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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
ACTA
UNIVERSITATIS
UPSALIENSIS
UPPSALA
2015
ISSN 1651-6214
ISBN 978-91-554-9337-0
urn:nbn:se:uu:diva-262239
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.
© Andrés J. Cortés 2015
ISSN 1651-6214
ISBN 978-91-554-9337-0
urn:nbn:se:uu:diva-262239 (http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-262239)
“Laughter is a sunbeam of the soul.”
― Thomas Mann, The Magic Mountain
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 diversity 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 microhabitats. Manuscript in revision
III
Cortés, A.J., Wheeler, J.A., Sedlacek, J., Lexer, C., Karrenberg, S. Genome-wide patterns of microhabitat-driven divergence 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 genomic conflict. Manuscript in revision
Reprints were made with permission from the respective publishers.
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., Lexer, C., Wipf, S., Karrenberg, S., van Kleunen, M., Rixen, C. (2015) The
Response of the Alpine Dwarf Shrub Salix herbacea to Altered Snowmelt 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 facilitation 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.
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 ERECTA 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., CarrasquillaGarcia, 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 Population 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 tolerance, 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 mapping in common bean. BMC Genetics, 12:48
•
Kelleher, C.T., Wilkin, J., Zhuang, J., Cortés, A.J., Perez, A.L., Gallagher, T.F., Bohlmann, J., Douglas, C.J., Ritland, K. (2012) SNP discovery, 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 Genetics, 123(5):827-45
Contents
Introduction ................................................................................................... 11
Gene Flow (I) ........................................................................................... 12
Traits under Selection (II & III) ............................................................... 13
Sex Ratio (IV) .......................................................................................... 14
Aims of the Thesis.................................................................................... 15
Materials and Methods .................................................................................. 16
Study System ............................................................................................ 16
Sampling Locations .................................................................................. 17
Plot Survey (I & II) .............................................................................. 17
Transect Survey (III & IV) .................................................................. 17
Phenotyping.............................................................................................. 17
Genotyping ............................................................................................... 18
Microsatellite Genotyping (I & II)....................................................... 18
Genotyping-by-Sequencing (III & IV) ................................................ 18
Analytical Approaches ............................................................................. 19
Results and Discussion ................................................................................. 21
Alpine snowbeds are sinks of genetic diversity (I) .................................. 21
Heritabilities and Selection (II) ................................................................ 22
Between-microhabitat genomic divergence (III)...................................... 23
Female-bias and polygenic sex determination (IV).................................. 24
Conclusion .................................................................................................... 27
Svensk sammanfattning ................................................................................ 28
Resumen en español ...................................................................................... 30
Acknowledgments......................................................................................... 32
References ..................................................................................................... 34
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 variation 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 composition (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 Ranunculus (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.
11
Figure 1. Snowbeds and ridges as seen early in the spring (May 2011) at Wannengrat, 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 seedmediated 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-
12
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 microhabitats, and thus persist in situ upon climate change. Apart from differentiation 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 welldifferentiated 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 regarded as porous in the sense that different regions present contrasting signatures 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 random drift (Keller et al, 2013), regions with low divergence may be due to
balancing or uniform selection, high gene flow or ancestral shared polymorphism (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 betweenmicrohabitat 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).
13
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, separate 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 ancestors 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).
14
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 abortion of seeds or embryos (Barrett et al, 2010).
Aims of the Thesis
The following questions were addressed in the alpine dwarf shrub Salix herbacea 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?
(III)
6. What is the mechanism leading to adult female bias? (IV)
15
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 ecosystems (Beerling, 1998). In the Swiss Alps, S. herbacea is an optimal species 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 25 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 populations 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.
16
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 primarily 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 mountains. 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 avalanches, 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.
17
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 immediately 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 (QIAGEN, 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 technology (Cuppen, 2007). Flanking sequences were extracted from the S. purpurea
assembly (Carlson et al, 2014), and were used in Primer-Picker (LGC Genomics, UK) to design allele-specific oligonucleotides. Fluorescence signals
were interpreted by the KlusterCaller 1.1 software (LGC Genomics).
18
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 subdivisions 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 (Ne) and pairwise migration rates (Nem) were estimated following coalescent 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 106 bps, 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). Linkagedisequilibrium (LD) and Tajima’s D (Tajima, 1989) were computed in the
same windows with the R package PopGenome (Pfeifer et al, 2014). Secondly, 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 epistatic interactions and dominance effects, and to confirm single trait-marker
associations (Gyenesei et al, 2012).
19
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 estimation (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 Gaussian 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 sexassociated 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. Random mating was further simulated by randomly pairing these gamete genotypes. The sex of the resulting zygotes was assigned using a principalcoordinates-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.
20
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 different microhabitats are not genetically differentiated (FST within- and between-microhabitat comparisons 0.028 ± 0.003 and 0.035 ± 0.004 for withinand between-microhabitat comparisons, P-value = 0.691. This is also supported 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. herbacea 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).
21
Figure 5. Estimates of the number of migrants per generation (Nem) between microhabitat 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 phenological, 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 generation 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, prevent early season frost damage.
22
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 interactions 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). Significance values are bolded.
Change in stem number
Proportion flowering stems
Fitness
Trait (standardized)
β
df
F
p
-0.023
0.029
-0.041
0.211
0.054
0.179
67
67
67
67
67
67
0.032
0.342
0.426
4.153
0.076
1.110
0.86
0.561
0.516
0.046
0.784
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
Leaf size
Interval snowmelt to leaf expansion
-5.232 116 -3.279 0.011
-3.655 116 -2.217 0.051
Thermal duration until leaf expansion
Thermal duration until flowering
Leaf size x MH
Interval snowmelt to leaf expansion x MH
-3.646
2.467
3.758
4.058
Leaf size
Interval snowmelt to leaf expansion
Thermal duration until leaf expansion
Thermal duration until flowering
Leaf size x MH
Interval snowmelt to leaf expansion x MH
116
116
116
116
-2.218 0.020
1.418 0.85
1.614 0.073
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 coincided 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 FST was computed at each
transect, and they overlapped with ‘valleys’ in the FST when this statistic was
calculated within microhabitats and across transects, indicating that these
divergent regions are not the result of genetic drift. The ten betweenmicrohabitat divergence regions spanned a total of 219 genes, which may
help inferring functional traits that diverge between microhabitats.
23
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 microhabitatdriven 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 FST differentiation. Interchromosomal 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). Females 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).
24
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 include 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, 0.680.92), respectively. These ratios are very similar and statistically indistinguishable 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 primary. This primary female bias cannot be maintained by polygenic sex determining 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), cytonuclear interactions or zygote abortion (Maurice, 1992; Werren and
Beukeboom, 1998).
25
Figure 8. Sex bias across different life history stages and generations. (A) Proportion of females with 95% confidence intervals across life history stages of the arcticalpine willow Salix herbacea determined by marker-assisted-sexing (seed and seedlings) or by field observation (adults). (B) Proportion of females with 95% confidence intervals over 25 generations of simulated mating with different levels of
segregation distortion; simulations started from observed genotypes at 24 sexassociated loci.
26
Conclusion
Even though the alpine dwarf willows Salix herbacea presented differences
in flowering time between microhabitats triggered by snowmelt time, subpopulations 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 sexdetermining 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 between 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 illustrates how small-scale environmental variability helps understanding the
way organisms may react to changing conditions.
27
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 responser 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 snösmältning. 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 genomsekvenser som kunde korreleras med tidpunkt för snösmältning är nya mutationer 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 honliga. 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 livsmiljö. Denna skeva könsfördelning fanns i hela det område som ingått i stu28
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)
29
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.
30
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.
31
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.
32
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 effort 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 Santiago 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 Svedbergh, 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.
33
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Acta Universitatis Upsaliensis
Digital Comprehensive Summaries of Uppsala Dissertations
from the Faculty of Science and Technology 1288
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