Context dependent variation in aggression and mating behaviour in the pygmy ( collettei):

a study of wild population

Piotr Michalak

Degree project in biology, Master of science (2 years), 2021 Examensarbete i biologi 30 hp till masterexamen, 2021 Biology Education Centre, Uppsala University, and Department of Zoology, Stockholm University Supervisor: John Fitzpatrick External opponent: Carolina Segami 1 Contents

1 Introduction 3

2 Methods 6 2.1 Study species ...... 6 2.2 Video collection and selection process ...... 7 2.3 Scoring ...... 9 2.4 Environmental variables ...... 10 2.5 Statistics ...... 10

3 Results 12 3.1 Group size and composition ...... 12 3.2 Aggression ...... 12 3.3 Mating ...... 17 3.4 General activity ...... 19

4 Discussion 21 4.1 Social factors and environment ...... 21 4.2 Conclusions ...... 24

Abstract

To understand behaviour, it is important to consider the environment in which it occurs. The environment, consisting of both abiotic factors and social context, is usually highly variable and leads to variation in individual’s and group’s behaviour. To better understand the environmental influences on behaviour of pygmy (Dermogenys collettei), a small live-bearing fish, I viewed videos of shoals of wild halfbeaks in Singapore. I investigated effects of environmental variation (water depth, canopy cover and water vegetation) and social environment (group size and male to female sex ratio) on halfbeaks’ aggression and mating be- haviours. I found that environment had little effect and most variation between studied shoals was probably due to social factors. I found some evidence for aggres- sion increase in larger shoals, primarily in males. Sex ratio had different relation with aggression for individual sexes and mating behaviours decreased when sex ra- tio became more male biased. This study shows that halfbeaks probably modify their behaviour in relation to social environment. I also show that these changes are similar to those described in other species, which strengthens the validity of using halfbeaks to study social interactions.

2 No animal or plant can live in a vacuum. John Maynard Smith (1975)

1 Introduction

When considering animal behaviour, it is paramount to understand the context in which it occurs. Without it, we can provide little insight into why animal behaves a certain way. The environment of the animal is a complex, variable net of abiotic and biotic factors. These factors generate selective pressures that manifest themselves on both the level of an individual and a whole populations. If consistent on the evolutionary timescale, it can lead to species divergence (Rundle and Nosil 2005). This can operate only if there is individual, heritable variation of the trait in the population. Additionally, because the environment is highly variable, individuals usually show a range of behavioural responses. Abiotic environment can have a direct influence on animal behaviour. Factors like light intensity, temperature and ground topography can all play a role in influencing animal behaviour. Importantly however, abiotic factors influence multiple species in the same are and in turn, these influence each other. For example, trees might block the sunlight desired by an animal, possibly influencing their general predation risk. As light intensity can lead to higher visibility of prey, it influences predator’s and in consequence, prey’s behaviour. Under higher possible predation risk in turn, prey might decrease its activity to become less conspicuous. (Poecilia reticulata) under higher light intensity show lower number of aggressive behaviours that could be visible to potential predators (Endler 1987). Multiple night foraging species decrease their activity under bright moonlight (Lima and Dill 1990). Other factors that reduces prey visibility, like water vegetation for fish, also has been showed to impact behaviour of prey. In bluegills (Lepomis macrochirus) and fathead minnows (Pimephales promelas), fish tended to prefer areas densely covered by plants when predators where present, presumably as a refuge (Savino and Stein 1989). However, in dense vegetation one might expect fewer social interactions, as forming groups becomes difficult and visibility of conspecifics is also reduced. Through modifying their behaviour depending on the environment, organisms try to maximize their fitness and find an evolutionary trade-off between negative factors (like predation susceptibility (Dill 1987)) and other activities. Predation can influence a scope of life-history traits (eg. Reznick and Endler 1982). Food distribution is also influenced by the environment and can have surprising effect on a fundamental aspect of species and populations ecology. Emlen and Oring (1977) showed, how different distribution of food can lead to different observed mating systems. If resources are not uniformly distributed and can be monopolised, potential for multiple matings increases as more individuals can be found in the same

3 region. This in turn might influence the potential for sexual selection and differential reproductive success observed in such groups. Environmental factors have also an impact on social conditions. In turn the social environment is a critical factor further modifying behaviour. Differences in group struc- ture and size lead to different levels of competition for food and mates, and different predation risk. Operational sex ratio (OSR), the ratio of sexually active males to females, is expected to be a result of various factors, like potential for resource monopolization and ability of to explore that potential (Emlen and Oring 1977). OSR influences social behaviour and strength of sexual selection. As the ratio becomes more skewed towards one sex, the frequency and intensity of intrasexual competition and aggression has been showed to increase (Grant and Foam 2002; Weir et al. 2011). Although the pat- tern, observed here in Japanese medaka Oryzias latipes (a shoaling fish), is true for both female-female and male-male aggression, the increase is usually stronger within the male sex(Grant and Foam 2002). The OSR and increased competition also leads to changes in courtship. More males competing seems to correlate with decrease intersexual inter- actions per individual male, but with increased number of female interactions per single female (Clark and Grant 2010). In extreme cases, in some fish, change in OSR can lead to the sex change of an individual - a complete overhaul of sexual strategy (Ross 1990). Group size is also an important factor influencing social and sexual behaviours. In gup- pies, bigger groups show increased levels of male-male aggression (Seghers and Magurran 1991; Price and Helen Rodd 2006). However, population of guppies that were more prone to schooling (forming uniform groups) showed decreased levels of aggression - as predicted if schooling is considered an anti-predator response (Seghers and Magurran 1991). As our understanding of the importance of environmental and social factors increases, it becomes more important to our understanding of behavioural variation to combine data both from laboratory experiments and observations in the wild. One of the organisms used to study social interaction in the laboratory is pygmy halfbeak (Dermogenys collettei). It is a small, live-bearing fish that shows high levels of male-male aggressions and acomplex courtship behaviour (Greven 2010). It is used to study female mate choice (Ogden et al. 2020; Reuland et al. 2019) and shoaling behaviours (Ho et al. 2015). Despite it being relatively common and being used as a model for studying behaviour, little is known about its behaviour in the wild (Greven 2010). Especially little is known about how the environmental factors that influence social behaviours in halfbeaks. It is also not known, precisely which factors influence halfbeak behaviour (for example, what type of predation halfbeaks face (Ho et al. 2015)), and therefore it is not clear which environmental factors are most relevant. Moreover, halfbeaks in the wild were shown to behave differently than in the laboratory (Greven 2010). In this study, I attempt to increase our understanding

4 of halfbeak behaviour in the wild by looking at videos of wild populations taken at twelve different sites in Singapore, in their natural habitat. These twelve sites differ bothin abiotic and biotic environment and I expected these factors to influence the differences in observed behaviour. I looked into aggressive and mating behaviours, because they can be easily discerned in the videos. I also try to estimate general levels of activity in the videos. These three aspects of behaviour can be viewed as similar to three facets of individuals temperament (aggression, sociability and activity), that are known to be influenced by environmental factors and stressors like, predation risk (Réale et al. 2007; Heinen-Kay et al. 2016). While the studies of wild halfbeaks are limited, they have been shown to share several life history traits with poeciliid fishes (e.g. guppies,Poecilia ( reticulata) (Reznick et al. 2007). Both groups of fish are live-bearing, and engage in frequent social interactions, establish dominance hierarchies and exhibit mate choice (Greven 2010). Drawing from knowledge of other species, I therefore expect environmental factors to have predictable effects in halfbeaks. One of the potential hypotheses is that halfbeaks in different environ- ments would encounter different predation risk influenced by environmental factors. As mentioned previously, light intensity causes a decrease in conspicuous activity of guppies due to potential increased predation risk (Endler 1987). In nature canopy cover, spanning over a waterbody, casts a shadow over water and might in turn lower light intensity. This might in turn modify the predation risk and halfbeak behaviour. Additionally, dense veg- etation might give fish refuge from predators (Savino and Stein 1989), I expect therefore halfbeaks to engage more in conspicuous behaviour in habitats with vegetation present. In poeciliids it is also predicted that predation risk might influence the use of shallow water, as well as that time spend on male-male aggression and sexual behaviour (Heinen et al. 2013). There is a possibility that similar effect might be seen in halfbeaks. Itis worth noting, that these responses in poeciliids are predicted due to piscivorous fish. It is not well known if halfbeaks are primary preyed on by fish or birds (Ho et al. 2015). Social factors, like group size and sex ratio, can have effect on aggressive and mating behaviours as well. Larger groups were shown in guppies to correlate with higher aggres- sion (Seghers and Magurran 1991; Price and Helen Rodd 2006), while more male skewed sex ratio usually corresponded to increase in male-male competition and aggression in Japanese medaka Oryzias latipes (Clark and Grant 2010). Highly male skewed sex ratio suggest that chances of encounter of potential mate would decrease, as more males com- pete for proportionately less females (Clark and Grant 2010), leading possibly to decrease in courtship.

5 2 Methods

2.1 Study species

Pygmy halfbeak (Dermogenys collettei) is a small, tropical, live-bearing fish, primar- ily found in streams, ponds and other freshwater bodies in Southeast Asia (including Malaysia and Singapore) (Meisner 2001). Halfbeaks measure around 4 cm in length. They possess elongated lower jaw (beak) that are used to capture on the water surface. Halfbeaks are sexually dimorphic (females are larger in length and width than males) and dichromatic (males’ fins are more colourful). Male colouration is used infe- male mate choice (Reuland et al. 2019). Females have an orange spot on the ventral side (known as the gravid spot), that plays a role in male mate choice (Ogden et al. 2020). Halfbeaks live in mixed-sex groups (shoals) of unknown stability, swimming a few cen- timetres below the water surface. They are usually found near the water edge. Within the group they interact frequently exhibiting complicated mating behaviour, and intra-sex aggression (Greven 2010). Males actively court females through elaborate courtship behaviour. First, male fol- lows a female and starts swimming around her in semi-circular pattern (circling). If female appears receptive (stops swimming or swims slowly) he swims under her, positions himself below the female’s genital pore. Male might start nipping (opening and closing lower jaw toward genital pore) and checking (touching the female with a beak). Males are thought to also assess the female’s gravid spot while swimming underneath. The swimming under a female might last around four minutes in the laboratory (Ogden et al. 2020). As male cannot feed during this time, it is presumed to be an energetically costly behaviour. Rela- tive to courtship, copulation is extremely short ( 40–80 ms) and inconspicuous. The male might bend it body in an “S” shape during the copulation and contact female genital pore with modified anal fin (andropodium) used for sperm transfer. It is difficult to determine if the copulation occurred or if it was successful based on behavioural observations. The fertilisation is internal, and female might store sperm for up to six monthly breeding cycles after one copulation (Reuland et al. 2019). They give birth to competent fry. Halfbeaks in the laboratory frequently engage in aggressive behaviours, especially male-male competition (Greven 2010). Agonistic behaviour consists of four stages - im- posing, threatening, fight, and flight. The halfbeaks might cause substantial injury tothe opponent. Because of the competition, in-group dominance hierarchies are established, with a large female being the dominant. Male-specific dominance hierarchies are also es- tablished, and dominant males prevent subordinate from access to females (Greven 2010). However, most of these observations has been made in the laboratory.

6 2.2 Video collection and selection process

Videos of halfbeak shoals were taken at three water bodies in Singapore (Jurong Lake Gardens 1°20’11.0”N 103°43’41.0”E; Seletar river, Thomson Road Springleaf Nature Park 1°24’01.0”N 103°48’59.6”E; unnamed stream, Windsor Nature Park, 1°21’36.3”N 103°49’ 36.6”E) during three days of observation (July-August 2019) by Erika F. Isaksson (EI) and Alessandro Devigilii (AD) (Department of Zoology, Stockholm University). GoPro cameras were set up over or underneath the water surface. Videos were takes at twenty- four sites at three different locations (n=9, Jurong Lake Gardens; n=7, Thomson Road Springleaf Nature Park; n=8, Windsor Nature Park). Shoals were identified by the ob- servers as semi-stable halfbeak groups. All shoals were observed within 2-3 meters from the water edge. The information on the environmental factors were recorded at each spot. 82 hours of film were obtained and given to the author for further observation. The film was split automatically by the GoPro camera into 572 individual videos (mean length 8.63±3.16 minutes). Each video was viewed sped up (2x-4x times the speed) to choose videos for further analysis. Out of initial 572, 59 videos had no halfbeak present, and as such were excluded from the analysis. 513 videos had a halfbeak present. As I was interested in social behaviour in a halfbeak group, I required more than one halfbeak and same individuals for prolonged period of time. I set up an arbitrary threshold of continuous 30 seconds of one halfbeak present at the video (60 videos had halfbeaks for less time and as such were excluded, n=453). In the third step of selection, two same individuals had to be present for at least 30 continuous seconds (194 were excluded at this stage, n=259). The remaining 259 videos were assessed for quality, i.e. can they be reliably scored (113 videos excluded, n=146). This is the most subjective step and potential source of bias (see Discussion) (Steps 1-4 Fig 1). After step 4, the remaining videos were not distributed equally among the sites. Each location had different numbers of observations as selected videos were not chosen based on the water body they came from. It resulted in uneven numbers between the locations (Windsor Park, n=17; Thomson Road, n=47; Jurong Lake, n=82; total n=146). To decrease bias of watching too many videos from the same site in a row, a Python script was created to randomly select a predetermined number of videos from each location. Although conducting focal observations on each individual was in early plans, it quickly appeared to be beyond the scope of this project. Individuals within the groups were mov- ing out of frame making consistent group observation difficult. To get an overview of social interactions, I opted for scan sampling method. The videos were therefore cut into 5 second clips in 60 seconds intervals using ffmpeg (except for the first videos that werecut into 30 second intervals in order to assess if behaviours need higher frequency of scoring to be detected). These clips were then randomised (using “randomize playlist” in MPC-HC

7 software), watched in VLC media player and scored. I counted all occurrences of known antagonistic and mating behaviours and noted group size and composition visible during the clips. At this stage additional selection took place, as some videos were deemed not possible to score. Those videos (-59) were excluded (see Step 5 in Fig 1), leaving a total of 87 videos. At each step the observer was blinded as to the location from which the videos were taken to minimize the effects of possible expectations. The locations were coded as letters and were disclosed only after the scoring and initial look at the data took place.

Videos obtained

572 all videos Step 1 -59

513 had halfbeaks recorded

Step 2 -60 ≥ 1 halfbeak for continuous ≥ 30 453 seconds Step 3 -194 ≥ 2 halfbeaks for continuous ≥ 30 259 seconds Step 4 -113

146 good quality

Step 5 -59

87 visibility allowing for scoring

Analysis

Figure 1: Video selection process

8 2.3 Scoring

The remaining videos (n=87) were scored for discernible behaviours (Tab 1). The num- ber of individuals of each sex involved in specific behaviours were counted, as well as absolute number of particular behaviours seen during the clip. The behaviours consist of aggressive behaviours divided into chasing (swimming and/or following another individ- ual with increased speed) and displacement (when halfbeak was chased away or moved away after contact by another individual) ; and mating behaviours (circling - swimming in semi-circular fashion around another individual, primarily exhibited by males; swim- ming under - swimming underneath another individual; mating attempt - rapid contact with member of another sex and move away, usually preceded by swimming underneath). Common occurrence of very fast swimming of undetermined cause was also recorder (dart- ing). In each clip the number of individuals and group composition was assessed. Sex was determined primarily by size and behaviour. The juveniles were not sexed as it was deemed impossible by the observer. Adult fish that could not be sexed were classified as unknown.

Table 1: Video scoring chart

Behaviour type Behaviour Definition swimming and/or following another individual Aggression Chasing with increased speed being chased away or moved away after contact Displaced by another individual swimming in semi-circular fashion around an- Circling other individual, primarily exhibited by males Mating Swimming swimming underneath another individual under contact with member of another sex and move Mating at- away, usually followed by swimming underneath tempt swimming with increased speed without an obvi- Other Darting ous cause

9 2.4 Environmental variables

I was interested in effects of the environment on halfbeak behaviour. The assessment of environmental conditions was conducted by EI and AD directly at each site. Three ecological characteristics at each location - canopy cover, aquatic vegetation and water depth on which the shoal was observed. Canopy, meaning here a plant cover over the water surface, creates shadow over the water, potentially decreasing light intensity. For my analysis, the canopy was analysed as two levels (presence-absence). Aquatic vegetation (presence-absence) was also assessed i.e. whether vegetation is present in the water or not. As mentioned in the introduction, aquatic vegetation can offer refuge to fish and generally reduces predation risk. The depth on which the shoal was observed was also noted (shallow (<25cm) – deep (>25cm), as the use of different water depth is correlated with predation risk in some fish.

2.5 Statistics

Out of 835 clips that ended in the analysis, many (655) showed none of the behaviours. This resulted in a data set skewed heavily towards zero. More importantly, clips are not independent of each other. Therefore, the data was collapsed to the means for each observation spot. Clips that did not contain halfbeaks (n=59) were excluded. For the mating behaviours and counts involving male to female ratio (average number of males divided by average number of females), means were taken only from the clips that had both males and females. As the number of sites is low (n=12), each factor was fitted with an individual model. This results in a possibly oversimplified analysis (see Discussion), but avoids overfitting the model. Site means were fitted to linear models using the lm function in R(basic structure - lm(response variable∼explanatory variable, data)). Models where residuals were assessed as normally distributed were then compared using an anova function on the model (R statistics package). Model residuals that did not conform to a normal probability distribution were tested using Mann-Whitney Test (wilcox.test function in R) (canopy cover, aquatic vegetation, water depth, sex; two levels each). Correlation of behaviour against the group size and male/female ratio assessed either by the anova function in R, for normally distributed data, or Spearman correlation otherwise. In some cases, additional test was run where the fraction of individuals of particular sex engaging in a behaviour was taken as a dependant variable. This was done to ask if the behaviour becomes more common among individuals, rather just more observed because of the increase in raw numbers. All analyses were conducted using R v4.0.2 (R Core Team 2020) using RStudio

10 v1.3.1073 (RStudio Team 2020), graphs were made with ggplot2 package in R (Wick- ham 2016).

11 3 Results

3.1 Group size and composition

Mean number of fish observed at the sites were 6.92 ± 7.64 individuals (median 4.56, interquartile range of 2.11, n=12). Considerable deviation comes from two sites in Ju- rong Lake (obtained means 15.0 and 28.6). On average (mean) 2.53±2.43 females and 2.39±1.52 males were counted. Mean of juveniles was 1.89±1.03 and of unidentified sex, 3.68±3.24. Mean male to female ratio was 1.17±0.44. Group size was not influenced by any environmental variables (depth W=14, p=0.81; canopy W=29 , p=0.07; vegetation W=20, p=0.82). Neither was the observed male to female ratio (depth F=0.47, p=0.51; canopy , F=0.01, p=0.91; vegetation F=0.14, p=0.71).

3.2 Aggression

Social effects

Shoal size effects The total number of antagonistic behaviours increased as the shoal becomes larger (F = 19.71, p<0.01) (Fig 2).

Figure 2: Shoal size had a relevant effect on the amount of aggressive behaviours. As the shoals grow larger, more aggressive behaviours happen in a group.

Of two elements included in aggressive behaviour, chasing and displacement, both were shown to be statistically significant with respect to shoal size (chasing F=22.37,

12 p<0.01; displacement F=16.06, p<0.01). The effects disappear however when considered number of behaviours per individual (i.e. number of behaviours divided by the group size) (aggression F=0.45, p=0.52; chasing F=0.42, p=0.53; displacement F=0.53, p=0.48). The effect of changes in overall aggression with size is not seen when single sexes are considered (males rs=0.31, p=0.32; females rs=-0.05, p=0.89). However, more males chase in bigger shoals (F=120.80, p<0.01, Fig 3a), as well as the behaviour becomes more common (rs=0.59, p=0.04, Fig 3b). Females showed no correlation, in either chasing

(rs=-0.03, p=0.94) or displacement (rs=0.30, p=0.34).

(a) (b)

Figure 3: More males exhibited chasing behaviours both when raw number of males (a) or fraction of males participating in behaviour (b) was considered.

Male to female ratio effects

Male to female ratio was not correlated with shoal size (rs =-0.43, p=0.16). It is therefore important to note, that shoals with male-skewed ratios probably have fewer females than a shoal with equal sex ratio. For that reason, the sex ratio effect was also tested against the fraction of individuals of particular sex engaged in the behaviour and not only in raw count. Total aggression showed no sex ratio related effect (F=1.15, p=0.31). No effect was found between sex ratio and either chasing (F=1.09, p=0.32) or displacement (F=1.31, p=0.28). For males, the number of individuals exhibiting aggressive behaviours was not related to sex ratio, both in raw number of individuals (rs=-0.22, p=0.48), and as fraction of all males present (F=1.84, p=0.2). When looking at the specific behaviours however, some patterns began to emerge. Fewer males showed chasing behaviour in shoals with

13 more male skewed ratios (rs=-0.65, p=0.02, Fig 4).

Figure 4: As the ratio becomes more male skewed, fewer males engage in chasing behaviour (red). Total aggression and displacement were not statistically significant.

This pattern remained when it was a fraction of males engaged in mating that was considered (F=6.00, p=0.03). No effect of sex ratio on displacement was found (F=1.71, p=0.22, for fraction F=3.52, p=0.09). Female participation in aggressive behaviour showed no effect of sex ratio (F=2.23, p=0.17, fraction F=4.30, p=0.07), however, when aggression was taken apart into specific behaviours, both chasing, and displacement proved to correlate with sex ratio, but having an opposite effect. In shoals with male- skewed ratios, more females chased (F=5.01, p=0.049, ”Chasing” Fig 5; fraction, F=5.52, p=0.04) and fewer were displaced (rs=-0.78, p<0.01, ”Displacement” Fig 5;fraction rs=- 0.77, p<0.01).

14 Figure 5: More females engage in chasing behaviour as the ratio becomes more male skewed (”Chasing”). It is possible that as more mates are available, females become more selective and chase more males away. Additionally, with more male biased sex ratio fewer females are being displaced (”Displacement”). As primary source of female displacement in halfbeaks is female-female competition, it is possible that as more males are present, females compete less and primarily chase away unwelcome males.

Environmental effects

Environment had little impact on observed behaviours. No effect of the environment was detected on total count of aggressive behaviours (depth F=3.46, p=0.09; canopy F=3.06, p=0.11; vegetation, F=0.2, P= 0.67, Fig 6).

15 Figure 6: Environment show no statistically significant effects on total aggression

There was no environmental effect for specific behaviours either (chasing: canopy F=3.27, p=0.1; depth F=3.21, p=0.1; vegetation F=0.19, p=0.68; displacement: canopy F=0.04, p=0.11; depth F=3.87, p=0.08; vegetation F=0.26, p=0.62). No effects of en- vironments has also been found for individual sexes (males: canopy W=17, p=1; depth F=2.28, p=0.16; vegetation W=19, p=0.93; females: canopy W=10.5, p=0.28; depth W=15, p=0.93; vegetation W=12, p=0.37). Specific antagonistic behaviours also showed no environmental influence when individual sexes were considered (male chasing: canopy W=21.5, p=0.54; depth W=10.5, p=0.36; vegetation W=25, p=0.27; male displacement: canopy W=19, p=0.87; depth W=6, p=0.09; vegetation W=17, p=0.93; female chas- ing: canopy W=16, p=0.86; depth W=8.5, p=0.2; vegetation W=15.5, p=0.0.73; female displacement: canopy W=18, p=1; depth W=18, p=0.78; vegetation W=19, p=0.93).

16 3.3 Mating

Social effects

Group size had no effect on the number of mating behaviours both in total occurrences

(rs=0.32, p = 0.32) and per an individual (rs=0.21, p=0.52). However, when sex ratio becomes more male biased, the number of mating behaviours decreases (rs=-0.70, p=0.01, Fig 7).

Figure 7: When sex ratio becomes more male biased, the number of mating behaviours de- creases.

This pattern remained and fewer males and females engaged in mating when sexes were considered separately (males rs=-0.69, p=0.01 ; females rs=-0.64, p=0.02; Fig 8). However, when considered as fraction of individuals, both become non-significant (males rs=-0.51, p=0.09; females rs=-0.41, p=0.19).

17 Figure 8: Increase in operational sex ratio is correlated with fewer males and fewer females engaging in mating behaviours.

Environmental effects

No effect of environment was observed on mating behaviours (depth W= 4,p=0.79; canopy W=24 , p=0.31; vegetation F=2.93, p=0.12, Fig 9). The number of mating females nor males changed based on the environment (males: W=21, p=0.62; W=17.5 p=0.86; F=1.94 p=0.19; females: W=20, p=0.74; W=18.5, p=0.73; F=1.76, p=0.21).

18 Figure 9: Environment show no statistically significant effects on mating behaviours

Additionally, no correlation between mating and antagonistic behaviour was found

(rs=0.10, p=0.76).

3.4 General activity

General activity, which was a sum of antagonistic, mating behaviours and darting (a quick, dart like swim without obvious cause) showed no correlation with the environ- ment (depth F= 0.45, p=0.52; canopy W= 18.50 , p=0.94; vegetation F= 0.96, p=0.35). General activity was not related with neither group size (F=1.61, p=0.23) nor sex ratio (F=0.70, p=0.42). When the sexes were considered separately, no correlation was de- tected with either environmental (males: canopy F=0.31, p=0.59; depth F=0.24, p=0.63; vegetation F=1.61, p=0.23; females: canopy F=0.02, p=0.9; depth F=0.01, p=0.92; veg-

19 etation F=0.4, p=0.54) or social factors (males: number of active males with shoal size

F=1.61, p=0.23; fraction of active males with shoal size rs=-0.11, p=0.74, number of ac- tive males with OSR, ratio F=1.77, p=0.21; females: number of active females with shoal size F=0.00, p=0.98; fraction of active females with shoal size F=1.74, p=0.22, number of active females with OSR, ratio F=1.02, p=0.34)

20 4 Discussion

4.1 Social factors and environment

In this study, it appears that the main factors that influence the halfbeak behaviour are social components of the environment. Contrary to the predictions, the environmental factors considered here, did not show statistically significant influences on any ofthe studied behaviours. Numbers of aggressive behaviours seen on the videos was influenced by the shoal size. More individuals in the group increase the number of antagonistic behaviours (Fig 2). This is also true for chasing and displacement. This conforms to predictions, that more individuals should present more behaviours. This effect however is not present when change is considered per single individual. This may suggest that in this population, individuals do not clearly modify the levels of aggression in relation to group size. When sexes were considered, effects mostly disappeared. However, males (only) showed the increase in chasing behaviours - both for general (Fig 3a) and per single individual (Fig 3b). This might suggest increased antagonism in males in larger groups. Indeed, increased aggression with larger shoal size has been shown by Devigili et al. (submitted) in focal observation of male halfbeaks from the same sites as in this study. In guppies bigger groups are also correlated with increased levels of aggression (Seghers and Magurran 1991; Price and Helen Rodd 2006). For halfbeaks, larger groups might create difficulty in creating a stable dominant hierarchy, which in turn, should lead to reduction of aggression, as noted by Greven (2010). Stability of halfbeak dominance hierarchy is still unknown and further studied in the laboratory and in nature are required to answer the question if hierarchy plays a role here. The effects of OSR showed to be interesting. No effects were detected whenthe behaviours were considered together. However, males showed a decrease in chasing be- haviour with more male skewed ratio (Fig 4). The expectation would be to increase the levels of aggression with higher competition. Aggression, especially towards potential competitors, generally increases with higher OSR (Clark and Grant 2010; Weir et al. 2011). It does, however, decline as potential costs of aggression and mate guarding out- weigh the benefits (Weir et al. 2011). This was observed for both males and femalesin Japanese medaka (Oryzias latipes) (Grant et al. 2000; Grant and Foam 2002). In this study, mean OSR was 1.17±0.44, less male biased than when aggression started to de- cline both in studies by Grant et al (2000; 2002) and Weir et al. (2011) (OSR around 2). Halfbeaks are however a different species, and therefore it is possible that their response to the OSR differs. Worth notice is also the fact that in guppies, similar lower levelsof aggression has been noted in males in groups with male-skewed OSR if individuals were

21 familiar with each other (Price and Helen Rodd 2006). Females also showed interesting changes in aggressive behaviours. When more males were present in relation to females, it seems females engaged more in chasing and were less displaced (Fig 5). It is known that female halfbeaks generally are aggressive towards males(Greven 2010) and although it cannot be clearly inferred from the data, is possible that with more male skewed ratio, female halfbeaks are more likely to chase males away. Indeed, it seems in accordance with the premise of effects of OSR as initially presented by Emlen and Oring (1977) asthe more abundant sex faces more competition and the less can become more prone to mate choice, as it is likely to find a mating partner. If the female aggression is predominantly male oriented, it could explain increase in chasing and decrease in displacement. In the studies on Japanese medaka, female-female aggression was shown to increase with more female-skewed ratio and start to decline after it reached certain threshold (Grant and Foam 2002). With the data from this study in its current form, it is difficult to discern exactly the direction of aggression. A certain level of caution has to be applied here however, as although these effects are statistically significant, they are based of low number of observation and considerable number of these are zeros. Indeed, removing just a single outlier from the chasing data makes the result statistically not significant and the correlation disappears entirely (rs=- 0.03, p=0.92). The mating behaviour seems to be influenced only by the operational sex ratio. This effect however is the most consistent in the whole dataset. As the ratio becomes more male skewed, the number of mating behaviours decreased (Fig 7). The effects of OSR on observed mating behaviours are the most consistent in this data as the decrease is seen in both males and females (Fig 8). The result however is not unexpected as similar results has been observed in Japanese medaka (Oryzias latipes) (Grant and Foam 2002; Clark and Grant 2010). There decreases in courtship interactions with male biased sex ratios was seen for males, however females showed an increase - something not observed here. Clark et all. (2010) attribute that effect to the encounter rate of conspecifics -the sex toward which the OSR is skewed, encounters members of the same sex more often than mates, which results in increased competition. Although it is tempting to postulate similar effect here, such an explanation is inconsistent with the observed lower levelsof male chasing with higher OSR. Possibly the biggest surprise is the lack of environmental effects. This can be due to technical limitations as mentioned below. However, environmental factors used in this study are often related to the risk of predation. A study of shoaling behaviour of halfbeaks in a population in Singapore postulated that predator recognition is generally low in that population (Ho et al. 2015). It is worth considering then that maybe halfbeaks do not

22 adjust their activity levels in response to potential dangers of fish predation. I also looked at halfbeaks already in groups. Shoaling itself is considered an anti-predatory response (Ho et al. 2015) and possibly the observed differences are all under similar predation stress. Ho et al. (2015) also speculated that main halfbeak predators might be birds rather than piscivorous fish. Importantly however, I have used environmental elements which donot measure predation directly. As no predation event has been observed and the nature of predation remains unclear, it is difficult to say to what extent the environmental factors impact predation risk.

Limitations There are certain limitations to this study. Although over eighty hours of film were available, the data was collapsed to per site basis. That, after exclusions, resulted in 12 independent data points (see Tab 1). It is a relatively small sample size that does not allow for using complex models and, possibly, strong conclusions. Some important interaction might have been lost in the process. Also, it is worth noting that the total count of behaviours of each sex has not been directly scored in this study. While the number of individuals of each sex engaged in particular behaviour has been counted as well as the total count of the behaviour that occurred has been used, how many individuals of each sex engaged in particular behaviour has not been directly scored. Sex specific behaviours were not common, and size is not fully reliable sex indicator. Considerable number of individuals had to be classified as of unknown sex. It is therefore possible that the sex ratio and sex specific behaviours in the shoals might be different from that noted in this study. Additionally, observed effects of sex ratio can be effects of different sex specific density, as was pointed in the studyof Oryzias latipes by Clark et all. (2010). Therefore, conclusions about effects of sex ratio should be treated with caution. The impact of video selection process also might play a role. As light intensity in the videos decreases, fish blend with background and they are difficult to see and score.As light intensity plays a role in frequency of behaviour in previous studies (Endler 1987) this limitation might influence detection of behavioural differences. It is possible that those clips in extreme shadows that would show difference were more excluded. Difference in canopy cover between used and unused sites was not detected in this study (χ2 =2.253, p = 0.133). It is also worth considering that, broad categories used (presence/absence) might not capture required range of within factors and higher resolution might show different results. This however would require a larger sample size.

23 4.2 Conclusions

Halfbeaks are still not a well understood as a species. In this study, I have tried to verify the predictions coming from known theory on effect of groups size and operational sex ratio, and potential environmental effects, and also compare these findings with those from other species. This study and that done by Devigili et al. submitted showed that halfbeaks might indeed increase levels of aggression with group size. Additionally, higher OSR leads to decrease in mating behaviours. This is worth noting for work in the laboratory, but more importantly, it might be interesting to modify the OSR of halfbeaks in the laboratory to see the effects in more controlled environment. Halfbeaks in recent years has been successfully used to study sexual selection in the laboratory (see Reuland et al. 2019; Ogden et al. 2020). Here, in the observational study, despite its limitations, influence of group size and sex ratio shown in other species have been reproduced here. In the past, halfbeaks were considered unfit to be a model organism (Greven 2010), but I believe recent evidence points otherwise.

24 References

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26 Weir, Laura K., James W. A. Grant, and Jeffrey A. Hutchings (2011). “The Influence of Operational Sex Ratio on the Intensity of Competition for Mates”. The American Naturalist 177.2, pp. 167–176. Wickham, Hadley (2016). ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York.

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