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).