Reliable biodiversity data are crucial for environmental research and management. Unfortunately, data paucity prevails for many regions and organismal groups such as aquatic invertebrates. High-throughput DNA-based identification, in particular DNA metabarcoding, has accelerated biodiversity data generation. However, in the process of metabarcoding, specimens are usually destroyed, precluding later specimen-based analyses. Metabarcoding of DNA released into the preservative ethanol has been proposed as a non-destructive alternative, but proof-of-principle studies have yielded ambiguous results, reporting variance in detection probability for various taxa and methodological biases. In this study, we tested the performance of preservative-based metabarcoding of aquatic invertebrates in comparison to a standard morpho-taxonomic assessment based on samples from one of Europe’s last free-flowing rivers, the Vjosa. Multi-habitat samples were collected at 43 sites in two seasons and stored in ethanol, after fixation in formaldehyde for morpho-taxonomic analyses. Preservative-based DNA metabarcoding detected three times more taxa but failed to detect other taxa found using the standard method. In addition to incomplete reference data and primer bias that likely precluded the detection of specific taxa, preservative-based DNA metabarcoding cannot provide accurate abundance estimates. However, the metabarcoding data revealed distinct small-scale and large-scale community patterns in the Vjosa river network, which were also recovered by quantitative data of the standard approach. Overall, our results indicate that preservative-based metabarcoding provides important biodiversity data, which could be further improved by quantitative validation. The method is robust and reliable, even though samples were taken under harsh field-conditions and stored without cooling. Further, our results emphasise the need for reliable DNA barcoding reference libraries. Building those may be supported by preservative-based metabarcoding that maintains intact vouchers for subsequent specimen-based analyses.

1. The mayfly Prosopistoma pennigerum (Müller, 1785) (Insecta: Ephemeroptera) once occurred in many European river networks. However, observations decreased in the last decades and the species can be considered largely extinct throughout Europe due to river alterations.2. Only three extant populations are known from Cabriel (southern Spain), Volga (Russia) and Vjosa (Albania) rivers.3. We recorded the species along a 150 km stretch in the Vjosa River in three sampling seasons (spring 2018, fall 2018 and fall 2019), counting up to 302 P. pennigerum per m 2 , the highest recorded abundance for the species to date. Moreover, we detected traces of environmental DNA in a newly designed targeted eDNA assay. 4. In our modelling approach we define the species' niche in a theoretically available niche space given by the Vjosa River network and predict a high probability of presence (θ) in downstream located sections of this river. Expected abundances (λ) could Gabriel Singer and Simon Vitecek shared senior authors.