Towards an Inerian DNA barcode reference library of freshwater macroinvertebrates and fishes

Freshwater macroinvertebrates and fishes are focal groups in major ecosystem biomonitoring programs in the Iberian Peninsula. Yet, their use as bioindicators is sometimes constrained by the time and cost needed for sorting macroinvertebrates specimens and their challenging taxonomic identification, and the huge sampling procedures for capturing rare or incipient fish species, respec- tively. Given the increasing cost-effectiveness of metazoan identification based on metabarcoding [i.e., high-throughput sequenc- ing (HTS) of DNA barcodes] and reliability of species-level identification and the high number of samples that can be processed, its use in biomonitoring of freshwater communities can provide an alternative to morphology-based approaches. However, the accuracy of species assignment in metabarcoding approaches relies on the availability of a comprehensive DNA barcode reference library. Because of the high level of endemicity in the Iberian Peninsula, current public repositories for DNA barcodes may not be informative enough to identify the Iberian fauna to species level. Here, we compiled the Iberian freshwater macroinvertebrates and fishes taxonomic list (including indigenous and non-indigenous species) and the available molecular data for the cytochrome oxidase I DNA barcode (cox1, COI-5P) in public repositories to assess the extent of DNA barcode coverage. The DNA barcode coverage was reported for DNA fragments within the Folmer region (658 bp). Given that HTS platforms provide DNA sequence in the range of 50-400 bp in length, we also reported the second half of the DNA barcode (313 bp, Leray region) and the first part of the Leray region (285 bp, Leray-285), which are short DNA barcodes useful to assign metabarcoding cox1 data. For macroin- vertebrates, the final taxonomic checklist comprises 3348 species including Mollusca (65 species), Crustacea (101 species) and Insecta (3182 species). We present an initial DNA barcode reference library, with an overall coverage of ~ 35 % of the Iberian taxa. Exploring this data, we find a strong taxonomic bias. Based on Leray-285, Odonata (43 of 79 species barcoded, 54.43 %) and Hemiptera (44 of 81 species barcoded, 54.32 %) were the best represented lineages. In contrast, Diptera (393 of 1693 species barcoded, 23.21 %), and Plecoptera (42 of 135 species barcoded, 31.11 %) were underrepresented. For fishes, the available DNA barcode data covered 98.11 % of the indigenous (76) and non-indigenous (30) species. By revealing and quantifying current gaps on the available data (~ 65 %), we aim to improve efficiency in designing the next steps towards the ambitious yet necessary goal of compiling a complete DNA barcode reference library for Iberian macroinvertebrates and fishes.