Arcellinida testate amoebae in environmental DNA-based bioindication, from taxonomy free to haplotypic level

Bioindication, evaluating biological responses to environmental disturbances, is crucial for assessing ecological status. While historical bioindication relied on macroscopic organisms, the introduction of environmental DNA (eDNA) techniques allows the application of protist without the necessity of morphological identification. In this study we propose a novel bioindication methodology utilizing Arcellinida, a group of top predators among protists, as bioindicators of freshwater ecosystems. In that purpose, we firstly characterized the Arcellinida diversity over one year at three different points of the Lake Sanabria, an ancient glacier lake known to be subjected to anthropogenic disturbances. We compared this diversity with an undisturbed control site. Secondly, we characterized the Arcellinida diversity in other ecosystems to generate the ecological background to test the connectivity between them. Results indicate limited connectivity between the different ecosystems, and an edge effect between terrestrial and aquatic ecosystems. Disturbed freshwater ecosystems exhibited reduced Arcellinida diversity at both specific and infraspecific levels, providing valuable insights into recent disturbances. Arcellinida-based bioindication provides a sensitive, accurate and easy to interpret protocol for monitoring disturbances in freshwater ecosystems. It represents a valuable tool for environmental assessments and conservation strategies.