Sample processing
After storage of ca. 8 months at -20°C, the 12 samples were shaken and
mixed with a spoon to suspend DNA, after which two times 200 ml ethanol
were filtered using a 100 ml microfunnel unit with a 0.45µM GN6 Metricel
membrane. Filters were stored at -80°C. Subsequently, bulk samples were
processed largely following the protocol outlined by Aylagas, Mendibil,
et al. (2016). In short, samples were six to 13 times decanted in the
lab using tap water. Decanting was repeated until no animals were
recovered from the samples. All animals remaining on a 1mm sieve were
collected and stored in ethanol. The remaining material after
decantation was sorted in a cleaned tray and heavier animals such as
bivalves were picked with clean tweezers and added to the decanted
material in ethanol. From each of the four locations, one replicate was
identified morphologically (ZVL-A, 120-B, 840-C, 330-C) to the lowest
taxonomic level possible prior to grinding. Specimens were identified up
to species level, except for juveniles which were identified to the
genus level and specimens belonging to Nemertea, Anthozoa and
Oligochaeta, which were only identified up to phylum, class and order
level, respectively, following the certified protocol for macrobenthos
identification (according to ISO16665:2014 and NMBAQCS’s ”Guidelines for
processing marine macrobenthic samples: a Processing Requirements
Protocol” Version 1.0). In total, 24 samples were available for further
molecular processing (12 ethanol samples and 12 bulk samples).