An alert model reporting tool that combines e-DNA metabarcoding and molecular ecology to study freshwater fish communities and identify new invasive species

The Internal Joint Initiative was given the green light by LifeWatch ERIC General Assembly in October 2019 to build the next generation Virtual Research Environment (VRE). Informaticians at the ICT-Core in Spain and the Service Centre in Italy have worked extensively since then with scientists from biodiversity and ecosystem communities across Europe to develop new platforms and tools that those researchers require to take their investigations to the next level.

Five separate validation cases on Non-indigenous Invasive Species (NIS) determined the technical specifications required, and guided the development of workflows that integrate data from myriad sources in multiple formats, allowing full interoperability in accordance with FAIR data principles. The Metabarcoding validation case on this page describes research carried out in assessing populations of freshwater fish in the Douro Basin in Portugal.


Freshwater ecosystems have been profoundly affected by habitat loss, degradation, and overexploitation, leaving them now especially vulnerable to biological invasions. Whether non-indigenous species are the key drivers or mere complementary factors of biodiversity loss is still debated among the scientific community, however biological invasions together with other anthropogenic stressors are determining population declines and homogenisation of biodiversity in freshwater ecosystems worldwide. For example, it has been demonstrated that river basins with greater numbers of non-indigenous species have higher extinction rates of native fish species. Consequently, the application of effective biomonitoring approaches to support protection actions of managers, stakeholders and policy-makers is nowadays essential.

Conventional methods of monitoring freshwater fish diversity are based on direct observation of organisms and are therefore costly, labour and resource intensive, require taxonomic expertise, and can be invasive. Obtaining information about species and communities by retrieving DNA from environmental samples has the ability to overcome some of these difficulties. The molecular investigation of environmental samples is known as environmental DNA (eDNA). Environmental DNA can be isolated from water, soil, air or faeces as organisms shed their genetic material in the surroundings through metabolic waste, damaged tissues, sloughed skin cells and decomposition. The analysis of eDNA consists of extracting the genetic material and subjecting it to a Polymerase Chain Reaction (PCR) which amplifies the target DNA. The use of high-throughput sequencing (HTS) allows the simultaneous identification of many species within a certain taxonomic group. This community-wide approach is known as eDNA metabarcoding and involves the use of broad-range primers during PCR that amplify a set of species. In recent years, the cost of this technology has drastically decreased, making it very attractive in conservation management and scientific research. A number of studies have demonstrated that eDNA metabarcoding is more sensitive than conventional biomonitoring methods for freshwater fish as it can detect rare or low-abundance taxa. As a result, eDNA metabarcoding can be used as an early-warning tool to detect new NIS at the initial stages of colonisation, when they are not yet abundant in the ecosystem.

This validation case regards eDNA metabarcoding fish sequences collected from the Douro Basin in Portugal. DNA sequences are processed through a bioinformatic pipeline wrapped in the first part of the analytical workflow which conducts a quality check and assigns the DNA sequences to produce a list of taxa. The analytical workflow developed can process DNA sequences of different kinds, depending on the genetic markers used for the analysis and so this workflow can be applied to different taxonomic groups and ecosystems. The taxa identified might include indigenous organisms as well as newly identified taxa within a certain geographical region.  For that reason, the national checklists of introduced and invasive species (GRISS) from GBIF are consulted to check if the organisms detected are recognised as NIS or if previously unrecorded NIS have been detected through eDNA metabarcoding analysis.

The metabarcoding workflow will soon be available on this page.

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The Bulgarian National Distributed Centre is represented by the  Agricultural University-Plovdiv.

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