COI barcoding provides reliable species identification and pinpoints cryptic diversity in Western Palearctic amphibians

Assembling DNA barcode reference libraries for various taxonomic groups allows researchers to use metabarcoding or environmental DNA approaches to gain a rapid understanding of diversity in given environments. However, our ability to use reference libraries depends on how accurately DNA barcodes are able to recover taxonomic boundaries and identify species, which is rarely considered. We constructed an extensive COI barcoding library for amphibians of the Western Palearctic and successfully recovered barcodes from 60 urodele and 73 anuran species (representing 94% and 98% of the nominal anuran and urodele species in the Western Palearctic, respectively), covering the intraspecific diversity of the majority of species in this region. We tested the effectiveness of our assembled DNA barcode dataset for species identification using barcoding gap, efficiency analyses, and two phylogenetic species delimitation methods. We obtained DNA barcodes for 1,251 specimens (691 anurans and 560 urodeles) with a high success rate (92-96%) of species identification. The absence of a barcoding gap in a number of samples was linked to species misidentifications, which suggest incipient speciation or cryptic diversity, or previously described mitochondrial introgression events. The phylogenetic species delimitation methods resulted in substantial oversplitting of currently accepted taxonomy. This COI barcoding library provides an almost complete and reliable reference library for Western Palearctic amphibians. We highlight the importance of generating comprehensive and well curated reference libraries that include intra- and interspecific genetic variability and the need of detailed taxonomic revision when ambiguous or incorrect DNA barcodes exist.