Freshwaters are considered among the most endangered ecosystems globally due to multiple stressors, which coincide in time and space. These local stressors typically result from land-use intensification or hydroclimatic alterations, among others. Despite recent advances on multiple stressor effects, current knowledge is still limited to manipulative approaches minimizing biological and abiotic variability. Thus, the assessment of multiple stressor effects in real-world ecosystems is required. Using an extensive survey of 50 stream reaches across North Portugal, we evaluated taxonomic and functional macroinvertebrate responses to multiple stressors, including marked gradients of nutrient enrichment, flow reduction, riparian vegetation structure, thermal stress and dissolved oxygen depletion. We analyzed multiple stressor effects on two taxonomic (taxon richness, Shannon-diversity) and two trait-based diversity indices (functional richness, functional dispersion), as well as changes in trait composition. We found that multiple stressors had additive effects on all diversity metrics, with nutrient enrichment identified as the most important stressor in three out of four metrics, followed by dissolved oxygen depletion and thermal stress. Taxon richness, Shannon-diversity and functional richness responded similarly, whereas functional dispersion was driven by changes in flow velocity and thermal stress. Functional trait composition changed along a major stress gradient determined by nutrient enrichment and oxygen depletion, which was positively correlated with organisms possessing fast-living strategies, aerial respiration, adult phases, and gathering-collector feeding habits. Overall, our results reinforce the need to consider complementary facets of biodiversity to better identify assembly processes in response to multiple stressors. Our data suggest that stressor interactions may be less frequent in real-word streams than predicted by manipulative experiments, which can facilitate mitigation strategies. By combining an extensive field survey with an integrative consideration of multiple biodiversity facets, our study provides new insights that can help to better assess and manage rivers in a global change context.This work was supported by the project STREAMECO: Biodiversity and ecosystem functioning under climate change: from the gene to the stream (PTDC/CTA-AMB/31245/2017), funded by the Portuguese Foundation for Science and Technology (FCT) and the European Regional Development Fund (ERDF) through the COMPETE2020—Programa Operacional Competitividade e Internacionalização (POCI). This work was also supported by the River2Ocean project (NORTE-01-0145- FEDER-000068), co-financed by the European Regional Development Fund (ERDF), through Programa Operacional Regional do Norte (NORTE2020); by CCDR-N (Norte Portugal Regional Coordination and Development Commission) and European Funds (FEDER/POCI/COMPETE2020) through the project AgriFoodXXI (NORTE-01-0145-FEDER-000041); and by the “ContratoPrograma” UIDB/04050/2020 funded by national funds through the FCT I.P., the Centre of Molecular and Environmental Biology (CBMA). This work had the support of national funds through Portuguese Foundation for Science and Technology (FCT), under the project LA/P/0069/ 2020 granted to the Associate Laboratory ARNET. CG-C was funded by ”la Caixa” Foundation (ID 100010434), by means of a Junior Leader Fellowship contract (LCF/BQ/PR22/11920005)
