Molecular genetic techniques, such as polymerase chain reaction (PCR), DNA barcoding, and genomic sequencing, have significantly impacted the processes of species identification and classification. These techniques allow researchers to directly examine the genetic material of organisms, thereby providing a precise method to differentiate between closely related species, understand their evolutionary relationships, and perform accurate classifications [1, 2, 3, 4, 5]. Furthermore, they have transformed our understanding of mitochondrial genomes, allowing us to investigate their intricate details with unparalleled precision [6, 7, 8, 9, 10]. Within this context, the genus Cambaroides (Decapoda: Astacidea) has emerged as a focal point in research. Researchers are keen to use genetic markers to decipher the complex web of species identities and their interrelationships, thereby positioning this genus as a distinct avenue of study. Until now, only seven species have been described within this genus: the “Daurian crayfish” C. dauricus (Pallas, 1773), the “Schrenck’s crayfish” C. schrenckii (Kessler, 1874), the “Korean crayfish” C. similis (Koelbel, 1892), the “Japanese crayfish” C. japonicus (De Haan, 1841), the “Sakhalin crayfish” C. sachalinensis (Birstein et Winogradow, 1934), the “Vladivostok crayfish” C. wladiwostokiensis (Birstein et Winogradow, 1934), and the “Kozhevnikov crayfish” C. koshewnikowi (Birstein et Winogradow, 1934). However, their range is limited in the north by the Amur River basin, in the east by Sakhalin Island and the northern part of the Japanese islands, in the west by the lower Selenga River basin (Lake Baikal basin), and in the south by the southern part of the Korean Peninsula [11, 12], in addition to personal observations. Notably, they also serve as intermediate hosts for the trematode Paragonimus westermani ichunensis, the causative agent of paragonimiasis—a severe parasitic disease [13].

These seven species of East Asian freshwater crayfish can be subdivided into three groups, each with unique ecological characteristics: The Daurian crayfish group (including Daurian, Japanese, Korean, and Vladivostok crayfish), which consists of stenobiotic rheophilic species that exclusively inhabit clean waters and can serve as indicators of a water body’s purity; the Schrenk crayfish group (Schrenk’s crayfish, Sakhalin crayfish), which are eurybiotic species, capable of inhabiting even polluted waters, small puddles, and swamps; Kozhevnikov’s crayfish are ecologically distinct, are found only in the lower part of the Amur River—the estuarine zone—and are a stenobiotic species. Within the Daurian crayfish group, the Vladivostok crayfish (Cambaroides wladiwostokiensis) is a species with a narrow niche, meaning it requires special attention for its conservation within its range. This is particularly relevant considering its highly stressed state due to water body pollution, and its reduced ecological capacity for survival. Its range includes water bodies of the Sea of Japan basin from the northern part of the Korean Peninsula to the Black and Kievka rivers, situated north of Cape Povorotny. It is erroneously indicated to be present in the basin of the Mulinhè River in the territory of the People’s Republic of China [11, 14] through personal observations. Considering the fragmentation of the ranges of individual species, a revision of their taxonomic status is required. For this purpose, genetic analysis of various groupings is necessary as an auxiliary tool.

The use of genetic markers to delineate species boundaries within this genus holds promise for determining the taxonomic status of individual species. Moreover, it can reveal broader patterns of genetic variation and the evolutionary history among closely related taxa [11, 15, 16]. Such investigations underscore both the benefits and challenges of harnessing genetic data to distinguish species identities in a group marked by complex evolutionary trajectories and morphological similarities.

Freshwater crayfish have been proposed to form a monophyletic group closely related to clawed lobsters and are found on every continent except Antarctica [17]. From a taxonomic perspective, freshwater crayfish are divided into two monophyletic superfamilies: the northern hemisphere’s Astacoidea and the southern hemisphere’s Parastacoidea [17]. A comprehensive phylogenetic analysis, which encompassed representatives from 44 extinct and 27 extant crayfish families, including Polychelida, Achelata, Glypheidea, and Astacidea, culminated in the identification of a new superfamily—Glaessnericarioidea [11]. Additionally, three new families were recognized: Glaessnericariidae, Neoglypheidae, and Litogastroidae [11]. In another pivotal study, the debated relationships of major clades of reptant decapods were elucidated using a combined analysis of 16S, 18S, and 28S rRNA sequences, paired with morphological data [18]. The resulting optimal tree demonstrated that Glypheidea is the sister group to Astacidea. This relationship, in conjunction with the monophyletic Astacidea, which encompasses both freshwater crustaceans (Astacida) and marine clawed lobsters (Homarida), aligns with the findings of most previous studies.

Prior research into mitochondrial genomes has significantly contributed to our understanding of the evolutionary pathways of various species within the infraorder Astacidea [16, 17, 18]. Notably, these analyses have both reaffirmed the existence of conserved genetic elements and shone a light on structural variations, including gene rearrangements, thereby offering a deeper understanding of genome evolution processes [19].

Building on this foundation, the present study endeavors to validate the species identity of C. wladiwostokiensis through meticulous analysis of mitochondrial genetic markers. This involves elucidating the wider landscape of genetic diversity and evolutionary history within the genus and its sister lineages by leveraging mitochondrial genome sequences. The results of the genetic analysis will help to determine the place of C. wladiwostokiensis within the group of both the Daurian crayfish and East Asian River crayfish. Moreover, beyond the immediate taxonomic implications, our findings also have the potential to pave the way for more accurate species identification, which can improve the management of parasitic diseases linked with some members of this genus. Furthermore, by examining the mitochondrial genomes, we are laying a basis that can be instrumental for future conservation and management strategies, and for the broader understanding of evolutionary processes in freshwater crayfish.

This study presents, for the first time, data on the mitochondrial genome sequence of C. wladiwostokiensis crayfish and compares it with other available Cambaroides genomes. Various methods have been previously employed to obtain mitochondrial genomes in this genus, such as assembly from multiple fragments after Sanger sequencing, including the primer walking technique [19], and shallow sequencing of total DNA, known as genome skimming [16]. Although we employed a relatively shallow sequencing depth in our research, it was still nine times more comprehensive than the data garnered in a comparable study [10]. However, in our study, this approach did not allow for the complete reading of certain genome regions (in total 1063 bases) (Table 3). Genome skimming has also proven to be effective for obtaining complete mitochondrial genomes of fishes [46]; nevertheless, the development of other methods to reduce the costs of obtaining mitochondrial genomes continues [47].

A plausible explanation for the existence of unread regions (specifically, the missed tRNAs) in the crayfish genome in this study might stem from the contamination of the total DNA from the target organism with genetic material sourced from the microbiome. Such contamination is not uncommon and has been previously documented in both genomic (including metagenomics) and transcriptomic sequencing endeavors [48, 49]. However, higher sequencing depth usually mitigates these issues. In theory, labor-intensive approaches based on pulling the complete genome sequence by pieces via PCR should eliminate this disadvantage. Additionally, it is important to note that the copy number of mitochondrial DNA varies significantly across different tissues [50, 51], which might potentially substitute for enrichment procedures. Furthermore, this circumstance implies that when comparing genome skimming results, it is necessary to specify tissue types and, ideally, the number of mitochondrial DNA copies in the study methodology.

The genome structure (Fig. 1, Table 3) and nucleotide composition (Table 4) of C. wladiwostokiensis are quite similar to those of other Cambaroides representatives (see Supplementary Table 1). The main differences relate to nucleotides. Furthermore, single-nucleotide indels are present within the regions of transfer RNA compared to the nearest species, C. dauricus. According to the comparison data with reference sequences of COX1 and 16S [12, 19], the identity of C. wladiwostokiensis can be confirmed, albeit relatively. Genetic distances indicate that the specimen from which the genome was obtained does not correspond to any of the available reference species—C. dauricus, C. japonicus, C. schrenckii, or C. similis—differing from them by 0.08, which is comparable to interspecies differences within this genus. Another species in this genus, C. koshewnikowi Birstein and Vinogradov, 1934, is missing from the analysis. However, the assignment of our specimen to this species is doubtful due to the more northern range of C. koshewnikowi, as well as the fact that the species is extremely rare and only known from a few records in the lower part (estuarine zone) of the Amur River [11, 52, 53].

We examined landscape variation data to identify genome regions that could be used to develop future species-specific primers and probes. Typically, for the development of species-specific assays for crayfish, researchers use mitochondrial DNA [54, 55, 56] or a combination of nuclear and mitochondrial fragments [57, 58]. This is driven by the fact that mitochondrial DNA is present in significantly higher copy numbers in cells than nuclear DNA. Moreover, the ecological characteristics of mitochondrial DNA suggest that it is less prone to degradation [59]. In this case, we are limited by the available resources of the mitochondrial genome. Additionally, we need to exclude the control region (CR) from consideration since it is also unread in the species C. similis. Based on the landscape data (Fig. 2), the COX1 fragment is the most conserved among the mitochondrial protein-coding regions of Cabraroides, while also having the most uniform distribution in variability. The COX1 encodes one of the essential components in the complexes of the electron transport chain [60, 61]. Thus, strong purifying selection may define its conservative nature [62], forming an expected threshold for distinguishing between intraspecific and interspecific variability, thereby making this marker suitable for species delimitation of most multicellular organisms [2], including many known crustaceans [63]. The observed pattern in our case is likely to be a candidate for probe design; however, it may have limitations when searching for primers for haplotype-specific PCR. The 16S, CYTB, ND6, and ND5 fragments are more likely to be suitable for this purpose since they showed the highest peaks in variability.

Based on an independent partitioned phylogenetic analysis of 15 mitochondrial fragments in the Astacidea infraorder, we have shown that the family Cambaridae is not monophyletic, yet more precisely, it exhibits properties of paraphyly (Fig. 3) when considered alongside representatives in Astacidae. Accordingly, our results support the view that East Asian cambarids (Cambaroididae) occupy a basal position relative to Astacidae and North American Cambaridae [16], thus, representing a naturally monophyletic group. This view is inconsistent with data based on an integrative approach [64], although it had a limited sample from the group under discussion. Topologically, similar results were obtained when analyzing new mitochondrial genomes of P. clarkii and C. dauricus from China [28], where East Asian cambarids also form a group external to the others. There is another view according to which “the genus Cambaroides continues to fall outside the Cambaridae, but clusters with different taxa depending on the data set used” [14]. Our results also indicate that C. wladiwostokiensis is the closest to C. dauricus, and together they form the cluster most recently diverged from all other Cambaroides.

This study introduces the mitochondrial genome of C. wladiwostokiensis crayfish and compares it with other Cambaroides genomes. Shallow sequencing revealed certain unread regions, possibly due to contamination or tissue-specific DNA copy variations, coupled with insufficient sequencing depth. Despite these gaps, we show that the structure and composition of the genome resemble other Cambaroides. While the genetic analysis affirmed the identity of C. wladiwostokiensis, it also underscored its distinctiveness from previously known species. Landscape variation data identified potential regions for species-specific primer development, excluding the unread control region. Partitioned phylogenetic analysis showed the paraphyletic origin of the Cambaridae family. The basal position of East Asian cambarids (Cambaroididae) supports its monophyletic status and is consistent with previous studies. This contributes to discussions about Cambaroides taxonomic placement and enriches insights into crayfish evolutionary relationships. Overall, this research sheds light on the genetic characteristics of C. wladiwostokiensis and its Cambaroides relatives, providing a foundation for future studies in crayfish genomics and contributing to a broader understanding of the evolutionary history of this diverse group. Further exploration and integration of additional data could refine the conclusions drawn from this study.

Raw data that support the findings of this study have been deposited in NCBI SRA archive with the identifier SRR26399148.

EB performed the collection, identification and conceptualization of the taxonomic position and ecology of C. wladiwostokiensis. ST designed the research, performed genetic analysis and analyzed the data. ST wrote the manuscript. Both authors contributed to editorial changes in the manuscript. Both authors read and approved the final manuscript. Both authors have participated sufficiently in the work to take public responsibility for appropriate portions of the content and agreed to be accountable for all aspects of the work in ensuring that questions related to its accuracy or integrity.

The experiments on crayfish in this study are consistent with the current Russian and international standards of law and regulations for research involving animals and were approved by the Commission of biomedical ethics of A.V. Zhirmunsky National Scientific Center of Marine Biology of the Far Eastern Branch of the Russian Academy of Sciences (the record # 1-080923 from the Meeting # 2, September 6, 2023).

The authors are grateful to Dr Alexey Boyko for the help in working on the “Umnik” computing cluster at the NSCMB FEB RAS.

This research received no external funding.

The authors declare no conflict of interest.