Assessing the Small Nuclear Ribosomal Subunit (18S rRNA) as a Potential Barcoding Gene for Amphipoda (Malacostraca: Peracarida) Along with the Design of a Unique Primer Set to Target the Amphipod 18S rRNA Gene.

The mitochondrial cytochrome oxidase I (COI) gene is frequently used as a barcoding gene for the identification of numerous taxa, including crustaceans such as amphipods. The gene's variability, which makes it useful for barcoding, can also create primer annealing issues that can inhibit amplification and sequencing, and nuclear copies of mitochondrial DNA (NUMTs) can sometimes preferentially amplify over the mitochondrial COI target locus. The small nuclear ribosomal gene (18S rRNA) can overcome issues of amplification and sequencing as primers can be designed to target highly conserved regions, making it useful across a wide variety of taxa. Due to its conserved nature, the 18S rRNA gene has not been considered a strong candidate for barcoding. Peracarid crustaceans have an insertion within the V4 hypervariable region, and amphipods are unique amongst the peracarids with an additional insertion in the V5 hypervariable region, which could make this part of the gene large and variable enough to target for barcoding. Herein, we present three primer sets designed for Amphipoda that amplify the majority of the 18S rRNA gene, including one pair (18S450F-18S1500R) that captures the V4/V5 hypervariable regions. Analyses utilizing tree-building algorithms and two species delimitation models demonstrate the potential utility of the gene region amplified by the 18S450F-18S1500R primers for barcoding. The utility of this gene for barcoding will depend on researchers incorporating this locus into future species descriptions and phylogenetic studies.