Brain morphology and behaviour in the guppy (Poecilia reticulata): Effects of plasticity and mosaic brain evolution

Brain morphology and behaviour in the guppy (Poecilia reticulata)

Effects of plasticity and mosaic brain evolution

Stephanie Fong

Stephanie Fong Brain morphology and behaviour in the guppy (Poecilia reticulata)

Department of Zoology

ISBN 978-91-7911-348-3

Stephanie Fong

Brain morphology and behaviour in the guppy (Poecilia reticulata)

Effects of plasticity and mosaic brain evolution

Stephanie Fong

Academic dissertation for the Degree of Doctor of Philosophy in Ethology at Stockholm University to be publicly defended on Thursday 17 December 2020 at 10.00 online via Zoom, public link is available at the department web site.

Abstract

Understanding how brains have evolved and subsequently culminated in the huge variation in brain morphology among contemporary vertebrate species has fascinated researchers for many decades. It has been recognized that brain morphology is both genetically and environmentally determined. Adaptations to ecological challenges, for one, has been proposed to be a major force in brain diversification processes. Considering the large energetic costs of neural tissue, it is believed that brain evolution is a highly complex process, involving a delicate balance between the corresponding costs and benefits.

Using the guppy (Poecilia reticulata) as the model organism, I first examined the conditions under which diversity in brain morphology is generated. This was done by investigating factors known to exert an influence on brain plasticity, namely environmental and cognitive effects (Paper I). Existing studies generally indicate that the provision of environmental enrichment lead to the enlargement of specific brain structures. While plastic alterations in brain morphology was found to respond to environmental complexity in my study, successful performance in two cognitive tasks did not produce any significant changes.

I next assessed the feasibility of the mosaic brain evolution hypothesis by artificially selecting for an increase and decrease in the relative size of the telencephalon (Paper II). Telencephalon size was shown to respond rapidly to divergent selection pressures, with no substantial changes in any of the other brain regions. A comparison with wild fish revealed that fish from the unselected control treatment had telencephalon sizes most similar to that of wild populations, whereas both up-selected and down-selected fish had considerably larger and smaller telencephalon, respectively.

I tested fish from the artificial selection lines in a test battery to determine if known differences in telencephalon size affects boldness (Paper III). Individuals were subjected to an emergence test, an open field test and a novel object test. I found no differences in boldness levels across selection treatments, but distinct sex differences were noted whereby males were more active and bolder.

The cognitive benefits associated with a larger telencephalon were examined in males in a test of self-control (Paper IV). Guppies from the up-selected lines attained a steeper learning curve and made more correct detours compared to their down-selected conspecifics.

In conclusion, I provide experimental evidence for the mosaic brain evolution hypothesis by showing that a specific brain region (telencephalon) can evolve rapidly and independently under directed selection. Future tests on other cognitive benefits as well as implicated costs, together with underlying neuronal changes would help to further unravel the factors governing brain evolution.

Keywords: brain plasticity, cognition, reversal learning, spatial learning, directed selection, mosaic brain, heritability, brain morphology, boldness, relative telencephalon size, inhibitory control, detour task, Poecilia reticulata.

Stockholm 2020

http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-186357

ISBN 978-91-7911-348-3 ISBN 978-91-7911-349-0

Department of Zoology

Stockholm University, 106 91 Stockholm

BRAIN MORPHOLOGY AND BEHAVIOUR IN THE GUPPY (POECILIA RETICULATA)

Stephanie Fong

Brain morphology and

behaviour in the guppy (Poecilia reticulata)

Effects of plasticity and mosaic brain evolution

Stephanie Fong

©Stephanie Fong, Stockholm University 2020 ISBN print 978-91-7911-348-3

ISBN PDF 978-91-7911-349-0

Printed in Sweden by Universitetsservice US-AB, Stockholm 2020

The thesis is based on the following articles, which are referred to in the text by their Roman numerals:

I Fong, S., Buechel, S.D., Boussard, A., Kotrschal, A., & Kolm, N. (2019). Plastic changes in brain morphology in relation to learning and environmental enrichment in the guppy (Poecilia reticulata). Journal of Experimental Biology, 222 (10), jeb200402.

II Fong, S., Rogell, B., Amcoff, M., Kotrschal, A., van der Bijl, W., Buechel, S., & Kolm, N.

(2020). Rapid mosaic brain evolution under artificial selection for relative telencephalon size in the guppy (Poecilia reticulata). Manuscript.

III Fong, S., Andersson, A., Amcoff, M., & Kolm, N. (2020). Relative telencephalon size does not affect boldness in the guppy (Poecilia reticulata). Manuscript.

IV

Triki, Z., Fong, S., Amcoff, M., & Kolm, N. (2020) Artificial mosaic brain evolution of relative telencephalon size improves cognitive performance in the guppy (Poecilia reticulata). Manuscript.

Candidate contributions to thesis articles*

* Contribution Explanation

Minor: contributed in some way, but contribution was limited.

Significant: provided a significant contribution to the work.

Substantial: took the lead role and performed the majority of the work.

I

II

III

IV

Conceived the study

Substantial Substantial Substantial Significant Designed the study

Substantial Substantial Substantial Significant Collected the data

Substantial Substantial Significant Minor Analysed the data

Substantial Substantial Substantial Minor Manuscript preparation

Substantial Substantial Substantial Significant

Contents

INTRODUCTION -3-

VARIATION IN BRAIN MORPHOLOGY -3-

MOSAIC BRAIN EVOLUTION HYPOTHESIS -4-

PHENOTYPIC PLASTICITY -5-

BRAIN MORPHOLOGY AND BEHAVIOUR -6-

AIMS -7-

STUDY SYSTEM -8-

METHODS -9-

BRAIN PLASTICITY -9-

ARTIFICIAL SELECTION EXPERIMENT -11-

BEHAVIOURAL TESTS USING SELECTION LINES -13-

RESULTS AND DISCUSSION -14-

PAPER I -14-

PAPER II -16-

PAPER III -20-

PAPER IV -21-

CONCLUSIONS AND FUTURE DIRECTIONS -22-

REFERENCES -25-

SVENSK SAMMANFATTNING -33-

ACKNOWLEDGEMENTS -35-

INTRODUCTION

Variation in brain morphology

The driving forces behind the immense variation in brain morphology has been the focus of research over decades. A classic, but reasonably outdated, view surrounding brain diversity pertains to the belief that species with larger brain volumes rank higher up on the intellectual scale in accordance with a theory known as Scala Naturae. According to this notion, brain evolution progresses in a unilinear manner, starting off with ‘simple’

organisms such as invertebrates and fish, and culminating with more ‘complex’ species such as primates and humans at the top of the hierarchy. This view has since been eradicated, and it has been acknowledged that brains have evolved in a non-linear manner and over several time points from the ancestral state (Northcutt 1981).

This subsequently raised the question of what common aspects of the physical or social environment led to the convergent evolution of brain morphology across species. This was in part due to the recognition that neural structures are shaped by adaptations to specific ecological selection pressures, shifting the focus towards understanding the relationship between brain size and cognitive benefits. In accordance with the cognitive buffer hypothesis (Sol 2009), larger brains provide greater information processing power which facilitates behavioural flexibility under changing or unpredictable conditions, thereby optimizing survival and/or fitness. For instance, a positive correlation between brain size and the ability of various mammalian species to colonize a novel environment was found, supporting the cognitive advantages offered by the possession of a larger brain (Sol et al. 2008).

Although the association between brain size and higher intellect has generally been accepted, it was later recognized that the sole comparison of brain size does not provide the complete narrative (Herculano-Houzel 2011). For instance, the positive correlation between brain size and neuron numbers, but not neuron density, was recently demonstrated by Marhounová et al. (2019), wherein the brains of females artificially selected for larger brains had higher number of neurons. These same large-brained females exhibited superior performance in a range of cognitive tests (e.g. Buechel et al.

2018; Corral-López et al. 2017; Kotrschal et al. 2015b; van der Bijl et al. 2015). However, this pattern may not necessarily hold across species; the neocortex and cerebellum have been shown to consist of similar number of neurons despite considerable interspecific volume differences (reviewed in Montgomery et al. 2016). All these suggest that underlying neural components and interconnectivity may be of equal if not higher importance when trying to understand the intricate relationship between brain morphology and cognition.

Mosaic brain evolution hypothesis

One prominent theory surrounding brain evolution postulates that selection pressures act on discrete functional brain components, resulting in the specific enlargement of targeted regions. Such independent changes in brain subdivisions characterize the heterogeneous brain pattern commonly referred to as the mosaic brain (Barton and Harvey 2000; Striedter 2005). According to this hypothesis, only functionally correlated brain regions evolve together, minimizing energy expenditure on the maintenance of unnecessary neural tissue. This is in agreement with the recognition that neural tissue is

metabolically expensive (Aiello and Wheeler 1995; Kotrschal et al. 2013b). For instance, comparative studies have shown that food caching avian species have an enlarged hippocampus in comparison to non-caching species (Garamszegi and Eens 2004; Sherry et al. 1989). Within-species comparisons in wild brown trout with different life history strategies have also presented evidence for the mosaic brain (Kolm et al. 2009). Trout that migrate to the sea and attain sexual maturity at a larger body size were found to have larger cerebellum relative to conspecifics that remain in their spawning grounds.

A leading opponent of the mosaic brain hypothesis is the concerted brain evolution hypothesis which states that developmental constraints restrict independent evolution of brain regions, resulting in the coordinated expansion or regression of the entire brain (Finlay and Darlington 1995). Accordingly, larger brains are merely ‘scaled-up’ versions of smaller brains, while retaining similar proportions of the different brain regions. Instead of questioning the validity of each theory, the current challenge lies in elucidating the conditions under which either predominates.

Phenotypic plasticity

Efforts to understand the mechanisms underlying brain evolution are further complicated by the fact that the vertebrate brain retains the ability for prolonged proliferation and neurogenesis, and thus remains susceptible to external influences throughout adulthood (Cayre et al. 2002; Gross 2000). Across vertebrate species, however, the turnover rate and consequent growth of neural tissue varies greatly. In particular, the brains of fish, amphibians and reptiles exhibit extensive adult neurogenesis relative to that of mammals and birds (Kaslin et al. 2008). This difference in brain growth has been proposed to be

related to the growth of various sensory systems and the corresponding neural substrates in non-mammalian vertebrates. Given this continued replacement and/or addition of neuronal cells, the brains of fish may be highly responsive to fluctuations in the surrounding environment.

Hence, apart from alterations in neural structures resulting from evolutionary selection pressures, vertebrate brains are able to undergo plastic changes as a form of adaptation to changes in local environment. Identified factors that are known to induce changes in brain morphology include environmental enrichment, hormones, temperature, light and learning (Brown et al. 2003; Fahrbach et al. 1995; Lerch et al. 2011; Moore et al. 2004;

Patel et al. 1997; Tramontin and Brenowitz 2000; van Praag et al. 2000). Seasonal changes in the size of the telencephalic nuclei in songbirds correspond to testosterone levels and intensity of song behaviour. During the breeding season, there is a positive correlation between elevated levels of testosterone and increase in the volume of the song control nuclei (Ball et al. 2002). Tackling the issue of phenotypic plasticity vs. evolutionary adaptations is therefore necessary to bridge this empirical gap in the study of brain evolution.

Brain morphology and behaviour

An important mediator of brain morphology is none other than the feedback received from behavioural responses. Ecological challenges imposed upon individuals tend to favour specific behavioural responses, which in turn act upon the underlying neural substrate mediating those functions. Evidence for the link between brain morphology and behaviour have been demonstrated through a series of comparative studies (e.g. Madden

2001; Overington et al. 2009; Reader and Laland 2002; Sol et al. 2005). However, one of the major drawbacks concerning comparative analyses is the inability to differentiate between correlation and causation. Unless all conditions are kept identical, it remains nearly impossible to rule out the effects from potential confounding factors yet to be unidentified. One way to ameliorate this issue is through the use of artificial selection experiments for either a specific behavioural or neural trait. And this approach has recently been successful in determining the costs and benefits of evolving a larger brain in the guppy (Poecilia reticulata) (e.g. Buechel et al. 2018; Corral-López et al. 2017;

Kotrschal et al. 2015a; Kotrschal et al. 2013b). This thesis deals with two of the major issues brought up, namely examining the effects of external influences (i.e. environmental enrichment and cognitive processes) on plastic changes in brain morphology; and the utilization of an experimental approach to test the mosaic brain evolution hypothesis in a vertebrate species.

AIMS

The main objective of this thesis was to understand brain evolution, namely what factors drive changes in brain morphology? Are brain regions free to evolve independently under directed selection? What are the consequences of the aforementioned selection process?

In Paper I, we investigated the effects of learning and environmental stimulation on plasticity changes in brain morphology. In Paper II, we artificially selected for relative telencephalon size (relative to the rest of the brain) in guppies over three generations to experimentally test the mosaic brain hypothesis. In Paper III, we examined whether the specific increase and decrease in a brain region (i.e. telencephalon) differentially influenced the level of boldness in our animals, given that this personality trait has the

potential to affect performance in future cognitive tests. Finally, in Paper IV, we experimentally tested the evolutionary consequences of an increased telencephalon size on inhibitory control in male guppies artificially selected for a larger or smaller telencephalon.

STUDY SYSTEM

Laboratory populations of guppies were originally obtained from Quare river high predation populations (i.e. populations where the fish predator Crenicichla alta, the pike cichlid, was present) in Trinidad and kept in large mixed-sex populations in Trondheim over several generations. All fish in the lab were kept under same conditions (i.e. 12:12 h light-dark schedule, with temperatures maintained at 25 ± 2°C) to minimize potential effects caused by the external environment. These lab-kept wild-type populations were used in Paper I to examine brain plasticity. In addition, these fish were also used as the starting populations for the selection experiment in Papers II, III and IV. The ease at which large samples of guppies can be kept in the lab, together with their relatively short generation time attests to its usefulness as a model system for studying brain evolution.

The extensive number of studies performed using fishes has revealed marked similarities between teleost brains and that of other vertebrates (O'Connell and Hofmann 2011; Salas et al. 2003; Vargas et al. 2006). For instance, the dorsal lateral pallium of the telencephalon has known implications in spatial learning, an aspect of cognition commonly attributed to the hippocampus in mammals (Vargas et al. 2000). More specifically, ecological niches typically occupied by wild guppies have also come under scrutiny, which have consequently led to the identification of some of the factors

responsible for the huge diversity in brain morphology in this species (Kotrschal et al.

2017; Kotrschal et al. 2012; Reddon et al. 2018).

METHODS

Brain plasticity

In Paper I, we wanted to examine whether learning processes and/or environmental enrichment had an effect on brain plasticity changes. To do so, lab-kept wild-type females were trained and tested in either a reversal learning task or a spatial learning task. These two learning paradigms were chosen given that they present ecologically relevant challenges to guppies (Buechel et al. 2018; Burns and Rodd 2008; Houde 1997; Kotrschal et al. 2015b). Two additional control groups were also included, one for each cognitive task, to account for any potential changes in brain morphology engendered by the test environment rather than learning per se (Figure 1). Barring training and testing, fish in the environment control group received identical handling and housing as their respective learning group.

Figure 1. Time progression of each group, reversal learning and spatial learning.

Individuals in the reversal learning treatment group were first tested on their ability to associate a coloured disc (red or yellow) with a food reward over 30 trials. Following the initial associative learning, the reward contingency was reversed and fish were given a maximum of 66 trials in this reversal learning phase. We recorded the first choice made by each individual as either correct or incorrect.

7 days 7 days

19-22 days 19-22 days

7 days 7 days

14 days 14 days

Fish allocated to the spatial learning treatment group were tested daily in a maze measuring 100 x 50 cm, over 14 consecutive days. Two dead-ends were included in the maze to increase task complexity, and the time taken to complete the maze were recorded for each individual over trials.

Following each test, fish in the treatment and their respective control groups were fixed in 4 % formalin and their entire brains subsequently dissected out. Quantification of brain size and the size of six major regions (i.e. telencephalon, optic tectum, cerebellum, dorsal medulla, hypothalamus and olfactory bulbs) were determined in accordance with the ellipsoid model (Pollen et al. 2007; White and Brown 2015): 𝑉= (𝐿 × 𝑊 × 𝐻)^'₍

We compared the brain morphology of the test group and their respective controls, with the aim of testing for the effects of learning vs. environmental stimulation on brain plasticity changes.

Artificial selection experiment

In Paper II, we performed an artificial selection experiment for relative telencephalon size in guppies. This procedure was adapted from a previous selection experiment for relative brain size (Kotrschal et al. 2013b). The starting population (F0) consisted of 225 breeding pairs, equally divided over three independent replicates, of lab-kept wild-type guppies which were allowed to produce at least two clutches prior to being euthanized and their brains fixed in 4 % formalin and subsequently dissected out. Total brain volume and volume of six major brain regions were similarly quantified as before. To rank the pairs according to their relative telencephalon size, we obtained the combined parental

residuals for each pair based on the regression of the telencephalon volume on the rest of the brain. From here, offspring from the top and bottom 15 pairs were used in the propagation of the following generation up- and down-selected lines, respectively (Figure 2). An unselected control line was also established, by the random pairing of offspring.

This line functioned as a reference to assess whether selection proceeded in a symmetrical manner. The selection process was repeated for three more generations, giving rise to the current generation F4.

Figure 2. Experimental design of the artificial selection experiment for relative telencephalon size in guppies. The different selection treatments are indicated by the different colours: a) blue (up-selected), b) yellow (control) and c) black (down-selected). Three independent replicates for each of the selection treatments were set up for every generation as indicated. Offspring from the top and bottom 15 pairs were used to generate 30 breeding pairs each for the next generation, respectively.

In addition, we also looked at how telencephalon size in the selected treatments related to that found in wild populations. To do so, the brains of wild indigenous female guppies previously collected from 16 different sites in Trinidad (Kotrschal et al. 2017) were

a) Offspring of 15 pairs with largest combined telencephalon residuals

c) Offspring of 15 pairs with smallest combined telencephalon residuals b) Random offspring to set up 30 new breeding pairs

Generation F0

3 replicates x 75 pairs

Generation F₃ 3 x (30U + 30C + 30D) Generation F₁

3 x (30pairs U + 30C + 30D)

Generation F₂ 3 x (30U + 30C + 30D)

measured. This comparison between selected vs. wild guppies facilitated the assessment of the rate of brain evolution under directed selection.

Behavioural tests using selection lines

Following the establishment of the selection lines, we were interested in examining for possible changes in boldness between individuals from the up- and down-selected lines.

Any detected differences between selection treatments could have implications for future behavioural tests performed in these fish and were hence examined in Paper III. A total of 156 guppies (78 males and 78 females) equally divided among the three separate replicates of up- and down-selected lines were subjected to a test battery consisting of an emergence test (ET), open field test (OFT) and novel object test (NO). Each trial was video recorded and subsequently analyzed. Given that all fish were only identifiable by running numbers based on a random allocation system in R, the selection treatment of each fish remained blind to the experimenter throughout the experiments and subsequent video analysis.

To identify possible personality trait dimensions, a principal component analysis (PCA) including all the recorded behaviours from the test battery was performed. We then extracted all the relevant principal components and investigated whether there was a difference in the identified traits between selection treatments. Considering known differences in boldness between males and females, we also analyzed for any sex-specific differences in the extracted components.

In Paper IV, we looked at how an evolutionary change in telencephalon size influenced cognitive performance by testing male guppies artificially selected for relative telencephalon size in a detour task. To maximize sample sizes of our most important test groups, we chose to only test males from the up- and down-selected lines. A total of 72 males, evenly distributed among selection line and replicate combination, were tested in their ability to correctly detour a transparent barrier to obtain a food reward.

Fish were individually housed and identified by running numbers, preventing any potential biases arising from knowledge of the treatment identity. Individuals were first habituated to feed from a green spot on a white Plexiglas plate placed at the front of the experimental tank. During the pre-training and test phases, a transparent plastic cup was placed upright over the green dot, in a manner that fish now had to detour the barrier to gain access to the food reward. For each phase, either the number of time-bins or the exact time taken to reach the reward was recorded. An important indicator of successful detour, whether individuals were able to swim past the barrier without coming into contact with it, was also noted.

RESULTS AND DISCUSSION

Paper I

We found an overall effect of treatment on brain size (F3,73 = 7.33, p < 0.001), but post hoc comparisons revealed absence of a learning effect on brain morphology within each cognitive task (reversal learning: t73 = -0.885, p = 0.758; spatial learning: t73 = -0.849, p = 0.758). Interestingly, we found that the learning environment seemed to have a strong effect on relative brain size, where individuals exposed to the spatial learning

environment had overall larger relative brain size in comparison to those assigned to the reversal learning setup (t73 = -3.30, p = 0.0074, Figure 3). Similarly, the relative optic tectum size of individuals subjected to the spatial learning environment were significantly larger in comparison to those in the reversal learning group.

Figure 3. Effects of the different treatment groups on relative brain size in female guppies. Shown on the figure are the estimated marginal means and standard errors (± sem) on the y-axis, with the different treatment groups on the x-axis. P-values were calculated following Holm’s adjustment for multiple comparisons. CRL, reversal learning environment control; TRL, reversal learning treatment; CSL, spatial learning environment control; TSL, spatial learning treatment.

The fact that no apparent changes in overall brain size and size of various brain regions were detected in response to learning could perhaps be due to the nature of the cognitive tasks chosen. Given that the tests were designed to mimic ecological challenges presented in the wild, it may very well be that individuals possess sufficient neural

0.675 0.700 0.725

CRL

Treatment

Relative brain size

TRL CSL TSL

***

**

**

processing capacity to succeed in both spatial and reversal learning. However, it remains to be tested whether more fine-scale alterations in neural processes have occurred.

We showed that the physical test environment had a considerable effect on both brain size and size of the optic tectum. The increased structural complexity of the spatial learning setup could have required the integration of multiple cues in order to navigate to the food reward. This is in contrast to the simpler test environment experienced by individuals in the reversal learning group. The fact that we only found an effect of the test environment on brain morphology changes could possibly be due to the intricate link between environmental variation in the wild and the associated cognitive challenges (Grether et al. 2001; Kelly et al. 1999; Kotrschal et al. 2017; Templeton 2004). Thus, the plastic aspects of the brains of guppies may be mainly targeted to environmental changes, potentially because they are most often intimately connected to cognitive demands in this species.

Paper II

After three generations of selection, relative telencephalon size was approximately 7 % and 5 % larger in the up-selected lines in comparison to the down-selected lines in females and males, respectively (Figure 4). This difference was significant in females (b = 0.031 [0.0052; 0.061], PMCMC = 0.040), and a non-significant trend in males (b = 0.024 [-0.00091;

0.056], PMCMC = 0.068). A non-significant trend for selection to progress in an asymmetrical manner was detected with a larger change in relative telencephalon size in the up- selected lines in relation to the unselected controls in both sexes (females up-selected vs.

controls: b = 0.016 [-0.0016; 0.036], PMCMC = 0.064; males up-selected vs. controls: b =

0.034 [-0.0076; 0.085], PMCMC = 0.088). Importantly, when comparing the selection lines directly to the starting population, there was a highly significant difference in relative telencephalon size between the starting population (F0) and the current generation (F3) in both females (down-selected: b = 0.084 [0.047; 0.11], PMCMC < 0.001; up-selected: b = 0.11 [0.077; 0.15], PMCMC < 0.001) and males (down-selected: b = 0.043 [0.011; 0.074], PMCMC = 0.012; up-selected: b = 0.067 [0.034; 0.097], PMCMC = 0.002). There were no differences in relative brain size between up- and down-selected lines for either sex (females: b = -0.0014, [-0.016; 0.017], PMCMC = 0.85; males: b = 0.011 [-0.013; 0.036], PMCMC

= 0.24).

Figure 4. Selection response for relative telencephalon size over three generations. P indicates the starting population. Illustrated are the means and standard error values (± sem) for standardized residuals of

telencephalon volume regressed on total brain remainder volume within each generation and replicate.

The figure shows the selection response across the three replicates, with the left and right panel illustrating the response in females and males, respectively.

When we examined the progression of brain evolution under directed selection by comparing the telencephalon sizes of our selection lines with those of wild-caught guppies, we found that the mean relative telencephalon size of our unselected control line was most similar to those possessed by the wild indigenous populations ((mean relative telencephalon volume calculated as the residuals from the regression of telencephalon on the brain remainder): down-selected: -0.41 ± 0.09; unselected control:

-0.04 ± 0.10; up-selected: 0.37 ± 0.09; wild fish: 0.03 ± 0.08) (Figure 5)). Selected lines possessed a smaller range of relative telencephalon sizes compared to those found under natural conditions (selection lines: range -0.41 to 0.37; wild populations: -0.93 to 1.02).

Figure 5. Comparison of relative telencephalon size in generation F3 female guppies and wild type fish obtained from 16 different sites in Trinidad. Plotted on the y-axis are the estimated marginal means with 95 % confidence intervals, obtained from standardized residuals of telencephalon volume regressed on total brain remainder volume. P-values were calculated following Holm’s adjustment for multiple comparisons. ***P<0.001, **P<0.01, *P<0.05.

Here, the first experimental support for the mosaic brain evolution hypothesis was provided by artificially selecting for relative telencephalon size in guppies. This independent evolution in a specific brain region without changes in overall brain size is suggestive of mosaic brain evolution being a possible evolutionary response to cognitive selection that may minimize energy expenditure on unnecessary neural tissue.

**

*

*

***

*

Paper III

We extracted three principal components which accounted for 65 % of the total variance and were subsequently interpreted as ‘boldness-activity’, ‘neophilia’ and ‘habituation’, respectively. Given the established role of the telencephalon in a multitude of cognitive processes, we expected that fish with known differences in telencephalon size could differ in their behavioural responses towards various tests of novelty. Contrary to initial predictions, we did not find an effect of telencephalon size on any of the identified boldness traits (PC1: χ²1 = 0.63, p = 0.43; PC2: χ²1 = 0.08, p = 0.78; PC3: χ²1 = 0.27, p = 0.60).

A possible explanation may be related to underlying neurological characteristics such as neuron numbers and interconnectivity, which may be of greater consequence for inter- individual variation in boldness, rather than the overall size of a specific brain region.

We did find that males tended to be more active and quicker to emerge from the start compartment in comparison to their female conspecifics (χ²1 = 10.11, p = 0.0015).

Conforming to this difference, sex-specific differences in activity budgets in guppies have shown that males tend to be more explorative under natural conditions, likely due to a higher motivation to search for mates.

It can be surmised that any potential differences in cognitive performance between selection treatments may not be simply motivated by differences in boldness levels.

However, we did find substantial individual variation in component scores for all identified traits. An appropriate next step would perhaps be to examine the link between telencephalon size and boldness at the individual level, to get a more thorough

understanding of how this brain region, presumably responsible for decision making and emotional control (Portavella et al. 2004), influences boldness in the guppy.

Paper IV

Differences in performance during the pre-training trials were detected between selection treatments, where a closer examination revealed that male guppies from the up-selected treatment achieved a steeper learning curve in comparison to individuals from the down-selected treatment (up-selected: emtrend = -0.39, p < 0.001; down- selected: emtrend = -0.153, p = 0.02). During the test trials, selection treatment did not have an effect on the latency to obtain the food reward (t1,70 = 0.18; p = 0.67). However, up-selected males outperformed their down-selected conspecifics in terms of the number of successful detours without touching the barrier (p = 0.031, Figure a).

Figure 6. Mean and 95 % confidence level of correct detours a) across trials and selection treatment and b) average over all trials for each selection treatment. *P<0.05.

0.0 0.2 0.4 0.6

1 2 3 4 5 6 7 8 9 Test trials

Correct detour

a

0.00 0.05 0.10 0.15 0.20

Selection line b

Selection line small large

*

Although the exact mechanisms responsible for the accelerated performance attained by up-selected males remains unknown, this is the first experiment providing direct support for the cognitive benefits provided by evolutionary changes in telencephalon size.

CONCLUSIONS AND FUTURE DIRECTIONS

The main theme surrounding this thesis deals with the evolvability of a specific brain region, the telencephalon, and the corresponding ramifications. More specifically, I aimed to test the mosaic brain evolution hypothesis by means of artificially selecting for an increase and decrease in the size of the telencephalon in guppies. To the best of our knowledge, this is the first such experimental undertaking to understand the processes governing brain evolution. I first investigated two possible factors that may influence plastic alternations in neural structures, namely environmental enhancement and learning processes (Paper I). The effects of environmental enrichment on brain plasticity have been profoundly examined over the years, due to the potential beneficial effects attributed to such enrichments (Hutchinson et al. 2012; Kempermann et al. 2002; Pereira et al. 2007; van Praag et al. 2000). The general consensus is that the provision of enrichment, be it social or asocial, tends to promote neurogenesis, dendritic branching or synaptogenesis in specific brain structures, although effects may vary across species. On the other hand, learning-related plasticity changes in neural structures have not been as extensively studied. By employing a cross-sectional approach in this study, we were therefore able to examine for the effect of cognitive processes on structural plastic alterations in the guppy brain, while accounting for potential changes pertaining to environmental influences. And although we show here that the guppy brain remains more amenable to plastic changes in response to the physical environment, the inclusion of

more challenging cognitive tasks in future studies may help to unveil how and whether learning affects brain morphology changes in this species, advancing our understanding of the neural underpinnings of brain plasticity.

Next, I performed an artificial selection experiment for relative telencephalon size in guppies and presented evidence for the mosaic brain evolution hypothesis after three generations of selection (Paper II). I hypothesized that this independent alteration of a single brain region could be due to unsubstantial physical and genetic correlations between brain regions, but this remains to be tested further. I found a trend for an asymmetrical response to selection, where the selection for a larger telencephalon progressed more rapidly than the selection for a small telencephalon. This suggests the potential existence of a cognitive threshold such that a minimum telencephalon size is required for fundamental processes. I also demonstrated that the relative telencephalon size of unselected control lines was not significantly different to that of wild fish. In contrast, both up-selected and down-selected females had larger and smaller telencephalon size in comparison to the wild populations, respectively.

In light of previous studies supporting the link between brain size and personality, I subsequently tested whether such differences in behavioural traits also exist in our selection lines (Paper III). A difference in boldness, for instance, may influence how individuals interact with test apparatus and correspondingly, their learning acquisition rate (Guenther and Brust 2017; Kareklas et al. 2017; Sneddon 2003). I found no such evidence for an effect of telencephalon size on boldness, yet perceptible sex-specific effects were noted. In a previous artificial selection experiment for overall brain size,

guppies with larger relative brains exhibited a more proactive personality (Kotrschal et al.

2013a). Although existing evidence for the association between brain size and boldness remains divided, no such study has delved into the effects of a specific brain region on this personality trait. Admittedly, our results argue against the role of the telencephalon in the mediation of boldness traits in guppies, but considering that guppies are a social living species, assays incorporating various social components would be a good extension to the current study.

The results from the last chapter (Paper IV) demonstrate a particular executive cognitive benefit (i.e. inhibitory control) associated with the evolutionary increase in the size of the telencephalon. Our finding that even a small difference in telencephalon size between the selection treatments resulted in a detectable improvement in performance for fish with a larger telencephalon lends further support for the cognitive benefits associated with mosaic brain evolution. As was formerly mentioned, current support for the mosaic brain evolution hypothesis are mainly derived from interspecific comparative analyses, wherein the enlargement of specific regions is coupled to various functional specializations such as food hoarding behaviour in birds (Garamszegi and Eens 2004).

The establishment of the artificial selection lines for telencephalon size in guppies provides the first experimental support for the mosaic brain hypothesis. Even though we show here that such selective increases in a specific brain region correspond to improved performance in a test of self-control, an important indication for mosaic brain evolution being an engine of cognitive evolution, much work remains with regards to the behavioural consequences of such a directed selection. We did not find any trade-offs in

terms of reproductive investment in our selection treatments even though the high energetic expenditure accrued to the development and maintenance of neural tissue has long been established. Hence, various other costs associated with the enlargement of the telencephalon merit closer examination. Given the implication of the telencephalon in various cognitive aspects, the use of these selection lines in behavioural assays such as reversal learning, social learning, spatial learning and memory, predator inspection and mate choice, among others, remain of great interest. In addition, examining changes in neuron numbers, metabolic rates as well as underlying gene expression levels in response to directed selection for telencephalon size would go a long way in understanding what drives mosaic brain evolution and how this has led to the huge diversity in brain morphology.

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SVENSK SAMMANFATTNING

Att förstå hur evolutionen av hjärnan resulterat i den enorma variation i hjärnmorfologi vi ser bland nu levande ryggradsdjur har fascinerat forskare i årtionden. Hjärnans morfologi bestäms både genetiskt och av omgivande miljöbetingelser. Till exempel anpassningar till ekologiska utmaningar har föreslagits vara en viktig drivkraft i hjärnans diversifieringsprocesser. Nervvävnad är mycket energikrävande och evolutionen av hjärnan är därför troligen en mycket komplex process som involverar en delikat balans mellan kostnader och fördelar.

Jag använde mig av guppyn (Poecilia reticulata) som modellorganism för att först undersöka under vilka förhållanden diversitet i hjärnmorfologi genereras. Jag gjorde detta genom att undersöka miljömässiga och kognitiva faktorer, som man sedan tidigare vet kan påverka hjärnplasticitet (Kapitel I). Tidigare studier har visat att ökad miljöberikning leder till en förstoring av vissa delar av hjärnan. Medan ökad miljökomplexitet ledde till plastiska förändringar i hjärnmorfologi fann jag att inga signifikanta skillnader efter framgångsrikt genomförande av två kognitiva test.

Jag undersökte sedan möjligheten för mosaikevolution av hjärnan genom att artificiellt selektera för ökad och minskad telencephalonstorlek (Kapitel II).

Telencephalonstorlek visade sig svara snabbt på divergerande selektionstryck medan övriga hjärnregioner var oförändrade. Telencephalonstorleken hos fisk från den icke- selekterade kontrollbehandlingen skiljde sig inte från den hos vildtypsfiskar, medan både upp- och nedselekterade fiskar hade större respektive mindre telencephalon.

Fisk från de artificiella selektionslinjerna testades sedan i tre olika tester för att undersöka om skillnader i telencephalonstorlek påverkar djärvhet (Kapitel III). Tiden det

tog för individer att komma ut från ett skydd och tendensen att undersöka en tom arena och ett okänt föremål undersöktes. Jag fann inga skillnader i djärvhet mellan de olika selektionslinjerna, men hanar visade sig vara mer djärva och aktiva än honor.

De kognitiva fördelarna med att ha en stor telencephalon undersöktes i ett självinhiberingstest (Kapitel IV). Guppies från de uppselekterade linjerna lärde sig snabbare och gjorde fler korrekta vägval jämfört med de nedselekterade linjerna.

Sammanfattningsvis har jag funnit experimentellt stöd för hypotesen om mosaikevolution av hjärnan genom att visa att olika hjärnregioner kan evolvera snabbt och oberoende av andra regioner under riktad selektion. Framtida test av kostnader, fördelar i andra kognitiva test och detaljerade undersökningar av nervvävnaden kommer att hjälpa oss att vidare reda ut vilka faktorer som ligger bakom evolutionen an hjärnan.

ACKNOWLEDGEMENTS

First and foremost, I would like to thank my supervisor, Niclas Kolm, for giving me this unique opportunity to be part of this once-in-a-lifetime project. Niclas, thanks for being a consistent supportive force, celebrating every little win with my selection experiment and helping me push forward throughout the past 4+ years. I faced many obstacles and setbacks over the years, but you believed in me and inspired me to never give up.

This taught me to be more resilient and to adopt a generally more positive outlook, something I am ultimately grateful for. I would also like to acknowledge my co-supervisor, Björn Rogell, who has no doubt assisted and taught me a lot regarding statistics, and for the constant encouragement.

I owe much gratitude to the rest of the Kolm lab: Mirjam, Annika and Zegni. Mirjam, you were always willing to lend a helping hand no matter how busy you were (which you always were), and I greatly appreciate the countless number of times I approached you for advice. Of course, a huge thank you for your help with the translation of my thesis summary as well. Annika, you were always around the department even before I started, so I knew I could turn to you for guidance in the lab. You have helped me more than you know and I thoroughly enjoyed all our conversations, be it work related or not, and of course attending our first conference together. Zegni, you have been a mentor to me during this past year, giving me valuable feedback on my manuscripts and experiments.

Thanks for all the fascinating discussions we have had, I have definitely learnt a lot from you.

Not forgetting previous members of the lab, Séverine, Alex, Wouter, Alberto and Teddy.

Séverine, you were nothing but welcoming when I first moved to Stockholm, helping make the move much easier. When I first started at the department, I knew I could always turn to you for help and you were always around to assist in any way you could. You have been a huge influence during my PhD journey, and I am thankful for that. Alex, if it weren’t for your constant guidance, I would have been entirely lost with my selection experiment.

You showed me the steps needed for such a huge undertaking, and taught me how to do my first dissection. Wouter, aka R guru, I owe a great debt to you for your willingness to help me with my analyses even after you moved to the faraway land of Canada. I have definitely learnt a lot about R from you. Alberto, thanks for providing advice with my experiments when needed and volunteering your time to help me resolve whatever problems I encountered in the lab. Teddy, you were not only someone I could turn to for advice during the start of my PhD, but you were an awesome office mate with all your jokes and constant discussions about Eurovision. I would also like to thank Vivien Holub, who was tasked with the huge responsibility of looking after thousands of my fish in the lab. I cannot emphasize enough how much easier you made my life but ensuring that my fish were well taken care of.

Like any other PhD student, I started my journey feeling extremely optimistic and excited.

But over the years, it was the encouragement and support from friends and colleagues that saw me through this journey. Erika, you were always bugging me to speak Swedish, correcting (and laughing at) me whenever I got it wrong. But I am grateful that you took the effort to help, and of course for our many outings/dinners/trips. Maddi, you have always been there with words of encouragement and believed in me. You have been a

true friend and continue to be a great help in the lab. Regina, I truly appreciate all the help and advice you have given me. You would always take time out and try your best to help me. Hannah, thanks for listening to me rant, for discussing statistics with me and for always making me laugh with your witty humour. Laura, thank you for being someone I knew I could always turn to. You have been a great friend, teammate, gossip companion and so on, and always had my back. You guys have always been there for me, through all the sweat and tears and to constantly encourage me to power though, and words simply cannot express how grateful I am for everything you have done for me. When I first moved to Stockholm, I was fortunate enough to be able to live together with two other students.

To Despina and Cathy (aka my pumpkins as affectionately labelled), thanks for always being so supportive and believing that I would make it to the end. And of course, to all my innebandy teammates in Sumpan, you guys have kept me sane. I always look forward to trainings after a stressful day at work and you have all helped to keep me positive and going strong.

To everyone in the department, each and every one of you have made working here much more enjoyable and memorable and I am extremely appreciative for everything. To my office mates, Malin, Ciaran, Amy, thanks for all the fun times we had pre-covid, and for making our shared office space one filled with productivity and laughter.

It goes without saying that I am exceptionally grateful for the support I have received from my family despite the distance and time difference. My parents, Catherine and Michael, have always supported me when I wanted to pursue my dream, even if it meant me moving overseas. Thanks for always believing in me and for pushing me to venture outside

of my comfort zone. To my sisters, Sam and Sarah, you two have been my constant support system through it all, and I am super grateful for having you both as my siblings.

And a special shout out to my amazing designer sister for helping me with the cover page of my thesis.