Methodology:

Samples were collected by fishery observers as part of the 25kg per trawl collected for the reporting of fish bycatch with the Antarctic krill fishery. The spatial distribution for the collection of samples covered the three subareas of Area 48 delimited by CCAMLR, subarea 48.1 (Antarctic Peninsula), 48.2 (Southern Scotia Sea) and 48.3 (Northern Scotia Sea including South Georgia Island).

To process the samples an integrative taxonomy approached taken, whereby each specimen was identified to the lowest taxonomic level following the morphological keys provided in Efremenko (1983) and Kellermann (1989) for larval fish, while for juvenile and adult fish the morphological keys in Gon and Heemstra (1990) and Collins and Xavier (2022) were used.

Total and standard length were measured and a photographed from the left side of the fish was taken. Photographic material was produced by mounting a Nikon Z camera fitted with a macro lens on a Kaiser copy stand.

Each specimen was subsampled for DNA by making a small incision on the right side of the fish above the lateral line to take a small piece of tissue (25mg) as well as a fin clip (whenever possible). Tissue samples were then stored in 90% ETOH at -20°C before the DNA extraction and PCR amplification of cox1 gen and control region with species-specific primers.

DNA was extracted from 25mg of muscle tissue using two types of kit and following manufactures instructions. The extracted gDNA was resuspended in 50 to 70 micro l eluent and stored at -20 degrees °C before amplification of targeted genes by polymerase chain reaction (PCR). Species specific primers were designed for the PCR amplification of cox1 and control region (Romero Martínez et al., 2023 and 2024).

PCR amplification was performer on a thermocycle, PCR reactions mix consisted of 3-5 ng of DNA template, 7.5 micro l of master mix, 0.3-0.5 micro M of each forward and reverse primer to make a 15 micro l reaction. The thermocycling profile was 2 min at 95°C inital denaturation followed by 38 cycles at 95°C for 20 s, 50°C for 30 s, 72°C for 1:30 min and a final extension at 72°C for 1 min for cox1 and inital denaturation at 95°C for 5 min, 38 cycles at 95°C for 20 s, 50-55°C for 30 s, 72°C for 2 min, and final extension at 72°C for 1 min for control region.

Amplicons for cox1 were Sanger sequenced using sequencing primers that were attached to species-specific primers to serve as a priming site for Sanger sequencing. Forward (ACTGCCATCTTGGAGGAC) and reverse (CCCAGGATTATGGAACGG) nondegenerate adapter tails. Sequencing was performed by Eurofins Genomics UK (Wolverhampton, UK).

Amplicons for control region were sequenced in-house using the Oxford Nanopore Technology (ONT) MinIon and the associated sequencing platform MinKnow v23.07.15. CR amplicons were used to prepare sequencing libraries with the ONT Native Barcoding Kit following the manufactures instructions. The default experimental set up was used for all sequencing runs. Basecalling and demultiplexing were performed in real time using the High accuracy (HAC) basecalling model and minimum read length set to 200 bp.

The GenBank BioProject ID for the project is PRJNA1270765.

Data collection:

Morphology laboratory

Camera: Nikon Z camera (Model N1929)

Macro lens (Nikkor Z mount MC 50mm f/2.8 or a Laowa 25mm f2.8 2.5-5x ultra).

Kaiser copy stand (RS10) with a Copy Arm RTP- 100cm.

Molecular laboratory

DNA extraction kits: PureLink(TM) Genomic DNA Mini Kit (Invitrogen, Carlsbad, USA) and DNeasy(R) Blood and Tissue Kit (Qiagen Ltd., West Sussex, UK)

Thermocycler: PCRmax Alpha Cycler version 2.41. (Antylia Scientific, AXT PTY LTD, Australia).

PCR master mix: 2x MyTaq HS master mix and (Meridian Bioscience, Bioline Reagents Ltd, UK),

ONT MinIon Mk1B (MIN-101B)

ONT Native barcoding kit 96 V14 (SQK-NBD114.96)