On The Big Challenges of a Small Shrub: Ecological Genetics of Salix herbacea L

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UNIVERSITATISACTA UPSALIENSIS

UPPSALA 2015

Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1288

On The Big Challenges of a Small Shrub

Ecological Genetics of Salix herbacea L.

ANDRÉS J. CORTÉS

ISSN 1651-6214 ISBN 978-91-554-9337-0 urn:nbn:se:uu:diva-262239

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Dissertation presented at Uppsala University to be publicly examined in Zootissalen, Evolutionsbiologiskt centrum (EBC), Norbyvägen 18, Uppsala, Wednesday, 28 October 2015 at 13:00 for the degree of Doctor of Philosophy. The examination will be conducted in English. Faculty examiner: PhD John Pannell.

Abstract

Cortés, A. J. 2015. On The Big Challenges of a Small Shrub. Ecological Genetics of Salix herbacea L. Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology 1288. 37 pp. Uppsala: Acta Universitatis Upsaliensis.

ISBN 978-91-554-9337-0.

The response of plants to climate change is among the main questions in ecology and evolution. Faced with changing conditions, populations may respond by adapting, going extinct or migrating. Fine-scale environmental variation offers a unique mosaic to explore these alternatives. In this thesis, I used ecological surveys, field experiments and molecular methods to study the range of possible responses at a very local scale in the alpine dwarf willow Salix herbacea L. Since gene flow may impact the potential for adaptation and migration, I first explored whether phenological divergence driven by snowmelt patterns impacts gene flow. I found that sites with late snowmelt work as sinks of the genetic diversity, as compared to sites with early snowmelt. I also used a combined approach that looked at the selection, heritability and genomic architecture of ecologically-relevant traits, as well as genomic divergence across the snowmelt mosaic. In this way, I was able to understand which genomic regions may relate to phenological, growth and fitness traits, and which regions in the genome harbor genetic variation associated with late- and early- snowmelt sites. I found that most of the genomic divergence driven by snowmelt is novel and is localized in few regions. Also, Salix herbacea has a strong female bias. Sex bias may matter for adaptation to climate change because different sexes of many dioecious species differ in several functions that may fluctuate with changing conditions. I found that the bias is uniform across environments and is already present at seeds and seedlings.

A polygenic sex determination system together with transmission distortion may be maintaining the bias. Overall, fast-evolving microhabitat-driven genomic divergence and, at the same time, genetically-based trait variation at a larger scale may play a role for the ability of S. herbacea to persist in diverse and variable conditions.

Keywords: Fine-scale environmental variation, migration, adaptation, snowmelt timing Andrés J. Cortés, Department of Ecology and Genetics, Plant Ecology and Evolution, Norbyvägen 18 D, Uppsala University, SE-752 36 Uppsala, Sweden.

urn:nbn:se:uu:diva-262239 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-262239)

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“Laughter is a sunbeam of the soul.”

― Thomas Mann, The Magic Mountain

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List of Papers

This thesis is based on the following papers, which are referred to in the text by their roman numerals.

I Cortés, A.J., Waeber, S., Lexer, C., Sedlacek, J., Wheeler, J.A., van Kleunen, M., Bossdorf, O., Hoch, G., Rixen, C., Wipf, S., Karrenberg, S. (2014) Small-scale patterns in snowmelt timing affect gene flow and the distribution of genetic di- versity in the alpine dwarf shrub Salix herbacea. Heredity, 113:233–239

II Sedlacek, J., Cortés, A.J., Wheeler, J.A., Bossdorf, O., Hoch, H., Klapste, J., Lexer, C., Rixen, C., Wipf, S., Karrenberg, S., van Kleunen, M. Can the dwarf willow Salix herbacea count on evolution in response to climate change? Heritabilities and selection in wild populations from different elevations and mi- crohabitats. Manuscript in revision

III Cortés, A.J., Wheeler, J.A., Sedlacek, J., Lexer, C., Karren- berg, S. Genome-wide patterns of microhabitat-driven diver- gence in the alpine dwarf shrub Salix herbacea L. Submitted manuscript

IV Cortés, A.J., Liu, X., Sedlacek, J., Wheeler, J.A., Lexer, C., Karrenberg, S. Maintenance of female-bias in a polygenic sex determination system is consistent with ge- nomic conflict. Manuscript in revision

Reprints were made with permission from the respective publishers.

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The following papers were written during the course of my doctoral studies as part of the Salix-Sinergia project, but are not included in this thesis.

• Little, C., Wheeler, J.A., Sedlacek, J., Cortés, A.J., Rixen, C. (2015) Small-scale drivers: the importance of snowmelt timing and nutrient availability on the performance of the alpine shrub Salix herbacea.

Oecologia, doi: 10.1007/s00442-015-3394-3

• Sedlacek, J.*, Wheeler, J.A.,* Cortés, A.J., Bossdorf, O., Hoch, G., Lex- er, C., Wipf, S., Karrenberg, S., van Kleunen, M., Rixen, C. (2015) The Response of the Alpine Dwarf Shrub Salix herbacea to Altered Snow- melt Timing: Lessons from a Multi-Site Transplant Experiment. PloS ONE, e0122395

• Wheeler, J.A., Schnider, F., Sedlacek, J., Cortés, A.J., Wipf, S., Hoch, G., Rixen, C. (2015) With a little help from my friends: community fa- cilitation increases performance in the dwarf shrub Salix herbacea. Basic and Applied Ecology, 16:202-209

• Sedlacek, J., Bossdorf, O., Cortés, A.J., Wheeler, J.A., van Kleunen, M.

(2014) What role do plant-soil interactions play in the habitat suitability and potential range expansion of the alpine dwarf shrub Salix herbacea?

Basic and Applied Ecology, 15(4):305–315

• Wheeler, J.A., Hoch, G., Cortés, A.J., Sedlacek, J., Wipf, S., Rixen, C.

(2014) Increased spring freezing vulnerability for alpine shrubs under early snowmelt. Oecologia, 175:219-229

* These authors contributed equally to this paper.

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The following papers were published during the course of my doctoral studies as part of other collaborative projects started before my PhD research, and are not included in this thesis. * These authors contributed equally.

• Blair, M.W.*, Cortés, A.J.*, This, D. (2015) Identification of an EREC- TA gene and its drought adaptation associations with wild and cultivated common bean. Plant Science, doi: 10.1016/j.plantsci.2015.08.004

• Blair, M.W., Cortés, A.J., Penmetsa, R.V., Farmer, A., Carrasquilla- Garcia, N., Cook, D.R. (2013) A high-throughput SNP marker system for parental polymorphism screening, and diversity analysis in common bean (Phaseolus vulgaris L.) Theoretical and Applied Genetics, 126(2):535-48

• Cortés, A.J., Monserrate, F.A., Ramírez-Villegas, J., Madriñan, S., Blair, M.W. (2013) Drought Tolerance in Wild Plant Populations: the Case of Common Beans (Phaseolus vulgaris L.) PloS ONE, e62898

• Madriñan S., Cortés, A.J., Richardson, J.E. (2013) Páramo is the world’s fastest evolving and coolest biodiversity hotspot. Frontiers in Genetics, 4(192)

• Blair, M.W., Soler, A., Cortés, A.J. (2012) Diversification and Popula- tion Structure in Common Beans (Phaseolus vulgaris L.) PloS ONE, e49488

• Cortés, A.J., Chavarro, M.C., Madriñan, S., This, D., Blair, M.W. (2012) Molecular ecology and selection of drought related Asr genes in wild and cultivated common bean (Phaseolus vulgaris) BMC Genetics, 13:58

• Cortés, A.J., This, D., Chavarro, M.C., Madriñan, S., Blair, M.W. (2012) Nucleotide diversity patterns at Dreb2 genes, candidate for drought tol- erance, in wild and cultivated common bean (Phaseolus vulgaris L.) Theoretical and Applied Genetics, 125(5):1069-85

• Galeano, C.H., Cortés, A.J., Fernandez, A.C., Soler, A., Franco-Herrera, N., Makunde, G., Vanderleyden, J., Blair, M.W. (2012) Gene-based single nucleotide polymorphism markers for genetic and association map- ping in common bean. BMC Genetics, 12:48

• Kelleher, C.T., Wilkin, J., Zhuang, J., Cortés, A.J., Perez, A.L., Gal- lagher, T.F., Bohlmann, J., Douglas, C.J., Ritland, K. (2012) SNP dis- covery, gene diversity, and linkage disequilibrium in wild populations of Populus tremuloides. Tree Genetics & Genomes, 8(4):821-29

• Cortés A.J., Chavarro, M.C., Blair, M.W. (2011) SNP marker diversity in common bean (Phaseolus vulgaris L.) Theoretical and Applied Genet- ics, 123(5):827-45

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Introduction

Understanding the responses of organisms to changing conditions is a major research focus in ecology and evolution. Organisms, populations or species react to environmental change by migrating, persisting at current locations or going extinct (Hoffmann and Sgro, 2011). Persistence of such populations in changing environments may be mediated by phenotypic plasticity, which is the range of phenotypes that a single genotype can express as a function of its environment (Nicotra et al, 2010), or by adaptation from standing varia- tion by increasing the frequency of existing variants that can cope with the new conditions (Bridle and Vines, 2007). Local adaptation to heterogeneous habitats has been recurrently documented (Gonzalo-Turpin and Hazard, 2009; North et al, 2011; Savolainen et al, 2013). However, its genetics is not always well understood (Savolainen et al, 2013).

Some of the largest impacts of climate change are expected in alpine environments, that are dominated by long-lived plant species and where snow cover and summer temperatures are the main drivers of vegetation composi- tion (Körner and Basler, 2010). Temperature increases over the past decades have already led upward migration in plant species (Walther et al, 2002).

The alpine zone is a highly heterogeneous environment that is characterized not only by strong altitudinal gradients but also by local depressions in which the snow accumulates and disappears very late in the summer (i.e.

snowbeds), and more exposed ridges with less snow and where the snow disappears several weeks to months earlier (Figure 1.). These local-scale differences have been shown to cause local adaptation in Dryas and in Ra- nunculus (Stanton and Galen, 1997). For species that occur in heterogeneous habitats, such small-scale variation can have important implications for the reaction to changing conditions. Small-scale topographic variability may provide new locations for migrants with suitable habitats within only a few meters of the current locations (Scherrer and Körner, 2011; Yamagishi et al, 2005). Alternatively, such small-scale habitat variability can lead to locally adapted subpopulations (Gonzalo-Turpin and Hazard, 2009), and such genotypes adapted to a more narrow range of conditions may respond poorly to future conditions. Therefore, in order to understand processes involved in potential reactions to changing condition, it is important to consider not only climate differences at different altitudes but also between microhabitats.

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Figure 1. Snowbeds and ridges as seen early in the spring (May 2011) at Wannen- grat, Switzerland.

Gene Flow (I)

The transfer of alleles across populations is known as gene flow. Variation in the timing of flowering between subpopulations in different microhabitats can restrict patterns of pollen-mediated gene flow (as compared to seed- mediated gene flow), and lead to small-scale genetic structure (Stanton et al, 1997) regardless of whether flowering time is genetically or environmentally regulated (Jump et al, 2009; Scherrer and Körner, 2011; Stanton and Galen, 1997; Stinson, 2004). Such small-scale genetic differentiation due to flowering-time divergence and restricted gene flow via pollen has been reported in the majority of studies on snowmelt-driven genetic differentiation (Hirao and Kudo, 2008; Shimono et al, 2009; Stanton et al, 1997; Yamagishi et al, 2005). Seed dispersal, however, can counteract isolation driven by barriers to pollen flow, because seed dispersal occurs later in the season when all winter snow has melted (Kudo and Hirao, 2006). Predominant gene flow via seed may, on the other hand, result in asymmetric source/sink-like patterns driven by wind, topology and the success of seed establishment (Nathan and Muller-Landau, 2000).

Understanding patterns of genetic variation and gene flow across early and late snowmelt microhabitats will help to predict the response of Alpine species to climate change. Upon climate warming, snowmelt is expected to occur generally earlier (Elmendorf et al, 2012; Molau et al, 2005), and cur-

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13 rent late snowmelt locations (snowbeds) likely develop season lengths more similar to current exposed ridges. Restricted gene flow and differentiation between sub-populations in different microhabitats can be associated with local adaptation (Giménez-Benavides et al, 2007). In this scenario, late snowmelt associated genotypes of long-lived species, such as the dominant shrub species, may have difficulties to persist during warming. Early snowmelt associated genotypes, in contrast, would need to establish in new localities, and this could be difficult in long-lived species even if suitable localities are nearby. Alternatively, a lack of differentiation between sub-populations in different microhabitats and unrestricted gene flow between them would be more compatible with an ability of most genotypes to grow in both micro- habitats, and thus persist in situ upon climate change. Apart from differentia- tion and gene flow, genetic variation contained in sub-populations in early and late snowmelt microhabitats could also differ, due to asymmetric gene flow for example, and this will determine the extent to which genetic variation is lost from one of the microhabitats.

Traits under Selection (II & III)

Genomic divergence has mostly been studied among species and well- differentiated populations (Nosil and Feder, 2011; Strasburg et al, 2011) but few of them have done so at a very local scale, such as at microhabitats driven by snowmelt, and from a genome-wide point of view. Genomes are re- garded as porous in the sense that different regions present contrasting signa- tures and levels of selection, isolation, drift and ancestral variation (Strasburg et al, 2011). Genome-wide patterns of divergence have recently been described with metaphors such as ‘islands’ and ‘continents’ of divergence, referring to peak-like or plateau-like regions of high genetic divergence surrounded by low-divergence regions (Nosil and Feder, 2011).

While divergence peaks may be caused by divergent selection from novel or standing genetic variation (Pritchard et al, 2010; Roesti et al, 2014) or ran- dom drift (Keller et al, 2013), regions with low divergence may be due to balancing or uniform selection, high gene flow or ancestral shared polymor- phism (Jones et al, 2012; Lexer et al, 2006).

A combined approach that explores selection gradients (Chapter II) and association mapping of ecologically-relevant traits, and between- microhabitat genomic divergence (Chapter III) allows understanding what regions in the genome are likely to differ between microhabitats, and therefore harbor genetic variation unique to each, and how these genomic regions may relate to phenological, growth and fitness traits (Barrett and Hoekstra, 2011; Evans et al, 2014; Poelstra et al, 2014; Stinchcombe and Hoekstra, 2008) (Figure 2).

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Figure 2. Connections between various approaches for studying the genetics of ecologically relevant variation. Numbers of chapters are indicated in roman numbers. Modified from Barrett & Hoekstra (2011).

Sex Ratio (IV)

Sex bias may matter for adaptation to climate change because sexes may differ in functions that can fluctuate with changing conditions. Ecological differences between sexes can lead to differential mortality and biased sex ratios at later life-history stages (Barrett et al, 2010). In higher plants, sepa- rate sexes are rare (dioecy, 7% of the species) but occur in diverse lineages, whereas hermaphroditic flowers or unisexual flowers on the same individual are common (Ashman et al, 2014). The sex determination system is known only for 14% of dioecious plant species (Ashman et al, 2014). Dioecy in plants is thought to have evolved multiple times from hermaphrodite ances- tors via stages with mixed sexual systems (Bachtrog et al, 2014; Beukeboom and Perrin, 2014). The evolution of sex determination mechanisms is thought to be driven by selection on sex ratios as well as by genetic conflict arising from sex-linked inheritance (Bachtrog et al, 2014; Beukeboom and Perrin, 2014; Bull, 1983; van Doorn, 2014; Werren and Beukeboom, 1998).

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15 In the absence of ecological differences, theory predicts unbiased primary sex ratios at birth when sex is determined through Mendelian segregation of nuclear loci (Bull, 1983; Fisher, 1915; Moore and Roberts, 2013). A bias in primary sex ratios is compatible with cyto-nuclear sex determination, environmental sex determination and biased transmission of sex determining alleles through meiotic drive (Bachtrog et al, 2014; Beukeboom and Perrin, 2014; Bull, 1983; Maurice, 1992). Determining the source of adult sex ratio bias is difficult because biased primary sex ratios can also arise after gamete formation, either due to pollen or sperm competition, or from selective abor- tion of seeds or embryos (Barrett et al, 2010).

Aims of the Thesis

The following questions were addressed in the alpine dwarf shrub Salix her- bacea L.

1. Are patterns of genetic differentiation and gene flow driven by small-scale differences in snowmelt timing? (I)

2. Do phenological, morphological and fitness-related traits show heritable variation? (II)

3. Is selection currently acting on any of these traits? (II)

4. What is the snowmelt-driven pattern of genomic divergence i.e.

localization, magnitude and origin of the divergent regions? (III)

5. What is the genomic architecture of ecologically-relevant traits?

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6. What is the mechanism leading to adult female bias? (IV)

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Materials and Methods

Study System

Salix herbacea L., Salicaceae (Figure 3), is a clonal, dioecious, prostrate dwarf shrub common in the circumpolar arctic, subarctic and in alpine eco- systems (Beerling, 1998). In the Swiss Alps, S. herbacea is an optimal spe- cies for studying the effects of climate change, as it occurs along a relatively long elevational gradient (2100-2800 m asl), and occupies a wide range of microsite types, from rocky, early-exposure ridges to late-season snowbeds.

It produces an extensive ramifying system with branched rhizomes. Seeds are wind dispersed. The aerial branches are woody and usually reach only 2- 5 cm above the ground surface. Clones are on average 16 cm in diameter (Häggberg, 2013) but clones of up to several meters in size have also been observed (Reisch et al, 2007). Clones can reach an age of over 100 years (De Witte et al, 2012) but most clones are younger (Häggberg, 2013). All popu- lations of S. herbacea in the Alps constitute a phylogeographic unique and distinct population (Alsos et al, 2009).

Figure 3. Female (left) and male (right) patches of the alpine dwarf shrub Salix herbacea.

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Sampling Locations

The series of studies here presented took place along three mountains near Davos, in the eastern Swiss Alps. Jakobshorn (46°46' N, 09°50' E, 2100 to 2600 m asl), Schwarzhorn (46°44' N; 09°57' E, 2380 to 2780 m asl) and Wannengrat (46°48' N, 09°46' E, 2280 to 2640 m asl), all had similar pri- marily north-east exposure and covered the main elevational range of our study species. In these mountains, two types of surveys were used, plot and transect surveys.

Plot Survey (I & II)

Salix herbacea was sampled at 12 sites on the three different mountains.

Two different altitudes (high and low, from 2100 to 2800 m asl, on average 1.2 km apart) were chosen to cover the altitudinal distribution of S. herbacea on each mountain. A snowbed and a ridge microhabitat (on average 35.2 ± 15.5 m apart) were chosen at each altitude based on topology and vegetation.

For chapter I, thirty 10 cm -diameter S. herbacea patches were randomly sampled within a 10m x 10m plot at each site yielding a total of 360 samples across the four microhabitat x altitude combinations through all three moun- tains. For chapter II, around one-hundred 10 cm -diameter S. herbacea patches were randomly sampled within the 10m x 10m plots at each site yielding a total of 1061 samples across the four microhabitat x altitude combinations through all three mountains.

Transect Survey (III & IV)

Three transects, covering the main elevational range of S. herbacea, were established on the three mountains. At 8 bands along transects on each of the three mountains, study plots (c. 3 x 3 m) were set up in two ridge microhabitat sites (early-season exposure from snow) and two late snowmelt microhabitat sites (late-season exposure), for a total of 96 plots. In each plot, two 10 cm -diameter S. herbacea patches were selected. One plot and 10 patches were lost during the course of the experiment, likely a consequence of ava- lanches, so the final number of patches, hereinafter referred to as individuals, was 180.

Phenotyping

Soil temperature recordings and field observations were used to estimate snowmelt timing as described in Wheeler et al. (2014). Monitoring of all individuals was carried out weekly from snowmelt until leaf senescence during the 2011, 2012 and 2013 growing seasons.

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Flowering incidence, plant sex, the proportion of stems flowering, leaf tissue damage (the proportion of leaves in a patch that were damaged by herbivores), the number of stems and mean leaf area (length x width) after full leaf expansion were recorded.

Genotyping

Five leaves from the same stem were sampled per patch and were immedi- ately stored in empty tea bags, and dried in silica gel Rubin (Sigma Aldrich, Germany). Genomic DNA was extracted from silica-dried leaf material using the QIAGEN DNeasy Plant Mini Kit (QIAGEN, Germany) following the manufacturer’s instructions. DNA concentration and purity was quantified using NanoDrop® spectrophotometer ND-1000 (Saveen & Werner AB, Limhamn, Sweden). DNA samples were stored at -18°C.

Microsatellite Genotyping (I & II)

Seven microsatellites (SSR) loci were used to access population structure (Chapter I) and estimate relatedness (Chapter II). The PCR reactions were multiplexed in two PCR runs using the QIAGEN Multiplex PCR Kit (QI- AGEN, Hilden, Germany) following the manufacturer’s instructions. The PCR products were pooled and separated by capillary electrophoresis at Uppsala University, Uppsala, Sweden, using an ABI 3130 DNA Analyzer and LIZ500 as ladder (Applied Biosystems, Foster City, CA, USA). Allele sizes were in base-pairs using GeneMapper v.3.7 (Applied Biosystems).

Genotyping-by-Sequencing (III & IV)

Two 96-plex genotyping-by-sequencing libraries were prepared according to Elshire et al. (2011) at Cornell University, USA. Library preparation with ApeKI digestions, genotyping and SNP calling was performed by the BRC Genomic Diversity Facility. The raw Illumina DNA sequence data (197,032,870 good barcoded reads per lane) was processed through the GBS analysis pipeline as implemented in TASSEL-GBS v3.0 (Glaubitz et al, 2014). Sequence tags were aligned to the female Salix purpurea reference genome (Carlson et al, 2014) using BWA-aligner (Li and Durbin, 2007).

Targeted genotyping of sex-associated SNP markers in seeds and seedlings (Chapter IV) was done by LGC Genomics, UK using KASP technolo- gy (Cuppen, 2007). Flanking sequences were extracted from the S. purpurea assembly (Carlson et al, 2014), and were used in Primer-Picker (LGC Ge- nomics, UK) to design allele-specific oligonucleotides. Fluorescence signals were interpreted by the KlusterCaller 1.1 software (LGC Genomics).

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Analytical Approaches

Linear models were used to assess the effect of snowmelt and altitude on flowering time, and how snowmelt varies between microhabitats. In order to assess whether snowmelt time and phenological differences trigger genetic isolation (Chapter I), pairwise FST values among the 12 low density 10m x 10m plots were obtained with GENEPOP v3.5 (Raymond and Rousset, 1995). The number of alleles and heterozygosity were compared between microhabitats using linear mixed models with microhabitat as fixed effect and mountain as a random effect (Venables and Ripley, 2002). Population structure was examined using the STRUCTURE v.2.3.3 software (Pritchard et al, 2000). A total of five independent runs were used for each K value from K=2 to K=12 using an admixture model and 100,000 iterations for the burn-in and 100,000 for the MCMC analysis. The optimal number of subdi- visions was determined based on the rate of change of the likelihood across different K values as described in Evanno et al. (2005). Effective population sizes (N_e) and pairwise migration rates (N_em) were estimated following coa- lescent theory and a maximum-likelihood based approach using MIGRATE v.3.0.3 (Beerli and Felsenstein, 1999).

Narrow sense heritability (h²) was estimated in the 12 high-density 10m x 10m plots using a multivariate animal model (Frentiu et al, 2008) with a marker-based relatedness matrix according to Lynch & Ritland (1999) (Chapter II). To test for selection on the phenological and morphological traits examined in the animal model, proxies of relative (i.e. relative to the mean across all sites) clonal (i.e. change in stem number) and sexual reproductive fitness (i.e. proportion of flowering stems) were fixed against the standardized phenotypic traits using multiple regression with linear mixed models as implemented in the “nlme” package (Pinheiro et al. 2014) to yield selection gradients (Lande and Arnold, 1983). Finally, the multivariate form of the breeder’s equation (Walsh, 2008) was used to predict the evolutionary response of each trait to selection over one generation (R).

Trait-marker associations were determined using three approaches. First, a sliding window analysis (window size = 1 x 10⁶bps, step size = 200 kb) was used to determine FST and the proportion of variable SNPs that are fixed between microhabitats (Chapter III) and sexes (Chapter IV) in ARLEQUIN v3.5 (Excoffier et al, 2005; Weir and Cockerham, 1984). Linkage- disequilibrium (LD) and Tajima’s D (Tajima, 1989) were computed in the same windows with the R package PopGenome (Pfeifer et al, 2014). Sec- ondly, a standard trait-marker association analysis was implemented in FaST-LMM (Lippert et al, 2011). As a third approach, the full dataset was interrogated through the BiForce algorithm in order to detect possible epi- static interactions and dominance effects, and to confirm single trait-marker associations (Gyenesei et al, 2012).

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Generalised linear mixed models were used to investigate ecological differences between sexes (Chapter IV). To test whether there is environmental-sex determination and males and females occur in different habitat we used a mixed model with sex treated as response variable and a binomial error distribution. Fixed effects were mean snowmelt day, elevation and temperature, all centered to a mean of zero to allow appropriate effect esti- mation (Schielzeth, 2011) and nested random effects were data logger, study plot and transect (Venables and Ripley, 2002). In order to test whether performance differed between sexes we used similar mixed models with Gauss- ian error distributions that treated the recorded traits as the response variable and sex as a fixed effect together with nested random effects as described above. Correlations among performance traits were weak. These statistical analyses were carried out in R v.2.15.1 (R Core Team), using the packages lme4 and lmerTest (Bates and Sarkar, 2007).

Simulations were used to explore whether segregation at sex-determining loci can lead to sex bias (Chapter IV). Multi-locus genotypes at sex- associated loci were used as proxies for sex determining loci. Multi-locus gamete genotypes were simulated by random allele selection from the observed male and female multi-locus genotypes at sex-associated loci. Ran- dom mating was further simulated by randomly pairing these gamete genotypes. The sex of the resulting zygotes was assigned using a principal- coordinates-analysis (PCoA). These simulations were repeated for 25 generations using 100 random offspring as parents for the following generation.

Segregation of one locus at a time was distorted in 10% (low), 40% (moderate), and 70% (high) of the female gametes, favoring transmission of gametes carrying the allele with major allele frequency. This procedure was repeated over all loci, 100 times each. In each generation, the sex ratio was recorded and 95% confidence intervals were generated by repeating the procedure 1,000 times.

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Results and Discussion

Alpine snowbeds are sinks of genetic diversity (I)

Even though there is phenological differentiation between microhabitats due to snowmelt timing (Figure 4), S. herbacea sub-populations growing in dif- ferent microhabitats are not genetically differentiated (F_ST within- and between-microhabitat comparisons 0.028 ± 0.003 and 0.035 ± 0.004 for within- and between-microhabitat comparisons, P-value = 0.691. This is also sup- ported by the lack of structure in a STRUCTURE analysis.

However, late-snowmelt microhabitats (snowbeds) are genetically more diverse than early-snowmelt sites (i.e. allelic richness: 8.93 ± 0.27 and 6.81

± 0.29 for snowbeds and ridges respectively, P-value = 0.007, number of alleles corrected by rarefaction: 6.76 ± 0.18 and 5.19 ± 0.20 for snowbeds and ridges respectively, P-value = 0.005, and expected heterozygosity: 0.733

± 0.009 and 0.690 ± 0.009 for snowbeds and ridges respectively, P-value = 0.042), and gene flow is asymmetric towards the snowbeds (Figure 5).

Overall, these results are consistent with snowbeds acting as sinks of genetic diversity, and seed dispersal preventing snowmelt-driven genetic isolation.

Figure 4. Day of snowmelt predicts when flowering starts for 274 female S. her- bacea patches growing in ridges (○) and snowbeds (●) and 85 male S. herbacea patches growing in ridges (Δ) and snowbeds (▲) surveyed in (A) 2011 and (B) 2012. Dashed lines are regression lines (R²= 0.827, P-value < 0.001).

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Figure 5. Estimates of the number of migrants per generation (Nem) between micro- habitat differing in snowmelt timing (ridges and snowbeds) in Salix herbacea from three mountains in the Swiss Alps.

Heritabilities and Selection (II)

Using the animal model with marker-based relatedness estimates in natural populations of S. herbacea, we found low to moderate heritabilities for phe- nological, morphological and fitness-related traits. There was negative selection on leaf size and thermal duration until leaf expansion, when using clonal reproduction as a fitness proxy, in both ridge and snowbed microhabitats (Table 1). There was positive selection on thermal duration until flowering in both ridge and snowbed microhabitats, when using sexual reproduction as a fitness proxy. However, there was selection on thermal duration until flowering in opposing directions in the two microhabitat types, when using the clonal reproduction as a fitness proxy. The latter suggests that with ongoing climate change selection pressures on phenology may change.

When using the multivariate form of the breeder’s equation to estimate potential evolutionary responses of traits, while accounting for genetic correlations among traits and selection on these traits (Walsh & Blows 2009;

Morrissey et al. 2010), the strongest predicted response was found for leaf size and the interval snowmelt to leaf expansion (R = -0.358 mm² per gener- ation and R = -5.238 days per generation) when using clonal reproduction as a fitness proxy. The adaptive potential found for these traits might enable S.

herbacea to adapt to changing selection pressures. Under a climate change scenario, with earlier snowmelt, evolutionary responses may shift towards responses that are currently observed on ridge microhabitats. Thus, longer thermal duration until flowering is expected, which might, for instance, pre- vent early season frost damage.

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23 Table 1. Standardized selection gradients (β) across all sites. Linear mixed models were run separately for the two relative fitness proxies: proportion of flowering stems (h² = 0.049) and change in stem number (h² = 0.071), and included the traits leaf size (h² = 0.386), interval snowmelt to leaf expansion (h² = 0.178), thermal duration until leaf expansion (h² = 0.469) and flowering (h² = 0.399), and their inter- actions with microhabitat type (MH), with plot nested within transect as random effect. Estimates of narrow-sense heritability (h²) are based on the multivariate animal model with a marker based relatedness matrix (Lynch & Ritland 1999). Signifi- cance values are bolded.

Fitness Trait (standardized) β df F p

Proportion flowering stems

Leaf size -0.023 67 0.032 0.86

Interval snowmelt to leaf expansion 0.029 67 0.342 0.561 Thermal duration until leaf expansion -0.041 67 0.426 0.516 Thermal duration until flowering 0.211 67 4.153 0.046

Leaf size x MH 0.054 67 0.076 0.784

Interval snowmelt to leaf expansion x MH 0.179 67 1.110 0.296 Thermal duration until leaf expansion x MH -0.095 67 0.376 0.542 Thermal duration until flowering x MH -0.075 67 0.163 0.688

Change in stem number Leaf size -5.232 116 -3.279 0.011 Interval snowmelt to leaf expansion -3.655 116 -2.217 0.051 Thermal duration until leaf expansion -3.646 116 -2.218 0.020 Thermal duration until flowering 2.467 116 1.418 0.85

Leaf size x MH 3.758 116 1.614 0.073

Interval snowmelt to leaf expansion x MH 4.058 116 1.308 0.184 Thermal duration until leaf expansion x MH 1.644 116 0.696 0.805 Thermal duration until flowering x MH -4.821 116 -2.047 0.043

Between-microhabitat genomic divergence (III)

Eight strong between-microhabitat divergence peaks and two weaker peaks were detected in seven different chromosomes (Figure 6). These peaks coin- cided with regions of low SNP density, extensive linkage disequilibrium and negative Tajima’s D values. This speaks for new genetic variation arising and being fixed in snowbeds and ridges separately, as compared to standing variation that is differentially recruited between microhabitats. The same peaks persisted when the between-microhabitat F_ST was computed at each transect, and they overlapped with ‘valleys’ in the F_ST when this statistic was calculated within microhabitats and across transects, indicating that these divergent regions are not the result of genetic drift. The ten between- microhabitat divergence regions spanned a total of 219 genes, which may help inferring functional traits that diverge between microhabitats.

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24

Figure 6. Between-microhabitat genomic divergence in Salix herbacea. Sliding window analysis (window size = 1 x 106 bps, step size = 200 kb) for the average between-microhabitat fixation index (FST). Results of all windowed analysis are plotted against window midpoints in millions of base pairs (bp). Black and orange colors highlight different chromosomes identified by roman numerals. The gray and red dashed horizontal lines indicate the genome-wide average and the threshold for identification of outliers. Significantly divergent regions are shown as gray columns.

The function of these genes suggests that the genetic difference between microhabitats could be related to unmeasured traits like osmotic stress, stem elongation and root growth. Overall, our results indicate that genomic divergence can occur in the presence of gene flow (Chapter I) and strong environmental differentiation at a very fine geographic scale.

In addition of the population genomic approach to study microhabitat- driven divergence and identify traits that may have diverged between environments, association mapping was performed to explore genetically based variation in ecologically relevant traits across microhabitats. A total of 57 regions comprising 66 GBS-derived SNP markers and distributed all across the genome were significantly associated with the explored traits for which heritabilities were computed (Chapter II). On average, they explain 19 % of the observed variation per trait. At least 10 regions enclose well-known candidate genes for 7 of the 9 surveyed traits.

Female-bias and polygenic sex determination (IV)

Ecological differences between sexes were not found and female sex bias was uniform across altitudes and microhabitats. Therefore, we explored the sex determination system and sexed seed and seedlings. Regions in different chromosomes were associated with sex in S. herbacea when looking at marker-sex association (Figure 7) and F_ST differentiation. Inter- chromosomal LD among the sex-associated markers was significantly higher than the average inter-chromosomal LD (inter-chromosomal R²: 0.7 ± 0.2 and 0.4 ± 0.1 for sex-associated and all markers, p < 0.001, Fig. 1B). Fe- males had higher heterozygosity in the sex-associated markers (Ho: 0.40 ± 0.02 for females and 0.34 ± 0.02 for males, p = 0.023). The sex-associated regions included regions in chromosomes XV and IX as reported for other Salix species (Hou et al, 2015; Pucholt et al, 2015; Semerikov et al, 2003).

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25 Figure 7. Sex-associated SNP-markers in the arctic-alpine willow Salix herbacea.

Gray dashed horizontal line marks the acceptance threshold after correction for multiple comparisons. Black and grey colors highlight different chromosomes identified by roman numerals.

The main sex-determining region in chromosome XV coincides with the strongest between-microhabitat divergence peak (Figure 6), likely due to reduced recombination. Furthermore, genes related to flower formation in- clude a bromodomain-associated transcription initiation factor and the WUSCHEL-like gene, an activator of flower patterning, matching to the sex associated region on chromosome VII, as well as a pentatricopeptide-protein involved in post-transcriptional processes within organelles and a microRNA with high similarity to the sex associated region on chromosome XV.

Seeds and seedlings were genotyped with 24 sex-associated SNP markers and sexed by means of a PCoA, showing that they are female-biased, with female proportions of 0.76 (95% CI, 0.64-0.88) and 0.80 (95% CI, 0.68- 0.92), respectively. These ratios are very similar and statistically indistin- guishable from each other and from the sex ratio of reproductive plants of 0.77 (95% CI, 0.66-0.88) (Figure 8A), which indicates that the bias is prima- ry. This primary female bias cannot be maintained by polygenic sex deter- mining systems with Mendelian segregation of nuclear alleles (Bachtrog et al, 2014; Bull, 1983; Fisher, 1915). When 25 generations of mating and gamete production were simulated, the female-bias decayed towards 50%, but when moderate segregation distortion was introduced by favoring transmission of the female-associated allele in 40% of the female gametes, female bias was stable (Figure 8B). In other words, when transmission distortion of sex determining alleles was introduced, female bias was maintained. This segregation distortion could be produced by three main different processes:

sex-locus meiotic drive (Beukeboom and Perrin, 2014; Jaenike, 2001), cyto- nuclear interactions or zygote abortion (Maurice, 1992; Werren and Beukeboom, 1998).

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26

Figure 8. Sex bias across different life history stages and generations. (A) Propor- tion of females with 95% confidence intervals across life history stages of the arctic- alpine willow Salix herbacea determined by marker-assisted-sexing (seed and seed- lings) or by field observation (adults). (B) Proportion of females with 95% confi- dence intervals over 25 generations of simulated mating with different levels of segregation distortion; simulations started from observed genotypes at 24 sex- associated loci.

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27

Conclusion

Even though the alpine dwarf willows Salix herbacea presented differences in flowering time between microhabitats triggered by snowmelt time, sub- populations growing in different microhabitats are not genetically differentiated. However, populations from the snowbeds are more diverse and may act as sinks of genetic diversity. In both microhabitats, there is selection for smaller leaves, shorter intervals between snowmelt and leaf expansion, and shorter thermal duration until leaf expansion. Significant heritabilities were found for leaf size, clonal and sexual reproductive traits. Multiple genomic regions were associated with the explored traits and some well-known candidate genes for phenological and tolerance traits were recovered. Multiple regions that diverged between microhabitats were detected suggesting that new genomic divergence can arise at very local geographic scales in the presence of gene flow and strong environmental differentiation. Regions of high genomic divergence are possibly related, based on the flanked genes, with selection on osmotic tolerance, stem elongation and root growth, as well as genomic constrains such as reduced recombination in sex- determining regions. Finally, sex bias may matter for adaptation to climate change because different sexes of many dioecious species differ in several traits and functions that may fluctuate with changing conditions. In alpine S.

herbacea, however, we found no evidence for ecological differences be- tween sexes and female sex bias was uniform across altitudes and microhabitats. Female bias in this species is likely maintained by a polygenic sex determination system together with transmission distortion, for instance via sex-locus meiotic drive or cyto-nuclear interactions. Fast-evolving microhabitat-driven genomic divergence and, at the same time, genetically-based trait variation at a larger scale may play a role for the ability of this species to persist in diverse and variable conditions. Ultimately, this research illus- trates how small-scale environmental variability helps understanding the way organisms may react to changing conditions.

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28

Svensk sammanfattning

Den ekologiska genetiken hos dvärgvide (Salix herbacea L.) i en värld som förändras

En av huvudfrågorna inom ekologi och evolution är hur växter reagerar på klimatförändringar. Populationer som möter ändrade livsvillkor kan antingen anpassa sig, utrotas eller migrera. Småskaliga variationer i miljö (olika mikrohabitat) ger en unik möjlighet att utforska dessa alternativ. Jag har i min avhandling använt mig av ekologiska kartläggningar, fältexperiment och molekylära metoder för att studera en rad möjliga sådana småskaliga respon- ser hos den alpina dvärgviden Salix herbacea L. Dvärgvide blev av Linné benämnt som världens minsta träd och de populationer jag valt att studera växer i Schweiziska alperna.

Då genflöde mellan populationer kan påverka den potential de har för anpassning och migration har jag först undersökt om den fenologiska variationen som beror på att snösmältningen sker vid olika tidpunkter påverkar genflödet. Jag såg att platser med sen snösmältning hade högre genetisk mångfald jämfört med platser med tidig snösmältning. Det fanns ett högt genflöde mellan populationer i olika mikrohabitat trots stora skillnader i snösmältning vilket sannolikt beror på att dess frön kan spridas över långa avstånd.

Jag har genom att kombinera olika metoder undersökt selektion, ärftlighet och genomisk arkitektur för ett antal ekologiskt relevanta egenskaper samt genomiska skillnader mellan områden med varierande tidpunkter för snö- smältning. Syftet med detta är att korrelera specifika områden i genomet med fenologiska egenskaper så som tillväxt och fitness, samt undersöka vart i genomet man kan hitta genetisk variation som kan associeras med platser med antingen sen eller tidig snösmältning. De flesta skillnader i genomse- kvenser som kunde korreleras med tidpunkt för snösmältning är nya mutat- ioner som finns i några få regioner. Detta indikerar att de uppkommit i en liten skala i närvaro av genflöde och stark miljödifferentiering, snarare än att ha ursprung i den ”stående” genetiska variationen.

Salix herbacea har en skev könsfördelning, vilket inte är ovanligt inom detta genus. Sjuttio procent av individerna i de vilda populationerna är hon- liga. Skillnader i könsfördelning kan ha betydelse för växtens anpassning till klimatändringar eftersom vissa funktioner hos de två könen hos många tvåkönade växter kan påverkas olika av förändrade förutsättningar i livs- miljö. Denna skeva könsfördelning fanns i hela det område som ingått i stu-

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29 dien och redan hos både frö och unga plantor. Processer som kan bidra till att bibehålla denna skeva fördelning är polygen könsbestämning och ojämn överföring av alleler till nästa generation, det senare kan bero på meiotiskt tryck, cytoplasmatisk reglering eller ökad dödlighet hos vissa zygoter. Fler experiment behövs för att ge klarhet i detta.

Sammanfattningsvis verkar både de småskaliga snabbt föränderliga genomiska skillnaderna som drivs av olika levnadsförhållanden i olika mikrohabitat och storskalig variation i genetiskt baserade egenskaper spela en roll då det gäller förmågan hos S. herbacea att leva och överleva i en föränderlig miljö.

(Översättare: Anna-Malin Linde)

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30

Resumen en español

Genética ecológica de Salix herbacea L. en un mundo cambiante

Como responden las plantas al cambio climático es hoy por hoy una de las preguntas esenciales en ecología y evolución. Dadas las condiciones ambientales cambiantes, las poblaciones podrían responder adaptándose, extinguiéndose o migrando. La variación ambiental a una escala local ofrece un mosaico único para explorar estas alternativas. En la presente tesis, he usado experimentos de campo y diversos métodos moleculares para estudiar el rango de posibles respuestas a una escala ambiental muy fina, modulada por los patrones de derretimiento de la nieve, en el sauce alpino enano Salix herbacea L., nombrado por Linneo como el árbol más pequeño del mundo ya que solo crece unos pocos centímetros por encima del suelo.

Debido a que el flujo genético puede impactar el potencial adaptativo y migratorio, primero que todo he explorado en qué medida la desincronización en la floración debido a los patrones de derretimiento de la nieve limita el flujo genético entre poblaciones con floración temprana respecto a poblaciones con floración tardía. He encontrado que sitios con derretimiento tardío de la nieve, usualmente depresiones, actúan como centros de diversidad cuando estos son comparados con sitios donde el derretimiento de la nieve es más temprano, usualmente riscos. Pese a las diferencias en tiempos de floración, no hay aislamiento genético, probablemente debido a que las semillas son dispersadas por el viento a grandes distancias independientemente de la topografía y del momento en el que se derrite la nieve.

Adicionalmente, he usado una combinación de genética cuantitativa, mapeo asociativo y escaneos genómicos para inferir la selección, heredabilidad y arquitectura genómica de caracteres relevantes ecológicamente a lo largo del gradiente de derretimiento de la nieve. De este modo, he podido entender qué regiones en el genoma están relacionadas con fenología, crecimiento y aptitud reproductiva, y qué regiones mantienen variación genética asociada con adaptación al derretimiento temprano y tardío de la nieve. He encontrado que la mayoría de la diversidad genómica causada por la variación en el derretimiento de la nieve es reciente y localizada en unas pocas regiones en distintos cromosomas. Ello sugiere que la divergencia genómica está actualmente surgiendo a una escala muy local, en presencia de flujo genético y fuerte diferenciación ambiental, en lugar de ser reclutada de variación ya existente.

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31 Finalmente, Salix herbacea, que es dioica, presenta un desequilibrio fuerte hacia hembras, lo cual no es raro dentro del género Salix. Setenta por ciento de los individuos en poblaciones silvestres son hembras. El desequilibrio en el radio sexual puede importar para adaptación al cambio climático porque diferentes sexos de especies dioicas varían en caracteres y funciones que fluctuarían con condiciones cambiantes. He encontrado que el desequilibrio es uniforme independientemente de los ambientes y que ya está presente al nivel de semillas. Esto indica que la desviación en el radio sexual es primaria. Un mecanismo de determinación sexual poligénico con distorsión en la segregación puede ser el responsable de mantener este patrón. La distorsión en la segregación se podría deber a desviación meiótica, regulación citoplasmática o mortalidad de los cigotos. Sin embargo, nuevos experimentos son necesarios para clarificar cuál de estos mecanismos es el responsable de generar la asimetría en la proporción sexual.

Para concluir, la reciente divergencia genómica debida a diferencias micro-ambientales, y la variación genética que regula diversos caracteres con importancia ecológica, juegan un rol importante en la habilidad de S.

herbacea para persistir en condiciones variables. A su vez, este estudio nos enseña cómo la variación ambiental a pequeña escala ofrece un mosaico ideal para estudiar las reacciones de las poblaciones a condiciones cambiantes.

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32

Acknowledgments

Special thanks to Sophie Karrenberg for her unparalleled advice, patience and encouragement during these years. I improved very much my critical thinking and my writing and oral skills thanks to her. I also want to acknowledge Julia Wheeler and Janosch Sedlacek (R.I.P.) for their professionalism, constancy and friendship during the long days of fieldwork and the weeks of R-coding. I very much appreciate Christian Lexer’s enthusiasm to co-supervise me during this time and always welcome me at Fribourg or at several expeditions by the Alps. I wish to thank the rest of the sinergia team, too - Oliver Bossdorf, Günter Hoch, Mark van Kleunen, Christian Rixen, and Sonja Wipf, for insightful discussions during the project meetings and the draft of manuscripts, and for making my stays in Konstanz and Davos very enjoyable.

I am also deeply grateful for the help of all the assistants who during the years made field and lab work more doable, they are: Julia Dankanich, Danielle Franciscus, Sofia Häggberg, Emilie Hallander, Günther Klonner, Chelsea Little, Magali Matteodo, Felix Prahl, Sofia Renes, Vytautas Rindzevicius, Philippe Roux-Fouillet (R.I.P.), Christian Scherrer, Flurina Schnider and Anja Zieger. Also, the people from the lab in Fribourg who made my stay there more enriching and also taught me petanque – Maria Amaral, Thelma Barbara, Celine Caseys, Dorothea Lindtke, Kai Stölting and Stephan Waeber. Help from Kerstin Jeppsson and Jenny Glans in the lab and the green house in Uppsala is also highly appreciated.

I am very much in debt with Steve DiFazio, Fredy Gouker and Larry Smart for allowing me access to the S. purpurea assembly, and with Sofia Berlin, Pascal Pucholt and Ann Rönnberg-Wästljung for letting me explore the S. viminalis resource as a potential candidate for reference genome. Also thanks to Richard Abbott, Inger Alsos, Alexander Antonelli, Christian Brochmann, Michael Donoghue, Jeff Doyle, Lutz Eckstein, Abel Gizaw, Bente Graae, Ary Hoffman and Brian Husband for insightful discussions and committed correspondence at different moments. I acknowledge the Institute for Genomic Diversity at Cornell University, especially Charlotte Acharya, Katie Hyma and Sharon Mitchell, and LGC Genomics, especially Jonathan Curry and Sam Gunn, for advice and commitment in genotyping, and the computation resources provided by the Swedish National Infrastructure for Computing (SNIC) through Uppsala Multidisciplinary Centre for Advanced Computational Science (UPPMAX) under project p2011044.

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33 This research was mostly funded by the Sinergia grant CRSI33_130409 from the Swiss National Science Foundation (SNSF). I was also supported during the three years of fieldwork in Switzerland by a Godfrey Hewitt scholarship and the Liljewalchs, Lundins, Sederholm and Tullberg funds, and during fieldwork in Scandinavia by Kungliga Vetenskapsakademien, Svenska Växtgeografiska Sällskapet and the Bjurzon, Extensus, Sernander and Regnell funds. Some lab/office expenses were covered by the Lundell and Dahlgren funds. I was encouraged to participate in several group meetings, workshops, excursions and lab visits by the Håkansson fund, the EBC Graduate School on Genomes and Phenotypes, and the Graduate Research School in Genomic Ecology (GENECO). I appreciate enormously the trust that all these agencies, funds and societies deposited on my commitment to carry out and successfully conclude with my scientific research, and the ef- fort of the two mentioned graduate schools, and in particular Kaz Armour, Ted Morrow, Jesper Nyström, Christina Rengefors, Joanna Rose, Helena Westerdahl and Jochen Wolf, to increase my academic network, and gave me tools to make my PhD studies more fruitful.

I appreciate the trust, support and inspiration that Matthew Blair and San- tiago Madriñan provided me in the earlier stages of my ongoing research, and the useful mentoring that Colin Kelleher and Åke Olson offered me. I also appreciate the feedback that Jon Ågren, Ingrid Ahnesjö, Ulf Lagercrantz, Martin Lascoux, Amy Parachnowitsch and the rest of the Plant Ecology and Evolution community at EBC gave me at different stages while disseminating my research. The same appreciation goes towards the people at APPS, CIAT, Uniandes and TSU. I also thank Tove Broberg, Frida Sved- bergh, Marie Swanberg and Ulla Johansson for dealing with paperwork.

Last but not least, life in Uppsala has been entertaining because of close colleagues and friends like Alberto, Amandine, Amelie, Anna-Malin (who, by the way, very kindly assisted me with the Swedish abstract), Camille, Charlie, Dmytro, Elham, Elodie, Fia, Froukje, Jesus, Ioana, Li, Lina, Maria, Marina, Matthew, Nannan, Rike (who generously commented on the Kappa as a green biologist who doesn't like plants), Tora and Xiaodong, and the swiss-swede girls, Janine, Jasmin, Judith and Reiko, always committed for fikas, dinners, movies, trips or parties. Sagda Correa is thanked for advice in the design. Great gratitude to the flatmate and friend who has been a daily companion - Gonçalo, and to Bello Brayan, who volunteered to be by my side in the last stages of this thesis. The funny guys Alex, Balint, Bo, Hector, João, Johnny, Mario, Niek, and Niklas, and the Colombian fellows Adriana, Alexandra, Andrés, Angela, Carolina, Catalina, Edwin, Giovanni, Ivan, Juan, Julie, Lizeth and Martha, are recalled pleasantly because of offering an air of familiarity and camaraderie in the foreign lands of Europe. Of course, the Feiroz’s staff will be remembered by their tastiness. Finally, I am very happy that Beba Boba and Pillo supported and accompany me unconditionally during my wanders by Colombia, Switzerland, Sweden and USA.