Triassic stem caecilian supports dissorophoid origin of living amphibians.

Living amphibians (Lissamphibia) include frogs and salamanders (Batrachia) and the limbless worm-like caecilians (Gymnophiona). The estimated Palaeozoic era gymnophionan-batrachian molecular divergence1 suggests a major gap in the record of crown lissamphibians prior to their earliest fossil occurrences in the Triassic period2-6. Recent studies find a monophyletic Batrachia within dissorophoid temnospondyls7-10, but the absence of pre-Jurassic period caecilian fossils11,12 has made their relationships to batrachians and affinities to Palaeozoic tetrapods controversial1,8,13,14. Here we report the geologically oldest stem caecilian-a crown lissamphibian from the Late Triassic epoch of Arizona, USA-extending the caecilian record by around 35 million years. These fossils illuminate the tempo and mode of early caecilian morphological and functional evolution, demonstrating a delayed acquisition of musculoskeletal features associated with fossoriality in living caecilians, including the dual jaw closure mechanism15,16, reduced orbits17 and the tentacular organ18. The provenance of these fossils suggests a Pangaean equatorial origin for caecilians, implying that living caecilian biogeography reflects conserved aspects of caecilian function and physiology19, in combination with vicariance patterns driven by plate tectonics20. These fossils reveal a combination of features that is unique to caecilians alongside features that are shared with batrachian and dissorophoid temnospondyls, providing new and compelling evidence supporting a single origin of living amphibians within dissorophoid temnospondyls.

Ongoing declines for the world's amphibians in the face of emerging threats.

Systematic assessments of species extinction risk at regular intervals are necessary for informing conservation action1,2. Ongoing developments in taxonomy, threatening processes and research further underscore the need for reassessment3,4. Here we report the findings of the second Global Amphibian Assessment, evaluating 8,011 species for the International Union for Conservation of Nature Red List of Threatened Species. We find that amphibians are the most threatened vertebrate class (40.7% of species are globally threatened). The updated Red List Index shows that the status of amphibians is deteriorating globally, particularly for salamanders and in the Neotropics. Disease and habitat loss drove 91% of status deteriorations between 1980 and 2004. Ongoing and projected climate change effects are now of increasing concern, driving 39% of status deteriorations since 2004, followed by habitat loss (37%). Although signs of species recoveries incentivize immediate conservation action, scaled-up investment is urgently needed to reverse the current trends.

Future temperature extremes threaten land vertebrates.

The frequency, duration, and intensity of extreme thermal events are increasing and are projected to further increase by the end of the century1,2. Despite the considerable consequences of temperature extremes on biological systems3-8, we do not know which species and locations are most exposed worldwide. Here we provide a global assessment of land vertebrates' exposures to future extreme thermal events. We use daily maximum temperature data from 1950 to 2099 to quantify future exposure to high frequency, duration, and intensity of extreme thermal events to land vertebrates. Under a high greenhouse gas emission scenario (Shared Socioeconomic Pathway 5-8.5 (SSP5-8.5); 4.4 °C warmer world), 41.0% of all land vertebrates (31.1% mammals, 25.8% birds, 55.5% amphibians and 51.0% reptiles) will be exposed to extreme thermal events beyond their historical levels in at least half their distribution by 2099. Under intermediate-high (SSP3-7.0; 3.6 °C warmer world) and intermediate (SSP2-4.5; 2.7 °C warmer world) emission scenarios, estimates for all vertebrates are 28.8% and 15.1%, respectively. Importantly, a low-emission future (SSP1-2.6, 1.8 °C warmer world) will greatly reduce the overall exposure of vertebrates (6.1% of species) and can fully prevent exposure in many species assemblages. Mid-latitude assemblages (desert, shrubland, and grassland biomes), rather than tropics9,10, will face the most severe exposure to future extreme thermal events. By 2099, under SSP5-8.5, on average 3,773 species of land vertebrates (11.2%) will face extreme thermal events for more than half a year period. Overall, future extreme thermal events will force many species and assemblages into constant severe thermal stress. Deep greenhouse gas emissions cuts are urgently needed to limit species' exposure to thermal extremes.

Protected areas slow declines unevenly across the tetrapod tree of life.

Protected areas (PAs) are the primary strategy for slowing terrestrial biodiversity loss. Although expansion of PA coverage is prioritized under the Convention on Biological Diversity, it remains unknown whether PAs mitigate declines across the tetrapod tree of life and to what extent land cover and climate change modify PA effectiveness1,2. Here we analysed rates of change in abundance of 2,239 terrestrial vertebrate populations across the globe. On average, vertebrate populations declined five times more slowly within PAs (-0.4% per year) than at similar sites lacking protection (-1.8% per year). The mitigating effects of PAs varied both within and across vertebrate classes, with amphibians and birds experiencing the greatest benefits. The benefits of PAs were lower for amphibians in areas with converted land cover and lower for reptiles in areas with rapid climate warming. By contrast, the mitigating impacts of PAs were consistently augmented by effective national governance. This study provides evidence for the effectiveness of PAs as a strategy for slowing tetrapod declines. However, optimizing the growing PA network requires targeted protection of sensitive clades and mitigation of threats beyond PA boundaries. Provided the conditions of targeted protection, adequate governance and well-managed landscapes are met, PAs can serve a critical role in safeguarding tetrapod biodiversity.

Assessing the illegal hunting of native wildlife in China.

Illegal harvesting and trading of wildlife have become major threats to global biodiversity and public health1-3. Although China is widely recognized as an important destination for wildlife illegally obtained abroad4, little attention has been given to illegal hunting within its borders. Here we extracted 9,256 convictions for illegal hunting from a nationwide database of trial verdicts in China spanning January 2014 to March 2020. These convictions involved illegal hunting of 21% (n = 673) of China's amphibian, reptile, bird and mammal species, including 25% of imperilled species in these groups. Sample-based extrapolation indicates that many more species were taken illegally during this period. Larger body mass and range size (for all groups), and proximity to urban markets (for amphibians and birds) increase the probability of a species appearing in the convictions database. Convictions pertained overwhelmingly to illegal hunting for commercial purposes and involved all major habitats across China. A small number of convictions represented most of the animals taken, indicating the existence of large commercial poaching operations. Prefectures closer to urban markets show higher densities of convictions and more individual animals taken. Our results suggest that illegal hunting is a major, overlooked threat to biodiversity throughout China.

A global reptile assessment highlights shared conservation needs of tetrapods.

Comprehensive assessments of species' extinction risks have documented the extinction crisis1 and underpinned strategies for reducing those risks2. Global assessments reveal that, among tetrapods, 40.7% of amphibians, 25.4% of mammals and 13.6% of birds are threatened with extinction3. Because global assessments have been lacking, reptiles have been omitted from conservation-prioritization analyses that encompass other tetrapods4-7. Reptiles are unusually diverse in arid regions, suggesting that they may have different conservation needs6. Here we provide a comprehensive extinction-risk assessment of reptiles and show that at least 1,829 out of 10,196 species (21.1%) are threatened-confirming a previous extrapolation8 and representing 15.6 billion years of phylogenetic diversity. Reptiles are threatened by the same major factors that threaten other tetrapods-agriculture, logging, urban development and invasive species-although the threat posed by climate change remains uncertain. Reptiles inhabiting forests, where these threats are strongest, are more threatened than those in arid habitats, contrary to our prediction. Birds, mammals and amphibians are unexpectedly good surrogates for the conservation of reptiles, although threatened reptiles with the smallest ranges tend to be isolated from other threatened tetrapods. Although some reptiles-including most species of crocodiles and turtles-require urgent, targeted action to prevent extinctions, efforts to protect other tetrapods, such as habitat preservation and control of trade and invasive species, will probably also benefit many reptiles.

A phylogeny-informed characterisation of global tetrapod traits addresses data gaps and biases.

Tetrapods (amphibians, reptiles, birds, and mammals) are model systems for global biodiversity science, but continuing data gaps, limited data standardisation, and ongoing flux in taxonomic nomenclature constrain integrative research on this group and potentially cause biased inference. We combined and harmonised taxonomic, spatial, phylogenetic, and attribute data with phylogeny-based multiple imputation to provide a comprehensive data resource (TetrapodTraits 1.0.0) that includes values, predictions, and sources for body size, activity time, micro- and macrohabitat, ecosystem, threat status, biogeography, insularity, environmental preferences, and human influence, for all 33,281 tetrapod species covered in recent fully sampled phylogenies. We assess gaps and biases across taxa and space, finding that shared data missing in attribute values increased with taxon-level completeness and richness across clades. Prediction of missing attribute values using multiple imputation revealed substantial changes in estimated macroecological patterns. These results highlight biases incurred by nonrandom missingness and strategies to best address them. While there is an obvious need for further data collection and updates, our phylogeny-informed database of tetrapod traits can support a more comprehensive representation of tetrapod species and their attributes in ecology, evolution, and conservation research.

Functional adaptive landscapes predict terrestrial capacity at the origin of limbs.

The acquisition of terrestrial, limb-based locomotion during tetrapod evolution has remained a subject of debate for more than a century1,2. Our current understanding of the locomotor transition from water to land is largely based on a few exemplar fossils such as Tiktaalik3, Acanthostega4, Ichthyostega5 and Pederpes6. However, isolated bony elements may reveal hidden functional diversity, providing a more comprehensive evolutionary perspective7. Here we analyse 40 three-dimensionally preserved humeri from extinct tetrapodomorphs that span the fin-to-limb transition and use functionally informed ecological adaptive landscapes8-10 to reconstruct the evolution of terrestrial locomotion. We show that evolutionary changes in the shape of the humerus are driven by ecology and phylogeny and are associated with functional trade-offs related to locomotor performance. Two divergent adaptive landscapes are recovered for aquatic fishes and terrestrial crown tetrapods, each of which is defined by a different combination of functional specializations. Humeri of stem tetrapods share a unique suite of functional adaptations, but do not conform to their own predicted adaptive peak. Instead, humeri of stem tetrapods fall at the base of the crown tetrapod landscape, indicating that the capacity for terrestrial locomotion occurred with the origin of limbs. Our results suggest that stem tetrapods may have used transitional gaits5,11 during the initial stages of land exploration, stabilized by the opposing selective pressures of their amphibious habits. Effective limb-based locomotion did not arise until loss of the ancestral 'L-shaped' humerus in the crown group, setting the stage for the diversification of terrestrial tetrapods and the establishment of modern ecological niches12,13.

Hidden hotspots of amphibian biodiversity in China.

Identifying and protecting hotspots of endemism and species richness is crucial for mitigating the global biodiversity crisis. However, our understanding of spatial diversity patterns is far from complete, which severely limits our ability to conserve biodiversity hotspots. Here, we report a comprehensive analysis of amphibian species diversity in China, one of the most species-rich countries on Earth. Our study combines 20 y of field surveys with new molecular analyses of 521 described species and also identifies 100 potential cryptic species. We identify 10 hotspots of amphibian diversity in China, each with exceptional species richness and endemism and with exceptional phylogenetic diversity and phylogenetic endemism (based on a new time-calibrated, species-level phylogeny for Chinese amphibians). These 10 hotspots encompass 59.6% of China's described amphibian species, 49.0% of cryptic species, and 55.6% of species endemic to China. Only four of these 10 hotspots correspond to previously recognized biodiversity hotspots. The six new hotspots include the Nanling Mountains and other mountain ranges in South China. Among the 186 species in the six new hotspots, only 9.7% are well covered by protected areas and most (88.2%) are exposed to high human impacts. Five of the six new hotspots are under very high human pressure and are in urgent need of protection. We also find that patterns of richness in cryptic species are significantly related to those in described species but are not identical.

Genetic transformation of the frog-killing chytrid fungus Batrachochytrium dendrobatidis.

Batrachochytrium dendrobatidis (Bd), a causative agent of chytridiomycosis, is decimating amphibian populations around the world. Bd belongs to the chytrid lineage, a group of early-diverging fungi that are widely used to study fungal evolution. Like all chytrids, Bd develops from a motile form into a sessile, growth form, a transition that involves drastic changes in its cytoskeletal architecture. Efforts to study Bd cell biology, development, and pathogenicity have been limited by the lack of genetic tools with which to test hypotheses about underlying molecular mechanisms. Here, we report the development of a transient genetic transformation system for Bd. We used electroporation to deliver exogenous DNA into Bd cells and detected transgene expression for up to three generations under both heterologous and native promoters. We also adapted the transformation protocol for selection using an antibiotic resistance marker. Finally, we used this system to express fluorescent protein fusions and, as a proof of concept, expressed a genetically encoded probe for the actin cytoskeleton. Using live-cell imaging, we visualized the distribution and dynamics of polymerized actin at each stage of the Bd life cycle, as well as during key developmental transitions. This transformation system enables direct testing of key hypotheses regarding mechanisms of Bd pathogenesis. This technology also paves the way for answering fundamental questions of chytrid cell, developmental, and evolutionary biology.

A global map of species at risk of extinction due to natural hazards.

An often-overlooked question of the biodiversity crisis is how natural hazards contribute to species extinction risk. To address this issue, we explored how four natural hazards, earthquakes, hurricanes, tsunamis, and volcanoes, overlapped with the distribution ranges of amphibians, birds, mammals, and reptiles that have either narrow distributions or populations with few mature individuals. To assess which species are at risk from these natural hazards, we combined the frequency and magnitude of each natural hazard to estimate their impact. We considered species at risk if they overlapped with regions where any of the four natural hazards historically occurred (n = 3,722). Those species with at least a quarter of their range subjected to a high relative impact were considered at high risk (n = 2,001) of extinction due to natural hazards. In total, 834 reptiles, 617 amphibians, 302 birds, and 248 mammals were at high risk and they were mainly distributed on islands and in the tropics. Hurricanes (n = 983) and earthquakes (n = 868) affected most species, while tsunamis (n = 272), and volcanoes (n = 171) affected considerably fewer. The region with the highest number of species at high risk was the Pacific Ring of Fire, especially due to volcanoes, earthquakes, and tsunamis, while hurricane-related high-risk species were concentrated in the Caribbean Sea, Gulf of Mexico, and northwestern Pacific Ocean. Our study provides important information regarding the species at risk due to natural hazards and can help guide conservation attention and efforts to safeguard their survival.

Virulence and Pathogenicity of Chytrid Fungi Causing Amphibian Extinctions.

Ancient enzootic associations between wildlife and their infections allow evolution to innovate mechanisms of pathogenicity that are counterbalanced by host responses. However, erosion of barriers to pathogen dispersal by globalization leads to the infection of hosts that have not evolved effective resistance and the emergence of highly virulent infections. Global amphibian declines driven by the rise of chytrid fungi and chytridiomycosis are emblematic of emerging infections. Here, we review how modern biological methods have been used to understand the adaptations and counteradaptations that these fungi and their amphibian hosts have evolved. We explore the interplay of biotic and abiotic factors that modify the virulence of these infections and dissect the complexity of this disease system. We highlight progress that has led to insights into how we might in the future lessen the impact of these emerging infections.

Clustered versus catastrophic global vertebrate declines.

Recent analyses have reported catastrophic global declines in vertebrate populations1,2. However, the distillation of many trends into a global mean index obscures the variation that can inform conservation measures and can be sensitive to analytical decisions. For example, previous analyses have estimated a mean vertebrate decline of more than 50% since 1970 (Living Planet Index2). Here we show, however, that this estimate is driven by less than 3% of vertebrate populations; if these extremely declining populations are excluded, the global trend switches to an increase. The sensitivity of global mean trends to outliers suggests that more informative indices are needed. We propose an alternative approach, which identifies clusters of extreme decline (or increase) that differ statistically from the majority of population trends. We show that, of taxonomic-geographic systems in the Living Planet Index, 16 systems contain clusters of extreme decline (comprising around 1% of populations; these extreme declines occur disproportionately in larger animals) and 7 contain extreme increases (around 0.4% of populations). The remaining 98.6% of populations across all systems showed no mean global trend. However, when analysed separately, three systems were declining strongly with high certainty (all in the Indo-Pacific region) and seven were declining strongly but with less certainty (mostly reptile and amphibian groups). Accounting for extreme clusters fundamentally alters the interpretation of global vertebrate trends and should be used to help to prioritize conservation efforts.

Two-speed genome evolution drives pathogenicity in fungal pathogens of animals.

The origins and evolution of virulence in amphibian-infecting chytrids Batrachochytrium dendrobatidis (Bd) and Batrachochytrium salamandrivorans (Bsal) are largely unknown. Here, we use deep nanopore sequencing of Bsal and comparative genomics against 21 high-quality genome assemblies that span the fungal Chytridiomycota. We discover that Bsal has the most repeat-rich genome of the Chytridiomycota, comprising 40.9% repetitive elements; this genome has expanded to more than 3× the length of its conspecific Bd, with autonomous and fully functional LTR/Gypsy elements contributing significantly to the expansion. The M36 metalloprotease virulence factors are highly expanded (n = 177) in Bsal, most of which (53%) are flanked by transposable elements, suggesting they have a repeat-associated expansion. We find enrichment upstream of M36 metalloprotease genes of three novel repeat families belonging to the repeat superfamily of LINEs that are implicated with gene copy number variations. Additionally, Bsal has a highly compartmentalized genome architecture, with virulence factors enriched in gene-sparse/repeat-rich compartments, while core conserved genes are enriched in gene-rich/repeat-poor compartments. Genes upregulated during infection are primarily found in the gene-sparse/repeat-rich compartment in both Bd and Bsal. Furthermore, genes with signatures of positive selection in Bd are enriched in repeat-rich regions, suggesting these regions are a cradle for the evolution of chytrid pathogenicity. These are the hallmarks of two-speed genome evolution, and this study provides evidence of two-speed genomes in an animal pathogen, shedding light on the evolution of fungal pathogens of vertebrates driving global declines and extinctions.

Evolutionary isolation of ryanodine receptor isoform 1 for muscle-based thermogenesis in mammals.

Resting skeletal muscle generates heat for endothermy in mammals but not amphibians, while both use the same Ca2+-handling proteins and membrane structures to conduct excitation-contraction coupling apart from having different ryanodine receptor (RyR) isoforms for Ca2+ release. The sarcoplasmic reticulum (SR) generates heat following Adenosine triphosphate (ATP) hydrolysis at the Ca2+ pump, which is amplified by increasing RyR1 Ca2+ leak in mammals, subsequently increasing cytoplasmic [Ca2+] ([Ca2+]cyto). For thermogenesis to be functional, rising [Ca2+]cyto must not interfere with cytoplasmic effectors of the sympathetic nervous system (SNS) that likely increase RyR1 Ca2+ leak; nor should it compromise the muscle remaining relaxed. To achieve this, Ca2+ activated, regenerative Ca2+ release that is robust in lower vertebrates needs to be suppressed in mammals. However, it has not been clear whether: i) the RyR1 can be opened by local increases in [Ca2+]cyto; and ii) downstream effectors of the SNS increase RyR Ca2+ leak and subsequently, heat generation. By positioning amphibian and malignant hyperthermia-susceptible human-skinned muscle fibers perpendicularly, we induced abrupt rises in [Ca2+]cyto under identical conditions optimized for activating regenerative Ca2+ release as Ca2+ waves passed through the junction of fibers. Only mammalian fibers showed resistance to rising [Ca2+]cyto, resulting in increased SR Ca2+ load and leak. Fiber heat output was increased by cyclic adenosine monophosphate (cAMP)-induced RyR1 phosphorylation at Ser2844 and Ca2+ leak, indicating likely SNS regulation of thermogenesis. Thermogenesis occurred despite the absence of SR Ca2+ pump regulator sarcolipin. Thus, evolutionary isolation of RyR1 provided increased dynamic range for thermogenesis with sensitivity to cAMP, supporting endothermy.

The regenerative capacity of neonatal tissues.

It is well established that humans and other mammals are minimally regenerative compared with organisms such as zebrafish, salamander or amphibians. In recent years, however, the identification of regenerative potential in neonatal mouse tissues that normally heal poorly in adults has transformed our understanding of regenerative capacity in mammals. In this Review, we survey the mammalian tissues for which regenerative or improved neonatal healing has been established, including the heart, cochlear hair cells, the brain and spinal cord, and dense connective tissues. We also highlight common and/or tissue-specific mechanisms of neonatal regeneration, which involve cells, signaling pathways, extracellular matrix, immune cells and other factors. The identification of such common features across neonatal tissues may direct therapeutic strategies that will be broadly applicable to multiple adult tissues.

Terrestrial reproduction and parental care drive rapid evolution in the trade-off between offspring size and number across amphibians.

The trade-off between offspring size and number is central to life history strategies. Both the evolutionary gain of parental care or more favorable habitats for offspring development are predicted to result in fewer, larger offspring. However, despite much research, it remains unclear whether and how different forms of care and habitats drive the evolution of the trade-off. Using data for over 800 amphibian species, we demonstrate that, after controlling for allometry, amphibians with direct development and those that lay eggs in terrestrial environments have larger eggs and smaller clutches, while different care behaviors and adaptations vary in their effects on the trade-off. Specifically, among the 11 care forms we considered at the egg, tadpole and juvenile stage, egg brooding, male egg attendance, and female egg attendance increase egg size; female tadpole attendance and tadpole feeding decrease egg size, while egg brooding, tadpole feeding, male tadpole attendance, and male tadpole transport decrease clutch size. Unlike egg size that shows exceptionally high rates of phenotypic change in just 19 branches of the amphibian phylogeny, clutch size has evolved at exceptionally high rates in 135 branches, indicating episodes of strong selection; egg and tadpole environment, direct development, egg brooding, tadpole feeding, male tadpole attendance, and tadpole transport explain 80% of these events. By explicitly considering diversity in parental care and offspring habitat by stage of offspring development, this study demonstrates that more favorable conditions for offspring development promote the evolution of larger offspring in smaller broods and reveals that the diversity of parental care forms influences the trade-off in more nuanced ways than previously appreciated.

Bending the curve: Simple but massive conservation action leads to landscape-scale recovery of amphibians.

Success stories are rare in conservation science, hindered also by the research-implementation gap, where scientific insights rarely inform practice and practical implementation is rarely evaluated scientifically. Amphibian population declines, driven by multiple stressors, are emblematic of the freshwater biodiversity crisis. Habitat creation is a straightforward conservation action that has been shown to locally benefit amphibians, as well as other taxa, but does it benefit entire amphibian communities at large spatial scales? Here, we evaluate a landscape-scale pond-construction program by fitting dynamic occupancy models to 20 y of monitoring data for 12 pond-breeding amphibian species in the Swiss state Aargau, a densely populated area of the Swiss lowlands with intensive land use. After decades of population declines, the number of occupied ponds increased statewide for 10 out of 12 species, while one species remained stable and one species further declined between 1999 and 2019. Despite regional differences, in 77% of all 43 regional metapopulations, the colonization and subsequent occupation of new ponds stabilized (14%) or increased (63%) metapopulation size. Likely mechanisms include increased habitat availability, restoration of habitat dynamics, and increased connectivity between ponds. Colonization probabilities reflected species-specific preferences for characteristics of ponds and their surroundings, which provides evidence-based information for future pond construction targeting specific species. The relatively simple but landscape-scale and persistent conservation action of constructing hundreds of new ponds halted declines and stabilized or increased the state-wide population size of all but one species, despite ongoing pressures from other stressors in a human-dominated landscape.

Into the danger zone: How the within-host distribution of parasites controls virulence.

Despite the importance of virulence in epidemiological theory, the relative contributions of host and parasite to virulence outcomes remain poorly understood. Here, we use reciprocal cross experiments to disentangle the influence of host and parasite on core virulence components-infection and pathology-and understand dramatic differences in parasite-induced malformations in California amphibians. Surveys across 319 populations revealed that amphibians' malformation risk was 2.7× greater in low-elevation ponds, even while controlling for trematode infection load. Factorial experiments revealed that parasites from low-elevation sites induced higher per-parasite pathology (reduced host survival and growth), whereas there were no effects of host source on resistance or tolerance. Parasite populations also exhibited marked differences in within-host distribution: ~90% of low-elevation cysts aggregated around the hind limbs, relative to <60% from high-elevation. This offers a novel, mechanistic basis for regional variation in parasite-induced malformations while promoting a framework for partitioning host and parasite contributions to virulence.

Skin microbiome disturbance linked to drought-associated amphibian disease.

The onset of global climate change has led to abnormal rainfall patterns, disrupting associations between wildlife and their symbiotic microorganisms. We monitored a population of pumpkin toadlets and their skin bacteria in the Brazilian Atlantic Forest during a drought. Given the recognized ability of some amphibian skin bacteria to inhibit the widespread fungal pathogen Batrachochytrium dendrobatidis (Bd), we investigated links between skin microbiome health, susceptibility to Bd and host mortality during a die-off event. We found that rainfall deficit was an indirect predictor of Bd loads through microbiome disruption, while its direct effect on Bd was weak. The microbiome was characterized by fewer putative Bd-inhibitory bacteria following the drought, which points to a one-month lagged effect of drought on the microbiome that may have increased toadlet susceptibility to Bd. Our study underscores the capacity of rainfall variability to disturb complex host-microbiome interactions and alter wildlife disease dynamics.