DOI: 10.5061/dryad.x69p8czqq
title: DNA barcoding is currently unreliable for species identification in Crayfish

description: 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 (NH) per species, and
 another that focused on total number of sequences (NS; 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 NH and NS 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.
description: 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).
description: # Mitochondrial DNA (COI gene) sequences downloaded from NCBI's
 nucloetide database, aligned, trimmed, and analyzed by Allison et al. One
 folder contains seqeunce alignments for crayfish genera that were used for
 analyses focused on the numbner of unique haplotypes (ie., the NH
 Datasets), and other other folder contains sequence alignments for
 crayfish genera that were used for analyses focused on the numbner
 sequences (i.e., the NS Datasets). All sequence alignments are in FASTA
 (.fas) file format. Folder: NH_Dataset This contains COI sequence
 alignments for each of seven genera. Haplotype names are arbitrary.
 -Cambarus_NH.fas: This alignment is 654-bp long and contains 266 unique
 haplotypes for members of the genus Cambarus. -Cherax_NH.fas: This
 alignment is 594-bp long and contains 208 unique haplotypes for the genus
 Cherax. -Creaserinus_NH.fas: This alignment is 579-bp long and contains
 105 unique haplotypes for the genus Creaserinus. -Euastacus_NH.fas: This
 alignment is 654-bp long and contains 103 unique haplotypes for the genus
 Euastacus. -Faxonius_NH.fas: This alignment is 626-bp long and contains
 245 unique haplotypes for the genus Faxonius. -Lacunicambarus_NH.fas: This
 alignment is 585-bp long and contains 99 unique haplotypes for the genus
 Lacunicambarus. -Procambarus_NH.fas: This alignment is 630-bp long and
 contains 173 unique haplotypes for the genus Procambarus. Folder:
 NS_Dataset This contains COI sequence alignments for each of eight genera.
 Sequence names are NCBI accession numbers. -Cambarus_NS.fas: This
 alignment is 654-bp long and contains 418 seqeunces for members of the
 genus Cambarus. -Cherax_NS.fas: This alignment is 594-bp long and contains
 365 seqeunces for members of the genus Cherax. -Creaserinus_NS.fas: This
 alignment is 579-bp long and contains 130 seqeunces for members of the
 genus Creaserinus. -Euastacus_NS.fas: This alignment is 654-bp long and
 contains 168 seqeunces for members of the genus Euastacus.
 -Faxonius_NS.fas: This alignment is 626-bp long and contains 831 seqeunces
 for members of the genus Faxonius. -Lacunicambarus_NS.fas: This alignment
 is 520-bp long and contains 131 seqeunces for members of the genus
 Lacunicambarus. -Procambarus_NS.fas: This alignment is 630-bp long and
 contains 689 seqeunces for members of the genus Procambarus.
 -Tenuibranchiurus_NS.fas: This alignment is 644-bp long and contains 87
 seqeunces for members of the genus Tenuibranchiurus.