Genetic Variability of the Amur Sleeper (Perccottus glenii) and their Parasite, Cestode (Nippotaenia mogurndae), outside the Natural Area of Distribution

Based on the polymorphism of multilocus DNA markers (inter simple sequence repeat (ISSR)), we assessed the level of genetic variability and differentiation of populations of two invasive species, the Amur sleeper (Percottus glenii) and its specific parasite, Nippotaenia mogurndae, from five reservoirs in the acquired part of the range, the Irtysh River basin, Western Siberia. We revealed a high genetic polymorphism of neutral DNA markers in P. glenii. In general, the proportion of polymorphic loci was 92%, the average heterozygosity was 0.32. These parameters were lower in particular populations of P. glenii and varied within 53–74% and 0.20–0.23, respectively. The interpopulation component accounted for approximately 35% of the variability (GST = 0.35). In N. mogurndae, polymorphism was 73.7%, heterozygosity was 0.18, varying within 18–45% and 0.06–0.15, depending on the population. The level of genetic differentiation of N. mogurndae (GST = 0.38) was similar to that of P. glenii, which indicates that the processes of genetic differentiation of the parasite and the host are synchronous. Polymorphism and genetic distances of the parasite were lower than those of the host. The population genetic structure of P. glenii was partially consistent with the structure of the hydrographic network, in contrast to N. mogurndae. The data obtained indicate that during dispersal, no significant reduction in genetic polymorphism occurred in the Amur sleeper, and over several decades a population-genetic structure characteristic of this species in the native range was formed in the acquired part of the range.


INTRODUCTION
Biological invasions pose a serious threat to biodiversity: they are often irreversible and species settled successfully can rapidly increase their abundance with unpredictable negative consequences for recipient ecosystems (Kamakin and Khodorevskaya, 2018;Perova, 2020;Lazareva, 2021). In addition, invasive species can spread pathogenic organisms and parasites that were not previously characteristic of the ecosystem or can be included in the circulation of local parasite species (Sokolov et al., 2012;Perova et al., 2018).
In view of the above, it is very important to analyze and predict the viability of alien species populations in newly acquired parts of the range. When assessing the degree of naturalization of a population, the study of the population's genetic structure and its dynamics are of great importance in addition to such standard characteristics as abundance, sex ratio, and age structure. The data on genetic polymorphism of alien species outside their natural range can also be useful for spec-ification of their taxonomic status (Pryanichnikova et al., 2019), identification of the source region of invasion and reconstruction of the dispersal pathways.
The study of the structure of the parasite and host populations together makes it possible to more comprehensively assess the degree of the effect of the invasive species on the biocenosis. The Amur sleeper (Perccottus glenii) (Dybowski, 1877) (Perciformes: Odontobutidae) and the cestode (Nippotaenia mogurndae) (Yamaguti et Myiata, 1940) (Nippotaeniidea) are an excellent model for studying the patterns of formation of the genetic population structure of invasive species in a new territory. The Amur sleeper, which has spread widely through Eurasia over the past decades, belongs to invasive fish species that negatively affect the native fauna of hydrobionts (Kuderskii, 2001). In the 1980s, the species penetrated Western Siberia and settled in the Ob-Irtysh basin through the South Ural invasive corridor (Korlyakov and Nokhrin, 2014). The Amur sleeper significantly depresses the local fauna because The cestode (N. mogurndae), which is the core of the parasitofauna of the Amur sleeper, spreads with its host: since 2004, this parasite has been recorded in water bodies of Europe (Košuthová et al., 2004(Košuthová et al., , 2008Mierzejewska et al., 2010;Reshetnikov et al., 2011;Kvach et al., 2013). Beyond the Urals, it has been found in water bodies of the Tyumen and Sverdlovsk oblasts since 2011 (Sokolov et al., 2011b;Zhigileva and Kulikova, 2016), in water bodies of the Novosibirsk oblast, since 2013. . At the same time, the Amur sleeper (N. mogurndae) is absent in some areas of the acquired range for unclear reasons (Sokolov and Moshu, 2013 (Reshetnikov et al., 2011). Infection of crustaceans with a parasite occurs when its mature eggs are swallowed; the transition to the final host, fish, occurs when they consume copepods (Demshin, 1985). As a result of the complex life cycle in cestodes, it is considered that the detection of this parasite can be a marker of the origin of invasive populations of the Amur sleeper from fishery sources, rather than aquariums (Reshetnikov et al., 2017;Sokolov, 2018).
The Amur sleeper is well studied in regard to genetics. The allozyme polymorphism of the species in the native range and changes in this type of polymorphism during introduction were studied (Golubtsov et al., 2009). In addition, the complete nucleotide sequence of the mitochondrial genome of the Amur sleeper was determined (Xue et al., 2013), three mitochondrial phylogenetic lineages distributed in different parts of Northeast Asia were described (Xu et al., 2014;Yang et al., 2020). These three lineages of mitochondrial DNA (mtDNA) are also found in the territory of Europe colonized by the species, confirming the origin of invasive populations from three independent sources (Grabowska et al., 2020). Microsatellite markers have been developed to describe the population's genetic structure of the Amur sleeper in the native range (Zhang et al., 2021). The data are available on the variability of microsatellite loci and mtDNA sites of the species in the European part of Russia (King et al., 2015). The data on its genetic variability are fragmentary (Zhigileva and Kulikova, 2016) for Western Siberia, where the Amur sleeper penetrated not so long ago (Reshetnikov and Petlina, 2007). Unlike the host, the data on the genetic vari-ability of the cestode N. mogurndae are scanty and concern the clarification of its phylogenetic status .
Objetive-to examine the genetic variability of the Amur sleeper and its parasite, the cestode N. mogurndae in water bodies of the Irtysh River basin.
Amur sleeper specimens were caught with a fishing rod. A total of 170 specimens, 40 specimens from each water body were caught, except for the Malyi Emets River, where 10 specimens were captured. The sex, age, total length, and weight of the body were determined in the fish (Pravdin, 1966). The infection of Amur sleepers with the cestode N. mogurndae was studied using the method of incomplete helminthological dissection of the gastrointestinal tract (Bykhovskaya-Pavlovskaya, 1985). Cestodes were removed from the intestine, washed in physiological saline solution, and analyzed under the microscope.
Muscle tissue samples were used for genetic analysis; they were fixed in 96% ethanol and stored at -20°С. The cestodes were frozen in 50 μL × 0.1 T Tris-Acetate-EDTA buffer. DNA of the fish and cestodes were extracted using alkaline lysis according to (Bender et al., 1983) with our modifications.
Fish were genotyped by inter simple sequence repeat polymerase chain reaction (ISSR-PCR) (Zietjiewicz et al., 1994). A total of 67 specimens of fish and 60 specimens of cestodes were genotyped.
Band frequencies, the proportion of polymorphic bands (P), genetic diversity (average expected heterozygosity) (h), the effective number of alleles (n e ), the Nei genetic similarity index (I N ), genetic distance (D), values of genetic differentiation (H T , H S , G ST ), and gene flow (N m ), were computed using the POPGEN software package (Yeh et al., 1999). Dendrograms were constructed based on Nei genetic distances by the UPGMA method using the POPGEN software   (Yeh et al., 1999). The Mantel test was used to check the isolation-by-distance model.

The size and age structure and infestation of Amur sleepers.
Specimens aged 3+ prevailed in the studied sample. The sex ratio varied from equal (1 : 1) in the Tobol River to one and a half to two-fold predominance of female or male specimens in other water bodies. Since sexual dimorphism is poorly expressed in the Amur sleeper in terms of size and weight parameters (Suslyaev et al., 2016), the average values of the absolute length and body weight of fish for the most common age group are given without taking into account the sex of fish (Table 1).
As a result of the helminthological study, the invasion of all studied Amur sleeper populations with cestode N. mogurndae was revealed. The prevalence varied within 88-95%, intensity of invasion was within 1-42, and the index of abundance was 5.1-11.8 (Table 2).
Genetic polymorphism of Amur sleepers. When genotyping using three ISSR primers, 41 amplified fragments were identified in the Amur sleeper (Table 3). Each population has a specific genetic profile due to the variation in the frequency of widespread variants, as well as the presence of unique fractions. One unique variant (p1-1) was identified in Amur sleepers from Lake Andreevskoye, two variants (p1-6 and p2-10), from Lake Sundukul, three variants (p2-4, p2-8, and p3-9) from the Malyi Emets River.
The total proportion of polymorphic loci for three ISSR markers for all samples of Amur sleepers, which combines all the studied populations reached 92%, the genetic diversity was 032.
These values were significantly lower in some samples of the Amur sleeper and varied within 53-74% and 0.20-0.23 at the average values of 61.7 ± 4.91% and 0.22 ± 0.01, respectively (Table 4). This feature indicates an expressed local differentiation of the Amur sleeper population in the study region that is evidenced by high interpopulation differentiation and low gene flow between populations (Table 5).
The smallest genetic distance (D = 0.06) in the Amur sleeper is found between populations of Lake Andreevskoye and the Tobol River, the largest genetic distance (D = 0.29) is between the populations of lakes Sundukul and Andreevskoye (Table 6). However, an analysis made based on the Mantel test did not show a significant correlation of geographical distances with genetic distances between the Amur sleeper populations, the relationship between these characters is weak (r = 0.07, p = 0.848).  The dendrogram of genetic distances of the Amur sleeper partly reflects the character of the hydrographic network (Fig. 3). One cluster combines samples from Lake Andreevskoye and the Tobol River, the distance between which is ~190 km (by water), the second cluster, samples from lakes Andreevskoye and Obrochnoye, are also closely located and belong to the basin of the same river. The exception is a sample from the Malyi Emets River, which is also part of the second cluster, although it is located at a distance of >1000 km.
Genetic polymorphism of cestode Nippotaenia mogurndae. In N. mogurndae, a total of 38 amplicons were described using four primers (Table 3); three unique variants were identified in the samples from the Tobol River (p2-1, p2-3, and p7-11) and Lake Obrochnoye (p3-1, p7-1, and p7-2) each.  The level of differentiation of parasite populations is comparable with similar parameters of the host: the proportion of interpopulation genetic variability (GST) is 0.38, the gene flow (N m ) is 0.81 (Table 5). However, the level of genetic polymorphism of the cestode is two times lower than that of the host, especially in particular hemipopulations (Table 4). The level of polymorphism of cestodes from fish in different water bodies varies between 18-45%, genetic diversity is within 0.06-0.15 with average values being 30.5 ± 6.07% and 0.11 ± 0.02, respectively. The genetic similarity between cestode samples from different water bodies is higher compared to the host, while the genetic distances are at least two times less than in fish (Tables 5, 6). The maximum genetic distance (D = 0.18) is found between populations of Lake Andreevskoye and the Tobol River, the smallest one (D = 0.0019), between the populations of the Malyi Yelets River and lakes Obrochnoye and Sundukul. Like the host, the correlation between geographic distances and genetic distances in the parasite is insignificant (r = 0.54, p = 0.110).
The dendrogram of genetic distances of N. mogurndae does not agree with the geographical distances between populations, and does not coincide with the dendrogram of the final host, the Amur sleeper (Fig. 3). At the same time, the average correlation was found between the genetic distances of Amur sleeper and the cestode (r S = 0.648, p = 0.847).

DISCUSSION
The Amur sleeper, first recorded beyond the Urals in the 1980s, for several decades has formed stable selfreproducing populations in water bodies and watercourses of the Irtysh River basin. According to the literature data (Reshetnikov and Chibilev, 2009;Korlyakov and Nokhrin, 2014), two waterways connect the Volga River basin with the Ob-Irtysh basin through the Chusovaya and Miass rivers. To determine the exact source of penetration of the Amur sleeper into   the Ob-Irtysh basin, additional studies of the populations of the alien species in the Sverdlovsk and Chelyabinsk oblasts, including using genetic markers, are required. Since both rivers are connected with the Iset River, it can be suggested that namely this river was the "entrance gate" of the Amur sleeper to Western Siberia, and its dispersal route eastward was in the Iset-Tobol-Irtysh-Ob direction. Based on this assumption, the populations from the Tobol River and its basin can be considered the closest to the original population in the region. The population from the Malyi Emets River, the most remote (>1000 km by water) from the initial population, should be considered the youngest of the studied ones.
The total parameters of genetic polymorphism of the Amur sleeper (P = 92%, h = 0.32) in the studied section of the Ob-Irtysh basin turned out to be quite high, despite its recent introduction, which usually implies the settlement of a small group (or groups) of specimens, and should inevitably be reflected in the level of polymorphism of the alien population due to the "founder effect." When compared with other fish species, the level of polymorphism for the ISSR markers of the Amur sleeper is not lower and in some cases even higher than in the widespread fish species of the Ob-Irtysh basin. Thus, in cyprinid fish (ide Leuciscus idus L., dace Leuciscus idus L. and roach Rutilus rutilus Pallas, 1811), polymorphism varies within 83-86%, heterozygosity, 0.26-0.36 according to a comparable set of markers (Zhigileva et al., 2013). The level of polymorphism in the pike Esox lucius L. and perch Perca fluviatilis L. was 69.6 and 85%, heterozygosity, 0.24 and 0.28, respectively (Zhigileva et al., 2017). The preservation of the initial genetic diversity of the Amur sleeper during dispersal is also reported in other publications. In particular, all three mtDNA haplotype lineages of this species known in the native range have been identified in Europe (Grabowska et al., 2020). A higher level of heterozygosity and genetic differentiation of the Amur sleeper in colonized territories compared to the native range was found according to the data of allozyme analysis (Golubtsov et al., 2009). Thus, summarizing our data and the data on other types of genetic markers, it can be stated that the reduction of genetic diversity during dispersal is not typical for this species.
At the same time, the level of polymorphism in local populations of the Amur sleeper is significantly lower, and the genetic differentiation is rather strongly expressed. High levels of differentiation and dominance of the interpopulation component of genetic variability recorded in the Amur sleeper in the studied section of the Ob-Irtysh basin are characteristic of this species in the initial range, as shown based on other types of genetic markers, microsatellites (Zhang et al., 2021) and mtDNA markers (Yang et al., 2020). Thus, the conclusion can be made that the genetic structure of populations typical for the Amur sleeper could have been formed in a relatively short time, several decades since the colonization of the south of Western Siberia.
The isolation-by-distance model evaluated using the Mantel test was not appropriate for the Amur sleeper. This may be due to the fact that during the active dispersal of the Amur sleeper, differentiation of its local populations seems to occur mainly as a result of genetic drift. The presence of unique genetic variants in the relatively recently formed populations of the alien species in lakes Andreevskoye, Sundukul, and Obrochnoye can also be explained by the effects of genetic drift.
The cestode N. mogurndae, a parasite of the Amur sleeper was detected in all studied water bodies of the Irtysh River basin with relatively high values of occurrence (88-95%) and abundance (5.1-11.8). Similar high rates of infection with this parasite have been described for the group of the Amur sleeper of the size of 80-130 mm in its initial range (Ermolenko, 2004). In our opinion, this indicates the successful naturalization of not only the Amur sleeper but also its parasite, N. mogurndae in the water bodies in the south of Western Siberia Taking into account that N. mogurndae has a complex life cycle, its incorporation in local food webs can be considered as a sign of successful naturalization of invasive species, both for the parasite and its host. The presence of N. mogurndae in the parasitofauna of the Amur sleeper in the Irtysh River basin also indicates a fishery, not an aquarium, as the source of the Amur sleeper invasion in the region, and confirms the hypothesis of Amur sleeper penetration through the South Ural invasive corridor from the Volga basin, where both invaders also successfully naturalized (Sokolov et al., 2011a;Kirilenko and Shemonaev, 2017).
The level of polymorphism in N. mogurndae is lower compared to the host, which may be due to its narrow host specificity, more stable living conditions of the parasite inside the host organism and, accordingly, less need to adapt to different living conditions in different water bodies. The highest levels of invasion and genetic polymorphism, as well as the presence of rare genetic variants, were found in a sample of N. mogurndae from the Tobol River. This may indicate the primacy of the Tobolsk focus of invasion of this cestode in the Irtysh River basin and confirms the previously identified pattern about the critical importance of this river as an "entrance gate" for many invasive species of the Ob-Irtysh basin: "Hydrobionts widely dispersed within the Ob basin are primarily found on the eastern slope of the Urals within the Tobol River basin, and subsequently disperse along the Ob River in an easterly direction" (Korlyakov and Nokhrin, 2014, p. 25).
In general, despite the synchronous introduction of the parasite and the host, as indicated by the close values of the genetic differentiation of populations of the Amur sleeper and cestode, they demonstrated differ- No. 2 2022 ent levels of polymorphism. The effect of genetic drift on the parasite populations is more pronounced. Unlike the Amur sleeper, there is not even a weak correlation between genetic distances and geographical distances between the populations of the cestode. Thus, the formation of the genetic structure of the populations of the Amur sleeper and cestode in the newly acquired range can be considered as an interesting example of the coevolution of two species with initially different levels of genetic variability, which, however, does not prevent these species from successfully colonizing new territories. It should be taken into account that the genetic structure of the cestode populations can also be affected by the population dynamics of its intermediate host, the cyclops Mesocyclops leuckarti (Dempshin, 1985;Košuthová et al., 2008). However, this aspect requires further study.
CONCLUSIONS Our data show that the Amur sleeper has a high level of genetic polymorphism in the acquired part of its range (south of Western Siberia). Over several decades, a population genetic structure with a pronounced interpopulation component of variability, characteristic of this species in the native range, has been formed. In the parasite of the Amur sleeper, the cestode N. mogurndae, the processes of genetic differentiation occur simultaneously with similar processes in the host despite the lower level of polymorphism compared to the host.

ACKNOWLEDGMENTS
We are grateful to E.O. Sprudzans (Tyumen State University) for assistance in sampling the material.

FUNDING
The study was partially supported by the Government of the Tyumen oblast within the project of the West Siberian Interregional Scientific and Educational Center, no. 89-DON (2).

COMPLIANCE WITH ETHICAL STANDARDS
Conflict of interest. The authors declare that they do not have a conflict of interest.
Statement on the welfare of animals. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.

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