch; dentigerous processes of vomers prominent, slightly larger than the choanae, oblique, situated posterior and median to choanae, triangular in outline, not separated medially, each processes bearing 5 to 6 teeth; tongue longer as wider, slightly notched posteriorly, posterior half not adherent to floor of mouth.
Skin on dorsum finely tuberculated with scattered larger tubercles (in life the skin tuberculated texture was more evident, Figs 2A, 2C and 3A); thin, low middorsal fold starting at tip of snout and ending at cloaca; dorsolateral folds absent; skin on chest, belly, and ventral surfaces of thighs coarsely areolate; thoracic and discoidal folds weak (Figs 2B and 3B); cloacal region bordered ventrally by several small tubercles.
Ulnar tubercles present; outer palmar tubercle prominent, partially divided into a larger (inner) and a smaller (outer) tubercles; thenar tubercle elliptical, larger than the inner palmar tubercle; subarticular tubercles prominent, round and subconical in section; supernumerary palmar tubercles rounded, large, slightly smaller than subarticular tubercles; fingers bearing lateral fringes (trait more evident in life); relative length of fingers I < II < IV < III; discs on fingers broadly expanded, truncate; all fingers bearing pads well defined by circumferential grooves (Fig 5A).
Hindlimbs long, slender; tibia length 51% of SVL; foot length 44% of SVL; heel with several small, rounded tubercles; outer edge of tarsus with a row of small, inconspicuous, tubercles (trait more visible in life); inner edge of tarsus bearing a long fold; inner metatarsal tubercle broadly ovoid, about 4x round and subconical (in profile) outer metatarsal tubercle; subarticular tubercles prominent, round and subconical in section; plantar supernumerary tubercles rounded, smaller than subarticular tubercles; toes bearing broad lateral fringes (trait more visible in life); webbing basal; discs on toes broadly expanded, truncate, about same size as those on fingers; toes with ventral pads well defined by circumferential grooves (Fig 5A); relative length of toes I <II < III < V < IV; Toe V much longer than Toe III (tip of Toe III reaches the middle of the penultimate subarticular tubercle on Toe IV, tip of Toe V extends beyond the distal edge of distal subarticular tubercle on Toe IV).
Coloration of holotype. In life (Fig 2): dorsum and snout reddish brown, flanks and dorsal surfaces of femurs light brown with dark brown bars; two black bordered, yellowish white blotches on dorsum (one larger and one smaller) and a wide interorbital bar with the same coloration; black supratympanic bars; dorsal surfaces of hindlimbs, arms, toes and fingers covered with reddish brown irregular markings; venter and throat yellowish white, ventral surfaces of hindlimbs and arms grey; iris bronze with a dark red, median horizontal streak and with black reticulations.
In preservative (Fig 3): dorsum brownish gray, flanks and dorsal surfaces of femurs yellowish white with brownish gray bars; the two black bordered blotches on the back and the wide interorbital bar became white; black supratympanic bars; venter, throat, ventral surfaces of hindlimbs and arms yellowish white. Body mass of holotype: 3.09 g.

Variation.
Morphometric variation is shown in Table 1. This species displays an evident sexual dimorphism, the females being significantly larger than the males (Fig 7). Both females and males vary greatly in the dorsal coloration, the majority of encountered individuals displaying various shades of brown or reddish brown, with or without whitish spots or blotches, with or without whitish interorbital bars. Female MUTPL 192 (Fig 8D) had a wide, dark brown middorsal band and contrasting yellowish white flanks, female MUTPL 935 (Fig 8F) was almost red (dark reddish brown with lighter stripes), and one male (Fig 9F) was greenish Table 1 with red spots on the dorsum. Only one individual (MUTPL 760, Fig 10A), from the many animals encountered by us, displayed some yellowish markings in the groin and concealed limb surfaces (but not like the widespread markings usually encountered in P. torresi). As for the iris, female MUTPL 177 ( Fig 8E) and male MUTPL 179 (Fig 9B) had an atypically light, whitish bronze coloration. In all specimens, the supratympanic fold was black. Advertisement cal. For the description of the P. lojanus call, we analyzed 7 recordings, two from Loja city, Parque Universitario de Educación Ambiental y Recreación "Francisco Vivar Castro" (from 2015 and 2016), one from Abra de Zamora (2018), one from Cerro Sacama (2020), and three from Loja city, Cerro Chiriaco (2021). Descriptive statistics of the acoustic variables are provided in Table 2 (the detailed information of each of the separate recordings is presented in the S3 Table). Pristimantis lojanus has an advertisement call characterized by a call series composed by whistles repeated over a period of time, somewhat similar to the calls of P. balionotus, P. jimenezi, or P. verrucolatus (Fig 11A-11G). The calls are composed usually by one, but sometimes two notes (frequency modulated tonal sounds). The calls are characterized by a mean duration of 0.367 s, a mean inter-call interval of 7.007 s and a mean call rate of 7.80 calls/min ( Table 2). In the case of the double-noted calls (Fig 11A), the call duration was 0.797-1.460 s (0.937 ± 0.192, n = 10) (S3 Table), the first note being longer than the second: 0.347-0.417 s (0.370 ± 0.021, n = 10) vs. 0.136-0.225 s (0.176 ± 0.028, n = 10). The mean dominant frequency of the call was 2598.0 Hz, with a mean 90% bandwidth of 2506.3-2674.3 Hz ( Table 2). The fundamental frequency is not recognizable, but usually 3 to 8 harmonics are visible. Table 2. Quantitative description of the advertisement calls (mean ± SD, range and n) of Pristimantis lojanus sp. nov. and P. torresi.

Pristimantis lojanus sp. nov.
Pristimantis torresi Notes per call 1- Similarly to some of the other species of the P. phoxocephalus group [7], the males can sometimes emit special, multi-noted calls, with up to 6 notes (Fig 11E-11G). These calls probably have some different function compared to the typical advertisement calls, and might be used in the case of social interactions, triggered by the presence of nearby females or competitive males.
In December 2020 we recorded four males of P. torresi, the sister species of P. lojanus, in its type locality, Guachanamá, El Apretadero, Southern Ecuador (S3 Table). The advertisement calls are somewhat similar to the one of P. lojanus and are composed by one-noted calls characterized by a mean duration of 0.229 s, a mean inter-call interval of 5.370 s and a mean call rate of 10.94 calls/min (Fig 11H-11N, Table 2). The mean dominant frequency of the call was 2446.0 Hz, with a mean 90% bandwidth of 2362.1-2576.5 Hz ( Table 2). The fundamental frequency is not recognizable, but usually 3 to 6 harmonics are visible.
The calls of these two species can be easily distinguished (even by ear) as they differ significantly in call duration, inter-call interval, call rate, and frequencies ( Table 2, Fig 11). Additionally, the multi-note calls emitted by these species are also different. In the case of P. lojanus, the short notes are produced immediately after one long note (Fig 11E) rather than a separate call from the call series like in the case of P. torresi (Fig 11H). They differ also in the note duration and inter-note interval ( Table 2).
The P. torresi male MUTPL 996 (FUTPL-A 256, 257) emitted in about 10 minutes 14 multinoted calls, probably triggered by the presence of two competitive males on the nearby branches. One of these males also responded by producing similar multi-noted calls.
Distribution. Pristimantis lojanus is known from the city of Loja and its close vicinity (all the streams that flow into the city, but also parks like Parque Universitario de Educación Ambiental y Recreación "Francisco Vivar Castro" and private protected areas like Reserva Madrigal del Podocarpus), as well as from several other localities (Fig 12). We also have confirmed records (with DNA samples or call recordings) from San Lucas and its vicinities, Bosque Servio Aguirre Villamagua, Cerro Sacama, Abra de Zamora, Cajanuma, Cristal, and San Antonio de Paycapamba. The species was encountered at an altitudinal range between 1937 m (Cristal) and 2782 m (Bosque Servio Aguirre Villamagua) a.s.l., in evergreen lower montane forest and evergreen upper montane forest ecosystems (sensu [33]).
Natural history. This is a common species sensu [7] (individuals were detected-seen or heard-in the adequate habitat, in large or moderate numbers, on 50-100% of the sampling days/nights). Most specimens were encountered during the night, on the vegetation (shrubs, branches of trees or grassy vegetation) from 10 cm above the ground up to 2-3 m. The majority of the individuals were encountered in habitats close to small streams. Calling males were encountered year-round, but more frequently on rainy nights. All the individuals were encountered in forested areas with some degree of human intervention (secondary forests). The species needs forested areas near the streams, as we could not detect the presence of P. lojanus on the stream segments from the city where the native vegetation was destroyed.
Conservation status. Pristimantis lojanus is known from about 25 localities, from an estimated area of about 400 km 2 . Even if this species is common (locally abundant) in the proper habitats, we recommend this species to be categorized as Endangered following the B1ab(iii,iv) + 2ab(iii,iv) IUCN criteria [34] because: (1) its Extent of occurrence (EOO) and Area of occupancy (AOO) are estimated to be less than 400 km 2 ; (2) the species distribution is severely fragmented; (3) none of its populations can be found in national protected areas; and (4) its habitats could be severely affected in the near future, as they are situated in the vicinity of densely populated areas.

Discussion
The distribution of Pristimantis lojanus is limited to a relatively small area, mostly included in the Upper Zamora River Basin, an area of approx. 620 km 2 delimited by the Guagrahuma-Acacana and Cajanuma orographic knots [35,36]. This region was a tertiary lake basin which opened around Salapa giving birth to the Zamora River [36,37]. The northernmost records of P. lojanus are from near San Lucas (Acacana), fieldwork carried out outside this area since 2016 failing to encounter it. In the south, the species reaches Cristal and San Antonio de Paycapamba; further south, around Vilcabamba, P. lojanus is replaced by a very similar, currently undescribed, species (pers. obs.). Since 2016, all sightings and collected specimens of the P. phoxocephalus group from the Loja basin can be assigned to P. lojanus. Due to the similar morphology and based on the available data, we consider that the specimens collected from Loja by Lynch in 1979 [20] and considered to be P. phoxocephalus are in fact P. lojanus.
Unfortunately, as the population of the city of Loja increased over the years, the situation of the amphibians from the city and neighboring areas deteriorated. Currently, there are 12 species of amphibians (including P. lojanus) reported from the city and its surroundings (upper montane forest ecosystems around the city, up to 2700 m, up to the mountain crests and subpáramo habitats). The last record of Rhinella amabilis from Loja was in 1971 (the Kansas University Herpetology Collections database, https://biodiversity.ku.edu/herpetology/collections), and we suspect it to be extinct (currently this species is listed as Critically Endangered in the IUCN Red List). Recently, we encountered a toad population inside the city, which turned out to be R. poeppigii (confirmed by DNA sampling); this is probably a recently introduced population, since this species has a lower altitudinal range (800-1670 m; [38]). Also, there is a large distribution gap between known records in the east, with the species not being detected even in well surveyed areas such as Abra de Zamora and San Francisco Scientific Research Station [7], between 1800-3000 m. The Loja Rocket Frog (Hyloxalus elachyhistus) was heard calling in 2019 and 2020 on the heavily transformed banks of Río Zamora, in a couple of locations in the center of the city. However, the population is facing extinction due to contamination and habitat change. For now, Gastrotheca elicioi is still common in Loja, but its survival depends on habitat availability, which is threatened by city expansion. In the case of G. lojana, no recent confirmation (using either molecular or call samples) of the species presence from Loja and its surroundings are available. On the other hand, we encountered G. pseustes in the northern outskirts of Loja (the species type locality is further north, close to San Lucas), living syntopically with G. elicioi. Gastrotheca pseustes is replaced by G. elicioi in Loja and further south.
As for the members of Strabomantidae (terrestrial-breeding frogs), there are seven species recorded for the area, three of which having an unclear taxonomic status: Noblella aff. heyeri, Pristimantis aff. andinognomus, P. atratus [20], P. aff. cajamarcensis, P. cryptomelas [20], P. lymani and P. lojanus. The most common species are P. lymani (which is the most tolerant to anthropic impact, being able to survive even in the green areas between the buildings), P. aff. cajamarcensis and P. lojanus. Pristimantis aff. andinognomus, P. atratus, and P. cryptomelas can be found only at higher elevations, over 2400 m, of the forested areas in the eastern flank of the city.
An unfortunate addition to Loja's fauna [39] is the Bullfrog, Lithobates catesbeianus [40], which inhabits several of the city ponds and can have a negative effect on the survival of the native species, especially of the pond-breeding G. elicioi. Since 2015 when Cobos et. al. [39] reported the presence of this species from Parque Pucará (4.0127˚S, 79.1951˚W, 2197 m), we encountered several other ponds in the city with Bullfrog populations: Laguna de la Cruz  found (e.g. >100/ hour in Parque Pucará) suggests that their control is increasingly difficult. Bullfrog eradication campaigns based on complete removal of individuals using manual methods (hand trapping, netting, electro-shocker, egg-mass removal) are time consuming, expensive and has had limited success in other habitats (e.g., [41][42][43]), although it can have an important impact on final bullfrog population size. Other methods, such as the use of native predatory fish in artificial ponds might be more effective [44], but there are no studies regarding their feasibility in the Tropical Andes. On the other hand, there is an urgent need for public awareness campaigns aimed at preventing further intentional Bullfrog introductions, as well as continued documentation of its dispersal.