Fish and macroinvertebrate assemblages reveal extensive degradation of the world's rivers.

Rivers suffer from multiple stressors acting simultaneously on their biota, but the consequences are poorly quantified at the global scale. We evaluated the biological condition of rivers globally, including the largest proportion of countries from the Global South published to date. We gathered macroinvertebrate- and fish-based assessments from 72,275 and 37,676 sites, respectively, from 64 study regions across six continents and 45 nations. Because assessments were based on differing methods, different systems were consolidated into a 3-class system: Good, Impaired, or Severely Impaired, following common guidelines. The proportion of sites in each class by study area was calculated and each region was assigned a Köppen-Geiger climate type, Human Footprint score (addressing landscape alterations), Human Development Index (HDI) score (addressing social welfare), % rivers with good ambient water quality, % protected freshwater key biodiversity areas; and % of forest area net change rate. We found that 50% of macroinvertebrate sites and 42% of fish sites were in Good condition, whereas 21% and 29% were Severely Impaired, respectively. The poorest biological conditions occurred in Arid and Equatorial climates and the best conditions occurred in Snow climates. Severely Impaired conditions were associated (Pearson correlation coefficient) with higher HDI scores, poorer physico-chemical water quality, and lower proportions of protected freshwater areas. Good biological conditions were associated with good water quality and increased forested areas. It is essential to implement statutory bioassessment programs in Asian, African, and South American countries, and continue them in Oceania, Europe, and North America. There is a need to invest in assessments based on fish, as there is less information globally and fish were strong indicators of degradation. Our study highlights a need to increase the extent and number of protected river catchments, preserve and restore natural forested areas in the catchments, treat wastewater discharges, and improve river connectivity.

Modelling technical and biological biases in macroinvertebrate community assessment from bulk preservative using multiple metabarcoding markers.

DNA metabarcoding from the ethanol used to store macroinvertebrate bulk samples is a convenient methodological option in molecular biodiversity assessment and biomonitoring of aquatic ecosystems, as it preserves specimens and reduces problems associated with sample sorting. However, this method may be affected by errors and biases, which need to be thoroughly quantified before it can be mainstreamed into biomonitoring programmes. Here, we used 80 unsorted macroinvertebrate samples collected in Portugal under a Water Framework Directive monitoring programme, to compare community diversity and taxonomic composition metrics estimated through morphotaxonomy versus metabarcoding from storage ethanol using three markers (COI-M19BR2, 16S-Inse01 and 18S-Euka02) and a multimarker approach. A preliminary in silico analysis showed that the three markers were adequate for the target taxa, with detection failures related primarily to the lack of adequate barcodes in public databases. Metabarcoding of ethanol samples retrieved far less taxa per site (alpha diversity) than morphotaxonomy, albeit with smaller differences for COI-M19BR2 and the multimarker approach, while estimates of taxa turnover (beta diversity) among sites were similar across methods. Using generalized linear mixed models, we found that after controlling for differences in read coverage across samples, the probability of detection of a taxon was positively related to its proportional abundance, and negatively so to the presence of heavily sclerotized exoskeleton (e.g., Coleoptera). Overall, using our experimental protocol with different template dilutions, the COI marker showed the best performance, but we recommend the use of a multimarker approach to detect a wider range of taxa in freshwater macroinvertebrate samples. Further methodological development and optimization efforts are needed to reduce biases associated with body armouring and rarity in some macroinvertebrate taxa.

Chironomidae traits and life history strategies as indicators of anthropogenic disturbance.

In freshwater ecosystems, Chironomidae are currently considered indicators of poor water quality because the family is often abundant in degraded sites. However, it incorporates taxa with a large ecological and physiological diversity and different sensitivity to impairment. Yet, the usual identification of Chironomidae at coarse taxonomic levels (family or subfamily) masks genus and species sensitivities. In this study, we investigate the potential of taxonomic and functional (traits) composition of Chironomidae to detect anthropogenic disturbance. In this context, we tested some a priori hypotheses regarding the ability of Chironomidae taxonomic and trait compositions to discriminate Mediterranean streams affected by multiple stressors from least-disturbed streams. Both taxonomic and Eltonian trait composition discriminated sites according to their disturbance level. Disturbance resulted in the predicted increase of Chironomidae with higher number of stages with hibernation/diapause and of taxa with resistance forms and unpredicted increase of the proportion of taxa with longer life cycles and few generations per year. Life history strategies (LHS), corresponding to multivoltine Chironomidae that do not invest in hemoglobin and lack strong spring synchronization, were well adapted to all our Mediterranean sites with highly changeable environmental conditions. Medium-size animals favored in disturbed sites where the Mediterranean hydrological regime is altered, but the reduced number of larger-size/carnivore Chironomids suggests a limitation to secondary production. Results indicate that Chironomidae genus and respective traits could be a useful tool in the structural and functional assessment of Mediterranean streams. The ubiquitous nature of Chironomidae should be also especially relevant in the assessment of water bodies naturally poor in other groups such as the Ephemeroptera, Plecoptera, and Trichoptera, such as the lowland rivers with sandy substrates, lakes, or reservoirs.

Adjusting the effect of seasonal variability in the bioassessment of streams.

Bioassessment tools should distinguish between the effects of anthropogenic degradation in communities and natural temporal changes. The present study tests the influence of natural seasonal variability on macroinvertebrate stream communities assessed by a predictive model (PORTRIV) and a multimetric index (IPtI) calibrated for spring. The scores of PORTRIV decreased significantly between spring and autumn, and between spring and winter (ca. 37 to 53%, respectively), while those of IPtI did not change significantly between seasons. For non-reference samples, the results of the predictive model also indicate no significant differences. A correction factor (CF) was calculated to adjust the existing differences in the model assessments between seasons, based on the percentage of variation of reference site scores from spring to autumn and winter. After the application of the CF to the OE50 scores of spring reference samples, the differences were no longer significant. Independent reference validation sites confirmed this tendency. This method has the advantage of avoiding large efforts required for the construction of databases from other seasons and the development of new models to allow the assessment of streams in seasons other than spring. Further tests with models developed in regions with more marked seasonal changes should be done to confirm its wider applicability.

Effect of environmental education on the knowledge of aquatic ecosystems and reconnection with nature in early childhood.

Blue and green ecosystems are considered a key for the improvement of cities sustainability, providing numerous ecosystem services and habitat for many species. However, urban streams are still neglected and degraded, specially in southern European countries. One important step towards the rehabilitation of these ecosystems is the awareness of their importance by citizens. This study aimed to assess the effect of 1-year of activities (field and laboratory) of an environmental education project on primary school children, in improving their knowledge on urban stream ecosystems and their problems. We analyzed students' questionnaires before and after field and laboratory activities, drawings and group interviews. Initially, most children had incipient contact with rivers and streams, showing fears and lack of knowledge about them. As the project progressed, their perceptions changed, with a clear increase in the proportion of students recognizing the biodiversity associated to rivers (e.g., names of riparian trees, aquatic plants and invertebrates). Also, their fears decreased significantly, while their awareness to the impacts of artificialization and lack of riparian vegetation increased. Our results show that direct contact with nature have a positive role in the way it is understood by children, as well as promoting responsible and sustainable behaviors, being effective from the early primary-school years.

Influence of river regulation and instream habitat on invertebrate assemblage' structure and function.

Dams modify geomorphology, water quantity, quality and timing of stream flows affecting ecosystem functioning and aquatic biota. In this study, we addressed the structural and functional macroinvertebrate community alterations in different instream mesohabitats of two Portuguese rivers impaired by dams. We sampled macroinvertebrates in riffles, runs and pools of river sites downstream of the dams (i.e. regulated; n = 24) and in sites without the influence of the dams (i.e. unregulated; n = 7), assessing a total of 64 mesohabitats, following late spring-early summer regular flows. We found a distinct taxonomic structure and trait composition of macroinvertebrate assemblages between regulated and unregulated flow sites, and also between mesohabitats in which the differences were more evident. When analysing each mesohabitat individually, the effect of flow regulation was detected only in run-type mesohabitats for both taxonomic and trait composition, leading us to infer that a selective macroinvertebrate assessment on run mesohabitats would be a valuable contribution to detect regulated flow effects on ecosystems impaired by dams. Additionally, there is evidence that respiration and locomotion traits could be effective tools to identify damming flow alterations. This study supports that the quality assessments of rivers impacted by dams could benefit from a sampling approach focused on run mesohabitats and the detection of some key traits, which would improve assessment accuracy.

Assessing changes in stream macroinvertebrate communities across ecological gradients using morphological versus DNA metabarcoding approaches.

Freshwater macroinvertebrates provide valuable indicators for biomonitoring ecosystem change in relation to natural and anthropogenic drivers. DNA metabarcoding is an efficient approach for estimating such indicators, but its results may differ from morphotaxonomic approaches traditionally used in biomonitoring. Here we test the hypothesis that despite differences in the number and identity of taxa recorded, both approaches may retrieve comparable patterns of community change, and detect similar ecological gradients influencing such changes. We compared results obtained with morphological identification at family level of macroinvertebrates collected at 80 streams under a Water Framework Directive biomonitoring program in Portugal, with results obtained with metabarcoding from the ethanol preserving the bulk samples, using either single (COI-M19BR2, 16S-Inse01, 18S-Euka02) or multiple markers. Metabarcoding recorded less families and different communities compared to morphotaxonomy, but community sensitivities to disturbance estimated with the IASPT index were more similar across approaches. Spatial variation in local community metrics and the factors influencing such variation were significantly correlated between morphotaxonomy and metabarcoding. After reducing random noise in the dissimilarity matrices, the spatial variation in community composition was also significantly correlated across methods. A dominant gradient of community change was consistently retrieved, and all methods identified a largely similar set of anthropogenic stressors strongly influencing such gradient. Overall, results confirm our initial hypothesis, suggesting that morphotaxonomy and metabarcoding can estimate consistent spatial patterns of community variation and their main drivers. These results are encouraging for macroinvertebrate biomonitoring using metabarcoding approaches, suggesting that they can be intercalibrated with morphotaxonomic approaches to recover equivalent spatial and temporal gradients of ecological change.

A taxonomy-free approach based on machine learning to assess the quality of rivers with diatoms.

Diatoms are a compulsory biological quality element in the ecological assessment of rivers according to the Water Framework Directive. The application of current official indices requires the identification of individuals to species or lower rank under a microscope based on the valve morphology. This is a highly time-consuming task, often susceptible of disagreements among analysts. In alternative, the use of DNA metabarcoding combined with High-Throughput Sequencing (HTS) has been proposed. The sequences obtained from environmental DNA are clustered into Operational Taxonomic Units (OTUs), which can be assigned to a taxon using reference databases, and from there calculate biotic indices. However, there is still a high percentage of unassigned OTUs to species due to the incompleteness of reference libraries. Alternatively, we tested a new taxonomy-free approach based on diatom community samples to assess rivers. A combination of three machine learning techniques is used to build models that predict diatom OTUs expected in test sites, under reference conditions, from environmental data. The Observed/Expected OTUs ratio indicates the deviation from reference condition and is converted into a quality class. This approach was never used with diatoms neither with OTUs data. To evaluate its efficiency, we built a model based on OTUs lists (HYDGEN) and another based on taxa lists from morphological identification (HYDMORPH), and also calculated a biotic index (IPS). The models were trained and tested with data from 81 sites (44 reference sites) from central Portugal. Both models were considered accurate (linear regression for Observed and Expected richness: R2 ≈ 0.7, interception ≈ 0.8) and sensitive to global anthropogenic disturbance (Rs2 > 0.30 p < 0.006 for global disturbance). Yet, the HYDGEN model based on molecular data was sensitive to more types of pressures (such as, changes in land use and habitat quality), which gives promising insights to its use for bioassessment of rivers.