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The Antillean manatee (Trichechus manatus) and the Amazonian manatee (Trichechus inunguis) are distributed in rivers in the Caribbean and Amazonian region of Colombia respectively. For 30 years, genetic information has been obtained from these populations in order to inform conservation programs for these endangered species and decide on the location to release them back to the wild. However, in previous studies, samples from rivers in some areas of the country were not included, given the difficulties to access these regions due to either logistic or safety issues. In this study, we analyzed mitochondrial DNA (mtDNA) control region (CR) sequences of from samples of T. manatus (n = 37) and T. inunguis (n = 4) (410 and 361 bp, respectively), obtained in new and previously unexplored rivers and bays in the country, including Santa Marta, Urabá Gulf, Ayapel Marsh (San Jorge River Basin), Meta River and Magdalena Medio and the low Magdalena River (Cesar Province and Canal del Dique) as well as additional samples from Puerto Nariño in the Colombian Amazon. Our results included the discovery of two newly described mtDNA CR haplotypes for T. manatus. In addition, we confirmed significant population differentiation at the mitochondrial level between the Magdalena and Sinú rivers and differentiation among areas of the same river, including the middle and low Magdalena River. This differentiation may be related to anthropic changes in the river since construction of the Canal del Dique in the XVI century. We also tested environmental DNA sampling and analyses techniques to evaluate its potential use for manatee detection and monitoring in bodies of water in Colombia, in order to evaluate new areas for future manatee conservation initiatives. We emphasize the need to continue using genetic information to provide evidence on the potential best locations to undertake animal release to prevent outbreeding depression.

Environmental DNA metabarcoding is a tool with increasing use worldwide. The uses of such technology have been validated several times for diversity census, invasive species detection, and endangered/cryptic/elusive species detection and monitoring. With the help of this technology, water samples collected (n = 37) from several main river basins and other water bodies of the northern part of Colombia, including the Magdalena, Sinú, Atrato, and San Jorge river basins, were filtered and analyzed and processed using universal 12S primers for vertebrate fauna and NGS. Over 200 native taxa were detected, the majority of them being fish species but also including amphibia, reptiles, and several non-aquatic species of birds and mammals (around 78, 3, 2, 9, and 8%, respectively). Among the matches, vulnerable, and endangered species such as the catfish Pseudoplatystoma magdaleniatum and the Antillean manatee (Trichechus manatus) were detected. The manual revision of the data revealed some geographical incongruencies in classification. No invasive species were detected in the filters. This is, to our knowledge, the first time this technique is used in rivers of the country and this tool promises to bring advances in monitoring and conservation efforts, since its low cost and fast deployment allows for sampling in small periods of time, together with the fact that it can detect a wide range of species, allows for a new way of censing the vertebrate diversity in Colombia. Diversity analysis showed how the species identified using this method point to expected community structure although still much needs to be improved in rates of detection and genomic reference databases. This technique could be used in citizen science projects involving local communities in these regions.