*3.1. Species Assessment*

The previous list of Costa Rican amphibians from 2011 included 196 species; however, we excluded the species *Pristimantis educatoris*[73] due to taxonomic uncertainty [74] for a total of 195 species. Our new list of amphibians in Costa Rica included a total of 215 species grouped in three orders, 16 families, and 48 genera (Table S3). This represented and addition of 20 species (10 anurans, 9 salamanders, and 1 caecilian; Table 3). The order Anura (frogs and toads) is the most diverse in Costa Rica, being 72% of the total species (13 families and 41 genera). Salamanders (order Caudata) are represented by only one family (Plethodontidae), three genera, and 53 species. Finally, caecilians (order Gymnophiona) are represent by two families, four genera, and eight species. A total of 63 species are endemic to Costa Rica (36 salamanders, 24 anurans, and 3 caecilians). We also included five anuran species that are not native to Costa Rica (*Eleutherodactylus coqui*, *E. johnstoni*, *E. planirostris*, *Lithobates catesbeianus*, and *Osteopilus septentrionalis*).


**Table 3.** List of new additions to the updated list of amphibians of Costa Rica. 


**Table 3.** *Cont.*

\**Eleutherodactylus planirostris* is an invasive species that have been found in the Caribbean Lowlands of Costa Rica.

Regionally (Figure 2a, Table S3), the Cordillera de Talamanca is the most diverse province in terms of species per area (88 species, 2.2 species/100 km2). It contains 23 species of amphibians that only occurred within this herpetological province (e.g., *Diasporus ventrimaculatus*, *Nototriton costaricense*). The Montane Slopes and Cordillera Central is the most diverse herpetological province (188 species, 1.3 species/100 km2), with 27 species that are exclusively found within this herpetological province (e.g., *Cochranella euknemos*, *Nototriton picadoi*). The Caribbean Lowlands (101 species, 0.7 species/100 km2) includes 20 species that are only found within this herpetological province (e.g., *Cruziohyla calcarifer*, *Caecilia volcani*). The Pacific Southwest (71 species, 0.9 species/100 km2), has five species that only occur within this province (e.g., *Craugastor taurus, Oophaga granulifera*). Finally, the Pacific Northwest (66 species, 0.5 species/100 km2) includes only two species that are found exclusively within this province (*Rhinophrynus dorsalis* and *Eleutherodactylus johnstonei*). A total of 20 species occur in all five herpetological provinces (e.g., *Craugastor fitzingeri*, *Diasporus diastema*, *Dendropsophus ebraccatus, Hyalinobatrachium fleischmanni*, *Lithobates warszewitschii*, *Rhinella horribilis*, *Smilisca sordida*).

Overall, 200 species have been classified into IUCN categories at the country level and 15 species need future assessment. A total of 155 species do not fulfill the criteria to be considered in the threatened categories, including 136 species listed as LC, 18 as DD, and one as NA (a category for taxa that occur in the region but have been excluded from the regional Red List for a specific reason). Within threatened categories, two species are listed as EX, 24 as CR, ten as EN, seven as VU, and two as NT. Regionally, lowlands exhibited the lowest percentage (0–2%) of DD species (Figure 2b). Similarly, about 75–80% of species occurring in lowlands are listed as LC (Figure 2c). In highlands, 6–10% of species are categorized as DD (Figure 2b) and 26% of species in Cordillera de Talamanca are classified within threatened categories (Figure 2d). According to EVS, a total of 81 species were classified as "no immediate risk," four species at "low vulnerability," 50 species at "medium vulnerability," and 48 species at "high vulnerability" (Table S3).

In our review, we found a total of 105 amphibian species (49%) that have been screened for *Bd* in Costa Rica (103 anurans and only 2 species of salamanders) (Table S4). In the field, the most common method used to detect *Bd* was qPCR, especially after 2005. Conventional PCR was used only in one study in the Caribbean Lowlands [64]. Histology and qPCR have also been used in retrospective studies on preserved specimens from declined and extinct species.

**Figure 2.** Map of Costa Rica showing (**a**) amphibian species richness for each herpetological province and percentage of amphibian species classified as (**b**) data deficient, (**c**) least concerned, and (**d**) threatened categories (near threatened, vulnerable, endangered, critically endangered, and extinct in the wild) for each herpetological province according to the Red List of Threatened Species from the International Union of Conservation of Nature (IUCN). Symbology: CL—Caribbean Lowlands, MSCC—Mountain Slopes and Cordillera Central, PN—Pacific Northwest, and PS—Pacific Southwest.

## *3.2. Endemic Dynamics*

Overall, *Bd* prevalence in Costa Rica was estimated to be 0.23 (60% of species tested positive for *Bd*) (Table S5). The most robust GLM found both herpetological province and the FRHI as significant predictors of *Bd* prevalence (AIC = 1700, *p* < 0.001, Table S6). Among herpetological provinces (Figure 3), the highest percentage of infected individuals was found in the Caribbean Lowlands (34%) and the lowest in the Pacific Northwest (4%). The Mountain Slopes, Cordillera Central, and Pacific Southwest had a similar percentage of infected individuals (≈23%). Furthermore, *Bd* was proportionally more prevalent in amphibians with terrestrial foraging and larval stage in phytotelma (ITF), pond-breeding treefrogs (IAP), and direct-developing species that breed in the forest (leaf-litter frogs DTT, rain frogs, DAT) (Figure 4a).

The species *Craugastor taurus* (the Golfito robber frog) was the species that had the highest average infection load (average *Bd* load of 11632.4 versus 571.6 genomic equivalents or 2.51 versus 1.18 after log transformation) (Table 4). We found an effect of the FRHI on infection load (F8342 = 7.91, *p* < 0.01, Table S6). Direct-developing frogs with terrestrial reproduction (robber frogs and leaf-litter frogs; DTR and DTT respectively) had the highest *Bd* loads (Figure 4b, Table 4).

**Figure 3.** Mean prevalence of infection with *Batrachochytrium dendrobatidis* (*Bd*) in amphibian assemblages at four herpetological provinces in Costa Rica (with 95% binomial CI). Means followed by a common letter are not significantly different according to the Tukey's honestly significant difference (HSD) test at the 5% level of significance. The plot does not display results for Cordillera de Talamanca because no sampling has been conducted for *Bd* in that province. Symbology: CL—Caribbean Lowlands, MSCC—Mountain Slopes and Cordillera Central, PN—Pacific Northwest, and PS—Pacific Southwest.

**Table 4.** Infection intensity in the 351 individuals where *Batrachochytrium dendrobatidis* (*Bd*) was quantified using qPCR in Costa Rica between 2000–2018. For every species, the table shows the foraging–reproduction habitat index (FRHI), the number of *Bd* positive swabs, the average (SE), and log10(SE) of genomic equivalents of *Bd* zoospores quantified per species estimated from the *Bd* + swabs. Symbology: First letter represents development: (I) indirect or (D) direct-developing amphibians. Second letter represents foraging habitat: terrestrial (T), arboreal (A), pond/puddle-dwellers (P), stream-breeders (R), and phytotelma (F). Third letter represents reproductive habitat: terrestrial (T), arboreal (A), pond/puddle-dwellers (P), stream-breeders (R), and phytotelma (F).


**Table 4.** *Cont.*


**Figure 4.** (**a**) Mean prevalence of infection with *Batrachochytrium dendrobatidis (Bd)* in amphibian assemblages (with 95% binomial CI) according to the foraging–reproduction habitat index (FRHI); (**b**) box plots with whiskers and notches that describe infection intensity for the 351 individuals where *Bd* was quantified using qPCR in Costa Rica between 2000–2018 according to the foraging–reproduction habitat index (FRHI). The box displays the inter-quantile range (25th–75th percentiles) with a center line representing the median (50th percentile). Notches show the median confidence region, and whiskers display the highest and lowest points. Means followed by a common letter are not significantly different according to the Tukey's honestly significant difference (HSD) test at the 5% level of significance. Symbology: First letter represents development: (I) indirect or (D) direct-developing amphibians. Second letter represents foraging habitat: terrestrial (T), arboreal (A), pond/puddle-dwellers (P), stream-breeders (R), and phytotelma (F). Third letter represents reproductive habitat: terrestrial (T), arboreal (A), pond/puddle-dwellers (P), stream-breeders (R), and phytotelma (F).
