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.

Cyanobacterial blooms are detrimental events that affect the quality of water and the normal functioning of ecosystems, especially when dominated by toxin-producing species. Although cyanobacteria and cyanotoxins have been reported in the land-sea interface since the late 80s, genetic evidence on how inland freshwaters influence the cyanobacterial communities in these systems is very scarce to date. This study aims to investigate the relationship between the cyanobacterial communities of an inland freshwater reservoir and an estuary located in an aquaculture-rich coastal area of NW Spain. During 2022 and 2023, a total of six sampling campaigns were carried out surrounding the blooming seasons. Cyanobacterial communities and their potential toxicity were analyzed through metabarcoding of the 16S rRNA gene and PCR-based screening of genes involved in the biosynthesis of cyanotoxins. Results demonstrate that the reservoir likely contributed significantly to the presence of potentially toxic cyanobacteria in the estuary, with more than 80 % of reservoir shared cyanobacterial ASVs (Amplicon Sequence Variants) belonging to potentially toxic genera. Genes related to microcystins and anatoxins were detected in both systems, accompanied by low toxin concentrations. Most key cyanobacterial genera were consistently dominated by specific ASVs, suggesting the presence of a few genotypes resistant to environmental gradients during transport. These findings highlight the influence of freshwater systems on estuarine cyanobacterial communities and demonstrate the potential of genetic tools for their high-resolution monitoring, useful in water management of ecological and economic risks. Further research is recommended to understand the global impacts of cyanobacterial bloom dispersion towards land-sea interface systems, particularly in economically relevant areas.

The negative effects associated with cyanobacterial blooms are of particular concern in protected ecosystems, as these areas are ecologically significant and attract a high number of visitors. This study aims to explore the cyanobacterial communities and associated toxicity in three reservoirs located within a Mediterranean National Park with a compromised situation at basin-level. Our results demonstrate the occurrence of dense toxic blooms containing microcystins (reaching values close to 280 μg L^-1) and low levels of anatoxin-a and saxitoxins (up to 0.02 μg L^-1). Comprehensive metabarcoding analyses based on cyanobacterial 16S rRNA and cyanotoxin-biosynthesis genes (mcyE, anaF and sxtA) unveiled three highly heterogeneous communities, despite the spatial proximity of reservoirs. Additionally, our results suggested the influence of water conductivity on the blooms composition. Among the diverse bloom-forming taxa found, Microcystis sp. and Planktothrix sp. were revealed as the microcystins-producer candidates, and Cuspidothrix issatschenkoi and Dolichospermum/Aphanizomenom sp. as the potential producers of anatoxin-a and saxitoxins, respectively. A polyphasic characterization confirmed the first report of the tropical-related species Planktothrix spiroides in Europe, showing elevated level of dominance. As a whole, we present the scenario of an ecologically important protected area facing significant challenges in the proper management of cyanobacterial blooms.

Most studies on toxic cyanobacteria focus on a limited range of aquatic environments, primarily large systems relevant to public health. However, few adopt a multi‐habitat perspective to examine their distribution across diverse waterbody types, including small systems such as natural/near-natural ponds or streams, where harmful cyanobacteria remain understudied. This study investigates the occurrence of toxic cyanobacteria across reservoirs and (temporary) natural/near‐natural ponds and streams within a Mediterranean protected area. By integrating metabarcoding of 16S rRNA and cyanotoxin‐biosynthesis genes with chemical analyses, we identified both planktonic and benthic toxic species. Microcystin‐producing Microcystis dominated lentic systems and anatoxin‐producing Microcoleus occurred in streams. Amplicon sequence variant (ASV) profiles derived from 16S rRNA and mcyE genes revealed fine‐scale genetic overlap of (toxic) populations across all studied systems, even between hydrologically unconnected reservoirs and ponds. Polyphasic characterization further enabled the attribution of microcystin concentrations quantified in wildlife-supporting ponds to Microcystis genetic variants shared with nearby bloom‐prone reservoirs. Among shared ASVs, Synechococcus‐like and Microcystis were the most widely distributed, contributing substantially to the compositional similarities observed among lentic systems, which shared 49-100% of their ASVs with at least one other waterbody. Together, our results reveal patterns suggesting local cyanobacterial connectivity potentially operating through non‐hydrologic pathways. Our findings highlight the applicability of genetic approaches for resolving the multi‐habitat distribution of toxic populations among hydrologically unconnected sites. This study underscores that monitoring in sensitive areas must extend beyond large bloom‐affected systems to include small waterbodies, which can function as hidden recipients and/or sources of toxic cyanobacteria.

El propósito de este trabajo es diseñar herramientas sencillas y eficaces en formato de árbol de decisiones que puedan ser aplicadas para la evaluación de los riesgos asociados a cianobacterias planctónicas potencialmente productoras de cianotoxinas, tanto en cuerpos de agua usados para abastecer agua de uso y consumo, como en aquellos usados para actividades recreativas. Este documento está dirigido a organismos, autoridades y/o empresas de naturaleza pública, privada y/o mixta con competencias en la gestión de los recursos hídricos continentales, así como a los usuarios consumidores de dichos recursos. Se proponen tres árboles de decisiones, uno para aguas destinadas a consumo y dos para aguas de uso recreativo, que pueden ser adaptados según las circunstancias locales de cada región. Además, se realiza una breve descripción de los diferentes parámetros usados en el diseño de estas herramientas y se describen de forma general las diferentes técnicas utilizadas habitualmente para la medición de dichos parámetros. Estas herramientas de alerta temprana y evaluación de riesgos deben ser adaptadas localmente para que la evaluación del riesgo (potencial o real) y la toma de decisiones se aproxime a la magnitud del riesgo existente, traduciéndose esto en un correcto aprovechamiento de los recursos.

Potentially toxic cyanobacterial communities are prolific in freshwater ecosystems and in national parks where vulnerable fauna tend to be found. This study focuses on the proliferation of toxic cyanobacteria and the risks they represent in the rivers and reservoirs of two Spanish national parks. As far as we know, this is the first time that an ecological, taxonomical and toxicological characterization of planktonic and benthic cyanotoxin-producing cyanobacteria is carried out in this type of protected areas in Spain. Our results, obtained during 2021 sampling campaigns, have confirmed the occurrence of cyanobacteria in these ecosystems and assessed the risks of these cyanobacteria to these ecosystems. An alarming occurrence of benthic mats, dominated mainly by Phormidium autumnale (also called Microcoleus autumnalis), a potentially anatoxin producer, has been observed in rivers from Sierra de Guadarrama National Park. In reservoirs from Monfragüe National Park, the planktonic communities have been dominated by Microcystis, Aphanizomenom, Arthrospira and Planktothrix. Genetic screening by PCR and sequencing have confirmed the presence of cyanotoxin biosynthesis genes (mcyE, anaF and sxtA) in all communities studied. The relationship among community diversity, the presence/concentration of cyanotoxins (microcystins, saxitoxins and anatoxins) and the environmental parameters measured is discussed. These results will contribute to preparing protocols for evaluating and managing the potential risk to visitors to, workers in and fauna of these protected ecosystems.