Mitogenome-Informed Metabarcoding for Non-Invasive Identification of Sympatric Flamingos in Northern Chile

The Chilean flamingo (Phoenicopterus chilensis) is a widely distributed South American species inhabiting diverse wetland ecosystems from Ecuador and Peru south to Chile and Argentina, and east to Brazil, Uruguay, and Paraguay, ranging from sea level to high-altitude Andean salars. Despite its broad distribution, local populations are increasingly exposed to anthropogenic pressures, raising conservation concerns at regional scales. In this study, we generated mitogenomic resources and developed a molecular framework for species identification using non-invasive samples. We sequenced genomic DNA from P. chilensis and assembled its complete mitogenome, enabling the in silico evaluation of commonly used metabarcoding markers. Subsequently, we collected eight fecal samples from a feeding site in a high-Andean lagoon where P. chilensis co-occurs with Phoenicoparrus andinus and Phoenicoparrus jamesi. As a positive control, nine additional fecal samples were collected from the Zoológico Nacional, Santiago, Chile, where only P. chilensis is maintained. DNA was extracted and initially screened via Sanger sequencing. To robustly assign taxonomic identity, we implemented a probabilistic framework combining maximum likelihood and Bayesian inference to estimate the posterior probability of species membership based on observed genetic variants. This approach revealed the presence of two genetically distinct groups not attributable to P. chilensis, indicating the coexistence and detectability of other flamingo taxa through fecal DNA. Our results demonstrate the value of integrating mitogenomic resources, metabarcoding, and statistical inference for non-invasive species identification in mixed-species systems. This framework provides a scalable tool for biodiversity monitoring and has direct implications for conservation strategies and evidence-based policy development in fragile high-Andean ecosystems.