Rutilus Rutilus) and Ide (Leuciscus Idus) in the Stream Oknebäcken, Sweden

Bachelor thesis

Phenotypic correlates of spawning migration behaviour for roach (Rutilus rutilus) and ide (Leuciscus idus) in the stream Oknebäcken, Sweden.

Author: Emma Lindbladh & Johanna Eriksson Supervisor: Anders Forsman Co-supervisor: Carl Tamario Examiner: Jarone Pinhassi Term: VT2020 Subject: Biology Level: Bachelor Course code: 2BIO1E

Abstract Migration occurs among many animal species for the purpose of, among other things, finding food or to reproduce. Spawning migration is a form of migration that occurs among many fish species where they move to another site for reproduction. The movement can be obstructed by migration barriers like road culverts. Barriers to migration pose one of the greatest threats to biodiversity and ecosystem functions in freshwater. They impair the connectivity of watercourses and may prevent fish from improving reproductive success or completing their life histories altogether. There are both benefits and costs with migration, benefits such as increased survival for the adults and offspring, and costs such as increased energy consumption and increased mortality. The costs are often dependent on the morphological traits of the individual, like body shape and size. In this study, the spawning migration of two species of fish of the family Cyprinidae, ide (Leuciscus idus) and roach (Rutilus rutilus) was investigated. Few studies have been made on ide or on roach compared to other cyprinids and salmonids. This study might therefore enhance the overall knowledge of these two species. The overall aims of this project are to study and compare phenotypic correlates of spawning migration behaviour of ide and roach. The field studies were performed in Oknebäcken, Mönsterås (SE632310-152985), Sweden in March and April 2020. To describe the watercourse and define the location and characteristics of different potential migration barriers, a simplified biotope mapping method was used. The fish were caught in a hoop net and then measured, weighted, sexed, and injected with passive integrated transponder using the bevel down method. In order to register in stream movement of fish, reading stations with antennas were placed, at two locations upstream from the marking station and one downstream at the estuary. The sex ratio differed from the expected 1:1 with a majority of females for both species. This might be a result of fluctuations in survival of spawn coupled with different age-at-maturity between sexes. We found that individuals that arrived early to the stream were larger for both study species, as other studies also reported. Also, male ide was both larger and arrived before female ide. There might be an energy cost associated with early arrival to the stream and therefore, larger individuals arrive first. For roach, there was no difference in arrival time between the sexes although female roach were larger. There was no difference in the time spent in the stream between the species. For ide, females stayed for a longer period of time in the stream than males. However, the opposite was true for roach. This may be because male roach might benefit from more fertilization events when staying longer. There might therefore be a trade-off between the energy cost in staying in the stream and the increased fitness advantage in fertilization events. We found no correlation between any of the morphological traits and migration distance. However, since very few individuals were registered at the upstream reading stations, there might be an effect of migration barriers on the spawning migration. The mortality after spawning was higher for roach than for ide. For ide, a larger proportion of females than males died. For roach, individuals that arrived early was

classified as alive to a greater extent than those who arrived late. Both similarities and differences between the species were discovered in this study which concludes that even closely related species might differ substantially from each other.

Key words Anadromous, cyprinids, diadromous, fish migration, ide, leuciscus idus, migration barriers, PIT-tag, roach, rutilus rutilus, sex ratio, spawning migration, Sweden.

Acknowledgments We want to thank Hanna Berggren and Petter Tibblin for help with the field work. Oscar Nordahl for instructions for PIT-tagging methods, expertise, and tips in general and help with the initial fieldwork setup. Kristofer Bergström for setup of reading stations and antennas. A big thank you to Carl Tamario, who has been with us all the way and have supported us in field work, the data management, and the writing of the manuscript. Carl worked hard with marking of the fish when we could not be present, and he also managed the antennas and collected the data from the reading stations. Finally, a thank you to our supervisor, Anders Forsman, who have showed us in the right direction, helping us interpret data and giving us comments on the manuscript.

Table of contents 1 Introduction 6 1.1 Description of study species 7 1.2 Aims and hypotheses 9 2 Methodology 11 2.1 Study area 11 2.2 Ethical permit 12 2.3 Catching of fish 12 2.4 Phenotypic measurements 12 2.5 Marking of individuals using PIT-tags 13 2.6 Reading stations 14 2.7 Statistics and calculations 15 2.7.1 Sex ratio 15 2.7.2 Body condition 15 2.7.3 Associations of arrival time with sex and morphological traits 16 2.7.4 Time spent in the stream 16 2.7.5 Migration distance 16 2.7.6 Mortality 16 3 Results 17 3.1 Biotope mapping of the stream 17 3.2 Sex ratio 18 3.3 Body condition 18 3.4 Temporal distribution of arrival 19 3.5 Associations of arrival time with morphological traits 19 3.5.2 Roach 20 3.5.3 Ide 21 3.6 Time spent in the stream 24 3.6.1 Roach 24 3.6.2 Ide 25 3.7 Migration distance 26 3.7.1 Roach 26 3.7.2 Ide 26 3.8 Mortality 27 3.8.1 Roach 27 3.8.2 Ide 28 4 Discussion 31 4.1 Body condition 31 4.2 Biotope mapping 31 4.3 Migration distance and phenotypic traits 32 4.4 Sex ratio 33 4.5 Dead or not registered? 34 4.6 Arrival time and time spent in the stream 35 5 Conclusion and suggestions for the future 37 6 Author contributions 38

7 References 39

Appendices Appendix 1: Biotope mapping report, part 1 Appendix 2: Biotope mapping report, part 2

1 Introduction Migration is a phenomenon that can be seen in a number of different animal taxa such as birds, insects, mammals and fish. To distinguish migration from dispersal, migration is periodical recurring movement between two distinctly different habitats and back again (Brönmark et al., 2014). Animals migrate for different reasons, for instance to find food, to reproduce or to avoid predators. Migrations can be annual as for birds that fly south in the winter, monarch butterflies that make long journeys to reproduce or caribou that lives in the sheltering forests during winter and up in the bare mountain fields during summer. There are also animals that make daily migrations. Zooplankton perform diel vertical migration (DVM) from the surface water, where they forage during night, to the profundal zone, were the darkness protects them against predators during the day (Hansson & Hylander, 2008). Migration comes with both benefits and costs. Some of the potential benefits are increased survival for the offspring, and enhanced growth and survival for both the adults and the offspring. Costs of migration include the energy consumption of the actual movement and an increased mortality as a consequence of being more exposed to predators. These costs often depend on the size and state of the individual (Haugen et al., 2008; Brönmark et al., 2014). For fish, the shape is an important factor, for example. A more streamlined body lowers the cost of swimming and has been shown to be correlated with long and challenging migrations in salmonids (Crossin et al., 2004; Fraser & Bernatchez, 2005; Fraser et al., 2007). The correlation between migration and body shape has also been studied in cyprinids, a study by Chapman and colleges (2015) found that body shape differed between individuals in a population of roach were partial migration occurred. Individuals that migrated had a more slender and shallow body shape than non-migrating individuals. Migration barriers pose one of the greatest threats to biodiversity and ecosystem functions in freshwater (Dudgeon et al., 2006). They impair the connectivity of watercourses and prevent fish from migrating upstream and downstream (Winemiller et al., 2016), leading to a reduction in the exchange of genes between populations (Raeymaekers et al., 2008; Wofford, Gresswell & Banks, 2005). Reduced genetic variation can lead to inbreeding and make the population more sensitive to environmental changes and pathogens. This may affect the whole population and possibly lead to extinction of species, which in turn would alter the species composition and potentially disrupt entire ecosystems (Tamario et al., 2019). Barriers also change the water flow, the transport of sediment and the aquatic habitat (Bunn & Arthington, 2002). Many fish species perform spawning migration, which is a well-studied phenomenon in salmonids (Brönmark et al., 2014). However, many other fish species migrate to find a suitable habitat for their offspring (Calles & Greenberg, 2007; Brönmark et al., 2014). Changes in the spawning-grounds used by fish and the addition of barriers such as culverts and dams make it difficult for the fish to

access the spawning-grounds and may thus impair their reproductive success (Tamario et al., 2019). Road culverts can pose a form of migration barrier. Displaced culverts can create high drops from the mouth of the pipe down to the surface of the watercourse, which is also dependent upon the water level that can make the fall more or less high. There might also be drops underneath the surface if the pipe is not placed on the bottom (Larsson, 2005). Both high and low water flows can cause the culvert to become a barrier. High water flow leads to high water velocities inside the pipe and low water flows can lead to low water levels inside the pipe or high drops at the outlet. At high water velocity the fish must put in a great swimming effort to be able to swim all the way through the pipe (Larsson, 2005). The velocity of the water also has an impact upon the bottom substratum of the pipe, high water flow may flush away all-natural substratum, if existent. Low water levels can have negative impact on fish, especially larger fish. If the fish is not completely submerged, the swimming ability is impaired resulting in energy loss and, if the gills are not covered by water, the fish will not be sufficiently oxygenated also resulting in poor swimming capacity and fatigue (Larsson, 2005). If water levels are low, this might increase the risk of injuries from obstacles on the bottom and the risk of predation. A natural bottom substratum inside the pipe creates “backwaters” behind boulders where water velocity is lower, for the fish to rest in. It also creates a more defined turbulent watercourse where it is easier for the fish to swim (Larsson, 2005). Some individuals may find it more difficult to get past migration barriers, which can then lead to a selection of certain phenotypes (Chapman et al., 2015). Larger and more stout-bodied individuals can have a harder time moving past barriers when water levels are low, as mentioned above. This could mean that large females with more and potentially higher-quality eggs cannot reach spawning-grounds in time, or even at all, and thus cannot reproduce. A study on brown trout (Haugen et al., 2008) showed migration barriers can select for different sizes. A fish ladder selected for medium size, it was more difficult for both larger and smaller individuals to get past. Another study by Calles & Greenberg (2007) showed that those who successfully passed nature-like fishways where for the most part larger individuals among 15 species of fish.

1.1 Description of study species In this study, two species of fish of the family Cyprinidae, ide (Leuciscus idus) and roach (Rutilus rutilus) were investigated. A literature search was performed on Web of science (2020-05-28), to explore how many studies that have been published on these two species. The search was made in “all databases” and in “Topic”. The results are shown in Table 1. These results indicate that not many studies have been made on ide or on roach compared to other cyprinids and salmonids. Therefore, we believe that it would be interesting to study these two species further and hopefully contribute with more information to the research field and expand the knowledge about spawning migration in cyprinids. There are advantages of studying more than one species because reoccurring patterns between species can then be identified.

Table 1: Results from a literature search performed in Web of Science (2020-05-28). The search was made in “All databases” and in “topic.

Search phrase Results Search phrase Results Search phrase Results “Leuciscus idus” 355 “Leuciscus idus” AND spawn* 26 “Leuciscus idus” AND migrat* 37

“Rutilus rutilus” 3603 “Rutilus” AND spawn* 256 “Rutilus rutilus” AND migrat* 276

Cyprinid* 47 955 Cyprinid* AND spawn* 2473 Cyprinid* AND migrat* 1534

Salmonid* 56 539 Salmonid* AND spawn* 5857 Salmonid* AND migrat* 6386

Both ide and roach are considered of least concern on the IUCN (International union for conservation of nature) red list of threatened species and have a native range covering large parts of Europe (Freyhof & Kottelat 2008). Both species live in fresh and brackish water and have their spawning areas in freshwater to which they migrate in spring. Both the populations of ide and roach in this study are anadromous, which means that they migrate from the brackish Baltic Sea to the freshwater in Oknebäcken to spawn. Both species are iteroparous, meaning that they spawn more than once in their lifetime (Winter & Fredrich, 2003; Kortet et al., 2004 b). Ide (L. idus) is the larger species of the two and can be up to 100 cm, but the average size is between 30-55 cm (Kullander & Delling, 2012). In Sweden, individuals can weigh up to 4 kg (Curry-Lindahl, 1985). They can become 10 to 15 years old and usually become sexually mature after 6 to 7 years. Females can lay up to 200 000 eggs that are sticky and easily attach to different substrates (Curry- Lindahl, 1985; Kullander & Delling, 2012). Eggs hatch after 2-4 week depending on the water temperature (Cala, 1970; Curry-Lindahl, 1985; Kullander & Delling, 2012). Ide usually migrates to spawn from the brackish water of the Baltic Sea into freshwater streams in April-May, sometimes in March or June. The spawning is preceded by a simultaneous migration of a larger number of fish, which can easily be seen from land in smaller streams. The spawning act takes place in shallow stream sections over gravel or grass bottoms (Kullander & Delling, 2012). Roach (R. rutilus) is generally smaller than the ide. A roach can be up to 50 cm long, but generally falls in the range of 15 to 30 cm (Kullander & Delling, 2012). Roach often weigh under 0,5 kg but can weigh as much as 1,4 kg in Sweden (Curry- Lindahl, 1985). They become sexually mature at an age of 3 to 5 years (Curry- Lindahl, 1985) and they can live for 5 to 15 years (Kullander & Delling, 2012). Females can lay from 5000 up to 200 000 eggs that are sticky and easily attach to different substrates (Curry-Lindahl, 1985; Kullander & Delling, 2012). Their eggs hatch after 4 to 12 days, also depending on temperature (Curry-Lindahl, 1985; Kullander & Delling, 2012). Roach migrate to spawn from the brackish water of the Baltic Sea into freshwater streams under a short period in April-June. The spawning act takes place during lively splashing near the beach, over vegetation (Kullander & Delling, 2012).

Although a small ide can be hard to tell apart from a roach, they do have different coloured eyes; ide have yellow eyes and the eyes of the roach are redder (Curry- Lindahl, 1985). Sexually mature individuals are easier to tell apart, especially during spawning (figure 1). Both species have breeding tubercles which during spawning makes it is easier to determine the sex of the individuals. Males generally have more breeding tubercles (Curry-Lindahl, 1985; Vøllestad & L'Abee-Lund, 1987) and rougher scales (Vankhede & Deshmukh, 2002). During the spawning period, fish can also be sexed by gently squeezing their gonads, to see if they have milt or eggs (Vøllestad & L'Abee-Lund, 1987; Hulthén et. al., 2014).

Figure 1: Pictures in the left column are of ide, one male (top) and two females (bottom two) and the right column are of roach (sex not known). Pictures have different scales and the size difference between species is not accurate, ides are bigger that roach. The fish on the pictures were caught and photographed during this study.

1.2 Aims and hypotheses The overall aims of this project are to study and compare phenotypic correlates of spawning migration behaviour of ide (L. idus) and roach (R. rutilus). Specifically, we investigate how spawning migration behaviours, such as arrival time, migration distance and time in the stream, is associated with fish morphological traits such as body length, body mass and body condition and whether it differs between species and according sex.

We will investigate the sex ratio of the study populations since several studies have reported various sex ratios for the studied species (Bateman, 1948; Jones, 2005; Holm, 2012; Cala 1970). Previous studies of ide (Cala, 1970) and roach (Vøllestad & L'Abee-Lund, 1987) show that larger individuals arrive earlier, and we will investigate if this pattern is expressed in our study populations as well. Cala (1970), also states that male ides arrive before the females. We will investigate if our study shows the same pattern for ide and if it is equivalent for roach. We predict that

males will stay for a longer period in the stream. It has been proposed that they arrive earlier than females and because of this they must wait for the females to arrive. Another reason could be that when females have laid all their eggs there is no point in staying in the stream. Males, on the other hand, would potentially benefit from staying for a longer period and fertilize more eggs. A study on threespine stickleback (Golovin et al., 2019) showed that females left before males. Their reproductive role is limited to the release of eggs, whereas males stay and care for the offspring. Further, we want to compare if there is a difference in time spent in the stream between the species. We also hypothesise that more slender individuals can and will migrate further upstream, based on the assumption that their more fusiform body makes it easier to swim past culverts and other barriers. Another interesting question that we want to address is if there is a difference in mortality after spawning between sexes and between the two species. Studies on cisco species have shown that mortality can differ between the sexes due to, among other things, age-at-maturity, predation, or competition for scarce resources (Pratt & Chong, 2012). In a population of ciscoes, females dominated the population and were larger than males and it was suggested that the higher mortality rate of males was due to predators being gape-limited and thereby making males more vulnerable to predation as they were of smaller size than females (Pratt & Chong, 2012).

2 Methodology

2.1 Study area The field studies were performed in Oknebäcken, Mönsterås (SE632310-152985) (figure 2). In Oknebäcken, a large number of fish migrate upstream to spawn. This location is therefore suitable, with regards to the amount of fish, to carry out the study.

Figure 2: Maps of where the study took place. The upper maps show where Mönsterås and the stream Oknebäcken are located in Sweden. The lower map shows the stream and where the marking station and the reading stations (antenna) are located.

To describe the watercourse and define the location and characteristics of different potential migration barriers, a simplified biotope mapping method was used. The biotope mapping is based on a standardized method retrieved from the County Administrative Board of Jönköping (County Administrative Board of Jönköping County, 2002). A map image of the biotope mapping was compiled using GIS. Biotope mapping is a method that involves creating a description of a watercourse regarding, among other things, physical conditions in and around the watercourse, different existing habitats, and the occurrence of migration barriers (County Administrative Board of Jönköping County, 2002). This information can be used in, for example, natural value assessments, action planning and environmental impact assessments. This method allows us to gather information about potential migration

barriers for fish and what measures may need to be taken to improve connectivity (County Administrative Board of Jönköping County, 2002).

Oknebäcken was observed during its course from the mouth to approximately three kilometres upstream. Potential barriers were located, measured, and photographed. Measurements of the stream width and water flow, and a description of the surrounding environment were obtained. The stream was first observed in February 2020, before the fish arrived and when water levels were high. The biotope mapping was performed in April after the marking of fish was completed. Reports from the biotope mapping can be seen in appendices 1 and 2 (in Swedish).

2.2 Ethical permit Since we in this study use fish for scientific research and are to perform operational interventions, it is classified as animal experiments according to chapter 1, 4 §, in the Swedish Animal Welfare Act (2018:1192). This means that permission is required to carry out this activity in accordance with chapter 7, 2 §, the Animal Welfare Act (2018:1192). Linnaeus University has applied for a permit from the Swedish Board of Agriculture and has been approved to conduct animal experiments (Diary number: 5.2.18-3833/19, date of the decision: 15-01-2020, valid until 15-01-2025). We have also undergone the training required for the purpose (such as practiced marking fish) according to chapter 7, 7 §, the Animal Welfare Act (2018:1192) in February 2020, before the fieldwork started. 2.3 Catching of fish The fish were caught in a hoop net that was placed 500 meters from the estuary, just upstream a wetland area (Oknebäcken, N 57.0199087, E 16.4471206), see marking station in figure 2. This location was chosen because the conditions were suitable for the workstation and because the fish caught there was probably heading for the stream and not the wetland. Usually, the hoop net was emptied in the morning the day after it was inserted in the stream. However, towards the end of the field study period, fish catches were so scarce in number that the net was not emptied as frequently. The hoop net was removed from the stream when more than two weeks had gone by with no catches of fish. The hoop net was operated from 2020-03-23 to 2020-05-13 (table 2). The fish that were caught in the hoop net were taken out and transferred to 65 litre plastic boxes with water and placed in the shade, just before it was time for processing, as described below. The fish were taken out of the hoop net in batches to avoid unnecessary stress and to avoid being stored too tightly in the boxes. When there was a large amount of fish in the hoop net, some fish were randomly released without being measured and marked. 2.4 Phenotypic measurements The fish was first placed on a workbench to measure the total body length, from the “nose” to the end of the tailfin. The sex was then determined by looking at whether the fish contained eggs or milt, or alternatively assessed based on roughness over the scales or breeding tubercles on the head. For both species, a male usually feels rougher and has more breeding tubercles on his head than a female (Curry-Lindahl, 1985; Kortet et al., 2004). It was also determined if the female had spawned or not. A female that has spawned is flat and shrunken along the abdomen, while a female

that has not yet spawned is relatively thick. All ides were weighed in a bag with a hanging scale (Berkley, 23 kg, 2 decimals) and roach in a plastic box with a kitchen scale (Biltema, range: 2-5000 g, accuracy: 2 g).

2.5 Marking of individuals using PIT-tags First, the fish was scanned with a hand-held reader (Biomark, HPR Lite reader) to make sure the fish was not already marked. The fish was injected with passive integrated transponder (PIT) tags (Biomark, HDX23; 23.1 mm long X 3.85 mm diameter, 0.6 g, 134.2 kHz). After the tag was injected, the fish was scanned again to confirm that the PIT-tag was detectable and to register the individual’s PIT-tag number.

The instrument used for injection of the PIT-tag was a syringe (Biomark, MK10) with a needle (Oregon RFID, lock needle, OD 4.5 mm, ID 3.9 mm, wall thickness 0.3 mm). The “bevel-down” method was used for marking, a method described and evaluated in a study by Cook and colleagues (2014), where the bevel of the needle is facing the abdominal wall to minimize damage to internal organs. The needle was first inserted under the scale as far as it could go. Second, the abdominal wall was penetrated at an angle of about 45 degrees, and then the angle was reduced so as not to damage the internal organs before the PIT-tag was injected. The injection was made between the anal opening and the pelvic fins, a bit up the side (figure 3). This method is better suited for cyprinid fish than penetration in front of the pelvic fins which is common for other species (Skov et al., 2005). The needle was disinfected in 90% ethanol between each injection to avoid spread of infection. The method of marking fish with PIT-tags has been evaluated in several studies carried out both in laboratories and in the field. A study by Skov and colleagues (2005) showed a low mortality rate when cyprinids were marked with 23 mm PIT-tags, the same tags that is used in this study. Another more recent study by Skov and colleagues (2020) showed no long-term effect of PIT-tag marking methods.

Figure 3: Injection of PIT-tag on ide using the “bevel-down” method. Date, PIT-tag numbers, length, weight, sex, information about if the fish had spawned or not and if the eggs ran out of the female was written down in a protocol. After processing, the fish was released back into the stream. The approximate handling time of each fish was 2-3 minutes. The field study period, with catching and marking of the fish took place in March-April 2020 (see table 2 for specific dates).

Table 2: Date of marking and the number of fish marked for each date, also divided by species.

Date Number of marked ide Number of marked roach Total number of marked fish 23-03-2020 4 0 4 24-03-2020 35 0 35 25-03-2020 130 0 130 26-03-2020 111 0 111 27-03-2020 45 0 45 01-04-2020 12 0 12 02-04-2020 47 1 48 03-04-2020 1 1 2 06-04-2020 9 22 31 07-04-2020 5 12 17 08-04-2020 45 113 158 09-04-2020 10 134 144 14-04-2020 0 46 46 15-04-2020 1 17 18 16-04-2020 0 38 38 17-04-2020 1 31 32 21-04-2020 1 52 53 23-04-2020 0 26 26 30-04-2020 0 0 0 01-05-2020 0 0 0 02-05-2020 0 0 0 04-05-2020 0 0 0 05-05-2020 0 0 0 07-05-2020 0 0 0 11-05-2020 0 0 0 13-05-2020 0 0 0 Total 457 493 950

2.6 Reading stations At two locations in the stream, upstream from the marking station, reading stations were constructed with readers and antennas in order to register passing fish (figure 2). The first and the second reading station was placed approximately 1150 meters (N 57.025054, E 16.434049) and 2240 meters (N 57.024561, E 16.419912) from the marking station, respectively. There was also a reading station at the estuary (N 57.0184081, E 16.4517072), downstream from the marking station, to detect fish leaving the stream after marking. Two readers (constructed by Dolby & Skov at DTU Aqua, National Institute of Aquatic Resources, Denmark) with antennas consisting of looped copper cables (speaker cable, Biltema/Ahlsels) covering the whole width of the stream, were placed at each location with approximately 2 meters between the antennas, to be able to detect at which direction the fish was swimming. The reading stations were powered by solar panels and 12V batteries. The PIT-tag is provided with an internal microchip with a unique code and an

electromagnetic coil (Castro-Santos et al., 1996). The reader emits an electromagnetic signal through the antenna and creates an electromagnetic field. When the tag encounters the electromagnetic field, the reader decodes the signal and transmits the unique code to a computer (Castro-Santos et al., 1996). The code is stored into a memory card together with date and time of passage. The data from the memory card were collected continuously from the day the PIT-tag markings started and then more sporadically. Data were collected until fish no longer migrated upstream, in the beginning of May. Before and after emptying the stations, the reading of the antenna was controlled with a PIT-tag attached to a stick, that was held at a distance from the antenna and was brought closer until it was detected.

The reading stations malfunctioned occasionally, often due to low battery levels. Most malfunctions occurred in the beginning, before the migrating of fish seemed to have started. Malfunctions after the migration started are not considered to have influenced the results of the study. Malfunctions only occurred during a couple of hours each time and therefore, fish that may have migrated upstream at this time should be registered when they migrate back downstream and the fish that may not have been registered at all were therefore probably few, and thus they should not affect the result or conclusions to any important degree.

2.7 Statistics and calculations Calculations were made using Microsoft Excel and statistical analyses were performed in R commander (version 3.6.1). All values from statistical analyses were rounded to two decimals except for P-values equal to or lower than 0.001, which were presented as P = 0.001 or < 0.001. To check whether data fulfilled the assumptions of ANOVAs, inspection of diagnostic plots were done before the test was performed.

2.7.1 Sex ratio The sex ratio was calculated in percentage to see the gender distribution for each species. To test whether the sex ratio differed significantly from an even distribution (1:1), a Pearson’s chi-squared test was performed.

2.7.2 Body condition To be able to compare the fishes’ body condition, the residuals from the least- squares linear regression of body mass on body length were estimated. For roach, both body mass and body length were log-transformed to obtain linearity prior to analysis and the equation from a linear regression was used to calculate an “ideal- body mass” at a certain length for individuals in this population. This value could then be used as a quantitative measure of whether the individual fish had a higher or lower body mass than the “ideal” that was expected from its’ body length. Separate calculations were performed for each sex. Linear regression equation of a straight line: y = k * x + m (1) Where k is the slope of the line and m is the intercept. Equation 1 calculates the “ideal-body mass”, y at a certain body length, x. Equation 1 can be used to calculate

a value for body condition, which is the difference between the individuals’ body mass and the “ideal-body mass” at the body length of the individual. Equations for body condition calculations: Ide: BC = BM - (k * BL - m) (2) Roach: BC = BM – 10^(k *log(BL) – m) (3) Where BC is body condition, BM is body mass and BL is body length, k is the slope of the line and m the intercept.

2.7.3 Associations of arrival time with sex and morphological traits A two-way ANOVA with interactions was used to investigate if there was an effect of species and sex on arrival time. To investigate the association between morphological traits and arrival time, the data was first plotted to make sure that the association was not curvilinear. Then, linear models were performed to investigate if there was an association between arrival time and any of the parameters body condition, body length or body mass for each species, subset by sex.

2.7.4 Time spent in the stream Number of days spent in the stream was calculated by the days from marking to the first registered passage through the estuary reading station. A two-way ANOVA with interaction was used to investigate the effects of species and sex on time spent in the stream. Linear models were used to investigate the interaction between sex and body condition on time spent in the stream, using separate analysis for each species.

2.7.5 Migration distance To quantify and compare migration distance, fish registered on any of the two upstream reading stations were classified “Upper” for passage through upper station, “Lower” for passage through lower station or “No” if no passage was registered. If an individual had been registered on both reading stations, it was grouped “Upper”. Three different two-way ANOVAs with interactions were performed for each species, to investigate the effects of body length, body mass or body condition and sex on migration distance.

2.7.6 Mortality Fish that were not registered on the estuary station were assumed dead. A Pearson’s chi-squared test was performed to test whether mortality differed between the two species. A binomial logistic regression was performed to investigate the associations of sex and arrival time with mortality, and of sex and body condition with mortality, using separate analysis for each species.

3 Results All parts of the result follow in detail below with a concluding summary (table 8) at the end.

3.1 Biotope mapping of the stream When the initial observation of the stream was performed in February, the high water levels made it difficult to evaluate with certainty whether the culverts posed as migration barriers. There was however, one particular culvert that was assumed to become a barrier as water levels dropped (figure 4). The downstream end of the pipes of this culvert were assumed to create a drop down to the stream at lower water levels. Also, the water levels inside the pipes were expected to be low which could be a problem for larger fish if they are not fully submerged. When the biotope mapping was performed in April, it was concluded that passage was possible for fish between the two pipes and, therefore, the culvert was not classified as a barrier for migrating fish (figure 4). Another barrier for migrating fish, that was identified during biotope mapping was culverts with blocked upstream openings. At some places, several branches and other things brought downstream with the water, was stuck across the pipes (figure 4). This might be a problem, especially for larger fish and when water levels are low. A more detailed description of the stream can be seen in the biotope mapping reports (appendices 1 and 2, in Swedish). Figure 4:

Top: The downstream end of the pipes of a culvert, water also flow between the two pipes.

Bottom: Branches blocking the upstream entrance of a culvert.

Pictures taken in April 2020, in the stream Oknebäcken (SE632310-152985).

3.2 Sex ratio The sex ratio for captured and marked roach was 22 % male and 78 % female. The sex ratio for ide was 44 % male and 56 % female (figure 5). The sex ratio differed significantly from an even distribution between the sexes, for both ide and roach 2 2 (Pearson’s chi-squared test, roach: X 1 = 155.64, P < 0.001, ide: X 1 = 7.11, P = 0.008) with a majority of females. Numbers of marked fish of each species and sex are reported in table 3.

Table 3: Number of marked fish of each species and sex and the calculated sex ratio. Roach Ide

22 44 % 56 Roach Ide % 78 % % Number of marked fish 493 457 Male 108 200 Male female Male female Female 385 257 Sex ratio M:F 22:78 44:56 Figure 5: Sex ratios for roach and ide.

3.3 Body condition The estimated parameter values from the fitted equations for the linear regression of “Ideal body mass” for females and males at a certain body length are reported in table 4. The relationships between body mass and body length are illustrated in figure 6.

Table 4: The equations for the linear regression of “Ideal body mass” for each species and sex Note that x in the roach equation should be log10-transformed values in cm and that the resulting y-values are body mass in kg in log10-space. Roach Ide Male equation Female equation Male equation Female equation y = 3,1421x - 5,1441 3,1411x-5,1022 y = 0,1026x - 3,4622 0,1162x - 3,9107

Roach body condition Ide body condition

Sex 3.5 Sex Female Female

Male Male

0.50

3.0

2.5

0.20

2.0

body.mass..kg.

0.10

Log (body mass (kg)) mass (body Log

1.5 0.05

15 20 25 30 35 40 46 48 50 52 54 56 58 Log (body length (cm)) body.length..cm. Figure 6: Least-squares linear regression of body mass on body length. For roach, body mass and body length are log-transformed.

3.4 Temporal distribution of arrival Ide arrived earlier to the stream than roach, there was a peak for ide on the 25th of March and on the 9th of April (2020) for roach (figure 7).

Figure 7: The temporal distribution of when fish, which were subsequently marked, arrived at the stream. On dates with “*” in front of the date, the hoop net was out of the water and no fishing occurred.

3.5 Associations of arrival time with morphological traits

3.5.1.1 Arrival time between species and sex There was no significant effect of the interaction between species and sex on arrival time (Two-way ANOVA, effect of interaction: F1;946 = 2.81, P = 0.09). Ide arrived significantly earlier to the stream than roach (Two-way ANOVA, effect of species: F1;946 = 1694.53, P < 0.001). Further, there was a significant difference in arrival time between sexes (Two-way ANOVA, sex: F1;946 = 9.98, P = 0.01). Male ide arrived before females (Two sample t-test, t455 = 3.46, P < 0.001). The mean day of the year of arrival for female ide was day 88.37 ± 5.45 days and for males 86.65 ± 4.42 days). For roach there was no significant difference in arrival time between

the sexes (Two sample t-test, t491 = 0.80, P = 0.42). The mean day of the year of arrival for female roach was day 102.36 ± 5.45 days and for males 101.91 ± 4.20 days (mean ± SD) (figure 8).

3.5.2 Roach

3.5.2.1 Body condition Fish that arrived early to the stream were heavier than expected from their body length, compared to fish that arrived late (figure 11). There was no significant effect of the interaction between body condition and sex on arrival time (Linear model, effect of interaction: t1;489 = -5.60, P = 0.55). There was a significant association between body condition and arrival time (Linear model: t1;489 = -2.39, P = 0.02).

3.5.2.2 Body Length Fish that arrived early to the stream were longer, compared to fish that arrived late, Figure 8: Mean arrival time for each species and independent of sex (figure 11). There was no sex. Bars showing standard deviations significant effect of the interaction between body length and sex on arrival time (Linear model, effect of interaction: t1;489 = 1.08, P = 0.28). There was a significant association between body length and arrival time (Linear model: t1;489 = -10.06, P < 0.001). Females (mean body length 29.73 ± 4.71 cm) were significantly longer than males (25.29 ± 3.98 cm, mean ± SD) (Welch two sample t-test: t199.15 = 9.82, P < 0.001) (figure 9).

3.5.2.3 Body mass Fish that arrived early to the stream had a higher body mass, compared to fish that arrived late, independent of sex (figure 11). There was no significant effect of the interaction between body mass and sex on arrival time (Linear model, effect of interaction: t3;489 = 0.09, P = 0.93). There was a significant negative association between body mass and arrival time (Linear model: t3;489 = -10.33, P < 0.001). Females had a higher mean body mass (0.36 ± 0.17 kg) than males (0.20 ± 0.11 kg) (mean ± SD), (Welch two sample t-test: t278.25 = 12.12 P < 0.001) (figure 9).

Figure 9: Body length and body mass frequency distribution for each sex of roach

3.5.3 Ide

3.5.3.1 Body condition There was a significant effect of the interaction between body condition and sex on arrival time (Linear model, effect of interaction: t3;451 = -2.73, P = 0.01). Males that arrived early to the stream were heavier than expected from their body length, compared to males that arrived late (figure 11). For females there was no association of body condition with arrival time (figure 11).

3.5.3.2 Body Length Fish that arrived early to the stream were longer, compared to fish that arrived late, independent of sex (figure 11). There was no significant effect of the interaction between body length and sex on arrival time (Linear model, effect of interaction: t1;452 = 0.22, P = 0.82). There was a significant association between body length and arrival time (Linear model: t1;452 = -2.89, P = 0.01). Mean body length of females (50.19 ± 2.15 cm) was significantly shorter than that of males (51.20 ± 1.81 cm, mean ± SD), (Welch two sample t-test t451.21=-5.47, P < 0.001) (figure 10).

3.5.3.3 Body mass Fish that arrived early to the stream had a higher body mass, compared to fish that arrived late, independent of sex (figure 11). There was no significant effect of the interaction between body mass and sex on arrival time (Linear model, effect of interaction: t1;452 = -1.88, P = 0.06). There was a significant association between body mass and arrival time (Linear model: t1;452 = -2.03, P = 0.04). Females had a higher mean body mass (1.93 ± 0.37 kg) than males (1.79 ± 0.27 kg) (mean ± SD) (Welch two sample t-test t452.91 = 4.54, P < 0.001) (figure 10).

Figure 10: Body length and body mass frequency distribution for each sex of ide.

Figure 11: Plots showing associations of body length, body mass and body condition with arrival time for ide. For body condition, 0 is the “ideal” weight at certain length for individuals in this population and the difference from zero are expressed in kg.

3.6 Time spent in the stream There was a significant effect of the interaction between species and sex on the time spent in the stream (Two-way ANOVA, effect of interaction: F1;443 = 28.41, P < 0.001). The significant interaction effect reflected that the difference between species varied according to sex (figure 12). Male roach stayed the longest (18.24 ± 20.05 days) and male ide the shortest amount of time (7.63 ± 5.11 days) in the stream. Female roach stayed significantly shorter (10.38 ± 11.34 days) than male roach (Two sample t-test t120 = -2.48, P = 0.02). Female ide stayed significantly longer in the stream (11.78 ± 6.64 days) than male ide (Two sample t-test t323=6.32, P < 0.001), (mean ± SD) (figure 12). Figure 12: Mean time spent in the stream for both species and sex. Bars showing standard deviations.

3.6.1 Roach The association between body condition at arrival and time spent in the stream was different in male and female roach (Linear model, effect of interaction: t1;118 = -2.73, P = 0.01). Females that were relatively heavy for their body length tended to stay for longer in the stream compared with females in poor condition (figure 13). Counter to intuition, males that were in poor condition instead stayed for a longer (not shorter) period in the stream (figure 13).

Sex

Female Male Roach

Days spent in the stream inthe Daysspent

10 0

-0.10 -0.05 0.00 0.05 0.10

Body condition

Figure 13: Body condition of female and male roach plotted against the number of days the individual stayed in the stream. Body condition is described as difference from 0, which is the “ideal” body mass.

3.6.2 Ide There was no significant effect of the interaction between sex and body condition on time spent in the stream (Linear model, effect of interaction: t3;320 = 0.21, P = 0.84). Females stayed longer in the stream than males, regardless of body condition (figure

14) (Linear model: t1;118 = -6.31, P < 0.001). There was no significant association between body condition and time spent in the stream (Linear model: t3;118 = 1.25, P = 0.21) (figure 14).

Sex

Female Male Ide

Days spent in the stream inthe Daysspent

5 0

-0.5 0.0 0.5

Body condition

Figure 14: Body condition of female and male ide plotted against the number of days the individual stayed in the stream. Body condition is described as difference from 0, which is the “ideal” body mass.

3.7 Migration distance

3.7.1 Roach No association between the morphological traits and migration distance was found. There was no significant interaction between sex and neither of the morphological traits body condition, body length nor body mass with migration distance (Two-way ANOVA, effect of interactions: body condition; F2;487 = 0.20, P = 0.82, body length: F2;487 = 0.52, P = 0.59, body mass: F2;487 = 0.25, P = 0.78 ). There was no association between neither of the morphological traits body condition, body length nor body mass with migration distance (Two-way ANOVA, body condition: F2;487 = 0.18, P = 0.84, body length: F2;487 = 2.34, P = 0.10, body mass: F2;487 = 1.70, P = 0.18). Mean body condition, body length and body mass for each sex at each station are reported in table 5.

Table 5: Mean values (±SD) of body condition (BC), body length (BL) and body mass (BM) for individuals at each reading station and the individuals that did not migrate. Mean No migration Lower station Upper station Female: 0.0012 (± 0.030) Female: -0.0019 (± 0.032) Female: 0.0038 (± 0.039) BC Male: 0.0018 (± 0.024) Male: 0.0029 (± 0.013) Male: -0.0038 (± 0.011)

BL Female: 29.69 (± 4.70) Female: 30.88 (± 4.76) Female: 26.83 (± 5.59) (cm) Male: 24.98 (± 4.12) Male: 27.09 (± 3.53) Male: 24.75 (± 3.18)

BM Female: 0.36 (± 0.17) Female: 0.40 (± 0.18) Female: 0.27 (± 0.22) (kg) Male: 0.19 (± 0.11) Male: 0.24 (± 0.11) Male: 0.18 (± 0.07)

3.7.2 Ide Because only one ide was registered on one of the upstream reading stations (table 6), the analysis of migration distance could not be performed.

Table 6: Number of migrating fish of each species and sex that passed the different reading stations.

Roach Ide

Number of marked fish 493 457 Male 108 200 Female 385 257 Number of migrating fish Passage lower reading station 42 1 Male 21 0 Female 21 1 Passage upper reading station 11 0 Male 9 0 Female 2 0 Passage estuary reading station 122 325 Male 21 164 Female 101 161

3.8 Mortality There was a significant difference in mortality between species (Pearson’s chi- 2 squared test χ 1 = 204.68 P < 0.001). There was a higher proportion of roach than ide that died (figure 15, table 7).

Figure 15: Mortality distributions in percent for each species. Red indicates dead and yellow indicates alive individuals

Table 7: The number of individuals that were classified as dead or alive for each species and sex and the percentage. Alive Dead % alive /sex % dead /sex Ide females 161 96 63 % 37 % Ide males 164 36 82 % 18 % Roach females 101 284 26 % 74 % Roach males 21 87 19 % 81 %

3.8.1 Roach There was no significant difference in mortality between the sexes (Binominal logistic regression: Z1;489 = -0.74, P = 0.46). For roach, 74% of the females and 81 % of the males were classified as dead, respectively (table 7).

3.8.1.1 Arrival time and mortality There was no significant effect of the interaction between sex and arrival time on mortality (Binomial logistic regression, effect of interaction: Z1;489 = 0.80, P = 0.42). There was an association between the time of arrival in the stream and mortality (Binomial logistic regression, Z1;489 = 2.97, P = 0.01), fish classified as alive arrived earlier to the stream. The fish classified as alive arrived on day 101.0 ± 4.7 of the year and on days 100.5 ± 3.3 of the year, for females and males respectively. Females classified as dead arrived at day 103.0 ± 5.6 of the year and males on day 102.8 ± 4.5 of the year (mean ± SD), (figure 16). Figure 16: Mean arrival time for individuals classified as dead and alive, for each sex. Bars 3.8.1.2 Body condition and mortality showing standard deviations. There was no significant effect of the interaction between sex and body condition on mortality (Binomial logistic regression, Z1;489 = -0.02, P = 0.98). There was no association between body condition and mortality (Binomial logistic regression Z1;489 = - 0.59, P = 0.56).

3.8.2 Ide There was a significant difference in mortality between the sexes (Binominal logistic regression: Z1;451 = -4.37, P < 0.001). For ide, 37% of the females and 18 % of the males were classified as dead, respectively.

3.8.2.1 Arrival time and mortality There was no significant effect of the interaction between sex and arrival time on mortality (Binomial logistic regression, effect of interaction: Z1;453 = -1.91, P = 0.06). There was no association between the time of arrival in the stream and mortality (Binomial logistic regression, Z = 1.58, P = 1;453 Figure 17: Mean arrival time for individuals 0.11). The fish classified as alive classified as dead and alive, for each sex. Bars arrived on day 87.92 ± 5.35 of the year showing standard deviations and on day 86.85 ± 4.54 of the year, for

females and males, respectively. Females classified dead arrived at day 89.11 ± 6.56 of the year and males on day 85.69 ± 3.69 of the year (mean ± SD), (figure 17).

3.8.2.2 Body condition and mortality There was no significant effect of the interaction between sex and body condition on mortality (Binomial logistic regression Z1;451 = 0.68, P = 0.49). There was no association between body condition and mortality (Binomial logistic regression Z1;451 = 0.69, P = 0.49).

Table 8: Comparisons of phenotypic correlates of spawning migration behaviours in roach and ide.

Roach Ide

Sex ratio M:F (%) 22:78 44:56

Associations of arrival time Species Arrived later than ide Arrived before roach Sex No difference in arrival time Males arrived first Body condition Fish that arrived early to the stream Males that arrived early to the stream were heavier than expected from their were heavier than expected from their body length, compared to fish that body length, compared to males that arrived late arrived late Body length Fish that arrived early to the stream Fish that arrived early to the stream were longer, compared to fish that were longer, compared to fish that arrived late, independent of sex arrived late, independent of sex Body mass Fish that arrived early to the stream Fish that arrived early to the stream had a higher body mass, compared to had a higher body mass, compared to fish that arrived late, independent of fish that arrived late, independent of sex sex Time spent in the stream Species The difference between species varied The difference between species varied according to sex according to sex Sex Males stayed longer than females Females stayed longer than males Body condition Females that were relatively heavy No association with time spent in the for their body length tended to stay stream longer in the stream compared with females in poor condition. Males that were in poor condition instead stayed for a longer period in the stream Migration distance Body condition No association with migration n.a. distance Body length No association with migration n.a. distance Body mass No association with migration n.a. distance Mortality Species Higher mortality than ide Lower mortality than roach Sex No difference between sexes Females died to a higher percentage Arrival time Fish classified as alive arrived earlier No association with mortality to the stream Body condition No association with mortality No association with mortality

4 Discussion The sex ratio differed from 1:1 with a majority of females for both species. We found that individuals that arrived early to the stream were larger for both study species. Also, male ide was both larger and arrived before female ide. For roach, there was no difference in arrival time between the sexes although female roach were larger. There was no difference in the time spent in the stream between the species. For ide, females stayed for a longer period of time in the stream than males. However, the opposite correlation was true for roach. We found no correlation between any of the morphological traits and migration distance. The mortality after spawning was higher for roach than for ide. For ide, a larger proportion of females than males died. For roach, individuals that arrived early was classified as alive to a greater extent than those who arrived late.

4.1 Body condition The body condition of roach in this study fell into the span of the mean body condition value of 20 other studies of roach (Svedäng, 1993). The value for the slope of the equations were between 2.9 – 3.6 and the mean was 3.27, in this study the slope value was 3.14. The intercept varied between -4.47 and -5.75 with a mean of -5.3 compared to -5.1 in this study.

4.2 Biotope mapping The methodology of biotope mapping is based on roach and trout, when assessing whether an object constitute a barrier. If a barrier is forceable/passable for roach it is assumed to be forceable/passable for most (all) other fish species. As demonstrated in this study, the size difference between adult individuals of ide and roach is quite large. Because the ide is larger, there is a potential risk that it is more difficult for it to get passed sections which have been deemed passable for roach. None of the culverts along the watercourse was determined as a definitive barrier at the time when the biotope mapping was conducted in April. At this point, the water level was determined as medium. Higher water levels were observed during the first inspection of the stream in February, but no observation have been made at low water levels and therefore there is no knowledge of how the stream's accessibility is influenced by low water levels. It is possible that some parts of the stream could pose as partial barriers. As mentioned earlier, larger fish can have a harder time getting past at low water levels, though it is not clear whether ide could have problems at medium levels as well. An important observation is that ide was visually observed on several locations along the watercourse, although only one ide was registered at the upstream reading stations. Observations were made both up- and downstream of the first station and as far up as just below the last culvert before the second uppermost station (figure 2). This clearly indicates that there is no definitive barrier for ide in the stream, though it does not exclude that it can be energy consuming for ide to migrate and that some individuals may refrain from doing so. There might be enough favourable spawning areas downstream the stations that do not make it worthwhile to migrate further. However, no investigation has been made as to where the most favourable areas for spawning are located in the stream, and thus it is only a speculation.

4.3 Migration distance and phenotypic traits Different studies have arrived at different results and conclusions when it comes to size of the fish and ability to get past obstacles when migrating. Therefore, it is hard to say that just one specific size is optimal. Optimal body size to successfully pass a fishway differs between species according to Calles & Greenberg (2007). Slender individuals may benefit from a larger body size and deep-bodied fishes may benefit from a smaller body size (Calles & Greenberg 2007). It can also be hypothesized that small individuals may not have the energy to deal with too high water flows and that they are also more exposed to predators, as some predators are gape-limited there should be a higher predation pressure on smaller fish. Large individuals might be stronger but might still have trouble getting through every part of the stream, especially at low water levels. There are probably both advantages and disadvantages with the different body sizes. In our study, marked ide did not migrate past the upstream stations and ide is the larger of the two species. Intuitively, one might conclude that the larger body size of ide makes the migration more difficult and therefore led to no registrations of ide at the reading stations. Our results however, showed no difference in either body condition, body length or body mass between roaches who migrated and roaches who did not migrate past any of the upstream reading stations. The fact that no associations between migration distance and any morphological traits were found for roach does not support the theory of size dependent migration distance. Perhaps the result would have been different if more roaches had migrated; in our study, the roaches that migrated were very few compared to the roaches that did not migrate past any of the upstream reading stations. The result of the ANOVAs might not be significant because the sample size is decreasing with migration distance (figure 18). Therefore, a statistical approach that takes this into account, such as a bootstrap, would be preferred. Unfortunately, such advanced statistical analysis is

above the level of competence of the authors of this report.

0.10

0.05

0.00

Body.condition..diff..in.kg.

-0.05 -0.10

No lower upper

Figure 18: Figure showing the body condition value for roach that migrated to the upper and lower stations and the individuals that did not migrate. The number of observations (n- value) are decreasing further upstream, making the results of simple statistical tests, such as an ANOVA uncertain.

In a study by Chapman and colleges (2015), they compare a population that contained both migrants and residents, a population where some individuals do not migrate at all but stay in the lake. This suggests that there should not be a difference in size between individuals that we marked, as our sample does not include any residents, according to the findings of the other study in which there is a difference in body size between the two groups mentioned above. However, there could still be variation within our population, as variation often occurs within populations. We compare individuals who have all begun to migrate up the stream, since the marking of fish took place next to the stream, 500 meters from the estuary. If some individuals only migrate just a bit up the stream, like the individuals we classified as not having migrated, and other migrate much longer, one can imagine that a difference in size-dependent migration distance could materialise. Those who migrate far might be favoured by a slenderer body shape than those who barely migrate at all. However, our results did not show a significant difference in size between migrants and non-migrants. Other factors than body size and shape can play a role when it comes to migration. Personality traits is one factor. A study on roach showed that bold fish migrate to a greater extent. Bolder individuals have a greater tendency to expose themselves to risks and they are more prone to discover new places. This likely makes them more exposed to predators, which also strengthens the reason for migrating (Chapman et al., 2011). An interesting task for future studies would be to investigate if this is true for our roach population, as well as for ide. However, it is not possible with the information obtained in our study to distinguish whether the migration distance is a result of migration barriers or behavioural differences between the species. The total amount of fish in the stream were lower this year compared to earlier years (Holm, 2012). This might explain the low number of migrating fish on our upstream antennas. The need to migrate might not be as high since a lower abundance of individuals in the stream will result in a lower competition for the spawning grounds.

4.4 Sex ratio The dominant sex was female for both roach and ide in our study populations. For ide the sex ratio was more evenly distributed between females and males than roach, for which the sex ratio was largely skewed with 78 % females. A potential reason for this uneven distribution may be that it is not necessary with as many males as females for fertilization, since one male can fertilize eggs from several females. Bateman’s principle states that the variation in the amount of offspring an individual receives in most species is greater for males than for females. The consequence of this is that males earn more from mating with more females than the other way around and that males therefore compete for mating opportunities (Bateman, 1948; Jones, 2005). Based on this principle, fewer males would reduce the competition for the fertilizing of eggs. However, if this were true, there would be a fitness advantage in having many sons, since male offspring have a higher reproductive success with lesser competition for mates. Because fewer individuals of one sex will lead to decreased intraspecific competition of mating partners (Emlen & Oring, 1977), a population will gravitate towards a 1:1 sex ratio. This relation was first described by

Ronald Fisher and is often called “Fisher’s principle (Hamilton, 1967). Hamilton defines the principle as: “Fisher’s principle of the sex ratio states that the sex ratio is in equilibrium when, in the population as a whole, the totals of effort spent producing the two sexes are equal. If the totals are not equal, producers of the sex corresponding to the lesser total have an advantage.” A study of roach (Tarkan, 2006) had a sex ratio of predominantly males. In the same study, a summary of studies on roach reports that in roach populations of northern latitudes, males mature before females, compared to lower latitudes were growth rate is generally higher and age-at-maturity is equal between the sexes. If the reproduction and survival of the offspring fluctuates from year to year, and age-at- maturity varies between sexes, then the sex ratios should fluctuate as well. Also, the survival of eggs and spawn in roach is generally low and large fluctuations in survival rate occur between years (Svedäng, 1993). In this study, it is likely that it only was the mature individuals that migrated to the stream. The total population sex ratio might therefore differ from the mature, spawning part of the population. To investigate if this pattern is the cause of an unequal sex ratio in the study populations, data from several years would be needed. The age-at-maturity could also be affected by competition for food and therefore maturity can be delayed after a year of spawning success with high survival of spawn (Svedäng, 1993). In addition to our speculations about the underlying causes of the sex ratios in our present study, there are many more factors that can affect the sex ratio in a population, which makes it difficult to draw conclusions about what factors is behind a prevailing ratio. Environmental conditions, food availability and adaptions to population dynamics is examples of what can cause the sex ratio to vary within a population, between populations and species from year to year (Oliveira et al., 2012; Brykov et al., 2008).

4.5 Dead or not registered? As there was no difference in body condition between those who survived and died for neither population of roach nor ide, there can be no conclusion that the deaths are due to poor body condition. However, there was a higher percentage of roach than ide that died. Given the size differences between roach and ide it may be wrong to discard the possibility that size matters to some extent when it comes to survival after spawning. It is conceivable that the roach died to a greater extent due to gape- limited predators, as roach is smaller than ide. This could also partially explain the shewed sex ratio since male roach are generally smaller than female roach and therefore more vulnerable to gape-limited predators (Pratt & Chong 2012). Although, no difference in mortality between sex was detected for roach in our present study. It is difficult to know whether all fish that were not registered at the estuary reading station have died, as no further follow-up of these has been carried out. A conceivable reason why fish were not registered is that they may have lost the PIT- tag along the way. This could be the reason why fish has not been registered at the

upstream reading station as well. However, in a study by Cook and colleagues (2014), the PIT-tag retention in Chinook salmon was over 90 % when using the bevel down method. In a laboratory study of roach and another cyprinid species, rudd (Skov et.al., 2005), there was no PIT-tag expulsions in the 400 fish studied. Although, Šmejkal and colleagues (2019) suggest that there might be a difference between sex in PIT-tag retention since females had a higher probability of expelling the PIT-tags with the gonads during spawning. No sign of this can be seen for roach in this study though, as there was no difference in mortality between sexes, but for ide lower fraction of the marked females than marked males were registered at the estuary station. Also, a problem with the reading stations is that if two tags pass by at the same time they may not be registered. So, if the fish swam by in large schools there is a possibility that all the individuals that passed were not registered. The low registration rate at the estuary station may also partially reflect that all fish had not migrated back to the sea before the time the field study and tracking of fish ended. It is not as likely for ide, as they started to migrate earlier, but it might be the case for roach. The result also shows that the survivors of roach were those who arrived first to the stream, and therefore might have swum out earlier, before registration ended. However, the estuary reading station was operational for more than 4 weeks after the last fish was marked, with no fish registered, before the data was compiled and analysed. Worth mentioning is that we did not se any large quantities of dead fish in the stream, which one could expect from the high mortality rates of roach. Although, considering the fact that a single female can lay up to 200 000 eggs (Curry-Lindahl, 1985; Kullander & Delling, 2012), there might be a possibility, if the survival of spawn is not considerably low, that the mortality rates after spawning are as high as reported in this study. Although, studies have shown mortality rates of spawn at over 90 % and in some conditions as high as 99 % (Svedäng, 1993). If the mortality of spawn is high, adults should aim to reproduce for several years to increase their fitness and therefore, the high mortality of roach is quite contradictive. Further studies are needed to be able to draw any certain conclusions about the mortality for roach.

4.6 Arrival time and time spent in the stream Cala (1970) states that larger individuals of ide spawned first and left the river first. A study of roach has also shown that larger individuals arrive first to the stream, Vøllestad, & L’Ab´ee-Lund (1987) found that both females and males of roach that arrived early in the season were larger than roach that arrived late. This pattern was evident in this study as well. Also, male ide arrived in the stream before females in our study population, as Cala (1970) also describes in his study of ide. Another study, by Šmejkal and colleagues (2017) found that male asp (Leuciscus aspius) arrived before females, suggesting that this pattern is due to intraspecific competition between males. Since larger individuals arrive earlier, there might be an energy cost coupled with presence in the stream and that larger individuals are better equipped with stored energy. This energy cost might be counterbalanced by a higher reproductive success in early arrival. A study of Atlantic salmon (Salmo salar) and Brown trout (Salmo trutta) (Dahl et al., 2004) showed that females arrive before males in both these species, to the Swedish stream Dalälven. They suggest a few explanations for this pattern such as a

skewed age structure between the sex, where females are older and larger. Our study, and several others have shown that larger individuals arrive earlier, however, we have not investigated the age structure in the study population. In the study by Dahl and collegues (2004) they also propose that females might prefer a certain spawning area and therefore, arrive before males to pick a spawning site. Šmejkal and colleagues (2017) described a pattern of daily spawning migration in asp (L. aspius), between calmer waters and more fast flowing waters at the spawning grounds. Male asp (L. aspius) arrived earlier to the spawning grounds and males also stayed for a longer period both daily as well as a seasonal, than female asp. This indicates that males put more energy into spawning, probably due to intraspecific competition. Producing eggs, especially of high quality could require more energy than producing milt (Bateman, 1948; Trivers, 1972), levelling out potential differences in energetic investments in spawning migration between sex. Vøllestad & L’Ab´ee-Lund (1987), found a correlation that larger and older individuals of roach had a higher fecundity, confirming that statement. For roach, there was no difference between the sexes in arrival time. Also, male roach stayed for a longer period in the stream than females. The reason for this could be that when females have laid their eggs, they have completed their mission and can leave the stream, but males would potentially benefit from staying longer and fertilize more eggs and by this improve their reproductive success. The reproductive success of females´ are for instance dependent on how many eggs they produce and the quality of them, whereas the reproductive success of males depends on the number of eggs they fertilize (Bateman, 1948; Trivers, 1972). The study on threespine stickleback (Golovin et al., 2019) showed a sex ratio where females were the dominant sex. The main reason for this was thought to depend on a higher depletion of energy reserves during the spawning period for males. This might be the case for ide in our study. The reason for female ide staying longer in the stream than male ide could be that intraspecific competition occurs between males. This is energy consuming for the males and they leave the stream earlier due to exhaustion. One sex staying for a longer period in the stream than the other sex could potentially be due to parental care as for example in the threespine sticklebacks (Golovin et al., 2019) and the cyprinid fathead minnow (Divino & Tonn, 2008). For the sticklebacks, the male stay after the female has left to care for the offspring. Parental care does not occur in all fish species and therefore it might not occur for roach and ide, or perhaps for just one of them. If parental care occurs it could potentially explain the difference in which sex stayed the longest in our study populations. Though it its more likely that parental care occurs for roach in our present study, as males stayed the longest in the stream to potentially care for the offspring as mentioned before. Whereas for ide, females stayed for the longest time. Paternal care, when the male is caring for the offspring is more common than maternal care among fish (Divino & Tonn, 2008; Klug, Bonsall & Alonso, 2013). In the study on fathead minnow, males were the ones that performed parental care by cleaning and protecting the eggs from predators (Divino & Tonn, 2008). However, a literature search on “roach”, “rutilus” and “parental care” performed on Web of science (2020-08-24) gave 4 results of which none seemed to concern roach. When a search

was made for “leuciscus” and “parental care”, no results were obtained. Therefore, it is not likely that parental care occurs in the study species. A study of roach report that the spawning period of that population lasted for 15-25 days (Vøllestad & L’Ab´ee-Lund, 1987), which was fairly similar to our study population where individuals stayed in the stream for a mean of 10 and 18 days with a deviation of 11 and 20 days for females and males respectively. In the study by Cala (1970), the conclusion was that most ide spawn during 2 to 3 days. In this study, ide stayed in the stream for much longer than that. The reason for this could be fluctuations in temperature. The temperature must be over 5ºC for hatching to occur and the mean temperature for ide to hatch were 13.8 ºC (Cala, 1970). If the temperature drops below favourable degrees, hatching can be interrupted and continue when the temperature rises again (Cala, 1970). Fluctuations in temperature can therefore contribute to an extended stay in the stream. Since the temperature was not recorded during field work, we cannot quantify the fluctuations that undoubtedly occurred. For example, some mornings during this study, the hoop net was covered in ice indicating low air and water temperatures.

5 Conclusion and suggestions for the future The unexpected results of the sex ratio might be a result of fluctuations in survival of spawn coupled with different age-at-maturity between sexes. For roach, the skewed sex ratio could also be a result of gape-limited predators in the Baltic, as male roach are generally smaller than female roach. Roach are the smaller of the two species, which make them more exposed to gape-limited predators and therefore roach might have higher mortality than ide. It is difficult to know if the fish classified as dead truly have died. Deficiencies in the reading stations and lack of supervisory control, might explain the low survival rates. The difference in mortality between species would be an interesting factor to investigate further. Also, it would be interesting to investigate the number of marked individuals that will return for spawning next year and if any of the fish classified as dead in this study will appear to be alive.

As other studies have reported, individuals who arrived early were larger. There might be an energy cost associated with early arrival to the stream and therefore, larger individuals arrive first. Males might benefit from more fertilization events when staying longer, as was the case for roach. There might therefore be a trade-off between the energy cost in staying in the stream and the increased fitness advantage in fertilization events. Parental care is a conceivable reason for one sex to stay longer in the stream than the other sex, although it is not likely that parental care occurs in our study populations.

Since very few individuals were registered at the upstream reading stations, there might be an effect of migration barriers on the spawning migration. There are many more factors that can be explored to learn more about the spawning migration and behaviour of the two species, including how migrating roach and ide are affected by predation. Do they profit from migrating far to avoid predation and, is the predation

pressure higher at the mouth of the stream? Is it beneficial to migrate for the survival and hatching success of the eggs? This last question might be addressed by placing plates in the stream with attached fish eggs to investigate whether and how the predation on eggs varies in the stream. In future studies, the temperature should be recorded to be able to monitor how the temperature can affect spawning behaviour when it comes to arrival time and time spent in the stream. Analysing pictures may be used to investigate the potential correlates of spawning migration behaviours with other morphological aspects of fish than size and condition that were studied here. Another suggestion for future work is to investigate associations with personality traits such as boldness, as mentioned before.

Both similarities and differences between the species were discovered in this study. One may not always be able to generalize and believe that they possess similar morphological, behavioural, or structural characteristics because they are closely related. The fact that they differ may be the reason for the differentiation and species formation.

6 Author contributions Both authors contributed equally to field work and writing. Johanna contributed slightly more with the data compilation and statistical analyses whereas Emma conducted additional literature searches and started off with the structure of the discussion and introduction.

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Appendix 1 Appendix 2