European River Lamprey Lampetra Fluviatilis in the Upper Volga: Distribution and Biology

After the construction of the Volga Hydroelectric Station and other dams, migration routes of the Caspian lamprey were obstructed. The ecological niches vacated by this species attracted another lamprey of the genus Lampetra to the Upper Volga, which probably came from the Baltic Sea via the system of shipways developed in the 18 th and 19 th centuries. Based on collected samples and observations from sites in the Upper Volga basin, we provide diagnostic characters of adults, and information on spawning behavior. Silver coloration of Lampetra uviatilis was noted for the rst time and a new size-related subsample of “large” specimens was delimited, in addition to the previously described “dwarf”, “small” and “common” adult resident sizes categories. The three water systems: the Vyshnii Volochek, the Tikhvin and the Mariinskaya, are possible invasion pathways, based on the migration capabilities of the lampreys. Dispersal and colonization of the Caspian basin was likely a combination of upstream and downstreams migrations. First, the lampreys migrated upstream along the rivers of the Baltic basin until they reached the water-parting line, followed by mostly downstream dispersal into rivers of the Caspian basin. Dispersal in the Volga River was similar, in accordance with the migration cycle of this opportunistic lamprey species.


Introduction
Four genera of lampreys (Petromyzontiformes) occur in the central part of the eastern Europe (European part of Russia, Fig. 1). Representatives of the genera Lampetra (the Northeast Atlantic Ocean and the rivers of Europe) and Lethenteron (drainages of the White and Barents seas of the Arctic Ocean) are represented by both migrant (anadromous, potamodromous) and freshwater (the so-called resident) The anadromous form of the European river lamprey Lampetra uviatilis inhabits the basins of the North, the Baltic, and the Mediterranean Sea. The freshwater form of the European river lamprey, traditionally referred to as the European brook lamprey Lampetra planeri, has been repeatedly registered in the Upper Volga, i.e. in the Caspian Sea basin, in the middle and the second half of the 20 th century [10,[14][15][16][17][18][19][20]. There are reasons to believe that the invasion of the European river lamprey into the Volga River has been associated with the development of inland shipping and the network of shipways in the 18 th -19 th centuries. The Upper Volga is thus of special interest for studies dealing with the dispersal of hydrobionts. It is connected with rivers of the Baltic Sea basin by three hydrological systems of shipways and with rivers of the White Sea basin by one hydrological system. Two navigable waterways from the Baltic Sea and one from the White Sea connect with the Rybinsk Reservoir, which is the zone of "accumulation of invaders" as well as the area of intergradation of the northern and the Ponto-Caspian hydrobionts [21].
The freshwater form of the European river lamprey is currently included in the sh fauna lists of the Rybinsk Reservoir with the status of a rare species decreasing in numbers [21][22][23]. Researchers from the Institute for Biology of Inland Waters of the Russian Academy of Sciences (IBIW RAS) note that the lamprey does not occur in the reservoir itself [21], surviving as small local populations in the tributaries [23]. Other experts from the same institute question the presence of the European river lamprey in the system of the Rybinsk Reservoir [24,25]. However, there is no detailed information about the distribution of the European river lamprey in the Rybinsk Reservoir and in the Upper Volga in general. Moreover, characteristics of its local populations are also unknown.
The aim of our study was to establish whether representatives of the order Petromyzontiformes occur in the rivers of the Upper Volga basin and to recon rm the taxonomic position of lampreys from the Upper Volga basin. A second objective was to analyze invasion pathways and dispersal mechanisms of lampreys from the Baltic Sea to the Upper Volga basin.

Results
Based on the analysis of the satellite images and the literature information [26][27][28][29], we calculated the distances between locations of lamprey samples and the Volga-Baltic water-parting line (Table 1).

Recon rmation of the taxonomic status
The external attributes and dentition allowed us to con rm that the adult specimens and larvae were European river lamprey Lampetra uviatilis: cloaca is posterior to the origin of the second dorsal n, under its rst half. Oral papillae very small, of uniform size along the entire perimeter of oral disc. Eyes dorsolateral. Teeth blunt in most specimens (Fig. 3). Exolateral rows without dentition. Three rows of right and three rows of left endolaterals. Second rows of endolaterals always with three cusps. Supraoral lamina with two unicuspid teeth separated by bridge without dentition. Marginal cusps of infraoral lamina larger than inner ones, usually bicuspid (sometimes asymmetric). In total, 6-10 cusps on infraoral lamina. Anterial rows bearing 0-2 teeth; if teeth present, 0-7 anterial teeth in the rst row. Posterial teeth absent. Trunk myomeres 58-77 in number. Relative size (% of the body length) in specimens with total length 123-151 mm: prebranchial length -8.6-13.9; branchial length -9.0-15; trunk length -45.9-56.4; tail length -25.2-31.4; disc length -4.5-6.7.
Coloration of the lampreys from the Vysochinsky Stream was dark dorsally and silvery on lateral sides, which is characteristic of juvenile European river lamprey during downstream migration (Fig. 4a). Coloration of males and females from the Kamenka River and males from the Saragozha River was dark brown dorsally and light on the ventral side (Fig. 4b, c). Females from the Saragozha River were olive or light brown on the dorsal side, olive or sandy on lateral sides. Dorsal part of the branchial area colored, ventral part light, oral disc not pigmented. Despite the coloration of the specimens from the Vysochinsky Stream, all the lampreys had well-expressed secondary sexual characteristics and developed reproductive products.

Discussion
The coloration of adults from the studied populations was diverse. Lampreys from the Saragozha and the Kamenka were, in general, colored similarly to adults of the European river lamprey from various rivers of Europe [3,7,30]. The coloration of adults from the Vysochinsky Stream deserves a special discussion.
Their silvery coloration is characteristic of post-metamorphic juveniles (transformers, macrophtalmia) during their downstream migration [31][32][33]. Sperone et al. [34] found what they took to be the southernmost resident population of the European river lamprey (they call it the European brook lamprey L. planeri) in the Lao River (Calabria, Italy). They caught two such specimens on October, 26 2018 with an electro sher (total length 170 and 175 mm). It is clear from the photo of the specimen with the silvery coloration (P. 133, and the gure in [34]) that it is not an adult but a post-metamorphic immature specimen (secondary sexual characteristics are absent). Most probably, it was a smolt of the European river lamprey. Of the other resident species of Lampetra, silvery coloration was also noted for Lampetra aepyptera [7]. It is unclear if the silvery coloration of lampreys is associated with their migratory activity and why lampreys from the Vysochinsky Stream do not lose this coloration upon maturation. These questions might open avenues for further research and the need for genetic analysis of archived specimens.
One of the hypotheses explaining the presence of the landlocked lampreys (e.g. resident European river lamprey in the Upper Volga or lake form of sea lamprey Petromyzon marinus in the Lake Ontario) assumes the impact of global climate changes on the ichthyofauna 70-10 thousand years ago. Lawrie [39] and Smith [40] believe that the sea lamprey inhabiting the lake and its drainages is a relict Pleistocene population in North America. Dorofeev et al. [41] and Slynko and Tereshchenko [23] suggest that the retreat of the Valdai glaciation in Europe under the in uence of the global warming (12-10 thousand years ago) resulted in the formation of numerous periglacial lakes and other water bodies of glacial origin. Owing to this, the entire Ponto-Caspian basin was populated by sh species of the Arctic freshwater and boreal-submontane faunistic complexes. The European river lamprey belongs to the latter. This is how the presence of the European river lamprey in the tributaries of the Upper Volga, the Ivankovo, the Uglich and the Rybinsk Reservoir is explained by Slynko and Tereshchenko [23].
If the European river lamprey had inhabited the Upper Volga for > 11000 years, its local populations would have mixed resulting in homogeneous phenotypes. In contrast, lampreys from the Vysochinsky Stream, the Saragozha River, and the Kamenka River show diverse phenotypes (Fig. 4). These water courses are located close to each other, are not separated by physical barriers, and have similar hydrological characteristics (all of them are lowland rivers). The observed differences indicate a recent invasion of the European river lamprey into this region. Therefore, we forward the invasion hypothesis (entry of the European river lamprey into the system of the Volga River via manmade shipways). Extremely low numbers of the European river lamprey in the entire Upper Volga are likely a consequence of the construction of dams for hydroelectric, which caused limni cation of the river and a decrease in the abundance of rheophilic species, including lampreys.
In the early 18 th century the Caspian, Baltic and White Sea basins were not yet connected (Fig. 5).
Shipways connecting the Upper Volga with the Baltic basin started with construction of the Vyshnii Volochek water system (1708, here and below we give the year of the completion of earthworks and construction activities on the arti cial canals connecting the rivers. Practically, the basins of different seas became connected after that. O cially, the water systems in question were opened later (max. 6 years later), one of the reasons was the need to build sluices for the passage of ships). This was followed by the Tikhvin water system (1805), and by the Mariinskaya water system (1808), which is now called the Volga-Baltic water system [26,28,29]. The North Dvina water system in 1828 connected the Caspian and the White sea basins [27]. The development of the system of shipways opened new water courses, along which various hydrobionts including sh and cyclostomes have been dispersing for more than 300 years. . This, probably, was made possible not only by the shipways but also because global warming causing northward displacement of a number of the species [43]. Dispersal in the opposite direction has been noted for fewer sh species, mainly due to their small adult sizes and short life cycle [44]. It may also be associated with the regulation of the run-off of the Volga River, which results in an increase in the water temperature, low oxygen conditions, a change in the water mineralization, demands on spawning substrate, and growing eutrophication [21]. The Tikhvin water system is closer than the other two to the system of the Saragozha River and to the Kamenka River. The Vyshnii Volochek water system is situated in direct proximity of the Vyazma River, the Malaya Dubenka River, the Malaya and the Bolshaya Kosha rivers, the Shutinka Stream and the sites of collection of the European river lamprey in the studies by Viktorov [19], Viktorov et al. [20] and Nezdolii & Kirillov [15]. We consider these two systems of shipways as the most probable invasion pathways of the European river lamprey from the Baltic to the Caspian basin in the corresponding parts of the distribution. However, distance cannot be the only criterion in this matter. To ascertain the pathways used by the European river lamprey for the invasion in the Caspian basin, phenogeographic studies are necessary.
The foregoing reasoning about the colonization pathways opened due to the human activity closely resembles the process underway in the North American Laurentian Great Lakes. While the situation with the Lake Ontario remains a topic of debate, the remaining invasion pathways are clear to most researchers. Sea lamprey (Petromyzon marinus) has spread throughout the system of the lakes due to the construction of channels, which has made it possible to bypass Niagara Falls. It took only 25 years for sea lamprey to get to the farthest Lake Superior [49]. Of note is that as both sea and European river lamprey penetrate more deeply into these freshwater systems, they reduce their adult sizes and fecundity, producing parasitic lake forms. The European river lamprey goes further to produce a nonparasitic resident form [3], which after metamorphosis has no need of feeding and can disperse wider and inhabit more types of habitats.
Anadromous lampreys are quite capable of covering the distances mentioned above during one upstream migration [3]. For instance, the presence of an anadromous lamprey from the Gdovka River in the collection of the Museum of the Zoological Institute of the Russian Academy of Sciences (ZISP 25430-25433) indicates that the European river lamprey could migrate upstream the Narva River and cross Lake Peipus, covering in total more than 3000 km. In water-abundant years, lampreys in the Luga River overcome the Kingisepp and the Sabsk rapids and entered the tributaries of the Luga, e.g. the Krupa River, ascending 150 km upstream (ZISP 26437; 26438).
A possible scenario of the dispersal of the European river lamprey into the Upper Volga, based on the evidence from the Tikhvin water system, is as follows. Anadromous lamprey adults were noted in the Syas River [50]. Berg  Earthworks on the Tikhvin water system were completed in 1805 [29], heralding the connection between the Baltic and the Caspian basin. After that, anadromous, lake adults (or resident adults from the Syas and the Tikhvinka) could cross the water-parting line and start migrating along the Volga slope. Further downstream dispersal along the rivers of the Tikhvin water system and the Volga River was achieved by the larval stages. Primary dispersal in the form of downstream migration is shown for lampreys aged 0+ [51,52], while downstream migration of older ammocoetes has been repeatedly recorded in various rivers throughout the year. During downstream migrations lampreys can cover considerable distances (tens of kilometers) over a short period of time. This means that it could take as little as several decades for the species to disperse across the Upper Volga. Upstream dispersal into rivers such as the Saragozha, the Kamenka and the Tunoshonka could be performed both by the resident adults and by the larvae of the European river lamprey [53].
Thus, the mechanism employed by the European river lamprey for colonization of the Caspian basin was a combination of upstream and downstream migrations. At the rst stage, the lampreys migrated upstream along the rivers of the Baltic basin until they reached the water-parting line. Reaching and crossing the water-parting line became possible owing to the anthropogenic interference: the construction of sluices on the rivers (allowing lampreys to navigate up the rapids) and on the water-parting line, and the opening of shipways. The second stage was represented by mass, mostly downstream migrations along the rivers of the Caspian basin. Dispersal along the system of the Volga River was also a combination of upstream and downstream migrations in accordance with the migration cycle of the European river lamprey.
The European river lamprey -a species capable of long-term and long-distance migrations both upstream and downstream -could disperse across the Caspian basin along corridors of anthropogenic origin. Considerable morphological diversity of its local populations reported in our study provides evidence for this hypothesis. The diversity of lampreys from the Vysochinsky Stream, the Saragozha River and the Kamenka River is probably associated with the fact these young (not more than 60 generations) local populations formed from a handful of pioneering adults.

Material And Methods
Animals. This study is based on the analysis of our own material: collections of lamprey larvae and adult lampreys during spawning as well as observations of the lamprey distribution in the Upper Volga (central European Russia, Fig. 1). All animals were captured and taken from the environment in accordance with guidelines approved by Institute of Ecology and Evolution, Russian Academy of Sciences. The lamprey larvae in all the rivers were caught in shallow areas with the help of a Kinalev net (8-mm rebar frame The Saragozha River is a 2 rd order tributary of the Rybinsk Reservoir. It ows from Lake Pavlovskoe, and is 53 km long. Together with the Vysochinsky Stream, the Saragozha belongs to a vast uvio-lacustrine system, which also includes Lake Ilovets, the Ilovets River, Lake Zastizhskoe, Lake Obretinskoe, the Zhelezinka River, etc. The width of the examined section of the river is ~15 m, the depth is 0.5-1 m, with pools up to 2 m deep. The bottom is sandy, with occasional small pebbly areas. It was in these pebbly areas that the lampreys were observed to spawn. The water has a light brownish (peaty) color and water temperature was 12°C at the time of sampling.
The Kamenka River is the 2 nd order tributary of the Rybinsk Reservoir. Its length is 14 km. The Kamenka River is a tributary of a large river, the Sit, into which it ows at a distance of 13 km from the reservoir. We examined a 2-km-long section of this river, situated at a distance of 1.25 km from the river mouth. At this location, the river is 10-12 m wide and depth varies from 30 cm to 1.8 m. The bottom is pebbly gravel, with numerous ratchels and boulders. Small and shallow inwashes of fallen leaves were noted in some places. The water has a light brownish (peaty) color and water temperature was 17°C at the time of sampling.
The Tunoshonka River (51 km long) is a rst-order tributary of the Volga River (Gorky Reservoir). It was examined in the lower reaches, where it is 30 m wide or even wider. Shallow areas with a depth of 20-50 cm alternate with deeper pools (≥2 m). Substrate is silty-sandy, with middle-sized pebble in some places.
The Vyazma River, which is a 2 nd -order tributary of the Volga River (Ivankovo Reservoir), is 43 km long. Near its mouth the Vyazma is 5-10 m wide and 20-50 cm deep. The bottom is smooth and stony. The Malaya Dubenka River (14 km long) drains into the Upper Volga Reservoir (Volgo lake). The lowermost 2-2.5 km of the river is a bay of the lake with no current, silty substrate, and abundant aquatic vegetation. Lamprey larvae were caught in the middle section where there was sandy/ pebble substrate, 0.5 m water depth and channel width of ~5m.
Cartography, meta-and digital data. Distances concerning the water bodies of the Baltic basin and the tributaries of the Upper Volga given in this paper were measured based on satellite images or topographic maps and are as near as possible to the actual ones. In the few instances when the distances were taken from the literature, the references are given.
Coastline on the gures in this study is based on SHP Small scale data [54]. Layer of the marine basins is developed on database Hydrosheds [55] 29], and Otsenka kachestva vody… [27].

Funding
The work was carried out with the nancial support of the Russian Science Foundation, Project No 19-14-00015.  Oral disc of the European river lamprey Lampetra uviatilis (Kamenka River) Figure 4