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.

In this paper we describe a simple approach using double peaks in chromatograms generated as artefacts in the vicinity of heterozygous indels, to identify the specific sequences present in individual strands of a given DNA fragment. This method is useful to assign bases in individuals that are heterozygous at multiple sites. In addition, the relative sizes of the double peaks help to determine the ploidy level and the relative contribution of the parental genomes in hybrids. Our interpretation was confirmed with the analysis of artificial mixtures of DNA of two different species. Results were robust with varying PCR and sequencing conditions. The applicability of this method was demonstrated in hybrids of the Squalius alburnoides complex and in heterozygotes of Chondrostoma oligolepis. Far from being limited to these fish models and the gene where it was tested (beta-actin), this sequence reconstruction methodology is expected to have a broader application.

Background.Worldwide predictions suggest that up to 75% of the freshwater fish species occurring in rivers with reduced discharge could be extinct by 2070 due to the combined effect of climate change and water abstraction. The Mediterranean region is considered to be a hotspot of freshwater fish diversity but also one of the regions where the effects of climate change will be more severe. Iberian cyprinids are currently highly endangered, with over 68% of the species raising some level of conservation concern.Methods.During the FISHATLAS project, the Portuguese hydrographical network was extensively covered (all the 34 river basins and 47 sub-basins) in order to contribute with valuable data on the genetic diversity distribution patterns of native cyprinid species. A total of 188 populations belonging to 16 cyprinid species ofSqualius, Luciobarbus, Achondrostoma, Iberochondrostoma, AnaecyprisandPseudochondrostomawere characterized, for a total of 3,678 cytochromebgene sequences.Results.When the genetic diversity of these populations was mapped, it highlighted differences among populations from the same species and between species with identical distribution areas. Factors shaping the contemporary patterns of genetic diversity were explored and the results revealed the role of latitude, inter-basin connectivity, migratory behaviour, species maximum size, species range and other species intrinsic traits in determining the genetic diversity of sampled populations. Contrastingly, drainage area and hydrological regime (permanent vs. temporary) seem to have no significant effect on genetic diversity. Species intrinsic traits, maximum size attained, inter-basin connectivity and latitude explained over 30% of the haplotype diversity variance and, generally, the levels of diversity were significantly higher for smaller sized species, from connected and southerly river basins.Discussion.Targeting multiple co-distributed species of primary freshwater fish allowed us to assess the relative role of historicalversuscontemporary factors affecting genetic diversity. Since different patterns were detected for species with identical distribution areas we postulate that contemporary determinants of genetic diversity (species’ intrinsic traits and landscape features) must have played a more significant role than historical factors. Implications for conservation in a context of climate change and highly disturbed habitats are detailed, namely the need to focus management and conservation actions on intraspecific genetic data and to frequently conduct combined genetic and demographic surveys.