Multiscale environmental analysis on autotrophic euglenid communities: insights from DNA metabarcoding.

Freshwater ponds are increasingly recognized as biodiversity hotspots for microorganisms, yet remain understudied compared to larger aquatic systems. Among protist communities, autotrophic euglenids play a significant role, especially in eutrophic, shallow water bodies. Despite their importance, they are understudied and often overlooked, mostly due to difficult morphological identification and the lack of relevant determination literature, as well as their unusual DNA structure and still unresolved taxonomy. To date, no comprehensive study has used metabarcoding to investigate ecological patterns of euglenids in freshwater ponds. This study employs high-throughput sequencing using euglenid-specific primers targeting the 18S rDNA V2 region, enabling detailed group-focused analysis focused on their ecology. It includes 190 samples from freshwater ponds collected over three years across three regions of Poland. Environmental parameters including physicochemical parameters of water, surrounding land use, temporal variation and spatial distribution were analysed in relation to euglenid read counts. Alpha diversity was assessed using global models, while Canonical Correspondence Analysis was employed to investigate how environmental conditions shape the composition of euglenid communities. Our findings highlight ecological variability within the group and shows the importance of species-specific approaches in studies of microbial ecology. This study provides the first integrative framework combining metabarcoding and ecological data to determine the drivers of euglenid diversity in freshwater pond ecosystems.