Metabarcoding protocol: Analysis of protists using the 18S rRNA gene and a DADA2 pipeline (Version 1)

This protocol has been prepared as part of the Interreg Alpine Space project Eco-AlpsWater (ASP569) - Innovative Ecological Assessment and Water Management Strategy for the Protection of Ecosystem Services in Alpine Lakes and Rivers, Activity A.T1.3, Deliverable D.T1.3.2 – 2, https://www.alpine-space.eu/projects/eco-alpswater/en/hom

Plankton DNA extraction from Sterivex filter units

The objective of this protocol is to provide a reliable and replicable method for the DNA extraction of lake micro-plankton to be used for downstream DNA analysis. This protocol is one of those proposed by the Eco-AlpsWater consortium to promote the implementation of High Throughput Sequencing (HT S) of environmental DNA (eDNA) in the biomonitoring and ecological assessment of water bodies. The extraction is performed from samples filtered through Sterivex cartridges (Sterivex™ GP 0.22μm) and stored at -20°C, as described in the protocol dx.doi.org/10.17504/protocols.io.xn6fmhe, and with the use of the DNeasy® PowerWater Sterivex Kit (QIAGEN) with specific modifications adapted to plankton DNA extraction. The application proposed here, in the context of EcoAlpsWater, aims at comparing DNA inventories to traditional phytoplanktonic inventories and at characterizing more broadly the micro-planktonic diversity through eDNA analysis (including bacteria). This protocol is part of the deliverables provided by the WP1 of the Eco-AlpsWater project. All members of the EcoAlpsWater consortium (http://www.alpine-space.eu/projects/eco-alpswater/en/home) contributed to the optimization of this protocol

Metabarcoding protocol: Analysis of Bacteria (including Cyanobacteria) using the 16S rRNA gene and a DADA2 pipeline (Version 1)

This protocol has been prepared as part of the Interreg Alpine Space project Eco-AlpsWater (ASP569) - Innovative Ecological Assessment and Water Management Strategy for the Protection of Ecosystem Services in Alpine Lakes and Rivers, Activity A.T1.3, Deliverable D.T1.3.2 – 1, https://www.alpine-space.eu/projects/eco-alpswater/en/hom

eDNA metabarcoding biodiversity of freshwater fish in the Alpine area

Environmental DNA (eDNA) based methods are proving to be a promising tool for freshwater fish biodiversity assessment in Europe within the Water Framework Directive (WFD, 2000/60/EC) especially for large rivers and lakes where current fish monitoring techniques have known shortcomings. Many freshwater fish are experiencing critical population declines with risk of local or global extinction because of intense anthropogenic pressure and this can have serious consequences on freshwater ecosystem functioning and diversity. Within the EU project Eco-AlpsWater, advanced high throughput sequencing (HTS) techniques are used to improve the traditional WFD monitoring approaches by using environmental DNA (eDNA) collected in Alpine waterbodies. An eDNA metabarcoding approach specifically designed to measure freshwater fish biodiversity in Alpine lakes and rivers has been extensively evaluated by using mock samples within an intercalibration test. This eDNA method was validated and used to study fish biodiversity of eight lakes and six rivers of the Alpine region including four EC countries (Austria, France, Italy, Slovenia) and Switzerland. More in detail, this metabarcoding approach, based on HTS sequencing of a section of the 12S rRNA gene, was used to assess freshwater fish biodiversity and their distribution in the different habitats. These data represent the first attempt to provide a comprehensive description of freshwater fish diversity in different ecosystems of the Alpine area confirming the applicability of eDNA metabarcoding analyses for the biomonitoring of fish inhabiting Alpine and perialpine lakes and rivers

Genotype × genotype interactions between the toxic cyanobacterium Microcystis and its grazer, the waterflea Daphnia

Toxic algal blooms are an important problem worldwide. The literature on toxic cyanobacteria blooms in inland waters reports widely divergent results on whether zooplankton can control cyanobacteria blooms or cyanobacteria suppress zooplankton by their toxins. Here we test whether this may be due to genotype × genotype interactions, in which interactions between the large-bodied and efficient grazer Daphnia and the widespread cyanobacterium Microcystis are not only dependent on Microcystis strain or Daphnia genotype but are specific to genotype × genotype combinations. We show that genotype × genotype interactions are important in explaining mortality in short-time exposures of Daphnia to Microcystis. These genotype × genotype interactions may result in local coadaptation and a geographic mosaic of coevolution. Genotype × genotype interactions can explain why the literature on zooplankton–cyanobacteria interactions is seemingly inconsistent, and provide hope that zooplankton can contribute to the suppression of cyanobacteria blooms in restoration projects

Local Expansion of a Panmictic Lineage of Water Bloom-Forming Cyanobacterium Microcystis aeruginosa

In previous studies, we have demonstrated that the population structure of the bloom-forming cyanobacterium Microcystis aeruginosa is clonal. Expanded multilocus sequence typing analysis of M. aeruginosa using 412 isolates identified five intraspecific lineages suggested to be panmictic while maintaining overall clonal structure probably due to a reduced recombination rate between lineages. Interestingly, since 2005 most strains belonging to one of these panmictic clusters (group G) have been found in a particular locality (Lake Kasumigaura Basin) in Japan. In this locality, multiple, similar but distinct genotypes of this lineage predominated in the bloom, a pattern that is unprecedented for M. aeruginosa. The population structure underlying blooms associated with this lineage is comparable to epidemics of pathogens. Our results may reveal an expansion of the possible adaptive lineage in a localized aquatic environment, providing us with a unique opportunity to investigate its ecological and biogeographical consequences