**4. Discussion**

## *4.1. Species Assessment*

We presented the first updated list of Costa Rican amphibians since 2011 [47]. Compared to the last list, we added 10 anurans, 9 salamanders, and 1 caecilian (Table 3) for a total of 215 species (Table S3). As is common throughout the world, anurans exhibited the highest amphibian species richness in Costa Rica, with 72% of listed species. However, the richness of salamander species is also high (25%). In Costa Rica, the diversity and endemism of amphibians (especially salamanders) increase with elevation and complex mountain topography [13]. Proportionally, in terms of number of species per unit area (km2), the richest herpetological province was the Cordillera de Talamanca. In this herpetological province, the number of species continued to increase and most of the newly described species in our report came from this remote and almost inaccessible province [69]. The Montane Slopes and Cordillera Central presented the highest number of species (158 species). Within this province, numerous mountain ranges provide multiple microhabitats for niche di fferentiation and further speciation [13,49]. In lowlands, the highest number of amphibian species occurred in the Caribbean Lowlands with 101 species. However, the Pacific Southwest presented more species per unit area. The Pacific Northwest only had 66 species, which was also the lowest number of species per unit area. This pattern may be attributed to the warm and dry conditions that occur in most part of this herpetological province [13,49].

According IUCN, the species *Craugastor escoces* and *Incilius periglenes* are classified as EX in Costa Rica. However, *C. escoces* was recently rediscovered [89]. Similarly, several species that remained undetected after the 1980s and 1990s such as *Incilius holdridgei* [20], *Craugastor taurus* [19], and *Atelopus varius* [18] have been rediscovered in peripheral populations during the last few years. However, the number of extinct species could be higher because multiple threatened species still remain undetected in the field (e.g., *Craugastor andi*, *Incilius fastidiosus*, *Atelopus senex*). We recommend expedition surveys to find populations of declined and data-deficient species [90] and captive-breeding for species where ex situ reproduction has been successful (e.g., harlequin frogs.) [91]. Although we acknowledge that there are limited funds available for these types of conservation e fforts, knowledge from these sites is essential to be able to identify conditions that favor the persistence of threatened species and identify species that should be targeted for future conservation e fforts [92].

Lowlands of Costa Rica exhibited the lowest proportion of DD species (0–2%; Figure 2b) and the highest proportion of LC species (75–80%; Figure 2c). On the other hand, highlands exhibited the highest percentage of DD species (6–10%; Figure 2b). Similarly, Cordillera de Talamanca had the highest percentage of threatened species (26%; Figure 2d). Based on these findings, we strongly recommend increasing the sampling e ffort in the montane and subalpine altitudinal belts (>2800 m) that exclusively occur in Cordillera de Talamanca and Montane Slopes and Cordillera Central. These herpetological provinces present the highest rate of endemism (especially for salamanders) and contain several of the recently described species [85,86]. Conducting expeditions and long-term studies in highlands will aid in monitoring threatened species and reducing information gaps, allowing for more accurate assessments of amphibian species.

To better evaluate the vulnerability of amphibian species, we utilized EVS (Table S3). This index relies on ecological information for categorizing threat levels, which makes application easy for most species in a specific region [52]. Unlike the IUCN Red List of Threatened Species, previous evaluations of threats are not considered by this index [49]. For that reason, species that are classified as LC by IUCN can be classified as highly vulnerable in this index (e.g., *Duellmanohyla rufioculis*). We categorized 48 species (24 salamanders, 21 anurans, and 3 caecilians) in "high vulnerability" (e.g., *Atelopus senex*, *Craugastor andi*, *Bolitoglossa pesrubra*, *Nototriton guanacaste*, and *Oscaecilia osae*). These species exhibited the highest EVS values because their habitats are restricted and because they exhibit complex reproductive modes. Quantifying environmental threats and combining information from both indexes will help policy-makers to prioritize conservation actions for threatened species.

In Central America, habitat destruction is the most important threat impacting amphibian populations [49,52]. Although approximately 30% of Costa Rica remains forested and protected, rapid urbanization, extensive agriculture, excessive pesticide use, illegal traffic, and inappropriate waste managemen<sup>t</sup> negatively affect numerous amphibian populations. However, even in seemingly pristine locations, amphibian declines have occurred [93]. Additionally, climate change has been associated with the decline of several amphibian species in Costa Rica by affecting their reproduction and likely increasing susceptibility to pathogens [28]. Although it has not been found in Central America, we recommend screening for the recently emerged fungus *Batrachochytrium salamandrivorans* [94], which causes chytridiomycosis in salamanders. Conveniently, swabbing methods and qPCR allow for accurate detection of both fungi species in the same assay, which may facilitate rapid population assessments. For a fully detailed review of the environmental threats for amphibian communities in Costa Rica, we recommend the work of Sasa et al. [49].
