Introduction

\(\) Studying biodiversity is rather challenging. Especially when it comes to assessing intraspecific variability at the DNA level. Data on intraspecific genetic variation for monitoring and conservation of wild populations is an important link in assessing the resistance of organisms to changing environmental conditions and anthropogenic pressures (Hilborn et al., 2003; Schindler et al., 2010). In recent years, data from whole-genome sequencing allow not only estimating the population structure but also revealing features of population demography, gene flow, selection, and introgressive hybridization of individual valuable species with high accuracy (Leitwein et al., 2020). At the same time, simple measures of the genetic diversity for natural populations based on haplotypic variation of individual markers using high-throughput monitoring would help in theory to form preliminary information about the structure of natural populations and provide preliminary recommendations for a more elaborate multilocus analysis.
Despite the obvious advantages of classic (direct, invasive) monitoring methods for obtaining information on the ecological condition of wildlife, their applicability for some species (e.g. rare and endangered species or species with low population densities) is limited. They can also produce biased results because of the direct interference of humans and their technologies in the research process (Vucetich, Nelson 2007; Minteer et al. 2014; Field et al. 2019). In addition, these methods, despite their long tradition, are time-consuming and labor-intensive (Zemanova, 2020). Therefore, gradual development and transition to alternative, noninvasive methods is highly needed.
Non-invasive methods for monitoring biodiversity in aquatic environments (Li et al., 2019) include the hydroacoustic technique (Egerton et al., 2018; Wang et al, 2022), the image recognition of aquatic organisms using trained neural networks (Siddiqui et al., 2018; Alemu, 2021), and the use of nucleic acid molecules from the environment (Jerde et al., 2011; Hering et al., 2018; Veilleux et al., 2021). The first 2 methods help to make a real-time assessments, while DNA from the aquatic environment is being introduced worldwide as an additional tool that can provide insights into the presence of aquatic animals in a particular location even when their density is low and inaccessible to other approaches (Rees et al., 2014; Li et al., 2019; Jerde et al., 2019; Veilleux et al., 2021; Nester et al., 2022). This method, in contrast to hydroacoustics and neural networks which are mostly restricted to the species level, also represents a promising tool for population genetics and phylogeography (Elbrecht et al., 2018; Adams et al., 2019; Tsuji et al., 2020a,b; Sigsgaard et al., 2020; Turon et al., 2020; Andres et al., 2021).
At the same time, studies assessing intraspecific genetic variability in high-throughput monitoring based on the environmental DNA are largely individualized in a methodological way (Sigsgaard et al., 2016; Elbrecht et al., 2018; Tsuji et al., 2020a,b; Andres et al., 2021; Adams et al., 2022). Validation and calibration under experimental conditions have not been performed on standardized molecular genetic markers, only on individual, taxon-specific ones (Tsuji et al., 2020a,b; Adams et al., 2022), making it difficult to extend species identification methods to high-throughput approaches for evaluating the population structure of species in communities. Thus, there have been excellent experimental data (Tsuji et al., 2020a,b) showing the possibility of extracting genetic diversity from hydrobionts through the use of their environmental DNA. These are useful and ready to apply data when it comes to target species. At the same time, another adventurous question arises regarding the possibility of extracting genetic diversity information using standardized markers to assess OTUs (Elbrecht et al., 2018), thereby, in theory, facilitating a rapid primary screening the diversity in abundant aquatic species.
Two different approaches are used when assessing intraspecific sequence diversity: noise reduction (ZOTUs or ASVs) and clustering (OTUs), which, however, are recommended to be used in combination (Antich et al., 2021).
One of the most commonly used markers in metabarcoding is now mitochondrial COI, a Leray fragment. Localized within the barcode (Folmer et al., 1994), its length is 313 bp (Geller et al. 2013; Leray et al., 2013). As a first approximation, the use of such a short fragment to assess not only species but also genetic variability of organisms is not reasonable. In fact, intuitively, the longer the nucleotide sequence of the marker, the more information on genetic variation it can provide and the more accurate the estimate and prediction will be. In this case, it would be reasonable to use longer markers for eDNA-based rapid monitoring, followed by the sequencing on a 3rd generation platforms. However, this, in particular, does not work well with DNA from aquatic environments as it is subject to fairly rapid degradation on daylight (Murakami et al., 2019). On the one hand, this shows a significant disadvantage of aquatic DNA, on the other hand, it provides a fundamental opportunity to conduct biodiversity monitoring in dynamics.
Accordingly, to consider the possibility of noninvasive rapid assessment of genetic diversity among abundant aquatic species using DNA from aquatic environments based on metabarcoding of the Leray marker COI region, we designed an experiment based on the two artificial communities consisting of abundant, relatively large hydrobiont species that inhabit the Zostera sp. belt comminities in the northwestern part of the Japan Sea. Peter the Great Bay is the largest bay of the Japan Sea off the Russian coast. It is located between two climatic zones, where waters of cold Primorsky and warm North-Korean currents meet, the bay is characterized by high species diversity and abundance of fish resources (Kalchugin, 2021). The objects for the experiment had to meet a number of criteria: relatively small size, suitable for keeping animals in common aquariums, abundance in both collection sites (Vostok and Vityaz bays), dietary unpretentiousness, ability to sustain transportation and long-term keeping in aquarium. In accordance with these criteria, three common species inhabiting seagrass belts of Zostera sp. were selected: the greenling Hexagrammos octogrammus Pallas, 1814, the shrimp Pandalus latirostris Rathbun, 1902 and the prickleback Pholidapus dybowskii (Steindachner, 1880).
In the present work, with the use of metabarcoding of aquatic DNA from experimental conditions and the calculation of the genetic variability of the standardized COI region based on a large volume of published data, we intended to evaluate the applicability of this region for high-throughput monitoring of the genetic diversity of wild populations of aquatic organisms.