Biodiversity monitoring via environmental DNA, particularly metabarcoding, is evolving into a powerful assessment tool for riverine systems. However, for metabarcoding to be fully integrated into standardized monitoring programmes, some current challenges concerning sampling design, laboratory workflow, and data analysis need to be overcome. Here, we review some of these major challenges and potential solutions. We further illustrate three potential pitfalls, namely the choice of suitable metabarcoding primers, the necessity of complete reference databases, and varying assay sensitivities, by a reappraisal of our‐own recently carried out metabarcoding study in the Volga headwaters. TaqMan qPCRs had detected catfish (Silurus glanis) and European eel (Anguilla anguilla), whereas metabarcoding had not, in the same samples. Furthermore, after extending the genetic reference database by 12 additional species and re‐analysing the metabarcoding data, we additionally detected the Siberian spiny loach (Cobitis sibirica) and Ukrainian brook lamprey (Eudontomyzon mariae) and reassigned the operational taxonomic units previously assigned to Misgurnus fossilis to Cobitis sibirica. In silico analysis of metabarcoding primer efficiencies revealed considerable variability among primer pairs and among target species, which could lead to strong primer bias and potential false‐negatives in metabarcoding studies if not properly compensated for. These results highlight some of the pitfalls of eDNA‐metabarcoding as a means of monitoring fish biodiversity in large rivers, which need to be considered in order to fully unleash the full potential of these approaches for freshwater biodiversity monitoring.

Translocations of Baltic whitefish (Coregonus sp.) into Austrian Alpine lakes have created ‘artificial hybrid zones’, threatening the genetic integrity of native lineages. We evaluate the genetic structure of Coregonus in Austrian lakes and characterize hybridization and introgression between native and introduced lineages. Fifteen populations (N = 747) were assessed for allelic variation at eight microsatellite loci and a reduced set (N = 253) for variation across two mtDNA genes (cyt b and NADH‐3). Bayesian approaches were used to estimate individual admixture proportions (q‐values) and classify genotypes as native, introduced or hybrids. q‐value distributions varied among populations highlighting differential hybridization and introgression histories. Many lakes revealed a clear distinction between native and introduced genotypes despite hybridization, whereas some locations revealed hybrid swarms. Genetic structure among lakes was congruent with morphological divergence and novelty raising speculation of multiple taxa, including a population south of the Alps, outside the putative native range of Coregonus. Although statistically congruent with inferences based on nuclear markers, mitochondrial haplotype data was not diagnostic with respect to native and non‐native lineages, supporting that the Alpine region was colonized post‐glacially by an admixture of mtDNA lineages, which coalesce >1 Ma. Mechanisms promoting or eroding lineage isolation are discussed, as well as a high potential to conserve native Alpine lineages despite the extensive historical use of introduced Baltic stocks.

Graylings (Thymallus) are among the less well-studied groups of salmonid fishes, especially across their Asian distribution range. Here we perform a comprehensive global review of their phylogeography, systematic diversity and range distributions, including biogeographic reconstruction and assessment of both conservation and taxonomic status of each species. Based on a mitogenomic phylogenetic analysis, three approaches to the delineation of molecular operational units, and evaluation of 15 a-priori defined species, we provide biological support for the recognition of 13 grayling species, plus two additional species tentatively. Several instances of paraphyly and its potential effect on systematic inferences are discussed. Overall, the genus displays increasing species diversity and decreasing range size from higher to lower latitudes and ancestral trait reconstruction supports an East Asian origin for extant diversity, most likely centred in the Amur River drainage. Europe’s colonization byThymallustook place as early as the late Miocene, at least two colonisations of North America are supported, and multiple dispersal events likely took place into Western Siberia. The conservation status for the 15 taxa was estimated to be: 6 least concern, 1 near-threatened, 2 vulnerable, 3 endangered and 3 data deficient.