Environmental DNA-based biomonitoring of Cuban Crocodylus and their accompanying vertebrate fauna from Zapata Swamp, Cuba

Crocodylians globally face considerable challenges, including population decline and extensive habitat modification. Close monitoring of crocodylian populations and their habitats is imperative for the timely detection of population trends, especially in response to management interventions. Here we use eDNA metabarcoding to identify the Critically Endangered Crocodylus rhombifer and the Vulnerable C. acutus, as well as vertebrate community diversity, in Cuba’s Zapata Swamp. We tested four different primer sets, including those used previously in Crocodylus population genetic and phylogenetic research, for their efficiency at detecting crocodylian eDNA. We detected C. rhombifer eDNA in 11 out of 15 sampled locations within its historical geographic distribution. We found that data analyses using the VertCOI primers and the mBRAVE bioinformatics pipeline were the most effective molecular marker and pipeline combination for identifying this species from environmental samples. We also identified 55 vertebrate species in environmental samples across the four bioinformatics pipelines— ~ 85% known to be present in the Zapata ecosystem. Among them were eight species previously undetected in the area and eight alien species, including known predators of hatchling crocodiles (e.g., Clarias sp.) and egg predators (e.g., Mus musculus). This study highlights eDNA metabarcoding as a powerful tool for crocodylian biomonitoring within fragile and diverse ecosystems, particularly where fast, non-invasive methods permit detection in economically important areas and will lead to a better understanding of complex human-crocodile interactions and evaluate habitat suitability for potential reintroductions or recovery programs for threatened crocodylian species.全球鳄类面临诸多严峻挑战，包括种群数量下降与栖息地大规模改造。对鳄类种群及其栖息地开展严密监测，以及时掌握种群动态、响应各类管理干预措施，是极为必要的。本研究借助环境DNA宏条形码（environmental DNA metabarcoding）技术，在古巴萨帕塔沼泽中检测极危物种菱斑鳄（Crocodylus rhombifer）与易危物种美洲鳄（Crocodylus acutus），同时分析该区域的脊椎动物群落多样性。本研究测试了4种不同的引物组，其中包括此前用于鳄属种群遗传与系统发育研究的引物组，以评估其检测鳄类环境DNA的效率。在菱斑鳄历史分布范围内的15个采样点位中，本研究在11个点位中检测到了菱斑鳄的环境DNA。研究结果显示，使用VertCOI引物与mBRAVE生物信息学分析流程的组合，是从环境样本中检测该物种的最优分子标记与分析流程方案。本研究通过4种生物信息学流程对环境样本进行分析，共鉴定出55种脊椎动物，其中约85%为萨帕塔沼泽生态系统中已记录的物种。其中包括8种此前未在该区域被记录的物种，以及8种外来物种，其中包含已知的鳄类幼体捕食者（如Clarias属物种）与鳄卵捕食者（如小家鼠Mus musculus）。本研究证实，环境DNA宏条形码技术是脆弱且多样的生态系统中开展鳄类生物监测的有力工具，尤其适用于那些需要快速、非侵入式监测的经济重要区域，有助于进一步厘清人类与鳄类之间的复杂相互作用，并为受威胁鳄类物种的再引入或种群恢复计划评估栖息地适宜性。