A look into salinity tolerance of the round Goby:

A tale of two salinities

Felix Englund Örn

Degree project for Master of Science in Biology

Animal Ecology, 60 hec, vt 2017 Department of Biological and Environmental Sciences University of Gothenburg

Supervisor: Charlotta Kvarnemo 1

Examiner: Staffan Andersson

Table of contents Abstract ...... 3 Introduction ...... 3 Aims & Predictions ...... 6 Method ...... 7 sampling, upkeep and acclimatization ...... 7 Figure 1...... 8 Table 1...... 9 Table 2 ...... 10 Figure 2 ...... 11 Behavioral Experiment 1: Activity ...... 11 Behavioral Experiment 2: Salinity Choice ...... 11 Figure 3...... 12 Statistics & null hypothesis ...... 12 Figure 4...... 13 DNA Extractions ...... 14 DNA Concentrations ...... 14 Results ...... 15 Behavioral Experiment 1: Activity ...... 15 Figure 5 ...... 15 Behavioral Experiment 2: Salinity Choice ...... 15 Figure 6...... 16 Discussion ...... 17 Figure 7...... 16 Figure 8...... 17 Acknowledgements ...... 20 References ...... 21

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Abstract

The round goby is a highly successful invasive of benthic fish. Transport via ballast water has led to its establishment in the , large harbors and rivers across Europe and the Laurentian Lakes of North America. Generalist behavior and wide tolerance to abiotic factors enables it to thrive in many environments and recent studies show salinity tolerance to be higher than previously reported, with suggestions that tolerance is linked to the salinity of origin. Establishment of round gobies in Gothenburg, Sweden, has provided a natural experiment with exposure, for the first time, to almost oceanic conditions. The aim of this study was to investigate Gothenburg round goby populations at two different salinities in order to reveal if variation in salinity tolerance is due to genotypic sorting of genetically pre-adapted individuals, or to phenotypic plasticity. This was addressed using behavioral activity experiments which find that round gobies are equally active across point of capture, suggesting that salinity tolerance is due to phenotypic plasticity. Importantly, the presence of a wild population, apparently unaffected by near oceanic conditions, suggests that high salinity is not a natural constraint on dispersal in the round goby. Furthermore, local findings indicate intraspecific competition may drive the fish to actively seek unpopulated areas with higher salinities.

as an invader stems in part from a generalist Introduction feeding behavior (Nurkse et al., 2015), multiple spawning’s per breeding season The round goby (Neogobius melanostomus) (Meunier et al., 2009), an ability to tolerate a is a species of benthic fish native to the wide range of salinities and temperatures as Caspian and Black seas. Through well as a tolerance to low oxygen levels, anthropogenic activities it has spread far and which enables it to thrive in many wide across Europe as well as to the Great environments (Kornis et al., 2012). Lakes of North America (Brown and Stepien, Throughout its history as an invader it has 2008) and has established itself as one of the established in a wide range of aquatic widest spread invasive fish species in the environments. Round goby populations have world (Kornis, 2012). As with any invasive previously been found in fresh to brackish species, it is of great concern how the native water (0 - approx. 15‰) and the species has ecosystem will acclimate to its introduction established itself in large European rivers and how future management should be (e.g. Rhine and Elbe) and in the Laurentian conducted. The round goby has been shown lakes of North America (freshwater) and the to affect ecosystems both negatively and Baltic Sea (brackish), but no populations positively and it has proven to be a complex have been observed in full oceanic matter affecting multiple trophic levels conditions (Charlebois et al., 2001; Kornis, et (Hirsch, 2015; Kornis et al., 2012). Its success al. 2012). However, a tolerance of up to 40‰

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has been reported (Kullander et al. 2012), An establishment in a new although Kornis et al. (2012) noted that environment, with new selective pressures, these results derive from the Caspian Sea such as high salinity, sets the stage for local and Aral Sea. In these oceans CaSO4 is the adaptation. Local adaptation, where locally dominant salt molecule, not NaCl like in most evolved genotypes have higher relative oceans where oceanic conditions of approx. fitness in the local environment compared 30-40 PSU is presumably above their the other environment (Kawecki and Ebert, tolerance. As the species continues to 2004; Savolainen, Lascoux and Merilä, 2013), expand, areas hosting such conditions will is one likely reason for the success of an are likely to be met. One such area is the invading species. Local adaptations for ongoing establishment in Swedish waters. dispersal abilities have been shown to be First recorded in the country in 2008 especially frequent at the front of expanding (Karlskrona, 56°9′39″N 15°35′10″E), the ranges, where individuals experience less round goby has subsequently established intraspecific competition due to low itself in various parts of Sweden, including population densities. This has been shown in the archipelago of Karlshamn a classic example of an invasive species the (56°10′12″N 14°51′47″E) and Stockholm cane toad (Rhinella marina). Studies have (59°19′46″N 18°4′7″E), the islands Gotland shown changes in skeletal morphology, (57°30′N 18°33′E) and Öland behavior and even a reduced immune (56°44′N 16°40′E) as well as the harbor of system response, facilitating further Gothenburg (57°42′25″N 11°57′59″E) dispersal (Hudson et al., 2016; Brown, (Brockmark, 2016). Phillips and Shine, 2014; Brown and Shine, The Gothenburg features a 2014). Front range specific changes also halocline where freshwater flows from the occur in the round goby as shown by Azour river Göta älv and saltwater pushes in from et al. (2015), with individuals at the forefront Kattegatt creating a complex and shifting of a distribution range exhibiting faster body of brackish water. Kattegat itself is growth and higher welfare due to less connected to the North Sea, which is an area intraspecific competition for food. Similarly, of higher oceanic salinities that is not yet Thorlacius et al. (2015) found personality hosting any known round goby populations. dependent dispersal, with round goby If individuals in the Gothenburg estuary and individuals caught at the invasion front being populations in similar areas were to disperse, more prone to disperse sooner and exhibit then salinity could be a factor limiting higher activity, whereas there was no dispersal, both to areas of higher and lower correlation between activity and dispersal salinities. This region therefore hosts a among individuals from old populations natural experiment where the potential for suggesting this behavior is lost as the round goby to spread to higher oceanic population gets older. conditions, e.g. the North Sea (approx. The individuals at the front of the 35‰), is likely to unfold, marking it as an invasion will be the first to face new salinities important region to track the round goby and thus we might expect them to develop establishment and spread.

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local adaptations to different salinities. But The Gothenburg population has yet what occurs when they enter into new to be investigated and could provide new salinities? Change in salinity will be a insights into the matter of salinity tolerance. physiological challenge due to the energetic Gothenburg features the largest harbor demands of a higher degree of among the Scandinavian countries with a osmoregulation conducted by the fish. This total of 11 000 ships making land at the higher demand is because of the discrepancy harbor each year (Göteborgs Hamn, 2013). between internal osmotic and external Proximity to large cargo ports has been environmental salt levels which the fish must linked to the round goby distribution pattern handle to keep in balance (Hempel and Thiel, in the Baltic Sea (Kotta et al., 2016) and 2015). Studies conducted by Ellis and studies have shown that certain populations MacIsaac (2009) and Karsiotis et al. (2012) in the region are genetically similar to those indicated that the round goby salinity from Kerson, Ukraine, which is a major port tolerance limit was at around 20‰, whereas in the (Ojaveer et al., 2015), all specimens in the experiment died within suggesting ballast water is a key vector of a month of exposure to salinities between invasion. It is thus presumed that the round 25‰ and 30‰. However, recent studies by goby introduction, or even multiple Hemple and Thiel (2015) and Behrens, van introductions, to Gothenburg also have Deurs and Christensen (2017) found occurred through ballast water. Through its different results with indications that the invasion and establishment across Europe, tolerance is as high as 30‰. An explanation, different round goby populations might have offered for their non-congruent results by become adapted to different salinities in Hempel and Thiel (2015), is that the fish in different ports over time since their first the three experiments were of different introduction. Individuals in the harbor of origins. The individuals unable to cope with Gothenburg might then already be adapted higher salinities in both Ellis and MacIsaac to a specific salinity range upon arrival. (2009) and Karsiotis et al. (2012) When discharged, larvae from divergent experiments were of freshwater origin whilst genetic backgrounds could experience a the individuals in Hemple and Thiel’s (2015) higher fitness if they manage to seek out experiment were from brackish waters. water in the river mouth more akin to their Behrens, van Deurs and Christensen (2017) native range of salinity. If so, a genetic offer the same explanation and also highlight sorting across the salinity gradient in the that their salinity acclimatization occurred at harbor would be expected to occur, meaning a slower pace than the experiments individuals establishing themselves in higher conducted by Ellis and MacIsaac (2009) and or lower salinities might differ genetically Kariotis et al. (2012). This therefore suggests due to differing origins. This is one of the that the origin of invading individuals is likely explanations that has been proposed to to influence the round gobies’ ability to occur when divergent genotypes are invade new regions. introduced into a new range and is reported to be a likely explanation for the expansion of the green shore crab (Carcinus maenas)

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along the North-East Pacific shoreline (Rius the round goby has undergone adaptation to et al., 2014). Alternative explanations why higher and lower salinities. If this is true the round goby might manage establishment between fish caught at different salinities in in different salinities are an unusually wide the estuary of Gothenburg, it would suggest individual tolerance to salinity or because of that genetic sorting has occurred. However, phenotypic plasticity; “the direct influence of if populations do not reveal genetic the environment on individual phenotypes differentiation or if it is not correlated with through developmental mechanisms” salinity, it suggests origin of invading fish (Chevin, Lande and Mace, 2010). Phenotypic matters less regarding success of plasticity has been argued to alleviate establishment. Unfortunately, due to the ecological pressures, permitting survival and time frame of this project, the genetic establishment in novel environments (Price, analysis could not be done in time for proper Qvarnström and Irwin, 2003, Moran and annotation of the genotypes. These results Alexander, 2014). are as such not presented here but will be included in future studies. Nevertheless, Aims & Predictions since a large part of my Master project was devoted to sampling and preparation for DNA sequencing, I have been advised by my The aim of this study was to investigate the supervisor to report the methods of this Gothenburg round goby population in hopes work in the method section below. to reveal more about the species relation to salinity, an abiotic factor that might limit Two different behavioral their dispersal. This matters specifically if the experiments were conducted to investigate salinity tolerance within the species is driven the preference of salinity and a measure of by genetic adaptations or phenotypic the general activity of the round goby. The plasticity. The study also offers a first view round goby used in these experiments were into the geographic range the round goby from two different locations in Gothenburg inhabits in the Gothenburg estuary, as the harbor that differ in salinity. These fish had species has not been investigated before in been acclimatized to a high (30 PSU) or a low this area. The methods with which I aimed to (0.1 PSU) salinity, resulting in four groups of achieve this were through a genetic analysis fish. Comparison between groups allowed and behavioral experiments. for analysis of the general activity, as measured by time spent moving over time, By producing Genotyping-By- depending on which salinity they had been Sequencing (GBS) data (as described by acclimatized to, between and within location Elshire et al. 2011) from two localities in of capture. My assumption was that fish Gothenburg and four other populations in caught in a relatively low salinity would show Europe, representing habitats of various a higher level of activity when acclimatized salinities, a bioinformatic analysis of the to a low salinity rather a high salinity due to genetic data could establish if there are the extra cost of osmoregulation and vice certain genotypes associated with either low versa. The same logic was applied to a or high salinity. If these exist it would suggest

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salinity choice test where a choice between preference correlated with point of high and low salinity represented the salinity capture it suggests a lack of genotypic where they had been caught and the salinity sorting. It also suggests that the at which they have been acclimatized to. The round goby has a tolerance to high activity and choice of salinity could indicate and low salinity through a if the coping mechanism to salinity is a phenotypically plastic response. genetic or phenotypic one. If both activity and choice are correlated to location rather Method than to which salinity they have been acclimatized to it would suggest the round Fish sampling, care and acclimatization goby seeks out a certain salinity. However, if Specimens of round goby were caught in the no correlation is found, it would suggest that river and river mouth of Göta älv in the city it is more likely that the round goby is able to of Gothenburg (57°42′25″N 11°57′59″E) on cope in both a high and low salinity due the west coast of Sweden. Since no study on plasticity. The information from these results round goby has previously been conducted will also be valuable upon interpretation of in the Gothenburg area, the extent of its the results from the genetic study in the near spread was not fully known. As such, eleven future. locations around Gothenburg of various salinities were fished at, four of which The two main hypotheses of the study yielded fish (Fig. 1). are the following: Fish from two of the locations were 1. Fish from a high saline point of used in the behavioral studies and a third capture should a) choose a high one was sampled for data collection in the salinity over a low salinity when form of fins clips (DNA sampling), weight, presented with the choice and b) length and sex recordings. show a higher level of activity when acclimatized to a high salinity than to The first location was Röda Sten a low salinity (vice versa for fish from (57°41'22.5"N 11°54'05.2"E) with an average a low saline point of capture). This salinity of 8.6 PSU. Specimens at this site could indicate that fish from the two were caught on the 26th and 27th of locations are adapted to a lower and September 2016. The second location, Volvo higher salinity, suggesting individuals Museum (57°41'41.1"N 11°48'57.5"E), had in the Gothenburg harbor might an average salinity of 24.3 PSU with differ genetically due to a genetic measurements reaching as high as 29.5 PSU. sorting event. Specimens at this site were caught on the 6th, 2. If the round goby does not show 10th, 14th and more activity or a salinity-based

20th of October 2016. No fish from the third 11°55'39.5"E), were kept alive, but caudal location, Sannegårdshamnen (57°42'10.4"N fins were sampled and the length, weight

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Figure 1. Map of all sites fished at for this study. The green arrows indicate spots which yielded fish and red where no fish was caught. The numbers inside the arrows on the map correspond to a number in Table 1 with more detailed information on each site.

and sex were recorded the same day they set at 11 °C. Before being transferred to the were caught, the 3th of September 2016. holding tanks (120 x 35 x 55 cm) of about 210 Sannegårdshamnen had an average PSU of liters, the fish were kept in water with a 5.2. All salinity measurements (Table 1 and 2) salinity of <30 PSU for 2 min and then in were taken from water close to bottom of freshwater for 30 s, to attempt to eliminate the areas (roughly 3-4 m below the potential parasites attached to the fish which surface) and measured with a Hach HQ30d would not survive such a high/low salinity. multimeter. The bottom surface of the holding tanks were filled with gravel as substratum with Fish were caught using hook and line, halved clay pots and stones to create a with frozen shrimp used as bait for both habitat featuring multiple hiding spots for methods. Live fish were transported in a the fish. A total of 12 tanks were set up in cooling box filled with water from the four sets, with three tanks in each set. In a sampling site to aquaria set up in the closed system, two of the three tanks hosted facility in the Zoology building at 7-10 fish, and were fitted with a foam filter the Department of Biological and with air driven circulation along with an air Environmental Sciences, University of stone. The third tank, placed beneath the Gothenburg. At arrival, the fish were briefly other two, acted as a sump, hosting a filter kept in buckets with air stones to acclimate which water from the above tanks flowed them to the temperature of the through before reaching the pump, environmentally controlled room, which was

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Table 1. Detailed information on each of the sampled location. Each number corresponds to a spot marked on the map in Figure 1.

circulating it to the top tank where it reached installed on the ceiling 2.4 m up in the middle a Vecton V2 600 Ultraviolet Water Steriliser of the room. Fish were feed once a day, 7 fitted with a 25W G25T8 UV Lamp to further days a week. During weekdays, they were clean the water Fig. 2). Water in the bottom fed 5 g of 2 mm fish pellets and one 3-g cube tank was replaced every week and filters of Ocean Nutrition Krill Pacifica and during were cleaned in order to keep nutrients from weekends they were fed 3 g of pellets. Tanks accumulating to hazardous levels. Water were cleaned for leftover food every temperature was determined by the air weekday. The water in the aquaria was temperature of the room, which as gradually changed (lowered or raised) to mentioned was set to 11 °C. The light cycle different salinities, creating four different was set to a 12-hour-light: 12-hour-dark treatments groups in total. One group of fish photoperiod with 4 fluorescent lights from the low salinity environment of Röda

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Table 2. Summary of the salinity measurements for the sampled sites, the date they were taken and their calculated average.

Sten was ramped to 30 PSU (High Salinity, aquaria was gradually changed (lowered or HS) and another group to 0.1 PSU (i.e. the raised) to different salinities, creating four salinity of the tap water at the facility) (Low different treatments groups in total. One Salinity, LS). The same was done for the fish group of fish from the low salinity from the higher salinity locality Volvo environment of Röda Sten was ramped to 30 museum creating the following groups: Röda PSU (High Salinity, HS) and another group to Sten HS (N = 18), Volvo Museum HS (N = 15), 0.1 PSU (i.e. the salinity of the tap water at Röda Sten LS (N = 18) and Volvo Museum LS the facility) (Low Salinity, LS). The same was (N = 15). Before the salinity of the water was done for the fish from the higher salinity changed to these two different treatments, locality Volvo museum creating the following fish from both localities were first kept at a groups: Röda Sten HS (N = 18), Volvo salinity akin to salinity where they were Museum HS (N = 15), Röda Sten LS (N = 18) caught, then the salinity was ramped to 15 and Volvo Museum LS (N = 15). Before the PSU for all fish and allowed to acclimatize, salinity of the water was changed to these before the final four groups were created. All two different treatments, fish from both salinity changes occurred with a maximum of localities were first kept at a salinity akin to 1 PSU a day, meaning a change to 30 or 0.1 salinity where they were caught, then the PSU took around 15 days at the minimum to salinity was ramped to 15 PSU for all fish and complete. Aquaforest Sea Salt mixed with allowed to acclimatize, before the final four tap water from a non-metallic pipe (thus free groups were created. All salinity changes from metal ions that could poison the fish) occurred with a maximum of 1 PSU a day, was used in all the tanks. The water in the meaning a change to 30 or 0.1 PSU took

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Figure 2. Illustration of the aquaria set up during storage of the fish at the Zoology building at the Department of Biological and Environmental Sciences, University of Gothenburg. Yellow arrows indicate water flow and the green boxes foam filters. around 15 days at the minimum to complete. HSM lens used at 20 mm) was set up 2 Aquaforest Sea Salt mixed with tap water meters above the tanks to give a clear top from a non-metallic pipe (thus free from down view of the fish inside the tanks (Fig. metal ions that could poison the fish) was 3). Recordings were shot in a resolution of used in all the tanks. 1080p at 25 frames per second.

Behavioral Experiment 1: Activity Behavioral Experiment 2: Salinity Choice In two rounds, 12 round gobies from each Six clear plastic tanks (47 x 26 x 22 cm) were location and treatment were filmed for 10 set up at an angel of roughly 15° to create a min (creating 20 min of recorded video for slope, enabling a halocline to form inside the each group of 12 individuals) and analyzed tanks. This was achieved by first adding 8 with the tracking software EthoVision XT12 liters of water of 25 PSU into the tank and (Noldus Inc., Netherlands). Sets of six glass then slowly (to avoid mixing) adding 8 more aquaria were filmed together, with one fish liters of freshwater. The angled plastic tank per aquarium, each one filled with water thus had a higher salinity at the bottom of from the storage aquarium the fish was the tank and a lower one at the top due to taken from. The sides of the aquaria were the heavier density of salt. Before forming covered to ensure no fish could observe the halocline, one PVC pipe with a suction other fish. A digital video camera (Canon 60D fitted with a Sigma 10-20 mm 1:4-5.6 DC

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Figure 3. Snapshot from the video recording of the activity experiment, showing the cameras point-of-view of the six tanks. cup was fitted at each end of the tanks in measurements in total). Fish from all four order to create two hiding spots at different treatment groups were tested (N = 48; 12 per salinities (Fig. 4). Water temperature and group). A control test with no halocline, in salinity of the top and bottom areas were water of the same salinity, was conducted to recorded before fish were put in and at the determine to what extent fish chose depth of end of the experiment, using a Hach HQ30d water, rather than salinity. In this case, only multimeter. The measurements were taken the 24 individuals that were kept in 0.1 PSU from just outside the PVC pipes (i.e. the were used, with 0.1 PSU as test water. This hiding spot). Average salinity inside the tank was not done with fish from the HS over the 60 min experimental runs was: Top, treatment since fish from that treatment 10.4 ± 0.5 PSU; Bottom, 25.6 ± 0.7 were used in other experiments and were PSU.Average temperature inside the tank at not freely available at the time. the two hiding spots was: Top, 13.0°C ± 0.4°C; Bottom, 13.5°C ± 0.3°C. Fish were Statistics & null hypothesis After 7 outliers and 5 fish whose sex could added to the aquaria by being dropped not be determined were removed, 35 of the through a plastic pipe placed in the middle of 48 recorded individuals were included in the the tank, to minimize the mix of water when analyses. Statistical analyses were carried releasing the fish. The salinity choice was out using IBM SPSS Statistics 22. measured as the position of the fish, in either high or low salinity, recorded at the start and The activity, measured as the log every 10 minutes for 60 minutes (7 transformed total time (to normalize data)

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Figure 4. Illustration of the tank used in the salinity choice experiment. The tank was set up at a 15° angel so that a slope was formed allowing the more dense, high saline water to settle at the bottom of the tank and keep the less dense, low saline water at the top creating a halocline inside the tank (illustrated here through the dark and light blue water). The two gray circles represent the two PVC-pipes acting as hiding spots on the bottom and top of the tank. the fish spent moving during 20 min of The salinity preference, as measured by recorded and analyzed observation, was the proportion time spent in the bottom tested with the following null hypotheses hiding spot (i.e. high salinity), was tested through a three-way analysis of variance between the four groups with a Scheirer- (ANOVA): Ray-Hare Test (Ennos, 2012) with the follow null-hypothesis: 1. There is no significant difference between any of the main effects (i.e. 1. There is no significant difference sex, treatment and location of between any of the main effects (i.e. capture). treatment and location) 2. There are no significant 2-way 2. There is no significant 2-way interactions between the main effects interaction between the main effects (treatment x location, treatment x (treatment X location). sex, location x sex) 3. There is no significant 3-Way The preference of hiding spot (i.e. depth) interaction between the main effects between the control groups was tested (treatment x location x sex). with a Mann-Whitney U test with following null-hypothesis: During calculations, all non-significant (P > 0.05) interactions were sequentially 1. There is no significant difference removed, starting from the highest order. between the two locations in preference of hiding spot.

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A Scheirer-Ray-Hare Test and a Mann- put into a new Eppendorf tube and stored in Whitney U test are the non-parametric 99.7 % ethanol until extractions took place. version of a two-way ANOVA and two- Extractions occurred according to the sample t-test (Ennos, 2012) and was applied manufacturers’ protocol with small due to data being heavily skewed to the right adjustments. Briefly, each sample was air and thus non-normally distributed. dried and put inside an Eppendorf tube to During calculations, all non- which 180 μl of Buffer ATL and 20 μl of significant interactions were removed Proteinase K were pipetted. These were (ruled under confidence limit of 0.05). immediately mixed for 15 seconds before being put into an incubator (ThermoStat DNA Extractions Plus, Eppendorf, Hamburg, Germany) at 56 Caudal fins sampled from fish from two °C for 4 hours (15-second mixings using an localities in Gothenburg, Sannegårdshamnen IKA® MS1 Shaker (IKA, Staufen, Germany) and Röda Sten, were used for genetic occurred at 1h 15 min and 2h 25 min). After extractions. Caudal fin sampling of Volvo incubation, samples were mixed again and Museum individuals first occurred after the 200 μl of Buffer AL and 99.7% ethanol was behavioral experiments were conducted due added and the sample was mixed again. This to low number of fish from this site. As there mixture was pipetted into a DNeasy Mini where there was a surplus available from Spin Column placed in a collection tube and Röda Sten there was enough to sample and centrifuged at 8000 rpm for 1 min after use in the experiments. Caudal fins which the collection tube was emptied and previously collected from the following four 500 μl Buffer AW1 added to the top of the locations were also extracted: Travemünde DNeasy Mini Spin Column and centrifuged at (53°53'44.4"N 10°47'52.7"E, average PSU: the same setting again. The collection tube 11.9), the rivers Rhine (51°51'39.2"N was then discarded and a new one put into 6°04'17.6"E, average PSU: 0) and Elbe place and 500 μl of Buffer AW2 pipetted into (53°32'56.9"N 9°59'10.3"E, average PSU 0) spin columns and centrifuged at 14 000 rpm all three of which are situated in Germany, for 3 min. The spin column was placed in a and the Danish strait Gullborgsund new Eppendorf tube and 200 μl Buffer AE (54°51'21.1"N 11°44'59.0"E, average PSU: was added and centrifuged at 8000 rpm for 1 12.9). min again. This step was repeated in a new Before DNA was extracted, fin-clips Eppendorf tube to create two elutions of that had been stored in ethanol in Eppendorf DNA. tubes were air dried and then weighed to DNA Concentrations ensure no fin sample exceeded 250 μg (as Since the GBS method requires 100 ng of specified in the manufacturers protocol, each sample, all extracted DNA sample Qiagen DNeasy Blood & Tissue Kit) or was concentrations needed to be checked below 100 μg. After weighing, fins were cut to ensure there was a) enough DNA in each into small pieces with sterilized scissors and extracted sample and b) to what level samples needed to be diluted to. Thus, the

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concentration of all the samples was Results measured with a NanoDrop 2000 (Thermo Fisher Scientific, Waltham, USA). However, Behavioral Experiment 1: Activity due to the risk of overestimating the actual Activity, measured as time spent moving concentration, a more sensitive machine was during the 20 min recorded, did not differ employed (Qubit Flourometer 3.0, Thermo significantly between locations, treatments Fisher Scientific, Waltham, USA) in order to or sex. There was, however, a weak trend double check the concentrations. By towards fish in low salinity being more active sampling six different samples and (Three-Way ANOVA: Location: F1,31 = 1.96, P measuring their concentration with a Qubit = 0.17; Treatment = F1,31 = 3.13, P = 0.08; Sex dsDNA HS Assay Kit (allowing quantification = F1,31 < 0.01, P = 0.94) (Fig. 5 and Fig. 6). All between 0.2 - 100 ng DNA) and comparing interactions were non-significant (all p > the concentrations between the two 0.05) and were thus removed from the machines, a factor by which the NanoDrop model. 2000 overestimated the concentrations Behavioral Experiment 2: Salinity could be calculated. This was then used to Preference calculate the estimates of all the other Salinity preference, as measured by the samples. With the estimated concentrations, proportion time spent in the high salinity the dilution to get 100 ng DNA per sample (bottom) part of the tank over the course of could be done. 60 mins, did not differ significantly between location or treatment (Scheirer-Ray-Hare Time Moving (log sec)

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1.5

1 Male (n = 19) min

time moving during 20 during time moving Female (n = 13)

- 0.5

0 Mean log Mean Low (n = 16) High (n = 19) Salinity

Figure 5. Graph shows the activity difference, as measured by the log-transformed means of the time spent moving during 20 min (±SE), between the Low and High salinity treatments divided by sex.

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Time Moving (log sec) 2

1.5

RS (n = 19) 1 Röda Sten (n =19) VM (n = 16)

0.5

0

Mean time moving during 20 min 20 during time moving Mean Low (n = 16) High (n = 19) Salinity Figure 6. Graph shows the activity difference, as measured by the log-transformed means of the time spent moving during 20 min (±SE), between the Low and High salinity treatments of both Röda Sten (RS) and Volvo Museum (VM). Test: Location: χ² (df = 1, N = 45) = 0.9, P > not differ significantly between locations in 0.05; Treatment = χ² (df = 1, N = 45) = 0.9, P the control group either (Mann-Whitney U: > 0.05)(Fig. 7). The interaction between the Location: U12,12 = 53, P = 0.17) (Fig. 8). two main effects was non-significant and Figure 7 shows the proportion time removed from the model. over 60 min that the fish of the four groups The preference of hiding spot, as spent in the top versus bottom hiding spot measured by proportion time spent in the (i.e. low or high salinity). Figure 8 shows the bottom (deeper) versus the top (shallow) same data but for the control groups (i.e. no hiding spot over the course of 60 mins, did salinity gradient) for the two locations and

Proportion time spent in Bottom vs Top 100%

80%

60%

40% Time in Top Time in Bottom 20%

Propotion time out of 60 min 60outof time Propotion 0% VM LS (n=12) VM HS (n=9) RS LS (n=12) RS HS (n=12) Location & Treatment Figure 7. Graph showing the average proportion time (±SE) spent in the bottom versus the top of the tank between the four different groups: Röda Sten Low Salinity (RS LS), Röda Sten High Salinity (RS HS), Volvo Museum Low Salinity (VM LS) and Volvo Museum High Salinity (VM HS).

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Proportion time spent in Bottom vs Top (Control)

100%

80%

60%

40% Time in Top Time in Bottom 20%

0%

Proportion time out of 60 min60 of out time Proportion VM (n = 12) RS (n = 12) Location

Figure 8. Graph showing the average proportion time (±SE) spent in the bottom versus top of the tank between for the two control groups: Volvo Museum (VM. High saline environment) and Röda Sten (RS. Low saline environment).

Table 3. The average time, over 60 min, the fish from the four groups and the two controls spent in the bottom (high salinity) hiding spot and Top (low salinity) hiding spot (Fig 7 & 8).

Table 3 shows the average time each group expressed the same preference for salinity spent in the Top and Bottom. regardless of where they were captured, or which salinity they had been acclimatized to, Discussion all choosing to stay in the high salinity when given the choice. This study found that activity level in round Studies have shown decreased goby from two different localities in physiological performance (reduced growth, Gothenburg was not affected by location, sex swimming performance, competitive ability) or acclimatization to high or low salinity. Still, in round gobies kept in salinities above 25 a non-significant trend indicated that PSU (Behrens, van Deurs and Christensen, low salinity treated individuals from both 2017; Hempel and Theil, 2015). However, the locations were more active. The round goby round gobies in this study, regardless of

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recent experience to higher salinities (i.e. careful and avoid being caught, move into slow salinity ramping and long deeper depths or new environments acclimatization period), were not adversely (preventing capture with the fishing affected, at least not expressed as reduced methods used in this study). activity. This result indicates that round The prediction that gobies should gobies from Röda Sten and Volvo Museum prefer a salinity similar to their 'home are not likely genetically adapted to specific salinity' does not find support in the salinity salinities. If so, we would expect individuals preference experiment, as Röda Sten to be more active in the salinity individuals did not differ in the proportion corresponding to their location (i.e. RS LS and time they spent in the high salinity water in VM HS individuals to be more active than RS the bottom of the choice tank, compared to HS and VM LS). My results instead suggest individuals from Volvo Museum. However, that a plastic response to a wide range of as seen in the control group, even with no salinity stress enables round goby to salinity cline present, the round gobies maintain the same level of activity across the exhibited a preference for the bottom board. Plastic responses to alleviate section of the tank. The preference to hide in environmental conditions have been the bottom might not then be due to a previously reported in the round goby, as preference of high salinity, but more likely transcriptional plasticity in individuals from one of depth. The round goby is a benthic the Great Lakes has been shown to improve fish species (Kornis, 2012) and might be temperate tolerance (Wellband and Heath, more inclined to hide in deeper water, thus 2017). showing a higher preference for the bottom Activity levels have also been hiding spot. This is a constraint in the linked to the age of the population in round experimental design, which is based upon gobies (Thorlacius et al., 2013), with salt being denser than water, meaning that individuals from newer population being the high salinity will always be at the deeper more active than older ones. The fact that I end. Many more refined salinity choice set found no difference in activity between the ups exist (e.g. Jutfelt et al., 2016) and populations therefore suggests that the sub- adjustments to create two separate tanks populations at Volvo Museum and Röda Sten with bodies of water at a stable low and high are likely to be of similar age. This notion is salinity and a pass through for the fish would to some extent supported by findings from be more accommodating to round gobies, an analysis of individuals from Röda such as the automated salinity choice system Sten and Volvo Museum by MSc student used by Serrano et.al. (2010). It might also be Andrew Holmes (2017, pers. Comm., 26 more relevant to test the salinity choice in May). He found Röda Sten and Volvo round goby larvae. Fish larvae have been Museum individuals to generally be of 2-3 shown in other species to recognize water years of age. However, this age structure that is either of riverine or estuarine origin could also suggest that fish do not survive (James et al., 2008). Since its been shown beyond this age, older individuals are more and generally assumed it is in this life stage

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round goby enters into ballast water to Sannegårdshamnen (catch per hour = 4, (Hensler and Jude, 2007; Kornis et al., 2012), Table 1) and 5 times lower than Röda Sten understanding larvae salinity preference (catch per hour = 10, Table 1) indicating based upon origin would provide further these two brackish areas have a higher insight into the feasibility of a genetic sorting abundance of round gobies. So, individuals occurring when round goby larvae arrive seeking habitats further out toward the through ballast water to environments ocean in the Gothenburg area are likely to featuring both high and low salinities. experience less intraspecific competition. However, at the Volvo Museum site there It is puzzling that no round gobies, might instead be more interspecific except for a single individual, were caught in competition. Fishing at this site revealed a the surveyed freshwater locations in the higher degree of biodiversity despite fishing Gothenburg area (Table 1.) Their tolerance with techniques specifically to catch round to freshwater is well established (Behrens, goby. More of the native black goby ( van Deurs and Christensen, 2017; Hempel niger) was caught accidentally than at any and Theil, 2015; Karsiotis, 2012; Ellis and other site and other species including MacIsaac, 2009), as is there their generalist goldsinny wrasse (Ctenolabrus rupestris), behavior to food and habitat (Jakubčinová et green shore crab (Carcinus maenas), al., 2017; Nurkse et al., 2015). Yet the eight european flounder (Platichthys flesus), freshwater sampled sites in the Gothenburg european plaice (Pleuronects platessa), area yielded just one single fish. This lack of atlantic (Gadus morhua) and european freshwater round gobies in Gothenburg but (Pollachius pollachius) were also the occurrence of a wild deme in near caught. The “biotic resistance hypothesis” oceanic conditions could mean the source (Levin and Adler, 2004) states that more population to Gothenburg is not freshwater diversity should increase the resistance to adapted. Arriving larvae would thus not seek invasion, since competition is higher and themselves to these areas upon adult fish thus colonizers have fewer resources would do worse in these conditions, limiting available to them (Levine and D’ Antonio, colonization to brackish to high saline 1999). Kalgoriou (2011) found potential waters. This would also give the prediction support for this hypothesis. He found sandy that activity levels to be equal across points and less diverse bottom habitats hosted of capture, as was found from the results. more non-indigenous species compared to Fish at the Volvo Museum location Posidonia oceanica (Mediterranean might represent a population formed by tapweed) meadows with higher levels of front range expanding individuals. One of the biodiversity. Zostera marina (common prerequisites for individuals to expand eelgrass) in Skagerrak (north of Kattegat) has further out from a source population is the been in decline with loss of meadows occurrence of less intraspecific competition impacting biodiversity negatively (Nyqvist et (Azour et al., 2015). Only 33 individuals were al., 2009). These areas are geographically caught at Volvo Museum with a catch per close to Gothenburg and could be hour at two fish. This is half as high compared subjugated and more sensitive to round goby

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invasions if eelgrass meadows continue to the potential that the species will be able to decline. In experimental and field studies on spread to the North Sea, unless hindered by , increased diversity was also other factors than salinity. Future shown to reduce survival and decrease management should consider that oceanic settlement indirectly of invaders, an affect salinity conditions may not necessarily be an attributed to the reduction in availability of efficient natural boundary for round goby open space (Stachowicz et al., 2002). The establishment. The local round goby range higher salinity at Volvo Museum might also also suggests that front range dispersers will affect reproductive success. Experiments on experience less intraspecific competition, the (Pomatoschistus minutus) which could be a contributing factor to why showed males had significantly lower the round goby would continue to expand reproductive success (spawning and into more oceanic conditions. The resilience hatching success), in non-native salinities. of a high biodiversity ecosystem might help This was presumably due to increased limit their establishment, highlighting the developmental failure of fertilized eggs or importance of ecosystem stability and the due to sperm being damaged when exposed importance of maintaining them. Having to non-native salinities, despite adults doing already shown successful spread and no well physically otherwise (C. Kvarnemo, signs of stopping, the round goby is likely to personal communication) join the ranks of species such as the pacific oyster (Crassostera gigas) and comb jelly, The round gobies' ability to handle Mnemiopsis leidyi in the public’s growing salinity is indicated by this study to be a awareness of invasive species at the Swedish tolerance stemming from plasticity. The west coast. occurrence of a genetic sorting is not therefore supported. However, the lack of Acknowledgements gobies found in freshwater systems and a wild population in salinities reaching oceanic conditions suggest the Gothenburg Special thanks to Andrew Holmes, Charlotta population might originate from a genotype Kvarnemo and Leon Green. I also want to adapted to brackish salinities, and that fish give thanks to Oliver Englund Örn, Alexander from a freshwater origin is simply not Hansson, Angela Pauliny, Mårten Klinth, present in the harbor. Future genetic work Svante Martinsson, Linda Hasselberg Frank, will help to sort this out. Volvo Museum, a Magnus Lovén Wallerius, Linda Werme, location with salinity recordings of up to 29.5 Sportfiskarna, Erica Leader & Jarl Andreas PSU, represents a wild population showing Amarkund at UiO.

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