DNA barcoding analysis of macrofauna
A total of 726 individuals were photographed. Primers amplifying thecytochrome c oxidase I (COI ) gene were used for DNA barcoding as this is the standard DNA barcoding marker (Hebert, Cywinska, Ball, & deWaard, 2003); it performs very well for species level discrimination and has a well-populated reference database (the Barcode of Life Data (BOLD) System) (Ratnasingham & Hebert, 2007). A piece of the tail-end was removed for DNA extraction, and the remaining sample was placed in a 1.5 mL tube or larger container as needed. DNA was extracted using DNeasy 96 Blood & Tissue Kits (Qiagen). The COI C_VF1LFt1 and C_VR1LRt1 primers were tried first (Ivanova, Zemlak, Hanner, & Hebert, 2007); samples that failed to amplify or sequence with these primers were amplified with polyLCO and polyHCO primers (Carr, Hardy, Brown, Macdonald, & Hebert, 2011).
To ensure polychaete taxonomic data generated here can be compared to other studies across spatial scales and over time, which is difficult with complex taxonomies, the BOLD Barcode Index Number (BIN) system was adopted to classify barcoded polychaetes (Ratnasingham & Hebert, 2013). When a barcode was too short for automatic inclusion into a BIN by BOLD, it was manually applied to the appropriate BIN via the identification portal of BOLD based on sequence similarity. There were sixteen instances where one morphospecies produced sequences that fell into multiple BINs. When this occurred, the sequenced specimens were examined and then all individuals for that morphospecies were re-examined to determine if they could be appropriately assigned to the identified BINs. There were six occasions where there were 2-5 genetic groups revealed by DNA barcoding that were not visually distinguishable upon re-inspection of specimens, such that it was not possible to assign them confidently into a BIN. In these cases, all specimens were ’lumped’ into what we refer to as a ’combined BIN’ representing that morphospecies. In a few instances there were individual specimens for which no sequence was obtained but for which a taxonomic name could be confidently assigned using traditional morpho-taxonomic methods. Two alpha diversity parameters describing species richness, Shannon and Simpson indices, were calculated for the macrofauna data using thevegan R package (Oksanen et al., 2017).