Preface. Ås, 9/ Torbjørn H. Kornstad - PDF

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Preface The intersection between ecology and genetics is an extremely interesting research field, where many connections and mechanisms are yet to be revealed. If we were able to understand more of the

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Preface The intersection between ecology and genetics is an extremely interesting research field, where many connections and mechanisms are yet to be revealed. If we were able to understand more of the genetic forces that drive adaptation and evolution, we would be better fit to meet ecological challenges like habitat destruction, invasive and alien species, climate change, and so on. Current research has come a long way, and only the last ten years molecular techniques have shown a rapid development, allowing a much better approach to the concept. At the same time, the more we get to know, the more we realize we do not understand. This thesis constitutes a very small piece of the complete picture, but hopefully this piece will fit in somewhere and give further information to the topic that future work can build upon. Many people have helped me along the path towards completing this thesis. First of all I would like to thank my main supervisor, Associate Professor Siri Fjellheim at the Department of Plant Sciences. She has made a huge effort in supporting me, answering my questions and providing funds, she has joined in fieldwork and she has filled in for me in experimental work when I have needed it. I would also like to thank my co-supervisor Professor Mikael Ohlson at the Department of Ecology and Natural Resource Management for good advice; PhD research fellow Siri Lie Olsen at the Department of Ecology and Natural Resource Management for statistical advice and good writing tips; researcher Marte Holten Jørgensen at the Department of Plant Sciences for statistical advice; engineer Jørn Medlien at the Centre for Plant Research in Controlled Climate and engineers Øyvind Jørgensen, Sylvia Sagen Johnsen and Anne Guri Marøy at the Department of Plant Sciences for help with experiments and lab work; and research technician Leidulf Lund at the University of Tromsø for providing both advice and seeds of diploid Arabidopsis thaliana. Lastly I would like to thank my family for support through the educational course towards a Master of Sciences degree, and my friends for a great time at the Norwegian University of Life Sciences. Ås, 9/ Torbjørn H. Kornstad I Abstract Species that possess more than two sets of chromosomes are denoted as polyploids. It is hypothesized that polyploids show high gene redundancy, hybrid vigour and masking of deleterious alleles, and that this make them better at adapting to novel environments because of wider phenotypic response range. It is also speculated that adaptive advantage of polyploidy contributes to invasiveness as there is a trend that polyploids are overrepresented within invasive species. The allopolyploid Arabidopsis suecica and its parent species A. arenosa and A. thaliana were chosen as a model system to investigate relationships between phenotypic plasticity, fitness and genetic variation. In this thesis I try to uncover genetic structures in the study species, and I investigate if A. suecica show higher plasticity and/or fitness than its parent species, if the different species show different levels of genetic diversity and whether A. suecica could work as a model for studying polyploidy and invasiveness. Three to four wild Norwegian populations of each species were analyzed for phenotypic responses to differences in availability of nutrient and light, while population structure and genetic diversity was assessed through analysis of AFLP markers. The species were separated into genetic and phenotypic clusters with A. suecica being intermediate between its parent species. Clear population structure was inferred in A. thaliana and A. arenosa, while no structure was inferred in A. suecica. The species exhibited similar phenotypic responses. A. arenosa seemed to have higher phenotypic plasticity and higher genetic diversity than the two other species, probably related to its outbreeding reproduction strategy. Furthermore, a general positive relationship between genetic diversity and phenotypic plasticity was found. Low genetic diversity and more population structure were found in the indigenous, inbreeding A. thaliana. Population spacing might explain the clear genetic structure in A. arenosa, while the lack of structure in A. suecica could be due to coherent populations. When it came to fitness measured as allocation of resources to reproduction, the trend pointed towards A. arenosa having lower fitness under poor environmental conditions. A. suecica, on the other hand, showed the ability to keep up fitness under different environmental conditions, which makes it a promising model for investigating invasiveness and polyploidy. Still, further studies are needed to confirm this. Keywords: Polyploidy, invasive species, phenotypic plasticity, fitness, genetic variation III IV Samandrag Artar som innehar meir enn eitt kromosomsett kallast polyploidar. Ein trur at polyploidar har høg grad av duplikerte gen i genomet, høg heterosis og maskerer skadelege allel, og at det gjer dei betre til å tilpasse seg til nye miljø fordi dei har eit vidare fenotypisk responsområde. Det er òg mogleg at adaptive fordelar ved polyploidi kan bidra til høgare invasibilitet, sia det er ein trend at polyploidar er overrepresentert blant invasive artar. Den allopolyploide arten Arabidopsis suecica og foreldreartane A. arenosa og A. suecica vart vald som eit modellsystem for å undersøke samanhengar mellom fenotypisk plastisitet, fitness og genetisk variasjon. I denne gradsoppgåva prøver eg å avdekke genetiske strukturar i studieartane, og eg undersøker om A. suecica har høgare plastisitet og/eller fitness enn foreldreartane, om dei ulike artane har ulike nivå av genetisk diversitet og korvidt A. suecica kan fungere som ein modell for studium av polyploidi og invasibilitet. Tre til fire ville norske populasjonar av kvar art vart analysert for fenotypiske responsar til ulik tilgjengelegheit på næring og lys, medan populasjonsstruktur og genetisk diversitet vart undersøkt gjennom analyse av AFLP-markørar. Artane delte seg i genetiske og fenotypiske klyngar, og A. suecica plasserte seg mellom foreldreartane. Det vart finni ein klar populasjonsstruktur i A. arenosa og A. thaliana, men ikkje i A. suecica. Artane viste liknande fenotypiske responsar. A. arenosa verka å ha høgare fenotypisk plastisitet og høgare genetisk diversitet enn dei to andre artane, truleg grunna ein utkryssande reproduksjonsstrategi. Vidare vart ein generell positiv samanheng mellom genetisk diversitet og fenotypisk plastisitet finni. Låg genetisk diversitet og meir populasjonsstruktur vart finni i A. thaliana som er stadeigen og innkryssande. Adskilte populasjonar kan kanskje forklara den klare genetiske strukturen i A. arenosa, medan manglande strukturar i A. suecica kan vera grunna samanhengande populasjonar. Når det kom til fitness målt som allokering av ressursar til reproduksjon, pekte trenden mot at A. arenosa kan ha lågare fitness under dårlege miljøtilhøve. A. suecica viste derimot evne til å halde oppe fitness under ulike miljøtilhøve, noko som gjer arten til ein lovande modell for å undersøke invasibilitet og polyploidi. Likevel trengs det ytterlegare forsking for å stadfeste dette. Nøkkelord: Polyploidi, invasive artar, fenotypisk plastisitet, fitness, genetisk variasjon V VI Table of contents 1. Introduction Materials and methods Study area Study species Analysis of phenotypic responses Experimental design Measurements of phenotypic variables Measurements of ploidy level and chromosomal numbers Analysis of genetic markers Data analysis Phenotypic responses Population structure and genetic diversity Comparison of genetic diversity and phenotypic plasticity Results Growth experiment Multivariate analysis of phenotypic responses Analyses of phenotypical responses Measurements of ploidy level and chromosomal numbers Analyses of population structure and genetic diversity Population structure Genetic diversity Analysis of molecular variance (AMOVA) Comparison of genetic diversity and phenotypic plasticity Discussion Arabidopsis suecica places between its parent species in both pheno- and genotype No clear population structure could be identified in Arabidopsis suecica Arabidopsis arenosa is most plastic and show the highest level of genetic diversity There is a positive relationship between genetic diversity and plasticity Phenotypic plasticity and high genetic diversity does not imply higher fitness Is Arabidopsis suecica a suitable model for studies of polyploidy and invasiveness? Conclusion Litterature Appendix 1: Pictures from the growth chamber experiment Appendix 2: Protocol for running AFLP Appendix 3: Figures used for inferring numbers of clusters in Structure VII VIII 1. Introduction Polyploidization, i.e. mutations leading to organisms that possess more than two sets of chromosomes, is recognized as a driving force for adaptation and ecology (Lynch 2007; Sobel et al. 2010). Polyploidy can be observed in numerous taxonomic groups, but is thought to be especially frequent in angiosperms (Wendel 2000; Otto 2007; Song et al. 2012). In fact, it is often assumed that all angiosperms have undergone polyploidization at some point during their evolution (De Bodt et al. 2005; Soltis & Soltis 2009). There are two main ways of gaining polyploidy, namely autopolyploidy where the genome is duplicated within a species, and allopolyploidy where a new species is formed from hybridization between two parent species combined with whole genome duplication (Soltis & Soltis 2000). Successful allopolyploidization results in rapid speciation in an evolutionary context. The overall polyploidization rate is about 1/10 th of the overall speciation rate (Meyers & Levin 2006; Otto 2007), meaning that over a longer time span it will not constitute the most important speciation force. Also, polyploids themselves show reduced speciation rates, partly due to the fact that their possibilities of undergoing new polyploidizations are lower than in diploids (Mayrose et al. 2011; Arrigo & Barker 2012). However, in a world with large ecological changes within short time spans, it is reasonable to believe that speciation as a result of polyploidization could have ecological consequences, and these consequences should be investigated. When a species is polyploid and possesses more than two sets of chromosomes, genetic forces act differently from what they do in diploids. A newly formed allopolyploid combines genes from two unrelated individuals, opening up for hybrid vigour and masking of deleterious alleles (te Beest et al. 2012). The combination of homeologous genes from two parent species often results in one of the genes being silenced, but it is proposed that subfunctionalization could work as a mechanism for retaining homeologous genes in the genome (Lynch & Force 2000; Hegarty & Hiscock 2008). A high gene redundancy due to the presence of homeologous loci suggests that allopolyploids could withstand inbreeding and population bottlenecks better than their diploid counterparts (Song et al. 2012; te Beest et al. 2012). Following this logic, allopolyploids could be better at adapting to new environments and sudden environmental changes, due to the underlying gene redundancy. The generation of new expressional patterns and novel epigenetic variation could also contribute to this (Comai 2005; Chen 2007). At the same time, there are genetic forces associated with polyploidization 1 that could be detrimental. The genome is notoriously unstable, and polyploidization is a process that changes the genome abruptly in just one generation. This can lead to problems in the mitosis and meiosis giving aneuploid cells, and problems with gene expression due to development of uneven relationships between genes and regulatory factors (Comai 2005). Epigenetic re-modelling could also cause instability in newly formed polyploids (Comai et al. 2003a). Though polyploidization is believed to imply both advantages and disadvantages, the view that polyploidization opens up for wider ecological and phenotypical variation and thus enable species to adapt quickly is widely accepted (Comai 2005; Otto 2007; Flagel & Wendel 2009; te Beest et al. 2012; but see Meyers & Levin 2006; Mayrose et al. 2011; Arrigo & Barker 2012). Summed up, a theoretical framework for a possible positive relationship between polyploidy and abilities to adapt is established (Flagel & Wendel 2009). An important task now is to find out whether causal relationships exist, and eventually uncover how they work. With this as a background, it is highly interesting to carry out an experiment where an allopolyploid species is compared with its parent species with regard to performance under different environmental conditions. If the proposed ideas on the benefits of being polyploid hold true, the allopolyploid should show better performance and keep up fitness across a range of environmental conditions. A study system with only three species will not provide results that can be directly generalized to all allopolyploids, but it is a good way of building up a model that can be expanded in later experiments. Further on, it has been proposed that polyploids tend to have higher probability of being invasive than diploids (Lee 2002; Pandit et al. 2011; te Beest et al. 2012). In this perspective, a model system for comparing an allopolyploid species with its parent species could also act as a model system for understanding some of the underlying mechanisms that lead to a species becoming invasive. The species complex chosen to assess these propositions consists of the allopolyploid species Arabidopsis suecica (Fr.) Norrl. ex O.E.Schulz and its two parent species, A. thaliana (L.) Heynh. and A. arenosa (L.) Lawalrée. Within the species complex, the model species A. thaliana is well investigated. Further on, the species are simple to grow and have a relatively short lifespan. This provides a good background for the thesis work. In Norway A. thaliana is regarded as an indigenous species, although it has the ability to behave like a weed (Elven 2005). The other two are regarded as alien species and classified in the risk category Potentially High risk (PH). This means that they show low or no impact on the Norwegian 2 nature per now, but it is believed that this could possibly change in the future (Gederaas et al. 2012). With this information as a background, it is investigated how the species complex could work as a model system for studying the genetic and phenotypic effects of allopolyploidization, also in an invasive species perspective. A. suecica is not viewed as a threat to Norwegian nature as of today, but it is here proposed that it could have the potential to work as a model species. Koch and Matschinger (2007) call for genetic research on nonmodel species in the Arabidopsis genus. Further on, the chosen species complex has been proposed as a model system for studying what effects polyploidization has on the genome itself (Chen et al. 2004). It is assumed that higher genetic diversity constitutes a foundation for higher fitness (Reed & Frankham 2003). Thus, a study on allopolyploidy and fitness in the Arabidopsis genus should include genetic investigations. While population structure and genetic diversity is well investigated in A. thaliana (e.g. Beck et al. 2008; Lewandowska Sabat et al. 2010), there is still a long way to go when it comes to A. suecica and A. arenosa. A study conducted by Lind-Hallden et al. (2002) compared genetic diversity in the three species, but otherwise little knowlegde is available. This thesis aims to contribute in filling the knowledge gaps by assessing and comparing population structure and genetic diversity between the three species based on genotyping from Amplified Fragment Length Polymorphism (AFLP) markers. Two terms are assessed specifically in the thesis: Phenotypic plasticity and fitness homeostasis. The first is the ability to exhibit a wide range of phenotypes across varying environmental conditions (Bradshaw 1965; Schlichting 1986), the second is the ability to keep fitness as equal as possible between varying environmental conditions (Richards et al. 2006; Hulme 2008). It is proposed that high phenotypic plasticity provides wider possibilities to adapt to new environments (Sultan 2000; Davidson et al. 2011), while high fitness homeostasis could imply better abilities at coping with and adapting to stressful environments (Richards et al. 2006; Hulme 2008). The terms are assessed through analysis of phenotypic variation as response to environmental conditions exhibiting different levels of stress. An attempt is done to compare the two terms in light of the results. 3 Based on the theoretical framework, the following research questions were formulated: 1. Do the allopolyploid A. suecica show higher phenotypic plasticity and/or fitness homeostasis than its parent species, and does this reflect a higher ability to adapt to different environments? 2. Do the study species show different levels of genetic diversity, and if so, is this related to phenotypic plasticity? 3. Is A. suecica suitable as a model species for studying relationships between polyploidy and invasiveness, even though the species currently does not behave in an invasive way? 4 2. Materials and methods 2.1. Study area Seeds from 10 wild populations of A. thaliana, A. suecica and A. arenosa were sampled. The number of sampled populations per species was three A. thaliana populations, three A. suecica populations and four A. arenosa populations. The seeds were sampled from three different geographic areas within Southern Norway, namely Drammen, Eidskog and Gudbrandsdal, so that seeds from at least one population of each species were sampled from each geographical area (Table 1, Fig. 1). Table 1: List of populations where seeds were sampled, specifying locality codes, locality names, what geographical areas the different localities belong to, species, collection date, latitude in degrees north (Lat ( N)) and longitude in degrees east (Long ( E)). Code Locality name Geographical Collection Lat Long Species area date ( N) ( E) T-EID1 Bakkeberget Eidskog A. thaliana S-EID3 Åbogen stasjon Eidskog A. suecica A-EID4 Pramhus Eidskog A. arenosa A-DRA1 Berskog Drammen A. arenosa S-DRA2 Drammen stasjon Drammen A. suecica T-DRA3 Åslyveien Drammen A. thaliana T-SFRO3 Kjorstad Gudbrandsdal A. thaliana S-NFRO3 Kvam stasjon Gudbrandsdal A. suecica A-NFRO4 Nymoen Gudbrandsdal A. arenosa A-GAU1 Steinslia Gudbrandsdal A. arenosa For each population, 20 randomly chosen individuals were sampled. If a population consisted of less than 20 individuals, as many individuals as possible were sampled. The plants were dried, and the seeds extracted and transferred to 2 ml tubes (Eppendorf, Hamburg, Germany). 5 Fig. 1: Map showing localities of populations where seeds were sampled for the experiment Study species The species collected all belong to the genus Arabidopsis, and they form a hybrid complex. A. suecica originates from an allypolyploid hybridization between the mostly diploid A. thaliana and the mostly autotetraploid A. arenosa (O Kane et al. 1996; Jakobsson et al. 2006), possibly within the eastern parts of A. thaliana s native range (Beck et al. 2008). The formation of the species probably happened through the fertilization of a female, unreduced A. thaliana gamete with a normal, male A. arenosa gamete (Säll et al. 2003). It is believed to have risen in a single event between and years ago, somewhere south of its present native distribution in Sweden and Finland (Säll et al. 2003; Jakobsson et al. 2006). It has been shown that out of A. suecica s 26 chromosomal pairs, 16 derive from A. arenosa and 10 derive from A. thaliana (Comai et al. 2003b). A. suecica exhibits bivalent, homologous pairing of its chromosomes in the meiosis (Comai et al. 2003b; Pecinka et al. 2011). Studies indicate that A.
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