Anthropogenic chemicals in freshwater environments contribute majorly to ecosystem degradation and biodiversity decline. In particular, anthropogenic organic micropollutants (AOM), a diverse group of compounds including pesticides, pharmaceuticals, and industrial chemicals, can significantly impact freshwater organisms. AOM were found to impact the genetic diversity of freshwater species, however, the degree to which AOM cause changes in population genetic structure and allelic richness of freshwater macroinvertebrates remains poorly understood. Here, the impact of AOM on the genetic diversity of the common amphipod Gammarus pulex (Linnaeus, 1758) (clade E) was investigated on a regional scale. The site-specific AOM levels and their toxic potentials were determined in water and G. pulex tissue sample extracts for 34 sites along six rivers impacted by wastewater effluents and agricultural run-off in central Germany. Population genetic parameters were determined for G. pulex from the sampling sites by genotyping 16 microsatellite loci. Genetic differentiation among G. pulex from the studied rivers was strongly associated with geographic distance between sites, but also with differences in site-specific concentrations of AOM. Thus, genetic diversity parameters of G. pulex were found to be related to site-specific AOM levels; allelic richness was significantly negatively correlated to levels of AOM in G. pulex tissue (p < 0.003) and was reduced by up to 22% at sites with increased levels of AOM. This was seen despite a positive relationship between allelic richness and the presence of waste-water effluent. In addition, the inbreeding coefficient of G. pulex from sites with toxic AOM levels was up to 2.5 times higher than in G. pulex from more pristine sites. These results indicate that AOM levels commonly found in European rivers significantly contribute to changes in the genetic diversity of an ecologically relevant indicator species.

AOM concentrations in water and G. pulex tissue samples were analyzed by a Thermo Ultimate 3000 liquid chromatography (LC) system coupled with a quadrupole-orbitrap high-resolution mass spectrometer (HRMS; Thermo QExactive Plus). To quantify AOM in the samples, raw data from the LC-HRMS analysis were converted into the .mzML format using ‘ProteoWizard v3.0.18265’. The peak list for each batch was generated by MZmine v2.32. The list was annotated for 523 compounds from water samples and 497 compounds from G. pulex tissue samples and corrected for blanks. The analyzed compounds comprised AOM with a wide spectrum of hydrophobicity and application categories including pesticides, pharmaceuticals, and household and industrial chemicals known to regularly occur in central-European rivers.

To assess population genetic structure and genetic diversity parameters of G. pulex, 16 microsatellite loci were genotyped in a total of 931 individuals from the different sampling sites (10–30 individuals per site). Genomic DNA was extracted from pereopods of each individual using the DNeasy Blood & Tissue kit (Qiagen, Hilden, Germany). DNA integrity was checked on an agarose gel, followed by DNA concentration quantification using a NanoDrop spectrophotometer (NanoDrop Technologies Inc.). Microsatellite loci were amplified and genotyped on 20 (Holtemme River sites) to up to 30 (all other sites) G. pulex DNA samples.

Supplementary table S3. List of concentrations of compounds detected in water samples from every site in ng/L, with information on types of application (Type), and minimal detection limits (MDL) of the detected compounds.

Supplementary table S4. List of concentrations of compounds detected in amphipod tissue samples from every site in ng/g wet tissue, with information on types of applications (Type), and minimal detection limits (MDL) of the detected compounds.

Tab_microsat_all_sites. Table in genepop format with Gammarus pulex microsatellite genotypes of 16 loci from six rivers and 34 sites in Central Germany. Genotypes are are listed for the Altenau River (A1-A5), the Eine River (E1-E6), the Holtemme River (H1-H6), the Parte River (P1-P5), the Saale River (S1-S5), and the Wipper River (W1-W7). Six zeros are listed when no value was available.