The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.

1The Segura River Basin is one of the most arid and regulated zones in the 2 Mediterranean as well as Europe that includes four hydrologic river types, according to 3 their natural flow regime: main stem rivers, stable streams, seasonal streams and 4 temporary streams. The relationships between flow regime and fluvial and riparian 5 habitats were studied at reference and hydrologically-altered sites for each of the four 6 types. Flow regime alteration was assessed using two procedures: 1) an indirect index, 7 derived from variables associated with the main hydrologic pressures in the basin, and 82) reference and altered flow series analyses using the Indicators of Hydrologic 9 Alteration (IHA) and the Indicators of Hydrologic Alteration in Rivers (IAHRIS). 10Habitats were characterized using the River Habitat Survey (RHS) and its derived 11Habitat Quality Assessment (HQA) score, whereas riparian condition was assessed 12 using the Riparian Quality Index (RQI) and an inventory of riparian native/exotic 13 species. Flow stability and magnitude were identified as the main hydrologic drivers of 14 the stream habitats in the Segura Basin. Hydrologic alterations were similar to those 15 described in other Mediterranean arid and semiarid areas where dams have reduced flow 16 magnitude and variability and produced the inversion of seasonal patterns. Additionally, 17 the Segura Basin presented two general trends: an increase in flow torrentiality in main 18 stems and an increase in temporality in seasonal and temporary streams. With the 19 indirect alteration index, main stems presented the highest degree of hydrologic 20 alteration, which resulted in larger channel dimensions and less macrophytes and 21 mesohabitats. However, according to the hydrologic analyses, the seasonal streams 22 presented the greatest alteration, which was supported by the numerous changes in 23 habitat features. These changes were associated with a larger proportion of uniform 24 banktop vegetation as well as reduced riparian native plant richness and mesohabitat 25 density. Both stream types presented consequent reductions in habitat and riparian 26 quality as the degree of alteration increased. However, stable streams, those least 27 impacted in the basin, and temporary streams, which are subject to great hydrologic 28 stress in reference conditions, showed fewer changes in physical habitat due to 29 hydrologic alteration. This study clarifies the relationships between hydrologic regime 30 and physical habitat in Mediterranean basins. The hydrologic and habitat indicators that 31 respond to human pressures and the thresholds that imply relevant changes in habitat 32 and riparian quality presented here will play a fundamental role in the use of holistic 33 frameworks when developing environmental flows on a regional scale. 34 2

A current challenge of biodiversity and conservation is the estimation of the spatial extent of habitat types across broad territories. In the absence of fine‐resolution maps, predictive modelling helps in assessing the spatial distribution of vegetation cover. However, such approaches are still uncommon in regional planning and management. Here, we present a framework for mapping the area of occupancy (AOO) of habitat types that allows highly suitable estimates at different scales. We model the potential AOO with abiotic variables related to topography and climate, resulting in broad AOO estimates that are subsequently downscaled to the local AOO with remote sensing. The combination of individual local AOO estimates allows the defining of the realized AOO, comprising locations with a high suitability and low uncertainty for each habitat. We applied this framework to mapping 24 protected habitat types of Natura 2000 sites in northern Spain. Local and realized AOO were highly accurate, with a 70% overall accuracy for the realized AOO. Remote sensing data, and especially LiDAR, were the most important predictors in habitat types related to forests and shrubs, followed by rock outcrops and pastures. Environmental variables were also relevant for specific habitats subject to abiotic constraints. The combination of ecological modelling with remote sensing offers multiple advantages over traditional field surveys and image interpretation, allowing the harmonization of habitat maps across large regions and through time. This is particularly useful for implementing conservation actions under Natura 2000 principles or assessing IUCN criteria for ecosystems.

ABSTRACT1. Direct and indirect utilization of coldwater springs produces a wide variety of benefits to human societies around the world, but this resource use may also be associated with significant costs to the environment, including biodiversity loss and deterioration of water quality. Despite the importance of springs little attention has been paid to their management and conservation.2. This review article draws on international literature and results from recent research on springs in New Zealand, to highlight the characteristic features of springs, identify the main human threats to their integrity, and review spring management strategies. The principal aim of the paper is to provide a management framework that can facilitate the protection, enhancement and restoration of springs.3. A key element in the management and conservation of springs is recognition of their position at the interface of three distinct ecosystems } groundwater, surface water and terrestrial. Human impacts on all three contributing ecosystems can have significant effects on spring habitat integrity.4. Effective management of springs must recognize the full range of environmental and societal values associated with them, understanding threats to the sustainability of these values and formulating strategies that provide a balance between potentially conflicting uses. As with any management strategy, the clear definition of management goals for springs is a precursor to effective conservation, protection and restoration.

Community assembly is determined by a combination of historical events and contemporary processes that are difficult to disentangle, but eco-evolutionary mechanisms may be uncovered by the joint analysis of species and genetic diversity across multiple sites. Mountain streams across Europe harbour highly diverse macroinvertebrate communities whose composition and turnover (replacement of taxa) among sites and regions remain poorly known. We studied whole-community biodiversity within and among six mountain regions along a latitudinal transect from Morocco to Scandinavia at three levels of taxonomic hierarchy: genus, species and haplotypes. Using DNA barcoding of four insect families (>3100 individuals, 118 species) across 62 streams, we found that measures of local and regional diversity and intraregional turnover generally declined slightly towards northern latitudes. However, at all hierarchical levels we found complete (haplotype) or high (species, genus) turnover among regions (and even among sites within regions), which counters the expectations of Pleistocene postglacial northward expansion from southern refugia. Species distributions were mostly correlated with environmental conditions, suggesting a strong role of lineage- or species-specific traits in determining local and latitudinal community composition, lineage diversification and phylogenetic community structure (e.g., loss of Coleoptera, but not Ephemeroptera, at northern sites). High intraspecific genetic structure within regions, even in northernmost sites, reflects species-specific dispersal and demographic histories and indicates postglacial migration from geographically scattered refugia, rather than from only southern areas. Overall, patterns were not strongly concordant across hierarchical levels, but consistent with the overriding influence of environmental factors determining community composition at the species and genus levels.