Protected high-mountain rivers harbor widespread toxic Microcoleus-dominated mats with distinct genetic profiles

Toxic Microcoleus remains poorly studied in mountain freshwaters, where cyanotoxins threaten public and ecological health, particularly in protected areas that hold high ecological value and attract a high density of visitors. This study investigated the proliferation of cyanobacterial mats and cyanotoxin production in two high-mountain rivers flowing through a National Park. Our results confirmed the widespread and recurrent proliferation of Microcoleus-dominated mats containing the neurotoxins anatoxin-a and homoanatoxin-a. Mat coverage showed potentially hazardous levels, reaching up to 40-50 % of the riverbed. Comprehensive genetic characterization through metabarcoding sequencing (targeting 16S rRNA and cyanotoxin-biosynthesis genes) revealed a significant differential distribution of Microcoleus genotypes between two adjacent high-mountain rivers, as well as the persistence of the anaF gene from Microcoleus. Although saxitoxins and microcystins were not detected, the sxtA gene closely related to Scytonema and mcyE from Nostoc-like species were occasionally found. This is the first report of abundant proliferations of anatoxin-producing Microcoleus in Spain, broadening research into Southern and Mediterranean Europe, where toxic Microcoleus remains underreported. These results reinforce the importance of genetic analyses as valuable tools for understanding Microcoleus population dynamics. Our findings highlight that toxic mats should be considered a potential threat in minimally impacted mountain freshwaters, especially those frequently visited for their high ecological value.