Human disturbance and higher temperatures are linked to amphibian chytrid fungus in Catalonia, Northeastern Spain

Abstract

Chytridiomycosis, caused by Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), threatens amphibian biodiversity worldwide. While Bd is widespread in Europe, its local epidemiology remains poorly understood. We conducted a five-year survey (2019–2023) of 3,253 amphibians from 14 species across 41 sites in Catalonia, Northeastern Spain, to assess chytrid occurrence and environmental drivers. Using duplex qPCR, Bd was detected in 141 individuals (4.3%) from seven species, while Bsal was absent. Infections were geographically widespread but varied among species: Alytes obstetricans larvae showed the highest prevalence (26.6%), indicating a potential reservoir role, and Hyla meridionalis also exhibited high prevalence (14.3%) and infection loads, suggesting Bd tolerance. Other species, including urodeles and some alpine taxa, showed low or no infection. Although no overt disease was observed, chytridiomycosis-related mortality was suspected in A. obstetricans. Binomial GLMMs identified anthropogenic disturbance and maximum summer temperature as significant predictors of Bd occurrence. The apparent endemicity of Bd and absence of Bsal reinforce the need for continued surveillance, an improved understanding of host roles in disease dynamics, and integration of key environmental drivers to inform mitigation measures and preserve amphibian biodiversity.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-45967-3.

Introduction

Emerging infectious diseases are increasingly recognized as key threats to biodiversity conservation¹. Amphibian chytridiomycosis, caused by the fungal agents Batrachochytrium dendrobatidis (Bd) and B. salamandrivorans (Bsal), is responsible for the greatest loss of biodiversity associated with any pathogen ever recorded^2–4. Both pathogens have been directly linked to the decline of at least 501 amphibian species, with 90 species confirmed or presumed extinct⁵. Despite over 25 years of intensive research since the discovery of Bd in 1998², effective management strategies suitable for use in wild amphibian populations are still lacking. As a result, amphibians are currently the most threatened vertebrates on Earth, with over 41% of species at risk of extinction and ongoing population declines caused by these agents^6,7.

Bd and Bsal are obligate non-hyphal chytridiomycete fungi that infect the stratified keratinizing epidermis of amphibians, specifically the skin of post-metamorphic individuals and the mouthparts of tadpoles^4,8. Both pathogens have life cycles that include motile aquatic zoospores as a dispersal stage and exhibit strong temperature dependence, with optimal growth ranging from 17 to 25 °C for Bd and 10–15 °C for Bsal^4,8. Growth is possible outside these ranges at reduced rates, but mortality occurs at temperatures exceeding 28 °C for Bd and 25 °C for Bsal^4,9. Both pathogens exhibit an exceptionally broad host range – Bd alone can infect over 700 species¹⁰– yet responses to exposure and infection vary significantly across species, populations and life stages^11–13.

Infection dynamics and outcomes are shaped by a complex interplay of host, pathogen and environmental factors. Host species identity and life stage are key determinants¹⁴, with Bsal mainly infecting and causing disease in urodeles. In Bd, tadpoles typically exhibit asymptomatic, localized infections, while recently metamorphosed amphibians suffer the most severe effects². Among pathogen-related factors, virulence varies across Bd lineages and strains¹⁴, and infection intensity is a major factor influencing individual and population-level outcomes^15,16. Bioclimatic environmental variables, particularly temperature and humidity, are crucial mediators of chytrid fungi epidemiology by affecting pathogen survival and transmission rates¹⁴. These factors can also be influenced by latitude and altitude and shift seasonally, especially in temperate areas. Moreover, several studies suggest that anthropogenic disturbances play a significant role in shaping the occurrence of chytrid fungi by facilitating pathogen introduction and spread, as well as altering ecosystem functioning^17,18.

Central to the understanding of pathogen dynamics and population outcomes is the variation in host defenses among species, populations and life stages. Animal defenses against infection involve two distinct but complementary mechanisms: tolerance and resistance. Tolerance refers to the host’s ability to carry a given pathogen burden with limited detrimental effects on fitness/survival, whereas resistance is the host’s ability to prevent infection or limit infection intensity after exposure¹⁹. Within a single amphibian species, tolerant and resistant individuals may be crucial long-term survivors contributing to the persistence of amphibian populations. Conversely, pathogen-tolerant reservoir species or life stages can facilitate pathogen maintenance, posing challenges for effective disease mitigation¹³. Overall, the devastating impact of chytridiomycosis is driven by its introduction into naïve host populations, the environmental persistence of infectious zoospores, and the presence of such amphibian reservoirs that sustain a high force of infection for susceptible hosts²⁰.

In Europe, chytridiomycosis has led to significant mortality in multiple species, driving population declines and local extinctions. Bd had particularly severe impacts on Alytes obstetricans populations in Central Spain, Portugal, and the Western French Pyrenees^21–24. Similarly, Bsal introduction had catastrophic consequences for Salamandra salamandra populations in Central Europe⁴. Although Bd is now widespread across Europe²⁵, research in its landscape epidemiology and host-pathogen dynamics within multi-host amphibian communities remains limited. Catalonia (NE Spain) covers an area of 32,108 km² and boasts remarkable ecological diversity. Its landscapes range from alpine ecosystems in the Pyrenees, reaching elevations of 3,143 masl, to Mediterranean habitats spanning coastal ranges and lowlands. This diversity is also reflected in its amphibians, with 15 species adapted to various habitats and altitudes, including two endemic species (Calotriton asper and C. arnoldi). Despite its ecological significance, little research has focused on the dynamics of chytrid fungi in Catalonia’s amphibian populations (but see^26–28). A notable gap in research is the absence of studies on A. obstetricans in the Catalan Pyrenees, despite the severe mortality events and the potential role of tadpoles as reservoirs in other montane systems^23,29. Additionally, in 2018, an outbreak of Bsal chytridiomycosis was detected in Montnegre i el Corredor Natural Park, 1,000 km from its nearest known occurrence in northern Europe³⁰. However, no further investigations were conducted to assess the presence of Bsal in surrounding areas or across Catalonia following the outbreak. Overall, long-term and region-wide studies on multi-host communities remain scarce, limiting our understanding of chytridiomycosis epidemiology at a more local scale. Consequently, there is a pressing need to analyze the distribution patterns of chytrid fungi and evaluate the influence of environmental factors and host species on infection dynamics.

In this study, we assessed chytrid fungi occurrence in amphibians from Catalonia over five years to evaluate its epidemiology across a broad latitudinal, altitudinal and taxonomic range. Using duplex qPCR assay results, we analyzed variations in infection prevalence and intensity among amphibian hosts and modeled Bd prevalence to identify environmental and anthropogenic factors influencing pathogen occurrence. This knowledge may provide sustainable prevention and mitigation strategies for high-risk populations, a key research priority in amphibian conservation^31,32.

Materials and methods

Ethics statement

Animal capture, manipulation and sampling protocols were approved by Departament d’Acció Climàtica, Alimentació i Agenda Rural (Generalitat de Catalunya, Spain) under the license SF/0013/23. Additional permits were also obtained for developing scientific activities within protected areas: Zona Volcànica de la Garrotxa Natural Park (NP) (2023PNATZVGAUT005), Aiguamolls de l’Empordà NP (2023PNATAAEAUT025), Capçaleres del Ter i del Freser NP (FUE-2023-03010204), Cadí-Moixeró NP (9015-117182/2023) and Alt Pirineu NP (9015-117558/2023). All methods are reported following ARRIVE guidelines (https://arriveguidelines.org) and were performed in accordance with the relevant institutional, national and international guidelines and regulations.

Study area and survey protocol

From 2019 to 2023, we sampled amphibians at 41 sites from Catalonia, NE Spain (Fig. 1). Sampling sites were selected taking into account areas of high amphibian diversity and abundance, various bioregions and altitudes (0–2,360 masl), protected areas (i.e., NPs) and baseline data on chytrid occurrence²⁶. Catalonia is home to 15 amphibian species – 9 anurans and 6 urodeles – all of which are protected regionally³³. One urodele species, C. arnoldi, is classified as Critically Endangered by the IUCN and is currently subject to several conservation management strategies, including a captive breeding program^34,35. For this study, we sampled post-metamorphic amphibians from 14 species representing all seven extant families found in Catalonia: Salamandra salamandra, Pleurodeles waltl, Calotriton asper, Triturus marmoratus, Lissotriton helveticus, Alytes obstetricans, Discoglossus pictus, Pelodytes punctatus, Hyla meridionalis, Pelophylax perezi, Rana temporaria, Pelobates cultripes, Epidalea calamita and Bufo spinosus. Additionally, we opportunistically sampled A. obstetricans tadpoles. Due to the conservation status of C. arnoldi and the recent detection of Bsal in Montnegre i el Corredor NP³⁰, this species and certain areas were excluded from sampling in the present study. As most amphibians are inactive during winter in Catalonia, we performed surveys during the active reproductive period (spring-summer) to maximize capture probability. Fieldwork was always conducted under moderate conditions, never exceeding 25 °C, and falling within the suitable thermal range for Bd survival and transmission.

Fig. 1.

Map showing the distribution of Batrachochytrium dendrobatidis (Bd) infection in amphibians studied in Catalonia (NE Spain) from 2019 to 2023. Bd-positive (red dots) and Bd-negative (yellow dots) sampling sites, along with the main bioregions, are depicted.

Animal capture and sampling

Amphibians were located through calls and visual encounters after sunset and caught by hand or using dipnets in the case of aquatic species and tadpoles. After capture, each animal was contained in a clean plastic bag and handled with a fresh pair of nitrile gloves for sampling. We then obtained a skin swab (Snappable Ps + cotton, Deltalab^®) from each amphibian by firmly drawing it five times each along both sides of the ventral abdomen, both ventral hind limbs and drink patch, and the plantar surface of both hind feet, for a total of 30 strokes³⁶. In urodeles, the 30 strokes were divided between the ventral abdomen and the plantar surface of all four limbs. In tadpoles, their mouthparts were swabbed by inserting a fine-tip swab (Aluminium + cotton, Deltalab^®) into the mouth and twirling it several times. All animals were thoroughly examined for signs of disease before being released back at their capture site. Skin swabs were stored at -20 °C within the first 24 h after collection until DNA extraction.

Strict disinfection and biosecurity protocols were followed to minimize cross-contamination of samples and the spread of pathogens within and between field sites. Any equipment in contact with the animals was single-use (e.g., gloves, bags, swabs) and personal clothing and equipment were thoroughly disinfected with 1% Virkon^® S (Zotal Laboratorios, Sevilla, Spain) between sampling sites.

Chytrid fungi diagnostics

We extracted DNA from skin swabs using 65 µL of PrepMan™ Ultra Sample Preparation Reagent (Applied Biosystems, USA) following Hyatt et al. (2007). We conducted a duplex qPCR assay to simultaneously detect Bd and Bsal and quantify infection intensity in each animal sample³⁷. A synthetic double-stranded 750 bp gBlocks^® (Integrated DNA Technologies, USA) gene fragment containing the target PCR sequences of both pathogens was used as a positive control and quantitation standard. Standard dilutions in log10 increments (10^− 1 to 10⁶ copies; in duplicate) were included in each qPCR plate to create a standard curve, allowing us to quantify the genomic equivalents (GE) per qPCR reaction. Negative controls (nuclease-free water) were also included in each plate. A sample was considered positive if the gene copy number was above 1. All qPCR assays were run on an Applied Biosystems™ 7500 Real-Time PCR System (Carlsbad, CA, US).

Spatiotemporal and host patterns of Bd infection

We calculated Bd prevalence from the ratio of positive animals to the total number of samples within a species with 95% Wilson confidence intervals. Differences in Bd prevalence among positive species were assessed using Fisher’s exact tests and pairwise comparisons with Benjamini-Hochberg correction to control for the false discovery rate. Log-transformed Bd loads were compared between species using a Kruskal-Wallis test.

We also evaluated temporal variation in Bd prevalence using binomial generalized linear mixed-effects models (GLMMs) including sampling site as a random intercept to account for repeated measures. Year was initially evaluated as a fixed factor to test for interannual differences and as a continuous predictor to assess monotonic trends.

To assess broad-scale spatial structure in infection probability, elevation and bioregion were additionally evaluated in exploratory binomial GLMMs, including sampling site as a random intercept and year as a fixed control variable.

Modeling Bd infection risk across Catalonia

We used an information-theoretic approach to compare multiple models and identify the best predictors of Bd infection occurrence in post-metamorphic amphibians in Catalonia. Alytes obstetricans larval data were excluded from this analysis because sampling was opportunistic and constrained by detectability, as larvae could only be sampled at sites where they were directly observable and catchable. Our model framework, therefore, focuses on post-metamorphic individuals, for which sampling effort was comparable across sites.

In the statistical modeling, we used eight bioclimatic, geographic, and anthropogenic factors as predictors, with Bd counts (i.e., Bd-positive swabs) by species and sampling events as the response variable. We intentionally separated counts by species to account for the independent variability introduced by the amphibian species in the approach. Site-species combinations with fewer than five sampled individuals were also excluded, as these low-sample events produced unstable variance estimates in preliminary analyses. This resulted in a dataset of 156 observations with a total count of 2,731 individuals sampled. Explanatory variables included anthropogenic disturbance, amphibian species richness, bioregion, habitat type, altitude, mean temperature, summer mean maximum temperature, and precipitation (see Supplementary Information for a detailed description of each variable and data sources). The variable anthropogenic disturbance was a composite index from the first component of a PCA (Principal Component Analysis) that included the percentage of urban land use, the density of roads, distance to human populations and total nitrogen from livestock production³⁸(see Supplementary materials for more information, Supplementary Tables S1 and S2). Geospatial variables were extracted from raster layers in buffers of 5 km around sampling points using “raster”, “sp”, “sf” and “geosphere” packages in R^39,41–43.

We initially explored data to investigate records, distributions, imbalances and the relationship among the response variables and predictors⁴⁴. We standardized the continuous predictors using the “scale” function in R and log-transformed the variable “anthropogenic disturbance” to improve the assumption of data normality. To avoid collinearity between explanatory factors, we used the variance inflation factor (VIF) with a correlation cut-off coefficient ≥ 0.7. Accordingly, we excluded “mean temperature”, “bioregion” and “elevation” from subsequent analyses. In addition, inference at the bioregion level was limited by sparse detections and quasi-complete separation in several regions (see Supplementary Table S4); therefore, bioregion was not retained in the Bd infection multivariate models. We retained summer mean maximum temperature (hereafter referred as temperature) instead of mean temperature, as it more directly represents the thermal conditions that can influence Bd infection dynamics during the warmest period of the year. Moreover, this variable reduced collinearity with other climatic predictors, improving model stability and parameter estimation.

Because our dataset consisted of aggregated observations with repeated measures across sampling points and years, we fitted binomial GLMMs using the glmmTMB package⁴⁵, including random intercepts for sampling point and year to account for spatial and temporal non-independence among observations. Given the low overall prevalence of infection and the high number of zeros, we evaluated whether a zero-inflated model structure was required by comparing standard and zero-inflated binomial GLMMs via likelihood-ratio tests. Although the zero-inflated models showed an improvement in likelihood, diagnostic tests based on DHARMa residual simulations⁴⁶ indicated no evidence of overdispersion or unaccounted zero inflation. Because the random intercepts already captured the zero-heavy structure, and given that Bd has been reported throughout Catalonia^26,27,47,48, we considered the excess zeros to be non-structural and therefore retained the simpler binomial GLMM.

Among the set of model candidates, we identified the most parsimonious models using the corrected Akaike Information Criterion for small sample sizes (AICc). We applied an information-theoretic multimodel inference approach using the MuMIn package⁴⁹. Models with ΔAICc < 2 relative to the best-fitting model were considered to have substantial support⁵⁰, and we obtained model-averaged parameter estimates using full averaging to account for model-selection uncertainty. We validated the top models using the DHARMa package, which employs simulation-based scaled residuals to assess model fit. Specifically, we tested for overdispersion, zero inflation, residual uniformity, and outliers to ensure appropriate model performance (Supplementary Figures S1 and S2). All statistical analyses were conducted in R version 4.1.4⁵¹, and statistical significance was assessed at p ≤ 0.05.

Results

Patterns of chytrid fungi occurrence

Over the study period, we collected skin swabs from 3,253 wild amphibians from 14 species across Catalonia (Fig. 1; Table 1). Infection with Bd was detected in 141 individuals from seven different species, including 55 A. obstetricans larvae, with an overall prevalence of 4.33 (CI _95% 3.69–5.09). Infection with Bd was detected at 12 of the 41 study sites, indicating a substantial geographical distribution across Catalonia. Bsal was not detected at any site throughout the study period.

Table 1.

Infection by Batrachochytrium dendrobatidis (Bd) across amphibian species sampled in Catalonia from 2019 to 2023. The table presents the species, order, number of Bd positive sites by total sites where the species was sampled, sample size, number of Bd-positive individuals, prevalence (%), 95% confidence interval (CI), median genomic equivalent (GE), and maximum GE detected. Different letters indicate significant statistical differences in prevalence between positive species.

Species	Order	Sites Bd positive /sampled	Sample size	Bd positive	Prevalence (%)	95% CI	Median GE	Max. GE
Alytes obstetricans larvae	Anura	2/5	207	55	26.57 a	21.02–32.97	55	12,580
Hyla meridionalis	Anura	6/10	265	38	14.34 b	10.63–19.07	188	63,000
Pelophylax perezi	Anura	7/23	589	40	6.79 c	5.03–9.12	22	5,880
Discoglossus pictus	Anura	1/4	39	1	2.56 bcd	0.13–13.18	6	6
Epidalea calamita	Anura	1/9	59	1	1.69 cd	0.09–8.99	1	1
Alytes obstetricans	Anura	2/18	247	3	1.21 d	0.41–3.51	812	70,383
Triturus marmoratus	Urodela	1/6	90	1	1.11 cd	0.06–6.03	88	88
Calotriton asper	Urodela	1/11	715	2	0.28 d	0.08–1.01	6	12
Lissotriton helveticus	Urodela	0/3	28	0	0	0.00–12.06	0	0
Pleurodeles waltl	Urodela	0/5	78	0	0	0.00 -4.69	0	0
Salamandra salamandra	Urodela	0/9	123	0	0	0.00 -3.03	0	0
Bufo spinosus	Anura	0/18	87	0	0	0.00 -4.23	0	0
Pelobates cultripes	Anura	0/1	3	0	0	0.00 -56.15	0	0
Pelodytes punctatus	Anura	0/3	10	0	0	0.00 -27.75	0	0
Rana temporaria	Anura	0/15	713	0	0	0.00 -0.54	0	0
Total			3,253	141	4.33	3.69–5.09	52	70,383

Unadjusted Bd prevalence was low across the study period (1.2–6.2% annually), with a modest peak in 2020. Temporal variation was evaluated using binomial GLMMs including sampling site as a random intercept. Including Year as a categorical predictor significantly improved model fit relative to a null model (LRT p < 0.001), indicating interannual variation in detection probability. However, no single year contrast was strongly supported, and models treating Year as a continuous predictor did not support a monotonic temporal trend. Thus, although prevalence slightly fluctuated among years, no consistent directional increase was detected.

Descriptively, Bd prevalence decreased across elevational bands, from 8.05% below 500 masl to ≤ 0.5% above 1500 masl, with no infections detected above 2000 masl. In exploratory year-controlled GLMMs, adding elevation significantly improved model fit relative to the year-only model (LRT p = 0.0018). Elevation was negatively associated with Bd detection probability (β = − 1.11 ± 0.36 per 500 m, z = − 3.05, p = 0.002), corresponding to an approximately 67% decrease in the odds of detection per 500 m increase in elevation (Supplementary Table S4).

Among the seven Bd-positive amphibian species, A. obstetricans larvae showed the highest Bd infection prevalence with 26.57%, followed by H. meridionalis with 14.34%, P. perezi with 6.79%, D. pictus with 2.56%, E. calamita with 1.69%, A. obstetricans with 1.21%, T. marmoratus with 1.11% and C. asper with 0.28% (Table 1). Among the positive species, significant differences were observed between A. obstetricans larvae and all other species, including A. obstetricans adults. Hyla meridionalis prevalence also had significant differences with all species except for D. pictus, and P. perezi had significant differences with C.asper and A. obstetricans adults (Supplementary Table S3). Infection loads in Bd-positive animals ranged from 1 to 70,383 GE (median = 52 GE) (Fig. 2; Table 1). Although some individuals exhibited high infection loads, most post-metamorphic amphibians sampled appeared healthy and showed no signs of disease. Depigmentation of the mouthparts was observed in 17 of the 51 Bd-positive A. obstetricans larvae, despite appearing otherwise healthy. The highest Bd infection load was detected in a deceased A. obstetricans metamorph exhibiting signs of dysecdysis. Moreover, another Bd-positive individual was also found dead, and the only three positive A. obstetricans detected were metamorphs with high infection loads (Fig. 2). No significant differences were detected in infection loads between Bd-positive species.

Fig. 2.

Batrachochytrium dendrobatidis (Bd) log-transformed infection loads across amphibian species sampled in Catalonia from 2019 to 2023. Individual data points are overlaid on boxplots (for species with > 3 observations) to show the distribution of Bd loads. Black points represent live individuals, while red points highlight Bd-positive individuals found dead during sampling.

Modeling Bd infection risk across Catalonia

Among the evaluated models, four competing binomial GLMMs fell within ΔAICc < 2 of the best model (AICc = 204.9; Supplementary Table S5). The top-ranked model explaining Bd occurrence across Catalonia included temperature (β = 1.20, SE = 0.59, z = 2.04, p = 0.042) and anthropogenic disturbance (β = 1.22, SE = 0.50, z = 2.44, p = 0.015) as significant predictors (AIC = 204.5, BIC = 219.7, logLik = − 97.2, deviance = 194.5, df.resid = 151). Other competitive models (ΔAICc < 2) included combinations of temperature, anthropogenic disturbance, species richness and precipitation, but in all cases, the effects of anthropogenic disturbance and temperature remained consistently positive and among the strongest predictors (Supplementary Table S5; Σw = 1.00 for anthropogenic disturbance, Σw = 0.82 for temperature, Σw = 0.27 for species richness and Σw = 0.17 for precipitation). In the best‐ranked model, random intercepts accounted for substantial spatial (sampling point: SD = 1.36, Var = 1.84) and moderate temporal (year: SD = 0.82, Var = 0.68) variability, indicating that spatial heterogeneity exceeded temporal variability in Bd infection probability.

The direction and magnitude of the averaged estimates were consistent with those of the best model, further supporting a positive association between Bd occurrence, anthropogenic disturbance, and temperature across sites and years (Fig. 3 and Supplementary Table S6). Among the top models (ΔAICc < 2), fixed effects explained on average about 60% of the variance in infection probability (AIC-weighted mean marginal R² = 0.60, ranging from 0.40 to 0.67). Exponentiated coefficients of the average model showed that a one-unit increase in anthropogenic disturbance was associated with a 3.68-fold increase in the odds of Bd infection (OR = 3.68, 95% CI = 1.14–11.89), whereas a one-unit increase in temperature was associated with a 2.93-fold increase (OR = 2.93, 95% CI = 0.66–12.97). Model-averaged predictions showed a positive relationship with observed infection probabilities, indicating that the averaged model adequately captured variation in Bd prevalence (Supplementary Figure S2). Detailed values of the explanatory variables at each sampling point are summarized in Supplementary Tables S7 and S8.

Fig. 3.

Model-averaged parameter estimates (± 95% confidence intervals) from the binomial GLMM explaining infection probability across sites and years. Estimates are shown on the logit scale, with the dashed line indicating no effect (0). Anthropogenic disturbance and maximum temperature showed positive effects, while precipitation and species richness were weakly supported.

Discussion

Our study provides valuable insights into the occurrence and epidemiology of chytrid fungi in amphibian populations across Catalonia, NE Spain. The findings reveal that Bd is broadly distributed across Catalonia, with prevalence differing significantly among species. These differences suggest varying levels of resistance and tolerance across species and life stages, providing an initial step toward identifying potential infection reservoirs. Additionally, our statistical modeling identifies anthropogenic disturbances and temperature as key predictors of Bd occurrence, reinforcing the growing concern that climate change and human activities have a role in shaping disease dynamics in amphibian communities.

Bd distribution and endemicity in Catalonia

The detection of Bd at 12 of the 41 surveyed sites indicates that the pathogen is well established in Catalonia. Bd was detected across a wide elevational gradient and multiple habitat types. Although exploratory mixed-effects models indicated a negative association between elevation and infection probability, Bd presence was nevertheless recorded from lowland areas up to > 1,500 masl, supporting its broad geographical distribution. Despite this wide spatial extent, Bd prevalence remained relatively low throughout the study period, and mixed-effects models did not detect consistent temporal shifts after accounting for repeated sampling at sites, suggesting a pattern more compatible with endemic persistence than with a recent epidemic expansion. These findings align with previous studies in Spain and other European regions, where Bd is widespread but exhibits spatial and temporal variability in infection dynamics⁵². In Europe, large-scale epidemics and mass mortalities have been restricted to montane areas of the Iberian Peninsula and the French Pyrenees^21,22,24.

Notably, Bd infection was not detected in alpine sites and was detected only rarely in subalpine areas during the study period. This contrasts with the findings of Martínez-Silvestre et al. (2020), who reported Bd-positive populations at multiple high-elevation sites in the Pyrenees, including infections in C. asper at one site that was also surveyed in the present study. Pathogen fade-out may occur when the local density of susceptible hosts is too low to sustain transmission or when environmental conditions reduce the survival of free-living pathogen stages⁵³. However, the latter is unlikely in Pyrenean areas, as Bd is known to survive and grow at temperatures below 4 °C⁵⁴. A more plausible explanation is that the host community has contributed to Bd’s disappearance at this site, likely due to the absence of pathogen-tolerant reservoirs. The current amphibian assemblage at this site is largely composed of C. asper and R. temporaria, with only a single A. obstetricans adult and no larvae ever detected during our surveys. These two species, thus, appear unable to support long-term pathogen persistence (see additional discussion on species patterns below). Additionally, we cannot rule out the possibility that the initial introduction of Bd triggered a chytridiomycosis outbreak, leading to the local decline or near-extirpation of A. obstetricans, as documented in other montane areas^21,24. The loss of the most susceptible host may have favored more resistant species, ultimately leading to pathogen fade-out.

On the other hand, our results confirmed the presence of Bd at sites where the pathogen had been previously detected^26,48. The persistence of Bd likely involves the presence of competent, pathogen-tolerant reservoirs and environmental conditions conducive to its survival. However, historical data were unavailable for most of our study sites (38 out of 41), and we documented Bd infection at 10 locations for the first time. Aside from the two deceased metamorphs of A. obstetricans, for which chytridiomycosis is a likely cause of mortality, no overt outbreaks have been reported before or during our study period in Catalonia. Instead, the low prevalence observed appears more compatible with sporadic and cryptic mortality without population declines, indicating a state of host-pathogen coexistence similar to that described in other European regions⁵⁵. Longitudinal studies examining seasonal and multi-year infection dynamics will be essential to clarify the long-term impacts of Bd on amphibian populations in Catalonia. In the following sections, we further discuss factors associated with host species, environmental factors and anthropogenic influences to gain a deeper understanding of Bd infection dynamics in Catalonia.

The absence of Bsal in our study area is encouraging, as this pathogen has been responsible for severe declines in S. salamandra populations in other parts of Europe⁴. Although we were unable to sample the index site where Bsal was detected in Catalonia³⁰, and cannot confirm its complete eradication, our results suggest that Bsal has not further spread into amphibian populations across Catalonia. Nevertheless, continued surveillance remains crucial due to its potential for introduction and spread.

Host-specific patterns of Bd infection

Our results reveal substantial interspecific variation in Bd prevalence and infection characteristics, emphasizing the importance of host species in disease dynamics. Alytes obstetricans larvae exhibited the highest prevalence, significantly differing from the rest of the studied species. Although not statistically significant, Bd loads in larvae were also relatively high despite the absence of generalized clinical disease. These findings suggest that A. obstetricans larvae exhibit a degree of tolerance to Bd infection, aligning with previous research identifying them as key reservoirs for Bd persistence²³. Alytes obstetricans larvae can undergo prolonged developmental periods and may overwinter for at least one year. These long-lived tadpoles with high infection prevalence can act as a continuous reservoir, sustaining a Bd infection source year-round – similar to patterns observed in other Bd systems^56,57. Moreover, this species is distributed throughout Catalonia, found across a broad range of habitats from sea level to over 2,500 masl, further enhancing its potential as a reservoir.

Conversely, we rarely detected Bd infections in postmetamorphic A. obstetricans, with only three metamorphs testing positive – two of which were found dead and had high Bd loads. Given the high prevalence detected in larvae, these findings suggest that Bd infections are cleared during metamorphosis or lead to mortality at this stage. The high susceptibility of A. obstetricans to chytridiomycosis is well-documented in the literature^21,22,24, with newly metamorphosed individuals recognized as the most vulnerable stage in susceptible species². Once metamorphosed, adults become fully terrestrial, and opportunities for Bd exposure are probably limited to mating and when males approach water during egg hatching. These life history traits may also explain the low prevalence observed in adults. While we did not observe large mortality outbreaks, our findings support previous literature indicating that this species is highly susceptible and may succumb to Bd^21,22,24.

We also documented significantly higher Bd prevalence and substantial infection intensities in H. meridionalis despite the absence of clinical disease. Such high prevalence was unexpected, given that aquatic species are generally more exposed to Bd infectious stages^5,58, while H. meridionalis has an arboreal lifestyle. This suggests that, even if exposure is rare, it tends to result in successful infections, indicating relatively low species resistance to Bd. These findings offer a novel contribution to the literature and identify this species as a potentially Bd-tolerant host that could play a significant role in the maintenance of Bd.

Although not statistically significant, P. perezi had the third-highest prevalence while showing lower Bd loads than A. obstetricans larvae or H. meridionalis. Considering its aquatic lifestyle, exposure to Bd zoospores is likely recurrent, which may indicate that this species has a comparatively higher resistance to infection. The potential role of Pelophylax species as Bd reservoirs has already been proposed in other European countries⁵⁹. While our results do not fully support a similar role for P. perezi in Catalonia, its widespread distribution – only absent in alpine areas – renders it a relevant species for future Bd research and surveillance efforts.

The other amphibian species studied exhibited very low prevalence or absence of Bd infection. This could indicate that they are either resistant to infection or highly susceptible to disease, meaning that infected individuals are rarely, if ever, found alive. Individual mortality has been reported in several of these species, including Bufo spp., S. salamandra, D. pictus, R. temporaria and P. perezi^23,27,48,60. Since Bd outcomes largely depend on infection intensity¹⁴, virtually any species may experience disease and mortality if pathogen load exceeds a critical threshold. However, such events are likely rare, as no mortality outbreaks have ever been reported for these European species. Additionally, previous research has documented asymptomatic infections in E. calamita and D. pictus in Catalonia⁶¹. Although some authors suggest that D. pictus could act as a Bd supershedder²⁷, high loads were observed only in dead individuals, indicating that infection is generally associated with mortality rather than tolerance. Together, the low occurrence of Bd infections in these species supports the hypothesis that they are relatively resistant hosts and unlikely to serve as effective reservoirs. Consistent with our findings, urodeles appear to exhibit greater resistance to Bd infection and disease across multiple studies^5,62. Nevertheless, the small sample size for some species may obscure true patterns and differences, warranting caution when interpreting our results.

Environmental and anthropogenic drivers of Bd occurrence

Our statistical modeling identified two significant predictors of Bd presence: anthropogenic disturbance and temperature. Temperature is widely recognized as the most important environmental factor influencing Bd occurrence in the wild^63–65. Laboratory studies indicate that Bd thrives in moderate temperatures and is generally intolerant of temperatures exceeding 28–30 °C, leading to the expectation that higher temperatures would reduce Bd prevalence⁵⁴. However, our results show a positive correlation between Bd prevalence and maximum temperatures, suggesting that factors beyond simple thermal limits in vitro influence disease dynamics^66,67. One potential explanation is that macroclimatic temperature measurements do not accurately capture the thermal conditions of amphibian microhabitats, where Bd may persist despite high air temperatures⁶⁸. Many amphibians exhibit behavioral thermoregulation by seeking cooler, moist refuges – such as leaf litter, shaded streams, or burrows – that maintain favorable conditions for Bd, even during hot periods when air temperatures peak^69,70. Another possibility is that increased temperature variability, rather than absolute temperature, influences infection patterns by inducing physiological stress in amphibians, weakening immune defenses and facilitating Bd infection^71,72. Moreover, temperature may indirectly influence Bd dynamics through host community composition. Species adapted to warmer, lowland and more Mediterranean environments appear to play a disproportionate role in Bd maintenance in our study area. Consequently, higher temperatures may favor host assemblages that sustain Bd transmission, even if Bd itself does not benefit directly from elevated temperatures. These findings highlight the need for further research into microclimate conditions and host behavior to elucidate the complex relationships between temperature and chytrid dynamics in natural populations. Overall, our results suggest that macroclimatic temperature may act as a proxy for broader environmental and community-level processes, rather than directly reflecting Bd thermal limits. While our observational data do not allow for direct causal inference, our analysis supports the hypothesis that climate change could facilitate Bd persistence and transmission, particularly in temperate regions^65,73.

Anthropogenic disturbance emerged as another key driver of Bd occurrence, supporting previous research on the role of human activities in the pathogen’s epidemiology^17,74,76,77. Our model incorporated an index reflecting various forms of human environmental alteration, with variation representing a gradient from relatively wild and inaccessible areas to densely populated or agriculturally dominated areas. However, we could not determine the relative contribution of specific human activities to Bd prevalence, as many of the variables used in our anthropogenic disturbance index are correlated. Human-assisted movements of infected amphibians, contaminated water, sediments or objects are known to drive Bd distribution, introducing or reintroducing the pathogen into previously uninfected areas^78–81. Additionally, human activities degrade natural ecosystems, reducing habitat quality for amphibians and increasing other stressors that may weaken host immunity and promote Bd infection and persistence^14,82,83. Given the ongoing expansion of human activities in Catalonia, conservation strategies should incorporate measures to mitigate anthropogenic impacts on amphibian populations.

While species richness is often thought to play a crucial role in regulating disease dynamics, our modeling approach shows that it does not significantly influence Bd epidemiology in Catalonia. Several authors have explored the concept of a dilution effect, where higher host diversity reduces disease risk, in the amphibian-Bd system. This effect is thought to occur because more diverse communities may limit the transmission of generalist pathogens by increasing the likelihood of encounters between susceptible and resistant hosts⁸⁴. Laboratory studies generally support the occurrence of the dilution effect in Bd systems^85,86, but field studies have produced mixed results, likely due to the variation in host composition, habitat complexity, and species-specific interactions^86,87. Our findings add to the growing body of evidence suggesting that the presence and identity of specific species, rather than overall species richness, play a more decisive role in shaping disease outcomes in complex natural systems. Thus, the presence of a particular species in a community can either suppress or amplify pathogen transmission depending on its susceptibility and ecological interactions^85,87. Understanding community-wide transmission dynamics and species-specific contributions is therefore essential for predicting disease outcomes in multi-host systems like amphibian chytridiomycosis.

Conclusion

Our study highlights the intricate relationships between host species, environmental factors, and anthropogenic disturbances in shaping Bd epidemiology in Catalonia. The identification of A. obstetricans larvae and H. meridionalis as key hosts suggests that targeted surveillance of these species could enhance monitoring efforts. Furthermore, understanding species-specific susceptibility and the potential reservoir role of certain hosts remains crucial for mitigating disease impacts. We acknowledge some limitations of our study, including heterogeneous sampling across sites and species, lack of seasonal coverage, and concentration of positive detections in a few locations. Nevertheless, the large overall sample size, consistent effort, and the fact that only a subset of species were repeatedly positive across multi-host communities support the robustness of our observed patterns. While infection dynamics vary across species, landscapes, and seasons, our findings indicate that factors influenced by climate change and anthropogenic disturbances contribute to Bd introduction and persistence, highlighting the need for integrated conservation strategies. Future studies incorporating seasonal sampling and experimental infections could further clarify species-specific roles and drivers of Bd-dynamics. As human activities and climate shifts continue to reshape ecosystems, proactive management, long-term monitoring, and preventative measures, particularly against the potential spread of Bsal, will be essential to safeguard amphibian biodiversity.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

MPR: conceptualization, methodology, validation, investigation, resources, writing–original draft; XFA: investigation, formal analysis, data curation, visualization, writing–review and editing; JE: methodology, investigation, writing–review and editing; LC: investigation, writing–review and editing; LP: formal analysis, data curation, writing–review and editing; IM: validation, investigation, supervision, writing–review and editing; OC: conceptualization, validation, investigation, resources, supervision, writing–review and editing. All authors have read and agreed to the published version of the manuscript.

Funding

MPR was funded by the 2021 FI Scholarship, Departament de Recerca i Universitats, Generalitat de Catalunya, Spain (FI_B 00171). XFA acknowledges support under a Ramón y Cajal contract (RYC2022‑036927‑I), funded by the Agencia Estatal de Investigación (Spanish Ministry of Science, Innovation and Universities). This work has been supported by Barcelona Zoo Foundation (2019 and 2020 scholarships) and Alt Pirineu Natural Park (Salvador Grau i Tort 2022 scholarship).

Data availability

All data generated or analyzed during this study are included in this published article and its Supplementary Information files.

Declarations

Competing interests

The authors declare no competing interests.