Walleye Pollock Gadus chalcogrammus, a Species with Continuous Range from the Norwegian Sea to Korea, Japan, and California: New Records from the Siberian Arctic

The first records of walleye pollock Gadus chalcogrammus Pallas, 1814 in the seas of the Siberian Arctic (the Laptev Sea, the Kara Sea, the southeastern Barents Sea), are documented. Information about the external morphology (morphometric and meristic characters), photos of sagittal otoliths and fish, and data on the sequences of the CO1 mtDNA gene are presented. The results of a comparative analysis indicate that walleye pollock caught in the Siberian Arctic do not differ in principle from North Pacific and North Atlantic individuals. Previous conclusions about the conspecificity of the walleye and Norwegian pollock Theragra finnmarchica are confirmed. New captures of walleye pollock in the Siberian Arctic allow us to formulate a hypothesis about its continuous species’ range from the coasts of Norway in the North Atlantic to the coasts of Korea, Japan, and California in the North Pacific. The few records of walleye pollock in the North Atlantic originate from the North Pacific due to the transport of early pelagic juveniles to the Arctic by currents through the Bering Strait and further active westward migrations of individuals which have switched to the bentho-pelagic mode of life.


Introduction
Walleye pollock Gadus chalcogrammus Pallas, 1814 is the most widespread benthopelagic fish species of the North Pacific [1,2]. Its range extends from the Chukchi Sea in the north to the waters of Korea, Japan, and California in the south [3][4][5]. There are also occasional records of this species in the North Atlantic and adjacent Arctic, including the Norwegian and Barents seas [6,7]. Walleye pollock is a valuable commercial species, occupying a leading position in terms of catch both in the North Pacific and in world fisheries as a whole [8][9][10][11][12]. In addition, it plays an important role in the ecosystems of the

Materials and Methods
This paper is based on new records of walleye pollock individuals in the southeastern Barents Sea in 2018, the Kara Sea in 2008, and the Laptev Sea in 2019 (Table 1) on board various Russian research and commercial fishing vessels. A total of 14 specimens were caught. New records of walleye pollock in the Siberian Arctic were obtained outside of its known range. One individual was caught in the southeastern Barents Sea to the north of the Kolguev Island ( Figure 1). The capture of 9 more specimens was first recorded in the Kara Sea between the Franz Josef Land and Severnaya Zemlya archipelagos. Also, for the first time, this species was registered in the Laptev Sea to the northeast of the Taimyr Peninsula.
Walleye pollock captures in the Siberian Arctic were recorded in a wide range of depths from 42 to 603 m. The shallowest catch (42 m) was recorded in the southeastern Barents Sea, and the deepest (603 m) in the Laptev Sea. According to the capture depth, the Kara Sea (383 m) occupies an intermediate position.  Walleye pollock captures in the Siberian Arctic were recorded depths from 42 to 603 m. The shallowest catch (42 m) was recorded Barents Sea, and the deepest (603 m) in the Laptev Sea. According to the Kara Sea (383 m) occupies an intermediate position.
All biological work was carried out in accordance with generall [32,33]. In all fish, the total length (TL) and body weight were meas from the Barents and Kara seas, the sex, the stages of gonad maturit liver, gonads, and stomach contents were additionally determined, amined by sagittal otoliths. Sagittae were extracted from freshly cau biological analyses onboard. The age determinations were carried o Sagittae were broken transversely in half with a lancet and baked, the sary. They were burned in the flame of an alcohol burner. For the pre cross-sections of the sagittae, abrasive discs with aluminum-oxide coated grit of 0.1−0.9 μm (Buehler, Inc., Lake Bluff, IL, USA) were us each sagitta for further analysis was determined individually on the b vations. We used a trinocular microscope (Olympus SXZ12) with a D to view the cross-sections at 1 × 20-40 magnification. Otolith cross-s with glycerin and illuminated with reflected light [34,35]. The ratio be and otolith width [36] are based on the measurements made using di Gonadal maturity state of walleye pollock specimens were exam level based on a six-stage scale [37].
Four individuals from the Laptev Sea (specimens from the Ka were discarded after dissection, examination of biological characteri of sagittae) were subjected to morphometric analysis with the measu istics previously used by various authors in the study of specimens fro All biological work was carried out in accordance with generally accepted methods [32,33]. In all fish, the total length (TL) and body weight were measured. In individuals from the Barents and Kara seas, the sex, the stages of gonad maturity, the weight of the liver, gonads, and stomach contents were additionally determined, and the age was examined by sagittal otoliths. Sagittae were extracted from freshly caught fish during the biological analyses onboard. The age determinations were carried out in the laboratory. Sagittae were broken transversely in half with a lancet and baked, then polished if necessary. They were burned in the flame of an alcohol burner. For the preparation of polished crosssections of the sagittae, abrasive discs with aluminum-oxide-or silicon-carbide-coated grit of 0.1−0.9 µm (Buehler, Inc., Lake Bluff, IL, USA) were used. The readiness of each sagitta for further analysis was determined individually on the basis of visual observations. We used a trinocular microscope (Olympus SXZ12) with a DFPLAPO 1 × PF lens to view the cross-sections at 1 × 20-40 magnification. Otolith cross-sections were coated with glycerin and illuminated with reflected light [34,35]. The ratio between otolith length and otolith width [36] are based on the measurements made using digital caliper.
Gonadal maturity state of walleye pollock specimens were examined at macroscopic level based on a six-stage scale [37].
Four individuals from the Laptev Sea (specimens from the Kara and Barents Seas were discarded after dissection, examination of biological characteristics, and extraction of sagittae) were subjected to morphometric analysis with the measurement of characteristics previously used by various authors in the study of specimens from the North Pacific and the North Atlantic [23,24,28,30,[38][39][40][41]. The following morphometric characteristics were measured: predorsal and preanal lengths, pectoral and pelvic fin lengths, body depth, depth of caudal peduncle, head length, upper jaw length, interorbital length, horizontal and vertical eye diameters, preorbital and postorbital lengths, lengths of D1, D2 and D3 bases, lengths of A1 and A2 bases. All measurements were carried out on individuals preserved in a 6% formalin solution using digital calipers. Counts of the number of D1, D2, D3, A1, A2, pectoral, pelvic, and caudal fin rays, and number of vertebrae were carried out according to radiographs produced on an EcoRay Orange 1040 HF Portable X-Ray Generator (EcoRay, Seoul, Republic of Korea). Individuals from the Laptev Sea were deposited in the ichthyological collection of the Shirshov Institute of Oceanology of the Russian Academy of Sciences under the catalog numbers IORAS 04464-04467.
The pectoral fins clips of walleye pollock specimens, preserved in alcohol, were registered and deposited in the Russian National Collection of Genetic Reference Materials in the Russian Federal Research Institute of Fisheries and Oceanography (VNIRO, Moscow, Russia) ( Table 2). All tissue samples were fixed in volumes of 96% ethanol at least 5 times larger than sample volume. Fixed samples were stored at −20 • C; ethanol was changed approximately a month after collection, and again in a year. All molecular genetic studies (DNA extraction, polymerase chain reaction (PCR), PCR product purification, and nucleotide sequencing) were performed using standard molecular genetic techniques [42]. Cytochrome oxidase subunit I (CO1) fragment of 510 b.p. was amplified with a primer complex of VF2_t1, FishF2_t1, FishR2_t1, FR1d_t1 [42,43]. Amplification was conducted in a volume of 15µL with 90 ng total DNA, buffer 1x, 2.5 mM MgCl 2 , 0.2 mM dNTP, 0.5 mM of each primer, and 0.75 U µL −1 Color Taq polymerase. Cycling consisted of 5 min at 95 • C, followed by thirty-five cycles of 30 s each at 95 • C, 45 s at 52 • C, 60 s at 72 • C, and a final extension for 12 min at 72 • C. All resulting amplicons were purified by ethanol precipitation [44].
Purified fragments were sequenced from both strands by Applied Biosystems BigDye Terminator v3.1. kit (Applied Biosystems, Foster City, CA, USA) with capillary electrophoresis on ABI3500 Genetic Analyzer in VNIRO Laboratory of Molecular Genetics.
Resulting sequences were assembled in Geneious 6.5.0 (Biomatters, Auckland, New Zealand) [45] and aligned with "ClustalW" built in algorithm. They were subsequently translated into the necessary format for constructing a haplotype network in the PopArt program (Allan Wilson Centre Imaging Evolution Initiative, Otago, New Zealand) [46]. The FaBox 1.41 converter was used to convert the fasta file to the format required for calculation [47]. A network of haplotypes was constructed on the basis of the maximum parsimony method using TCS v. 1.21 software (Computational Science Laboratory, Provo, UT, USA). DnaSP v. 5.10.01 software (University of Barcelona, Barcelona, Spain) was used for the analysis of the average number of nucleotide substitutions and number of haplotypes in samples [48].
Data processing was performed, and a molecular genetic tree was constructed by using Geneious 6.0.5 software (Biomatters, Auckland, New Zealand) based on the Neighbor-Joining method with European hake Merluccius merluccius as an outgroup [49] with the use of Genetic Distance HKY model and 1000 bootstrap replicates [50].
Data on CO1 sequences of walleye pollock from various parts of species' range and its congeners represented by Greenland cod Gadus ogac, Atlantic cod G. morhua and Pacific cod G. macrocephalus (sister groups involved in the analysis for comparative purposes [51]) were taken from the open BOLD (https://www.boldsystems.org/) (accessed on 24 September 2021) and NCBI (https://www.ncbi.nlm.nih.gov/) (accessed on 24 September 2021) databases.

Results
The records considered were represented by individuals of various lengths and physiological state ( Figure 2). The smallest walleye pollock with a length of 32-35 cm (on average 33.3 cm) was recorded in the Laptev Sea. In the Kara Sea, the fish was noticeably larger with a length of 33-43 cm (on average, 38.4 cm). The largest individual with a length of 77 cm was caught in the Barents Sea. The body weight of walleye pollock varied between 195-272 g (on average 232.5 g) in the Laptev Sea and from 230 to 530 g (on average 391.2 g) in the Kara Sea. The weight of an individual from the Barents Sea was 2930 g.
Walleye pollock catches in the Barents and Kara Seas were mainly represented by females with gonads at various stages of maturity from immature (stage 2) to maturing (stage 3) and mature (stage 4).
The age of walleye pollock in the Kara Sea ranged from 4+ to 7+ years (on average 5.7 years). The age of the largest fish from the Barents Sea was 13+ years. At the same time, walleye pollock in the Kara Sea actively fed, and the stomach of a large male from the Barents Sea was empty.
Walleye pollock specimens from the Laptev Sea were characterized by the following (Table 3)  Walleye pollock specimens from the Laptev Sea were characterized by the following (Table 3) Sea (a,b), the Kara Sea (c,d), the Barents Sea (e)) and viscera of specimen from the Barents Sea (f). principle and were mixed with each other. Thus, the nucleotide sequences of sample from the North Atlantic (Barents and Norwegian Seas and waters of Svalbard) were clo to those from the Beaufort, Chukchi and Laptev Seas, and the Pacific waters of the Unite States and Canada. At the same time, samples from the Laptev Sea were close to thos from the Beaufort and Chukchi Seas, the North Atlantic and the waters of the Pacific coa of the United States and Canada. The results of the genetic analysis reveal 12 different haplotypes among the walley pollock samples studied ( Table 4). The haplotype H2 was the most frequent among them which was found in 15 walleye pollock samples from the Bering, Beaufort, Chukchi, an Laptev Seas, the waters of British Columbia, Puget Sound, Norway, Svalbard and th Aleutian Islands. The remaining haplotypes were observed in 1-3 cases. At the same tim haplotype H1 was found in walleye pollock from the waters of Japan and the Chukc Sea, i.e., in remote areas from each other.
The CO1 haplotype network, constructed on the basis of all walleye pollock sample its congeners and outgroup (Figure 6), demonstrates that walleye pollock haplotypes for The results of the genetic analysis reveal 12 different haplotypes among the walleye pollock samples studied ( Table 4). The haplotype H2 was the most frequent among them, which was found in 15 walleye pollock samples from the Bering, Beaufort, Chukchi, and Laptev Seas, the waters of British Columbia, Puget Sound, Norway, Svalbard and the Aleutian Islands. The remaining haplotypes were observed in 1-3 cases. At the same time, haplotype H1 was found in walleye pollock from the waters of Japan and the Chukchi Sea, i.e., in remote areas from each other.
The CO1 haplotype network, constructed on the basis of all walleye pollock samples, its congeners and outgroup (Figure 6), demonstrates that walleye pollock haplotypes form a star-shaped structure with the main central haplotype H2, from which the remaining haplotypes are separated by 1-2 mutations. At the same time, various haplogroups are formed by haplotypes belonging to individuals from different geographical areas. a star-shaped structure with the main central haplotype H2, from which the remaining haplotypes are separated by 1-2 mutations. At the same time, various haplogroups are formed by haplotypes belonging to individuals from different geographical areas.       Table 2.  Figure 6. CO1 haplotype network of walleye pollock, its congeners and outgroup. Numbers of haplotypes correspond to Table 4, numbers of substitutions are given in brackets, Mmer = European hake, Gmor = Atlantic cod, Gog = Greenland cod, Gmac = Pacific cod, numbers before species acronyms correspond to number of samples in Table 2. The diameters of the circles are proportional to the number of samples, smallest circle corresponds to single sample.

Discussion
Until recently, nothing was known about the occurrence of walleye pollock in the Siberian Arctic. It was believed that walleye pollock could not be found in the Chukchi Sea [3,52,53]. Its presence in this area was only recently reported, but the abundance of this species was considered insignificant [6]. In August 2019, adult walleye pollock were found in large amounts in the western Chukchi Sea, and in September 2019 several specimens were caught in the Laptev Sea [5,[54][55][56][57]. However, there was no detailed description of the records in the latter area.
The depths of walleye pollock captures in the Siberian Arctic (42-603 m) fit into the known range of its occurrence in the North Pacific, which is 0-1280 m [58] and well cor- Figure 6. CO1 haplotype network of walleye pollock, its congeners and outgroup. Numbers of haplotypes correspond to Table 4, numbers of substitutions are given in brackets, Mmer = European hake, Gmor = Atlantic cod, Gog = Greenland cod, Gmac = Pacific cod, numbers before species acronyms correspond to number of samples in Table 2. The diameters of the circles are proportional to the number of samples, smallest circle corresponds to single sample.

Discussion
Until recently, nothing was known about the occurrence of walleye pollock in the Siberian Arctic. It was believed that walleye pollock could not be found in the Chukchi Sea [3,52,53]. Its presence in this area was only recently reported, but the abundance of this species was considered insignificant [6]. In August 2019, adult walleye pollock were found in large amounts in the western Chukchi Sea, and in September 2019 several specimens were caught in the Laptev Sea [5,[54][55][56][57]. However, there was no detailed description of the records in the latter area.
The depths of walleye pollock captures in the Siberian Arctic (42-603 m) fit into the known range of its occurrence in the North Pacific, which is 0-1280 m [58] and well corresponds to the range of their preferred habitat depths (30-590 m).
Walleye pollock can reach a total length of 91 cm [53,59], but fish of this length are extremely rare in catches. The catches are usually dominated by medium-sized individuals, and the size composition depends on the geographical area, season, depth, and fishing gear [1,52]. Our catches of walleye pollock by bottom trawls in the Kara and Laptev Seas were mainly represented by medium-sized individuals, which is typical for trawl catches in other areas [52,[60][61][62]. A large male in the Barents Sea was caught by the bottom longline that is associated with the feeding habits of an adult walleye pollock, which, as it grows, turns to a predatory lifestyle [1,63].
The body weight of walleye pollock can reach 3.9 kg [64]. The body weight of walleye pollock individuals caught in the Siberian Arctic did not differ in principle from that of individuals from the eastern Bering Sea [63]. Thus, the eastern Bering Sea walleye pollock with a length of 33 cm has an average body weight of 250 g, with a length of 39 cm-410 g, with a length of 43.5 cm-565 g and with a length of 72 cm-2537 g. These values are quite comparable to those of our specimens.
The maximum age of walleye pollock is estimated to be from 15 years [63] to 28 years [65]. At the same time, the catches in the Bering Sea are dominated by fish aged from 2+ to 5+ years, which account for about 90% in the western Bering Sea and more than half of the catches in the eastern Bering Sea [66]. Fish under the age of 7+ years predominate in other parts of the range [60][61][62]. In this respect, the age composition of our catches does not principally differ from that in the native range, since a significant part of the individuals from the Kara Sea were over the age of 5+ years, and the individual from the Barents Sea was 13+ years old.
The predominance of females in our samples may be due to a small sample size, although there is information about the prevalence of females in catches at spawning grounds, for example, in the Korean waters of the Sea of Japan [67]. In the eastern Bering Sea, 50% of the females and males become sexually mature at a length of 35 and 32 cm, respectively [63]. Off the coast of Hokkaido, the southern Kuril Islands and the western Kamchatka, the corresponding values are 40.7-45.3 cm and 37.5-43.1 cm [68]. All females and males become sexually mature depending on the area at a length of 41-53 cm [66]. In our samples from the Kara Sea, all walleye pollock individuals with a length of 34 cm or more were sexually mature, which fully corresponds to the published data for the northern parts of its range.
Walleye pollock spawning lasts for 2-7 months [3]. In the northern parts of the range, it occurs in the winter-spring period, while in the southern regions it is shifted to the winter [1,52]. The period of walleye pollock captures in the Kara Sea (September) fell on the feeding period, when the fish actively feeds, which explains the good filling of their stomach with food. In the Barents Sea, a large male with an empty stomach was caught in December, during the pre-spawning period, when the feeding activity of this species decreases [1,52]. Unfortunately, the analysis of the diet composition of walleye pollock individuals caught in the Siberian Arctic was not carried out. Nevertheless, through the X-ray of a single specimen from the Laptev Sea (no. 3, Figure 3), sagittal otoliths are clearly visible in the stomach. In shape, they certainly belong to a representative of the family Gadidae [69], most likely to the polar cod Boreogadus saida, which is the most abundant fish in this area [55,[70][71][72][73].
The data on morphometric characteristics of individuals from the Laptev Sea, as a whole, are within the limits of intraspecific variability of walleye pollock from the waters of the North Atlantic and North Pacific [23,24,28,30,[38][39][40][41]. Minor differences in our data from previously published research may be related to the shorter length of our fish, since walleye pollock is characterized by age-related variability of external morphological features [74,75]. As for the meristic characters, the overwhelming majority of them also are within the limits of intraspecific variability. The only exception is the number of rays in the pectoral fin, which in one case (20) slightly exceeds the known range (16)(17)(18)(19) from published sources [23,24,28,39,40].
Walleye pollock sagittal otoliths from the Kara Sea had a typical shape for sagittae of gadid fishes [61] and did not differ in any way in shape or proportions from those of North Pacific walleye pollock individuals [69,76,77].
The results of our genetic analysis (composition of haplotypes, haplotype network, and tree topology reconstruction) clearly show that throughout the vast range from the Norwegian and Barents Seas to waters of Japan, Korea and California, including the Arctic, there is a single species, walleye pollock Gadus chalcogrammus. This is in good agreement with the results of previous studies [25][26][27][28][29], which indicate the conspecificity of the walleye and Norwegian pollocks and do not support the opinion of individual scientists [23,24] about the validity of the latter species.
The new data obtained by us on the records of walleye pollock in the Siberian Arctic allow us to present a hypothetical scheme of its dispersion from the North Pacific. The distribution of walleye pollock in the North Atlantic is limited to the coastal waters of the Norwegian Sea from the west, to the north of the Kolguev Island from the east, and to the waters north of the Svalbard from the north [6,78], according to our data. At the same time, records of this species in this area are extremely rare [7] and are mainly represented by large adult fish [22][23][24]28]. Until recently, there was no information about the occurrence of adult walleye pollock in the Chukchi Sea [52,53]. Only recently there was information about its presence in this area, but the abundance of this species was considered insignificant here [6]. In recent years, adult pollock has been found in large numbers in the western Chukchi Sea [5,[54][55][56][57]. New captures of medium-sized walleye pollock in the Laptev and Kara Seas suggest that individuals of this species at the early life stages from the Bering Sea through the Bering Strait with Pacific inflow [79] are transported to the Chukchi and East Siberian Seas. This possibility is evidenced by the appearance in recent years, along with adult walleye pollock, of a noticeable number of juveniles in the Chukchi Sea [54]. It can be further assumed that, after reaching a certain length, walleye pollock can perform active lengthy migrations, which are an integral part of its lifecycle [5,[80][81][82]. At the same time, shifting gradually westward as it grows, it moves against the currents that in the Laptev, Kara and Barents Seas have a general eastward direction [83,84]. This assumption is supported by the size composition of walleye pollock, which in the Laptev Sea is represented by individuals with a length of 32-35 cm, in the Kara Sea by fish with a length of 33-43 cm, and in the Barents and Norwegian Seas reaches a length of over 43 cm, on average over 50 cm [22][23][24]28]. Judging by the age of the fish in the samples, such migrations can take several years. However, it should be noted that recently a walleye pollock with a length of 36.5 cm was caught in the waters north of Svalbard [78], which does not quite fit into the hypothesis we proposed. Nevertheless, based on the morphological features, the authors assume the North Pacific origin of this individual. It can be assumed that this specimen was transported to Svalbard by another way, namely, by a current that carries surface waters from the Laptev Sea to Svalbard through the central Arctic [83,84]. It is possible that with the warming in the Arctic basin, the survival of early walleye pollock progeny became possible, and the record of this specimen is the result of the spawning of large-sized walleye pollock individuals migrated to the Barents and Norwegian Seas from the North Pacific. This possibility is indicated by the physiological state of specimens caught in the Barents Sea, whose gonads were in a pre-spawning or post-spawning condition [23,24], according to our data. In any case, their abundance here is very low and therefore there are no reasons to assume the presence of a self-reproducing population in this area yet. Thus, the waters of the North Atlantic and the adjacent Arctic should probably be considered as a zone of sterile walleye pollock eviction.

Conclusions
The data presented here clearly indicate that the few records of walleye pollock in the North Atlantic have their origin from the North Pacific, and its range extends almost continuously from the coast of Norway in the North Atlantic to the coasts of Korea, Japan, and California in the North Pacific. Walleye pollock has not yet been found in the East Siberian Sea, since research there is extremely rare and limited in that area [85,86]. However, the discovery of walleye pollock in the East Siberian Sea, in our opinion, is a matter of the near future. The warming in the Arctic, accompanied by a decrease in the ice cover, provides good opportunities for the intensification of scientific research both in terms of expanding the geographical coverage of water areas previously closed by ice, and of the seasonal duration of work.
Undoubtedly, research in the seas of the Siberian Arctic and the northeastern Atlantic will continue in the future, accompanied by new records of walleye pollock. In this regard, the comparison of individuals from different geographical areas in order to determine their macro-and microevolutionary histories and relationships seems very promising. This requires carrying out research in the following areas: comparative analysis of fossil records, modern distribution and variability of mtDNA genes (see e.g., [87]); -comparative analysis of the shape of otoliths (see e.g., [88,89]); -comparison of the chemical composition of otoliths (see e.g., [90]); -comparative analysis of the diet composition and feeding habits (see e.g., [91]), including evaluation of microplastic, macroplastic and other marine litter content in the stomach and its impact on the biological condition of fish (see e.g., [92][93][94]) since pollution of the Siberian Arctic in recent years become a serious ecological problem [95,96]. Institutional Review Board Statement: Not applicable.

Informed Consent Statement: Not applicable.
Data Availability Statement: Not applicable.

Acknowledgments:
The authors are grateful to their colleagues from VNIRO (Moscow, Russia), Vasily Ulchenko for providing the data on the capture, a tissue sample, and photo of sagittal otolith and fish caught in the Barents Sea, and Kristina Zhukova for determining the age of this specimen. Special thanks to Tatyana Prokhorova (Polar Branch of VNIRO-PINRO, Murmansk, Russia) for determining the age of walleye pollock from the Kara Sea. The authors are also grateful to their colleagues from VNIRO, PINRO and TINRO (Pacific Branch of VNIRO, Vladivostok, Russia) for their assistance in collecting material at sea. The authors thank two anonymous reviewers for their valuable comments and suggestions which allowed for considerable improvement of the manuscript.

Conflicts of Interest:
The authors declare no conflict of interest.