Conservation of Cuban crocodiles in Zapata Swamp: an environmental DNA baseline biomonitoring perspective

Conservation efforts for crocodylian species are confronted with noteworthy challenges, including population decline and extensive habitat modification over time. Consequently, close monitoring of crocodylian populations and their habitats is imperative for the timely detection of any alterations in their aquatic ecosystems and for implementing effective conservation actions. This study is the first to investigate eDNA metabarcoding methods to identify the critically endangered Crocodylus rhombifer and the vulnerable C. acutus in their natural environments, to recover information on the vertebrate community diversity present in their megadiverse ecosystems, and to explore the coexistence of Cuban Crocodylus with endemic fauna and exotic vertebrate species. Using four mitochondrial markers (Croc_COI, Croc_CR, Vert_COI, and 12S) and the most extensive sampling effort in the wild for Crocodylus in Cuba, the presence of eDNA from C. rhombifer was detected in 11 out of 15 sampled locations within its historical geographic distribution. Data analyses using the Vert_COI and the mBRAVE bioinformatics pipeline resulted in the most effective molecular marker and pipeline combination for Cuban Crocodylus identification. In total, 56 vertebrate species were identified, distributed among 43 genera, 29 families, 25 orders, and five classes - 61% of the species listed by traditional methods- were molecularly identified using four target regions and four bioinformatic pipelines. Fifteen new species previously undetected in the area were reported using eDNA metabarcoding techniques. Similarly, this approach allowed for detecting invasive alien species in Zapata Swamp National Park areas. The present results highlight eDNA metabarcoding as a powerful tool for Crocodylus identification and biodiversity monitoring within a fragile and diverse ecosystem. This approach allows for faster and non-invasive Crocodylus biomonitoring methods, where early identification of crocodiles in areas of economic importance permits the reduction of complex human-crocodile interactions. Last, evaluating habitat suitability for potential reintroductions or recovery programs into the wild directly impacts Crocodylus conservation.