Brian Gratwicke1*
Jorge Guerrel2Orlando Garcés2Estefany Illueca2
Nathanial J. Weisenbeck1
Jessica L. Deichmann1,3
Roberto Ibáñez2,4- 1Center for Species Survival, Smithsonian’s National Zoo and Conservation Biology Institute, Front Royal, VA, United States
- 2Panama Amphibian Rescue and Conservation Project, Smithsonian Tropical Research Institute, Panama City, Panama
- 3Liz Claiborne & Art Ortenberg Foundation, New York, NY, United States
- 4Sistema Nacional de Investigación, Secretaría Nacional de Ciencia, Tecnología e Innovación (SENACYT), Panama City, Panama
Passive acoustic monitoring offers promising solutions for monitoring elusive amphibian species, but the method’s effectiveness for detecting rare or potentially extinct amphibian species remains poorly evaluated. We conducted observer surveys along transects and deployed autonomous recording units (ARUs) at 19 stations across three localities in Panama during two rainy seasons (2022, 2024) to detect species of conservation concern that were thought to have been extirpated following disease related declines in 2005-2008, including the potentially extinct Rabb’s treefrog Ecnomiohyla rabborum. Using template-based pattern matching and expert validation of acoustic data, we documented four conservation-priority species: Silverstoneia nubicola, Oophaga vicentei, Triprion spinosus, and Ecnomiohyla veraguensis. Three of these species were previously presumed extirpated from the study localities following chytridiomycosis-related declines; one (E. veraguensis) was a new distribution record for the species. ARUs detected common species at twice as many stations compared to observers on transects, though pattern-matching efficiency varied substantially (35-97%) depending on call characteristics with repetitive patterns performing better than single notes. Bd prevalence in the amphibian community was 21% with continuing evidence of Bd-related frog deaths. The current amphibian abundance (mean 5.82 amphibians/100m transect) may indicate partial recovery of amphibian communities approximately 20 years post-epizootic decline. While we found no evidence of Ecnomiohyla rabborum, our results validate ARUs as an effective tool for detecting rare arboreal Ecnomiohyla species and for monitoring amphibian recovery. The persistence of presumed extirpated populations highlights the value of continued acoustic monitoring and suggests the potential evolution of disease resistance in some species.
Introduction
Amphibian populations are experiencing rapid global declines, with much of our current understanding derived from expert opinion (Gratwicke et al., 2016; Luedtke et al., 2023; Scheele et al., 2019). While rigorous monitoring approaches are essential for understanding these declines (Lambert et al., 2020; Nowakowski et al., 2024), limited funding often constrains implementation of comprehensive surveillance programs (Gratwicke et al., 2012). Passive acoustic monitoring, which uses autonomous recording units (ARUs) to document sounds in the environment, presents an increasingly cost-effective method for collecting extensive temporal data, particularly for rare, nocturnal, and cryptic species (Deichmann et al., 2018; Lapp et al., 2023). These data also provide valuable insights into anuran natural history and behavior of common species (Crump and Houlahan, 2017; Hilje and Aide, 2012; Wood et al., 2023) and can be used to analyze soundscapes through acoustic indices, species richness indices and species activity profiles (Boullhesen et al., 2021; Duarte et al., 2019), although frog chorus activity creates a significant challenge to identify individual species (Brodie et al., 2020).
The continuous deployment capability of ARUs enables real-time monitoring and determination of species-specific historical and shifting baselines (Aide et al., 2013; Deichmann et al., 2018). However, detection efficacy varies among species due to differences in vocalization behavior and the challenges of isolating individual calls within multispecies choruses and from other sounds in the acoustic environment (Campos-Cerqueira and Aide, 2016; Lapp et al., 2021; LeBien et al., 2020; Wood et al., 2023). A significant constraint in processing ARU data is the limited availability of comprehensive call reference libraries, particularly for tropical anurans (Crump and Houlahan, 2017; Lapp et al., 2023; Van Horn et al., 2021). While this bottleneck has largely been overcome for avian acoustic monitoring through machine learning approaches (Kahl et al., 2021), template-based pattern-matching remains a useful intermediate step for anuran call identification and may be less challenging than the melodic structures of some bird songs (LeBien et al., 2020). Even without species-level resolution, archiving soundscapes in a recovering amphibian community allow acoustic activity indices to understand and compare the recovery at a community level (Pieretti et al., 2011).
Panamanian amphibians experienced catastrophic decline when Batrachochytrium dendrobatidis arrived in 1996 (Crawford et al., 2010; Lips et al., 2008, Lips et al., 2006). Out of 230 known amphibian species in Panama 65 are listed by the IUCN as Endangered or more threatened (Amphibiaweb, 2025), and at least 9 species are presumed extinct (Gratwicke et al., 2016), including the Panamanian endemic, Ecnomiohyla rabborum (Rabb’s treefrog). The last captive individual died in 2016, and it has not been sighted in the wild since 2008 (IUCN, 2020a). This study aimed to deploy ARUs at three localities within the known or potential range of E. rabborum and evaluate the effectiveness of autonomous sound recording units using sound pattern-matching tools with the Arbimon platform (https://arbimon.org) to detect other species of conservation concern and document any potential post-decline recovery of populations. We also conducted an analysis of poison dart and rocket frogs (Dendrobatoidea) that are not considered threatened. We selected this group because we have a good library of calls for this group (Ibáñez et al., 1999), historical call survey data and distribution collected by R.I (Ibáñez et al., 1996), and they have clear calls suitable for acoustic analyses to better understand and validate these methods.
Methods
Historical acoustic surveys
RI conducted an acoustic survey of five species of frogs (Allobates talamancae, Colostethus flotator, Colostethus pratti, Silverstoneia flotator, Silverstoneia nubicola) in September 1981 at one locality - Cerro Campana, along a 650m audio transect (Heyer et al., 1994; Lips et al., 1999). These species were common, diurnal, and terrestrial, each associated with streams to varying degrees. The transect was located along a single-track dirt road (8.68212°N, 79.92871°W, 864 masl - 8.68441°N, 79.93233°W, 878 masl), traversing forest and stream habitats. R.I. counted heard males at every 30 steps, avoiding double counting. These counts were conducted in the rainy season, three times during daylight hours. On average, each transect count was completed in 30 min. These data have not previously been published.
Contemporary passive acoustic surveys
We deployed AudioMoths v1.1.0 (equipped with 32GB SD cards in IPX7 waterproof cases) at 19 stations (5 stream stations and 14 terrestrial stations) across three high-biodiversity localities historically known for rare and possibly extinct amphibians (Figure 1). These localities are covered by tropical premontane forests in the mountainous region of the central cordillera in the Cocle and Panama provinces, where naive populations of amphibians first experienced Bd outbreaks around 2005 (Woodhams et al., 2008). Cerro Gaital is a National Monument and Altos de Campana is a National Park, while Altos de Maria is a low-density urban development. Sampling periods were May 17-November 23, 2022, and July 18-August 25, 2024, coinciding with Panama’s rainy season when most frog species breed and vocalize. AudioMoths were installed at breast height in trees and programmed to record 1-minute segments every 10 minutes for a 24-hour duty cycle (144 recordings/day) at 48kHz sampling rate and medium gain. Station coordinates were recorded using a Garmin GPS. Approximately 128,700 recordings were collected and uploaded to the “Searching for Species of Conservation Concern” project1 for analysis and annotation (Aide et al., 2013; LeBien et al., 2020).
Figure 1. Map of sampling localities in Panama in relation to protected area boundaries (green): Monumento Natural Cerro Gaital, and Parque Nacional y Reserva Biológica Altos de Campana. AudioMoths were deployed and transect monitoring was conducted with a total of 19 sampling stations (5 in streams, 14 terrestrial). About 128,700–1 min sound recordings were used for acoustic analysis.
Contemporary observer surveys
At each station and season where audiomoths were deployed, two observers conducted minimum of one diurnal and one nocturnal survey along a 100m transect, searching approximately 2m on either side of the transect line or stream (Heyer et al., 1994; Lips et al., 1999). All amphibians detected visually or acoustically were recorded, and when possible, captured and swabbed for Batrachochytrium dendrobatidis (Bd) analysis using an MW113 swab five times on each foot, 10 times on the belly, 10 times on each flank, and 10 times on each thigh for a total of 60 passes (Hyatt et al., 2007; Kriger et al., 2006). Swabs were placed into 1.5-mL microcentrifuge tubes and stored in a –20 °C freezer until processing (Klocke et. al 2023). Bd prevalence rates and 95% Bayesian credible intervals (Jaynes and Kempthorne, 1976) were calculated using an online calculator https://www.causascientia.org/math_stat/ProportionCI.html. Difficult to identify frog specimens were photographed and genetically identified following buccal swabs (Advantage Bundling SP, MW100) and analyzed a fragment of the 16S ribosomal RNA gene (Crawford et al., 2012). We placed the swab in a 1.5 mL tube with 30–40 mg of zirconia/silica beads (Cat. No. 11079105z), added 50 µl of PrepMan Ultra Sample Preparation Reagent (Life Technologies, product No. 4318930), and agitated the tube for 1 min in a Mini-Beadbeater-96 (BioSpec, Cat. No. 1001). Then, we diluted the extracted DNA to 1:10 with DNase- RNase-free molecular grade water (G-Biosciences, Cat. No. 786-292) and PCR-amplified the 16S marker with GoTaq Green Master Mix (Promega, Cat. No. M7122), using an initial denaturation at 95 °C for 720 s and 35 cycles of denaturation at 95 °C for 30 s, annealing at 45 °C for 30 s, extension at 72 °C for 80 s, and a final extension at 72°C for 420 s. PCR products were cleaned with ExoSAP-IT PCR Product Cleanup Reagent (Life Technologies, Cat. No. 78201.1.ML), prepared for sequencing using BigDye Terminator v3.1 Ready Reaction Mix (Applied Biosystems, Cat. No.43-374-55) and BigDye Xterminator Purification Kit (Applied Biosystems, Cat. No.43-764-86), and Sanger sequenced on an Applied Biosystems 3500XL Genetic Analyzer. To determine the species, we used the Basic Local Alignment Search Tool (BLAST) to compare the 16S nucleotide sequences against known sequences in the GenBank database (https://www.ncbi.nlm.nih.gov/genbank/), in addition to morphological features.
Analyses
We assembled a library of template calls 10 out of 14 species of conservation concern known to occur at the localities surveyed (Table 1). We did not have template calls for 4 of these species Craugastor punctariolus, Craugastor tabasarae, Hemiphractus elioti and Pristimantis museosus (Table 1). However, one sound recording from a single captive individual Ecnomiohyla rabborum exists and presents a rare opportunity to evaluate the utility of template-matching approaches for “rediscovering” lost species (IUCN, 2020a; Mendelson et al., 2008). To validate our acoustic monitoring approach for the rare species we first tested detection of common dendrobatoid species (poison dart and rocket frogs) against observer survey results. We uploaded available template recordings for both conservation-priority species and common dendrobatoid frogs present at the study localities (Table 1). We uploaded reference call recordings to their own “reference call site” using the required date time naming format and added the species under the explore tab. We then visualized the reference calls in Arbimon visualizer (0–24 kHz frequency) and created time-frequency bounding boxes around known calls. Template optimization involved analyzing small data subsets, adjusting to include multiple repeated notes or exclude harmonics as needed to improve true positive ratios. Species with long repetitive calls including four to six notes in the bounding box seemed to be optimal and harmonic notes were not generally helpful to include in the template. These templates are publicly available to all Arbimon users. With templates established, we conducted pattern matching analyses using an initial similarity threshold of 0.3 for pattern matching to minimize false positives (LeBien et al., 2020). For species yielding few true positives, we lowered the threshold to 0.2; for species with very high numbers of matches like Andinobates minutus, Craugastor evanesco and Gastrotheca cornuta, we increased it to 0.4 to facilitate manual validation. Two experts (R.I. and B.G.) validated each pattern-matched annotation by examining audio clips, sonograms, and associated metadata (locality, timestamp). The total number of validated calls from the pattern-matching exercise was divided by the number of days of audiomoth deployment to generate an ‘calls per day’ metric to use as a relative index of abundance to compare stations. The validated dataset was exported as a.csv file for descriptive analysis using ggplot in R (R Development Core Team, 2021).
Table 1. Species of conservation concern with an IUCN redlist status of Near Threatened (NT), Vulnerable (VU), Endangered (EN), Critically Endangered (CR) or higher and dendrobatoid species of Least Concern (LC) that are known to occur at least one of the 3 localities.
Results
Species detection and identification
Template-based sonogram analysis effectively differentiated between frog species (Figure 2). ARUs and pattern matching successfully detected four conservation-priority species: Silverstoneia nubicola, Oophaga vicentei, Triprion spinosus, and Ecnomiohyla veraguensis (Table 2, Figure 3). Detections of these four species were acoustic only, except for one juvenile Ecnomiohyla captured during observer surveys and genetically identified as E. veraguensis (Figure 3). Due to a hand malformation potentially affecting survival, this individual was retained in captivity and provided the first recorded call of the species upon maturity, providing a template for our acoustic detection approach. We found no evidence of Ecnomiohyla rabborum, or Atelopus zeteki that are considered potentially extinct, or 4 other species of conservation concern for which template calls were sourced (Table 2).
Figure 2. Calls identified and validated from pattern-matching, actual sonograms from wild individuals are shown.
Table 2. Locality-level summary showing all the positive validated matches divided by the total days of AudioMoth deployment.
Figure 3. The following species thought to have disappeared from the 3 survey localities were documented through acoustic analysis, that were validated by subsequent unconstrained observer surveys outside regular visual transects: Oophaga vicentei (a), Silverstoneia nubicola (b), Triprion spinosus (e). Agalychnis lemur (c) is thought to have disappeared, and was not found acoustically, but was rediscovered during unconstrained searches connected to observer surveys. Ecnomiohyla veraguensis (d) was discovered during a visual transect and was previously unknown from this locality; the juvenile male specimen was raised in captivity and, when matured, its call was recorded and used as a template for subsequent acoustic surveys. Photographs of the actual specimens.
ARUs detected Least Concern dendrobatoid frogs at twice as many stations compared to the observer surveys (Table 2). Pattern-matching efficiency varied substantially by species, ranging from 35% for Andinobates minutus to 97% for Colostethus panamansis (Table 3). Notably, C. pratti, historically the most abundant dendrobatoid surveyed in Cerro Campana (Table 4), was absent from this locality despite over 2,000 validated acoustic detections at the other two study localities.
Table 3. Pattern-matching efficiency varied widely depending on the frog species, true positives were validated by two experts and the successfully matched sample proportion with 95% confidence intervals (CI).
Table 4. Historical acoustic surveys conducted pre-Bd declines. Individual calling males were counted on a 650m transect at the three times of day.
Diel activity
Species exhibited distinct calling patterns (Figure 4). Dendrobatoid frogs showed exclusively diurnal activity but with species-specific peaks: S. nubicola displayed crepuscular behavior (peaks 6–7 am, 5 pm); S. flotator showed bimodal activity (peaks 10 am, 4 pm); C. panamansis and A. minutus demonstrated strong early morning activity (6–7 am) with continued calling throughout the day. A. talamancae and O. vicentei calling peaked at midday, while C. pratti exhibited trimodal activity (peaks 8 am, 1 pm, 4 pm). These diel calling patterns of dendrobatoid frogs were mirrored by the historic acoustic call surveys, but they lacked the fine scale temporal resolution of the passive acoustic arrays (Table 4, Figure 4). E. veraguensis vocalized primarily at dusk (7 pm) and pre-dawn (5 am), and T. spinosus called mainly between 7–9 pm but these graphs were based on a very small number of calls, 18 and 10 calls respectively.
Figure 4. Variation in call frequency by time of day among poison dart and rocket frog species (Andinobates minutus, Allobates talamancae, Colostethus panamansis, Colostethus pratti, Oophaga vicentei, Silverstoneia flotator, Silverstoneia nubicola and two rare treefrogs Ecnomiohyla veraguensis and Triprion spinosus detected across all localities. For locality-level summaries see Table 2.
Disease prevalence
The amphibian community showed moderate Bd prevalence (21%, 95% Bayesian credible intervals [14.6%, 28.1%], Table 5), with 251 amphibians from 35 species visually detected across 31 transects with mean abundance of 8.1 amphibians visually encountered per 100m transect. The physical searches represent 66.8 hours of search effort amounting to 3.75 frogs per hour (Table 5). Only frog species were Bd positive with infection loads ranging between 2 to 7,377,000 zoospore equivalents (median = 807 zoospore equivalents, Table 5).
Table 5. Summary of amphibians seen or heard in transect samples, bold = at least one individual was swabbed and had a high Bd load above 10,000 zoospores.
Anecdotal observations
Additional anecdotal observations outside formal sampling in Cerro Campana included observations of T. spinosus and S. nubicola and Agalychnis lemur individuals (Figure 3). A physical specimen of O. vicentei was encountered outside of these surveys near Bajo Bonito (04/30/2025, 8.67582411°N, 80.05116264°W, 905 masl), but it validates the calls detected nearby at Cerro Gaital, especially considering that the IUCN portion of this range was documented as extirpated (IUCN, 2020b). We documented a new locality record for Pristimantis gretathunbergae in Altos de María (05/20/2022, 8.63509°N, 80.07840°W, 1042 masl), based on a recently deceased frog found on the forest floor (STRI Collection of Herpetology, specimen CH-10577) with a Bd load of 63.8 x 106 zoospore equivalents. Several species of conservation concern lacking template calls (Craugastor punctariolus, C. tabasarae, C. evanesco, Pristimantis museosus, Hemiphractus elioti) were also not detected anecdotally, or during observer surveys.
Discussion
ARU effectiveness and limitations
The effectiveness of ARU detection varied significantly among species. Pattern-matching proved highly effective for species with distinctive, repetitive calls like Dendrobatoidea, but less reliable for those with simple or quiet vocalizations (Figure 2). For Ecnomiohyla veraguensis and Triprion spinosus, the use of pattern matching had low ratios of validated pattern matches, but at least they have loud, distinctive and repetitive patterns in their call. This may partly be explained by the fact that they are very rare, rather than the lack of a suitable call for employing this approach. The use of different thresholding for different species was a tradeoff for managing post-pattern matching manual validation effort. While this may raise concerns that very different numbers of positive calls may be found based on those thresholds, one must inherently assume that the detectability of each species using this method is very different. As such, inter-species call frequency comparisons should be avoided, but within a species detectability should be the same as long as thresholding was consistent across all sites. The high false-positive rate for Agalychnis lemur, Craugastor evanesco and Gastrotheca cornuta, who have very simple and loud but monosyllabic calls without a consistently repeated call pattern highlights the need for more sophisticated machine learning approaches as more training data becomes available. For example, machine-learning approaches have been successfully deployed for A. lemur in another study in Costa Rica that used template optimization pattern recognition combined with machine learning to reject false positives (Chirino et al., 2025). However, these require large volumes of annotated training data beyond the scope of this study given the large numbers of species and paucity of training data.
While we failed to detect E. rabborum, our successful acoustic documentation of E. veraguensis validates ARU methodology for sampling rare, arboreal Ecnomiohyla species. The genus’s elusive nature, canopy-dwelling habits, and infrequent calling patterns (Kubicki and Salazar, 2015) suggest continued ARU monitoring may be valuable for detecting remnant populations. One barrier to broader implementation of this method is the paucity of reference calls. While captive recordings exist for several threatened Panamanian species (Gratwicke et al., 2016), and reference calls are available through established sources (Ibáñez et al., 1999; www.fonozoo.com), platforms such as www.inaturalist.org and www.xeno-canto.org are emerging as important crowd-sourced repositories for wild frog vocalizations.
Diel activity
The passive acoustic monitoring method has revealed a fine scale diurnal calling frequency pattern for the first time for several species. Similar efforts to replicate this type of analysis in the past required highly trained experts and an extraordinary field effort (RI pers. obs.; e.g., Navas, 1996). This information is invaluable to design appropriate survey strategies and timing of effort especially for very rare species. For example, S. nubicola is identifiable by its call, but its crepuscular nature does not coincide with the hours when diurnal or nocturnal surveys are often conducted and may explain partially why earlier search efforts did not find them. As research on soundscapes considers the impacts of anthropogenic disturbance on reproduction-related biophony is becoming more relevant to environmental mitigation work (Deichmann et al., 2017).
Post-decline recovery patterns
To make historical comparisons of amphibian abundance, we used visual encounters as the response variable to be consistent with past methodology (Crawford et al., 2010; Kilburn et al., 2010). Current amphibian abundances (3.6-11.3 amphibians seen/100m transect, Table 5) suggest partial recovery compared to post-decline levels (0.2-6.6 frogs/100m) reported by Crawford et al. (2010) from El Copé National Park but these numbers are still well below pre-decline abundances in El Copé (23–32 frogs/100m) (Crawford et al., 2010). When looking at search effort (number of individual amphibians per person hour search), visual encounters survey abundances of 1.7 - 2.8 amphibians/hour of search effort (Table 5) are higher than the 1 amphibian/hour of search effort documented for El Copé two years post-epizootic, but are below pre-epizootic abundances documented for Cerro Campana of 4 amphibians/hour of search effort (Kilburn et al., 2010).
The prevalence of Bd in amphibians has decreased compared to historical records obtained during chytridiomycosis outbreaks in 2006–2007 at Altos de María and Cerro Campana (i.e., 78% and 47%, respectively; Woodhams et al., 2008; Kilburn et al., 2010). Nonetheless, the overall persistence of Bd (21% prevalence) with continued mortality indicates ongoing disease pressure despite the apparent recovery of some populations. While counting calls repeatedly at a single station is not necessarily a good indicator or abundance of animals, it offers a high resolution and accurately measurable relative index of species-specific reproductive effort in a geographic location as a baseline for future comparisons. Establishing baseline sampling protocols, archiving calls and making them publicly accessible through platforms such as Arbimon will be an asset for future recovery monitoring.
Conservation implications
Our acoustic surveys documented the persistence of three conservation-priority species: O. vicentei and T. spinosus were presumed extirpated from most these localities following chytridiomycosis-related declines, and S. nubicola was last documented from Campana National Park in 2016 (Crawford et al., 2010; IUCN, 2020c, IUCN 2020d, IUCN, 2020b; Woodhams et al., 2008). The detection of over 400 O. vicentei calls confirms this endangered species’ presence in the El Valle region, where it was thought extinct (IUCN, 2020b). Similarly, T. spinosus recordings provide the first evidence of Panamanian populations in a decade (IUCN, 2020d), though limited detections (10 positive sound files) suggest a small population size. The documentation of S. nubicola, severely impacted by chytridiomycosis at the study localities around 2006-2007 (Kilburn et al., 2010; Woodhams et al., 2008), indicates a potentially recovering population.
Some species that disappeared from Cerro Campana (Woodhams et al., 2008) have reappeared (i.e. Pristimantis caryophyllaceus, Pristimantis cruentus, Pristimantis pardalis, Agalychnis lemur, Smilisca sila, Andinobates minutus, Sachatamia albomaculata, Cochranella euknemos, Allobates talamancae, Colostethus panamensis, Silverstoneia nubicola, Rhinella horribilis) and appear to be recovering. However, C. pratti, remains missing even though it has recovered at other localities. This presents an opportunity to experimentally translocate animals from the other recovered populations of this species that we documented in this survey, which is a recommended intervention strategies for amphibian disease recovery that assumes evolved resistance in the source population (Knapp et al., 2024; Mendelson et al., 2019). These findings also present opportunities for other targeted conservation actions recommended by the IUCN global amphibian conservation action plan (Bletz et al., 2024). Specifically, augmentation with captive releases and in-situ breeding site supplementation strategies that have proved successful in Costa Rica (IUCN, 2020d, IUCN, 2020e). However, any conservation actions should be carried out on a small scale, accompanied by a responsible adaptive management framework with monitoring of host communities and Bd prevalence in order to better understand the effects before being applied broadly (Bletz et al., 2024).
Conclusions
Our findings demonstrate the value of combining passive acoustic monitoring with traditional surveys for detecting rare and recovering amphibian populations. While pattern-matching approaches show promise, particularly for species with distinctive calls, our current methods are limited and underscore the need for expanded call libraries and focus on developing advanced machine-learning techniques. The fact that we do not have template calls for pattern-matching efforts of five species of conservation concern highlights the need for publicly available sources of calls to be archived for research purposes. The documented persistence of previously missing species at our localities in Panama highlights the importance of long-term monitoring and suggests that some species may be developing disease resistance, offering hope for amphibian conservation in the region.
Data availability statement
The original contributions presented in the study are included in the Supplementary Material Data S1, and on Arbimon https://arbimon.org/p/searching-for-frog-species-of-conservation-concern/insights. Further inquiries can be directed to the corresponding author.
Ethics statement
The animal study was reviewed and approved by the Smithsonian Tropical Research Institute ACUC SI-21013 and SI-24045 and was conducted under the permit No. ARG-0067-2021 from the Panamanian Ministry of Environment (MiAmbiente). The study was conducted in accordance with the local legislation and institutional requirements.
Author contributions
BG: Writing – original draft, Conceptualization, Funding acquisition, Visualization, Data curation, Formal analysis. JG: Investigation, Writing – review & editing, Data curation, Supervision, Project administration. OG: Writing – review & editing, Methodology, Data curation, Investigation. EI: Formal analysis, Writing – review & editing, Investigation. NW: Writing – review & editing, Validation. JD: Conceptualization, Writing – review & editing, Formal analysis, Methodology. RI: Writing – original draft, Funding acquisition, Investigation, Project administration, Conceptualization, Formal analysis, Supervision, Validation, Visualization, Methodology, Data curation.
Funding
The author(s) declared that financial support was received for this work and/or its publication. This study was funded by the Shared Earth Foundation in honor of George and Mary Rabb for whom Ecnomiohyla rabborum were named, the Anele Kolohe Foundation, the Bezos Earth Fund and an Anonymous Foundation. RI received support from the Sistema Nacional de Investigación, SENACYT, and for the historical surveys from an EXXON fellowship, Smithsonian Tropical Research Institute. This work is a product of the Panama Amphibian Rescue and Conservation Project, which is a partnership between Cheyenne Mountain Zoo, Zoo New England, the Smithsonian’s National Zoo and Conservation Biology Institute, and the Smithsonian Tropical Research Institute.
Acknowledgments
We thank MiAmbiente for permits and their park staff for kindly assisting us during field visits to the Parque Nacional y Reserva Biológica Altos de Campana and the Monumento Natural Cerro Gaital. We thank Juan Polo and the staff at Altos de María for facilitating permission and access to these localities. We are grateful to Mark Mandica of the Amphibian Foundation who provided the call template of the Rabbs treefrog and the Atlanta Botanical Garden where the Rabbs Treefrog call was recorded. We are grateful to RainforestX and Arbimon for hosting the call storage and analysis tools.
Conflict of interest
The authors declared that this work was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
Generative AI statement
The author(s) declared that generative AI was not used in the creation of this manuscript.
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Supplementary material
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/famrs.2025.1736880/full#supplementary-material
Footnotes
References
Aide T. M., Corrada-Bravo C., Campos-Cerqueira M., Milan C., Vega G., and Alvarez R. (2013). Real-time bioacoustics monitoring and automated species identification. PeerJ 1, e103. doi: 10.7717/peerj.103
Amphibiaweb (2025). Amphibiaweb [WWW Document]. IUCN, Gland, Switzerland. Available online at: http://www.amphibiaweb.orghttp://www.amphibiaweb.org (Accessed 12/12/202512.12.25).
Bletz M. C., Gratwicke B., Belasen A. M., Catenazzi A., Duffus A. L. J., Lampo M., et al. (2024). “Infectious diseases: Advances and challenges in addressing emerging pathogens,” in The amphibian conservation action plan (ACAP): A status review and roadmap for global amphibian conservation (IUCN).
Boullhesen M., Vaira M., Barquez R. M., and Akmentins M. S. (2021). Evaluating the efficacy of visual encounter and automated acoustic survey methods in anuran assemblages of the Yungas Andean forests of Argentina. Ecol. Indic. 127, 107750. doi: 10.1016/j.ecolind.2021.107750
Brodie S., Allen-Ankins S., Towsey M., Roe P., and Schwarzkopf L. (2020). Automated species identification of frog choruses in environmental recordings using acoustic indices. Ecol. Indic. 119, 106852. doi: 10.1016/j.ecolind.2020.106852
Campos-Cerqueira M. and Aide T. M. (2016). Improving distribution data of threatened species by combining acoustic monitoring and occupancy modelling. Methods Ecol. Evol. 7, 1340–1348. doi: 10.1111/2041-210X.12599
Chirino F., Willink B., Elizondo-Calvo J., Chaves-Acuña W., Salas-Solano D., Salazar-Zúñiga J. A., et al. (2025). Environmental drivers of calling activity in the critically endangered lemur leaf frog, Agalychnis lemur (Hylidae: Phyllomedusinae). Philos. Trans. B 380, 20240050. doi: 10.1098/rstb.2024.0050
Crawford A. J., Cruz C., Griffith E., Ross H., Ibáñez R., Lips K. R., et al. (2012). DNA barcoding applied to ex situ tropical amphibian conservation programme reveals cryptic diversity in captive populations. Mol. Ecol. Resour. 13, 1005–1018. doi: 10.1111/1755-0998.12054
Crawford A. J., Lips K. R., and Bermingham E. (2010). Epidemic disease decimates amphibian abundance, species diversity, and evolutionary history in the highlands of central Panama. Proc. Natl. Acad. Sci. 107, 13777–13782. doi: 10.1073/pnas.0914115107
Crump P. S. and Houlahan J. (2017). Designing better frog call recognition models. Ecol. Evol. 7, 3087–3099. doi: 10.1002/ece3.2730
Deichmann J. L., Acevedo-Charry O., Barclay L., Burivalova Z., Campos-Cerqueira M., d’Horta F., et al. (2018). It’s time to listen: there is much to be learned from the sounds of tropical ecosystems. Biotropica 50, 713–718. doi: 10.1111/btp.12593
Deichmann J. L., Hernández-Serna A., Delgado C. J. A., Campos-Cerqueira M., and Aide T. M. (2017). Soundscape analysis and acoustic monitoring document impacts of natural gas exploration on biodiversity in a tropical forest. Ecol. Indic. 74, 39–48. doi: 10.1016/j.ecolind.2016.11.002
Duarte M. H. L., Caliari E. P., Viana Y. P., and Nascimento L. B. (2019). A natural orchestra: how are anuran choruses formed in artificial ponds in southeast Brazil? Amphibia-Reptilia. doi: 10.1163/15685381-20191079
Gratwicke B., Lovejoy T. E. T. E., and Wildt D. E. (2012). Will amphibians croak under the Endangered Species Act? BioScience 62, 197–202. doi: 10.1525/bio.2012.62.2.13
Gratwicke B., Ross H., Batista A., Chaves G., Crawford A. J., Elizondo L., et al. (2016). Evaluating the probability of avoiding disease-related extinctions of Panamanian amphibians through captive breeding programs. Anim. Conserv. 19, 324–336. doi: 10.1111/acv.12249
Heyer R., Donnelly M. A., Foster M., and McDiarmid R. W. (1994). Measuring and monitoring biological diversity: standard methods for amphibians (Washington DC: Smithsonian Institution Press) 384pp.
Hilje B. and Aide T. M. (2012). Calling activity of the common tink frog (Diasporus diastema) (Eleutherodactylidae) in secondary forests of the Caribbean of Costa Rica. Trop. Conserv. Sci. 5, 25–37. doi: 10.1177/194008291200500104
Hyatt A. D., Boyle D. G., Olsen V., Boyle D. B., Berger L., Obendorf D., et al. (2007). Diagnostic assays and sampling protocols for the detection of. Batrachochytrium dendrobatidis 73, 175–192. doi: 10.3354/dao073175
Ibáñez R., Jaramillo A C. A., Frank A., and Jaramillo F. E. (1996). Inventario de anfibios y reptiles Parque Nacional Altos de Campana: fase inicial para la conservación de estas especies en el Parque Nacional Altos de Campana. Final Report G-9516. (Círculo Herpetológico de Panamá & Fundación Natura). Available online at: https://repository.si.edu/handle/10088/31959 (Accessed September 1, 2025).
Ibáñez R., Rand S. A., Ryan M. R., and Jaramillo A C. A. (1999). Vocalizaciones de ranas y sapos del Monumento Natural Barro Colorado, Parque Nacional Soberanía y áreas adyacentes/Vocalizations of frogs and toads from Barro Colorado Nature Monument, Soberanía National Park and adjacent areas (Círculo Herpetológico de Panamá & Smithsonian Tropical Research Institute. Sony Music: Fundación Natura).
IUCN (2020a). Ecnomiohyla rabborum [WWW Document]. IUCN Red List of Threatened Species. URL. Available online at: https://www.iucnredlist.org/species/85341796/54357363 (Accessed April 16, 2020).
IUCN (2020b). Oophaga vicentei [WWW Document]. IUCN Red List of Threatened Species. Available online at: https://www.iucnredlist.org/species/55209/54344862 (Accessed September 1, 2025).
IUCN (2020c). Silverstoneia nubicola [WWW Document]. IUCN Red List of Threatened Species. Available online at: https://www.iucnredlist.org/species/55121/3024457 (Accessed September 1, 2025).
IUCN (2020d). Triprion spinosus [WWW Document]. IUCN Red List of Threatened Species. Available online at: https://www.iucnredlist.org/species/55296/3028482 (Accessed September 1, 2025).
IUCN (2020e). Agalychnis lemur [WWW Document]. IUCN Red List of Threatened Species (10.12.24). Available online at: https://www.iucnredlist.org/ja (Accessed September 1, 2025).
Jaynes E. T. and Kempthorne O. (1976). “Confidence intervals vs bayesian intervals,” in Foundations of probability theory, statistical inference, and statistical theories of science: proceedings of an international research colloquium held at the university of western ontario, london, Canada, 10–13 May 1973, vol. Volume II . Eds. Harper W. L. and Hooker C. A. (Foundations and Philosophy of Statistical Inference. Springer Netherlands, Dordrecht), 175–257. doi: 10.1007/978-94-010-1436-6_6
Kahl S., Wood C. M., Eibl M., and Klinck H. (2021). BirdNET: A deep learning solution for avian diversity monitoring. Ecol. Inf. 61, 101236. doi: 10.1016/j.ecoinf.2021.101236
Kilburn V. L., Ibáñez R., Sanjur O., Bermingham E., Suraci J. P., and Green D. M. (2010). Ubiquity of the pathogenic chytrid fungus, Batrachochytrium dendrobatidis, in anuran communities in Panamá. EcoHealth 7, 537–548. doi: 10.1007/s10393-010-0634-1
Klocke B., Estrada A., Mataya M., Medina D., Baitchman E., Belden L., et al. (2023). Movement and survival of captive-bred Limosa harlequin frogs (Atelopus limosus) released into the wild. Front. Amphibian Reptile Sci. 1. doi: 10.3389/famrs.2023.1205938
Knapp R. A., Wilber M. Q., Joseph M. B., Smith T. C., and Grasso R. L. (2024). Reintroduction of resistant frogs facilitates landscape-scale recovery in the presence of a lethal fungal disease. Nat. Commun. 15, 9436. doi: 10.1038/s41467-024-53608-4
Kriger K. M., Hines H. B., Hyatt A. D., Boyle D. G., and Hero J. M. (2006). Techniques for detecting chytridiomycosis in wild frogs: Comparing histology with real-time Taqman PCR. Dis. Aquat. Organisms 71, 141–148. doi: 10.3354/dao071141
Kubicki B. and Salazar S. (2015). Discovery of the Golden-eyed fringe-limbed treefrog, Ecnomiohyla bailarina (Anura: Hylidae), in the Caribbean foothills of southeastern Costa Rica. Mesoamer Herpetol 2, 76–86.
Lambert M. R., Womack M. C., Byrne A. Q., Hernández-Gómez O., Noss C. F., Rothstein A. P., et al. (2020). Comment on “Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity. Science 367, aay1838. doi: 10.1126/science.aay1838
Lapp S., Rhinehart T., Freeland-Haynes L., Khilnani J., Syunkova A., and Kitzes J. (2023). OpenSoundscape: an open-source bioacoustics analysis package for Python. Methods Ecol. Evol. 14, 2321–2328. doi: 10.1111/2041-210X.14196
Lapp S., Wu T., Richards-Zawacki C., Voyles J., Rodriguez K. M., Shamon H., et al. (2021). Automated detection of frog calls and choruses by pulse repetition rate. Conserv. Biol. 35, 1659–1668. doi: 10.1111/cobi.13718
LeBien J., Zhong M., Campos-Cerqueira M., Velev J. P., Dodhia R., Ferres J. L., et al. (2020). A pipeline for identification of bird and frog species in tropical soundscape recordings using a convolutional neural network. Ecol. Inf. 59, 101113. doi: 10.1016/j.ecoinf.2020.101113
Lips K. R., Brem F., Brenes R., Reeve J. D., Alford R., Voyles J., et al. (2006). Emerging infectious disease and the loss of biodiversity in a Neotropical amphibian community. Proc. Natl. Acad. Sci. United States America 103, 3165–3170. doi: 10.1073/pnas.0506889103
Lips K. R., Diffendorfer J., Mendelson J. R., and Sears M. W. (2008). Riding the wave: reconciling the roles of disease and climate change in amphibian declines. PloS Biol. 6, e72. doi: 10.1371/journal.pbio.0060072
Lips K. R., Reaser J. K., Young B. E., and Ibáñez R. (1999). El monitoreo de anfibios en América Latina. Un manual para coordinar esfuerzos. The nature conservancy. United States National Science Foundation. Smithsonian Tropical Research Institute, Ciudad de Panamá, Panamá. Colegio de la Frontera Sur, Chetumal, México. Pontificia Universidad Católica de Ecuador, Quito, Ecuador. 142pp.
Luedtke J. A., Chanson J., Neam K., Hobin L., Maciel A. O., Catenazzi A., et al. (2023). Ongoing declines for the world’s amphibians in the face of emerging threats. Nature 622, 308–314. doi: 10.1038/s41586-023-06578-4
Mendelson III J. R., Savage J. M., Griffith E., Ross H., Kubicki B., and Gagliardo R. (2008). Spectacular new gliding species of Ecnomiohyla (Anura: Hylidae) from central Panama. Journal of Herpetology, pp.750–759.
Mendelson J. R., Whitfield S. M., and Sredl M. J. (2019). A recovery engine strategy for amphibian conservation in the context of disease. Biol. Conserv. 236, 188–191. doi: 10.1016/j.biocon.2019.05.025
Navas C. A. (1996). The effect of temperature on the vocal activity of tropical anurans: a comparison of high and low-elevation species. J. Herpetol 30, 488–497. doi: 10.2307/1565691
Nowakowski A. J., Deichmann J. L., Barata I. M., Basham E. W., Bower D. S., Lau M. W. N., et al. (2024). “Surveys and monitoring: challenges in an age of rapid declines and discoveries,” in Amphibian conservation action plan: A status review and roadmap for global amphibian conservation, IUCN SSC occasional paper (Gland, Switzerland: IUCN).
Pieretti N., Farina A., and Morri D. (2011). A new methodology to infer the singing activity of an avian community: The Acoustic Complexity Index (ACI). Ecol. Indic. 11, 868–873. doi: 10.1016/j.ecolind.2010.11.005
R Development Core Team (2021). R: A language and environment for statistical computing. Available online at: https://doi.org/ISBN%25203-900051-07-0.
Scheele B. C., Pasmans F., Skerratt L. F., Berger L., Martel A., Beukema W., et al. (2019). Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity. Science 363, 1459–1463. doi: 10.1126/science.aav0379
Van Horn G., Cole E., Beery S., Wilber K., Belongie S., and Mac Aodha O. (2021). “Benchmarking representation learning for natural world image collections,” in Presented at the Proceedings of the IEEE/CVF Conference on Computer Vision and Pattern Recognition. 12884–12893. Available online at: https://openaccess.thecvf.com/content/CVPR2021/papers/Van_Horn_Benchmarking_Representation_Learning_for_Natural_World_Image_Collections_CVPR_2021_paper.pdf (Accessed 1.9.25).
Wood C. M., Kahl S., Barnes S., Van Horne R., and Brown C. (2023). Passive acoustic surveys and the BirdNET algorithm reveal detailed spatiotemporal variation in the vocal activity of two anurans. Bioacoustics 32, 532–543. doi: 10.1080/09524622.2023.2211544
Keywords: amphibian conservation, Panama, passive acoustic monitoring, pattern matching, rare species
Citation: Gratwicke B, Guerrel J, Garcés O, Illueca E, Weisenbeck NJ, Deichmann JL and Ibáñez R (2026) Rediscovery of frogs of conservation concern in Panama using passive acoustic monitoring and pattern-matching analysis. Front. Amphib. Reptile Sci. 3:1736880. doi: 10.3389/famrs.2025.1736880
Received: 31 October 2025; Accepted: 11 December 2025; Revised: 09 December 2025;
Published: 21 January 2026.
Edited by:
Ilaria Bernabò, University of Calabria, ItalyReviewed by:
Roberto Alonso, University of Havana, CubaMauricio Diaz Vallejo, Instituto de Ecología (INECOL), Mexico
Luis Castillo, GRUPO RANA NGO, Peru
Copyright © 2026 Gratwicke, Guerrel, Garcés, Illueca, Weisenbeck, Deichmann and Ibáñez. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
*Correspondence: Brian Gratwicke, Z3JhdHdpY2tlYkBzaS5lZHU=