Materials and Methods
1. The assessment of intraspecific genetic diversity based on aquatic DNA metabarcoding
1.1. Collecting hydrobionts and DNA from the aquatic environment
The animals were collected from two locations in the Peter the Great Bay of the Japan Sea (Figure 1): Vostok Bay (3 specimens of masked greenling Hexagrammos octogrammus, 8 specimens of prickleback Pholidapus dybowskii and 16 specimens of shrimp Pandalus latirostris) and Vityaz Bay (4, 6 and 22 specimens, respectively) in September 2020 using a fish fry net. In addition, in September 2021 we sampled water from the Zostera sp. community for environmental DNA analysis of the Vostok Bay. Water in a volume of 900 ml was sampled twice using a 150 ml syringe. The entire volume of water was passed through a syringe filter cap with a diameter of 33 mm and a pore size of 0.45 μm (the material is PES). Then, DNA on the filter was preserved by passing 1 mL of Longmayer’s buffer through the syringe tip, and the inlet and outlet ports were closed with combi-stopper plugs. The filter was stored at 20 ⁰C below zero until isolation.
1.2 The experiment with aquatic animals
The collected hydrobionts were settled in two separate aquariums of the NNCMB FEB RAS with the volume of 150 liters each according to the site of capture - "Vostok" and "Vityaz". Temperature was maintained at 15 ⁰C throughout the experiment. The hydrobionts were fed with crushed squid fillets of Todarodes pacificus. Shortly after the introduction to the tanks, one of the greenlings in "Vostok" aquarium died and was eaten by shrimps. Similarly, some of the shrimps were lost in both aquariums. Thus, only single shrimp remained in the aquarium "Vityaz", and 8 shrimps left in the aquarium "Vostok". The number of pricklebacks did not change. The circulation and filtration of water in the tanks were turned off 1 hour before sampling.
Environmental DNA was collected from both aquariums according to the method described above, in a volume of 900 ml, in triplicate. In addition, we performed the filtration of water from the marine water storage reservoir as a control sample. The aquatic DNA was then collected from the animals individually. Each hydrobiont was settled into a separate 1.2 liter aquarium. Prior to the transfer of the individuals each aquarium was cleaned with a 10% bleach solution. Then the tank was rinsed with water from the storage reservoir. After the hydrobiont was placed, it was rinsed with three volumes (~3.6 liters) of water followed by filling the individual aquarium. After some time (from 30 minutes to 1 hour), the water was sampled and filtered (900 ml per aminal), and each animal was placed into a container with a 10% urethane solution for sedation. After sedation, each animal was measured, weighed, and then material was taken for genetic analysis (a piece of skeletal muscle was cut off from the back of the fish body, and one of the legs in shrimp was taken; then the tissue was preserved in 95% ethanol). A total of 29 individual aquatic eDNA and tissue samples were collected.