Summary DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few large‐scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which identification was feasible in the routine national monitoring. DNA metabarcoding identified more than twice the number of taxa than the morphology‐based protocol, and also yielded a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R2 values were low. With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as expensive as morphology‐based identification. Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems with current laboratory protocols and reference databases.

18 19 1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few 20 large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 212) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. 22The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. 23Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which 24 identification was feasible in the routine national monitoring. 253) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded 26 a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological 27 method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of 28 specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R 2 were low. 29With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding 30 datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as 31 expensive as morphology-based identification. 32 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment 33 results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable 34 method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological 35 identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock 36 the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems 37 with current laboratory protocols and reference databases. 38 39 40

An experiment in >1000 river and riparian sites found spatial patterns and controls of carbon processing at the global scale.

1.Many studies have shown traditional species diversity indices to perform poorly in discriminating anthropogenic influences on biodiversity. By contrast, in marine systems, taxonomic distinctness indices that take into account the taxonomic relatedness of species have been shown to discriminate anthropogenic effects. However, few studies have examined the performance of taxonomic distinctness indices in freshwater systems. 2. We studied the performance of four species diversity indices and four taxonomic distinctness indices for detecting anthropogenic effects on stream macroinvertebrate assemblages. Further, we examined the effects of catchment type and area, as well as two variables (pH and total phosphorus) potentially describing anthropogenic perturbation on biodiversity. 3. We found no indications of degraded biodiversity at the putatively disturbed sites. However, species density, rarefied species richness, Shannon's diversity and taxonomic diversity showed higher index values in streams draining mineral as opposed to peatland catchments. 4. Of the major environmental gradients analysed, biodiversity indices showed the strongest relationships with catchment area, lending further support to the importance of stream size for macroinvertebrate biodiversity. Some of the indices also showed weak linear and quadratic relationships to pH and total phosphorus, and residuals from the biodiversity index-catchment area regressions (i.e. area effect standardized) were more weakly related to pH and total phosphorus than the original index values. 5. There are a number of reasons why the biodiversity indices did not respond to anthropogenic perturbation. First, some natural environmental gradients may mask the effects of perturbation on biodiversity. Secondly, perturbations of riverine ecosystems in our study area may not be strong enough to cause drastic changes in biodiversity. Thirdly, multiple anthropogenic stressors may either increase or decrease biodiversity, and thus the coarse division of sites into reference and altered streams may be an oversimplification. 6. Although neither species diversity nor taxonomic distinctness indices revealed anthropogenic degradation of macroinvertebrate assemblages in this study, the traditional species diversity and taxonomic distinctness indices were very weakly correlated. Therefore, we urge that biodiversity assessment and conservation planning should utilize a number of different indices, as they may provide complementary information about biotic assemblages.

18 19 1) DNA metabarcoding holds great promise for the assessment of macroinvertebrates in stream ecosystems. However, few 20 large-scale studies have compared the performance of DNA metabarcoding with that of routine morphological identification. 212) We performed metabarcoding using four primer sets on macroinvertebrate samples from 18 stream sites across Finland. 22The samples were collected in 2013 and identified based on morphology as part of a Finnish stream monitoring program. 23Specimens were morphologically classified, following standardised protocols, to the lowest taxonomic level for which 24 identification was feasible in the routine national monitoring. 253) DNA metabarcoding identified more than twice the number of taxa than the morphology-based protocol, and also yielded 26 a higher taxonomic resolution. For each sample, we detected more taxa by metabarcoding than by the morphological 27 method, and all four primer sets exhibited comparably good performance. Sequence read abundance and the number of 28 specimens per taxon (a proxy for biomass) were significantly correlated in each sample, although the adjusted R 2 were low. 29With a few exceptions, the ecological status assessment metrics calculated from morphological and DNA metabarcoding 30 datasets were similar. Given the recent reduction in sequencing costs, metabarcoding is currently approximately as 31 expensive as morphology-based identification. 32 4) Using samples obtained in the field, we demonstrated that DNA metabarcoding can achieve comparable assessment 33 results to current protocols relying on morphological identification. Thus, metabarcoding represents a feasible and reliable 34 method to identify macroinvertebrates in stream bioassessment, and offers powerful advantage over morphological 35 identification in providing identification for taxonomic groups that are unfeasible to identify in routine protocols. To unlock 36 the full potential of DNA metabarcoding for ecosystem assessment, however, it will be necessary to address key problems 37 with current laboratory protocols and reference databases. 38 39 40