An optimised protocol for barcoding museum collections of decapod crustaceans: a case-study for a 10 - 40-years-old collection

Abstract

The sequencing of the crustacean collection of the MNHN, Paris, constitutes a promising yet very challenging barcoding project. For the collection’s crustacean specimens preserved in ethanol, some of which were collected up to 40 years ago, the conventional COI barcoding procedure of amplification with Folmer primers failed for more than half of the specimens (58%, n = 1920). We hypothesised that this failure may have been due to incompatible mismatches between the crustaceans targeted and the Folmer primer sequences and/or the amount of degradation of the DNA extracted from museum specimens. The comparison of the Folmer primers against the COI sequences from GenBank complete decapod mitochondrial genomes revealed that the annealing regions were, in fact, rather conserved, suggesting that the amplification failures were due more likely to the low quality of the DNA isolated. Using an alignment of all available decapod sequences we designed two internal primers in the middle of the barcoding COI region and also selected two additional external primers to be used as alternative to the standard Folmer primers. Using a two-overlapping-fragments amplification strategy and different primer combinations, our new protocol significantly increased the amplification success rate of the collection material from 42% with the Folmer primers to 84%, recovering an additional 364 complete barcodes and 443 minibarcodes (i.e. fragments of less than 400 base pairs), and expanding the species coverage from 254 to 397 barcoded crustaceans.