Characterization of culturable microbiota associated with the skin of amphibians (Anura) in the southern Andes Mountains of Ecuador

Characterization of culturable microbiota associated with the skin of amphibians (Anura) in the southern Andes Mountains of Ecuador | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Characterization of culturable microbiota associated with the skin of amphibians (Anura) in the southern Andes Mountains of Ecuador Jazmin M. Salazar, Juan Carlos González Rojas, Romel Riofrío, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5921108/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 22 May, 2025 Read the published version in Microbial Ecology → Version 1 posted 10 You are reading this latest preprint version Abstract Ecuador is recognized for having a high diversity of anuran spp., which are distributed mainly south of the Andes mountains. However, due to its geographic location and accessibility, there are few studies related to these amphibians. The objective of this study was to explore the bacterial and fungal biodiversity present on the skin of wild anuran spp. in the locations of Zamora Chinchipe, Loja, Cañar Azuay, and Morona Santiago through MALDI-TOF mass spectrometry. This analysis revealed the presence of 29 bacterial taxa and 9 fungal taxa, consisting mainly of: Pseudomonas chlororaphis (28%), Acinetobacter iwoffii (14%), Pseudomonas fluorescens (14%), and Hortaea werneckii (26.4%), Fusarium solani (20.5%), S yncephalastrum spp. (20.5%), respectively. Diversity varied across the five sampling locations, with geographic location proving to be a significant driver of diversity. Some of the most abundant bacterial and fungal genera have important associations with skin diseases. This work represents the first glimpse into the complex biodiversity of bacteria and fungi inhabiting this understudied substrate, and further studies will be needed to better understand bacterial and fungal biodiversity at these locations, along with the development of necessary animal protection and conservation measures. Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Figure 14 Figure 15 Introduction Ecuador is one of the countries with the greatest diversity of amphibians, with approximately 653 described species [ 1 , 2 ], of which 13% are critically endangered, 23% are endangered, and 20% are vulnerable [ 3 ]. Most of these species inhabit aquatic and terrestrial environments, which determine their physiological processes [ 4 ], making them susceptible to environmental changes that alter their habitat. It is worth noting that 40% of species is in decline in recent years [ 5 ]. The increase in anthropogenic activities such as ecosystem pollution [ 6 , 7 ] and the expansion of the agricultural frontier [ 8 ] have had observable effects on amphibian populations, which are severely threatened and in decline [ 9 , 10 , 11 ]. The microbiota, which represents the set of microorganisms that inhabit both the surface and the interior of organisms, can modulate host health by affecting their development, behavior, metabolism, and inflammatory responses [ 11 ]. In amphibians, bacterial communities present on the skin could offer protection against infection by synthesizing antifungal metabolites, acting as an integral part of the animal's immune system [ 12 ]. Some bacteria on the skin of amphibians are capable of inhibiting the growth of pathogens in vitro [ 13 , 14 ], and supplementing amphibian microbiomes with inhibitory bacteria can increase survival in laboratory assays [ 15 , 16 ]. Furthermore, the composition of bacterial communities in frogs and the persistence of the host population are often correlated [ 17 , 18 ]. In addition to the knowledge about bacterial microbiomes, there are few studies on fungal microbiomes [ 19 ]. Relatively few studies have examined the fungal microbiomes of vertebrate wildlife [ 20 – 23 ]. Furthermore, while these studies provide valuable starting points, they have often had limitations as they were carried out in captivity [ 21 , 24 ], which disrupts the microbiomes [ 25 , 26 ]. Previous studies have described that fungi inhabiting the skin of some amphibians are capable of producing antimicrobial compounds such as penicillin [ 27 ], although little is known about their effects on amphibian health and how they interact with host immune defenses. It should be mentioned that few studies have been conducted on cutaneous fungal communities in amphibians and a growing literature has been observed, highlighting the potential of bacteria for probiotic applications [ 28 , 29 ]. Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied in the field of microbiology for a many years. With the development of new technologies and method optimization, new rapid and accurate approaches have been developed to improve the accuracy of targeted identification. MALDI-TOF spectroscopy is not limited to identifying strains grown on solid media, or in vitro, but can also directly identify them from blood culture samples, cerebrospinal fluid, urine, and skin samples [ 30 – 34 ]. Identification by MALDI-TOF spectroscopy has been used to identify Gram-negative and Gram-positive bacteria, aerobes, anaerobes, mycobacteria, nocardia, yeasts, filamentous fungi, and viruses [ 35 – 42 ]. MALDI-TOF MS is a reliable, simple, and readily available technology [ 43 , 44 ]. There is currently a need to understand the diversity of the cultivable microbial communities that contribute to host resistance to disease. The objective of this study was to characterize the composition of the bacterial and fungal skin microbiota in anuran spp. using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and to establish the biodiversity in this environment. Materials and Methods Ethics Statement This study was conducted in strict accordance with the guidelines for the use of live amphibians and reptiles in field research developed by the American Society of Ichthyologists and Herpetologists, the League of Herpetologists, and the Society for the Study of Amphibians and Reptiles. Specific collecting permits for this study were obtained under authorization from the Ministry of Environment, Water, and Ecological Transition of Ecuador (MAATE), number MAAE-ARSFC-2021-1564. The samples did not include endangered or protected animal or plant species. Sampling Locations The study was conducted in eleven locations located from south to north in the foothills of the Andes Mountains during the months of September and November 2021. These locations include: (A) Zamora-Chinchipe Province; (1) the "provincial boundary" sector located in the southeastern part and classified as montane cloud forest, with species from the Areaceae, Poaceae, and Orchidaceae families; (2) the "Padmi sector," characterized as a lowland evergreen forest, located in the central-eastern part. It has shrub species such as Sapium and Grias peruviana , secondary forests with Dictyoloma peruviana , and also includes sections of tropical rainforest and premontane forest; (3) the "Piuntza sector," which exhibits premontane forest vegetation characterized by high tree and shrub diversity. Volcanic soil conditions, high humidity, and moderate altitude favor endemic communities and ecological transitions between tropical forests and montane ecosystems. (B) Loja Province; (1) “Cerro Pucará Park”, adjacent to the northwestern part of Podocarpus National Park. Its reference ecosystem is described as a montane cloud forest, located between 1,500 and 2,900 m a.s.l.; (2) “Abra del Zamora sector”, located on the northern periphery, between Podocarpus National Park and Pucará Park, classified as a montane cloud forest ecosystem. (C) Cañar Province; (1) “Laguna de Guabizhum sector”, located in the Soldados parish, belonging to the Déleg canton, has a montane cloud forest, with the presence of Cyperaceae species such as Barnadesia parviflora , Juglans neotropica , and Myrcianthes spp. (D) Azuay Province; (1) “Guangarcucho sector”, with vegetation formation characterized by the appearance of humid montane scrub, includes relatively humid valleys between 2,000 and 3,000 m.a.s.l. in the inter-Andean alley, where native vegetation has been devastated and replaced by agricultural crops and forests of Eucalyptus globulus , Salix humboldtiana and Acacia farnesiana ; (2) “Lazareto sector”, located in the urban area, which includes the banks of the Milchichig River, here the introduced forests host species of the genera Junglas neotropica , Baccharis latifolia and Spartium junceum ; (3) “Chanlud sector”, classified as a shrubby páramo, composed mainly of grasslands and shrubs, in an altitudinal range of 2600 to 3600 m a.s.l., characterized by the presence of the genus Calamagrostis and shrub species of the genera Baccharis, Gynoxys, and Brachyotum; (4) “Quimsacocha sector”, with an altitude of 3100 m a.s.l., is characterized by having characteristics of a shrubby páramo with a significant presence of Cortaderia nitida . (E) Morona Santiago Province; (1) “Wapú sector”, has an ecosystem classified as an evergreen piedmont forest with an altitude of 800 and 1300 m a.s.l. It has a tree composition native to the area where the canopy can reach 30 meters in height (Fig. 1 ). Sample Collection Sampling was carried out on amphibian spp. observed at the sampling sites, in water bodies, and in the surrounding vegetation. Specimens were located using the "visual encounter" method and subsequently morphologically characterized according to [ 45 , 46 ]. Specimens were handled using nitrile gloves, and a skin swab was subsequently performed in situ by skin rubbing according to [ 47 ] (Fig. 2 ). Two skin swabs were taken from each specimen. One swab was subsequently stored in a Falcon™ tube containing 2 ml of Brain Heart culture medium (Merck®) and the second with TGhL medium with Tryptone (SIGMA®), Hydrolyzed Gelatin (TM MEDIA®), and Lactose (SIGMA®). The samples were kept in a cooler with dry ice for 24 to 48 hours and transferred to the Microbial Ecology and Active Ingredients Laboratory of the Center for Research, Innovation and Technology Transfer (CIITT) of the Catholic University of Cuenca for processing. Isolation, Culture Conditions, and Activation Bacterial Isolation and Culture Bacterial cultures were prepared using a solid medium (95.5% blood agar) dispensed into 9 cm diameter Petri dishes. The medium was sterilized via autoclave (121°C, 15 psi, 15 minutes) and allowed to cool under a laminar flow cabinet. Samples, previously stored at 4°C, were aseptically inoculated onto the agar surface using a sterile inoculation loop, followed by streaking to isolate individual colonies. Plates were incubated at 37°C for 16 hours under anaerobic conditions. After incubation, colony growth was assessed, and a representative colony from each sample was selected for downstream analysis. Fungal Isolation and Culture Fungal cultures were prepared with 2% potato dextrose agar (PDA), sterilized via autoclave (121°C, 15 psi, 15 minutes), and poured into 9 cm Petri dishes. Mycelial plugs (5 mm diameter) were excised from stock cultures using a sterile punch and transferred to the PDA plates. Inoculated plates were incubated at 25–28°C under a 12/12 h light-dark photoperiod for 5 days. Mycelial growth was monitored daily, with expansion patterns and morphological features recorded to confirm viability and purity. MALDI-TOF MS Bacteria All samples were analyzed using an Axima™ Confidence MALDI-TOF MS spectrometer (Shimadzu-Biotech Corp., Kyoto, Japan) in positive linear mode (m/z = 2000–20,000). A small number of colonies from each pure culture were transferred to a FlexiMass™ destination well using a disposable loop, covered with 0.5 µl of 2,5-dihydroxybenzoic acid (DHB; 10 mg/ml in acetonitrile/0.1% trifluoroacetic acid 1:1) matrix solution, and air-dehydrated for 1–2 min at 24–27°C. The reference strain Escherichia coli K12 (genotype GM48) was used as a calibration standard and as a reference for quality control. Sample information such as medium and culture conditions were imported into Shimadzu Biotech Launchpad™ software, v.2.8 (Shimadzu-Biotech Corp., Kyoto, Japan). Protein mass profiles were obtained with linear positive mode detection at a laser frequency of 50 Hz and within a mass range of 2000–20,000 Da. The acceleration voltage was 20 kV and the extraction delay time was 200 ns. A minimum of 20 laser shots per sample were used to generate each ion spectrum. For each bacterial sample, 50 protein mass fingerprints were averaged and processed. The spectra were analyzed using SARAMIS™ (Spectral Archive and Microbial Identification System, AnagnosTec GmbH, Potzdam, Germany) with a reliability of 99% [ 48 ]. Fungi Samples were taken from previously stored at 4°C, and a portion of mycelium was extracted. Each sample was then homogenized using glass beads in 1 mL of 70% ethanol. Protein extraction was carried out by suspending the mycelium in 300 µL of ultrapure water, then mixed with 900 µL of 70% ethanol. The mycelium was then centrifuged at 13,000 × g for 2 min. The supernatant was discarded and resuspended in 50 µL of 70% formic acid and 50 µL of acetonitrile. The supernatant was then centrifuged, and the supernatant was collected. 1 µL of the protein extract was then spotted onto a MALDI plate by applying it to a stainless steel plate. It was mixed with 1 µL of matrix solution (α-cyano-4-hydroxycinnamic acid [HCCA] in acetonitrile/water/trifluoroacetic acid [50:47.5:2.5]) and dried at room temperature. Data acquisition was carried out using a MALDI-TOF MS spectrometer (Shimadzu-Biotech Corp., Kyoto, Japan) in reflectron mode, with a mass range of 2,000–20,000 Da. 240 laser shots were performed for each sample (40 shots in 6 positions). The obtained spectra were compared with the Bruker MBT Filamentous Fungi Library 2.0 database. The spectra were analyzed using correlation algorithms (logarithmic score: ≥2.0 indicating reliable identification) and with a fidelity of 99%. How positive control was used Candida albicans ATCC 10231 (spectrum of reference in databases). And as a negative control, a matrix without sample was used to rule out contamination. Statistical Analysis All statistical analyses were performed in R Studio (v. 4.4.3). Taxonomic relative abundances were preprocessed by calculating the mean abundance of each taxon across all sampling locations. Alpha diversity metrics (observed species, Shannon index, and Simpson index) were computed using the phyloseq R package (v. 1.50.0). Differences in alpha diversity between groups were evaluated using the non-parametric Kruskal-Wallis test, with post-hoc Dunn’s test for pairwise comparisons (adjusted via the Benjamini-Hochberg method, p < 0.05). Beta diversity was assessed using Bray-Curtis dissimilarity matrices. Non-metric multidimensional scaling (NMDS) was applied to visualize community dissimilarities, and statistical significance of observed differences was tested with PERMANOVA (999 permutations) using the vegan R package (v. 2.6.10). To further resolve community patterns, a heatmap was generated with the pheatmap package (v. 1.0.12), clustering samples based on Bray-Curtis distances. Intersection patterns of taxa across sampling locations were visualized using an UpSet plot (UpSetR package, v. 1.4.0), chosen over traditional Venn diagrams due to its enhanced readability for complex datasets. Results Characterization of Anurans The collection sites yielded a total of 20 different species of amphibians, distributed across the five sampling locations (Zamora Chinchipe, Loja, Cañar, Azuay, and Morona Santiago). The genera more characterized were Pristimantis (16 records), Gastrotheca (5 records), Hyloxalus (3 records), Ctenophryne (3 records), Lithobates (3 records), Adenomera (1 record), Chiasmocleis (1 record), Scinax (1 record), Dendropsophus (1 record) and Dendropsophus (1 record) (Table 1 ). Table 1 Spatial distribution and diversity of anuran species according to location and sector in Zamora Chinchipe, Loja, Cañar, Azuay and Morona Santiago. Location Sector Anuran spp. Coordinates Latitude Longitude Zamora Chinchipe Límite provincial Pristimantis andinognomus -3,992947 -79,145022 Pristimantis vidua -3,992947 -79,145022 Criadero Piuntza Lithobates catesbeianus -3,870451 -78,881814 Padmi Adenomera hylaedactyla -3,737577 -78,619146 Pristimantis diadematus -3,737577 -78,619146 Pristimantis conspicillatus -3,737577 -78,619146 Loja Parque cerro Pucará Lithobates catesbeianus -4,012724 -79,195073 Lithobates catesbeianus -4,012724 -79,195073 Abra del Zamora Pristimantis versicolor -3,985234 -79,145307 Pristimantis balionotus -3,985234 -79,145307 Pristimantis samaniegoi -3,985234 -79,145307 Pristimantis colodactylus -3,985234 -79,145307 Pristimantis matildae -3,985234 -79,145307 Cañar Laguna de Guabizhun Hyloxalus vertebralis -2,803048 -78,936164 Gastrotheca cuencana -2,803048 -78,936164 Azuay Guangarcucho Gastrotheca cuencana -2,843274 -78,885605 Gastrotheca cuencana -2,843274 -78,885605 Ctenophryne aequatorialis -2,843274 -78,885605 Ctenophryne aequatorialis -2,843274 -78,885605 Ctenophryne aequatorialis -2,843274 -78,885605 Lazareto Gastrotheca cuencana -2,882491 -79,008925 Hyloxalus vertebralis -2,882491 -79,008925 Hyloxalus vertebralis -2,882491 -79,008925 Chanlud Pristimantis erythros -2,895228 -78,957036 Pristimantis erythros -2,895228 -78,957036 Pristimantis lutzae -2,895228 -78,957036 Pristimantis lutzae -2,895228 -78,957036 Quimsacocha Gastrotheca pseustes -2,682961 -79,033192 Morona Santiago Wapú Pristimantis conspicillatus -2,682961 -79,033192 Pristimantis conspicillatus -2,682961 -79,033192 Chiasmocleis bassleri -2,682961 -79,033192 Scinax cruentommus -3,034273 -79,222145 Pristimantis conspicillatus -2,843274 -78,885605 Dendropsophus bifurcus -2,843274 -78,885605 General composition of the anuran spp., bacteria, and fungi assemblage A notable overall species diversity was observed within the five study areas (Zamora, Chinchipe, Loja, Cañar, Azuay, and Morona Santiago), where Pristimantis conspicillatus (11.8%) and Gastrotheca cuencana (11.8%) were the most abundant species, followed by Hyloxalus vertebralis (8.8%), Ctenophryne aequatorialis (8.8%), and Lithobates catesbeianus (8.8%), which presented high proportions compared to other identified species. Likewise, species of the genus Pristimantis , such as P. erythros (5.8%), P. lutzae (5.8%), P. matildae (2.9%), and P. colodactylus (2.9%), are well represented in the sample, demonstrating the high richness of this genus in the study locations (Fig. 3 A). Microbial associations were identified in Gastrotheca cuencana (Azuay) with the prevalence of Pseudomonas antarctica and Pseudomonas fluorescens . In contrast, Hyloxalus vertebralis (Azuay) presented Acinetobacter iwoffii and Staphylococcus xylosus . In Lithobates catesbeianus (Loja), a dominance of Lactobacillus curvatus and Chryseobacterium joostei was observed. Pristimantis conspicillatus (Morona Santiago) revealed Serratia marcescens . Furthermore, in Pristimantis diadematus (Zamora), co-occurrence of Bacillus pumilus and Pseudomonas brassicacearum was recorded. However, in Pristimantis samaniegio (Loja) and Pristimantis vidua (Zamora Chinchipe) no bacterial species were detected (Fig. 4 ). The total relative abundance of bacteria in anurans was mainly composed of species of the genera Pseudomonas, Bacillus, Pantoea, Corynebacterium and Acinetobacter. Within these, Pseudomonas chlororaphis was present (28%), followed by Acinetobacter iwoffii and Pseudomonas fluorescens (14%). The species Pseudomonas Antarctica was present (12%), followed by Pseudomonas orientalis , Pantoea agglomerans and Pseudarthrobacter oxydans in an equal percentage of (10%). While Pseudomonas jensenii and Rahnella aquatilis was evidencied (8%). The species with the lowest percentage were Pseudomonas kilonensis (6%), Pseudomonas thivervalensis , Bacillus pumilus and Kluyvera ascorbata (6%). We must highlight that the bacterial species with intermediate prevalence percentage were Bacillus infantis , Pseudomonas azotoformans , Staphylococcus xylosus , Pseudomonas rhodesiae , Pseudomonas taetrolens , Lactobacillus curvatus and Aeromonas bestiarum in similar values ​​of (4%). The least prevalent species were Corynebacterium striatum , Chryseobacterium joostei , Pseudomonas japonica , Serratia marcescens , Comamonas testosteroni , Proteus mirabilis , Pseudomonas brassicacearum , Pseudomonas extremerientalis , and Providencia rettgeri in (2%) (Fig. 3 B). On the other hand, the relative abundance of total fungal species in anurans showed a higher prevalence of Hortaea werneckii (26.4%), recognized for its ability to colonize the skin of amphibians and that could be involved in cutaneous infection processes, followed by Fusarium solani (20.5%), Syncephalastrum spp. (20.5%), Fusarium concentricum (8.8%). Species such as Fusarium oxysporum , Fusarium proliferatum and Aspergillus niger (5.8%) showed a notable abundance. Finally, the species that showed less abundance were represented by Fusarium spp. (2.9%) and Bipolaris spp. (2.9%) (Fig. 3 C). Relative composition of the bacterial population The total number of detections was 92, distributed among 29 bacterial species on the skin of anurans spp. at the sampling sites was 29, distributed in the provinces of Zamora, Chinchipe, Loja, Cañar, Azuay and Morona Santiago ( Supplementary Fig. 1 ). Subkingdom Negibacteria This subkingdom comprised a total of 79.31% of all observed species, including Acinetobacter iwoffii (3.45%), Chryseobacterium joostei (3.45%), Pseudomonas antarctica (3.45%), Pseudomonas azotoformans (3.45%), Pseudomonas chlororaphis (3.45%), Pseudomonas fluorescens (3.45%), Pseudomonas japonica (3.45%), among others. Subkingdom Posibacteria A total of 6 species were observed (20.69%). They were represented by: Bacillus infantis (3.45%), Corynebacterium striatum (3.45%), Staphylococcus xylosus (3.45%), Bacillus pumilus (3.45%), Pseudarthrobacter oxydans (3.45%), and Lactobacillus curvatus (3.45%). Phylum Proteobacteria This phylum was the dominant one in the samples from the studied locations, exhibiting 22 species and representing 75.86% of the bacterial composition. Species such as Acinetobacter iwoffii (3.45%), Pseudomonas Antarctica (3.45%), Pseudomonas azotoformans (3.45%), Pseudomonas chlororaphis (3.45%), Pseudomonas fluorescens (3.45%), Pseudomonas japonica (3.45%), Pseudomonas jensenii (3.45%), Pseudomonas kilonensis (3.45%), and Pseudomonas orientalis (3.45%) stood out here (Fig. 4 ). Firmicutes These covered of a total of 4 species that together represented 13.79% of the samples and consisted of Bacillus infantis (3.45%), Staphylococcus xylosus (3.45%), Bacillus pumilus (3.45%), and Lactobacillus curvatus (3.45%) (Fig. 5 ). Actinobacteria This phylum presented intermediate abundance among the total species with a (6.90%). Corynebacterium striatum (3.45%) and Pseudarthrobacter oxydans (3.45%) (Fig. 5 ). Bacteroidetes This phylum showed the lowest abundance of species, with a total representation of 3.45%), with Chryseobacterium joostei (3.45%) (Fig. 5 ). Composition of the Fungal Assemblage A total of 41 fungal detections were obtained on the skin of anuran spp., distributed across 9 species, in the provinces of Zamora Chinchipe, Loja, Cañar, Azuay, and Morona Santiago (Supplementary Fig. 2). Subkingdom Dikarya Ascomycota Within Ascomycota, Sordariomycetes was the dominant class at the sampling locations, with an average abundance of 44.12%, followed by Dothideomycetes (29.41%), and Eurotiomycetes in the lowest range (5.88%). The predominant genus was Fusarium (44.12%), represented by Fusarium solani (20.59%), Fusarium concentricum (8.82%), Fusarium oxysporum (5.88%), Fusarium proliferatum (5.88%), and Fusarium spp. (2.94%). The genus Hortaea showed average values ​​of 26.47%, with Hortaea werneckii (26.47%). The genus Aspergillus (5.88%) was represented Aspergillus spp. Finally, the genus Bipolaris was the least predominant with (2.94%) with Bipolaris spp. (Fig. 6 ). Mucoromycotina Mucromycotina was the least predominant class at the sampling locations (20.59%) where Syncephalastrum showed (20.59%), which was represented by Syncephalastrum spp. (Fig. 6 , 7 ). Alpha Diversity Patterns Across Localities Bacteria Bacterial alpha diversity, quantified using the Shannon, Simpson, and Chao1 indices (Fig. 8 ), demonstrated marked variability across localities. Azuay exhibited the highest overall diversity (Shannon = 2.36 ± 0.08; Simpson = 0.88 ± 0.01) and richness (Chao1 = 17.7 ± 3.3), closely followed by Zamora (Shannon = 2.20 ± 0.17; Simpson = 0.87 ± 0.02; Chao1 = 15.4 ± 3.1). Loja showed intermediate diversity (Shannon = 2.05 ± 0.15; Simpson = 0.86 ± 0.02) but lower richness (Chao1 = 9.3 ± 1.6). In contrast, Morona displayed reduced diversity (Shannon = 1.66 ± 0.07; Simpson = 0.77 ± 0.02) and the lowest richness (Chao1 = 7.4 ± 1.3). Notably, Cañar presented stark contrasts: one sample showed high richness (Chao1 = 21) with moderate diversity (Shannon = 1.79; Simpson = 0.83), while others were species-poor (Chao1 = 4.3 ± 0.6; Shannon = 1.33 ± 0.002; Simpson = 0.72 ± 0.01). Kruskal-Wallis tests confirmed significant differences across localities for Shannon and Simpson indices (p < 0.05), with post-hoc Benjamini-Hochberg adjustments highlighting Cañar’s differences from Azuay. Fungi Fungal alpha diversity, revealed pronounced contrasts among localities (Fig. 9 ). Loja exhibited the highest richness (Chao1 = 7.4 ± 3.0) and diversity (Shannon = 1.62 ± 0.10; Simpson = 0.79 ± 0.02), followed by Azuay (Chao1 = 6.9 ± 2.3; Shannon = 1.55 ± 0.17; Simpson = 0.76 ± 0.03). Zamora displayed moderate diversity (Shannon = 1.34 ± 0.32; Simpson = 0.70 ± 0.11) but lower richness (Chao1 = 4.8 ± 1.8). In contrast, Morona showed reduced diversity (Shannon = 1.02 ± 0.05; Simpson = 0.61 ± 0.03) and minimal richness (Chao1 = 3 ± 0). Notably, Cañar was statistically distinct (p < 0.05, Kruskal-Wallis with Benjamini-Hochberg adjustment), with no detectable fungal diversity (Shannon = 0; Simpson = 0) and extremely low richness (Chao1 = 1 ± 0), indicating a near-absence of viable fungal communities. Post-hoc analyses confirmed Cañar’s divergence from all other localities, which exhibited overlapping but variable profiles. Beta Diversity and Community Structure Bacteria Pairwise PERMANOVA analysis, based on Bray-Curtis dissimilarity, revealed distinct patterns of bacterial community differentiation between localities. While adjusted p -values ( p -adjusted = 1) did not reach statistical significance after Benjamini-Hochberg correction, effect size metrics (R²) and F-statistics highlighted biologically meaningful trends. The NMDS ordination plot, based on Bray-Curtis dissimilarity, corroborated pairwise PERMANOVA results, revealing distinct spatial clustering of bacterial communities (Fig. 10 ). Zamora, Loja, and Morona formed well-separated clusters, with 95% confidence ellipses showing minimal overlap, underscoring their unique taxonomic assemblages. This segregation aligns with their divergent alpha diversity profiles and suggests strong environmental filtering or niche specialization. The hierarchical clustering patterns observed in the Bray-Curtis-derived heatmap (Fig. 11 ) further validated the spatial structuring of bacterial communities. An UpSet plot analysis showed the distribution of bacterial species found on anuran skin at the study locations (Morona Santiago, Azuay, Cañar, Loja, and Zamora Chinchipe). It revealed the presence of eight bacterial species shared by all locations. This finding suggests the existence of widely distributed taxa, potentially adapted to a diverse range of environmental conditions. Certain locations shared certain bacterial species, and in several cases, taxa existed that were unique to specific combinations from two locations. Furthermore, each location harbored potentially unique bacteria, demonstrating a degree of microbial endemism. This differentiation could be associated with variation in the composition of the amphibian community (Fig. 12 ). Fungi Pairwise PERMANOVA analysis, based “n” Bray-Curtis dissimilarity, revealed biologically meaningful differentiation in fungal community composition across localities, despite the lack of statistical significance after Benjamini-Hochberg correction (all adjusted p-values = 1). Several comparisons showed high R² values, and large F-statistics, indicating strong effect sizes and potential ecological relevance. The NMDS ordination plot (based on Bray Curtis dissimilarity; Fig. 13 ) supported these trends, revealing partial spatial separation of fungal communities. Although some confidence ellipses overlapped, notable clustering was observed, particularly among the Zamora, Loja, and Morona samples, suggesting underlying ecological or host-driven factors shaping fungal assemblages. Complementarily, the UpSet plot (Fig. 14 ) highlighted the distribution of fungal taxa across localities. Three fungal taxa were shared among Morona, Zamora, Azuay and Loja, suggesting the presence of a core mycobiome possibly adapted to a broad range of environmental or host-related conditions. However, numerous taxa were exclusive to specific site combinations, and each locality also harbored unique taxa, indicating a high degree of microbial endemism and potential ecological specialization. The heatmap based on Jaccard dissimilarity (Fig. 15 ) further reinforced the observed spatial structure. Hierarchical clustering grouped localities according to similarities in fungal composition, reflecting consistent biogeographic patterns. Together, these findings underscore a non-random distribution of skin-associated fungi in anurans across the Andean and Amazonian transition zone. Discussion This study provides an overview of the diversity of bacterial and fungal communities on the skin of anuran spp., where each habitat and geographic location can serve as a selective filter determining local microbial diversity, a phenomenon known as the Baas-Becking principle [ 50 ]. Previous studies have shown that the skin microbiota profile is influenced by the phylogenetic identity of the host amphibian [ 51 ]. Our understanding of the composition and role of the microbiota associated with plants and animals, including humans, is increasing due to the application of technologies such as MALDI-TOF MS. Microbial communities living on animal skin represent an interesting scenario, as they are continuously exposed to the influence of the external environment. However, these studies have largely been limited to the human skin microbiome [ 52 ]. Among wild animals, amphibians, due to their absence of fur or feathers, provide an excellent model system to study skin-associated microbial communities, which are thought to mediate disease susceptibility by providing the first line of defense against pathogens [ 53 – 55 ]. Knowledge about host-associated microbial communities can assist with conservation actions for endangered species, as well as play a role as a bioindicator of a pathogen-free population [ 56 , 57 ]. Therefore, the aim of this study was to analyze the diversity of bacteria and fungi on the skin of anuran spp. in wild habitats using MALDI-TOF MS mass spectrometry in five locations: Azuay, Cañar, Loja, Morona Santiago and Zamora Chinchipe in the Republic of Ecuador. The diversity of anurans in Ecuador is recognized as one of the highest in the world due to its complex topography and habitats heterogeneity [ 58 ], presenting unique biogeographic patterns that vary significantly between provinces. Analysis of amphibian species composition at Zamora Chinchipe, Loja, Cañar, Azuay, and Morona Santiago highlighted endemism and anthropogenic pressures. The observed data showed a marked heterogeneity in anuran species richness, with emphasis on genera such as Pristimantis , Gastrotheca , and Hyloxalus , and the presence of invasive species such as Lithobates catesbeianus . In Zamora Chinchipe, the presence of species such as Pristimantis andinognomus and Pristimantis vidua , which are endemic to the montane forests of southern Ecuador [ 58 ], suggests a high specialization to humid microhabitats between 1,800–2,500 m. The coexistence of P. diadematus and P. conspicillatus , both species associated with lower vegetation strata, and Adenomera hylaedactyla , which is typical of floodable soils [ 59 ], reflected the ecological heterogeneity of the region. However, the detection of Lithobates catesbeianus , an invasive species [ 60 ], in riparian areas indicates possible anthropogenic alterations, since this anuran competes with native species for resources. In the province of Loja, the dominance of Pristimantis with the species versicolor , balionotus , and samaniegoi highlights the role of páramos and montane forests as centers of speciation [ 61 ]. Pristimantis matildae , recently described in the Tapichalaca Reserve [ 58 ], evidences the presence of microendemisms critical for conservation. However, the recurrence of Lithobates catesbeianus in multiple records suggests a worrying expansion of this species, associated with aquaculture activities [ 62 ]. In Cañar and Azuay, the presence of Gastrotheca cuencana , an ovoviviparous species endemic to the central Andes [ 63 ] and Hyloxalus vertebralis associated with ravines in cloud forests [ 64 , 65 ] reflects adaptations to cold and humid environments (> 3,000 m). The abundance of Ctenophryne aequatorialis in Azuay, a cryptically red microhylid, suggests evolutionary strategies to avoid predators in fragmented habitats [ 65 ]. The absence of Lithobates catesbeianus in these sites could be related to thermal limitations, although physiological studies are required to confirm this. Meanwhile, in Morona Santiago, the coexistence of Pristimantis conspicillatus shared with Zamora Chinchipe with Scinax cruentomma (typical of the Amazonian lowlands) [ 65 ], indicates a transition zone between the Andes and the Amazon. Chiasmocleis bassleri , a fossorial microhylid, highlights the importance of non-flooded soils in primary forests [ 66 ]. However, the absence of records of high Andean species suggests a biogeographic boundary defined by an altitude < 1,500 masl in this region. When comparing sampling locations, significant differences were observed where Zamora Chinchipe and Loja shared a higher richness of Pristimantis (6 and 5 species, respectively), while Azuay stands out for the diversity of Gastrotheca and Ctenophryne . This reflects altitudinal gradients where Zamora Chinchipe (800–2,500 masl) and Loja (1,500–3,000 masl) host mid-mountain species, while Azuay (> 3,000 masl) presents high Andean taxa. The presence of Lithobates catesbeianus in Zamora Chichipe and Loja, but not in Azuay, suggests that its invasion is limited by climatic or anthropogenic factors. Microendemic species, such as Pristimantis matildae (Loja) and Gastrotheca cuencana (Azuay-Cañar), face critical risks from deforestation and the effects of climate change. For example, 30% of the cloud forests in Azuay have been converted to grasslands, reducing habitats for Hyloxalus vertebralis [ 67 ]. The environmental microbiota is a critical component of ecosystem health, particularly in amphibians, where skin infections contribute significantly to global population decline. In Azuay, the bacterial community identified on the skin of anuran spp. was characterized by the presence of 15 species distributed in relative percentages ranging. The most abundant taxon was Acinetobacter iwoffii (n = 7), followed by Pseudomonas fluorescens (n = 6), Pseudomonas chlororaphis (n = 4), and Serratia marcescens (n = 1). The high frequency of A. iwoffii is relevant, since species of the genus Acinetobacter have been associated with skin infections in anuran spp., particularly under conditions of altered natural microbiome [ 68 – 70 ]. In anurans, an imbalance in the cutaneous microbiota could facilitate colonization by this pathogen, leading to dermatitis or systemic infections. P. fluorescens has been reported to cause necrotic skin lesions in Lithobates catesbeianus , especially in eutrophic environments [ 71 ]. Its high prevalence suggests a risk for anurans in altered habitats. However, studies by [ 72 ] on Gastroteca spp. showed that P. fluorescens had inhibitory functions on the chytrid fungus Batrachochytrium dendrobatidis (Bd), which is linked to many declines in anuran populations. Serratia marcescens has been reported to induce deep and extensive ulcers in the tree frog ( Litoria caerulea ) and is considered an important pathogen. Likewise, the various representatives of the genus Pseudomonas , which include P. azotoformans , P. japonica , P. jensenii , P. kilonensis , P. orientalis , and P. thivervalensis , constituted an important part of the microbial community in Azuay. This genus is known for its metabolic versatility and its ability to produce bioactive metabolites. However, in scenarios where there is an imbalance in the microbial community, some species of Pseudomonas can act as opportunistic pathogens, generating skin infections, which, in combination with environmental stress, can trigger complex clinical pictures in anurans [ 73 ]. In Zamora Chinchipe, anuran populations presented bacterial communities composed of 13 taxa, where the Pseudomonas genera predominated along with other environmental taxa. This location highlighted the presence of Pseudomonas chlororaphis (n = 3) and Pseudomonas antarctica (n = 2), as well as Pantoea agglomerans (n = 2) and Rahnella aquatilis (n = 2). The presence of Pantoea agglomerans is of particular interest because despite being a common inhabitant of the environment, it bacteria has been implicated in opportunistic infections in animals and humans, and can cause skin irritations [ 74 ]. P. chlororaphis has been reported to secrete phenazines, antifungal compounds that inhibit Bd [ 75 ]. However, in Zamora Chinchipe, its high abundance could displace commensal microbiota, increasing susceptibility to secondary infections. Furthermore, the diversity of Pseudomonas in Zamora Chinchipe suggests a dynamic bacterial ecosystem where competition between commensal and pathogenic species can determine the health status of anuran skin. The presence of Pseudomonas orientalis, Pseudomonas rhodesiae and Pseudomonas taetrolens in anuran spp. in smaller proportions reinforces the idea that microbial dysbiosis could be related to infectious outbreaks under conditions of environmental alteration, such as changes in temperature or pollution [ 76 , 77 ]. On the other hand, Loja presented a community composed of 9 taxa, with a predominance of bacteria belonging to the genera Pseudomonas and Pseudarthrobacter , as well as lactobacilli. An equitable distribution of bacterial species was also observed, where Pseudomonas antarctica , Pseudomonas chlororaphis and Pseudarthrobacter oxydans . Additionally, Lactobacillus curvatus (n = 2) and Lactobacillus jensenii (n = 1) were detected. The presence of lactobacilli on the skin of anurans could have a protective effect, since these microorganisms are known to produce lactic acid and bacteriocins, which inhibit the growth of pathogens and modulate the host immune response [ 78 ]. However, the coexistence with potential pathogens such as P. chlororaphis (a pathogen that causes dermatitis in amphibians) suggests that an imbalance in the bacterial community could trigger skin infections [ 12 ]. Similarly, Aeromonas bestiarum is a recognized pathogen in fish and amphibians, responsible for causing septicemia and dermatitis, which could pose a high risk to anuran populations in this region, under adverse environmental conditions. The coexistence of these pathogenic taxa with others that act as commensals raises the need to evaluate the microbial balance in anuran skin, since dysbiosis can facilitate the transition from symbiotic relationships to pathological states. The presence of bacteria with biotechnological potential, such as Pseudomonas fluorescens in Azuay and Pseudarthrobacter oxydans in Loja and Cañar, represents an opportunity for the development of bioremediation and biological control strategies. Pseudomonas fluorescens has been the subject of numerous studies due to its ability to produce natural antibiotics and toxic compound-degrading enzymes, which could be exploited for the decontamination of environments affected by agrochemicals and other pollutants [ 82 ]. Similarly, Pseudarthrobacter oxydans has demonstrated potential in the degradation of hydrocarbons and in promoting plant growth in contaminated soils, making it an attractive candidate for biotechnological applications in environmental contexts [ 83 ]. On the other hand, some studies have shown that alterations in bacterial composition can facilitate the invasion of external pathogens, such as Batrachochytrium dendrobatidis , the etiological agent of chytridiomycosis, which has significantly contributed to the global decline of amphibians [ 84 ]. It has also been suggested that variability in bacterial composition can influence the immune response of anurans, affecting their ability to resist secondary infections caused by opportunistic bacteria [ 85 ]. Regarding the observed fungal communities, these revealed seven fungal taxa in Azuay: Aspergillus niger (n = 1), Bipolaris sp . (n = 1), Fusarium concentricum (n = 2), Fusarium solani (n = 1), Hortaea werneckii (n = 3), Syncephalastrum sp. (n = 1) and Fusarium proliferatum (n = 1). The high prevalence of Hortaea werneckii is significant, since this fungus, in addition to being involved in the pathogenesis of tinea nigra in humans, can cause skin disorders in amphibians, promoting the appearance of hyperpigmented spots and keratosis [ 86 , 87 ]. The presence of Fusarium concentricum , Fusarium solani and Fusarium concentricum in Azuay, highlights the possibility of fusariosis, a disease characterized by the formation of erythematous and ulcerated lesions that can affect both the skin and underlying structures in amphibians [ 88 ]. The detection of Bipolaris spp., a fungus that in humans is associated with photoreaction and subcutaneous mycosis, reinforces the need to consider its role as a potential pathogen in the skin of anurans [ 89 ]. Furthermore, the presence of Aspergillus niger in Azuay not only indicates its biotechnological potential (given its ability to produce pectins and industrial enzymes), but also alerts to the risk of cutaneous aspergillosis in immunocompromised situations [ 90 ]. In Loja, six fungal taxa were reported: Fusarium oxysporum (n = 1), Fusarium solani (n = 1), Fusarium spp. (n = 1), Hortaea werneckii (n = 1), Syncephalastrum spp. (n = 2), and Aspergillus niger (n = 1). In this locality, the highest abundance corresponds to Syncephalastrum spp. indicating that this taxon could be playing a predominant role in the cutaneous fungal community. Although Syncephalastrum spp. It is generally considered a saprophytic fungus, its involvement in invasive mycoses in contexts of immunosuppression is cause for alarm, because skin infections can be complicated in environments with high humidity and stress in the host. On the other hand, the presence of Fusarium oxysporum and Fusarium solani , reinforces the concern about the possible incidence of fusariosis [ 88 ]. The detection of Aspergillus niger is relevant, since, in contexts of microenvironmental imbalance, it can act as an opportunistic pathogen, causing cutaneous and systemic aspergillosis in compromised individuals [ 90 ]. In the locality of Cañar, the fungal community was summarized in two taxon: Fusarium concentricum and Fusarium proliferatum with a representation of 50% each. Although the isolation of a two taxon could be interpreted as low diversity, the exclusive presence of F. concentricum and F. proliferatum is of great importance, since these fungus has been associated with skin infections and can produce mycotoxins that affect skin integrity. Furthermore, studies have shown that certain members of the Fusarium complex have a high invasive capacity, which could lead to dermatomycosis in anurans, especially under conditions of environmental stress or previous lesions in the epidermis [ 91 ]. In the locality of Zamora Chinchipe, the data indicated the presence of six fungal taxa: Syncephalastrum spp. (n = 1), Hortaea werneckii (n = 1), Fusarium oxysporum (n = 1), Fusarium solani (n = 3), Fusarium concentricum (n = 1) and Fusarium proliferatum (n = 1). The high prevalence of Fusarium solani is particularly relevant since this fungus is known to be an etiological agent in cutaneous fungal infections including amphibians, producing keratomycosis and cutaneous fusariosis, conditions that worsen in the presence of environmental stress or skin lesions [ 88 ]. Furthermore, Fusarium proliferatum , was present in Zamora chinchipe, this is an opportunistic pathogen that produces mycotoxins and can contribute to dermatomycosis, affecting the cutaneous integrity of anurans [ 89 ]. The presence of Hortaea werneckii , a halophytic fungus, suggests that under specific conditions it could contribute to alterations in the cutaneous barrier of amphibians, generating hyperpigmentation or irritation [ 86 ]. Likewise, Syncephalastrum spp., although less reported in skin infections, has been documented as a causative agent of mycosis in immunocompromised patients, which opens the possibility that a similar picture may manifest in debilitated anurans [ 92 ]. Finally, in Morona Santiago, the fungal community was composed of six taxa: Fusarium solani (n = 2), Hortaea werneckii (n = 4), Syncephalastrum spp. (n = 3), Aspergillus Niger (n = 1), Bipolaris spp. (n = 2) and Fusarium proliferatum . The predominance of Hortaea werneckii in Morona is of particular interest, since its high abundance may predispose to the appearance of skin infections [ 86 ]. The joint presence of Fusarium solani in and Syncephalastrum spp. in Morona Santiago suggests a complex fungal ecosystem, in which the interaction between pathogenic and saprotrophic fungi could influence the susceptibility of anurans to diseases such as fusariosis and syncephalasrosis, conditions that have been documented in clinical and experimental studies [ 88 ]. The detection of fungal taxa with biotechnological potential, such as Aspergillus niger in Azuay and Loja, and the presence of Fusarium species in various locations, offer interesting opportunities for the development of industrial and environmental applications. Aspergillus niger , for example, is widely recognized for its ability to produce enzymes, organic acids and secondary compounds of industrial relevance, which has allowed its application in the production of pectins, citrates and other biotechnological products [ 93 , 94 ]. Similarly, some Fusarium isolates have shown the ability to degrade toxic compounds and participate in bioremediation processes, which could be used for the treatment of contaminated environments, always considering the duality of these fungi as pathogens and potential agents in biotechnological processes [ 95 , 96 ]. These findings highlight the importance of multimethod approaches, which are critical for biodiversity studies, particularly in underexplored substrates and habitats. Specifically, MALDI-TOF MS analysis of skin swabbing in anurans is considered only the first step toward revealing the bacterial and fungal biodiversity inhabiting the skin of these amphibians in the Azuay, Cañar, Loja, Morona Santiago, and Zamora Chinchipe locations. Future field campaigns could provide more data through metabarcoding studies to observe potential biotrophic symbionts and parasites. Regarding alpha and beta diversity patterns, differences were found between some sampling locations that varied significantly by geographic location. Therefore, it is essential to continue research to establish the causal relationship between bacterial and fungal composition, the presence of pathogens, and their possible relationship with the development of skin diseases in amphibians. This research, in turn, can guide intervention and risk mitigation strategies in ecosystems affected by human activity and climate change. This work represents a first look at bacterial and fungal diversity in a little-studied substrate: the skin of wild anurans. Additional studies are needed to better assess this diversity, along with the development of necessary measures for its protection and conservation. Declarations Supplementary Information: The online version contains supplementary material available at xxxxxxxxxx. Acknowledgments: We would like to thank the Research Department of the Catholic University of Cuenca, associated with the project "Characterization and potential use of the active principles of amphibian secretion through the rescue of ancestral knowledge disclosed No. PICCIITT19-11. We thank Dr. Sergio Covarrubias of the Autonomous University of Zacatecas for his support. Author Contribution: J.S. and J.G. designed the study. F.S., J.S., J.G., R.F., collected the samples. R.F., A.M., M.C., performed the laboratory cultures and isolation. J.S., J.G., and A.V-T. performed MALDI - TOF MS. 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Supplementary Files Supplementarymaterial.docx Cite Share Download PDF Status: Published Journal Publication published 22 May, 2025 Read the published version in Microbial Ecology → Version 1 posted Editorial decision: Revision requested 30 Apr, 2025 Reviews received at journal 29 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 04 Apr, 2025 Reviews received at journal 03 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviewers agreed at journal 02 Apr, 2025 Reviewers invited by journal 01 Apr, 2025 Submission checks completed at journal 01 Apr, 2025 First submitted to journal 31 Mar, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5921108","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":437013576,"identity":"730e1138-9daa-4c3a-99e8-e9d9ee787eec","order_by":0,"name":"Jazmin M. 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Lircay s/n, Talca 360000, Chile","correspondingAuthor":true,"prefix":"","firstName":"Adrian","middleName":"","lastName":"Valdez-Tenezaca","suffix":""}],"badges":[],"createdAt":"2025-01-28 22:38:11","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5921108/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5921108/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s00248-025-02555-8","type":"published","date":"2025-05-22T15:56:52+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":79790206,"identity":"33267dee-94b2-4c17-bcfc-45db31235169","added_by":"auto","created_at":"2025-04-02 18:31:08","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":600036,"visible":true,"origin":"","legend":"\u003cp\u003eThe left side shows the location of the sampling sites in the areas corresponding to the provinces of Zamora Chinchipe, Loja, Cañar Azuay and Morona Santiago. At the bottom, a map of Ecuador is shown.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/50c6c4cbeb5e067bcd163415.png"},{"id":79790499,"identity":"e8b4b185-9400-42d0-8c4f-1592052302df","added_by":"auto","created_at":"2025-04-02 18:39:08","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":120959,"visible":true,"origin":"","legend":"\u003cp\u003eSkin swabbing procedure for microbiological sampling in anurans. The arrows, from left to right, indicate the application of a sterile swab to the dorsal skin and limbs of the anuran species \u003cem\u003ePristimantis erythros\u003c/em\u003e.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/f2ced9cb21e93a6d14a66f32.png"},{"id":79790208,"identity":"658acb76-bc80-477c-9758-da7261785c6a","added_by":"auto","created_at":"2025-04-02 18:31:08","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":338768,"visible":true,"origin":"","legend":"\u003cp\u003eTotal abundance of the species observed at the sites of Zamora Chinchipe, Loja, Cañar, Azuay, and Morona Santiago. \u003cstrong\u003e(A)\u003c/strong\u003e anuran spp., \u003cstrong\u003e(B)\u003c/strong\u003e bacteria spp., and \u003cstrong\u003e(C)\u003c/strong\u003e fungi spp.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/551db58645307620ee6d1092.png"},{"id":79790210,"identity":"828f06f5-c637-402e-b201-a2959cde5205","added_by":"auto","created_at":"2025-04-02 18:31:08","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":467826,"visible":true,"origin":"","legend":"\u003cp\u003eGeneral abundance at the species level recorded on the skin of anuran spp. in the locations of \u003cstrong\u003e(A)\u003c/strong\u003eAzuay, \u003cstrong\u003e(B)\u003c/strong\u003e Morona Santiago, \u003cstrong\u003e(C)\u003c/strong\u003e Loja, \u003cstrong\u003e(D)\u003c/strong\u003e Zamora Chinchipe and \u003cstrong\u003e(E)\u003c/strong\u003eCañar (Ecuador).\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/d6f99f2e2112a522aaaebef2.png"},{"id":79790500,"identity":"c5869892-c969-4948-b332-64bc9028fc1d","added_by":"auto","created_at":"2025-04-02 18:39:08","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":297507,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances of bacteria at the phylum level, recorded on the skin of anuran spp. in the locations of Azuay, Morona Santiago, Loja, Cañar and Zamora Chinchipe (Ecuador).\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/0809c77ba34fe382966bb728.png"},{"id":79790943,"identity":"6c21235d-eee6-4b3c-a423-368cb9c49f18","added_by":"auto","created_at":"2025-04-02 18:55:08","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116960,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances at the class level, recorded on the skin of anuran spp. in the locations of Azuay, Morona Santiago, Loja, Cañar and Zamora Chinchipe (Ecuador).\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/6937fdf8e918ea44cb17abb9.png"},{"id":79790717,"identity":"5f74a7d3-615e-461f-9eb8-2b10b3d0daf2","added_by":"auto","created_at":"2025-04-02 18:47:09","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":137701,"visible":true,"origin":"","legend":"\u003cp\u003eRelative abundances of fungi at the species level recorded on the skin of anuran spp. at sampling locations in the Republic of Ecuador.\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/e6a2803bdf27051552f6823c.png"},{"id":79790233,"identity":"c06ae936-2882-469e-88fd-cddabd8ab8da","added_by":"auto","created_at":"2025-04-02 18:31:09","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":162680,"visible":true,"origin":"","legend":"\u003cp\u003eEstimated alpha diversity of bacterial communities at the five sampling locations. (**) indicates significant differences between locations according to the Kroskal-Wallis test for each index. Observed variability values ​​(\u003cem\u003eP \u003c/em\u003e= \u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/2ebefc37315d7a43266b3f53.png"},{"id":79790224,"identity":"7630c2f0-90b9-4453-8116-f8235382f8bb","added_by":"auto","created_at":"2025-04-02 18:31:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":154845,"visible":true,"origin":"","legend":"\u003cp\u003eEstimated alpha diversity in fungi for the sampling locations (Azuay, Cañar, Loja, Morona Santiago and Zamora Chinchipe). (**) show significant differences between the three groups according to the Kruskal-Wallis test for each index. Observed variability values ​​(\u003cem\u003eP\u003c/em\u003e = \u0026lt;0.05).\u003c/p\u003e","description":"","filename":"floatimage9.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/43887607381e7bb3c864971d.png"},{"id":79790503,"identity":"ce927115-a05a-46d4-aa23-d7542630fd8f","added_by":"auto","created_at":"2025-04-02 18:39:08","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":107697,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Coordinate Analysis (PCoA) based on Bray-Curtis distances of bacterial communities on the skin of anuran spp. divided according to the sampling location (Azuay, Cañar, Loja, Morona Santiago and Zamora Chinchipe).\u003c/p\u003e","description":"","filename":"floatimage10.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/d9df4a480400f14f0ef10703.png"},{"id":79790516,"identity":"5467d6c5-5131-4d90-94f7-92bc1955e9b9","added_by":"auto","created_at":"2025-04-02 18:39:09","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":191912,"visible":true,"origin":"","legend":"\u003cp\u003eComparative heatmap of the bacterial community in the localities of Azuay, Cañar, Loja, Morona Santiago, and Zamora Chinchipe. Color intensities (light, low dissimilarity - dark, high dissimilarity) reflect variations in the relative abundance of taxa, highlighting clusters through dendrograms.\u003c/p\u003e","description":"","filename":"floatimage11.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/77a6f08442f96ec5ef125373.png"},{"id":79790218,"identity":"c884d35f-0086-4b29-bc95-bdbbf9726412","added_by":"auto","created_at":"2025-04-02 18:31:08","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":128000,"visible":true,"origin":"","legend":"\u003cp\u003eUpSet plot illustrates the intersection of bacterial species associated with the skin of anurans in Morona Santiago, Azuay, Cañar, Loja, and Zamora Chinchipe. The bars indicate the number of shared species in each intersection, while the lower matrix indicates the included localities.\u003c/p\u003e","description":"","filename":"floatimage12.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/29a55ca1cbf2ec829ce8fd12.png"},{"id":79790222,"identity":"3f9ae8f8-d12d-4f65-a113-6d68b0335e77","added_by":"auto","created_at":"2025-04-02 18:31:08","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":113959,"visible":true,"origin":"","legend":"\u003cp\u003ePrincipal Coordinate Analysis (PCoA) based on Bray-Curtis distances of the fungal communities on the skin of anuran spp. divided according to sampling location (Azuay, Cañar, Loja, Morona Santiago and Zamora Chinchipe).\u003c/p\u003e","description":"","filename":"floatimage13.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/d4d0de9920ec66419adaa479.png"},{"id":79790945,"identity":"0ab912ea-1ec2-438a-bc6e-5b265d9e852a","added_by":"auto","created_at":"2025-04-02 18:55:09","extension":"png","order_by":14,"title":"Figure 14","display":"","copyAsset":false,"role":"figure","size":127436,"visible":true,"origin":"","legend":"\u003cp\u003eThe UpSet plot illustrates the intersection of fungal species associated with anuran skin at the following locations (Morona Santiago, Azuay, Cañar, Loja, and Zamora Chinchipe). The bars indicate the number of shared species at each intersection, while the lower matrix indicates the included locations.\u003c/p\u003e","description":"","filename":"floatimage14.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/fae49780a748a01f62f037c7.png"},{"id":79790946,"identity":"b612c1f5-7b84-4bb0-891b-7d82e6e6e227","added_by":"auto","created_at":"2025-04-02 18:55:09","extension":"png","order_by":15,"title":"Figure 15","display":"","copyAsset":false,"role":"figure","size":262833,"visible":true,"origin":"","legend":"\u003cp\u003eThe comparative Bray-Curtis dissimilarity matrix (Heatmap) illustrates the variation in fungal composition among localities (Azuay, Cañar, Loja, Morona Santiago, and Zamora Chinchipe). The color gradient, from light (low dissimilarity) to dark (high dissimilarity), reflects differences in the abundance and presence of taxa.\u003c/p\u003e","description":"","filename":"floatimage15.png","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/966c97833ba7d5b35b444071.png"},{"id":83459943,"identity":"dbc3c043-2e78-41d1-99ca-196680d8c1ed","added_by":"auto","created_at":"2025-05-26 16:02:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4612461,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/89dbcfaf-9c68-4c2d-b2ef-382794900ef4.pdf"},{"id":79790204,"identity":"926f95da-dcb8-401c-86ff-2e652eac6e5c","added_by":"auto","created_at":"2025-04-02 18:31:08","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":684037,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-5921108/v1/37643e7f469902066172d515.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Characterization of culturable microbiota associated with the skin of amphibians (Anura) in the southern Andes Mountains of Ecuador","fulltext":[{"header":"Introduction","content":"\u003cp\u003eEcuador is one of the countries with the greatest diversity of amphibians, with approximately 653 described species [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], of which 13% are critically endangered, 23% are endangered, and 20% are vulnerable [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Most of these species inhabit aquatic and terrestrial environments, which determine their physiological processes [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], making them susceptible to environmental changes that alter their habitat. It is worth noting that 40% of species is in decline in recent years [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. The increase in anthropogenic activities such as ecosystem pollution [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e] and the expansion of the agricultural frontier [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] have had observable effects on amphibian populations, which are severely threatened and in decline [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe microbiota, which represents the set of microorganisms that inhabit both the surface and the interior of organisms, can modulate host health by affecting their development, behavior, metabolism, and inflammatory responses [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In amphibians, bacterial communities present on the skin could offer protection against infection by synthesizing antifungal metabolites, acting as an integral part of the animal's immune system [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Some bacteria on the skin of amphibians are capable of inhibiting the growth of pathogens in vitro [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], and supplementing amphibian microbiomes with inhibitory bacteria can increase survival in laboratory assays [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Furthermore, the composition of bacterial communities in frogs and the persistence of the host population are often correlated [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. In addition to the knowledge about bacterial microbiomes, there are few studies on fungal microbiomes [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Relatively few studies have examined the fungal microbiomes of vertebrate wildlife [\u003cspan additionalcitationids=\"CR21 CR22\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Furthermore, while these studies provide valuable starting points, they have often had limitations as they were carried out in captivity [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], which disrupts the microbiomes [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. Previous studies have described that fungi inhabiting the skin of some amphibians are capable of producing antimicrobial compounds such as penicillin [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], although little is known about their effects on amphibian health and how they interact with host immune defenses. It should be mentioned that few studies have been conducted on cutaneous fungal communities in amphibians and a growing literature has been observed, highlighting the potential of bacteria for probiotic applications [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMatrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been applied in the field of microbiology for a many years. With the development of new technologies and method optimization, new rapid and accurate approaches have been developed to improve the accuracy of targeted identification. MALDI-TOF spectroscopy is not limited to identifying strains grown on solid media, or in vitro, but can also directly identify them from blood culture samples, cerebrospinal fluid, urine, and skin samples [\u003cspan additionalcitationids=\"CR31 CR32 CR33\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. Identification by MALDI-TOF spectroscopy has been used to identify Gram-negative and Gram-positive bacteria, aerobes, anaerobes, mycobacteria, nocardia, yeasts, filamentous fungi, and viruses [\u003cspan additionalcitationids=\"CR36 CR37 CR38 CR39 CR40 CR41\" citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. MALDI-TOF MS is a reliable, simple, and readily available technology [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere is currently a need to understand the diversity of the cultivable microbial communities that contribute to host resistance to disease. The objective of this study was to characterize the composition of the bacterial and fungal skin microbiota in anuran spp. using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and to establish the biodiversity in this environment.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eEthics Statement\u003c/h2\u003e \u003cp\u003eThis study was conducted in strict accordance with the guidelines for the use of live amphibians and reptiles in field research developed by the American Society of Ichthyologists and Herpetologists, the League of Herpetologists, and the Society for the Study of Amphibians and Reptiles. Specific collecting permits for this study were obtained under authorization from the Ministry of Environment, Water, and Ecological Transition of Ecuador (MAATE), number MAAE-ARSFC-2021-1564. The samples did not include endangered or protected animal or plant species.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eSampling Locations\u003c/h3\u003e\n\u003cp\u003eThe study was conducted in eleven locations located from south to north in the foothills of the Andes Mountains during the months of September and November 2021. These locations include: (A) Zamora-Chinchipe Province; (1) the \"provincial boundary\" sector located in the southeastern part and classified as montane cloud forest, with species from the Areaceae, Poaceae, and Orchidaceae families; (2) the \"Padmi sector,\" characterized as a lowland evergreen forest, located in the central-eastern part. It has shrub species such as Sapium and \u003cem\u003eGrias peruviana\u003c/em\u003e, secondary forests with \u003cem\u003eDictyoloma peruviana\u003c/em\u003e, and also includes sections of tropical rainforest and premontane forest; (3) the \"Piuntza sector,\" which exhibits premontane forest vegetation characterized by high tree and shrub diversity. Volcanic soil conditions, high humidity, and moderate altitude favor endemic communities and ecological transitions between tropical forests and montane ecosystems. (B) Loja Province; (1) \u0026ldquo;Cerro Pucar\u0026aacute; Park\u0026rdquo;, adjacent to the northwestern part of Podocarpus National Park. Its reference ecosystem is described as a montane cloud forest, located between 1,500 and 2,900 m a.s.l.; (2) \u0026ldquo;Abra del Zamora sector\u0026rdquo;, located on the northern periphery, between Podocarpus National Park and Pucar\u0026aacute; Park, classified as a montane cloud forest ecosystem. (C) Ca\u0026ntilde;ar Province; (1) \u0026ldquo;Laguna de Guabizhum sector\u0026rdquo;, located in the Soldados parish, belonging to the D\u0026eacute;leg canton, has a montane cloud forest, with the presence of Cyperaceae species such as \u003cem\u003eBarnadesia parviflora\u003c/em\u003e, \u003cem\u003eJuglans neotropica\u003c/em\u003e, and \u003cem\u003eMyrcianthes\u003c/em\u003e spp. (D) Azuay Province; (1) \u0026ldquo;Guangarcucho sector\u0026rdquo;, with vegetation formation characterized by the appearance of humid montane scrub, includes relatively humid valleys between 2,000 and 3,000 m.a.s.l. in the inter-Andean alley, where native vegetation has been devastated and replaced by agricultural crops and forests of \u003cem\u003eEucalyptus globulus\u003c/em\u003e, \u003cem\u003eSalix humboldtiana\u003c/em\u003e and \u003cem\u003eAcacia farnesiana\u003c/em\u003e; (2) \u0026ldquo;Lazareto sector\u0026rdquo;, located in the urban area, which includes the banks of the Milchichig River, here the introduced forests host species of the genera \u003cem\u003eJunglas neotropica\u003c/em\u003e, \u003cem\u003eBaccharis latifolia\u003c/em\u003e and \u003cem\u003eSpartium junceum\u003c/em\u003e; (3) \u0026ldquo;Chanlud sector\u0026rdquo;, classified as a shrubby p\u0026aacute;ramo, composed mainly of grasslands and shrubs, in an altitudinal range of 2600 to 3600 m a.s.l., characterized by the presence of the genus Calamagrostis and shrub species of the genera Baccharis, Gynoxys, and Brachyotum; (4) \u0026ldquo;Quimsacocha sector\u0026rdquo;, with an altitude of 3100 m a.s.l., is characterized by having characteristics of a shrubby p\u0026aacute;ramo with a significant presence of \u003cem\u003eCortaderia nitida\u003c/em\u003e. (E) Morona Santiago Province; (1) \u0026ldquo;Wap\u0026uacute; sector\u0026rdquo;, has an ecosystem classified as an evergreen piedmont forest with an altitude of 800 and 1300 m a.s.l. It has a tree composition native to the area where the canopy can reach 30 meters in height (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eSample Collection\u003c/h3\u003e\n\u003cp\u003eSampling was carried out on amphibian spp. observed at the sampling sites, in water bodies, and in the surrounding vegetation. Specimens were located using the \"visual encounter\" method and subsequently morphologically characterized according to [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Specimens were handled using nitrile gloves, and a skin swab was subsequently performed in situ by skin rubbing according to [\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e] (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). Two skin swabs were taken from each specimen. One swab was subsequently stored in a Falcon\u0026trade; tube containing 2 ml of Brain Heart culture medium (Merck\u0026reg;) and the second with TGhL medium with Tryptone (SIGMA\u0026reg;), Hydrolyzed Gelatin (TM MEDIA\u0026reg;), and Lactose (SIGMA\u0026reg;). The samples were kept in a cooler with dry ice for 24 to 48 hours and transferred to the Microbial Ecology and Active Ingredients Laboratory of the Center for Research, Innovation and Technology Transfer (CIITT) of the Catholic University of Cuenca for processing.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003eIsolation, Culture Conditions, and Activation\u003c/h3\u003e\n\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eBacterial Isolation and Culture\u003c/h2\u003e \u003cp\u003eBacterial cultures were prepared using a solid medium (95.5% blood agar) dispensed into 9 cm diameter Petri dishes. The medium was sterilized via autoclave (121\u0026deg;C, 15 psi, 15 minutes) and allowed to cool under a laminar flow cabinet. Samples, previously stored at 4\u0026deg;C, were aseptically inoculated onto the agar surface using a sterile inoculation loop, followed by streaking to isolate individual colonies. Plates were incubated at 37\u0026deg;C for 16 hours under anaerobic conditions. After incubation, colony growth was assessed, and a representative colony from each sample was selected for downstream analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eFungal Isolation and Culture\u003c/h2\u003e \u003cp\u003eFungal cultures were prepared with 2% potato dextrose agar (PDA), sterilized via autoclave (121\u0026deg;C, 15 psi, 15 minutes), and poured into 9 cm Petri dishes. Mycelial plugs (5 mm diameter) were excised from stock cultures using a sterile punch and transferred to the PDA plates. Inoculated plates were incubated at 25\u0026ndash;28\u0026deg;C under a 12/12 h light-dark photoperiod for 5 days. Mycelial growth was monitored daily, with expansion patterns and morphological features recorded to confirm viability and purity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eMALDI-TOF MS\u003c/h3\u003e\n\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eBacteria\u003c/h2\u003e \u003cp\u003eAll samples were analyzed using an Axima\u0026trade; Confidence MALDI-TOF MS spectrometer (Shimadzu-Biotech Corp., Kyoto, Japan) in positive linear mode (m/z\u0026thinsp;=\u0026thinsp;2000\u0026ndash;20,000). A small number of colonies from each pure culture were transferred to a FlexiMass\u0026trade; destination well using a disposable loop, covered with 0.5 \u0026micro;l of 2,5-dihydroxybenzoic acid (DHB; 10 mg/ml in acetonitrile/0.1% trifluoroacetic acid 1:1) matrix solution, and air-dehydrated for 1\u0026ndash;2 min at 24\u0026ndash;27\u0026deg;C. The reference strain Escherichia coli K12 (genotype GM48) was used as a calibration standard and as a reference for quality control. Sample information such as medium and culture conditions were imported into Shimadzu Biotech Launchpad\u0026trade; software, v.2.8 (Shimadzu-Biotech Corp., Kyoto, Japan). Protein mass profiles were obtained with linear positive mode detection at a laser frequency of 50 Hz and within a mass range of 2000\u0026ndash;20,000 Da. The acceleration voltage was 20 kV and the extraction delay time was 200 ns. A minimum of 20 laser shots per sample were used to generate each ion spectrum. For each bacterial sample, 50 protein mass fingerprints were averaged and processed. The spectra were analyzed using SARAMIS\u0026trade; (Spectral Archive and Microbial Identification System, AnagnosTec GmbH, Potzdam, Germany) with a reliability of 99% [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eFungi\u003c/h2\u003e \u003cp\u003eSamples were taken from previously stored at 4\u0026deg;C, and a portion of mycelium was extracted. Each sample was then homogenized using glass beads in 1 mL of 70% ethanol. Protein extraction was carried out by suspending the mycelium in 300 \u0026micro;L of ultrapure water, then mixed with 900 \u0026micro;L of 70% ethanol. The mycelium was then centrifuged at 13,000 \u0026times; g for 2 min. The supernatant was discarded and resuspended in 50 \u0026micro;L of 70% formic acid and 50 \u0026micro;L of acetonitrile. The supernatant was then centrifuged, and the supernatant was collected. 1 \u0026micro;L of the protein extract was then spotted onto a MALDI plate by applying it to a stainless steel plate. It was mixed with 1 \u0026micro;L of matrix solution (α-cyano-4-hydroxycinnamic acid [HCCA] in acetonitrile/water/trifluoroacetic acid [50:47.5:2.5]) and dried at room temperature. Data acquisition was carried out using a MALDI-TOF MS spectrometer (Shimadzu-Biotech Corp., Kyoto, Japan) in reflectron mode, with a mass range of 2,000\u0026ndash;20,000 Da. 240 laser shots were performed for each sample (40 shots in 6 positions). The obtained spectra were compared with the Bruker MBT Filamentous Fungi Library 2.0 database. The spectra were analyzed using correlation algorithms (logarithmic score: \u0026ge;2.0 indicating reliable identification) and with a fidelity of 99%. How positive control was used Candida albicans ATCC 10231 (spectrum of reference in databases). And as a negative control, a matrix without sample was used to rule out contamination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eAll statistical analyses were performed in R Studio (v. 4.4.3). Taxonomic relative abundances were preprocessed by calculating the mean abundance of each taxon across all sampling locations. Alpha diversity metrics (observed species, Shannon index, and Simpson index) were computed using the phyloseq R package (v. 1.50.0). Differences in alpha diversity between groups were evaluated using the non-parametric Kruskal-Wallis test, with post-hoc Dunn\u0026rsquo;s test for pairwise comparisons (adjusted via the Benjamini-Hochberg method, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Beta diversity was assessed using Bray-Curtis dissimilarity matrices. Non-metric multidimensional scaling (NMDS) was applied to visualize community dissimilarities, and statistical significance of observed differences was tested with PERMANOVA (999 permutations) using the vegan R package (v. 2.6.10). To further resolve community patterns, a heatmap was generated with the pheatmap package (v. 1.0.12), clustering samples based on Bray-Curtis distances. Intersection patterns of taxa across sampling locations were visualized using an UpSet plot (UpSetR package, v. 1.4.0), chosen over traditional Venn diagrams due to its enhanced readability for complex datasets.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eCharacterization of Anurans\u003c/h2\u003e \u003cp\u003eThe collection sites yielded a total of 20 different species of amphibians, distributed across the five sampling locations (Zamora Chinchipe, Loja, Ca\u0026ntilde;ar, Azuay, and Morona Santiago). The genera more characterized were \u003cem\u003ePristimantis\u003c/em\u003e (16 records), \u003cem\u003eGastrotheca\u003c/em\u003e (5 records), \u003cem\u003eHyloxalus\u003c/em\u003e (3 records), \u003cem\u003eCtenophryne\u003c/em\u003e (3 records), \u003cem\u003eLithobates\u003c/em\u003e (3 records), \u003cem\u003eAdenomera\u003c/em\u003e (1 record), \u003cem\u003eChiasmocleis\u003c/em\u003e (1 record), \u003cem\u003eScinax\u003c/em\u003e (1 record), \u003cem\u003eDendropsophus\u003c/em\u003e (1 record) and \u003cem\u003eDendropsophus\u003c/em\u003e (1 record) (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eSpatial distribution and diversity of anuran species according to location and sector in Zamora Chinchipe, Loja, Ca\u0026ntilde;ar, Azuay and Morona Santiago.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLocation\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSector\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eAnuran spp.\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c5\" namest=\"c4\"\u003e \u003cp\u003eCoordinates\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eLatitude\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eLongitude\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eZamora Chinchipe\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eL\u0026iacute;mite provincial\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis andinognomus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,992947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,145022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis vidua\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,992947\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,145022\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCriadero Piuntza\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLithobates catesbeianus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,870451\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,881814\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePadmi\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eAdenomera hylaedactyla\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,737577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,619146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis diadematus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,737577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,619146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis conspicillatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,737577\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,619146\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eLoja\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eParque cerro Pucar\u0026aacute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLithobates catesbeianus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-4,012724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,195073\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eLithobates catesbeianus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-4,012724\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,195073\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eAbra del Zamora\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis versicolor\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,985234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,145307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis balionotus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,985234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,145307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis samaniegoi\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,985234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,145307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis colodactylus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,985234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,145307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis matildae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,985234\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,145307\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCa\u0026ntilde;ar\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLaguna de Guabizhun\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHyloxalus vertebralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,803048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,936164\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGastrotheca cuencana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,803048\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,936164\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAzuay\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGuangarcucho\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGastrotheca cuencana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,843274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,885605\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGastrotheca cuencana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,843274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,885605\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCtenophryne aequatorialis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,843274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,885605\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCtenophryne aequatorialis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,843274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,885605\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eCtenophryne aequatorialis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,843274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,885605\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLazareto\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGastrotheca cuencana\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,882491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,008925\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHyloxalus vertebralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,882491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,008925\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eHyloxalus vertebralis\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,882491\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,008925\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eChanlud\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis erythros\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,895228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,957036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis erythros\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,895228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,957036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis lutzae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,895228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,957036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis lutzae\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,895228\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,957036\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eQuimsacocha\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eGastrotheca pseustes\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,682961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,033192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eMorona Santiago\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eWap\u0026uacute;\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis conspicillatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,682961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,033192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis conspicillatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,682961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,033192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eChiasmocleis bassleri\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,682961\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,033192\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eScinax cruentommus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-3,034273\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-79,222145\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003ePristimantis conspicillatus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,843274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,885605\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e\u0026nbsp;\u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e\u003cem\u003eDendropsophus bifurcus\u003c/em\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e-2,843274\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e-78,885605\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eGeneral composition of the anuran spp., bacteria, and fungi assemblage\u003c/h2\u003e \u003cp\u003eA notable overall species diversity was observed within the five study areas (Zamora, Chinchipe, Loja, Ca\u0026ntilde;ar, Azuay, and Morona Santiago), where \u003cem\u003ePristimantis conspicillatus\u003c/em\u003e (11.8%) and \u003cem\u003eGastrotheca cuencana\u003c/em\u003e (11.8%) were the most abundant species, followed by \u003cem\u003eHyloxalus vertebralis\u003c/em\u003e (8.8%), \u003cem\u003eCtenophryne aequatorialis\u003c/em\u003e (8.8%), and \u003cem\u003eLithobates catesbeianus\u003c/em\u003e (8.8%), which presented high proportions compared to other identified species. Likewise, species of the genus \u003cem\u003ePristimantis\u003c/em\u003e, such as \u003cem\u003eP. erythros\u003c/em\u003e (5.8%), \u003cem\u003eP. lutzae\u003c/em\u003e (5.8%), \u003cem\u003eP. matildae\u003c/em\u003e (2.9%), and \u003cem\u003eP. colodactylus\u003c/em\u003e (2.9%), are well represented in the sample, demonstrating the high richness of this genus in the study locations (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Microbial associations were identified in \u003cem\u003eGastrotheca cuencana\u003c/em\u003e (Azuay) with the prevalence of \u003cem\u003ePseudomonas antarctica\u003c/em\u003e and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e. In contrast, \u003cem\u003eHyloxalus vertebralis\u003c/em\u003e (Azuay) presented \u003cem\u003eAcinetobacter iwoffii\u003c/em\u003e and \u003cem\u003eStaphylococcus xylosus\u003c/em\u003e. In \u003cem\u003eLithobates catesbeianus\u003c/em\u003e (Loja), a dominance of \u003cem\u003eLactobacillus curvatus\u003c/em\u003e and \u003cem\u003eChryseobacterium joostei\u003c/em\u003e was observed. \u003cem\u003ePristimantis conspicillatus\u003c/em\u003e (Morona Santiago) revealed \u003cem\u003eSerratia marcescens\u003c/em\u003e. Furthermore, in \u003cem\u003ePristimantis diadematus\u003c/em\u003e (Zamora), co-occurrence of \u003cem\u003eBacillus pumilus\u003c/em\u003e and \u003cem\u003ePseudomonas brassicacearum\u003c/em\u003e was recorded. However, in \u003cem\u003ePristimantis samaniegio\u003c/em\u003e (Loja) and \u003cem\u003ePristimantis vidua\u003c/em\u003e (Zamora Chinchipe) no bacterial species were detected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe total relative abundance of bacteria in anurans was mainly composed of species of the genera Pseudomonas, Bacillus, Pantoea, Corynebacterium and Acinetobacter. Within these, \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e was present (28%), followed by \u003cem\u003eAcinetobacter iwoffii\u003c/em\u003e and \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e (14%). The species \u003cem\u003ePseudomonas Antarctica\u003c/em\u003e was present (12%), followed by \u003cem\u003ePseudomonas orientalis\u003c/em\u003e, \u003cem\u003ePantoea agglomerans\u003c/em\u003e and \u003cem\u003ePseudarthrobacter oxydans\u003c/em\u003e in an equal percentage of (10%). While \u003cem\u003ePseudomonas jensenii\u003c/em\u003e and \u003cem\u003eRahnella aquatilis\u003c/em\u003e was evidencied (8%). The species with the lowest percentage were \u003cem\u003ePseudomonas kilonensis\u003c/em\u003e (6%), \u003cem\u003ePseudomonas thivervalensis\u003c/em\u003e, \u003cem\u003eBacillus pumilus\u003c/em\u003e and \u003cem\u003eKluyvera ascorbata\u003c/em\u003e (6%). We must highlight that the bacterial species with intermediate prevalence percentage were \u003cem\u003eBacillus infantis\u003c/em\u003e, \u003cem\u003ePseudomonas azotoformans\u003c/em\u003e, \u003cem\u003eStaphylococcus xylosus\u003c/em\u003e, \u003cem\u003ePseudomonas rhodesiae\u003c/em\u003e, \u003cem\u003ePseudomonas taetrolens\u003c/em\u003e, \u003cem\u003eLactobacillus curvatus\u003c/em\u003e and \u003cem\u003eAeromonas bestiarum\u003c/em\u003e in similar values ​​of (4%). The least prevalent species were \u003cem\u003eCorynebacterium striatum\u003c/em\u003e, \u003cem\u003eChryseobacterium joostei\u003c/em\u003e, \u003cem\u003ePseudomonas japonica\u003c/em\u003e, \u003cem\u003eSerratia marcescens\u003c/em\u003e, \u003cem\u003eComamonas testosteroni\u003c/em\u003e, \u003cem\u003eProteus mirabilis\u003c/em\u003e, \u003cem\u003ePseudomonas brassicacearum\u003c/em\u003e, \u003cem\u003ePseudomonas extremerientalis\u003c/em\u003e, and \u003cem\u003eProvidencia rettgeri\u003c/em\u003e in (2%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eOn the other hand, the relative abundance of total fungal species in anurans showed a higher prevalence of \u003cem\u003eHortaea werneckii\u003c/em\u003e (26.4%), recognized for its ability to colonize the skin of amphibians and that could be involved in cutaneous infection processes, followed by \u003cem\u003eFusarium solani\u003c/em\u003e (20.5%), \u003cem\u003eSyncephalastrum\u003c/em\u003e spp. (20.5%), \u003cem\u003eFusarium concentricum\u003c/em\u003e (8.8%). Species such as \u003cem\u003eFusarium oxysporum\u003c/em\u003e, \u003cem\u003eFusarium proliferatum\u003c/em\u003e and \u003cem\u003eAspergillus niger\u003c/em\u003e (5.8%) showed a notable abundance. Finally, the species that showed less abundance were represented by \u003cem\u003eFusarium\u003c/em\u003e spp. (2.9%) and \u003cem\u003eBipolaris\u003c/em\u003e spp. (2.9%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eRelative composition of the bacterial population\u003c/h2\u003e \u003cp\u003eThe total number of detections was 92, distributed among 29 bacterial species on the skin of anurans spp. at the sampling sites was 29, distributed in the provinces of Zamora, Chinchipe, Loja, Ca\u0026ntilde;ar, Azuay and Morona Santiago (\u003cb\u003eSupplementary Fig.\u0026nbsp;1\u003c/b\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eSubkingdom Negibacteria\u003c/h2\u003e \u003cp\u003eThis subkingdom comprised a total of 79.31% of all observed species, including \u003cem\u003eAcinetobacter iwoffii\u003c/em\u003e (3.45%), \u003cem\u003eChryseobacterium joostei\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas antarctica\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas azotoformans\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas japonica\u003c/em\u003e (3.45%), among others.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eSubkingdom Posibacteria\u003c/h2\u003e \u003cp\u003eA total of 6 species were observed (20.69%). They were represented by: \u003cem\u003eBacillus infantis\u003c/em\u003e (3.45%), \u003cem\u003eCorynebacterium striatum\u003c/em\u003e (3.45%), \u003cem\u003eStaphylococcus xylosus\u003c/em\u003e (3.45%), \u003cem\u003eBacillus pumilus\u003c/em\u003e (3.45%), \u003cem\u003ePseudarthrobacter oxydans\u003c/em\u003e (3.45%), and \u003cem\u003eLactobacillus curvatus\u003c/em\u003e (3.45%).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003ePhylum\u003c/h2\u003e \u003cdiv id=\"Sec20\" class=\"Section3\"\u003e \u003ch2\u003eProteobacteria\u003c/h2\u003e \u003cp\u003eThis phylum was the dominant one in the samples from the studied locations, exhibiting 22 species and representing 75.86% of the bacterial composition. Species such as \u003cem\u003eAcinetobacter iwoffii\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas Antarctica\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas azotoformans\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas japonica\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas jensenii\u003c/em\u003e (3.45%), \u003cem\u003ePseudomonas kilonensis\u003c/em\u003e (3.45%), and \u003cem\u003ePseudomonas orientalis\u003c/em\u003e (3.45%) stood out here (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003eFirmicutes\u003c/h2\u003e \u003cp\u003eThese covered of a total of 4 species that together represented 13.79% of the samples and consisted of \u003cem\u003eBacillus infantis\u003c/em\u003e (3.45%), \u003cem\u003eStaphylococcus xylosus\u003c/em\u003e (3.45%), \u003cem\u003eBacillus pumilus\u003c/em\u003e (3.45%), and \u003cem\u003eLactobacillus curvatus\u003c/em\u003e (3.45%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003eActinobacteria\u003c/h2\u003e \u003cp\u003eThis phylum presented intermediate abundance among the total species with a (6.90%). \u003cem\u003eCorynebacterium striatum\u003c/em\u003e (3.45%) and Pseudarthrobacter oxydans (3.45%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e \u003ch2\u003eBacteroidetes\u003c/h2\u003e \u003cp\u003eThis phylum showed the lowest abundance of species, with a total representation of 3.45%), with \u003cem\u003eChryseobacterium joostei\u003c/em\u003e (3.45%) (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003eComposition of the Fungal Assemblage\u003c/h2\u003e \u003cp\u003eA total of 41 fungal detections were obtained on the skin of anuran spp., distributed across 9 species, in the provinces of Zamora Chinchipe, Loja, Ca\u0026ntilde;ar, Azuay, and Morona Santiago (Supplementary Fig.\u0026nbsp;2).\u003c/p\u003e \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e \u003ch2\u003eSubkingdom Dikarya\u003c/h2\u003e \u003cdiv id=\"Sec26\" class=\"Section4\"\u003e \u003ch2\u003eAscomycota\u003c/h2\u003e \u003cp\u003eWithin Ascomycota, Sordariomycetes was the dominant class at the sampling locations, with an average abundance of 44.12%, followed by Dothideomycetes (29.41%), and Eurotiomycetes in the lowest range (5.88%).\u003c/p\u003e \u003cp\u003eThe predominant genus was Fusarium (44.12%), represented by \u003cem\u003eFusarium solani\u003c/em\u003e (20.59%), \u003cem\u003eFusarium concentricum\u003c/em\u003e (8.82%), \u003cem\u003eFusarium oxysporum\u003c/em\u003e (5.88%), \u003cem\u003eFusarium proliferatum\u003c/em\u003e (5.88%), and \u003cem\u003eFusarium\u003c/em\u003e spp. (2.94%). The genus Hortaea showed average values ​​of 26.47%, with \u003cem\u003eHortaea werneckii\u003c/em\u003e (26.47%). The genus Aspergillus (5.88%) was represented \u003cem\u003eAspergillus\u003c/em\u003e spp. Finally, the genus Bipolaris was the least predominant with (2.94%) with \u003cem\u003eBipolaris\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e \u003ch2\u003eMucoromycotina\u003c/h2\u003e \u003cp\u003eMucromycotina was the least predominant class at the sampling locations (20.59%) where Syncephalastrum showed (20.59%), which was represented by \u003cem\u003eSyncephalastrum\u003c/em\u003e spp. (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, \u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec28\" class=\"Section2\"\u003e \u003ch2\u003eAlpha Diversity Patterns Across Localities\u003c/h2\u003e \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e \u003ch2\u003eBacteria\u003c/h2\u003e \u003cp\u003eBacterial alpha diversity, quantified using the Shannon, Simpson, and Chao1 indices (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e), demonstrated marked variability across localities. Azuay exhibited the highest overall diversity (Shannon\u0026thinsp;=\u0026thinsp;2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08; Simpson\u0026thinsp;=\u0026thinsp;0.88\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01) and richness (Chao1\u0026thinsp;=\u0026thinsp;17.7\u0026thinsp;\u0026plusmn;\u0026thinsp;3.3), closely followed by Zamora (Shannon\u0026thinsp;=\u0026thinsp;2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17; Simpson\u0026thinsp;=\u0026thinsp;0.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02; Chao1\u0026thinsp;=\u0026thinsp;15.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.1). Loja showed intermediate diversity (Shannon\u0026thinsp;=\u0026thinsp;2.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15; Simpson\u0026thinsp;=\u0026thinsp;0.86\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) but lower richness (Chao1\u0026thinsp;=\u0026thinsp;9.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1.6). In contrast, Morona displayed reduced diversity (Shannon\u0026thinsp;=\u0026thinsp;1.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07; Simpson\u0026thinsp;=\u0026thinsp;0.77\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02) and the lowest richness (Chao1\u0026thinsp;=\u0026thinsp;7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;1.3). Notably, Ca\u0026ntilde;ar presented stark contrasts: one sample showed high richness (Chao1\u0026thinsp;=\u0026thinsp;21) with moderate diversity (Shannon\u0026thinsp;=\u0026thinsp;1.79; Simpson\u0026thinsp;=\u0026thinsp;0.83), while others were species-poor (Chao1\u0026thinsp;=\u0026thinsp;4.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.6; Shannon\u0026thinsp;=\u0026thinsp;1.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.002; Simpson\u0026thinsp;=\u0026thinsp;0.72\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01). Kruskal-Wallis tests confirmed significant differences across localities for Shannon and Simpson indices (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05), with post-hoc Benjamini-Hochberg adjustments highlighting Ca\u0026ntilde;ar\u0026rsquo;s differences from Azuay.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e\n\u003ch3\u003eFungi\u003c/h3\u003e\n\u003cp\u003eFungal alpha diversity, revealed pronounced contrasts among localities (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e). Loja exhibited the highest richness (Chao1\u0026thinsp;=\u0026thinsp;7.4\u0026thinsp;\u0026plusmn;\u0026thinsp;3.0) and diversity (Shannon\u0026thinsp;=\u0026thinsp;1.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10; Simpson\u0026thinsp;=\u0026thinsp;0.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02), followed by Azuay (Chao1\u0026thinsp;=\u0026thinsp;6.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2.3; Shannon\u0026thinsp;=\u0026thinsp;1.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17; Simpson\u0026thinsp;=\u0026thinsp;0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03). Zamora displayed moderate diversity (Shannon\u0026thinsp;=\u0026thinsp;1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32; Simpson\u0026thinsp;=\u0026thinsp;0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11) but lower richness (Chao1\u0026thinsp;=\u0026thinsp;4.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1.8). In contrast, Morona showed reduced diversity (Shannon\u0026thinsp;=\u0026thinsp;1.02\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05; Simpson\u0026thinsp;=\u0026thinsp;0.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03) and minimal richness (Chao1\u0026thinsp;=\u0026thinsp;3\u0026thinsp;\u0026plusmn;\u0026thinsp;0). Notably, Ca\u0026ntilde;ar was statistically distinct (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, Kruskal-Wallis with Benjamini-Hochberg adjustment), with no detectable fungal diversity (Shannon\u0026thinsp;=\u0026thinsp;0; Simpson\u0026thinsp;=\u0026thinsp;0) and extremely low richness (Chao1\u0026thinsp;=\u0026thinsp;1\u0026thinsp;\u0026plusmn;\u0026thinsp;0), indicating a near-absence of viable fungal communities. Post-hoc analyses confirmed Ca\u0026ntilde;ar\u0026rsquo;s divergence from all other localities, which exhibited overlapping but variable profiles.\u003c/p\u003e \u003cdiv id=\"Sec31\" class=\"Section2\"\u003e \u003ch2\u003eBeta Diversity and Community Structure\u003c/h2\u003e \u003cdiv id=\"Sec32\" class=\"Section3\"\u003e \u003ch2\u003eBacteria\u003c/h2\u003e \u003cp\u003ePairwise PERMANOVA analysis, based on Bray-Curtis dissimilarity, revealed distinct patterns of bacterial community differentiation between localities. While adjusted \u003cem\u003ep\u003c/em\u003e-values (\u003cem\u003ep\u003c/em\u003e-adjusted\u0026thinsp;=\u0026thinsp;1) did not reach statistical significance after Benjamini-Hochberg correction, effect size metrics (R\u0026sup2;) and F-statistics highlighted biologically meaningful trends. The NMDS ordination plot, based on Bray-Curtis dissimilarity, corroborated pairwise PERMANOVA results, revealing distinct spatial clustering of bacterial communities (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eZamora, Loja, and Morona formed well-separated clusters, with 95% confidence ellipses showing minimal overlap, underscoring their unique taxonomic assemblages. This segregation aligns with their divergent alpha diversity profiles and suggests strong environmental filtering or niche specialization. The hierarchical clustering patterns observed in the Bray-Curtis-derived heatmap (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003e) further validated the spatial structuring of bacterial communities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAn UpSet plot analysis showed the distribution of bacterial species found on anuran skin at the study locations (Morona Santiago, Azuay, Ca\u0026ntilde;ar, Loja, and Zamora Chinchipe). It revealed the presence of eight bacterial species shared by all locations. This finding suggests the existence of widely distributed taxa, potentially adapted to a diverse range of environmental conditions. Certain locations shared certain bacterial species, and in several cases, taxa existed that were unique to specific combinations from two locations. Furthermore, each location harbored potentially unique bacteria, demonstrating a degree of microbial endemism. This differentiation could be associated with variation in the composition of the amphibian community (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cdiv id=\"Sec33\" class=\"Section4\"\u003e \u003ch2\u003eFungi\u003c/h2\u003e \u003cp\u003ePairwise PERMANOVA analysis, based \u0026ldquo;n\u0026rdquo; Bray-Curtis dissimilarity, revealed biologically meaningful differentiation in fungal community composition across localities, despite the lack of statistical significance after Benjamini-Hochberg correction (all adjusted p-values\u0026thinsp;=\u0026thinsp;1). Several comparisons showed high R\u0026sup2; values, and large F-statistics, indicating strong effect sizes and potential ecological relevance. The NMDS ordination plot (based on Bray Curtis dissimilarity; Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e) supported these trends, revealing partial spatial separation of fungal communities.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eAlthough some confidence ellipses overlapped, notable clustering was observed, particularly among the Zamora, Loja, and Morona samples, suggesting underlying ecological or host-driven factors shaping fungal assemblages. Complementarily, the UpSet plot (Fig.\u0026nbsp;\u003cspan refid=\"Fig14\" class=\"InternalRef\"\u003e14\u003c/span\u003e) highlighted the distribution of fungal taxa across localities. Three fungal taxa were shared among Morona, Zamora, Azuay and Loja, suggesting the presence of a core mycobiome possibly adapted to a broad range of environmental or host-related conditions. However, numerous taxa were exclusive to specific site combinations, and each locality also harbored unique taxa, indicating a high degree of microbial endemism and potential ecological specialization. The heatmap based on Jaccard dissimilarity (Fig.\u0026nbsp;\u003cspan refid=\"Fig15\" class=\"InternalRef\"\u003e15\u003c/span\u003e) further reinforced the observed spatial structure. Hierarchical clustering grouped localities according to similarities in fungal composition, reflecting consistent biogeographic patterns. Together, these findings underscore a non-random distribution of skin-associated fungi in anurans across the Andean and Amazonian transition zone.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides an overview of the diversity of bacterial and fungal communities on the skin of anuran spp., where each habitat and geographic location can serve as a selective filter determining local microbial diversity, a phenomenon known as the Baas-Becking principle [\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Previous studies have shown that the skin microbiota profile is influenced by the phylogenetic identity of the host amphibian [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. Our understanding of the composition and role of the microbiota associated with plants and animals, including humans, is increasing due to the application of technologies such as MALDI-TOF MS. Microbial communities living on animal skin represent an interesting scenario, as they are continuously exposed to the influence of the external environment. However, these studies have largely been limited to the human skin microbiome [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Among wild animals, amphibians, due to their absence of fur or feathers, provide an excellent model system to study skin-associated microbial communities, which are thought to mediate disease susceptibility by providing the first line of defense against pathogens [\u003cspan additionalcitationids=\"CR54\" citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. Knowledge about host-associated microbial communities can assist with conservation actions for endangered species, as well as play a role as a bioindicator of a pathogen-free population [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Therefore, the aim of this study was to analyze the diversity of bacteria and fungi on the skin of anuran spp. in wild habitats using MALDI-TOF MS mass spectrometry in five locations: Azuay, Ca\u0026ntilde;ar, Loja, Morona Santiago and Zamora Chinchipe in the Republic of Ecuador.\u003c/p\u003e \u003cp\u003eThe diversity of anurans in Ecuador is recognized as one of the highest in the world due to its complex topography and habitats heterogeneity [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], presenting unique biogeographic patterns that vary significantly between provinces. Analysis of amphibian species composition at Zamora Chinchipe, Loja, Ca\u0026ntilde;ar, Azuay, and Morona Santiago highlighted endemism and anthropogenic pressures. The observed data showed a marked heterogeneity in anuran species richness, with emphasis on genera such as \u003cem\u003ePristimantis\u003c/em\u003e, \u003cem\u003eGastrotheca\u003c/em\u003e, and \u003cem\u003eHyloxalus\u003c/em\u003e, and the presence of invasive species such as \u003cem\u003eLithobates catesbeianus\u003c/em\u003e. In Zamora Chinchipe, the presence of species such as \u003cem\u003ePristimantis andinognomus\u003c/em\u003e and \u003cem\u003ePristimantis vidua\u003c/em\u003e, which are endemic to the montane forests of southern Ecuador [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], suggests a high specialization to humid microhabitats between 1,800\u0026ndash;2,500 m. The coexistence of \u003cem\u003eP. diadematus\u003c/em\u003e and \u003cem\u003eP. conspicillatus\u003c/em\u003e, both species associated with lower vegetation strata, and \u003cem\u003eAdenomera hylaedactyla\u003c/em\u003e, which is typical of floodable soils [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e], reflected the ecological heterogeneity of the region. However, the detection of \u003cem\u003eLithobates catesbeianus\u003c/em\u003e, an invasive species [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e], in riparian areas indicates possible anthropogenic alterations, since this anuran competes with native species for resources. In the province of Loja, the dominance of \u003cem\u003ePristimantis\u003c/em\u003e with the species \u003cem\u003eversicolor\u003c/em\u003e, \u003cem\u003ebalionotus\u003c/em\u003e, and \u003cem\u003esamaniegoi\u003c/em\u003e highlights the role of p\u0026aacute;ramos and montane forests as centers of speciation [\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. \u003cem\u003ePristimantis matildae\u003c/em\u003e, recently described in the Tapichalaca Reserve [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e], evidences the presence of microendemisms critical for conservation. However, the recurrence of \u003cem\u003eLithobates catesbeianus\u003c/em\u003e in multiple records suggests a worrying expansion of this species, associated with aquaculture activities [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. In Ca\u0026ntilde;ar and Azuay, the presence of \u003cem\u003eGastrotheca cuencana\u003c/em\u003e, an ovoviviparous species endemic to the central Andes [\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e] and \u003cem\u003eHyloxalus vertebralis\u003c/em\u003e associated with ravines in cloud forests [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e, \u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e] reflects adaptations to cold and humid environments (\u0026gt;\u0026thinsp;3,000 m). The abundance of \u003cem\u003eCtenophryne aequatorialis\u003c/em\u003e in Azuay, a cryptically red microhylid, suggests evolutionary strategies to avoid predators in fragmented habitats [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. The absence \u003cem\u003eof Lithobates catesbeianus\u003c/em\u003e in these sites could be related to thermal limitations, although physiological studies are required to confirm this. Meanwhile, in Morona Santiago, the coexistence of \u003cem\u003ePristimantis conspicillatus\u003c/em\u003e shared with Zamora Chinchipe with \u003cem\u003eScinax cruentomma\u003c/em\u003e (typical of the Amazonian lowlands) [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e], indicates a transition zone between the Andes and the Amazon. \u003cem\u003eChiasmocleis bassleri\u003c/em\u003e, a fossorial microhylid, highlights the importance of non-flooded soils in primary forests [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. However, the absence of records of high Andean species suggests a biogeographic boundary defined by an altitude\u0026thinsp;\u0026lt;\u0026thinsp;1,500 masl in this region. When comparing sampling locations, significant differences were observed where Zamora Chinchipe and Loja shared a higher richness of \u003cem\u003ePristimantis\u003c/em\u003e (6 and 5 species, respectively), while Azuay stands out for the diversity of \u003cem\u003eGastrotheca\u003c/em\u003e and \u003cem\u003eCtenophryne\u003c/em\u003e. This reflects altitudinal gradients where Zamora Chinchipe (800\u0026ndash;2,500 masl) and Loja (1,500\u0026ndash;3,000 masl) host mid-mountain species, while Azuay (\u0026gt;\u0026thinsp;3,000 masl) presents high Andean taxa. The presence of \u003cem\u003eLithobates catesbeianus\u003c/em\u003e in Zamora Chichipe and Loja, but not in Azuay, suggests that its invasion is limited by climatic or anthropogenic factors. Microendemic species, such as \u003cem\u003ePristimantis matildae\u003c/em\u003e (Loja) and \u003cem\u003eGastrotheca cuencana\u003c/em\u003e (Azuay-Ca\u0026ntilde;ar), face critical risks from deforestation and the effects of climate change. For example, 30% of the cloud forests in Azuay have been converted to grasslands, reducing habitats for \u003cem\u003eHyloxalus vertebralis\u003c/em\u003e [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe environmental microbiota is a critical component of ecosystem health, particularly in amphibians, where skin infections contribute significantly to global population decline. In Azuay, the bacterial community identified on the skin of anuran spp. was characterized by the presence of 15 species distributed in relative percentages ranging. The most abundant taxon was \u003cem\u003eAcinetobacter iwoffii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;7), followed by \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;6), \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4), and \u003cem\u003eSerratia marcescens\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1). The high frequency of \u003cem\u003eA. iwoffii\u003c/em\u003e is relevant, since species of the genus \u003cem\u003eAcinetobacter\u003c/em\u003e have been associated with skin infections in anuran spp., particularly under conditions of altered natural microbiome [\u003cspan additionalcitationids=\"CR69\" citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e]. In anurans, an imbalance in the cutaneous microbiota could facilitate colonization by this pathogen, leading to dermatitis or systemic infections. \u003cem\u003eP. fluorescens\u003c/em\u003e has been reported to cause necrotic skin lesions in \u003cem\u003eLithobates catesbeianus\u003c/em\u003e, especially in eutrophic environments [\u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. Its high prevalence suggests a risk for anurans in altered habitats. However, studies by [\u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e] on \u003cem\u003eGastroteca\u003c/em\u003e spp. showed that \u003cem\u003eP. fluorescens\u003c/em\u003e had inhibitory functions on the chytrid fungus \u003cem\u003eBatrachochytrium dendrobatidis\u003c/em\u003e (Bd), which is linked to many declines in anuran populations. \u003cem\u003eSerratia marcescens\u003c/em\u003e has been reported to induce deep and extensive ulcers in the tree frog (\u003cem\u003eLitoria caerulea\u003c/em\u003e) and is considered an important pathogen. Likewise, the various representatives of the genus \u003cem\u003ePseudomonas\u003c/em\u003e, which include \u003cem\u003eP. azotoformans\u003c/em\u003e, \u003cem\u003eP. japonica\u003c/em\u003e, \u003cem\u003eP. jensenii\u003c/em\u003e, \u003cem\u003eP. kilonensis\u003c/em\u003e, \u003cem\u003eP. orientalis\u003c/em\u003e, and \u003cem\u003eP. thivervalensis\u003c/em\u003e, constituted an important part of the microbial community in Azuay. This genus is known for its metabolic versatility and its ability to produce bioactive metabolites. However, in scenarios where there is an imbalance in the microbial community, some species of \u003cem\u003ePseudomonas\u003c/em\u003e can act as opportunistic pathogens, generating skin infections, which, in combination with environmental stress, can trigger complex clinical pictures in anurans [\u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e]. In Zamora Chinchipe, anuran populations presented bacterial communities composed of 13 taxa, where the \u003cem\u003ePseudomonas\u003c/em\u003e genera predominated along with other environmental taxa. This location highlighted the presence of \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3) and \u003cem\u003ePseudomonas antarctica\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), as well as \u003cem\u003ePantoea agglomerans\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) and \u003cem\u003eRahnella aquatilis\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2). The presence of \u003cem\u003ePantoea agglomerans\u003c/em\u003e is of particular interest because despite being a common inhabitant of the environment, it bacteria has been implicated in opportunistic infections in animals and humans, and can cause skin irritations [\u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. \u003cem\u003eP. chlororaphis\u003c/em\u003e has been reported to secrete phenazines, antifungal compounds that inhibit Bd [\u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e]. However, in Zamora Chinchipe, its high abundance could displace commensal microbiota, increasing susceptibility to secondary infections. Furthermore, the diversity of \u003cem\u003ePseudomonas\u003c/em\u003e in Zamora Chinchipe suggests a dynamic bacterial ecosystem where competition between commensal and pathogenic species can determine the health status of anuran skin. The presence of \u003cem\u003ePseudomonas orientalis, Pseudomonas rhodesiae\u003c/em\u003e and \u003cem\u003ePseudomonas taetrolens\u003c/em\u003e in anuran spp. in smaller proportions reinforces the idea that microbial dysbiosis could be related to infectious outbreaks under conditions of environmental alteration, such as changes in temperature or pollution [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e]. On the other hand, Loja presented a community composed of 9 taxa, with a predominance of bacteria belonging to the genera \u003cem\u003ePseudomonas\u003c/em\u003e and \u003cem\u003ePseudarthrobacter\u003c/em\u003e, as well as lactobacilli. An equitable distribution of bacterial species was also observed, where \u003cem\u003ePseudomonas antarctica\u003c/em\u003e, \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e and \u003cem\u003ePseudarthrobacter oxydans\u003c/em\u003e. Additionally, \u003cem\u003eLactobacillus curvatus\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2) and \u003cem\u003eLactobacillus jensenii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1) were detected. The presence of lactobacilli on the skin of anurans could have a protective effect, since these microorganisms are known to produce lactic acid and bacteriocins, which inhibit the growth of pathogens and modulate the host immune response [\u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. However, the coexistence with potential pathogens such as \u003cem\u003eP. chlororaphis\u003c/em\u003e (a pathogen that causes dermatitis in amphibians) suggests that an imbalance in the bacterial community could trigger skin infections [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Similarly, \u003cem\u003eAeromonas bestiarum\u003c/em\u003e is a recognized pathogen in fish and amphibians, responsible for causing septicemia and dermatitis, which could pose a high risk to anuran populations in this region, under adverse environmental conditions. The coexistence of these pathogenic taxa with others that act as commensals raises the need to evaluate the microbial balance in anuran skin, since dysbiosis can facilitate the transition from symbiotic relationships to pathological states.\u003c/p\u003e \u003cp\u003eThe presence of bacteria with biotechnological potential, such as \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e in Azuay and \u003cem\u003ePseudarthrobacter oxydans\u003c/em\u003e in Loja and Ca\u0026ntilde;ar, represents an opportunity for the development of bioremediation and biological control strategies. \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e has been the subject of numerous studies due to its ability to produce natural antibiotics and toxic compound-degrading enzymes, which could be exploited for the decontamination of environments affected by agrochemicals and other pollutants [\u003cspan citationid=\"CR82\" class=\"CitationRef\"\u003e82\u003c/span\u003e]. Similarly, \u003cem\u003ePseudarthrobacter oxydans\u003c/em\u003e has demonstrated potential in the degradation of hydrocarbons and in promoting plant growth in contaminated soils, making it an attractive candidate for biotechnological applications in environmental contexts [\u003cspan citationid=\"CR83\" class=\"CitationRef\"\u003e83\u003c/span\u003e]. On the other hand, some studies have shown that alterations in bacterial composition can facilitate the invasion of external pathogens, such as \u003cem\u003eBatrachochytrium dendrobatidis\u003c/em\u003e, the etiological agent of chytridiomycosis, which has significantly contributed to the global decline of amphibians [\u003cspan citationid=\"CR84\" class=\"CitationRef\"\u003e84\u003c/span\u003e]. It has also been suggested that variability in bacterial composition can influence the immune response of anurans, affecting their ability to resist secondary infections caused by opportunistic bacteria [\u003cspan citationid=\"CR85\" class=\"CitationRef\"\u003e85\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding the observed fungal communities, these revealed seven fungal taxa in Azuay: \u003cem\u003eAspergillus niger\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eBipolaris sp\u003c/em\u003e. (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eFusarium concentricum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eFusarium solani\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eHortaea werneckii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3), \u003cem\u003eSyncephalastrum\u003c/em\u003e sp. (n\u0026thinsp;=\u0026thinsp;1) and \u003cem\u003eFusarium proliferatum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1). The high prevalence of \u003cem\u003eHortaea werneckii\u003c/em\u003e is significant, since this fungus, in addition to being involved in the pathogenesis of tinea nigra in humans, can cause skin disorders in amphibians, promoting the appearance of hyperpigmented spots and keratosis [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e, \u003cspan citationid=\"CR87\" class=\"CitationRef\"\u003e87\u003c/span\u003e]. The presence of \u003cem\u003eFusarium concentricum\u003c/em\u003e, \u003cem\u003eFusarium solani\u003c/em\u003e and \u003cem\u003eFusarium concentricum\u003c/em\u003e in Azuay, highlights the possibility of fusariosis, a disease characterized by the formation of erythematous and ulcerated lesions that can affect both the skin and underlying structures in amphibians [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. The detection of \u003cem\u003eBipolaris\u003c/em\u003e spp., a fungus that in humans is associated with photoreaction and subcutaneous mycosis, reinforces the need to consider its role as a potential pathogen in the skin of anurans [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. Furthermore, the presence of \u003cem\u003eAspergillus niger\u003c/em\u003e in Azuay not only indicates its biotechnological potential (given its ability to produce pectins and industrial enzymes), but also alerts to the risk of cutaneous aspergillosis in immunocompromised situations [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. In Loja, six fungal taxa were reported: \u003cem\u003eFusarium oxysporum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eFusarium solani\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eFusarium\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eHortaea werneckii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eSyncephalastrum\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;2), and \u003cem\u003eAspergillus niger\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1). In this locality, the highest abundance corresponds to \u003cem\u003eSyncephalastrum\u003c/em\u003e spp. indicating that this taxon could be playing a predominant role in the cutaneous fungal community. Although \u003cem\u003eSyncephalastrum\u003c/em\u003e spp. It is generally considered a saprophytic fungus, its involvement in invasive mycoses in contexts of immunosuppression is cause for alarm, because skin infections can be complicated in environments with high humidity and stress in the host. On the other hand, the presence of \u003cem\u003eFusarium oxysporum\u003c/em\u003e and \u003cem\u003eFusarium solani\u003c/em\u003e, reinforces the concern about the possible incidence of fusariosis [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. The detection of \u003cem\u003eAspergillus niger\u003c/em\u003e is relevant, since, in contexts of microenvironmental imbalance, it can act as an opportunistic pathogen, causing cutaneous and systemic aspergillosis in compromised individuals [\u003cspan citationid=\"CR90\" class=\"CitationRef\"\u003e90\u003c/span\u003e]. In the locality of Ca\u0026ntilde;ar, the fungal community was summarized in two taxon: \u003cem\u003eFusarium concentricum\u003c/em\u003e and \u003cem\u003eFusarium proliferatum\u003c/em\u003e with a representation of 50% each. Although the isolation of a two taxon could be interpreted as low diversity, the exclusive presence of \u003cem\u003eF. concentricum\u003c/em\u003e and \u003cem\u003eF. proliferatum\u003c/em\u003e is of great importance, since these fungus has been associated with skin infections and can produce mycotoxins that affect skin integrity. Furthermore, studies have shown that certain members of the \u003cem\u003eFusarium\u003c/em\u003e complex have a high invasive capacity, which could lead to dermatomycosis in anurans, especially under conditions of environmental stress or previous lesions in the epidermis [\u003cspan citationid=\"CR91\" class=\"CitationRef\"\u003e91\u003c/span\u003e]. In the locality of Zamora Chinchipe, the data indicated the presence of six fungal taxa: \u003cem\u003eSyncephalastrum\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eHortaea werneckii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eFusarium oxysporum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eFusarium solani\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;3), \u003cem\u003eFusarium concentricum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1) and \u003cem\u003eFusarium proliferatum\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1). The high prevalence of \u003cem\u003eFusarium solani\u003c/em\u003e is particularly relevant since this fungus is known to be an etiological agent in cutaneous fungal infections including amphibians, producing keratomycosis and cutaneous fusariosis, conditions that worsen in the presence of environmental stress or skin lesions [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e]. Furthermore, \u003cem\u003eFusarium proliferatum\u003c/em\u003e, was present in Zamora chinchipe, this is an opportunistic pathogen that produces mycotoxins and can contribute to dermatomycosis, affecting the cutaneous integrity of anurans [\u003cspan citationid=\"CR89\" class=\"CitationRef\"\u003e89\u003c/span\u003e]. The presence of \u003cem\u003eHortaea werneckii\u003c/em\u003e, a halophytic fungus, suggests that under specific conditions it could contribute to alterations in the cutaneous barrier of amphibians, generating hyperpigmentation or irritation [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. Likewise, \u003cem\u003eSyncephalastrum\u003c/em\u003e spp., although less reported in skin infections, has been documented as a causative agent of mycosis in immunocompromised patients, which opens the possibility that a similar picture may manifest in debilitated anurans [\u003cspan citationid=\"CR92\" class=\"CitationRef\"\u003e92\u003c/span\u003e]. Finally, in Morona Santiago, the fungal community was composed of six taxa: \u003cem\u003eFusarium solani\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;2), \u003cem\u003eHortaea werneckii\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;4), \u003cem\u003eSyncephalastrum\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;3), \u003cem\u003eAspergillus Niger\u003c/em\u003e (n\u0026thinsp;=\u0026thinsp;1), \u003cem\u003eBipolaris\u003c/em\u003e spp. (n\u0026thinsp;=\u0026thinsp;2) and \u003cem\u003eFusarium proliferatum\u003c/em\u003e. The predominance of \u003cem\u003eHortaea werneckii\u003c/em\u003e in Morona is of particular interest, since its high abundance may predispose to the appearance of skin infections [\u003cspan citationid=\"CR86\" class=\"CitationRef\"\u003e86\u003c/span\u003e]. The joint presence of \u003cem\u003eFusarium solani\u003c/em\u003e in and \u003cem\u003eSyncephalastrum\u003c/em\u003e spp. in Morona Santiago suggests a complex fungal ecosystem, in which the interaction between pathogenic and saprotrophic fungi could influence the susceptibility of anurans to diseases such as fusariosis and syncephalasrosis, conditions that have been documented in clinical and experimental studies [\u003cspan citationid=\"CR88\" class=\"CitationRef\"\u003e88\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe detection of fungal taxa with biotechnological potential, such as \u003cem\u003eAspergillus niger\u003c/em\u003e in Azuay and Loja, and the presence of \u003cem\u003eFusarium\u003c/em\u003e species in various locations, offer interesting opportunities for the development of industrial and environmental applications. \u003cem\u003eAspergillus niger\u003c/em\u003e, for example, is widely recognized for its ability to produce enzymes, organic acids and secondary compounds of industrial relevance, which has allowed its application in the production of pectins, citrates and other biotechnological products [\u003cspan citationid=\"CR93\" class=\"CitationRef\"\u003e93\u003c/span\u003e, \u003cspan citationid=\"CR94\" class=\"CitationRef\"\u003e94\u003c/span\u003e]. Similarly, some \u003cem\u003eFusarium\u003c/em\u003e isolates have shown the ability to degrade toxic compounds and participate in bioremediation processes, which could be used for the treatment of contaminated environments, always considering the duality of these fungi as pathogens and potential agents in biotechnological processes [\u003cspan citationid=\"CR95\" class=\"CitationRef\"\u003e95\u003c/span\u003e, \u003cspan citationid=\"CR96\" class=\"CitationRef\"\u003e96\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThese findings highlight the importance of multimethod approaches, which are critical for biodiversity studies, particularly in underexplored substrates and habitats. Specifically, MALDI-TOF MS analysis of skin swabbing in anurans is considered only the first step toward revealing the bacterial and fungal biodiversity inhabiting the skin of these amphibians in the Azuay, Ca\u0026ntilde;ar, Loja, Morona Santiago, and Zamora Chinchipe locations. Future field campaigns could provide more data through metabarcoding studies to observe potential biotrophic symbionts and parasites. Regarding alpha and beta diversity patterns, differences were found between some sampling locations that varied significantly by geographic location. Therefore, it is essential to continue research to establish the causal relationship between bacterial and fungal composition, the presence of pathogens, and their possible relationship with the development of skin diseases in amphibians. This research, in turn, can guide intervention and risk mitigation strategies in ecosystems affected by human activity and climate change.\u003c/p\u003e \u003cp\u003eThis work represents a first look at bacterial and fungal diversity in a little-studied substrate: the skin of wild anurans. Additional studies are needed to better assess this diversity, along with the development of necessary measures for its protection and conservation.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information:\u003c/strong\u003e The online version contains supplementary material available at xxxxxxxxxx.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We would like to thank the Research Department of the Catholic University of Cuenca, associated with the project \u0026quot;Characterization and potential use of the active principles of amphibian secretion through the rescue of ancestral knowledge disclosed No. PICCIITT19-11. We thank Dr. Sergio Covarrubias of the Autonomous University of Zacatecas for his support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution:\u003c/strong\u003e J.S. and J.G. designed the study. F.S., J.S., J.G., R.F., collected the samples. R.F., A.M., M.C., performed the laboratory cultures and isolation. J.S., J.G., and A.V-T. performed MALDI - TOF MS. A.V-T. performed the bioinformatics analyses of the data. G.V-G. and A.V-T. performed the statistical analysis of the data. AV-T. analyzed and discussed the results and wrote the article. All authors have read and agreed to the published version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancing:\u003c/strong\u003e Catholic University of Cuenca PICCITT19 call.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability:\u003c/strong\u003e No datasets were generated or analyzed during the current study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest:\u003c/strong\u003e The authors declare no competing interests\u003c/p\u003e\n"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eScheele BC, Pasmans F, Skerratt LF, Berger L, Martel AN, Beukema W, Canessa S (2019) Amphibian fungal panzootic causes catastrophic and ongoing loss of biodiversity. Science 363:1459-1463. https://doi.org/10.1126/science.aav0379 \u003c/li\u003e\n\u003cli\u003eHuang G, Qu Q, Wang M, Huang M, Zhou W, Wei F (2022) Global landscape of gut microbiome diversity and antibiotic resistomes across vertebrates. 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Journal of Fungi 7:943 https://doi.org/10.3390/jof7110943 \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5921108/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5921108/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eEcuador is recognized for having a high diversity of anuran spp., which are distributed mainly south of the Andes mountains. However, due to its geographic location and accessibility, there are few studies related to these amphibians. The objective of this study was to explore the bacterial and fungal biodiversity present on the skin of wild anuran spp. in the locations of Zamora Chinchipe, Loja, Ca\u0026ntilde;ar Azuay, and Morona Santiago through MALDI-TOF mass spectrometry. This analysis revealed the presence of 29 bacterial taxa and 9 fungal taxa, consisting mainly of: \u003cem\u003ePseudomonas chlororaphis\u003c/em\u003e (28%), \u003cem\u003eAcinetobacter iwoffii\u003c/em\u003e (14%), \u003cem\u003ePseudomonas fluorescens\u003c/em\u003e (14%), and \u003cem\u003eHortaea werneckii\u003c/em\u003e (26.4%), \u003cem\u003eFusarium solani\u003c/em\u003e (20.5%), S\u003cem\u003eyncephalastrum\u003c/em\u003e spp. (20.5%), respectively. Diversity varied across the five sampling locations, with geographic location proving to be a significant driver of diversity. Some of the most abundant bacterial and fungal genera have important associations with skin diseases. This work represents the first glimpse into the complex biodiversity of bacteria and fungi inhabiting this understudied substrate, and further studies will be needed to better understand bacterial and fungal biodiversity at these locations, along with the development of necessary animal protection and conservation measures.\u003c/p\u003e","manuscriptTitle":"Characterization of culturable microbiota associated with the skin of amphibians (Anura) in the southern Andes Mountains of Ecuador","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-02 18:31:03","doi":"10.21203/rs.3.rs-5921108/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-30T09:16:51+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-29T18:10:22+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T13:40:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"171216944057137463922145139537064337217","date":"2025-04-04T08:04:24+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-04T01:00:49+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"163500262453858469819505891769737685355","date":"2025-04-03T17:06:47+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"308889699711923869718087415335953292878","date":"2025-04-02T15:45:20+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-04-01T16:51:13+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-04-01T13:07:48+00:00","index":"","fulltext":""},{"type":"submitted","content":"Microbial Ecology","date":"2025-03-31T20:41:09+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"microbial-ecology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"meco","sideBox":"Learn more about [Microbial Ecology](https://www.springer.com/journal/248)","snPcode":"248","submissionUrl":"https://submission.nature.com/new-submission/248/3","title":"Microbial Ecology","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"bacb5444-46f3-4cdc-85af-ddbd6c9064ed","owner":[],"postedDate":"April 2nd, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-05-26T15:59:03+00:00","versionOfRecord":{"articleIdentity":"rs-5921108","link":"https://doi.org/10.1007/s00248-025-02555-8","journal":{"identity":"microbial-ecology","isVorOnly":false,"title":"Microbial Ecology"},"publishedOn":"2025-05-22 15:56:52","publishedOnDateReadable":"May 22nd, 2025"},"versionCreatedAt":"2025-04-02 18:31:03","video":"","vorDoi":"10.1007/s00248-025-02555-8","vorDoiUrl":"https://doi.org/10.1007/s00248-025-02555-8","workflowStages":[]},"version":"v1","identity":"rs-5921108","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5921108","identity":"rs-5921108","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}