Unlocking rivers' hidden diversity and ecological status using DNA metabarcoding in Northwest Spain

Abstract

Rivers are crucial ecosystems supporting biodiversity and human well-being, yet they face increasing degradation globally. Traditional river biomonitoring methods based on morphological identification of macroinvertebrates present challenges in terms of taxonomic resolution and scalability. This study explores the application of DNA metabarcoding analysis in both bulk and environmental DNA (eDNA) samples for comprehensive assessment of macrozoobenthic biodiversity, detection of invasive and endangered species, and evaluation of river ecological status in northwestern Spain. DNA metabarcoding of homogenized bulk samples and water eDNA revealed a mean of 100 and 87 macrozoobenthos species per sample respectively. However, the specific composition was significantly different with only 27.3% of the total species being shared. It was not possible to identify all the OTUs to species level; only 17.43% and 49.4% of the OTUs generated could be identified to species level in the bulk and eDNA samples, respectively. Additionally, a total of 11 exotic species (two first records for the Iberian Peninsula and another three first records for Asturias region) and one endangered species were detected by molecular tools. Molecular methods showed significant correlations with morphological identification for EQR values (Ecological Quality Ratio) of IBMWP index, yet differences in inferred river ecological status were noted, with bulk samples tending to indicate higher status. Overall, DNA metabarcoding offers a promising approach for river biomonitoring, providing insights into biodiversity, invasive species, and ecological status within a single analysis. Further optimization and intercalibration are required for its implementation in routine biomonitoring programmes, but its scalability and multi-tasking capabilities position it as a valuable tool for integrated monitoring of river ecosystems.

Keywords: IBMWP; bulk samples; eDNA; ecological status; exotic species; macroinvertebrates.

FIGURE 1

Map showing sampling locations in rivers from Northwest Spain. The colors and shapes vary depending on the sampling technique applied at each locality: Orange Circles: Morphological identification and bulk metabarcoding; Blue Squares: Morphological identification, bulk metabarcoding, and eDNA metabarcoding.

FIGURE 2

Species composition and number of species per sampling location. (a) Bulk samples. (b) eDNA samples. Chironomidae were the dominant IBMWP taxa in both bulk and eDNA samples. The second and third most dominant taxa varied between methods and were Acariformes and Oligochaeta in the bulk samples and Leuctridae and Simuliidae in the eDNA samples. In addition, several families (Anthomyiidae, Crambidae, and Thaumaleidae) were found in eDNA samples but not in bulk samples, suggesting sampling or amplification bias.

FIGURE 3

Comparison between (a) EQR and (b) ecological status obtained for the same sampling locations using different methods: Molecular (eDNA and bulk samples) and morphological. The linear model of EQR values for the eDNA samples does not differ significantly in either slope or intercept from the morphological method. However, the bulk samples have a significantly higher intercept which, as can be seen in Figurea, means that the EQR values are higher than those calculated for the same points using the morphological method. It can also be seen in (b) where the states inferred from the eDNA samples are around the visectrix (same value as morphological), but the bulk samples are mainly above it (better status than morphological).