Sampling sites

Sample analysis

Morphological analysis

DNA extraction and PCR amplification

Preparation and High-Throughput sequencing of DNA

Sequence data processing

Ecological status class assessment

Statistical analysis

Software

Comparison of methods for environmental assessment – Ecological status class

Comparison of methods for environmental assessment – Polluosensibilité spécifique index (IPS)

Deviations link to Environmental variables

General deviations in species lists

Shannon diversity and taxa missing from reference databases

Causes for differences in the species list between morphological and molecular assessments

Main species causing differences in IPS

Comparative use of the rbcL and 18S markers

Different ecological status resulted from the morphological and the molecular assessments

The reference database incompleteness leads both to non-identification and misidentification of species

The detection of species is also affected by primer bias and the choice of the pipeline

The metabarcoding detect additional species

Not only the presence of species had an impact on the IPS scores, species abundances were also affecting the index calculations

Different markers give different ecological status assessment