J. Limnol., 2016; 75(1): 101-108 ORIGINAL ARTICLE DOI: 10.4081/jlimnol.2015.1253 Double origin of the racer goby (Babka gymnotrachelus) in Poland revealed with mitochondrial marker. Possible implications for the species alien/native status Michał GRABOWSKI,1* Kamil HUPAŁO,1 Aneta BYLAK,2 Krzysztof KUKUŁA,2 Joanna GRABOWSKA3 1Department of Invertebrate Zoology and Hydrobiology, University of Lodz, Banacha 12/16, 90-237 Lodz; 2Department of Environmental Biology, University of Rzeszow, Zelwerowicza 4, 35-601 Rzeszow; 3Department of Ecology and Vertebrate Zoology, University of Lodz, Banacha 12/16, 90-237 Lodz, Poland *Corresponding author: [email protected] ABSTRACT In mid-1990s racer goby (Babka gymnotrachelus) penetrated to the Vistula and Western Bug rivers in Poland through the canal connecting the Baltic and the Black Sea basins, namely the Vistula and Dnieper drainages. In early 2000s, the species was reported from Polish section of the Strwiąż River that is an affluent of the Dniester River that drains to the Black Sea basin. According to the Polish legislation, the racer goby has been enlisted as an alien invasive species that may pose threat to local biota. Our analysis of the mtDNA cytochrome b diversity revealed that the Vistula/Western Bug and Strwiąż populationsonly are different genetic units. First one orig- inated from the Dnieper River, while the second derives from the Dniester River. According to the results of mismatch analysis, both are in the stage of demographic and spatial expansion. The haplotype frequencies in population from the Vistula/Western Bug differ signif- icantly from those in the source population in Dnieper, suggesting founder effect, possibly due to human-mediated introduction of low number of individuals. On the other side, the population in Strwiąż does not differuse in structure from the one in Dniester, providing a hint towards spontaneous range expansion. Interpretation of our results in light of historical data lead to the conclusion that presence of racer goby in Strwiąż was probably overlooked in previous, spatially limited, studies. Thus, in Strwiąż the species should not be treated as alien, contrary to its status in the Vistula drainage. This double origin of racer goby populations in Poland creates a peculiar situation for national legislation procedures as one population is alien and invasive, while the other one is not. In light of our findings, the population from Strwiąż should be recognised as a special case. Steps should be undertaken to raise public awareness to prevent translocation of the gobies between the rivers to prevent deterioration of the evolutionarily isolated genetic pools of the Dnieper and of the Dniester basins. Our results illustrate the need for case studies upon genetic population structure, following appearance of new species in previously unoccupied water bodies, even on local scale. Key words: Alien aquatic species; population genetic structure; legal status; Ponto-Caspian invaders. Received: May 2015. Accepted: August 2015. INTRODUCTION routes have been used by five species of Ponto-Caspian gobies - the most expansive alien fish from that region The territory of Poland is shared largely between the (Copp et al., 2005). The racer goby, Babka gymnotra- Vistula and Oder river systemsNon-commercial and 99.7% of the country chelus (Kessler, 1857) (syn. Neogobius gymnotrachelus area belongs to the Baltic Sea drainage basin. Only 0.1% (Kessler, 1857)), colonised the middle and the upper parts of the area, in south-western Poland is drained to the of Danube (Roche et al., 2013), from where it migrated North Sea via the Elbe river system. The remaining 0.2% to Rhine via the Rhine-Main-Danube Canal (Borcherding of the area in south-eastern Poland belongs to the Black et al., 2011). Already in mid-1990s the species was found Sea basin, draining largely to the Strwiąż River, which is in the Vistula river system - first in the Western Bug and a tributary of the upper Dniester. Due to the artificial soon after, in 2000, in the Vistula itself (Danilkiewicz, canals connecting the main rivers belonging to the Baltic 1996; Kostrzewa and Grabowski, 2001). It was the first basin and further joining them to the basin of the Black Ponto-Caspian gobiid recorded in the Baltic stretch of the Sea and of the North Sea, the hydrographic network in central corridor (Kostrzewa and Grabowski, 2003; Poland is an important stepping stone area for numerous Grabowska et al., 2008). Apparently, the species reached alien and invasive aquatic species migrating from the Poland through the Pripyat-Bug canal that connects the Ponto-Caspian region to Western Europe. Three main Black and Baltic basins, namely the Dnieper and Vistula routes of their expansion have been defined and named drainages, since the end of the 18th century. Such invasion by Bij de Vaate et al., (2002) as the northern, central and route was initially concluded from the first record locality southern migration corridor. Particularly the two latter of the racer goby in the Western Bug and the known his- 102 M. Grabowski et al. tory of its previous expansion in Ukraine and Belarus data upon genetic composition of the native populations (Danilkiewicz, 1996; Grabowska et al., 2008, Se- from the Dniester and Dnieper. Finally, we discussed our menchenko et al., 2011). Ohayon and Stepien (2007) re- results with literature on the distribution of the racer goby vealed that the invasive population from the Vistula River in the Dniester basin and presented implications of our is genetically identical to the native one inhabiting the findings for the species invasive/native status of the pop- Dnieper River, proving the Pripyat-Bug canal as the in- ulations of racer goby in Poland. vasion route of racer goby to the Baltic basin. Besides, the authors reported that the populations inhabiting the METHODS Danube, Dniester and Dnieper (i.e. the species native Individuals of racer goby were collected in 2013 area) are genetically distinct units. from several localities along Western Bug, Vistula and Soon after its first record in the Vistula river system, from the Strwiąż River (Fig. 1, Tab. 1). Samples of fins in 2003, two individuals of racer goby were found in the or muscle tissue were fixed in situ in 96% ethanol and Polish section of the Strwiąż River belonging to the then stored at room temperature. Each sample was ho- Black Sea basin (Amirowicz and Kukuła, 2005). Already mogenized in a 1.5 mL tube containing 200 mL of in 2009, the racer goby dominated the local fish assem- 2 Queen’s lysis buffer (Seutin et al., 1991) and incubated blage with a density of 3.4 ind m and since then has re- overnight at 55°C with 5 mL of proteinase K (20 mained very abundant (Kukuła and Bylak, 2010, 2013). mg/mL). The total DNA was then extracted based on a The earlier ichthyological surveys did not reveal presence standard phenol-chloroform method as described by of that species in the Polish section of Strwiąż (Rolik, Hillis et al. (1996). Air-driedonly DNA pellets were re-sus- 1967, 1971), whereas fish fauna in the Ukrainian part of pended in 100 mL of TE. The mitochondrial cytochrome this river has never been surveyed. The racer goby is re- b gene was amplified with the polymerase chain reaction ported as native to Dniester and its tributaries, such as: (PCR) in 20 µL reactions containing 2 µL (c. 100 ng) of Zbruch, Zwanchik, Smotrych, Reut and Bystrica template DNAuse and 18 µL of mix containing 10 µM Tris- (Smirnov, 1986; Khudyi, 2002; Romanescu, 2010; HCl buffer pH 8, 400 nM of each primer, 200 µM of Manilo, 2014). Thus, the Dniester River would seem as each dNTP and 1/2 units of Taq polymerase. Primers the most obvious donor area. However, the origin of the were AJG15 (Akihito et al., 2000) and H15343g population in Strwiąż cannot be judged with certainty. (Ohayon and Stepien, 2007). A negative control contain- The upper Strwiąż in Poland neighbours with the Vistula ing no template DNA was used in each run. PCR reac- drainage system. Although the distance between Strwiąż tions comprised 35 cycles of 45 s at 94°C (denaturation), and San, a major tributary of Vistula, is ca. 10 km there 30 s at 52°C (annealing) and 60 s at 72°C (extension), are no natural or artificial connections between the two followed by a final extension step for 3 min. PCR prod- rivers. So far there are no reports on presence of racer ucts were cleaned up by exonuclease I (20 U µL–1, Fer- goby in the San River. Yet, its introduction from the Vis- mentas, Vilnius, Lithuania) and alkaline phosphatase tula basin, e.g., as live bait, cannot be excluded. Knowing FastAP (1 U µL–1, Fermentas) treatment according to the that the Dnieper population (being the source for the in- manufacturer’s guidelines. Sequencing of the PCR prod- vasion of Vistula) is distinct in genetic terms from the ucts was performed using BigDye terminator technology one in the Dniester basin, its translocation to the latter by Macrogen Inc. (Seoul, Korea) using the same primers one could be treated as pollutionNon-commercial of the local genetic pool as at the amplification stage. Sequences were edited and of the racer goby. Furthermore, if indeed the population aligned using Bioedit, ver. 7.2.4 (Hall, 1999; in Strwiąż is of the Dniester origin then it cannot be un- http://www.mbio.ncsu.edu/BioEdit/bioedit.html) with equivocally defined as alien unless proven as ClustalW plug-in. Haplotypes were identified by eye. expanded/introduced due to human activity. Then, if this To test for the genetic dissimilarities between popula- population is not alien (contrary to the one in Vistula and tions sampled at different localities along the Dnieper-Vis- Bug rivers), it would create a very peculiar situation and tula and Dniester-Strwiąż routes, we used the Fst estimator need for redefining the legal status of the species in as well as the exact test of population differentiation (χ2 Poland.