updated version- Lake biofilms sampling for both downstream DNA analysis and microscopic counts v1
Authors: Rimet Frederic , Kurmayer Rainer , Salmaso Nico , Capelli Camilla , Chardon Cecile , Vautier Marine , Gueguen Julie , Bouchez Agnès , Domaizon Isabelle 
Published: February 2021

The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-phytobenthos and associated microbes in biofilms, to be used in both downstream DNA analysis and algal microscopic counts. The field protocol is optimized for routine sampling and is in agreement with CEN guidance (NF EN 13946) and CEN technical report (CEN/TR 17245) for the analysis of benthic diatoms from rivers and lakes. The application proposed here in the context of EcoAlpsWater aims at comparing DNA inventories to traditional inventories (microscopy). The sampling strategy has been designed to assess localized hotspots pollutions/perturbations impacts on the shoreline, taking into account shoreline’s heterogeneity. This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium have contributed to the optimization of this protocol. The use of microalgae to assess ecological quality of lakes is traditionally based on phytoplankton that allow to evaluate the trophic status. A single depth-integrated water sample from one sampling site above the deepest point of the lake is chosen, and samplings are carried out in the euphotic zone at different seasons. Though not always included as biological quality indicator in lake monitoring, phytobenthos has relevance for lake ecological quality assessment and might be used as a complementary indicator beside phytoplankton in the future (e.g. DeNicola & Kelly 2013). Phytobenthic communities of the lake’s shorelines are generally heterogeneous. The reason for this heterogeneity can be due to many factors, including substrate characteristics as well as anthropogenic factors such as eutrophication, the presence of pollution hotspots, like arrivals of polluted rivers in the lakes or arrivals of wastewaters through drains (e.g. Crossetti et al. 2013, Rimet et al. 2015/2016/2018, Cicek & Yamuc, 2017). But it can also be caused by the nature of the neighboring terrestrial habitats and land use (for instance closed forest vs open fields, or pasture vs steep rock falls). The objective of this protocol is to provide a reliable and replicable method for the sampling of lake micro-phytobenthos and associated microbes biofilms to be used in both downstream DNA analysis and algal microscopic counts. Examples of applications to assess localized hotspots pollutions impact on the shoreline, taking into account shoreline’s heterogeneity, will be provided. The field protocol is based on routine methods used for biofilms sampling and is in agreement with : - CEN 2014. Water quality - NF EN 13946 - Guidance for the routine sampling and preparation of benthic diatoms from rivers and lakes. Afnor, 1-23. - CEN 2018. Water quality - CEN/TR 17245 - Technical report for the routine sampling of benthic diatoms from rivers and lakes adapted for metabarcoding analyses. CEN, 1-8.