DOI: 10.5061/dryad.5qfttdz92
title: Arctic nekton uncovered by eDNA metabarcoding: diversity, potential range expansions and benthopelagic coupling

description: The Arctic Ocean is home to a unique fauna that is disproportionately
 affected by global warming but that remains under-studied. Due to their
 high mobility and responsiveness to global warming, cephalopods and fishes
 are good indicators of the reshuffling of Arctic communities. Here, we
 established a nekton biodiversity baseline for the Fram Strait, the only
 deep connection between the North Atlantic and Arctic Ocean. Using
 universal primers for fishes (12S) and cephalopods (18S), we amplified
 environmental DNA (eDNA) from seawater (50–2700 m) and deep-sea sediment
 samples collected at the LTER HAUSGARTEN observatory. We detected twelve
 cephalopod and 31 fish taxa in the seawater and seven cephalopod and 28
 fish taxa in the sediment, including the elusive Greenland shark
 (Somniosus microcephalus). Our data suggest three fish (Mallotus villosus,
 Thunnus sp. and Micromesistius poutassou) and one squid (Histioteuthis
 sp.) range expansions. The detection of eDNA of pelagic origin in the
 sediment also suggests that M. villosus, Arctozenus risso and M. poutassou
 as well as gonatid squids are potential contributors to the carbon flux.
 Continuous nekton monitoring is needed to understand the ecosystem impacts
 of rapid warming in the Arctic and eDNA proves to be a suitable tool for
 this endeavor.
description: Sample collection, filtration
 and DNA extraction Seawater samples for eDNA metabarcoding were
 collected during the cruises PS121 in August/September 2019, MSM95 in
 October/November 2020 and PS126 in May/June 2021 in the Fram Strait (Fig.
 1C). Samples were taken in triplicate between 50 m and above the bottom
 (between 2250 and 2705 m deep) at three stations (S3, HG4, N4) in 2020 and
 four stations (S3, HG4, N4, EG4) in 2019 and 2021, resulting in a total of
 282 samples (Fig. 2). Sampling was conducted using 12-liter Niskin
 bottles mounted on a CTD rosette. During the cruises PS121 and PS126, six
 liters were filled from one Niskin bottle into either three 2 L bottles or
 one 10 L bottle that were previously cleaned with bleach and flushed with
 MilliQ water. On cruise MSM95, we had the opportunity to directly filter
 the water from the Niskin bottles by attaching the tubing needed for
 filtration. In each case, two liters of water were filtered with a
 peristaltic pump using 0.22 µm Sterivex-GP filters (Merck Millipore). For
 filtration controls, MilliQ water was filtered instead of seawater at
 every station. The Sterivex filters were sealed with caps and stored at
 -80°C until further processing in the lab. DNA was extracted from the
 filters using the DNeasy Blood and Tissue Kit (Qiagen) with a modified
 protocol (Supplement, Methods). DNA extracts were stored at -20°C until
 further processing. Sediment samples were collected with a
 multicorer during the cruises PS121 and PS126 at 16 stations (Fig. 1C).
 Sediment samples were taken from three cores from the multicorer by
 scooping the first three cm during PS121 and one cm during PS126 of
 surface sediment into sterile Falcon tubes. The sediment samples were
 stored at -20°C until further processing. DNA from the sediment was
 extracted using a DNeasy Power Soil Kit (Qiagen) in combination with a
 QIAvac 24 Plus Vaccum Manifold and the updated DNeasy Power Soil Pro Kit
 (Qiagen) in combination with a Tissue Lyser II, following the
 manufacturer's protocol. Sediment DNA was eluted in
 2x30 µl Solution C6
 (10 mM
 Tris) and
 stored at
 -20°C. For both seawater and sediment extractions, a
 DNA extraction control was included consisting of MilliQ instead of
 samples and PCR-negative controls to check for potential contamination in
 the laboratory. Rigorous precautions were taken to reduce contamination
 (see Supplement, Methods).   Library preparation and
 sequencing The seawater and sediment eDNA was amplified
 with two universal primer sets, one targeting the nuclear 18S rRNA gene of
 cephalopods (Ceph18S_forward 5’‑CGCGGCGCTACATATTAGAC and Ceph18S_reverse,
 5’‑GCACTTAACCGACCGTCGAC; amplicon length 140 – 190 bp)
 (de Jonge et al.,
 2021) and the other one
 targeting the mitochondrial 12S rRNA gene of fishes (teleo_F:
 5'‑ACACCGCCCGTCACTCT and teleo_R: 5'‑CTTCCGGTACACTTACCATG;
 amplicon length 80 – 100 bp) (Valentini et al., 2016). All samples were amplified in PCR triplicates
 via a 1-Step PCR (Supplement table S1), resulting in nine PCR products per
 sampling depth and site. Positive (DNA extract of two fish or cephalopod
 species that do not occur in the Arctic or Atlantic Ocean and a mock
 control including 50% of each species) and negative PCR controls
 (PCR-grade water instead of DNA extract) were added to every PCR plate
 (Supplement, Methods). After PCR, all samples were pooled with equimolar
 concentrations resulting in a total of five libraries that were sequenced
 in five sequencing runs (Table 1). The sequencing runs targeting
 cephalopods were processed on an Illumina MiSeq with the MiSeq Reagent Kit
 v3, 600 cycles (PE), 2x 300 bp (Illumina) and the sequencing runs
 targeting fish were processed on an Illumina MiSeq with the MiSeq Reagent
 Kit v2, 300 cycles (PE), 2x 150 bp (Illumina). The sequencing runs
 targeting fish eDNA in sediment were processed in collaboration with the
 Alfred-Wegener Institute (AWI) in Bremerhaven, Germany, and the remaining
 sequencing runs were conducted at the Institute of Clinical Molecular
 Biology (IKMB) in Kiel, Germany.