UnknownDigital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2043
Ecology and evolution of local adaptation in Arabidopsis thaliana
GIULIA ZACCHELLO
ISSN 1651-6214 ISBN 978-91-513-1206-4 urn:nbn:se:uu:diva-440264
Dissertation presented at Uppsala University to be publicly examined in Zootissalen, EBC, Villavägen 9, Uppsala, Thursday, 10 June 2021 at 10:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: Dr. Xavier Picó (Estación Biológica de Doñana, CSIC, The Spanish National Research Council).
Abstract Zacchello, G. 2021. Ecology and evolution of local adaptation in Arabidopsis thaliana. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 2043. 50 pp. Uppsala: Acta Universitatis Upsaliensis. ISBN 978-91-513-1206-4.
In this thesis, I investigate the ecological genetics of local adaptation in the annual herb Arabidopsis thaliana with the aim to identify key traits, agents of selection, and genomic regions contributing to adaptive differentiation. The thesis focuses on adaptive differentiation in timing of germination and seed dormancy between populations located close to the southern and northern range margins in Europe. I aimed to: (1) determine how much a particular genomic region contributes to local adaptation between an Italian and a Swedish population, (2) characterize selection on timing of germination in a Mediterranean climate, (3) quantify variation in seed dormancy and identify potential agents of selection on seed dormancy within Italy and Fennoscandia, and (4) examine the genetic basis of the seed dormancy cycle and the adaptive value of seed banks.
Differences in a genomic region at the end of chromosome 5 explained a large proportion of differences in fitness, and in germination and flowering time between the Italian and Swedish population.
A field experiment at the site of the Italian population, indicated strong stabilizing selection on timing of germination with an optimum coinciding with the time of germination in the local population.
Seed dormancy of Italian populations was stronger than that of Fennoscandian populations, but also varied considerably within regions, indicating that this trait has considerable evolutionary flexibility. In Fennoscandia, variation in seed dormancy was related to climatic conditions in summer, suggesting that differences are at least partly adaptive.
The seed dormancy cycle in the soil differed between the focal Italian and Swedish population, and matched seasonal changes in conditions for seedling establishment at their sites of origin. Differences in the genomic region at the end of chromosome 5 could explain a large proportion of the difference in the seed dormancy cycle. Mortality of seeds was much higher in Italy than in Sweden, indicating that the importance of the seed bank for population dynamics differs between the two sites.
Overall, the results suggest that differences in a genomic region on chromosome 5 and in early life stages can play a key role in local adaptation in A. thaliana.
Keywords: Arabidopsis thaliana, germination time, natural populations, Near isogenic lines, reciprocal transplants, seed bank, seed dormancy
Giulia Zacchello, Department of Ecology and Genetics, Plant Ecology and Evolution, Norbyvägen 18 D, Uppsala University, SE-752 36 Uppsala, Sweden.
© Giulia Zacchello 2021
Alla mia famiglia
List of Papers
This thesis is based on the following papers, which are referred to in the text by their Roman numerals.
I Zacchello, G., Oakley, C.G., Ågren, J. Disentangling effects of linked
fitness QTL on local adaptation (Manuscript)
II Zacchello, G., Vinyeta, M., Ågren, J. (2020) Strong stabilizing selec- tion on timing of germination in a Mediterranean population of Ara- bidopsis thaliana. American Journal of Botany, 107: 1518-1526
III Zacchello, G., Bomers, S., Böhme, C., Postma, F., Ågren, J. Seed dor-
mancy varies widely among Arabidopsis thaliana populations both between and within Fennoscandia and Italy (Submitted manuscript)
IV Zacchello, G., Ågren, J. Seed survival in the soil and genetic differ-
ences in seed dormancy cycle in Arabidopsis thaliana (Manuscript)
Reprints were made with permission from the respective publishers.
Contents
Introduction ..................................................................................................... 9 Local adaptation ......................................................................................... 9 Genetic basis of local adaptation .............................................................. 10 Selection on timing of germination .......................................................... 11 Regulation of timing of germination: seed dormancy .............................. 13 Seed banks ................................................................................................ 15 My project ................................................................................................ 16
Material and Methods ................................................................................... 17 Study species ............................................................................................ 17 Study system ............................................................................................ 17 Reciprocal transplant experiments (I) ...................................................... 19 Manipulation of timing of germination (II) .............................................. 20 Quantification of regional variation in seed dormancy and correlations with environmental conditions at sites of origin (III) ............................... 21 Reciprocal seed burial experiment (IV) ................................................... 22
Results and Discussion ................................................................................. 24 Strong effects of genomic regions towards the end of chromosome 5 on local adaptation (I) .............................................................................. 24 Strong stabilizing selection on timing of germination (II) ....................... 25 Within-region variation in seed dormancy is related to differences in summer conditions (III) ............................................................................ 27 Genetic and environmental factors contribute to intraspecific variation in seed bank traits (IV) ............................................................................. 28
Conclusions ................................................................................................... 30
Local adaptation: The process through which populations show higher fitness in their home environment than populations originating from other locations (Kawecki and Ebert 2004).
Near Isogenic Lines (NILs): Lines in which specific genes or genomic regions have been introgressed from a donor parental line into an oth- erwise homogeneous genetic background (Keurentjes et al. 2007).
Phenotype – environment association: Co-variation between popula- tion-mean phenotypes and environmental variables measured at the sites of origin (Michel et al. 2014).
Quantitative Trait: Trait that varies continuously. Examples are hu- man height, plant size or day of flowering. Quantitative traits are often affected by a large number of genes, by environmental variation and by genotype-by-environment interactions (Rifkin 2012).
Quantitative Trait Locus (QTL): A section of DNA that correlates with phenotypic variation of a quantitative trait (Rifkin 2012).
Recombinant Inbred Lines (RILs): Collection of lines derived from a cross between genetically divergent lines and used to pinpoint quan- titative trait loci (Pollard 2012).
Seed dormancy: A mechanism that prevents germination under good germination conditions. Primary : dormancy imposed by the mater- nal plant and expressed by seeds at the time of maturation and release. Secondary : dormancy acquired by seeds after seed dispersal (Baskin and Baskin 2014).
Soil seed bank: Reserve of viable non-germinated seeds in the soil (Baskin and Baskin 2014).
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Introduction
One of the most fascinating aspects of the natural world is the vast diversity that it hosts. Variation among living organisms can be observed among spe- cies, populations within a species, and individuals within populations. Under- standing the processes that shape such diversity remains a key challenge in evolutionary biology and is essential for predicting effects of climate change on biodiversity. In this thesis, I focus on the processes that generate diversity within species.
Local adaptation Populations of the same species can experience different selective pressures due to environmental heterogeneity. As a result of this divergent selection, populations may evolve traits that well suit their local environment and be- come locally adapted (Kawecki and Ebert 2004). Local adaptation can be de- tected using reciprocal transplant experiments, in which individuals of differ- ent populations are transplanted into each other’s environment (Clausen et al. 1940). Populations are considered locally adapted when local populations have higher fitness than non-local populations in such experiments (Fig. 1; Kawecki & Ebert, 2004; Lascoux et al., 2016).
Divergent selection and local adaptation contribute to the maintenance of genetic variation within species (Linhart and Grant 1996), can lead to specia- tion (Kawecki and Ebert 2004), and can affect species’ responses to climate change (Hoffmann and Sgrò 2011). Being fundamental in many aspects of biology, local adaptation has been widely studied. Since the introduction of its basic concept by Turesson in 1922, widespread evidence of local adapta- tion has been documented in both animals and plants (Leimu and Fischer 2008; Hereford 2009). However, the ecological and genetic mechanisms un- derlying local adaptation is still a field of active research.
The traits mediating local adaptation have often not been identified (Kawecki and Ebert 2004), and we have limited information about the envi- ronmental factors that lead to local adaptation (MacColl 2011; Wadgymar et al. 2017) and the genetic basis of local adaptation (Kawecki and Ebert 2004; Mitchell-Olds and Schmitt 2006; Savolainen et al. 2013). In this project, I investigate the ecological genetics of local adaptation using the model
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organism Arabidopsis thaliana with the aim to identify key traits, agents of selection, and genomic regions contributing to adaptive differentiation.
Genetic basis of local adaptation The genetic basis of local adaptation has received increasing interest in the last decades thanks to the increase in available tools for studying genomic variation. One approach used to study the genetics of local adaptation is to reciprocally transplant hybrid mapping populations (Gardner and Latta 2006; Verhoeven et al. 2008; Lowry et al. 2009; Ågren et al. 2013; Leinonen et al. 2013; Postma and Ågren 2016; Lowry et al. 2019). In these experiments, gen- otypes from different habitats are crossed, the resulting segregating popula- tions are exposed to the native environment of the parental genotypes and fi- nally, quantitative trait loci (QTL) for fitness and putatively adaptive traits are mapped. The segregating populations used are typically F2 hybrids (Verhoeven et al. 2008; Leinonen et al. 2013) or Recombinant Inbred Lines (RILs; Gardner and Latta 2006; Lowry et al. 2009; Ågren et al. 2013; Dittmar et al. 2014; Postma and Ågren 2016), in which the effects of QTL are evalu- ated against a wide range of genomic backgrounds (Fig. 2).
A complementary tool to F2 and RILs are Near Isogenic Lines (NILs), in which specific genomic regions are introgressed into a homogeneous genomic background (Fig. 2). By comparing trait expression of NILs that differ in the introgression segments they carry, it is possible to evaluate the effect of well- defined genomic regions and thus disentangle the effects of neighboring QTL.
In experiment (I), I test the extent to which a genomic region including previously identified fitness QTL contributes to fitness and trait differences between two locally adapted populations by using NILs.
Figure 1. Signal of local adaptation. In reciprocal transplant experiments, populations are reciprocally trans- planted to their sites of origin. If the local population has higher fitness than the non-local population, popu- lations are said to be locally adapted.
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Figure 2. Schematic representation of one pair of chromosomes during the produc- tion of Near Isogenic Lines (NILs) in a selfing organism. Two initial inbred strains, A and B, represented in orange and green, respectively, are crossed to produce an F1 hybrid that will have received one chromosome from each parent. The F1 is selfed and recombinant inbred lines are produced by selfing and single-seed decent. After 9-10 generations, lines will be almost completely homozygous, and have chromo- somes combining different parts of the genomes of the parental lines (Broman and Sen 2009). Finally, to obtain Near Isogenic Lines (NILs), RILs with introgressions at the loci of interests are selected. RILs with introgression segments from parent A are backcrossed with parent B, while RILs with introgression segments from parent B are backcrossed with parent A. The resulting offspring are propagated through selfing for several generations to obtain a homozygous and as narrowly defined in- trogression as possible. The final lines will be homozygous and will be identical to one of the parents except for a specific portion of interest which derives from the other parent.
Selection on timing of germination Germination is a key life-history transition in plants, which represents the pas- sage from seed to seedling. Timing of germination can be expected to be under strong selection in most systems because it can affect fitness both directly, by determining conditions during seedling establishment (Kalisz 1986; Miller et
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al. 1994; Lu et al. 2016), and indirectly, by influencing other fitness-related traits expressed later in the life cycle (Evans and Cabin 1995; Weinig 2000; Donohue et al. 2010; Burghardt et al. 2015, 2016). Timing of germination can differ greatly among populations (Heide 1974; Thompson 1975; Shimono and Kudo 2003; Yilmaz 2008; Montesinos-Navarro et al. 2012) and such variation can have both environmental and genetic causes (Donohue et al. 2005). Di- vergent selection can lead to the evolution of genetically based differences in germination time, and this should contribute to adaptive differentiation among populations. However, early life stages are often not included in studies of local adaptation which may cause the strength of divergent selection to be un- derestimated (e.g., Antonovics and Primack 1982; Schmid 1985; Wang and Redmann 1996; Fournier-Level et al. 2011; Ågren and Schemske 2012; Wadgymar et al. 2018; Ferris and Willis 2018; Lowry et al. 2019).
A full understanding of the adaptive significance of timing of germination requires a characterization of the effects of variation in germination time on fitness. In seasonal environments, optimal timing of germination is expected to be the result of conflicting selection through different components of fit- ness: early germination is usually hypothesized to increase adult survival and fecundity, because it allows for a long period of growth before reproduction (Donohue et al. 2010), but also to decrease seedling survival because of un- stable conditions for seedling establishment at the beginning of the germina- tion season (Donohue et al. 2005). As a result, selection for an intermediate timing of germination is expected (Fig. 3).
Differences in timing and rate of seasonal changes among environments are likely to shift the optimal timing of germination as well as to affect the time frame when germination is possible. In addition, the effects of germina- tion time on fitness may vary also among genotypes due to inherent differ- ences in traits other than timing of germination (Mercer et al. 2011). Infor- mation about possible genotypic differences in the optimal timing of germi- nation is important for assessments of results of common-garden and recipro- cal transplant experiments, in which germination of different genotypes is usually synchronized (e.g., Antonovics and Primack 1982; Schmid 1985; Wang and Redmann 1996; Fournier-Level et al. 2011; Ågren and Schemske 2012; Wadgymar et al. 2018; Ferris and Willis 2018; Lowry et al. 2019). However, there is a scarcity of studies experimentally examining the fitness consequences of germination time, their correlation with environmental fac- tors and differences among genotypes.
The relationship between variation in specific traits and fitness can be char- acterized by experimentally manipulating trait expression (e.g. Andersson 1982; Boquet and Clawson 2009). Compared to the documentation of trait- fitness correlations in natural populations, this approach can increase statisti- cal power by increasing the range of trait variation and by removing correla- tions among traits (Sinervo et al. 1992; Sinervo and Basolo 1996).
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In experiment (II), I use an experimental approach to examine how varia- tion in timing of germination affects fitness at the site of a natural population of Arabidopsis thaliana using two different genotypes.
Regulation of timing of germination: seed dormancy Seed release is often followed by environmental conditions that are not suita- ble for seedlings to survive. About 90% of the angiosperms in the temperate region produces seeds with ‘primary seed dormancy’, a mechanism imposed by the mother plant that prevents germination under temporary good germi- nation conditions (Baskin & Baskin, 2014; Fig. 4). To be released from dor- mancy, a seed must experience certain environmental factors for minimal lengths of time, such as ‘chilling’ over winter or dry after-ripening over sum- mer (Bewley et al. 2013). In this way, seed dormancy may increase the chance that seed germination occurs when conditions are suitable for seedling estab- lishment.
Optimal strength of seed dormancy should be positively correlated with the length of the period following seed release that is unfavourable for seedling establishment and survival (Meyer and Monsen 1991; Allen and Meyer 1998; Llorens et al. 2008; Wagmann et al. 2012), and may thus vary among popula- tions inhabiting contrasting environments. Genetic differentiation in seed dor- mancy along geographic gradients has been found in several species (e.g., Hacker 1984; Allen and Meyer 2002; Kronholm et al. 2012; Debieu et al. 2013). In order to unravel the possible drivers of selection, such variation can be related to differences in environmental factors. Phenotype-environment as- sociations, i.e., assessment of covariation between genetic differences in pop- ulation mean phenotype and environment of origin, is a common approach to identify potential agents of selection (Wadgymar et al. 2017).
Figure 3. Expected relationship be- tween timing of germination and fit- ness in seasonal environments. Early timing of germination is expected to increase adult survival and fecundity, while later germination is expected to increase seedling survival.
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Phenotype-environment associations may arise from natural selection, phe- notypic plasticity, or both. In particular, primary seed dormancy is known to be affected by strong maternal environment effects and to the interaction be- tween maternal environment and genotype (Schütz and Rave 2003; Donohue 2009; Fernández-Pascual et al. 2013; Postma and Ågren 2015; Kerdaffrec and Nordborg 2017). Yet, most studies that document within-species variation in seed dormancy have grown populations in the greenhouse (e.g., Allen & Meyer, 2002; Debieu et al., 2013; Kronholm et al., 2012; Vidigal et al., 2016; Wagmann et al., 2012), in which the environmental conditions are usually far from those experienced by plants in natural populations. Seeds of different populations should therefore ideally be produced in multiple relevant field en- vironments in order to disentangle the role of genetic differentiation, environ- mental effects and their interaction for variation in primary seed dormancy and its association with climate (Young et al. 1991; Schütz and Milberg 1997).
In experiment (III), I investigate regional variation in seed dormancy among natural populations of A. thaliana in southern and northern Europe, and investigate phenotype-climate associations.
Figure 4. Seed dormancy cycle. Freshly dispersed seeds show primary dormancy (1), which impedes immediate germination. Primary dormancy is gradually released (2), and if the seed is exposed to the appropriate conditions (e.g., light, water availa- bility), germination is triggered (3). Alternatively, if seeds are not exposed to germi- nation cues, seeds can acquire secondary dormancy and enter the seed-bank (4). Sec- ondary seed dormancy can be released (5) and re-acquired (4) for several cycles, un- til germination is triggered or the seed dies (6).
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Seed banks Release of primary seed dormancy does not ensure germination. If seeds are not exposed to conditions that trigger germination, they can acquire ‘second- ary dormancy’ and enter the so-called soil seed bank (Fig. 4; Finch-Savage & Leubner-Metzger, 2006; Wagmann et al., 2012; Saatkamp et al., 2014). Sec- ondary dormancy can be broken and re-acquired for several cycles if germi- nation is not triggered and seeds do not die (Fig. 4; Finch-Savage & Leubner- Metzger, 2006; Baskin & Baskin, 2014).
Formation of seed banks can have several advantages. Seed banks can de- crease density-dependent mortality (Ellner 1986, 1987; Nilsson et al. 1994; Venable and Brown 1998; Venable 2007), spread the risk of seedling mortal- ity over time by adopting a bet-hedging strategy (Venable 2007; Saatkamp et al. 2014), and ensure persistence in risky environments such as areas prone to flooding and fires (Aikio et al. 2002; Venable 2007; Saatkamp et al. 2014). Despite their likely important role for the evolutionary dynamics of popula- tions, intraspecific variation of traits of seeds in the seed bank is little studied.
Genetic variation in primary seed dormancy has been well characterized in several species, in particular in crops (e.g., Li et al. 2004; Ogbonnaya et al. 2008; Magwa et al. 2016; Yuan et al. 2020) and in the model organism Ara- bidopsis thaliana (Alonso-Blanco et al. 2003; Bentsink et al. 2010; Montesinos-Navarro et al. 2012; Debieu et al. 2013; He et al. 2014; Vidigal et al. 2016; Kerdaffrec et al. 2016). In A. thaliana, one genomic region at the end of chromosome 5 has been consistently found to contribute strongly to differences in primary seed dormancy among lines of different origin (Alonso- Blanco et al. 2003; Bentsink et al. 2010; Silady et al. 2011; Postma and Ågren 2015; Footitt et al. 2020). This genomic region harbors the gene DELAY OF GERMINATION 1 (DOG1), whose expression is known to promote dormancy (Bentsink et al. 2006). Variation in DOG1 among natural populations corre- lates with climatic gradients within Europe, suggesting that this variation is adaptive (Kronholm et al. 2012). Because seed banks are often a major source of seedlings and because seeds in the seed bank can show a seed dormancy cycle finely tuned with the external environment (Fenner and Thompson 2005; Footitt et al. 2011; Finch-Savage and Footitt 2017), secondary dormancy can also be expected to show adaptive differentiation. However, little is known about intraspecific genetic differentiation in secondary dormancy. In particu- lar, we do not know if the same genomic regions that contribute to variation in primary dormancy are also responsible for variation in secondary dor- mancy. NILs can be used to quantify the contribution of specific genomic re- gions to phenotypic differences between two lines (e.g., Keurentjes et al. 2007; Ding et al. 2011; Wang et al. 2019).
The contribution of seed banks to population dynamics depends not only on the capacity of seeds to synchronize germination with seasonal changes, but also on the capacity of seeds to survive in the soil. Variation among species in seed survival in the soil has been well documented (Telewski and Zeevaart
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2002; Baskin and Baskin 2014). However, when populations inhabit con- trasting environments, we can expect also within-species variation in seed sur- vival in the soil. Although rates of seed mortality in the soil may strongly af- fect the adaptive value of a seed bank, little is known about intraspecific var- iation in seed survival.
In experiment (IV), I investigate genetically based differences in the seed dormancy cycle between two natural populations of A. thaliana, and quantify seed survival in the soil at the populations’ sites of origin.
My project In my PhD project, I examined the adaptive significance and genetic basis of differences in timing of germination and seed dormancy among natural popu- lations in two regions of the native range of the annual, selfing herb Arabidop- sis thaliana.
The aim of my work was to: (a) Assess the fitness and trait effects of a genomic region including pre-
viously identified fitness QTL (I), (b) characterize the adaptive significance of timing of germination in the
field (II), (c) document regional variation in seed dormancy and identify agents of
selection on this trait (III), and (d) assess genetic and environmental effects on seed dormancy cycle and
survival in the soil (IV).
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Material and Methods
Study species Arabidopsis thaliana (L.) (Brassicaceae), thale cress, is an annual, highly-self- ing herb, native to Eurasia and Africa (Durvasula et al. 2017). The species produces seeds with physiological dormancy and is known to form soil seed banks (Baskin and Baskin 1983). A. thaliana is a pioneer species, and it grows on rocky, sandy and disturbed soils.
It has become a plant model organism thanks to its short genome of about 135 Mb organized in 5 chromosomes, its fast life-cycle, and relatively small size. The multiple molecular tools available and its wide distribution made A. thaliana an ideal species for this project.
Study system All experiments included genotypes sampled in two locally adapted natural populations of Arabidopsis thaliana, one at Rödåsen in north-central Sweden (6248’N, 1812’E) and the other at Castelnuovo di Porto in central Italy (4207’N, 1229’E; Ågren and Schemske 2012). The populations are located close to the northern and southern limit of the species’ European range, re- spectively. The climatic conditions at the two sites differ strongly. At the Swe- dish site, winters are characterized by a long period with sub-freezing temper- atures, while summers are short and relatively wet (Fig. 5D, F). At the Italian site, summers are long and dry, while winters are mild and wet (Fig. 5C, E).
Both populations have a winter annual life cycle, but their phenologies dif- fer (Ågren & Schemske, 2012). The Swedish population germinates in Au- gust-September, flowers in May and sheds seeds in late June (Fig. 5B), whereas the Italian population germinates in November, flowers in February- March and disperse seeds in late April (Fig. 5A). As a consequence, the seed stage is much longer in the Italian than in the Swedish population. The Italian population produces seeds with stronger dormancy than the Swedish popula- tion (Postma and Ågren 2016).
A previous QTL mapping of fitness in a RIL population derived from a cross between the Italian and Swedish population and planted as seedlings at the two sites detected 15 fitness QTL, of which two were located at the end of chromosome 5, i.e., fitness QTL 5:4 and fitness QTL 5:5 (Fig. 6; Ågren et al.,
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2013). Fitness QTL 5:4 overlapped with the major fitness QTL identified in a similar experiment that transplanted seeds (fitness QTL Q9; Postma and Ågren 2016). Moreover, both fitness QTL overlapped with several QTL for putative adaptive traits: Fitness QTL 5:4 overlapped with a major seed dor- mancy QTL (Postma and Ågren 2015), a germination time QTL (Postma and Ågren 2018) and the dormancy locus DELAY OF GERMINATION 1 (DOG1; Postma and Ågren 2015; Fig. 6). Fitness 5:5 co-localized with QTL for flow- ering time (Ågren et al. 2017), freezing tolerance (Oakley et al. 2014) and photosynthetic traits (Oakley et al. 2018; Fig. 6).
Figure 5. Timing of life cycle stages (A, B), monthly mean soil temperature (C, D) and mean monthly precipitation (E, F) at the sites of origin of an Italian (A, C, E) and a Swedish (B, D, F) natural population of Arabidopsis thaliana, at Castelnuovo di Porto and Rödåsen, respectively. Monthly mean soil temperature was averaged across the period 2003-2018 (see Ågren and Schemske, 2012 for more details). Mean monthly precipitation was averaged across the period 1970-2000 (Data from Fick and Hijmans, 2017).
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Figure 6. Genomic composition of chromosome 5 of an Italian (in red) and a Swe- dish genotype (in blue) of Arabidopsis thaliana, and of 4 Near Isogenic Lines (NILs) derived from a cross between the Italian and Swedish genotype. NILs C3 and C7 were produced by crossing recombinant inbred lines with Swedish introgression segments to the Italian ecotype, and NILs R11 and R13 by crossing recombinant in- bred lines with Italian introgression segments to the Swedish ecotype. Introgression segments included previously identified QTL for fitness in seed and seedling plant- ings at the sites of origin of the parental lines (Ågren et al. 2013; Postma and Ågren 2016), germination time in Sweden (Postma and Ågren 2018), dormancy of seeds matured at the two sites (Postma and Ågren 2015), flowering time at the two sites (Ågren et al. 2017), and the seed dormancy gene DELAY OF GERMINATION 1 (DOG1; Postma & Ågren, 2015). QTL were mapped in a RIL mapping population derived from a cross between the Italian and Swedish ecotype. For each QTL, the filled circle indicates map position, and the horizontal line its 95% Bayesian credible interval.
Reciprocal transplant experiments (I) To quantify the effects of introgressions including one or two of the fitness QTL at the end of chromosome 5 on timing of germination, flowering start, and fitness, we used near isogenic lines (NILs) in which Swedish genomic segments including fitness QTL 5:4 or both fitness QTL 5:4 and 5:5 had been introgressed into the Italian genetic background (NILs C3 and C7, respec- tively), and NILs in which corresponding Italian genomic segments had been
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introgressed into the Swedish genetic background (NILs R11 and R13, respec- tively; Fig. 6).
We reciprocally planted seeds and seedlings of both parental lines and NILs at the two field sites. In three consecutive years, seeds were planted at the time of seed dispersal (late April in Italy, and late June in Sweden), and seedlings were planted at the time of seedling establishment in the local population (No- vember in Italy, and September in Sweden). We monitored timing of germi- nation, flowering start, establishment (number of seedlings at the end of the germination period divided by number of viable seeds sown), survival (from the end of the germination period to fruit maturation in the seed transplants, and from transplant until fruit maturation in the seedling transplants), fecun- dity (number of fruits per surviving plant) and fitness (establishment × sur- vival × fecundity in the seed transplants, survival × fecundity in the seedling transplants). In the seed transplants, we used seeds matured at the field sites, because maternal environment may markedly affect primary seed dormancy (Donohue et al. 2010, Chiang et al. 2011, Postma and Ågren 2015).
For each site, we used generalized linear models (GLM) to assess the effect of genotype on seedling establishment, adult survival, fecundity, total fitness, timing of germination and flowering start.
Manipulation of timing of germination (II) To examine how germination time affects survival, fecundity, and the relative fitness of two genotypes differing in time to first flower, we manipulated tim- ing of germination and transplanted young seedlings of the Italian and Swe- dish genotype to the Italian site once a month from August to December 2017.
For this experiment, we used seeds matured in the greenhouse in February 2017. Before each transplant, seeds were sown on agar and stratified to induce germination. Ten-day old seedlings were transferred into a randomized block design at the site. Each month, 480 seedlings (240 of each genotype) were transplanted, for a total of 2400 seedlings in the experiment. Once seedlings were in the field, we monitored seedling survival (survival before winter), adult survival (survival between winter and time of reproduction), and fecun- dity (number of fruits per surviving individual). Total fitness was quantified as the number of fruits per transplanted seedling (seedling survival × adult survival × fecundity).
We assessed the effect of month of transplant, genotype (Italian or Swe- dish) and the interaction between these two factors on fitness, seedling sur- vival, adult survival and fecundity using linear and generalized linear models.
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Figure 7. Map of the study populations in Fennoscandia (in blue) and Italy (in red), and of the two field common gardens, one located in Sweden (Rödåsen) and one lo- cated in Italy (Castelnuovo di Porto).
Quantification of regional variation in seed dormancy and correlations with environmental conditions at sites of origin (III) To examine variation in seed dormancy within geographical regions, we trans- planted seedlings of lines collected in 28 Fennoscandian populations and 17 Italian populations, including the two focal Italian and Swedish populations used in the other experiments (Fig. 7).
Seeds were collected in natural populations between 2005 and 2012. To produce plants for the experiment, seeds of five maternal lines from each pop- ulation were sown on agar and stratified for 7 days and then moved to a growth room for 8 days. The generated seedlings were transplanted in three different environments: in the greenhouse and at the sites of origin of the two focal populations (Fig. 7). In the greenhouse, plants were grown at 20 °C 16 h light
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and 16 °C 8 h dark, while at the two field sites, plants were exposed to natural field conditions until harvest.
Once seeds were collected, we assessed their dormancy level by conducting germination tests. We tested dormancy levels of seeds matured at the field sites 12 weeks after harvest, and of seeds matured in the greenhouse 1, 3, 12, 30 and 53 weeks after harvest. In all tests, 50-100 seeds of each line were sown on watered filter papers in petri dishes and kept in growth rooms (20 °C 16 h light with photosynthetically active radiation of 150 μmol m-2 s-1 and 16 °C 8 h dark) for one week. At the end of the week, the proportion of germinated seeds was determined.
We examined the effect of region of origin, maternal environment and their interaction on germination proportions of populations using ANOVAs. In ad- dition, we determined whether among-population variation in primary dor- mancy within regions is associated with climate at the sites of origin. We first used principal component analysis (PCA) to identify the main axes of varia- tion in temperature and precipitation variables, and then used linear models to examine the relationship between the first three Principal Components (PCs) and germination proportion. All climatic data were obtained from WorldClim database (Fick and Hijmans 2017).
Reciprocal seed burial experiment (IV) To examine differences in seed bank traits between the two focal populations and their genetic basis, we performed two seed burial experiments. In one ex- periment conducted at the Swedish site, we assessed the effect of the genomic region including the major primary seed dormancy QTL and DOG1 at the end of chromosome 5 on the seed dormancy cycle in the soil. We used seeds of the Italian and Swedish genotype, and of NIL C3 and NIL R11 (described above; Fig. 6) produced in the greenhouse. Seeds were placed in fine-meshed polyester bags and buried at the time of seed dispersal in the local population at end of June in 2019. Each bag contained 100 seeds and bags were buried approximately 5 cm below soil surface. We excavated 8 bags per genotype on 14 occasions during the following 1.5 years. After each excavation, seeds were brought to the lab to test their dormancy by conducting germination tests. We sowed seeds on two layers of watered filter papers and kept them in a growth room (22 C 16 h light, 16 C 8 h dark). After one week in the growth room, we quantified germination proportion as the number of seedlings that had emerged per seed sown. To test whether seed dormancy differed between gen- otypes, we used GLM with germination proportion as response variable, and time since burial (categorical variable), genotype and their two-way interac- tion as independent variables. We ran separate tests for each year (2019 and 2020).
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In the second experiment, we tested whether seed survival in the soil differs between the Italian and the Swedish site and/or between genotypes. We estab- lished an 8-year long reciprocal seed burial experiment in Italy and Sweden in 2017. Here, the first three years of the experiment are presented. We produced seeds of the Italian and Swedish ecotype in the greenhouse, and the mature seeds were placed in fine-meshed polyester bags. At each site, we buried 96 bags per genotype approximately 5 cm below the ground surface. Each bag contained 100 seeds, for a total of 38,400 experimental seeds. A subset of 12 bags per genotype was excavated at each site once a year at the time of seed- ling establishment in the local population (September in Sweden; November in Italy). Bags were brought to the lab and we estimated the number of pre- dated seeds by counting empty seed coats or partially eaten seeds, and the number of unviable seeds by counting black shriveled seeds, previously shown to be dead (Postma et al. 2016) and by testing viability of the remaining seeds. In the viability assays, seeds were sown on agar and stratified for 2 weeks (4 C in the dark). Seeds were then exposed to growth room conditions for one week and the number of seeds that did not germinate was recorded. We estimated seed survival as: 1 – (proportion of predated seeds + proportion of unviable seeds), where unviable seeds were either black or non-germinated tested seeds. To test whether seed survival differs between sites and geno- types, we used GLM with seed survival as response variable, and time since burial (categorical variable), site, genotype and their two- and three-way in- teractions as independent variables.
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Results and Discussion
Strong effects of genomic regions towards the end of chromosome 5 on local adaptation (I) When we reciprocally transplanted seeds and seedlings of the Italian and Swe- dish genotype, we found strong selection against the non-local genotype at both sites (Fig. 8). The effects of introgressions of genomic regions towards the end of chromosome 5 corresponded to 15-36% and 0-54% to the fitness differences observed between the Italian and the Swedish parent in the seed and seedling transplants, respectively (Fig. 8). Introgression of the local al- leles into the non-local genotype tended to increase fitness, while introgres- sion of the non-local alleles into the local genotype tended to decrease fitness (Fig. 8).
The results were largely consistent with those predicted from previous es- timates of effects of fitness QTL 5:4 and 5:5 in a RIL mapping population derived from a cross between the Italian and the Swedish ecotype (Ågren et al. 2013). Importantly, we detected signals of epistatic interactions at the level of fitness QTL 5:5 in Sweden, as previously observed using the RIL popula- tion. However, we found two main differences using NILs instead of RILs. First, in the seedling transplant, we observed stronger fitness effects of the introgression including both fitness QTL 5:4 and 5:5 than the introgression including only fitness QTL 5:4 in Italy in only one year (C7 vs. C3, and R13 vs. R11; Fig. 8A-C). By contrast, both fitness QTL were detected in all three study years in the former RIL seedling transplant at the Italian site, indicating possible redundancies in fitness effects of the two QTL regions. Secondly, we observed that the local fitness QTL 5:4 region was favored in Sweden (Fig. 8D, E), while previously, no or negative fitness effects of this QTL were ob- served in the RIL seedling transplant, suggesting among-year variation in se- lection on the region of fitness QTL 5:4.
Introgressions had also effects on the phenology of the Italian and Swedish ecotype. We observed that introgression of Italian alleles in the Swedish ge- netic background tended to delay germination in the seed transplant and to result in an earlier flowering start in the seedling transplant, while introgres- sion of Swedish alleles in the Italian genetic background tended to delay flow- ering. Such results largely match with previous observations based on the RIL mapping population.
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Figure 8. Mean ± SE overall fitness (number of fruits produced per seedling planted) of the Italian genotype, the Swedish genotype and 4 Near Isogenic Lines (NILs) in the seedling transplant experiment at the sites of origin of the Italian (A, B, C) and the Swedish (D, E) population in three and two consecutive years, respec- tively. The significance of genotypic effects on fitness, tested with Likelihood Ratio χ2-tests in GLMs, is reported. Different letters indicate statistically significant differ- ences in means based on Tukey’s HSD test.
Using a multiyear transplant approach, we thus demonstrated that a rela-
tively small part of the genome can disproportionally contribute to fitness dif- ferences between two locally adapted populations. To further disentangle the effects of fitness QTL 5:4 and 5:5, experiments that use NILs with introgres- sions including only fitness QTL 5:5 are needed. Furthermore, NILs with mul- tiple introgressions should be used in the future to further investigate the fit- ness effects of targeted epistatic interactions.
Strong stabilizing selection on timing of germination (II) When we transplanted seedlings of the Italian and Swedish genotype at the Italian site between August and December, we found evidence of strong sta- bilizing selection on timing of germination. All seedlings germinating in Au- gust, September, and October died within the first month of transplant (Fig. 9C), and the fitness of the November cohort was 32 and 50 times higher than that of the December cohort in the Italian and Swedish genotype, respectively (Fig. 9A). The optimal timing of germination thus coincided with the main germination period at the study site.
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Figure 9. The effects of timing of germination on (A) overall fitness (number of fruits produced per seedling planted), (B) seedling survival, (C) adult survival, and (D) fecundity (number of fruits per reproducing plant) of the Italian (red bars) and the Swedish (blue bars) genotypes of Arabidopsis thaliana at the site of the Italian population. Means ± SE are given.
There was no evidence of conflicting selection on germination timing through seedling survival and through adult survival and fecundity, respec- tively. Instead, seedlings transplanted in November were superior with regards to all fitness components (Fig. 9B-D). The steep fitness function for germina- tion time shows that conditions for seedling establishment and plant develop- ment changed rapidly across the season with a narrow window of opportunity for seedling establishment. By contrast, a former similar experiment per- formed at the Swedish site found that the optimum germination timing was in August, the time window available to seedling establishment was longer and differences in fitness among cohorts smaller than observed in the current study (Akiyama and Ågren 2014). The shorter period when soil moisture and tem- perature are suitable for seedling establishment in Italy compared to Sweden is likely to explain the between-site difference in steepness of the fitness func- tions for germination time.
We observed strong selection against the non-local Swedish genotype, and the strength of selection did not differ between the November and the Decem- ber cohort (Fig. 9A).
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Overall, our results suggest that the rate of seasonal change should be an important aspect to consider for an understanding of spatio-temporal variation in selection on phenological traits. Our findings also highlight the importance of planting time in common garden and reciprocal transplant experiments such that the phenology and growth of transplants is similar to that of local popu- lations.
Within-region variation in seed dormancy is related to differences in summer conditions (III) We found strong and consistent differences in seed dormancy between natural populations of Arabidopsis thaliana from Italy and Fennoscandia across three different maternal environments. Populations from Italy had on average stronger seed dormancy than populations from Fennoscandia (Fig. 10A, B), in line with our expectations based on climatic differences between the two regions. In Italy, seed release is followed by a long period unfavorable for seedling establishment, whereas in Fennoscandia this period is much shorter. As a consequence, stronger seed dormancy is expected to be favored in Italy compared to Fennoscandia. Our findings are consistent with a previously ob- served latitudinal gradient in primary seed dormancy of A. thaliana across Europe (Kronholm et al. 2012; Debieu et al. 2013).
Seeds collected in the greenhouse had the strongest seed dormancy among the tested maternal environments, and seeds produced at the Swedish field site showed stronger seed dormancy than seeds produced at the Italian field site (Fig. 10A). No maternal environment × region of origin effects on seed dor- mancy was detected (Fig. 10A). We observed large variation in seed dor- mancy among populations within regions, and the effect of maternal environ- ment on seed dormancy 12 wk after maturation varied among populations (significant population × maternal environment interaction; Fig. 10A). Our findings are in line with a well-known effect of temperature during seed mat- uration on dormancy (Chiang et al. 2011; Footitt et al. 2011, 2013; Fenner 2018), although further studies are needed to unravel the relative importance of this and other environmental variables.
Among Fennoscandian populations, variation in seed dormancy was asso- ciated with differences in summer temperature and precipitation at the site of origin, while no such correlation could be detected in the smaller sample of populations from Italy. Among Fennoscandian populations, seed dormancy tended to increase with increasing summer temperature and decreasing sum- mer precipitation, consistent with seed dormancy clines observed across Eu- rope (Kronholm et al. 2012) and the Iberic peninsula (Vidigal et al. 2016).
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Figure 10. Mean germination proportion of natural populations of Arabidopsis thali- ana sampled in Italy (in red) and Fennoscandia (in blue) (A) 12 weeks after seed harvest in three maternal environments: greenhouse, an Italian field site (Castel- nuovo di Porto), and a Swedish field site (Rödåsen), and (B) 1, 3, 12, 30 and 54 weeks after seed maturation in the greenhouse. Regional means are indicated in bold.
These findings taken together suggest that seed dormancy is an evolution-
ary flexible trait subject to strong divergent selection across the European range. We suggest that future studies should investigate (i) the contribution of this among-population variation in seed dormancy to local adaptation, and (ii) the response of seed dormancy to climate change.
Genetic and environmental factors contribute to intraspecific variation in seed bank traits (IV) When we characterized the seed dormancy cycle in the soil of the Italian and the Swedish population, we found strong genetic differences, which matched the germination schedule in the respective natural population. The Italian eco- type showed stronger primary and secondary seed dormancy than the Swedish ecotype, and lost secondary seed dormancy nearly 2 months later than did the Swedish ecotype (Fig. 11). By including NILs in this experiment, we could show that the genomic region that contributes most to the difference in pri- mary dormancy between the two genotypes (Postma and Ågren 2015), also strongly affects secondary seed dormancy. Introgressions of the Italian ge- nomic segment into the Swedish genetic background delayed secondary dor- mancy loss, while introgression of the Swedish segment into the Italian geno- type resulted in an earlier and greater loss of secondary dormancy (Fig. 11). Overall, effects of introgressions corresponded to 37-41% of the difference in time of secondary dormancy loss between the two ecotypes, and introgression
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of the Swedish genomic segment into the Italian genetic background resulted in a fivefold increase in the magnitude of secondary dormancy loss.
Seed survival was very high at the Swedish site after 3 y of burial (mean seed survival >95%), while most of the seeds buried in Italy died between the first and the second year in the soil regardless of the genotype (mean seed survival year 2: 6%). Predation of buried seeds was very low at both sites. The difference in seed survival suggests that the contribution of the seed banks to population dynamics differs markedly between the Italian and Swedish site.
In conclusion, we demonstrated a large difference in seed dormancy cycle and striking variation in seed mortality in the soil between two natural popu- lations of A. thaliana. Additional studies should be conducted to examine the extent to which such differences contribute to local adaptation.
Figure 11. The proportion of Arabidopsis thaliana seeds of four genotypes germi- nating in tests conducted at the time of seed burial (data points to the far left) and af- ter different duration of burial at the Swedish site (data points connected with full line; mean ± SE) of four genotypes. Germination proportions were recorded after one week in a growth room under standard conditions.
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Conclusions
In this thesis, I have investigated the ecology and evolution of local adaptation in A. thaliana. I have demonstrated that a relatively small part of the genome that controls timing of germination and flowering can explain a large propor- tion of fitness differences between two locally adapted populations (I). I found that selection on timing of germination can differ strongly between locally adapted populations inhabiting contrasting climates, which should contribute to the maintenance of population differences in this trait (II). In line with this finding, seed dormancy, which controls timing of germination, was shown to differ strongly between populations in Fennoscandia and Italy, but also to vary widely among populations within each of the two regions, indicating substan- tial evolutionary flexibility of seed dormancy (III). Variation in summer tem- perature and precipitation were identified as likely environmental factors driv- ing divergent selection on seed dormancy within Fennoscandia (III). In addi- tion, I showed that differences in a region on chromosome 5 contributes strongly to differences in the seed dormancy cycle between an Italian and a Swedish ecotype. The mortality schedule of seeds in the soil can differ mark- edly between populations of the same species, which should affect selection on traits expressed in the seed bank (IV). Taken together, the results highlight the importance of including the early-life stages and seed bank dynamics in studies of local adaptation.
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Svensk sammanfattning
Hur uppstår biologisk mångfald? Biologisk mångfald kommer till uttryck på flera sätt. Det finns fler än 300 000 växtarter beskrivna, från encelliga alger till träd som kan leva mer än 1000 år. Också inom arter kan många gånger en omfattande ge- netisk variation observeras. Vad är det för processer som ligger bakom denna variation och som gör att den upprätthålls? Hur har växter an- passat sig till olika klimatförhållanden? För att söka svar på frågor av detta slag har jag studerat populationer av den ettåriga örten backtrav i Skandinavien och Italien.
Lokal anpassning är ett utbrett fenomen hos växter, men också hos djur, bakterier och svampar. Det innebär att vid odling i en gemensam miljö har lokala populationer en överlevnadsfördel jämfört med populationer från andra miljöer. Det kan yttra sig i högre överlevnad, högre avkommeproduktion, eller bådadera. Trots att lokal anpassning har påvisats hos ett stort antal arter, är vår kunskap om de genetiska skillnader och de skillnader i egenskaper som är viktiga för lokal anpassning fortfarande begränsad. Detsamma gäller kun- skapen om vilka skillnader i miljön som är avgörande för olika genotypers överlevnadsförmåga. Vad är det som gör att vissa genotyper klarar sig bättre än andra i den här miljön, men sämre än andra i en annan miljö? Det här är frågor som jag studerat i min doktorsavhandling och som är viktiga för att förstå de processer som styr biologisk mångfald.
Jag har använt nord- och sydeuropeiska populationer av den ettåriga växten backtrav, Arabidopsis thaliana, som studiesystem, och har särskilt intresserat mig för egenskaper som påverkar växters etablering, överlevnad och tillväxt tidigt i livet. Backtrav är en ört som i Europa förekommer i en rad olika miljöer från Medelhavsområdet till norra Skandinavien (Fig. 1A). Möjligheterna att odla backtrav på lab, och dess snabba livscykel gjorde att den tidigt identifie- rades som modellsystem för att studera de molekylära processer som styr väx- ters utveckling från frö till blomning. Idag är backtrav den genetiskt bäst ka- raktäriserade växten av alla. Det sammantaget med dess förekomst i vitt skilda klimat gör backtrav idealisk för studier av hur växter anpassat sig till olika miljöförhållanden.
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Jag har med fältexperiment undersökt den genetiska basen för lokal anpass- ning mellan en svensk och italiensk population av backtrav, hur groningstid- punkt påverkar överlevnad och reproduktion, hur fröegenskaper varierar mel- lan populationer i Italien och i norra Europa, och hur frögroningsförmåga ut- vecklas under säsongen hos frö som hamnat i marken och inte grott redan första året.
Studiesystem För att studera mekanismer bakom lokal anpassning har jag utfört fältstudier i en svensk och en italiensk population av backtrav som växer under markant olika klimatförhållanden. Den italienska populationen växer i ett medelhavs- klimat som kännetecknas av varma, torra somrar och milda, regniga vintrar, medan den svenska populationen växer på en plats med borealt klimat som kännetecknas av milda somrar, kalla vintrar och med nederbörd som är mer jämnt fördelad över året.
Både den italienska och den svenska populationen har en ettårig livscykel, men tidpunkter för frögroning och blomning skiljer kraftigt. Den svenska po- pulationen blommar i maj, sprider mogna frön i slutet av juni, och frön gror i augusti-september (Fig. 1B). Den italienska populationen blommar betydligt tidigare, i februari-mars, sprider frön i slutet av april, och frön gror först i
Figure 1. Arabidopsis thaliana eller backtrav. Livscykel hos den svenska (B) och den italienska (C) populationerna.
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november (Fig. 1C). Det innebär att tiden mellan fröspridning och groning är betydligt längre i den italienska populationen: cirka 6 månader i Italien, jäm- fört med cirka 2 månader i Sverige. Tidigare fältexperiment har påvisat stark lokal anpassning mellan de två populationerna. Genom att korsa de två popu- lationerna och producera genotyper som kombinerar italienska och svenska gener på olika sätt har det varit möjligt att identifiera egenskaper liksom reg- ioner på var och en av backtravens fem kromosomer som bidrar till lokal an- passning. Bland annat skiljer sig de två populationerna i hur lång tid det tar för frön att bli groningsdugliga efter att de spridits. Den italienska populat- ionen producerar frö med en betydligt djupare frövila. Frövilan förhindrar frö att gro även om ljus- och vattentillgång är god. När frövilan hos det nyprodu- cerade fröet är stark, dröjer det länge innan det är redo att gro.
Jag har undersökt hur genetiska skillnader mellan den italienska och svenska populationen påverkar fröegenskaper, groningstidpunkt, överlevnad och fröproduktion. För att ta reda på om skillnader mellan de två population- erna i fröegenskaper speglar generella skillnader mellan populationer i Italien och norra Europa har jag också gjort en jämförande studie av ett större antal populationer från de två områdena.
Förändringar i kromosom 5 påverkar växters fenologi och lokal anpassning Lokal anpassning bygger på att populationer ackumulerat mutationer som är gynnsamma för överlevnad i sin hemmamiljö. För att undersöka hur skillnader inom en begränsad del av kromosom 5 bidrar till skillnader i fröegenskaper, överlevnad och fröproduktion när de två populationerna växer i Sverige och Italien har jag använt mig av en uppsättning framkorsade genetiska linjer. Dessa linjer är genetiskt identiska med den italienska respektive svenska ge- notypen förutom i en begränsad del av kromosom 5, där den andra genotypens genuppsättning har blivit inkorsad. Det gör det möjligt att i fältexperiment kvantifiera hur mycket skillnader i just denna region påverkar backtravens egenskaper när den växer i Italien respektive Sverige.
Vi fann att den undersökta regionen starkt påverkade backtravens gronings- och blomningstidpunkt, liksom överlevnad och fröproduktion. Generellt sett ökade det inkorsade kromosomsegmentet den främmande populationens över- levnad och fröproduktion, men minskade den lokala populationens överlevnad och fröproduktion. Det inkorsade kromosomsegmentet påverkade gronings- tidpunkt och blomningstid i en riktning som kunde förväntas utifrån tidigare genetiska studier. Resultaten visar att förändringar i en tämligen liten del av en växts genuppsättning kan kraftigt påverka växters egenskaper och därmed starkt bidra till lokal anpassning.
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Groningstidpunkt är av avgörande betydelse De flesta växter producerar stora mängder frö, men bara en bråkdel av dessa gror och lyckas överleva groddplantsstadiet. Det innebär att egenskaper som påverkar etableringsframgång bör vara utsatta för stark selektion och potenti- ellt bidra till lokal anpassning. Kunskapen om hur fröegenskaper och gro- ningstidpunkt påverkar etableringsframgång i olika miljöer är emellertid mycket begränsad.
Groningstidpunkt är kopplad till hur stark frövilan är. Jag har i ett fältexpe- riment undersökt hur groningstidpunkt påverkar överlevnad och fröprodukt- ion där den italienska populationen växer. Jag planterade ut unga groddplantor en gång i månaden mellan september och december och registrerade överlev- nad och fruktproduktion fram till tidpunkt för fröspridning följande vår.
Resultaten indikerade mycket stark selektion på groningstidpunkt. Optimal groningstidpunkt var i november och sammanföll med den period då groning sker i den lokala populationen. Alla groddplantor som planterades ut i augusti- oktober dog inom en månad. Dessutom var fruktproduktion per planterad groddplanta många gånger högre i november- än i decemberplanteringen.
Ett liknande experiment har tidigare utförts i den svenska populationen. I det experimentet, inföll optimal groningstidpunkt i augusti, men skillnaderna mellan groning i augusti, september och oktober var inte lika dramatiska som skillnaderna mellan olika groningstidpunkter i Italien. Mindre dramatiska skillnader kan kopplas till en långsammare förändring i etableringsbetingelser på den svenska växtplatsen. Resultaten styrker hypotesen att skillnader i mil- jöförhållanden och optimal groningstidpunkt har bidragit till de genetiska skillnader i frövila och groningstidpunkt som dokumenterats mellan de två populationerna.
Frövilans styrka skiljer mellan norra och södra Europa I tidigare studier har vi visat att frövilan är betydligt starkare hos en italiensk än hos en svensk backtravspopulation och att detta är förknippat med en fördel under etableringsfasen i den italienska respektive svenska populationen. Hur representativ är då skillnaden mellan dessa två populationer för variation i frövila mellan backtravspopulationer i Italien och norra Europa? Den varma, torra perioden som oftast följer efter backtravens frömognad är ogynnsam för groddplantsetablering. Eftersom den perioden är betydligt längre i medelhavs- klimat än i norra Europa, kan vi förvänta oss att frö producerat av italienska populationer generellt ska ha starkare frövila än frö producerat av nordeuro- peiska populationer. Men också inom geografiska regioner kan sommarens längd variera markant och frövilans styrka kan förväntas korrelerad med som- marperiodens längd och nederbörd.
För att testa dessa hypoteser jämförde vi frövilans styrka hos frön som pro- ducerats av 28 nordeuropeiska populationer (provtagna i Sverige, Norge och
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Finland) och 17 italienska populationer. Frövilans styrka påverkas av miljö- förhållandena där frön mognar, och vi undersökte därför frö som producerats i tre olika miljöer: i växthus, och i fältexperiment i Italien respektive Sverige.
Frövilan var generellt svagare i de nordliga populationerna än i de itali- enska populationerna oavsett var de producerats, men vi kunde dessutom do- kumentera en avsevärd variation i frövila bland populationer från samma geo- grafiska region. Bland de nordliga populationerna var frövilan starkare i po- pulationer från växtplatser med varmare och torrare somrar, medan motsva- rande samband inte kunde detekteras bland de italienska populationerna som inte uppvisade lika stor variation i miljöförhållanden som de nordliga popu- lationerna. Resultaten visar att frövilans styrka är en egenskap som uppvisar omfattande genetisk variation, och att denna variation är korrelerad med skill- nader i sommarklimat mellan olika miljöer där backtrav förekommer.
Har frön som inte grott en andra chans? Alla frön som sprids och överlever gror inte nödvändigtvis första året. En del frön blir kvar i marken och kan där gå in i så kallad sekundär frövila och bilda en fröbank. Fröbanken kan ses som en buffert som gör det möjligt för en po- pulation av ettåriga växter att fortleva även om fröproduktion och etablering helt uteblir under ett eller flera år. Hos backtrav genomgår frön i marken en årlig cykel i frövila. Frön som inte grott en given säsong går in i sekundär frövila och blir groningsdugliga på nytt först när den har släppt. För att det här ska fungera väl är det viktigt att frövilecykeln är väl synkroniserad med sä- songsmässiga förändringar i förutsättningar för framgångsrik groddplantseta- blering. Vidare kan betydelsen av fröbanken förväntas starkt påverkas av hur länge frön kan överleva i marken.
För att undersöka i vilken utsträckning skillnader i frövilecykel i marken mellan den italienska och svenska populationen kan förklaras av genetiska skillnader i den region av kromosom 5 som tidigare visats starkt påverka frövila vid frömognad (primär frövila) anlade vi ett experiment på den plats där den svenska populationen växer. Vi grävde ner frön av den italienska och svenska genotypen, och av två av de framkorsade linjer som beskrivits ovan, och registrerade sedan hur frövilans styrka förändrades under 1.5 år. Vi fann att den italienska populationens sekundära frövila var starkare och släppte se- nare under säsongen än den svenska populationens frövila, och att dessa skill- nader i stor utsträckning kunde förklaras av genetiska skillnader i den del av kromosom 5 som också starkt påverkar primär frövila. I ett andra experiment, undersökte vi överlevnad hos frö i marken på den italienska och den svenska populationernas växtplatser under tre år. Vi fann att frönas överlevnad på båda lokalerna var väldigt hög under det första året. Frööverlevnaden var fortsatt hög under de följande åren i Sverige, medan den sjönk till 5% efter två år i Italien.
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Det första experimentet visar att egenskaper som kommer till uttryck i frö- banken kan variera kraftigt mellan populationer av en och samma art, och att skillnaderna mellan de två studiepopulationerna i stor utsträckning kan hänfö- ras till skillnader inom en begränsad del av en av backtravs fem kromosomer. Den stora skillnaden i frööverlevnad i marken mellan de två växtplatserna in- dikerar att möjligheterna för fröbanken att påverka populationsutveckling också skiljer markant.
Slutsatser Sammanfattningsvis fann jag att: Förändringar i en liten del av en växts genuppsättning kan starkt bidra till
lokal anpassning. Groningstidpunkt kan vara av avgörande betydelse för en växts överlev-
nadschans och reproduktionsförmåga. Genetiska skillnader mellan populationer i fröegenskaper och gronings-
tidpunkt kan kopplas till skillnader i klimatförhållanden. Skillnader mellan populationer i groningsegenskaper hos frö i marken kan
bidra till lokal anpassning.
Riassunto in italiano
Come nasce la biodiversità? Solo nel mondo vegetale, si contano migliaia di specie dalle alghe ai mu- schi, dalle erbe agli alberi. Queste specie si sono differenziate le une dalle altre nel corso delle ere. E non solo. A ben guardare si può notare che esiste un’enorme diversità anche all’interno di una stessa specie. Ma cosa fa nascere la diversità? Per rispondere a questa domanda ho viaggiato tra Italia e Svezia dove crescono popolazioni a prima vista uguali, ma in realtà molto diverse, di una stessa specie chiamata Arabidopsis thaliana.
Arabidopsis thaliana, o arabetta comune, è una piccola pianta erbacea (Fig. 1A) che si può trovare in tutti i continenti ad eccezione dell’Antartide. Grazie al suo genoma relativamente piccolo suddiviso in soli 5 cromosomi, alla sua facile reperibilità e alla sua rapidità di crescita, A. thaliana è diventata una specie studiata in tutto il mondo.
Nei miei studi ho focalizzato la mia attenzione in particolare su due popo- lazioni di questa specie, una localizzata nel centro Italia e l’altra in centro Sve- zia. Studi precedenti hanno mostrato che se piante della popolazione italiana vengono fatte crescere nel luogo di origine della popolazione svedese e, vice versa, piante svedesi vengono fatte crescere nel luogo di origine della popola- zione italiana, solamente poche piante sopravvivono, e quelle che riescono a farlo producono molti meno frutti rispetto alla popolazione locale. Quando la popolazione straniera ha prestazioni (ossia tasso di sopravvivenza e riprodu- zione) più basse rispetto alla popolazione del luogo significa che si è in pre- senza di adattamento locale. Questo significa che le due popolazioni sono state sottoposte a diverse pressioni ambientali, nel tempo hanno accumulato diffe- renze e si sono così adattate a vivere nello specifico luogo in cui crescono, tanto che se spostate in un altro ambiente hanno difficoltà a sopravvivere e riprodursi. È bene notare, però, che non tutte le differenze che si osservano tra popolazioni sono causate da adattamento locale, ma possono essere causate, ad esempio, dal caso.
L’adattamento locale è un fenomeno diffuso nel mondo vegetale, ma anche in quello animale, dei batteri e dei funghi. Nonostante ciò, si conosce ancora poco delle cause e modalità di questo fenomeno. Molte sono infatti le do- mande che si pongono i biologi evoluzionistici, tra cui: quanti e quali cambia- menti devono avvenire nel genoma perché si verifichi adattamento locale? Quali tratti si devono differenziare tra popolazioni? Quali solo le pressioni
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ambientali che contribuiscono all’insorgere dell’adattamento locale? Durante i miei studi, ho cercato una risposta a queste domande.
Sistema di studio Le due popolazioni, italiana e svedese, sono state inizialmente scelte per via delle forti differenze climatiche nei loro rispettivi luoghi d’origine – ed io ne so qualcosa! La popolazione italiana è soggetta a clima mediterraneo, caratte- rizzato da estati calde e secche ed inverni miti e piovosi. La popolazione sve- dese, invece, proviene da un clima boreale, contraddistinto da estati miti, in- verni rigidi e da precipitazioni constanti lungo tutto l’arco dell’anno.
Sia la popolazione italiana che quella svedese hanno un ciclo di vita an- nuale. Tuttavia, le due popolazioni seguono tempistiche diverse durante l’anno. Nella popolazione svedese, i semi germinano già ad agosto-settembre, le piante fioriscono solo a maggio e i nuovi semi vengono dispersi a giugno (Fig. 1B). I semi della popolazione italiana, invece, germinano a novembre, le piante fioriscono a febbraio-marzo e i nuovi semi prodotti vengono dispersi ad aprile (Fig. 1C). Questo significa che la durata dello stadio di seme è par- ticolarmente diversa tra le due popolazioni (circa 6 mesi in Italia, 1-2 mesi in Svezia). Nei miei studi ho quindi voluto indagare quanto e come questa parti- colare differenza possa contribuire all’insorgere dell’adattamento locale.
Figura 1. A) Arabidopsis thaliana o arabetta comune. Cicli di vita nella popolazione svedese (B) e italiana (C).
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Un piccolo passo per il cromosoma 5, un gigantesco balzo per Arabidopsis thaliana Per fare in modo che si verifichi adattamento locale, le popolazioni devono accumulare differenze nel genoma. Se questa è una certezza, lo stesso non si può dire di come ci si aspetta avvengano questi cambiamenti nel genoma. A lungo si è dibattuto se debbano susseguirsi numerosi ma piccoli cambiamenti, oppure pochi ma grandi cambiamenti o ancora se debba avvenire un misto tra i due scenari.
Studi precedenti hanno mostrato che una particolare area del genoma loca- lizzata alla fine del cromosoma 5 è particolarmente importante nel determinare svariate differenze tra la popolazione italiana e quella svedese, incluse diffe- renze nella durata dello stadio di seme. Per capire quanto quest’area da sola possa contribuire all’adattamento locale, abbiamo incrociato piante italiane con quelle svedesi e creato delle piante con un genoma ibrido. In queste nuove piante, l’intero genoma è identico a quello di una delle due popolazioni eccetto che per l’interessante area alla fine del cromosoma 5, identica invece al ge- noma dell’altra popolazione. Abbiamo poi messo le piante ibride e quelle ori- ginali nei luoghi d’origine delle popolazioni italiana e svedese.
Abbiamo osservato che cambiare il genoma alla fine del cromosoma 5 comporta grandi conseguenze per le piante. Abbiamo visto che le piante con genoma italiano a cui era stato inserito il segmento svedese sopravvivevano e si riproducevano peggio delle piante con solo genoma italiano in Italia, mentre miglioravano le loro prestazioni in Svezia. Al contrario, l’inserimento del seg- mento italiano nel genoma svedese ha aumentato le prestazioni rispetto alle piante con solo genoma svedese in Italia ma le ha peggiorate in Svezia. In totale, le differenze nell’area del genoma alla fine del cromosoma 5 spiega- vano fino al 54% delle differenze di prestazioni tra popolazione italiana e sve- dese. In aggiunta, le piante ibride modificavano i loro tempi di germinazione e fioritura rispetto alle piante con solo genoma italiano o svedese. Questi ri- sultati dimostrano che cambiamenti in una parte relativamente piccola del ge- noma possono avere enormi ripercussioni e contribuire così fortemente all’adattamento locale.
Chi ben comincia è a metà dell’opera La selezione durante i primi stadi di vita è solitamente molto forte. Basti pen- sare che la stragrande maggioranza della mortalità nelle piante avviene tra gli stadi di seme e plantula. È quindi facile intuire che differenze nei primi stadi di vita hanno il potenziale di dare un contributo fondamentale all’adattamento locale. Tuttavia, i primi stadi di vita sono poco studiati in studi di biologia evoluzionistica proprio perché sono di esito troppo incerto.
Per capire quanto è forte la selezione durante i primi stadi di vita abbiamo osservato cosa succede se la popolazione italiana cambia momento in cui
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germina, ossia se cambia la durata dello stadio di seme. Per farlo abbiamo trapiantato giovani plantule nel luogo di origine della popolazione italiana una volta al mese tra agosto e dicembre e monitorato le prestazioni delle piante nei mesi successivi. Tutte le piante trapiantate tra agosto e ottobre sono morte poco dopo il trapianto. La maggior parte delle piante trapiantate a novembre, invece, sono sopravvissute fino alla riproduzione, mentre solo un terzo delle piante trapiantate a dicembre ha fatto altrettanto. In aggiunta, le piante di no- vembre hanno prodotto circa 10 volte più frutti delle piante di dicembre.
Questi risultati mostrano che la selezione sui primi stadi di vita è estrema- mente forte nel sito di origine della popolazione italiana. Un simile esperi- mento era stato fatto precedentemente nel sito di origine della popolazione svedese. In quel caso, agosto era risultato il mese migliore per germinare, ma la selezione era risultata più mite probabilmente perché il clima in Svezia cam- bia più lentamente tra estate ed autunno rispetto all’Italia. Il fatto che nei loro luoghi d’origine le popolazioni italiana e svedese vadano a germinare rispet- tivamente proprio a novembre e ad agosto-settembre è indicativo del fatto che le differenze che si osservano nei tempi di germinazione tra le due popolazioni non siano dovute al caso bensì a diverse pressioni ambientali.
Al posto giusto nel momento giusto Essendo il tempo di germinazione così importante, nelle piante esistono dei meccanismi molto fini che lo regolano. Uno tra questi è la dormienza dei semi, la quale garantisce che la germinazione non avvenga nella stagione sbagliata. Circa il 90% delle angiosperme nelle zone temperate producono semi con dor- mienza poiché la stagione che segue la dispersione dei semi spesso non con- sentirebbe alle giovani plantule di sopravvivere. La dormienza mostrata dai semi non appena rilasciati viene definita dormienza primaria. Più a lungo per- sistono le condizioni avverse alla sopravvivenza delle plantule dopo il rilascio dei semi, più forte possiamo aspettarci essere la dormienza primaria.
In Europa, A. thaliana produce semi in primavera ma, nelle popolazioni a ciclo invernale, i semi potranno germinare solo quando le condizioni estive sono passate. Poiché la durata del periodo estivo caldo e secco è molto mag- giore nel sud rispetto al nord Europa, possiamo aspettarci che la dormienza sarà maggiore nei semi delle popolazioni invernali meridionali. Lo stesso trend dovrebbe verificarsi anche su scala locale, all’interno di queste due re- gioni: le popolazioni provenienti da luoghi con estati più lunghe produrranno semi con dormienza più forte all’interno dell’Italia e della Fennoscandia.
Per testare queste ipotesi, abbiamo raccolto semi da 28 popolazioni fenno- scandinave e 17 popolazioni italiane. Poiché la dormienza dei semi è solita- mente influenzata dal luogo in cui le piante materne crescono, abbiamo dun- que fatto crescere semi delle popolazioni campionate in tre ambienti: in serra, e nei siti di origine delle popolazioni italiana e svedese usate negli esperimenti
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descritti precedentemente. Una volta che le piante sono arrivate a maturazione, abbiamo raccolto i semi da loro prodotte e ne abbiamo testato la dormienza.
I semi prodotti in serra avevano la dormienza più forte, seguiti dai semi prodotti in Italia, a dimostrazione del fatto che il luogo di crescita materno influenza la dormienza. I semi delle popolazioni fennoscandinave avevano una dormienza più debole rispetto alle popolazioni italiane indipendentemente dall’ambiente materno. In aggiunta, abbiamo osservato un’enorme variazione nella dormienza dei semi anche tra popolazioni della stessa regione. Le popo- lazioni fennoscandinave da siti più caldi e secchi hanno prodotto semi con maggiore dormienza. Non abbiamo però trovato nessuna relazione tra clima e dormienza nelle popolazioni italiane. Il fatto che almeno in una regione ab- biamo trovato una correlazione tra ambiente e dormienza, e che entrambe le regioni abbiano mostrato ampia diversità nella dormienza tra popolazioni sug- gerisce che questo tratto abbia un alto potenziale per contribuire all’adatta- mento locale.
Chi dorme non piglia pesci… o forse sì! Non sempre i semi germinano alla prima occasione. Alcuni semi rimangono dormienti nel suolo, acquisiscono la cosiddetta dormienza secondaria e vanno a formare la banca dei semi del suolo. La banca dei semi è una sorta di piano B per le popolazioni: assicura che in caso di disastro o condizioni molto av- verse, la popolazione possa ristabilirsi e permette alle piante di distribuire il rischio di mortalità negli anni. I semi nel suolo hanno livelli di dormienza se- condaria che oscillano durante l’anno a seconda delle stagioni e possono così germinare negli anni successivi alla produzione, sempre a patto che non muoiano prima. Seppur una sostanziale parte delle plantule possa nascere dalla banca dei semi, poco si conosce di come i semi del suolo possano con- tribuire all’adattamento locale. In particolare, sono poco studiate differenze tra popolazioni nelle oscillazioni annuali di dormienza secondaria e nella mor- talità dei semi nel suolo.
Per studiare se queste due caratteristiche possano variare all’interno di una stessa specie, abbiamo condotto due diversi esperimenti. In un primo esperi- mento, abbiamo sotterrato semi italiani e svedesi nel luogo di origine della popolazione svedese. In aggiunta, per sapere se la porzione del genoma alla fine del cromosoma 5 contribuisce anche a differenze nella dormienza secon- daria, abbiamo utilizzato anche semi ibridi derivati dall’incrocio delle due po- polazioni come descritto in precedenza. Abbiamo poi recuperato i semi dal suolo ad intervalli regolari nel corso di un anno e mezzo e controllato i livelli di dormienza. Abbiamo così osservato che la popolazione italiana mostra li- velli di dormienza secondaria sempre maggiori e tende a perdere la dormienza secondaria più tardi durante l’anno rispetto alla popolazione svedese. Le dif- ferenze nell’area genomica alla fine del cromosoma 5 spiegavano gran parte delle differenze osservate tra le due popolazioni.
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In un secondo esperimento, abbiamo sotterrato semi italiani e svedesi nel luogo di origine di entrambe le popolazioni e monitorato la mortalità dei semi una volta l’anno per tre anni. Abbiamo osservato che dopo un anno, la soprav- vivenza dei semi era molto alta in entrambi i Paesi, e tale è rimasta per gli anni successivi in Svezia. In Italia, invece, la mortalità è aumentata fino al 95% dopo due anni dall’inizio dell’esperimento.
Queste osservazioni dimostrano che può esserci enorme variazione all’in- terno di una specie nelle caratteristiche della banca dei semi. Abbiamo osser- vato che popolazioni diverse hanno evoluto diversi cicli di dormienza nel suolo, e che gran parte di queste differenze sono spiegate da differenze a li- vello dell’area genomica alla fine del cromosoma 5. In aggiunta, la banca dei semi può avere durata molto diversa tra popolazioni e contribuire così diver- samente alle dinamiche demografiche.
Conclusioni Nei miei studi ho osservato che l’adattamento locale: Può essere spiegato ampiamente da cambiamenti in una piccola parte del
genoma; Può essere fortemente causato da cambiamenti nei primi stadi di vita,
quali la durata dello stadio di seme e le oscillazioni di dormienza dei semi; Può essere influenzato dalle condizioni climatiche durante i primi stadi di
vita; Può essere potenzialmente influenzato da caratteristiche diverse dei semi
nel suolo.
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Acknowledgements
Many great people were directly involved in this project or have contributed to this enriching experience that the PhD has been.
I am deeply grateful to my supervisor, Jon Ågren, for his incessant teaching during these past years. You have thaught me to never stop being critical to- wards my work and to push my limits. The passion you put into your work has been inspiring. Thanks also to my co-supervisor, Martin Lascoux, for the moral support and insightful comments on my work. An honorary mention goes also to Christopher Oakley, whose work, comments and suggestions have been fundamental to this thesis.
During these years, I had the opportunity to spend quite some time in the field and it would not have been so fun if it wasn’t for all the people that came with me. Special thanks go to Linus V, who made long road trips and trans- plants much funnier than expected and helped me since day 1. I am very thank- ful also to Mariona, who was such a helpful co-worker and friend during the busiest field season of all. Thanks also to Maria U for her precious help in the field, the saved trips to the High Coast and the mushroom hunting. I want to thank also Maurizio, Emil, Mattias and the numerous other field companions that I met at the High Coast and in Rome.
When I wasn’t in the field, I could enjoy working at the Plant Ecology pro- gram. I am very grateful to past and current Ågren lab members, in particular to Tom R and Tom E for their statistical and writing support. Thanks also to Maria G for being such a helpful colleague and understanding friend. Tack till Linus S för att vara en trevlig kontorskompis och för att driva mig att prata svenska. Thanks to Nina and Sophie for having been such great teaching men- tors and for their support during the PhD. It was an honor to work together with all the past and current PhD students and colleagues at the program, which I will remember as a stimulating and friendly working environment. I will always have great memories of my PhD thanks to each one of you.
Tack också till det svenska samhället för att ha lärt mig att det bästa alter- nativet alltid är att vara snäll.
I want to thank also my family and my friends based in Uppsala, Italy and around the world for being there for me regardless of how far we live from each other.
E, infine, grazie a Simone per essere una persona così bella.
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