a “ the journal of a Ch eck Lis t seu data i) NOTES ON GEOGRAPHIC DISTRIBUTIO Range expansion for the Critically Endangered poison-dart frog Leucostethus bilsa Vigle et al., 2020 (Amphibia, Dendrobatidae), demonstrating the importance of small forest reserves in the Ecuadorian Choco S. Holden Jones', Daniela Franco-Mena’, Darwin Zambrano Cantos’, Julio César Loor Vélez’, Carlos Aulestia’, Rakan A. Zahawi"™, Juan M. Guayasamin? 1 School of Life Sciences, University of Hawaii at Manoa, Honolulu, Hawaii, USA 2 Laboratorio de Biologia Evolutiva, Instituto Bidsfera, Colegio de Ciencias Bioldgicas y Ambientales, Universidad San Francisco de Quito (USFQ), Quito, Ecuador 3 Fundacion para la Conservacion de los Andes Tropicales (FCAT), Quito, Ecuador 4 Charles Darwin Foundation, Puerto Ayora, Galapagos, Ecuador Corresponding author: S. Ho/den Jones (jonessh@hawaii.edu) Abstract. The Ecuadorian Choco is a global conservation hotspot with high levels of biodiversity and endemism but is threatened by extensive deforestation. Here, we report the discovery of an additional locality for the Critically Endangered poison-dart frog Leucostethus bilsa (Amphibia, Dendrobatidae) in a privately protected forest reserve in Esmeraldas, Ecuador. This may represent a distinct IUCN location for the species, thereby downgrading its IUCN threat level to Endangered. This finding increases the extent of occurrence for L. bilsa from 0.9 to 4.78 km?, and the area of occupancy from 8.0 to 16.0 km?, demonstrat- ing the importance of small forest reserves for the conservation of forest-dependent amphibians. Further surveys are warranted to determine whether the species is present in additional forest fragments in the region, the degree of connectivity, if any, between individuals inhabiting different fragments, and whether the species’ population is increasing, stable, or in decline. Key words. Amphibians, connectivity, conservation, forest-dependence, fragmentation Jones SH, Franco-Mena D, Cantos DZ, Loor Vélez JC, Aulestia C, Zahawi RA, Guayasamin JM (2024) Range expansion for the Critically Endangered poison-dart frog Leucostethus bilsa Vigle et al., 2020 (Amphibia, Dendrobatidae), demonstrating the importance of small forest reserves in the Ecuador- ian Choco. Check List 20 (4): 853-858. https://doi.org/10.15560/20.4.853 INTRODUCTION Dendrobatidae is a Neotropical family of anurans containing approximately 16 genera and 205 species (Frost 2024). Known colloquially as the poison-dart frogs, dendrobatids are diurnal, lay eggs on land or vegetation, can exhibit bright coloring, and contain powerful neurotoxins, hence their common name. Leucostethus Grant, Rada, Anganoy-Criollo, Batista, Dias, Jeckel, Machado, & Rueda-Almonacid, 2017 is a OPEN Qaccess cryptic, forest-dwelling genus of Dendrobatidae with 11 species that range from the rainforests of western Amazonia to the Chocé and Gorgona Island (Vigle et al. 2020; Grant and Bolivar-Garcia 2021). Leucostethus bilsa Vigle, Coloma, Santos, Hernandez-Nieto, Ortega-Andrade, Paluh & Read, 2020 Academic editor: Luisa Diele-Viegas was recently described and is known only from a small area of forest within the 3,000 ha Bilsa Biological Received: 13 February 2024 Reserve in the Choco of northwestern Ecuador (Figure 1) (Ortega-Andrade et al. 2010; Vigle et al. 2020). Accepted: 26 June 2024 The species is classified as Critically Endangered by the International Union for the Conservation of Nature Published: 11 July 2024 (IUCN) due to its extremely limited range (a single location) in a highly fragmented habitat (IUCN 2022). The Chocdé-Darien Ecoregion has been identified as a top 25 global hotspot for conservation action due to its high levels of biodiversity and endemism, and the rapid rate and extent of habitat loss (Myers et al. 2000; Copyright © The authors. This is an open-access Berlin and Leguizamén 2024). Here we report the discovery of additional L. bilsa individuals in the nearby article distributed under terms of the Creative ' de, 2 . Commons Attribution License (Attribution 4.0 privately protected reserve of the Fundacion para la Conservacion de los Andes Tropicales (FCAT) and International — CC BY 4.0) provide an updated distribution map for this species. 853 Jones etal. - Leucostethus bilsa range expansion 854 79.7100°W 79.6800°W L. bilsa coordinates : | Range expansion Vigle et al. 2020 FCAT Reserve boundary Bilsa Reserve boundary 0.3600°N = o 5 oO a = ‘ \ 79.7100/W 79.6800W Figure 1. Range expansion for the Critically Endangered Leucostethus bilsa in the distinct Fundacion para la Conservacion de los Andes Tropicales (FCAT) Forest Reserve, 5.1 km NE of the Bilsa Biological Reserve, Esmeraldas province, Ecuador. METHODS The FCAT Reserve system encompasses several isolated patches of primary forest totaling 650 ha in the Mache-Chindul mountains between the Cube lagoon (00.3924°N, 079.6473°W, 300 m a.s.l.) and the Bilsa Biological Reserve (00.3555°N, 079.6895°W, 500 m a.s.l.) in Esmeraldas province, Ecuador. The FCAT Reserve primarily comprises tropical rainforests (Jongsma et al. 2014) with an average annual precipitation of 2,500—3,500 mm and average temperature between 23—25.5 °C (Clark et al. 2006). The intervening agricultural mosaic primarily consists of pasture and cacao plantations. Deforestation in the surrounding landscape began in the 1980s and has accelerated in recent years, greatly reducing the number and extent of isolated forest patches between the Bilsa and FCAT Reserves (Perlin and Leguizamoén 2024). We found the first Leucostethus bilsa individual incidentally during a visual encounter transect in the core FCAT Reserve as part of a Separate seven-month study. The sampling effort comprised 298 survey-hours during the wet and dry seasons of 2022-2023 across 32 sites to compare the amphibian communities of cacao plantations and nearby remnant forests. This first individual was photographed in the field and collected as a voucher specimen. We confirmed the specimen’s identification at the Laboratorio de Biologia Evolutiva of the Universidad San Francisco de Quito (USFQ), and subsequently deposited the specimen at the Museo de Zoologia, USFQ, Quito, Ecuador (ZSFQ). We shared this finding with FCAT work- ers, who informed us that they had seen similar individuals at various locations within the FCAT Reserve. We asked them to take GPS coordinates and photographs of subsequent L. bi/sa encounters during their normal field activities throughout the FCAT Reserve using GPS devices and cell phones. We later confirmed these sightings as L. bi/sa. We note that nobody at FCAT has encountered or heard of L. bi/sa being found outside of the FCAT or Bilsa Reserves, even though most FCAT employees have lived and worked in the region for decades. In addition, the species has never been reported outside of intact primary forest, despite extensive sampling effort of the amphibian community across the region’s agricultural habitats (Mazanec 2022). We used QGIS v. 3.30.2 (QGIS.org 2024) to map the updated species distribution alongside previously known occurrences as described in Vigle et al. (2020) (Figure 1). We reviewed all available online databases to ensure that L. bi/sa had not been previously reported from the FCAT Reserve. While additional L. bilsa records from the Museo de Zoologia at the Pontificia Universidad Catolica del Ecuador (QCAZ) exist, we Check List 20 (4) - https://doi.org/10.15560/20.4.853 Jones etal. - Leucostethus bilsa range expansion 855 Figure 2. /eucostethus bilsa individual in the Fundacion para la Conservaci6n de los Andes Tropicales (FCAT) Reserve (individual not collected). A. Frontal view. B. Dorsal view. C. Lateral view. D. Ventral view. Note the diagnostic cream dorsolateral stripe and mustard-yellow flash coloration. Photos by Daniela Franco-Mena. did not add them to Figure 1 because we were unable to confirm their identity. Similarly, we did not add locations from Ortega-Andrade et al. (2010), who report finding individuals that appear to be Leucostethus bilsa, but which they describe as Co/ostethus sp. The authors report finding individuals near the Scientific Station, along the Red, Monkey and Aguacatal trails, and along the Rompefrente and Duchas streams but do not provide precise coordinates for these encounters; as such they were not included in Figure 1 or in Our area of occupancy and extent of occurrence calculations. Nevertheless, the locations from both the QCAZ database and Ortega-Andrade et al. (2010) closely align with those reported by Vigle et al. (2020) and are all located within a small area of the Bilsa Biological Reserve. We used the GeoCAT tool (Bachman et al. 2011) to calculate the species’ updated extent of occurrence and area of occupancy following IUCN recommendations (IUCN 2022). Despite using the most recent, available, and cloud-free satellite imagery for our base map (taken in 2017), the imagery overestimates current forest cover in the landscape between the FCAT and Bilsa Reserves. All collection and field research was conducted under permit MAATE-CMARG-2022-0575, issued by the Ministerio del Ambiente, Agua y Transicion Ecoldgica (MAATE) of Ecuador. RESULTS Leucostethus bilsa Vigle, Coloma, Santos, Hernandez-Nieto, Ortega-Andrade, Paluh & Read, 2020 Figure 2 New records. ECUADOR — ESMERALDAS « Fundacion para la Conservacion de los Andes Tropicales (FCAT) Reserve, Guayacan Trail; 00.3748°N, 079.6675°W; 480 m a.s.l.; 8.1X. 2022; S. Holden Jones leg.; 1 sex indet.; found at night (22:30), in leaf litter of mature forest near steep embankment with seasonal stream; ZSFQ 5382 « FCAT Reserve, Guayacan Trail; 00.3754°N, 079.6688°W; 490 m a.s.1.; 15.Il. 2023; Darwin Zambrano Cantos obs.; 1 sex indet.; found during day, in leaf litter of mature forest with steep slope « FCAT Reserve, Guayacan Trail; 00.3754°N, 079.6683°W; 490 m a.s.l.; 15.Il. 2023; Daniela Franco-Mena obs.; 1 sex indet.; found during day, in leaf litter of mature forest with steep slope « FCAT Reserve, Guayacan Trail; 00.3753°N, 079.6690°W; 480 ma.s.l.; 15.1. 2023; Julio César Loor Vélez obs.; 1 sex indet.; found during day, in leaf litter of mature forest with steep slope « FCAT Reserve, below station; 00.3732°N, 079.6645°W; 450 ma.s.l.; VIII. 2023; Carlos Aulestia obs.; 1 sex indet.; found during day, next to small seasonal stream + FCAT Reserve, mature forest north of Palacios restoration site; 00.3775°N, 079.6630°W; 500 m a.s.|.; 15.IIL. 2023; Daniela Franco-Mena obs.; 1 sex indet.; found during day, in leaf litter of mature forest with steep slope (Table 1). The individuals of L. bi/sa in the FCAT Reserve appear to represent a distinct IUCN location from those reported by Vigle et al. (2020), Ortega-Andrade et al. (2010), and the QCAZ database due to the indepen- dent ownership and management of the FCAT and Bilsa Reserves, and the scarcity of suitable mature forest habitat between them (IUCN 2024). This range expansion increases the species’ extent of occurrence from 0.90 to 4.78 km’, and the area of occupancy from 8.0 km? to 16.0 km?. We highlight that despite extensive sampling effort for amphibians in the surrounding agricultural landscape (298 survey-hours, S. Jones et al. Check List 20 (4) - https://doi.org/10.15560/20.4.853 Jones etal. - Leucostethus bilsa range expansion 856 Table 1. Records of Leucostethus bilsa in the Fundacion para la Conservacién de los Andes Tropicales (FCAT) and Bilsa Biological Reserves as reported in this study and Vigle et al. (2020), including a specimen deposited at the Museo de Zoologia, USFQ, Quito, Ecuador (ZSFQ). No. Country Province Locality Latitude (°N) Longitude (°W) Source/reference | Ecuador Esmeraldas FCAT Reserve 00.3748 079.6675 ZSFQ 5382, this study 2 Ecuador Esmeraldas FCAT Reserve 00.3732 079.6645 Obs., this study 3 Ecuador Esmeraldas FCAT Reserve 00.3753 079.6690 Obs., this study 4 Ecuador Esmeraldas FCAT Reserve 00.3754 079.6688 Obs., this study 3) Ecuador Esmeraldas FCAT Reserve 00.3754 079.6683 Obs., this study 6 Ecuador Esmeraldas FCAT Reserve 00.3775 079.6630 Obs., this study 7 Ecuador Esmeraldas __Bilsa Reserve 00.3583 079.7083 Vigle et al. 2020 8 Ecuador Esmeraldas _Bilsa Reserve 00.3558 079.7033 Vigle et al. 2020 9 Ecuador Manabi Bilsa Reserve 00.3478 079.7164 Vigle et al. 2020 10 Ecuador Manabi Bilsa Reserve 00.3451 079.7123 Vigle et al. 2020 11 Ecuador Manabi Bilsa Reserve 00.3481 079.7166 Vigle et al. 2020 unpublished), we did not find any additional individuals of L. bi/sa, suggesting that the species is restricted to mature forest. Identification. All individuals were identified as L. bilsa following the original description by Vigle et al. (2020): (i) bright mustard-yellow flash marks in axillae, groin, and anteroventral surfaces of the hindlimbs; (ii) cream oblique dorsolateral stripe; and (iii) white venter (Figure 2). No other species from the lowlands of northwestern Ecuador shares these traits. DISCUSSION These new records demonstrate the existence of Leucostethus bilsa within the privately protected FCAT Reserve in the Ecuadorian Chocé, which may represent a distinct population separate from the one found at the type locality. Although these populations were likely historically linked, the low degree of remain- ing forest cover between the FCAT and Bilsa Reserves following decades of deforestation (Perlin and Leguizamon 2024; Figure 1) and the high degree of forest-dependence demonstrated by L. bilsa has likely isolated the individuals in the FCAT Reserve from the original population described in Vigle et al. (2020). While these new records increase the species’ extent of occurrence to 4.78 km? and area of occupancy to 16.0 km’, both metrics still suggest the species is at an elevated risk of extinction. Nevertheless, our discovery of an additional location for the species will likely no longer qualify it as Critically Endangered in future assessments (IUCN 2024). Although a detailed survey of the distribution and demography of L. bilsa was outside the scope of this study, we have observed a decline in the area, extent, and quality of potential mature forest habitat between the Bilsa and FCAT Reserves. Future research should seek to address whether L. bilsa is present in additional forest fragments in the region and to better understand the species’ population demographics to determine if these populations are isolated or declining. This finding underscores the importance of conserving the few remaining primary forest patches in the Ecuadorian Choco for forest-dependent species like L. bilsa, as it is likely that additional individuals may be discovered in other smaller forest patches. Additionally, many nearby fragments have been shown to harbor high avian, mammalian, and palm tree diversity (Browne and Karubian 2016; Cook et al. 2020), although amphibians are under sampled in all but the largest patches of forest like the Bilsa and FCAT Reserves (Jongsma et al. 2014). Matrix composition often has a large effect on the degree of connectivity between fragmented herpetofauna communities which can help sustain seemingly isolated populations (Almeida-Gomes and Rocha 2014; Mendenhall et al. 2014). Even though L. bilsa displays a high degree of forest dependence, the abundance of riparian corridors, live fences, and shade-grown cacao in the surrounding landscape provides a framework whereby connectivity may increase following a sufficient degree of forest restoration. Surveying additional forest fragments would help determine the true extent of L. bilsa’s range, and better inform conservation and restoration efforts to create a biologically meaningful corridor between the Bilsa and FCAT Reserves, thereby benefiting L. bilsa as well as other forest-dependent species. Check List 20 (4) - https://doi.org/10.15560/20.4.853 Jones etal. - Leucostethus bilsa range expansion 857 ACKNOWLEDGEMENTS We thank the FCAT team for their assistance throughout the fieldwork portion of this project, and for their work conserving remnant forest in the Ecuadorian Choco. We would also like to thank Hugo Andrade and an additional anonymous reviewer whose feedback improved the manuscript. ADDITIONAL INFORMATION Conflict of interest The authors declare that no competing interests exist. Ethical statement No ethical statement is reported. Funding This work was financially supported by Fulbright Ecuador, The Rufford Foundation, and the ARCS Founda- tion Honolulu Chapter. Author contributions Conceptualization: SHJ, DFM, RAZ, JMG. Data curation: SHJ, DFM. Formal analysis: SHJ,. Funding acqui- sition: SHJ, RAZ, JMG. Investigation: SHJ, DFM, DZC, JCLV, CA, RAZ, JMG. Methodology: SHJ, DFM, RAZ, JMG. Resources: JMG. Supervision: RAZ, JMG. Visualization: SHJ, DFM. Project administration: SHJ, JMG. Software: SHJ. Validation: JMG, DFM. Writing — original draft: SHJ. Writing — review and editing: SHJ, DFM, RAZ, JMG. Author ORCID iDs S. Holden Jones © https://orcid.org/0000-0001-5790-5175 Daniela Franco-Mena © https://orcid.org/0000-0003-3655-2678 Rakan A. Zahawi © https://orcid.org/0000-0002-5678-2967 Juan M. 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