Data Sheet 1_Dynamic responses of gut microbiota to agricultural and wildfire ash: insights from different amphibian developmental stages.docx

Combustion by-products—specifically wildfire ash and rice-straw ash—are emerging contaminants in freshwater ecosystems. However, their impacts on amphibian survival and gut microbiota across various developmental stages remains largely unclear, thereby limiting evidence-based conservation strategies in fire-affected habitats. This study evaluated the effects of artificial water (control, C) and aqueous extracts of ash (AEAs) derived from wildfire ash (W) and rice straw ash (S) on the survival and gut (G) microbiota of Rana dybowskii tadpoles (T) and adult frogs (F). Exposure to wildfire ash significantly reduced tadpole survival compared to rice straw ash, whereas no significant differences were observed in adult frogs. Alpha diversity of the gut microbiota differed significantly among tadpole groups but not among adult groups. Bray-Curtis and weighted UniFrac analyses revealed significant differences in the gut microbiota of adult frogs and tadpoles across different treatment groups. Linear discriminant analysis effect size (LEfSe) identified a significant enrichment of specific bacterial genera across treatment groups. BugBase analysis indicated that in the TCG, TSG, and TWG groups, notable variations in the TCG, TSG, and TWG groups, there were notable differences in Forms-Biofilms and Potentially-Pathogenic, while in the FCG, FSG, and FWG groups, significant differences were observed in Aerobic, Gram-Positive, Potentially-Pathogenic, and Stress-Tolerant. These findings suggest that wildfire ash exhibits greater toxicity than rice straw ash to both life stages of R. dybowskii, with tadpoles being more vulnerable. By elucidating the link between ash-derived pollutants and amphibian gut health, this study underscores the growing threat of routine straw burning and intensifying wildfires to global freshwater biodiversity and advocates for ash-specific mitigation measures and microbiota-informed conservation strategies.

燃烧副产物——尤其是野火灰与稻草灰——是淡水生态系统中日益受到关注的新兴污染物。然而目前学界对这类污染物在不同发育阶段对两栖动物存活率及肠道菌群（gut microbiota）的影响仍知之甚少，这极大限制了受火灾影响栖息地的循证保护策略制定。本研究评估了人工水溶液（对照组，C）以及源自野火灰（wildfire ash, W）、稻草灰（rice straw ash, S）的水相灰提取物（aqueous extracts of ash, AEAs）对中国林蛙（Rana dybowskii）蝌蚪（tadpoles, T）与成蛙（adult frogs, F）的存活率及肠道（G）菌群的影响。与稻草灰处理组相比，野火灰暴露显著降低了蝌蚪的存活率，但成蛙存活率无显著组间差异。肠道菌群的α多样性（alpha diversity）在蝌蚪各组间存在显著差异，但成蛙各组间无显著差异。Bray-Curtis距离分析与加权UniFrac分析（weighted UniFrac analysis）结果显示，不同处理组的成蛙与蝌蚪的肠道菌群结构均存在显著差异。线性判别分析效应大小（linear discriminant analysis effect size, LEfSe）结果表明，各处理组中特定细菌属的丰度存在显著富集。BugBase分析结果显示，在蝌蚪对照组（TCG）、稻草灰处理蝌蚪组（TSG）与野火灰处理蝌蚪组（TWG）中，生物被膜形成型（Forms-Biofilms）和潜在致病菌（Potentially-Pathogenic）相关菌群的丰度存在显著差异；而在成蛙对照组（FCG）、稻草灰处理成蛙组（FSG）与野火灰处理成蛙组（FWG）中，需氧菌（Aerobic）、革兰氏阳性菌（Gram-Positive）、潜在致病菌与耐胁迫菌（Stress-Tolerant）相关菌群的丰度均存在显著差异。上述研究结果表明，相较于稻草灰，野火灰对中国林蛙两个发育阶段的个体均表现出更强的毒性，且蝌蚪对污染物的敏感性更高。本研究通过阐明灰基污染物与两栖动物肠道健康之间的关联，凸显了常规秸秆焚烧与频发野火对全球淡水生物多样性的日益严峻的威胁，并呼吁制定针对灰污染的缓解措施以及基于菌群信息的保护策略。