DNA barcoding is commonly used for species identification. Despite this, there has not been a comprehensive assessment of the utility of DNA barcoding in crayfishes (Decapoda: Astacidea). Here we examined the extent to which local barcoding gaps (used for species identification) and global barcoding gaps (used for species discovery) exist among crayfishes, and whether global gaps, if present, met a previously suggested 10 × threshold.

Using publicly available mitochondrial COI sequence data from the National Center for Biotechnology Information’s nucleotide database, we created two versions of the COI datasets used for downstream analyses: one focused on the number of unique haplotypes (N_H) per species, and another that focused on total number of sequences (N_S; i.e., including redundant haplotypes) per species. Ultimately, a total of 81 species were included, with 58 species and five genera from family Cambaridae and 23 species from three genera from family Parastacidae.

We found that local barcoding gaps were present in only 30 species (20 members of Cambaridae and 10 Parastacidae). Global barcoding gaps were detected in only four genera (Cambarus, Cherax, Euastacus, and Tenuibranchiurus), and they were all well below the previously suggested 10× threshold. We propose that a ~5x threshold could act as a more appropriate working hypothesis for species discovery. While the N_H and N_S datasets yielded largely similar results, there were some discrepant inferences.

Currently, the utility of DNA barcoding for species identification and discovery in crayfish is quite limited, and caution should be exercised when molecular approaches are used in place of taxonomic expertise.

Assessment of the evidence for local and global barcoding gaps is important for understanding the reliability of molecular species identification and discovery, but outcomes are dependent on the current state of taxonomy. As this improves (e.g., via resolving species complexes, possibly elevating some subspecies to the species-level status, and redressing specimen misidentifications in natural history and other collections), so too will the utility of DNA barcoding.

We searched the NCBI nucleotide database for crayfish COI sequences using the following search terms and Boolean operators: “genus name" AND “cytochrome” OR “COI” OR “COX1”. All searches were conducted, and sequences downloaded, between March and December 2022. We aligned sequences from each genus separately using MUSCLE (Edgar, 2004), implemented in MEGA v.7.026 (Kumar, Stecher, & Tamura, 2016).