Florida State University Libraries
Electronic Theses, Treatises and Dissertations
The Graduate School
2005
The Role of Local Adaptation in the
Evolution of Reproductive Isolation in
Diodia Teres
Joe Hereford
Follow this and additional works at the FSU Digital Library. For more information, please contact [email protected]
FLORIDA STATE UNIVERSITY
COLLEGE OF ARTS AND SCIENCES
THE ROLE OF LOCAL ADAPTATION IN THE EVOLUTION OF
REPRODUCTIVE ISOLATION IN DIODIA TERES
By
JOE HEREFORD
A Dissertation submitted to the
Department of Biological Science
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
Degree awarded:
Fall Semester 2005
The members of the Committee approve the dissertation of Joe Hereford
defended on August 3rd 2005.
__________________________
Alice A. Winn
Professor Directing Dissertation
__________________________
J. Anthony Stallins
Outside Committee Member
__________________________
Hank Bass
Committee Member
__________________________
David Houle
Committee Member
__________________________
Joseph Travis
Committee Member
Approved:
____________________________
Timothy S. Moerland, Chair, Department of Biological Science
The Office of Graduate Studies has verified and approved the above named
committee members.
ii
To my mother Dorothy and my two sisters Angie and Amy
iii
ACKNOWLEDGEMENTS
I thank my advisor Alice A. Winn without who was tireless in teaching me
to think critically, design experiments, and present my work. Though I did not
work on a project that directly benefited her research program, Alice gave me
anything I wanted including over half of the lab and all the greenhouse space I
needed. She has seen me through some tough times both professionally and
personally and has always been unwavering in her support of myself and my
work. Much of this work is done in collaboration with Dr. Winn, specifically
chapter 3.
I thank David Houle who showed me alternate ways of doing things. His
door was always open to me, despite the number of students and projects
circulating through his lab.
I thank Joe Travis for taking the time to think about my project, talk to me
about experimental design, and taking the time to write extensively about the
merits of different metrics of adaptive divergence. I would strongly encourage
any student in ecology and evolutionary biology to have as much exposure to
these three people as possible.
I also thank my other committee members Hank Bass and Tony Stallins
for making me think of better ways to present my results. I want to thank Lei Li
and Tony Arnold for serving on my committee in the past.
Don Levitan and Tom Miller provided lab space and tools/materials for
many of the projects that I worked on. Don Levitan and Thomas Hansen
provided many helpful discussions on topics ranging from reproductive isolation
to measurement of natural selection.
I thank Ken Moriuchi for providing discussions of just about every phase of
this project, and for being a catalyst for other projects that he and I have worked
on. Likewise I thank past and present students in the ecology and evolution
department for providing many critical comments on all the presentations and
papers that I presented to them.
iv
Sharon Herman, Chuck Martin, Doug Scott, Greg Seamon, and the
Florida Department of Environmental Protection provided access to all the field
sites in this study.
This project was funded by National Science Foundation grants to Alice A.
Winn, and by a National Science foundation dissertation improvement. I also
received funding from the department from the Godfrey award in Botany, and
funding from the university from a dissertation research grant.
v
TABLE OF CONTENTS
List of Tables
vii
List of Figures
x
Abstract
xii
1. Introduction
1
2. Phenotypic Selection and Local Adaptation in Diodia teres (Rubiaceae)
4
3. Quantitative Measurement of Parallel Local Adaptation in Six Populations of
an Annual Plant
20
4. Differences in the Strengths of Alternate Modes of Reproductive Isolation in
the Annual Plant Diodia teres (Rubiaceae)
48
5. Are There Positive Relationships Between Divergence and Reproductive
Isolation within a Species?
75
6. Conclusions
94
References
97
Biographical Sketch
108
vi
LIST OF TABLES
Table 2.1 Generalized linear model of size at planting, planting site, source
population, genotype nested within source population, and planting site by
source population interaction on fruit production. Fruit production was assumed
to be Poission distributed.
17
Table 2.2. Analysis of selection on three traits in the three planting sites.
Selection coefficients were calculated for genotype means, and N refers to the
number of genotypes. Means are means of genotype mean for each trait.
Standard deviations are given in parentheses. Sµ is the univariate selection
elasticity, βµ is the multivariate elasticity for the trait, and β is the selection
gradient. The bootstrap calculated confidence intervals for β are also presented.
Selection estimates in bold are significantly different from zero.
18
Table 3.1. Mean (standard deviation) percent cover of D. teres, heterospecific
herbs, and canopy, and percent sand and clay in the soil at the six planting sites.
Numbers in bold are habitat means. The χ2 values quantify the effect of habitat
type on each variable in Kruskal-Wallis tests. All tests were significant at P <
0.01 with 2 degrees of freedom.
37
Table 3.2. Mahalnobis distances between all pairs of sites based on percent
cover of D. teres, hebaceous plants, tree canopy, and percent sand in the soil.
Boldface type indicates distances between sites within a habitat type.
38
Table 3.3. Generalized Linear Model for A effects of year, planting habitat type,
source habitat type, planting site nested within planting habitat type, source
population nested within source habitat type, and their interactions on fruit
number, and B the same model excluding effects of habitat.
39
Table 3.4. Comparisons by planting site of fruit number produced by plants in a
foreign planting site, but their native habitat type versus all plants from foreign
habitats. The native source populations were excluded from these tests. All
tests are Generalized Linear models as described in the text with 1 degree of
freedom. Tests are one tailed.
40
Table 3.5. Mean (standard deviation and sample size) number of fruits produced
by transplants from each source population transplanted into each planting site.
Means in bold are the fruit production of populations in their native planting site.
Boxes enclose groups of means from the same habitat type. Row marginals are
means (standard deviations and sample sizes) for each source population across
all planting sites, and column marginals are means for each planting site.
Marginal means that share superscript are not significantly different. Asterisks
refer to results of pairwise comparisons of source population means within each
planting site .
41
vii
Table 3.6. Mean (standard deviation and sample size) of the maternal family
means of number of fruits produced in the 2003 reciprocal translplant
experiment. Symbols as in Table 3.5.
42
Table 3.7. Comparisons of mean fruit number produced for plants from the
native population and plants from all other populations pooled. All tests are
Generalized Linear models as described in the text with 1 degree of freedom.
Tests are one tailed.
43
Table 3.8. Measures of Fitness Divergence between all possible population pairs
based on fruit numbers from the 2002 and 2003 reciprocal transplants.
44
Table 4.1. Generalized Linear Model of effects of pollen donor source
population, pollen recipient population and their interaction on probability of
successful hand pollination. Denominator degrees of freedom for all sources of
variation is 98.
65
Table 4.2. Mean (standard deviation, number of pollen recipients) rate of
successful hand pollination between populations as pollen donors and pollen
recipients. Rates in bold are means of purebred crosses. Marginal values are
mean pollination success of populations when acting as pollen donors or pollen
recipients. Marginals that share the same letter superscript are not significantly
different. Asterisks indicate significance of comparisons between a specific
hybrid pollination and the pooled hand pollination success rate of the purebred
crosses of its parental populations.
66
Table 4.3. Generalized Linear Model of effects of date of planting, initial size at
planting, block nested within planting site, planting site and their interactions on
number of fruit produced by F2 progeny from hand pollinations within and
between populations. Denominator degrees of freedom for all sources of
variation is 2882.
67
Table 4.4. Mean (standard deviation, number of fullsib families) number of fruits
produced by fullsib families of hybrid and purebred progeny. Means in bold
indicate purebred progeny at their native planting site. Marginal row totals are
means of all hybrids and the native purebreds at each planting site, and column
marginal totals are means of each population as a hybrid parent across planting
sites. Marginal means that share the same letter superscript are not significantly
different. Asterisks indicate significance of comparisons of the fruit set of hybrids
with the fruit set of the native parental purebred at each planting site.
68
Table 4.5. Matrix of pairwise estimates of postmating / prezygotic reproductive
isolation (above) and postzygotic reproductive isolation (below) between
populations. Negative estimates of postmating/prezygotic isolation indicate that
hybrids crosses were more successful than purebred crosses, and positive
values indicate the opposite. Values of postzygotic isolation less than one
viii
indicate that hybrids had lower relative fitness than purebreds, and values
greater than one indicate that hybrids had greater relative fitness. Only one
hybrid family between the Sandhills populations, and between Dunes population
1 and Sandhills population 1 survived into the F2 generation. Thus no estimate of
postzygotic reproductive isolation is given for these populations.
69
Table 4.6. Generalized Linear Model of the effects of date of planting, initial size
at planting, planting site, experimental block within planting site, planting site,
cross combination, and genotype nested within cross combination on hybrid fruit
number. The model included the fruit production of hybrids only. Denominator
degrees of freedom for all sources of variation is 1154.
70
Table 5.1. Descriptive statistics of population genetic structure by locus and
population. "Allele/loci" column gives the number of alleles segregating at each
locus, or the proportion of loci that are polymorphic within each population.
Lower bound and upper bound refer to 95% confidence intervals of overall
estimates of F-statistics calculated by bootstrapping 700 replicates.
88
Table 5.2. Allele frequencies at each locus within each population.
89
Table 5.3. Estimates of Nei's (1978) genetic distance between all pairs of
populations. Habitat abbreviations are, IN = Inland, SA = Sandhills, DU = Dunes.
Distances in bold are between populations from the same habitat type.
90
ix
LIST OF FIGURES
Figure 2.1. Mean number of fruits produced at each planting site by each source
population. The Dunes source population is represented by the solid circles, the
Sandhills by the open circles, and the Inland source population is represented by
the solid triangles. Means that share the same letter are not significantly different
within planting sites after correction for multiple comparisons.
19
Figure 3.1. Map of the locations of the source populations and planting sites in
northern Florida and south Georgia. Dunes populations are abbreviated DU,
Inland abbreviated IN, and Sandhills abbreviated SA.
45
Figure 3.2. Mean fruit production of transplants by habitat type for the 2002 and
2003 experiments. Means within a habitat type that share the same letter are not
significantly different based on Generalized Linear Model of the effect of source
habitat on fruit set. Error bars are one standard error.
46
Figure 3.3. The relationship between squared environmental distance and
Fitness Divergence from the 2002 experiment and 2003 experiments. The
mantel correlation was 0.37 (p = 0.10) in 2002 and -0.03 (p = 0.51) in 2003.
47
Figure 4.1. A diagram of a flower of D. teres (taken from Zomlefer 1994).
71
Figure 4.2. Meanpollination success of purebred crosses, intra-habitat crosses,
and inter habitat crosses Error bars are one standard error of the mean. Points
that share the same letter superscript are not significantly different.
72
Figure 4.3. Meanfruit number of purebred, intra-habitat, and inter habitat
progeny. Points that share the same letter superscript are not significantly
different. Error bars are one standard error of the mean.
73
Figure 4.4. The relationship between pairwise postmating/prezygotic
reproductive isolation and postzygotic isolation for all pairs of populations. The
rank correlation between measures is -0.03, p>0.90.
74
Figure 5.1 A representative gel of the locus PGM. The lane that is marked
shows the "fast" allele in an individual native to Inland population 1. All other
lanes contain the “slow” allele.
91
Figure 5.2. Phenogram of the six study populations using allozyme loci and
obtained by cluster analysis (UPGMA).
Figure 5.3. Relationships between each of the two modes of reproductive
isolation, and the two measures of divergence. The Pearson correlation
x
92
coefficients are A = -0.05, B = 0.05,C = 0.20, and D = -0.47. Only the correlation
between postzygotic isolation and genetic distance (section D) was at least
marginally significant (p = 0.10).
93
xi
ABSTRACT
Numerous studies of local adaptation have shown that populations can
adapt quickly to local environmental conditions. Other studies have shown that
sister species tend to occur in different environments. Recent work has gone a
long way toward showing that adaptation can directly result in reproductive
isolation under some circumstances, but few studies have attempted to measure
the effects of local adaptation on the degree of reproductive isolation. Here I
have attempted to bring together the research on local adaptation and studies of
reproductive isolation to test hypotheses of the ultimate causes and mechanisms
of local adaptation, and their consequences for the evolution of reproductive
isolation between locally adapted populations. In the first chapter I describe a
test of the underlying mechanism of local adaptation between populations of the
annual plant Diodia teres. In the second chapter (written in collaboration with
Alice A. Winn), I test the hypothesis that the degree of adaptive divergence
between six populations of this species is correlated with the degree of
environmental variation between populations. The third chapter is a test of
hypotheses about the differences between the strength of prezygotic and
postzygotic reproductive isolation. In the fourth chapter I test hypotheses that
reproductive isolation is correlated with the degree of adaptive and nonadaptive
divergence between populations. I found evidence of local adaptation between
some populations, but was not able to identify traits that were responsible for that
adaptation. The degree of adaptation was not correlated with environmental
differences, suggesting that divergent selection is not the only force acting on
reproductive isolation. I found that postmating/prezygotic isolation was stronger
than postzygotic isolation, and that the degree of divergence was not correlated
with any measure of reproductive isolation. Overall this study shows that
adaptation to local conditions can be associated with the evolution of
reproductive isolation, but it also shows that divergent selection alone does not
account for all adaptive divergence and isolation. It is one of the few studies to
quantify reproductive isolation early in divergence, and to examine the
xii
relationships between divergence and reproductive isolation over a range of
variation, not limited to a single pair of nearly completely isolated populations.
xiii
CHAPTER 1
INTRODUCTION
The primary goal of the field of evolutionary biology is to understand the
processes that gave rise to the diversity of organisms on the planet. The study of
speciation seeks to explain this diversity by determining how a single species
evolves into multiple species. This field has a long history in evolutionary
biology, but has often been mired in debates that are tangential to the rules that
govern the evolution of new species.
One of the most controversial issues of speciation is the question of the
relative importance of sympatric and allopatric speciation (Coyne and Orr 2004).
Most would agree that speciation occurs in most often in alloparty, but there
remains much contention about the frequency of sympatric speciation (Mayr
1963; Bush 1975; Coyne and Orr 2004). Proponents of allopatric speciation
contend that limited gene flow is essential to the formation of new species (Mayr
1963; Coyne and Orr 2004) while proponents of sympatric speciation contend
that if selection is strong enough sympatric speciation is possible, and that it
occurs in nature (Bush 1975). These arguments were largely driven by
biogeographic patterns of species distributions (Coyne and Orr 2004), without
any formal development of theory about the underlying mechanisms of
speciation.
Research that focused on mechanisms was largely theoretical
(Felsenstein 1981; Barton and Charlesworth 1984; Carson and Templeton 1984;
Gavrilets 2003), and there were few empirical tests of these theories. For
example, Carson and Templeton (1984) argued that genetic drift was responsible
for rearranging variation within populations and promoting reproductive isolation
between populations, while Barton and Charlesworth (1984) argued that
divergent selection was largely responsible for generating reproductive isolation
between populations, and that effective population sizes in nature were not small
enough to allow drift to be an important factor in the evolution of reproductive
1
isolation. Throughout these arguments there was little disagreement that
adaptation to different environments could lead to speciation, but there were few
tests of this idea (but see Moore 1946). Despite limited empirical interest in
adaptation from the speciation literature, there was a wealth of studies of
adaptation to different environments in the ecological genetic literature.
There is also a long history in evolutionary biology of interest in the
evolution of adaptation to local environments within a species (Sumner 1932;
Clausen et al 1940; Mooney and Billings 1961; Schmidt and Levin 1985). This
work has shown that different populations within a species can adapt rapidly to
their native environments, and that these differences can have a complex genetic
basis (Sumner 1932; Clausen et al 1940). However, few of these studies looked
for a relationship between adaptation to different environments and reproductive
isolation between populations. Therefore, there was a disconnect between the
literature of speciation which suggested that adaptation to new environments was
important for the evolution of new species, and ecologists studying local
adaptation but not determining the consequences of local adaptation for the
evolution of reproductive isolation.
Recent work on ecological speciation has shown that adaptation to
different environments can confer reproductive isolation (Schluter 1995; Nosil et
al. 2002; Via and Hawthorne 2002). These studies have shown that adaptation
to different environments can directly result in reproductive isolation because
offspring do not fit into the ecological niche of either of their parents. Schluter
(1995) showed that hybrids between populations of three spine sticklebacks
adapted to different limnetic or benthic environments had relatively lower growth
rates in both parental environments. Other studies have shown that reproductive
isolation increases with divergence (Coyne and Orr 1989; Tilley et al. 1990;
Mendelson 2003; Moyle et al 2004) without any reference to ecological
differences between species. The ecological speciation work and the reviews of
the relationship between reproductive isolation and divergence have shown that
reproductive isolation is associated with higher divergence between species, and
that there is a quantitative relationship between the degree of divergence and the
2
level of reproductive isolation between species. But these studies are typically
conducted between different species with nearly complete reproductive isolation.
Though little reproductive isolation is expected during the early stages of
divergence (Mendelson et al. 2004), there is evidence that populations within a
species can vary in the degree of reproductive isolation. Studies that have
shown optimal outbreeding distance (Waser and Price 1994; Fenster and
Galloway 2000) suggest that reproductive isolation can evolve between
populations of the same species. However, these studies have not determined
the relationship between geographic distance and genetic divergence.
Therefore, we do not know if divergence associated with adaptation to local
conditions between populations of a single species can lead to reproductive
isolation.
Here I describe a set of experiments designed to test for a role of
adaptation among populations of the annual plant Diodia teres on the evolution of
reproductive isolation. In the first chapter I describe a test of local adaptation
among three populations and I attempt to identify traits that might confer
adaptation. In the second chapter I test the hypothesis that there is 'parallel local
adaptation' (Kawecki and Ebert 2004) between six populations of this species. In
the third chapter I quantify reproductive isolation between each of the six study
populations to test the hypothesis that adaptation to different environments has
an effect on the evolution of reproductive isolation. Finally in the fourth chapter I
test the hypothesis that there is a quantitative relationship between the degree of
reproductive isolation and divergence between populations.
3
CHAPTER 2
PHENOTYPIC SELECTION AND LOCAL ADAPTATION IN DIODIA TERES
(RUBIACEAE)
Abstract
Theory predicts that local adaptation or specialization can promote
speciation through the effects of a genetic trade-off between adaptation to
different environments. While many studies have tested the hypothesis of local
adaptation only a few have measured selection on traits thought to confer local
adaptation. In this study I sought to test the hypothesis of local adaptation, and
to measure selection in three environments of the annual plant Diodia teres.
Plants were reciprocally transplanted into three planting sites to test for local
adaptation. Differences in directional selection were estimated using standard
regression based methods. The reciprocal transplant demonstrated local
adaptation between one population pair out of the three possible comparisons.
Though there was evidence of differential selection on some traits, the pattern of
selection did not meet my expectations. I conclude that there is evidence of local
adaptation of some populations, but I have not determined which traits are
responsible for this adaptation.
Introduction
Since the pioneering work of Clausen et al. (1940), it has been repeatedly
shown that population divergence can be a response to environmental variation
and is often adaptive (e.g. Kindell et al. 1996; Nagy and Rice 1997; reviewed in
Linhart and Grant 1996). A few studies have shown that locally adapted
populations are under divergent selection for traits that confer adaptation to
different environments (Jordan 1991; Bennington and McGraw 1995; Etterson
2004a), establishing a link between selective pressures and divergence
associated with environmental variation. The link between differential selective
pressures and local adaptation is widely recognized as a potential driving force of
speciation (Erlich and Raven 1969; Schluter 2000). Populations may vary in
4
fitness as a result of nonadaptive processes such as inbreeding depression or
founder effects, but it is trade-offs associated with adaptation to different
environments that are thought to influence the evolution of reproductive isolation.
Local adaptation is thought to promote speciation or reproductive isolation
through a trade-off in which adaptation to one habitat causes lower fitness in
another habitat as a result of a cost of adaptation (Schluter 2000). This trade-off
usually takes the form of selection for a trait in one environment, and selection
against the trait in another. In this scenario, a hybrid between two locally
adapted populations would have low fitness in both parental environments
because its phenotype would be incompatible with the ecological niches of both
parents (e.g. Nagel and Schluter 1998; Ramsey et al. 2003). Given frequent
reports of local adaptation (reviewed by Linhart and Grant 1996; Schluter 2000),
it is tempting to conclude that speciation due to trade-offs associated with local
adaptation is common as well, and if this argument were taken to its extreme
every population of a species that occurs in different environments should be
reproductively isolated.
Local adaptation results from selection on traits that confer high fitness in
different environments. This selection could take the form of selection in
opposite directions on the same traits in different environments. Or it could take
the form of selection on different traits in different environments. For example,
Nagy (1997) found that selection in opposing directions on petal shape and petal
color in Inland and Coastal sites in hybrids between subspecies of Gilia capitata.
Though the differences in selection were not significant, the trend was for
selection for the native phenotype in each of the environments. In a study of
natural selection on floral morphology (Aigner 2004) found evidence of selection
for narrower flowers under hummingbird pollination, but no selection favoring
wider flowers when pollinated by bees. In these examples, directional selection
confers adaptation to different environments. Opposing selection on the same
trait in different environments implies that local adaptation results from a tradeoff, while selection on different traits in different environments implies that local
adaptation does not result from a trade-off. Instead local adaptation results from
5
independent selection on the different traits in different environments. Of course,
both these mechanisms will be important, but we need to quantify the pattern of
selection on individual traits to determine their relative importance.
Here I describe a study of differentiation among three populations of the
annual plant, Diodia teres and selection on traits that might underlie this
differentiation. I performed a complete reciprocal transplant experiment among
three populations, with clonal replicates of genotypes from each population
planted at all planting sites. I then measured the fruit production of each
replicate to test for the presence of local adaptation. I estimated selection
coefficients for traits that might confer adaptation to different environments to
quantify the pattern of selection. I performed these experiments to address three
questions. What is the pattern of local adaptation among the three study
populations, which traits influence adaptation to different environments, and is
the pattern of selection on the traits consistent with a local adaptation due to a
trade-off or to independent evolution of different traits in different environments?
Materials and Methods
Experimental methods
Diodia teres (Rubiaceae) is a self-compatible annual, found from Panama
to the northeastern United States, the northwestern edge of its range extends to
Michigan, USA (Kearney and Peebles, 1964). In northern Florida, D. teres
occurs in contrasting habitats including coastal sand dunes with sandy soils, little
herbaceous cover and no tree canopy, in Inland sites characterized by clay soils,
more herbaceous growth and a partial tree canopy, and in Sandhills habitats with
sandy soils and more herbaceous growth than Dunes sites (Hereford and Winn
2005). Jordan (1992) demonstrated local adaptation of D. teres to similar
habitats in North Carolina.
In December of 1998, seeds from 20-22 individuals of D. teres were
collected from one source population in each of the three habitat types described
above. The Dunes population seeds were collected at Saint Joseph Bay State
Park in Bay County, Florida. The Sandhills population seeds were collected at
Dog Lake in Leon County, Florida. The seeds from the Inland population were
6
collected at Pebble Hill Plantation in Thomas County, Georgia. All seeds were
germinated and one individual from each original maternal plant was grown to
adult size in an environmentally controlled growth chamber under a15/9 hr.
light/dark cycle and a 30º/20º temperature cycle.
Individual genotypes were replicated by making cuttings from plants grown
in the growth chamber. There was a total of 62 genotypes with 21 from the
Dunes population, 22 from the Inland population, and 19 from the Inland
population. The cuttings were 5 to 8 cm in length and always included at least
two internodes. To aid in the establishment of new roots, each cutting was
treated with a commercial rooting agent. Cuttings were planted in potting soil in
72-well flats and allowed to take root in the greenhouse. Approximately 95% of
the cuttings established roots within 14 days. After making sure that the cuttings
had taken root and had begun to grow new leaves, I planted clonal replicates of
each genotype into the sites of two of the populations from which seeds were
collected, Pebble Hill Plantation and Dog Lake. Cuttings could not be planted at
St. Joseph Bay State Park because there was limited space where human
disturbance could be avoided. Consequently, I planted cuttings at a different
Dunes site, St. George Island State Park, Franklin County, Florida, which is
similar to St. Joseph Bay in soil texture, and D. teres density (J. Hereford
unpublished data).
A total of 718 cuttings were planted with an average of three replicates
from each genotype at each planting site in May of 1999. I estimated the initial
size of each cutting by counting the number of internodes on the day they were
planted. In December of 1999 when about 90% of plants had senesced, I
counted the number of fruits on each plant. D. teres fruits always produce only 2
seeds, therefore fruit number is always half of seed production. I used the
number of fruits at the end of the growing season as the measure of lifetime
fitness.
In choosing traits for the selection analysis I focused on traits that might
be under opposing or differential selection in the different planting sites. The
planting sites differ in environmental variables that may favor different traits or
7
may result in opposing selection on the same traits. Dunes sites have sandy
soils, and little herbaceous cover of plants of the same stature as D. teres (Table
3.1). This type of environment will select for plants that are adapted to dry, well
drained soils with little above ground competition. The Inland sites have clay
based soils with more cover of herbaceous plants of the same stature of D. teres.
This environment will select for plants adapted to wetter soils and shading due to
above ground competition. The Sandhills site has intermediate soil texture and
similar herbaceous cover to the Inland site (Hereford unpublished data). These
environments may select for plants adapted to dry environments, but also will
favor traits that confer adaptation to above ground competition.
I returned the plants to the laboratory and measured three additional traits
to identify differences in the pattern of directional selection between planting
sites. I measured stem elongation, leaf length, and the number of branches.
Stem elongation has been shown to be favored in environments with more
shaded environments. It has been shown in other plant species that longer
stems are favored in high-density environments and shorter stems are favored in
low density environments (Dudley and Schmitt 1996). I expect longer stems to
be favored in more crowded environments such as Inland and Sandhills planting
sites and shorter stems to be favored in Dunes sites where there is less shading.
I expect smaller leaves to be favored in dryer environments such as Dunes
environments because previous studies have shown that smaller leaves are
adaptive in dryer environments (Dudley 1996). Given that previous studies have
shown a trend towards divergent selection for degree of branching in high and
low density environments (Dorn et al. 2000), I expect selection for increased
branching in the Dunes sites and selection against branching or no selection on
branching in the Inland and Sandhills sites.
I measured stem elongation and leaf length at the fourth internode from
the apical meristem of the longest branch because the basal nodes developed
when the plants were in the growth chamber. I calculated elongation by dividing
the length of the internode by the width of the internode at its widest point. Leaf
length was measured at the same node that stem elongation was measured by
8
recording the length of the longest leaf at that node. Number of branches was
measured as the total number of branches produced by each plant. I did not
measure all traits that might be under opposing or differential selection because it
was not possible to obtain accurate measures of all traits. For example, rootshoot ratio should be under opposing selection in sand and clay soil
environments, but I was not able to measure retrieve enough of the root tissue
from plants grown in the Inland site to measure this trait accurately.
Data analysis
The purpose of these analyses was to test for local adaptation between
population pairs, to calculate AEC for fitness, and to compare estimates of
selection coefficients between planting sites. Fitness was measured as fruit
production at senescence. The fruits of plants that died before the end of the
growing season were counted as well so that a plant that senesced before most
others in a planting site was not necessarily considered to have a fitness of zero.
Many plants died before producing fruits, and these were assigned a fitness of
zero. The frequency of observations with zero fruits made it impossible to
transform fruit number to fit a normal distribution.
Because the distribution of fruit number consisted of a range of positive
whole numbers with many zeros, I tested the overall distribution of fruit number
against the expectation of the Poisson distribution using deviance/degrees of
freedom as an indicator of goodness of fit. Fitting a Poisson model to the
distribution of fruit number gave a deviance / degrees of freedom of 12.5. By
comparison, the deviance / degrees of freedom of fitness assuming a normal
distribution was 5134. Consequently, fruit number was assumed to fit a Poisson
distribution for significance tests.
To test for local adaptation, I quantified the effects of planting site, source
population, and their interaction on fitness. A significant planting site effect
indicates environmental effects on fitness, a significant source population effect
indicates that populations differ in the number of fruits produced regardless of
where they are planted. A significant planting site by source population
interaction shows that the performance of a source population depends on where
9
it was planted, a necessary but not sufficient condition for local adaptation. To
test for local adaptation, I compared differences in fruit production between
populations within planting sites after accounting for effects of initial size at
planting. The full analysis was carried out with a Generalized Linear Model
(GLiM), using a log link function and a Poisson distribution function. The model
included effects of initial size at planting, planting site, source population,
genotype nested within source population, and planting site by population
interaction on fruit production. Tests of local adaptation, pairwise comparisons of
fruit number of the three populations in each planting site, were carried out by
limiting GLiM to two populations and a single planting site. These tests were
carried out for all pairs of populations at all planting sites. I corrected the
likelihood ratio tests of all GLiM's for overdispersion, by dividing the likelihood
ratio statistic by the an estimate of dispersion (McCullagh and Nelder 1989).
I estimated selection gradients (Lande and Arnold 1983) and selection
elasticities (van Tienderen 2000; Hereford et al. 2004), by regressing trait values
on relative fruit number within each planting site. Selection elasticities are
selection gradients standardized by the mean of the trait. Selection coefficients
calculated in this way produce measures in units of mean trait value as opposed
to a standardized selection gradient, which measures selection in units of
standard deviations of the traits. Elasticities are useful in determining the
strength of selection because an elasticity of 1 implies that selection on a trait is
as strong as selection on relative fitness. With a standardized selection gradient,
description of the strength of selection depends on the variance in the trait and
relative fitness, but because mean relative fitness is always 1, elasticities of
different traits can be compared and the strength of selection can be more easily
interpreted.
I regressed fitness on genotype mean trait values to avoid environmental
covariance, which can bias estimates of phenotypic selection (Rausher 1992;
Stinchcombe et al. 2002; Winn 2004). Only directional selection was estimated,
because I am interested in describing divergent selection between different
10
planting sites. Consequently, no second-order terms were included in the
regression models.
I compared estimates of univariate and multivariate selection among
planting sites. Univariate selection measures the total effect of the trait on fitness
including the influence of correlated characters, and thus describes the total
selection pressure on the trait. Multivariate selection measures the direct effect
of the trait on fitness by holding variation in correlated characters constant, and
thus describes the selection pressure if the trait were to act in isolation from other
traits. To measure the effect of multicollinearity among the traits on selection
gradients, I quantified Variance Inflation Factors for each trait in each multiple
regression. The Variance Inflation Factor measures the amount by which each
regressor inflates the error in the estimate of the regression coefficient (Belsley et
al. 1980). As this statistic increases from 1 the regression coefficient becomes
less reliable due to multicollinearity.
Because the distribution of fruit number was not normal, I determined
statistical significance of directional selection estimates with the bootstrap. I
randomly resampled genotypes and recalculated regressions of fruit number on
the traits. This process was repeated 1000 times for the data from each planting
site to calculate 95% confidence intervals for each regression coefficient or
selection gradient. Selection gradients were considered significantly different
from zero if their confidence intervals did not overlap zero, and selection
gradients for the same trait in different planting sites were considered different
from each other if their confidence intervals did not overlap.
The tests of Goodness of Fit and GLiM for genotype effects on fruit
production were analyzed with the procedure GENMOD in the SAS statistical
package (SAS Institute 1994, Cary NC). I performed regressions and calculated
Variance Inflation Factors using the SAS procedure REG. All bootstraps were
programmed in SAS Macro language (SAS Institute 1994, Cary NC).
Results
Local Adaptation
11
Initial size at planting had a large positive effect on final number of fruits
(Probit regression β=0.038 d.f.=1 p<0.0001), and was significant in the GLiM on
fruit production as well (Table 2.1). The GLiM indicated significant effects of
planting site and source population on fruit production (Table 2.1). On average,
more fruits were produced at the Inland site than at the other planting sites
(mean number of fruits at the Inland site was 59.54 ± 2.78 SE and 53.70 ± 2.41
SE at the Dunes site). Transplants at the Sandhills site produced the fewest
fruits with a mean of 40.52 ± 2.21 SE. The Dunes and Sandhills populations
produced similar numbers of fruits across all planting sites (61.39 ± 2.36 SE and
60.85 ± 2.72 SE, respectively), and the Inland population produced about half the
fruits of the other populations (33.86 ± 2.81 SE).
The GLiM on fruit production showed a significant planting site by
population interaction (Table 2.1). The Inland population was outperformed by all
populations at all planting sites, but the Dunes and Sandhills populations showed
local adaptation to their native sites (Fig. 2.1). The Dunes population
outperformed the Sandhills population at the Dunes planting site and the
Sandhills population outperformed the Dunes population at the Sandhills site.
These populations produced similar numbers of fruits at the Inland site, where
they significantly outperformed the Inland population.
Selection
The multiple regression of traits on fitness showed little evidence that
multicollinearity affected estimates of selection. Variance Inflation Factors of all
traits at each planting site were close to 1, with a maximum of 1.36 for leaf length
in the Dunes site. The largest correlation between any two variables was 0.43
for stem elongation and number of branches in the Dunes planting site.
Selection elasticities suggest strong selection on the three traits in all planting
sites, but the direction of selection on the traits did not meet the expectations
based on opposing directional selection in different environments. Multivariate
selection gradients were significant for all of the traits in at least one of the sites
(Table 2.2). Multivariate selection elasticities were large, with selection on leaf
length at the Sandhills site almost twice as large as the expected directional
12
selection on fitness. The pattern of univariate selection was qualitatively similar
to the multivariate pattern. Selection on stem elongation was always negative,
and selection on number of branches was always positive. The pattern of
selection on leaf length varied among planting sites. There was strong negative
selection on leaf length in the Sandhills site, and positive nonsignificant selection
in the Dunes planting site. These were the populations that showed local
adaptation (Fig. 2.1). The confidence intervals for multivariate estimates of
selection on leaf size in the Dunes and Sandhills planting sites did not overlap,
but the confidence intervals of univariate selection on this trait in these planting
sites did (Table 2.2). Thus, selection on leaf size suggests that smaller leaves
are favored in the Sandhills planting site, and that there is a trend of selection for
larger leaves in the Dunes planting site, but this is the opposite of the pattern that
I predicted. Multivariate estimates of selection on leaf length at the Dunes and
Sandhills sites are significantly different from each other.
Discussion
The Dunes and Sandhills populations were locally adapted, but the Inland
population was not and had the lowest fitness in all planting sites. The lack of
local adaptation of the Inland source population is surprising given that most
differentiation in plant populations is thought to be adaptive (Linhart and Grant
1996). However, populations that do not show local adaptation to environmental
differences may be common. The majority of studies of a single population pair
report local adaptation (Linhart and Grant 1996), but those that include more than
one population pair often report that not all populations are locally adapted
(Antonovics and Primack 1982; Schmidt and Levin 1985; Rice and Mack 1991).
The conclusion that the Inland population of D. teres is not locally adapted is in
agreement with other experiments with this species. Jordan (1992) showed that
an Inland population was locally adapted in one year, but not in the next, and
subsequent reciprocal transplant experiments among some of the populations in
the present study have shown year to year variation in the expression of local
adaptation of Inland populations as well (Table 3.5 and 3.6).
13
The magnitude and direction of selection I observed did not match the
predictions of opposing selection at different planting sites. There was more
evidence of selection on different traits in different environments. I found
significant selection gradients and elasticities against stem elongation in the
Inland site, but no significant selection in the Dunes site. However, univariate
selection against elongation was significant in the Dunes site as well. Therefore,
it is likely that there is selection against elongation in all sites, but the lack of
significant selection gradients and elasticities at the Dunes site results from a
lack of statistical power to detect the smaller selection gradient. I expected
selection for increased elongation at the Inland and Sandhills sites and
decreased elongation at the Dunes site. I also expected no selection for
branching at the Inland and Sandhills sites. These predictions were not born out.
Resource allocation-acquisition theory may provide an explanation for
uniform selection in these environments. van Noordwijk and de Jong (1986)
showed that variation in resource acquisition leads to positive correlations
between life-history traits. A similar mechanism could lead to the positive
relationship between branch number and fruit number. Plants that can acquire
the resources to produce many branches may also be those that produce many
fruits, and plants that cannot acquire resources may produce few branches and
few fruits. Even if branching per se is not favored, plants that are able to grow
many branches will also be those that produce many fruits, and as a result there
will be a positive relationship between branch number and fruit number. The
same mechanism could explain the consistent negative relationships between
stem elongation and fruit number.
The observed pattern of selection on leaf length was the opposite of the
predicted pattern of selection for smaller leaves in the Dunes sites and larger
leaves in other sites. Selection on leaf length was significantly negative at the
Sandhills site, and nonsignificant at the Dunes and Inland sites. It has been
shown that selection favors small leaves in dryer environments (Dudley 1996),
and it is possible that even though the Dunes site has more well drained, sandier
14
soils, the Sandhills site may have been dryer for unexpected reasons, such as
lower rainfall or more below ground competition.
Though the pattern of selection on leaf length was opposite what I
expected, selective pressures on this trait at the Dunes and Sandhills sites were
significantly different (Table 2.2). This pattern suggests a possible trade-off
mediated by leaf length in the two sites, although strict interpretation of
significance tests would lead to the conclusion that there is selection for
decreased leaf length in the Sandhills planting site and no selection in the Dunes.
Negative selection on leaf length in the Sandhills and no selection in the Dunes
would support the mechanism of local adaptation predicted by the absence of a
negative AEC, that adaptation to different sites occurs as a result of selection on
different traits in different environments. Other studies that have quantified local
adaptation and selection have shown that selection tends to act on different traits
in different environments not in different directions on the same traits in different
environments. Bennington and McGraw (1995) and Etterson (2004a) showed
that selection often favored different traits in different environments in locally
adapted populations, and Jordan (1991) found evidence of the same pattern in
populations of D. teres in North Carolina, though in that study statistical
differences between estimates of selection were not evaluated.
Although traits I measured experienced strong selection, I did not find
convincing evidence of opposing patterns of selection. This does not rule out the
possibility of opposing patterns of selection on other traits such as water use
efficiency and specific leaf area that could differ adaptively between populations
(e.g. Dudely 1996), but it does suggest that the traits that I have chosen, are
adaptive, but do not contribute to local adaptation. It is likely that I did not
choose traits that were experiencing opposing selection or traits that were under
differential selection among planting sites. Had I chosen such traits I may have
found more evidence that the pattern of selection was based on trade-offs or
based on adaptations involving different traits in different environments. With the
traits that I have chosen I cannot make either claim.
15
This experiment was carried out for only one growing season, and the
results may differ between years (e.g. Rice and Mack 1991). In addition, the
Dunes population was not planted in its native site, but in an environment that
was similar to its native site. While these conditions may give misleading results,
there is evidence that Dunes and Sandhills populations are locally adapted over
multiple years and planting Dunes plants in a non-native site did not influence the
expression of local adaptation. Subsequent experiments have shown that Dunes
and Sandhills populations are locally adapted, while local adaptation of Inland
populations varies across years (Fig. 3.3). It has also been shown that plants of
the Dunes source population in this study consistently had higher fitness than
plants from other source populations at the Dunes site, therefore
they are adapted to Dunes habitat types (Table 3.5 and 3.6).
Models of reproductive isolation based on the effects of adaptation and
divergent selection suggest that pairs of populations that are locally adapted may
also be reproductively isolated, while there may be less reproductive isolation
between pairs of populations that are not locally adapted. (Rice 1984; Schluter
2000). If these theories are correct I would expect more reproductive isolation
between Dunes and Sandhills populations than between either of these and
Inland populations. More generally, I would expect that populations of a species
that are reciprocally locally adapted will be more reproductively isolated than
pairs of populations where one population has higher fitness in both
environments, or where populations have equal fitness.
16
Table 2.1 Generalized linear model of size at planting, planting site, source
population, genotype nested within source population, and planting site by
source population interaction on fruit production. Fruit production was assumed
to be Poission distributed.
Source of variation
d.f.
Chi square
p-value
Initial size at planting
Planting site
Source population
Genotype within source population
Planting site X source population
1
2
2
60
4
154.42
67.48
119.25
143.81
29.63
0.0001
0.0001
0.0001
0.0001
0.0001
17
Table 2.2. Analysis of selection on three traits in the three planting sites.
Selection coefficients were calculated for genotype means, and N refers to the
number of genotypes. Means are means of genotype mean for each trait.
Standard deviations are given in parentheses. Sµ is the univariate selection
elasticity, βµ is the multivariate elasticity for the trait, and β is the selection
gradient. The bootstrap calculated confidence intervals for β are also presented.
Selection estimates in bold are significantly different from zero.
β
β
95% C. I. of
0.79 0.662
0.63 0.564
-0.59 -0.224
0.058
0.214
-0.034
(-0.015, 0.122)
(0.113, 0.284)
(-0.063, 0.001)
(1.90)
(1.80)
(5.52)
(0.52)
-0.09 -0.249
0.79 0.785
-0.47 -0.393
-0.016
0.238
-0.025
(-0.057, 0.017)
(0.195, 0.280)
(-0.045, -0.001)
(1.79)
(1.40)
(7.72)
(0.61)
-1.42 -1.70
0.39 0.353
-.051 -0.444
-0.108
0.164
-0.026
(-0.21, -0.045)
(0.244, 0.444)
(-0.055, -0.012)
Planting Site/ Trait
N
X
s.d.
Sµ
Dunes
Leaf length (mm)
Number of branches
Stem elongation
Relative fruit number
50
63
63
63
11.42
2.64
6.61
1.0
(1.51)
(1.48)
(3.20)
(0.55)
Inland
Leaf length (mm)
Number of branches
Stem elongation
Relative fruit number
55
63
63
63
15.56
3.30
15.72
1.0
Sandhills
Leaf length (mm)
Number of branches
Stem elongation
Relative fruit number
59
63
61
63
15.77
2.15
17.07
1.0
µ
β
Figure 2.1 Mean number of fruits produced at each planting site by each source
population. The Dunes source population is represented by the solid circles, the
Sandhills by the open circles, and the Inland source population is represented by
the solid triangles. Means that share the same letter are not significantly different
within planting sites after correction for multiple comparisons (α set at 0.05 ÷ 9).
19
CHAPTER 3
QUANTITATIVE MEASUREMENT OF PARALLEL LOCAL ADAPTATION IN SIX
POPULATIONS OF AN ANNUAL PLANT
Abstract
Tests of local adaptation can demonstrate the role of selection in natural
populations, but because most studies have been carried out with only one pair
of populations, the effects of other forces such as genetic drift cannot be ruled
out. Furthermore, studies of local adaptation are interpreted qualitatively, and do
not take into account quantitative differences in fitness between populations.
Here we report the results of a 6 X 6 reciprocal transplant study conducted over
two years, in six populations of an annual plant. We quantified differences in fruit
set between populations from three habitat types, and introduce a method of
quantifying differences in fitness between populations in reciprocal transplant
studies. In addition, we quantify some of the environmental variation between
planting sites. We found evidence for local adaptation at the level of habitat, and
variation between populations in the extent of local adaptation. There was no
significant relationship between environmental variation and the degree of local
adaptation between populations. The pattern of local adaptation and the lack of
a relationship between fitness differences between populations and
environmental variation suggests that adaptation to local conditions is influenced
by both divergent selection and nonadaptive divergence.
Introduction
Common reports of local adaptation suggest that populations frequently
respond to selection to become locally adapted to their native habitat (reviewed
by Linhart and Grant 1996; Schluter 2000). By local adaptation I mean
adaptation to the native environment such that the native population has greater
fitness in its home site than members of a foreign population. However, many
examples of local adaptation are limited to a pair of populations native to strongly
contrasting environments. Systems in which local adaptation is evaluated are
20
often chosen because of an expectation of contrasting selection between
habitats such as natural soils versus mine tailings (Jain and Bradshaw 1966;
Macnair 1993), or serpentine soils (Kruckeberg 1951), or different host plant
species (Funk 1998). Although genetic drift can also contribute to divergence
between populations, any advantage of a population in its native site is typically
assumed to reflect response to natural selection.
In their review of local adaptation in plants, Linhart and Grant (1996)
concluded that there is little influence of gene flow or genetic drift on local
adaptation, but there have been few tests that could have demonstrated a role of
drift in local adaptation. Genetic drift can influence local adaptation through
effects of population bottlenecks, founder effects, or through its influence on
gene frequencies in small populations. Consider a pair of populations derived
from the same ancestral population. If members of these populations colonize
new environments they could diverge from each other by a variety of
mechanisms. First there is the commonly cited mechanism of divergent selection
(Jordan 1991; Bennington and McGraw 1995). Newly founded populations may
also diverge due to founder effects if one population is founded by a small
number of individuals, or by population bottle-necks if one of the populations
suffers a period of increased mortality. With these last effects, divergence could
occur even between populations in similar environments (Travisano et al. 1995).
The founder effects and bottlenecks could cause one population to be better
adapted to the same environment than the other, and all these mechanisms
could result in differences in fitness between populations in the same
environments.
Testing the hypothesis of local adaptation for replicate populations from
alternate habitats can provide stronger support for the hypothesis of local
adaptation to specific types of environments. If only a single population from a
given type of habitat were studied, then it would be impossible to conclude
whether home site advantage resulted from adaptation to a specific environment
or home site advantage occurred by chance. If replicate populations native to
the each of multiple habitat types are reciprocally transplanted, consistent home
21
site advantage will mean that populations have adapted to the specific
environmental conditions of their native habitat type. Variation among
populations of the same habitat type most likely will represent chance differences
in fitness between populations. These chance differences may occur through
effects of genetic drift such as population bottlenecks or founder events.
Examples of 'parallel adaptation', in which local adaptation is demonstrated in
replicate populations native to the same habitat types (Kawecki and Ebert 2004)
are rare in the literature (but see Clausen et al. 1940), but they can reveal how
both divergent selection and genetic drift influence local adaptation.
No two environments or populations will be exactly alike given continuous
environmental variation and polygenic variation. Local adaptation is usually
interpreted qualitatively, populations are said to be locally adapted or not, but
local adaptation can be quantified. Members of the native population have some
advantage over foreign individuals, and this advantage can vary continuously.
With the exception of Schmidt and Levin (1985), few studies have attempted to
quantify the degree of local adaptation, and no study has quantified pairwise
local adaptation between populations. A pairwise measure of the degree of local
adaptation can quantify the extent to which a pair of populations has diverged in
adaptation to specific habitats. Such a measure would permit tests of the
quantitative degree to which populations are adapted to their native
environments, and the effects of factors that influence the degree of adaptation,
such as environmental differences within habitats or the availability of genetic
variation.
We measured the degree of pairwise local adaptation in two populations
of an annual plant from each of three habitat types in a 6 X 6 reciprocal
transplant experiment to test for parallel adaptation to the three habitats and for
pairwise local adaptation to native planting sites. We also quantified differences
in percent cover and soil texture among all planting sites. We calculate a
quantitative measure of the degree of difference in fitness between environments
between population pairs and examine its correlation with our measure of
22
environment differences to ask if populations from more similar environments
show smaller differences in fitness than populations from different environments.
Materials and Methods
Diodia teres (Rubiaceae) is a self-compatible annual plant ranging from
tropical to temperate climates. It occurs from Panama, north to the northeastern
United States with the western edge of its range extending to Michigan (Kearney
and Peebles 1964). In the southeastern United States it is found in a range of
habitat types from coastal sand dunes to inland forests, where it occurs along
roadsides, and in canopy gaps. Previous studies have demonstrated local
adaptation between populations of D. teres from inland agricultural and coastal
habitats in North Carolina (Jordan 1992) and between populations from Dunes
and Sandhills habitats in north Florida. The present study includes two
populations from each of three habitat types designated Dunes, Sandhills, and
Inland (Fig. 3.1). These habitats span an environmental gradient from the coastal
plain (Dunes) to the piedmont (Inland) along which we expected soil type to
change from sandy to clay based, and the vegetation to become more dense and
dominated more by overhead canopy.
Habitat designation
To determine if the locations of our study populations can confidently be
placed into different habitat types, we measured soil particle size and three
aspects of the plant communities at the site of each population. We estimated
percent cover of other individuals of D. teres, of all other herbaceous plant
species, and of canopy coverage from woody vegetation along 15 randomly
located 1m transects. The proportion of the transect tape covered by leaves of a
given type of vegetation was converted to percent cover of that type of vegetation
(Barbour et al. 1987). At the same 15 locations, we took soil samples and used
gravitational analysis (Cox 1978) to estimate the percent of the soil made up by
sand, silt, and clay. The proportion of these components has a large effect on
the water holding capacity of the soil (Barbour et al. 1987).
Ecotypic differentiation
23
To determine the pattern of local adaptation among our populations, we
performed a 6 X 6 reciprocal transplant experiment among the populations in
each of two years. In winter of 2000, we collected seeds from 30-45 individual
plants in each population. In March of 2001, we planted one seedling from each
of the 179 field maternal genotypes (between 18 and 41 seedlings per population
depending on germination) in the greenhouse, and allowed them to mature and
produce seeds in a common maternal environment. In March of 2002, these
seeds produced in the greenhouse were germinated in sand, and seedlings were
transferred to flats filled with potting soil as they emerged. Approximately 20
seedlings from each source population, each representing a different original
maternal family, were planted at each planting site between April 2002 and May
2002, coinciding with the timing of natural seedling emergence. Planting
seedlings instead of seeds circumvents natural selection on seeds, but given low
rates of germination (0.21; Hereford and Moriuchi 2005), we planted seedlings to
insure sample sizes sufficient to detect population differences. A large number of
seedlings were lost at the Sandhills 1 site to unknown causes a few days after
planting and were re-planted a week later. All other seedlings that were
transplanted to a site were planted on the same day and were of similar size (2-4
true leaves). Seedlings were transplanted directly into the soil, with minimal
disturbance to natural vegetation. In December of 2002, when most plants had
senesced at all sites, we counted the total number of fruits produced by each
individual as an estimate of fitness.
We repeated the reciprocal transplant in 2003, but with seeds collected
from a different group of maternal families. In winter of 2000 we collected seeds
from an additional 80-90 maternal plants per population as part of another
experiment. Between 7 and 9 individuals of this collection were grown from seed
in the greenhouse and allowed to self-fertilize and set seeds. Seeds were
collected from these plants and grown in the greenhouse for another generation,
resulting in plants that had undergone two generations of growth in the
greenhouse and at least two generations of self-fertilization. We germinated
seeds from these plants in February and March of 2003 and transplanted 10
24
seedlings from each maternal family into each planting site in the field between
March and May using the same protocol as in 2002. Seedlings were of similar
size when planted, and because the order in which seeds were germinated was
haphazard, individual plants were equally likely to be planted on any given
planting day. Because the seedlings of a given maternal family are not
independent replicates, we performed all analyses of the 2003 experiment on
family mean fruit number.
Data analysis
We performed Kruskal-Wallis tests to compare the means of
environmental variables among the three habitat types because these variables
did not meet the assumptions of parametric one-way ANOVA. To determine if
planting sites from the same habitat types were more similar than planting sites
from different habitats, we calculated Mahalanobis distances (McLachlan 1992),
hereafter environmental distance, between all pairs of planting sites. This
method scales the difference in environmental variables between sites by the
overall variances of the variables and the covariation between them. If planting
sites from the same habitat type are more similar, the sites with the lowest
environmental distances will be from the same habitat. We calculated
environmental distances based on percent cover of conspecifics, herbaceous
cover, canopy cover, and percent sand in the soil analysis. We did not include
percent clay and silt because these are necessarily correlated with percent sand.
The three soil texture variables were significantly correlated inter se, with
correlation coefficients of at least 0.47.
We tested for habitat-level ecotypic differentiation and pairwise local
adaptation by comparing the fruit production of native and non-native transplants
in each planting site. If there is ecotypic differentiation, then plants from the
same habitat type will produce similar numbers of fruits in each others’ native
planting site, and will have greater average fruit set than plants native to the
alternate habitat types. Similarly, if plants are locally adapted to their native
planting sites, they will have greater fruit production in their native site than plants
from foreign populations.
25
Because the distribution of fruit production was not normal at any planting
site and contained many zero values, and neither natural log nor square root
transformations improved the fit to a normal distribution, we tested whether fruit
number fit a Poisson distribution. We found a reasonable fit to a Poisson with a
scaled deviance (deviance/degrees of freedom) of approximately 15 for both the
2002 and 2003 experiments. In contrast, the scaled deviance for a normal
distribution was 286 at all sites for 2002, and 479 for the 2003 experiment.
Consequently, effects of year, habitat type of the planting site, habitat type of the
source population, source population, planting site, and interactions on fruit
production were analyzed with Generalized Linear Models (GLiM), using a log
link function and a Poisson distribution function. Poisson distributed variables
will typically be overdispersed (McCullagh and Nelder 1989), meaning the
variance will not be exactly equal to the mean, but will be greater than the mean.
To correct for possible effects of overdispersion, all likelihood ratio tests in
GLiM's were divided by an estimate of dispersion (McCullagh and Nelder 1989).
All analyses of fruit number were carried out with GLiM of this form.
Adaptation at the level of habitat would be indicated by a significant
interaction between planting site habitat type and source population habitat type
on fruit production, and a pattern in which populations of the native habitat type
produce significantly more fruits than populations from foreign habitats. We
quantified this interaction in addition to the main effects of planting habitat type,
source habitat type, and year on fruit production. We also quantified effects of
planting site nested within planting habitat type and source population nested
within source habitat type. Though nested effects are typically treated as
random, interpretation of these effects is obviously limited to the set of
populations in this study (Sokal and Rohlf 1995). We quantified interactions
between main effects and year and the interactions between nested effects and
year to measure the influence of temporal variation on fruit production.
If there is adaptation to habitat, we would also expect that populations
planted in a foreign planting site, but of their native habitat type should outperform plants from foreign habitat types. We tested for an advantage of these
26
"foreign -natives" by comparing the mean fruit set of each source population in
the alternate planting site of its native habitat type to the combined mean of the
foreign populations at that site. The native population was excluded from this
test.
To reveal patterns of local adaptation at the population level, we
conducted a second analysis of fruit production that did not include the effects of
habitat. For each planting site, we compared the fruit set of native plants to that
of transplants from each foreign source population to test whether each foreign
population produced fewer fruits than the native population. If a native
population sets more fruits than a foreign population, the native population is
locally adapted relative to the foreign population. We corrected the p-values of
all pairwise tests to account for multiple comparisons by dividing the alpha-level
(0.05) by the number of comparisons (30) to yield a corrected alpha of 0.0017.
Separate analyses and corrections were performed for each year.
Quantifying local adaptation
To quantify total differences in fitness between populations so that we can
compare them to the differences in the environments, we summed the absolute
value of the difference in relative fitness between each pair of populations, and
refer to this quantity as Fitness Divergence (FD). This metric quantifies the
absolute value of the differences in fitness between a pair of populations in
different environments. It is calculated with the following formula,
FD =
W11 − W 21 + W 22 − W12
where W is fitness standardized by the mean fitness of the population with the
higher fitness at a given planting site, and the subscripts refer to the relative
fitness of the ith populations at the jth planting site.
This metric does not differentiate between the many qualitative outcomes
of a reciprocal transplant experiment (Lortie and Aarssen 1996), but does
quantify differences in fitness in different environments. For the questions
presented above, we are interested mainly in total differences in fitness. For
example, if we were interested in the extent of home site advantage specifically
27
we would have subtracted foreign fitness from native fitness in both planting
sites.
We used the following computer packages to perform the analyses
described above. Kruskal-Wallis tests were performed with the SAS procedure
NPAR1WAY (SAS Institute 1994, Cary NC) to compare environmental variables
among planting sites. Environmental distances between pairs of sites were
calculated using the procedure DISCRIM in the SAS statistical package with the
METRIC=FULL option. We used the procedures GENMOD and CORR (SAS
Institute, Cary, NC) to perform Generalized Linear Models and correlations,
respectively. Dispersion was estimated and accounted for with the
SCALE=PEARSON option in the Procedure GENMOD. Mantel tests were
carried out to test the hypothesis that there was a relationship between FD and
environmental differences using the software package R 4.0 (Philippe Casgrain;
http://ProgicielR.webhop.org/).
Results
Environmental differences
Our planting sites sorted into the three habitat types based on soil texture
and vegetative cover. Planting sites belonging to the different habitat types
differed significantly in percent cover of D. teres, percent cover of heterospecific
herbs, percent canopy cover, and percent sand (Table 2.1). Dunes habitats were
open and sandy, Inland habitats had canopy cover and clay based soils, and the
Sandhills sites were intermediate to these.
Planting sites belonging to the same habitat type were more similar to each other
than to planting sites from other habitat types. Environmental distances between
planting sites from the same habitat were substantially lower than those between
populations from different habitats (Table 2.2). The environmental distances
between Dunes and Inland planting sites were the largest, and distances
between planting sites from these habitats and Sandhills planting sites were
intermediate.
Habitat level divergence
28
Year, planting habitat type, and source habitat type all had significant
effects on fruit production (Table 3.3). The significant planting habitat effect
supports the conclusion that the three habitats are perceived as distinct
environments by D. teres. The significant source population habitat effect
suggests genetic differentiation for average fruit production among populations
from the different habitats (Table 3.3). The possibility of adaptation at the level of
habitat type is supported by the significant planting habitat by source population
habitat type interaction. Although the year by planting habitat type by source
population habitat type interaction was not significant (Table 3.3), inspection of
the mean fruit number of source habitats and planting habitat types in different
years indicates differences between years in the pattern of habitat level
adaptation (Fig. 3.2). In 2002, populations from the native habitat type produced
the most fruit in all habitats supporting the conclusion of habitat-level adaptation.
In 2003, plants from the dunes and Sandhills habitats again produced the most
fruit in their native habitat types, but plants from the Sandhills habitat also
produced the most fruits in the inland habitat (Fig. 3.2).
If the habitat types reflect different selective environments that result in
repeatable responses to selection, then plants that are planted at a foreign
planting site of their native habitat type will have greater fruit production than
plants from foreign habitats at that site. We found that these "foreign-natives"
produced significantly more fruit than the combined mean of all foreign plants in
five of six comparisons in 2002, although only two of six comparisons were
significant in 2003 (Table 3.4).
Planting site and source population level divergence
We also found significant variation in fruit production among the planting
sites and between source populations within habitat types (Table 3.3). The
number of fruits set in different habitats differed between years, but the number
of fruits set by populations from each source habitat was similar between years
(Table 3.3). In addition to the significant effect of year on fruit production, there
was a significant year by planting site nested within habitat type interaction
(Table 3.3). This result suggests that planting sites within habitats differed in fruit
29
set between years, providing further evidence for temporal variation in the quality
of habitats.
In our analysis of fruit production at the population level, we detected
variation between years and among planting sites and source populations (Table
3.3). More fruits were set on average in 2002 (mean fruit number 18.6) than in
2003 (mean fruit number 14.9). In both years, mean fruit number varied more
among planting sites than among source populations (Table 3.5 and 3.6)
suggesting greater effects of environmental than genetic sources of variation.
Mean fruit production ranged from 15.9 to 25.5 among source populations and
from 5.9 to 25.1 among planting sites in 2002. In 2003, the range was from 9.7
to 25.6 among source populations and from 2.9 to 31.5 among planting sites.
The mean fruit production was greatest for plants planted at the Inland 1 site in
both years of the study (Table 3.5 and 3.6).
The significant source population by planting site interaction is necessary
but not sufficient for local adaptation. To test for local adaptation, we compared
the fruit production of the native population to the combined mean fruit production
of all foreign populations at each planting site. The fruit production of the native
population was significantly greater in three of six planting sites in 2002 and in
two sites in 2003 (Table 3.7).
To test for reciprocal local adaptation between each pair of populations,
we compared the fruit production of plants of each foreign population to the fruit
production of the native population at each planting site in each year. There was
a strong pattern of overall local adaptation in 2002. The native population
produced more fruits than foreign populations 24 out of 30 times, and in 2003 18
out of 30 times. Therefore, there was an overall pattern of local adaptation in
2002, but this was less pronounced in 2003. These results support the patterns
of adaptation to habitat type (Fig. 3.2). To compare the fruit set of individual
pairs of populations, it is necessary to test for significant differences in fruit set
between native and foreign populations. The native population produced
significantly more fruits than foreign populations in six of thirty comparisons in
2002 (one significant after correction for multiple comparisons; Table 3.5), and 10
30
of thirty comparisons in 2003 (two significant after correction for multiple
comparisons; Table 3.6). We detected one consistent case of reciprocal local
adaptation, in which there was a home-site advantage in both planting sites; the
Dunes 1 and Sandhills 2 populations exhibited reciprocal local adaptation in both
years of the study (Table 3.5 and 3.6). Plants from both Inland populations
produced fewer fruits than Sandhills population 1 in both years. The Inland
populations and Sandhills population 2 set fewer fruits than Dunes population 1
at the Dunes 1 planting site in both years. There were three instances in 2003
where the native population produced significantly fewer fruits than foreign
populations, one at the Inland 1 site and two at the Dunes 2 site (Table 3.6).
Relationship between Fitness divergence and environmental variation
The magnitude of FD between populations, was highly variable and did
not appear to reflect differences between habitat types (Table 3.8). Fitness
divergence ranged from 0.21 to 1.03 in 2002 and from 0.18 to 1.20 in 2003.
There was no suggestion that FD is lower between populations of the same
habitat type than between populations from different habitats (Table 3.8). For
example, FD between the two Dunes populations was 0.88 and 0.84 in 2002 and
2003, respectively. FD between populations from other habitats and Dunes
populations was as low as 0.21 in 2002 and 0.18 in 2003.
There was a trend toward a positive correlation of FD between years
(Mantel correlation 0.40, p = 0.07). There was a trend for a correlation between
FD and environmental distance in 2002 (Mantel correlation 0.37, p = 0.10), but
the correlation was weak and not significant in 2003 (Mantel correlation -0.03, p =
0.51).
Discussion
Planting sites of the same habitat types were more similar in
environmental variables than planting sites from different habitats (Table 3.1),
and there was evidence of local adaptation at the level of habitat type. There
was evidence of local adaptation at the level of source populations in both years
(Table 3.5 and 3.6), though comparisons of mean fruit set between specific
populations were not often significant, especially in 2003. We found no
31
significant relationships between FD and environmental variation (Fig. 3.3).
These results suggest that populations adapt at the level of habitat, but there is
less evidence of adaptation at the level of individual populations. In addition,
there is temporal variation in relative fruit set of populations.
We found that planting sites within habitat types represent replicate
environments (Table 3.1 and 3.2) and that our source populations of D. teres
have differentiated into ecotypes (Fig. 3.2). Ecotypic differentiation has been
identified in a variety of organisms (Sumner 1932; Clausen et al 1940; Bocher
1949; Mooney and Billings 1961) and parallel adaptation has been identified in
others (Via 1991; Schluter 1995; Berglund et al. 2004), and it is not surprising
that populations that occupy habitats as different as those in this study have
differentiated into ecotypes.
The habitats that we describe here are quantitatively different in soil
texture and vegetative cover (Table 3.1), and planting sites with the lowest
environmental distances were those of the same habitat type (Table 3.2). The
differences in soil texture may be particularly important because there was less
variation within planting sites in soil texture than in vegetation, and differences in
soil composition are a common environmental basis for plant population
differentiation (Linhart and Grant 1996).
The conclusion that the source populations in this study are replicates of
three ecotypes is supported by the observation of significant adaptation at the
level of habitat. In 2002, the native ecotype always produced significantly more
fruits, and a weaker but similar pattern held in 2003 (Fig. 3.2). Although the
native population had the greatest fruit production at two of three habitats in
2003, native fruit production was significantly greater only for the Sandhills
habitat. We also found that in 2002, most "foreign-natives" produced more fruits
than the combined mean fruit number of the foreign populations (Table 3.4). This
result was less common in 2003, when only the Dunes populations produced
more fruit at their foreign-native planting site. Studies that have measured
selection in locally adapted species have typically not estimated selection or
measured local adaptation in replicate populations of different ecotypes (Jordan
32
1991; Bennington and McGraw 1995; Etterson 2004). We do not know how
similar the selective pressures at different sites of the same habitat type are, but
given that "foreign-native" populations often outperformed foreign populations,
particularly at the Dunes sites, selective pressures are likely to be more similar
within habitat types than between habitat types.
It is difficult to determine why there was less evidence for local adaptation
at the level of habitat and individual populations in 2003 than in 2002. Some of
the difference results from lack of home site advantage of Inland population 2
(Table 3.6), but there were fewer examples of home site advantage in other
populations as well. In another study of D. teres in North Carolina, Jordan (1992)
found evidence for local adaptation in one year, but no significant local
adaptation in the next. An Inland population in that study occupied an
environment that was similar in soil type to the Inland habitat of this study. Other
reciprocal transplant studies in other species conducted over multiple years
found yearly variation as well (Schmidt and Levin 1985; Rice and Mack 1991)
The Inland populations in this study may not have been locally adapted in 2003
for two reasons. First the plants that were sampled for the 2003 experiment may
have experienced more inbreeding depression than the plants in 2002 because
they underwent an additional generation of selfing. Second there may have been
differences in the environments between years that affected the expression of
local adaptation. The explanation of inbreeding depression unlikely, because all
populations underwent an additional generation of selfing and fruit number was
lower for all source populations. Thus, inbreeding depression was similar across
all populations. In addition, the year by source population interaction was not
significant (Table 3.3), which suggests that populations did not differ in the extent
of inbreeding depression. The second possibility is more probable. Other
studies have shown that temporal variation in the environment can alter the
expression of local adaptation (Schmidt and Levin 1985; Rice and Mack 1991).
Without site-specific rainfall, temperature, or other environmental data it is
difficult to explain why the Inland populations were not locally adapted in 2003.
Although we cannot explain the differences in the expression of local adaptation
33
between years, it is clear that reciprocal transplant experiments need to be
conducted over multiple seasons whenever possible because the pattern of local
adaptation can be subject to temporal environmental variation.
Planting site and source population level divergence
Analyses at the planting site and population levels revealed that native
populations did not always produce the most fruits, and rarely set significantly
more fruits than foreign populations (Table 3.5 and 3.6), though "foreign-native"
populations usually had the greatest fruit number when the native population did
not (Table 3.4). We detected only one case of significant pairwise local
adaptation; the Sandhills 2 and Dunes 1 populations showed consistent local
adaptation relative to each other in both years of the study (Table 3.5 and 3.6). If
local adaptation were complete at every site, we would have found significant
local adaptation between all pairs of populations. Complete, reciprocal local
adaptation would probably have resulted in a stronger relationship between
environmental distance and FD than we detected, assuming our environmental
variables were reflect important selective factors responsible for local adaptation.
If populations are not at equilibrium, we expect this relationship to strengthen
over time because responses to divergent selection can be fast (Bone and
Farres 2001; Reznick and Ghalambor 2001).
Sandhills population 1 had a large influence on the extent of local
adaptation of other populations to their native planting sites. Plants from this
population often produced more fruits than the native population at all sites
(Table 3.5 and 3.6), and never produced significantly fewer fruits than the native
population. The environment at Sandhills site 1 may provide some explanation
of its relatively high fruit number at all sites. Relative to the other habitats, the
soil at this site is intermediate in percent sand, and percent cover of trees and
other herbs is similar to that in the Dunes (Table 3.1). This environment may
select for a generalist that can maintain high fruit number at all planting sites as a
result of unique selective pressures. Other reciprocal transplant studies have
found populations with high fitness in many environments (Schmidt and Levin
1895; Rice and Mack 1991), and Stanton and Galen (1997) showed that
34
dispersal from a population with high fitness lead to maladaptation of a near-by
population with low fitness. The occurrence of populations with high fitness in
multiple environments could indicate that low gene flow among populations
allows a genotype with high fitness in many environments to exist without
invading other populations.
Genetic drift could influence the degree of local adaptation through
founder events or population bottle-necks. Populations that undergo these
effects may have a lower effective population size, and as a result, lower genetic
variation than populations that do not encounter such effects. If a pair of
populations are adapting to similar environmental conditions the population with
smaller effective size may not be as well adapted as the population with the
larger effective size. Furthermore, in a reciprocal transplant experiment, the
better adapted population could have greater fitness in both home sites. This
effect of genetic drift could be responsible for the large difference in fitness of the
two Dunes populations (Table 3.5 and 3.6). Cohan (1984) showed that if
populations were under weak uniform selection divergence will be faster with
both drift and selection than under drift alone. Such effects of population history,
through the influence of bottlenecks have been demonstrated experimentally
(Travisano 1995), and are thought to influence subsequent population adaptation
(Langerhans and DeWitt 2004).
Our results demonstrate that patterns of local adaptation reflect a mixture
of selection and genetic drift. The role of selection is apparent in adaptation at
the level of habitat type. The lack of a relationship between environmental
differences and FD, and the observation that populations from similar
environments did not always perform similarly in their native habitat types
suggests that local adaptation is influenced not only by selection, but also by
stochastic processes such as founder effects or population bottlenecks. Genetic
drift probably influences local adaptation by limiting genetic variation within
populations, and slowing their response to selection, or by promoting divergence
between populations adapting to similar environmental conditions. The temporal
variation and variation between source populations of the same habitat type we
35
observed demonstrates that reciprocal transplant experiments need to be
conducted over multiple seasons and should be replicated at the level of habitat
to determine the relative importance of selection and stochastic forces in local
adaptation.
36
Table 3.1. Mean (standard deviation) percent cover of D. teres, heterospecific
herbs, and canopy, and percent sand and clay in the soil at the six planting sites.
Numbers in bold are habitat means. The χ2 values quantify the effect of habitat
type on each variable in Kruskal-Wallis tests. All tests were significant at P <
0.01 with 2 degrees of freedom.
Planting site/Habitat
% D. teres
% Herbs
% Canopy
% Sand
% Clay
Inland
5.80
(7.32)
24.45
(23.99)
25.83
(39.70)
0.59
(0.06)
0.17
(0.07)
Inland 1
3.67
(3.75)
15.63
(14.75)
34.33
(45.39)
0.63
(0.06)
0.18
(0.07)
Inland 2
7.93
(9.34)
33.27
(28.43)
17.33
(32.40)
0.55
(0.04)
0.10
(0.04)
Sandhills
3.67
(5.52)
8.40
(10.40)
25.83
(38.82)
0.84
(0.04)
0.07
(0.02)
Sandhills 1
0.13
(0.52)
8.93
(9.98)
0
(0)
0.81
(0.03)
0.06
(0.02)
Sandhills 2
7.2
(6.01)
7.87
(11.11)
51.67
(41.13)
0.86
(0.02)
0.06
(0.01)
Dunes
1.67
(2.93)
23.50
(27.16)
0
(0)
0.97
(0.01)
0.01
(0.01)
38.13
(29.63)
0
(0)
0.97
(0.01)
0.01
(0.01)
2.40
(3.71)
8.87
(13.81)
0
(0)
0.97
(0.01)
0.01
(0.01)
(χ2 = 11.32)
(χ2 = 13.20)
(χ2 = 16.57)
(χ2 = 80.03)
(χ2 = 67.38)
Dunes 1
Dunes 2
0.93
(1.67)
37
Table 3.2. Mahalnobis distances between all pairs of sites based on percent
cover of D. teres, hebaceous plants, tree canopy, and percent sand in the soil.
Boldface type indicates distances between sites within a habitat type.
IN1
IN2
SA1
SA2
DU1
IN2
SA1
SA2
DU1
DU2
9.22
32.57
71.54
51.20
97.26
7.71
104.66
163.54
23.76
14.81
108.50
171.44
23.51
14.58
2.31
38
Table 3.3. Generalized Linear Model for A effects of year, planting habitat type,
source habitat type, planting site nested within planting habitat type, source
population nested within source habitat type, and their interactions on fruit
number, and B the same model excluding effects of habitat.
A
χ2
p-value
1
2
2
3
4
2
2
4
4
3
3
11.19
15.50
9.47
95.00
20.54
52.16
2.87
30.39
3.17
25.30
3.18
0.0009
0.0005
0.0090
0.0001
0.0004
0.0001
0.2387
0.0001
0.5298
0.0001
0.3646
1
5
5
5
5
25
25
8.50
109.65
21.03
56.14
5.97
48.53
14.78
0.0036
0.0001
0.0009
0.0001
0.3101
0.0032
0.9464
Source of Variation
d.f.
Year
Planting habitat type
Source habitat type
Planting site within planting habitat type
Source pop. within source habitat type
Year X planting habitat type
Year X source habitat type
Planting habitat X source habitat type
Year X planting habitat X source habitat type
Year X planting site within planting habitat type
Year X source population within source habitat type
B
Year
Planting site
Source population
Year X planting site
Year X source population
Planting site X source population
Year X planting site X source population
39
Table 3.4. Comparisons by planting site of fruit number produced by plants in a
foreign planting site, but their native habitat type versus all plants from foreign
habitats. The native source populations were excluded from these tests. All
tests are Generalized Linear models as described in the text with 1 degree of
freedom. Tests are one tailed.
2002
Planting site
Source population
χ2
sig.?
Inland 1
Inland 2
Sandhills 1
Sandhills 2
Dunes 1
Dunes 2
Inland 2
Inland 1
Sandhills 2
Sandhills 1
Dunes 2
Dunes 1
3.69
4.05
1.06
3.28
5.38
26.02
yes
yes
no
yes
yes
yes
Planting site
Source population
χ2
sig.?
Inland 1
Inland 2
Sandhills 1
Sandhills 2
Dunes 1
Dunes 2
Inland 2
Inland 1
Sandhills 2
Sandhills 1
Dunes 2
Dunes 1
0.33
0.08
0.06
2.46
8.81
5.10
no
no
no
no
yes
yes
2003
40
Table 3.5. Mean (standard deviation and sample size) number of fruits produced
by transplants from each source population transplanted into each planting site.
Means in bold are the fruit production of populations in their native planting site.
Boxes enclose groups of means from the same habitat type. Row marginals are
means (standard deviations and sample sizes) for each source population across
all planting sites, and column marginals are means for each planting site.
Marginal means that share superscript are not significantly different. Asterisks
refer to results of pairwise comparisons of source population means within each
planting site.
Source population
IN1
Planting site
IN2
SA1
SA2
DU1
DU2
IN1
IN2
SA1
SA2
DU1
DU2
31.54
(42.67)
24
36.76
(30.42)
17
32.86
(37.28)
21
22.07
(26.79)
27
17.41
(23.07)
22
16.65
(21.35)
31
Site
mean
25.12A
(31.10)
142
10.37
(17.34)
16
6.44
(8.58)
18
8.05
(10.90)
21
5.27
(7.04)
22
3.78
(6.24)
23
3.77
(6.08)
31
5.88E
(9.53)
131
9.30*
(5.21)
10
5.50*
(2.88)
6
33.13
(34.89)
15
15.42
(16.72)
12
32.50
(18.12)
10
26.00
(18.10)
20
22.64B,C
(22.54)
73
17.10
(11.76)
10
24.08
(18.29)
12
20.72
(12.60)
25
22.09
(12.96)
31
13.86*
(13.92)
21
11.90
(8.27)
31
17.87B
(13.17)
130
13.69**
(10.90)
16
16.33*
(9.19)
12
23.26
(11.40)
19
17.76*
(14.29)
25
28.78
(15.30)
27
24.19
(13.34)
36
21.84A,C
(13.83)
135
7.00
(13.38)
12
6.83
(13.35)
12
5.5
(12.42)
22
13.20
(31.25)
25
55.04
(72.63)
26
15.21
(30.19)
41
19.33D
(42.00)
138
17.42A,B
(21.14)
77
19.81A,B
(24.04)
123
16.59B
(20.81)
142
25.53A
(39.07)
129
15.86B
(19.97)
190
18.60
25.97
749
Population 16.93A,B
(26.13)
mean
88
'*' p < 0.05
'**' p < 0.0017
41
Table 3.6. Mean (standard deviation and sample size) of the maternal family
means of number of fruits produced in the 2003 reciprocal translplant
experiment. Symbols as in Table 3.5.
Source population
DU1
DU2
68.03** 24.65
(32.02) (14.48)
8
7
31.52
(43.23)
7
17.62
(14.96)
9
Site
mean
31.45A
(29.21)
48
15.92
(10.72)
9
24.96
(13.45)
8
21.47
(17.57)
7
13.42
(7.19)
7
10.66
(5.76)
9
16.91B
(11.27)
49
11.35*
(5.76)
9
13.96*
(9.59)
9
26.52
(15.73)
9
13.89*
(8.68)
8
20.50
(13.44)
7
14.55*
(3.98)
9
16.71B
(11.06)
51
SA2
3.27**
(1.37)
9
11.16
(13.69)
9
11.46
(3.13)
8
9.48
(2.92)
8
6.08*
(2.60)
7
5.08*
(4.63)
9
7.72C
(6.93)
50
DU1
5.28**
(1.42)
9
11.25*
(8.09)
8
16.87
(8.80)
8
12.62*
(6.33)
8
20.91
(5.05)
7
20.24
(4.29)
9
14.26B
(8.05)
50
DU2
1.37
(1.45)
9
0.35
(0.72)
9
5.67*
(4.97)
8
2.95
(5.49)
8
7.36*
(6.73)
7
1.41
(2.56)
9
2.97D
(4.58)
50
13.22B
(14.22)
53
25.60A
(25.42)
49
13.79B
(12.04)
46
16.63A,B
(19.95)
42
11.59B
(9.66)
54
14.89
(16.63)
298
IN1
Planting site
IN2
SA1
IN1
IN2
SA1
20.77
(13.46)
9
28.35
(21.93)
8
16.13
(6.76)
9
Population 9.70B
(9.52)
mean
54
SA2
'*' p < 0.05
'**' p < 0.0017
42
Table 3.7. Comparisons of mean fruit number produced for plants from the
native population and plants from all other populations pooled. All tests are
Generalized Linear models as described in the text with 1 degree of freedom.
Tests are one tailed.
Native population
2002
sig.?
χ
χ
Inland 1
Inland 2
Sandhills 1
Sandhills 2
Dunes 1
Dunes 2
1.19
0.07
4.25
4.03
8.30
0.63
1.74
0.08
9.51
0.56
5.17
1.60
2
no
no
yes
yes
yes
no
43
2
2003
sig.?
no
no
yes
no
yes
no
Table 3.8. Measures of Fitness Divergence between all possible population pairs
based on fruit numbers from the 2002 and 2003 reciprocal transplants.
2002
IN2
SA1
SA2
DU1
DU2
IN1
IN2
SA1
SA2
DU1
0.52
0.76
1.03
0.53
0.26
0.59
0.97
0.83
0.21
0.75
1.01
0.96
0.86
0.59
0.88
2003
IN2
SA1
SA2
DU1
DU2
IN1
IN2
SA1
SA2
DU1
0.30
1.26
0.83
0.82
0.41
0.65
1.09
0.62
0.42
0.76
0.18
1.08
1.20
0.98
0.84
44
Figure 3.1. Map of the locations of the source populations and planting sites in
northern Florida and south Georgia. Dunes populations are abbreviated DU,
Inland abbreviated IN, and Sandhills abbreviated SA.
45
2002
2003
Figure 3.2. Mean fruit production of transplants by habitat type for the 2002 and
2003 experiments. Means within a habitat type that share the same letter are not
significantly different based on Generalized Linear Model of the effect of source
habitat on fruit set. Error bars are one standard error.
46
Figure 3.3. The relationship between squared environmental distance and
Fitness Divergence from the 2002 experiment and 2003 experiments. The
mantel correlation was 0.37 (p = 0.10) in 2002 and -0.03 (p = 0.51) in 2003.
47
CHAPTER 4
THE STRENGTHS OF ALTERNATE MODES OF REPRODUCTIVE ISOLATION
IN THE ANNUAL PLANT DIODIA TERES (RUBIACEAE)
Abstract
Many studies have shown that prezygotic isolation evolves faster than
postzygotic isolation. At a given level of divergence prezygotic isolation is
expected to be stronger than postzygotic isolation. Postzygotic isolation is
usually measured in the laboratory, where it may be weaker than under natural
conditions due to benign laboratory environments. If prezygotic isolation evolves
faster than postzygotic isolation, then it is expected to be a more effective barrier,
and there should be evidence of assortative mating, but little evidence of low
fitness of hybrids. In this study, I tested the hypothesis that postzygotic isolation
in the field is as strong as postmating/prezygotic isolation in six populations of the
annual plant Diodia teres. I conducted hand pollinations between all pairs of
populations and planted their F2 progeny into both parental field sites.
Postmating/prezygotic isolation was stronger than postzygotic isolation, and
there was evidence of strong heterosis between some population pairs. Crosspollination success was negatively associated with differences in parental
habitat, but inter-habitat hybrids had greater fitness than purebred plants. These
results suggest that even when measured in the field postzygotic isolation is not
as strong as prezygotic isolation, and that studies should focus on the ecology
and genetics of prezygotic mechanisms given their emerging role as the
dominant modes of reproductive isolation.
Introduction
Many definitions of species rely on reproductive isolation, the inability of
different groups or populations to successfully interbreed (Coyne and Orr 2004).
It has been long recognized that reproductive isolation can evolve as a byproduct of adaptive evolution (Dobzhansky 1951), and more specifically, the
48
Ecological Speciation model shows that adaptation to different ecological niches
can promote the evolution of reproductive isolation between taxa (Schluter 1998;
Dobeli and Dieckmann 2003; Rundle and Nosil 2005). Other studies have shown
that modes of isolation evolve at different rates. Meta-analyses of the
relationship between divergence and reproductive isolation often report that
prezygotic isolation evolves faster than postzygotic isolation (Coyne and Orr
1989; Mendelson 2003; but see Moyle et al. 2004). Taken together, these
findings suggest that reproductive isolation should increase with differences in
selective environments of two taxa, and that at a given level of divergence,
prezygotic isolation should be stronger than postzygotic isolation.
Studies have repeatedly shown that for a given level of divergence,
prezygotic isolation is stronger than postzygotic isolation based on a stronger
relationship between prezygotic isolation and genetic distance than between
postzygotic isolation and genetic distance (Coyne and Orr 1989; Mendelson et al.
2003). This result may occur because postzygotic isolation is usually measured
in the laboratory where hybrids may be able to maintain higher fitness than in
harsher field environments to which organisms have evolved. Laboratories
usually exclude natural enemies and provide plentiful resources, whereas field
environments contain natural enemies and often resources are limiting. If
hybrids have intermediate phenotypes, they may be unfit in either parental
environment, but may not have reduced fitness in the laboratory because they
would not be subjected to selection in parental environments. With the exception
of plant-pollinator interactions (Schemske and Bradshaw 1999), prezygotic
isolation is also typically measured in the lab. It will be necessary to measure
isolation in the laboratory if field measurement is intractable. For example,
measurement of behavioral isolation in Drosophila would be almost impossible in
the field. Thus, measurement of postzgotic isolation is usually carried out in the
laboratory, but we do not know if it would be stronger if measured in the field.
If reproductive isolation increases with differences in selective
environments and prezygotic isolation evolves faster than postzygotic isolation,
then we would expect that at the early stages of divergence, prezygotic isolation
49
should be a stronger barrier than postzygotic isolation. Studies typically report
reproductive isolation between populations adapted to different environments in
the form of assortative mate choice or hybrid disadvantage (Coyne and Orr
2004). If adaptation to different environments contributes to reproductive
isolation, then populations adapted to similar environments should be less
isolated than populations that are adapted to different environments.
Furthermore, if prezygotic isolation evolves faster than postzygotic isolation, then
prezygotic barriers should be stronger between populations from different
habitats than postzygotic barriers. Therefore, successful mating between
individuals belonging to populations from different habitats should be rare.
Although postzygotic isolation is often quantified as a discreet variable
(Coyne and Orr 1989; Sasa 1998; Presgraves 2002; Moyle et al. 2004), it can
vary continuously. Measuring postzygotic isolation in the field introduces
additional complications because natural environments are heterogeneous, and it
may be necessary to weight hybrid fitness by variation within environments. In
addition, to achieve the most general measure of isolation, hybrid fitness should
be measured in both parental environments.
For sessile organisms such as plants, the most effective prezygotic
isolating barrier will necessarily be geographic separation. After that, the ability
to fertilize and to be fertilized by heterospecifics is most important. In selfcompatible plant species, postmating/prezygotic isolation, which occurs after
mating but before formation of a zygote, has been shown to be a rapidly evolving
and strong barrier to reproduction (Levin 1976). In motile species that exhibit
mating behaviors, it is appropriate to quantify prezygotic isolation as differences
in mate choice or mating frequency. These measures are less relevant to sessile
species without behavior, where mechanisms such as pollination ability are most
important.
Here I describe tests of the hypotheses that postzygotic isolation
expressed in the field is as strong as postmating/prezygotic isolation, and that
postmating/prezygotic barriers are stronger than postzygotic barriers. I focus on
six populations of an annual plant, that have been shown to be adapted to their
50
native habitats. I conducted hand pollinations within and between all populations
to measure postmating/prezygotic isolation and I raised the progeny of these
hand pollinations to the F2 generation and planted them into each parental field
site to measure postzygotic isolation.
Materials and Methods
Diodia teres (Rubiaceae) is an annual plant with small (~2mm in diameter)
self-compatible flowers (Fig. 4.1). It is found from tropical to temperate climates,
and ranges from Panama north to the northeastern United States, with a
weastern border extending to Michigan (Kearney and Peebles 1964). In the
southeastern United States, it is found in a variety of habitats, from coastal sand
dunes to inland forests where it occurs along roadsides and in canopy gaps. The
focal populations of this study, which are located in northern Florida and southern
Georgia, have been described previously (Fig. 2.1). The populations are
designated Dunes 1 and 2, which occur in coastal sand dunes, Sandhills 1 and 2,
which occur in Sandhills habitats, and Inland 1 and 2, which occur in the margins
of inland forests. Seeds are gravitationally dispersed, and there is abundant
overlap in flowering time among populations (Hereford pers. obs.). The study
populations occur in discreet patches separated by at least 20km, so it is unlikely
that pollinators or seeds will often travel between them. There are no noticeable
differences in floral morphology between populations, and thus little evidence
that pollinators would differentially pollinate plants from different populations.
Though there is little evidence of pairwise local adaptation between the six study
populations, there is evidence of local adaptation at the level of habitat type.
Pollination success and postmating/prezygotic reproductive isolation
I measured postmating/prezygotic isolation by performing hand
pollinations between plants from different populations, henceforth hybrid crosses,
and plants from the same source population, henceforth purebred crosses. In
December of 2000 I collected seeds from 50 - 60 maternal plants in each of the
six populations. The original design of this experiment called for crosses
between nine populations, but because I was unable to obtain permits to work at
51
three of the planting sites, the number of crosses conducted between populations
varied due to the removal of some cross combinations from the design.
In March of 2001, I germinated the field-collected seeds and raised
seedlings from each maternal family for which seeds had germinated in the
greenhouse. After the plants began flowering, I haphazardly assigned four plants
from each population to be pollen donors and the remainder to be pollen
recipients. In total, 8-9 pollen recipients from each population were included in
the study. The hand pollinations were conducted within and among all six
populations, generating crosses between each pollen recipient and plants of
three degrees of divergence. The pollen recipient was pollinated by an individual
from the same population, a plant from a different population but the same
habitat type, and by two plants from populations of a different habitat type. Each
cross between two individuals was attempted up to five times. Because D. teres
is self-compatible, all flowers that were to be outcrossed were sliced open with
forceps the night before anthesis and the anthers were removed while they were
below the stigma. I used a 3x magnifying glass to examine the stigma of flowers
to be hand pollinated, and rejected any flowers that had pollen attached to the
stigma. I used different colors of acrylic paint to mark the calyx of the flower to
indicate different types of crosses. The next morning, pollen from the assigned
pollen donor plant was placed on the stigma of the marked flower of the pollen
recipient.
Approximately three weeks after pollinations were performed, I checked
for the presence of developing fruits. D. teres always produces always two
seeds per fruit. I considered a pollination successful if seeds were developing,
and placed a small amount of Elmer's glue between the two seeds and the calyx
to keep the fruit in place so that the seeds could be collected later. For each of
the five hand pollination attempts, I recorded whether or not the pollination was
successful, and used these rates of successful hand pollination to calculate
postmating/prezygotic isolation. To make sure that there was no apomixis or
other means of self-fertilization, I performed the above emasculation procedure
52
but did not hand pollinate the flowers. None of ten flowers that received this
treatment produced fruits.
Hybrid fitness and postzygotic reproductive isolation
In March of 2002, I germinated F1 seeds from the successful hand
pollinations in the greenhouse and allowed the seeds that germinated to grow to
maturity and self-fertilize to produce F2 seeds. Some seeds of F1 progeny did
not germinate, which reduced the number of F2 families in the study. Seeds
were collected from the F1 generation throughout the season and stored until
March of 2003. Progeny from each hand pollination in the previous generation
were replicated as offspring of the same maternal full-sib family. The pollen
recipient was the maternal plant, and all her F2 progeny from each pollen donor
composed a family. Throughout this paper "family" refers to the set of F2
progeny derived from a particular pollen donor and recipient pair. A pollen
recipient could produce up to four families, one purebred, one intra-habitat
hybrid, and two inter-habitat hybrid families.
In February of 2003, I germinated seeds from each hybrid and purebred
family. Because there was limited space available in the growth chamber, I could
not germinate all the seeds simultaneously, and haphazardly chosen seeds were
germinated. As a result, members of the same maternal family were not
germinated and planted at the same time. Seedlings that germinated were
immediately planted in flats and transferred to the greenhouse and eventually
transplanted to the field.
Although planting seedlings instead of seeds circumvented some potential
selection in the field, the advantages of planting seedlings outweighed this
disadvantage. Germination fraction is small (proportion germinated = 0.21;
Hereford and Moriuchi 2004), and previous attempts to germinate seeds in the
field were largely unsuccessful. I planted enough seedlings to insure that up to
10 seedlings per family could be planted into each parental planting site, and that
purebred families could be planted at all planting sites. The seedlings were
planted in the field when they had from two to six true leaves between March and
May of 2003. At each planting site, the seedlings were planted in a randomized
53
block design with minimal disturbance to natural vegetation. Planting gardens
were up to 30m X 25m, and experimental blocks were separated by
approximately 4m. For each seedling, I recorded the date it was planted and its
initial size, measured as the total area of leaf material. In December of 2003
after nearly all plants had senesced and growth had essentially stopped, I
recorded the number of fruits produced by each plant and used this as the
measure of fitness in calculations of postzygotic reproductive isolation.
Data analysis
The outcome of a hand pollination was either successful (fruit present) or
not successful (no fruit set). Consequently, analyses of effects on the rates of
successful hand pollinations were performed assuming a binomial response. I
used Generalized Linear Models (GLiM) with logit link functions and binomial
distribution functions to compare probabilities of successful hand pollination in
purebred, and hybrid crosses and to analyze the effects of pollen recipient
population and pollen donor population on probability of successful hand
pollination. In this analysis, the set of attempts to pollinate a pollen recipient with
a pollen donor was considered a single observation because separate attempts
of the same cross are not independent.
I used a GLiM with logit link functions and binomial distribution functions to
analyze effects of pollen donor population, pollen recipient population, and their
interaction on the probability of successful hand pollination. A significant pollen
recipient by pollen donor population interaction is necessary but not sufficient to
support reproductive isolation between populations. A significantly lower rate of
successful hand pollination in hybrids than in purebreds suggests
postmating/prezygotic reproductive isolation. To test the hypothesis that specific
pairs of populations were reproductively isolated, I compared the mean
pollination success of the purebred crosses to the mean pollination success of
the two reciprocal hybrid crosses between each pair of populations. These tests
were also performed using a GLiM with a logit link function and a binomial
distribution function. The significance of the pairwise tests was corrected for
54
multiple comparisons by dividing the alpha level (0.05) by the total number of
comparisons (30) to yield a corrected alpha of 0.0017.
Analyses of postzygotic reproductive isolation required comparing the
lifetime fitness of hybrid and purebred progeny in the field. I used fruit production
in the F2 generation as the measure of lifetime fitness. The residuals for fruit
production from a model that included effects of planting date, initial size,
experimental block nested within planting site, pollen donor population, and
pollen recipient population, and all interactions between discrete variables did not
fit a normal distribution, and were heteroscedastic. Numerous observations had
fruit set equal to zero, and the response variable consisted of counts. The
residuals of fruit production fit a Poisson distribution better than the Normal
distribution. Goodness of fit measured by the deviance divided by the degrees of
freedom of residuals of fruit production was ~10 for the Poisson and ~ 500 for the
Normal distribution. All analyses of fruit production were performed using a GLiM
with a log link function and a Poisson distribution function, which does not
assume equal variances (McCullagh and Nelder 1989). A variable will usually be
overdispersed relative to the expectation of a Poisson distribution (McCullagh
and Nelder 1989), meaning that the variance of the variable will be outside the
nominal variance of the Poisson. I corrected for overdispersion in all GLiM of
fruit production by estimating the dispersion parameter and dividing all likelihood
ratio statistics by this parameter (McCullagh and Nelder 1989).
If the planting environment affects the expression of postzygotic
reproductive isolation between populations, progeny fruit set will depend on the
interaction of planting site, pollen donor source population, and pollen recipient
source population. I estimated the main effects of planting site, pollen donor
source population, and pollen recipient source population on seed production
while accounting for variation in date of planting, initial size at planting, and
experimental block nested within planting site. The main effect of planting site is
confounded with the selection of families planted at a site because not all hybrids
were planted at all planting sites. I estimated the pollen donor by pollen recipient
population interaction on fruit number, a necessary but not sufficient condition for
55
postzygotic isolation. The interaction between planting site, pollen donor
population, and pollen recipient population measures the extent to which fruit set
of progeny from different combinations of pollen donor and pollen recipient
depended on where they were planted. I used a GLiM as described above with a
log link function and Poisson distribution function, adjusting for overdispersion, to
quantify these effects.
If there is postzygotic reproductive isolation between specific population
pairs, the fruit number of the hybrids between them will be less than the number
of fruits produced by the native purebred plants at a given planting site. To test
individual hypotheses of postzygotic reproductive isolation between each pair of
populations, I compared the mean fruit set of families of hybrids between a pair
of populations to the mean fruit set of the native purebreds at each parental
planting site. Though the individual observations in these tests were fullsib family
means, the residuals fit a Poisson distribution better than the Normal distribution.
The deviance assuming a Poisson distribution was ~10 and assuming a Normal
distribution the deviance was ~300. To test for isolation between each
population pair, I used GLiM similar to those described above for Poisson
distributed response, and accounted for overdispersion in each test. The
significance level of comparisons between purebred and hybrid progeny fruit
production was corrected for multiple comparisons by dividing the alpha level
(0.05) by the number of comparisons (30) to yield a corrected alpha level of
0.0017.
If postmating/prezygotic isolation arises as a by-product of adaptation to
different habitats, the average hand pollination success of inter-habitat crosses
should be lower than that of purebred crosses, and the hand pollination success
of intra-habitat crosses should be similar to purebred crosses. I tested this
hypothesis by comparing the hand pollination success of purebred crosses, intrahabitat crosses, and inter-habitat crosses. I used a GLiM similar to my previous
models of pollination success, with logit link function and binomial distribution
function, and to test the hypothesis that there were differences in successful
56
pollination between specific types of crosses I performed a logistic regression of
cross success on type of cross.
To determine if postzygotic reproductive isolation evolves as a by-product
of adaptation to different habitats, I tested the hypothesis that there were
differences in mean fruit set among purebred, intra-habitat hybrid, and interhabitat hybrid progeny grown in the field. Observations in this analysis were the
mean fruit numbers produced by each fullsib family. I used a GLiM to compare
mean fruit production, with log link function and assumed a Poisson distribution
and accounted for overdispersion by dividing likelihood ratios by the estimated
dispersion parameter as in other tests.
I calculated postmating/prezygotic reproductive isolation between each
pair of populations following Coyne and Orr (1989) as one minus the ratio of
successful hand pollination of hybrid crosses divided by the success rate
purebred pollinations. Note that these rates are rates of successful fertilization
because some hand pollinations may have resulted in fertilizations that were
aborted and no seeds were produced.
 Rate

hybrids
Postmating/prezygotic = 1 - 

 Rate purebreds 
I calculated postzygotic reproductive isolation, as the mean relative fitness
of hybrid progeny between a pair of populations divided by the mean relative
fitness of the purebred progeny of both populations.
W

Postzygotic isolation = 1−  Hybrids 
 W Purebreds 
I calculated least square mean fruit set as hybrid and purebred fitness. Least
squares means were computed after accounting for variation due to date of
planting, initial size at planting, experimental block within planting site, and
planting site. I calculated the postzygotic isolation between a pair of populations
using the least square mean fruit set of all their hybrids and the purebreds at both
parental planting sites.
I used the SAS (SAS Institute, Cary NC) procedure PROC GENMOD to
perform GLiM and logistic regressions, with the SCALE=PEARSON option in
57
models of fruit number. I used the SAS procedure CORR to estimate correlations
with the spearman option. Linear regressions were performed to quantify the
direction of effects of F2 progeny planting time and size at planting on fruit
production using the procedure REG.
Results
The mean rate of successful hand pollination between all pairs of
populations was 0.44 with a standard deviation of 0.35. The main effects of
pollen donor and pollen recipient population on pollination success were
significant, which suggests that populations differed in how often they were
successful when they acted as pollen donors and pollen recipients, respectively
(Table 4.1). If there is postmating/prezygotic reproductive isolation between
populations, then the likelihood of successful hand pollination between plants will
depend on the identity of the two parental populations, resulting in a significant
interaction between source populations when acting as pollen donors and pollen
recipients. In accordance with the expectation for postmating/prezygotic
isolation, the interaction between pollen donor and pollen recipient population
was significant (Table 4.1).
If a pair of populations show postmating/prezygotic reproductive isolation,
then purebred pollinations will be successful more often than hybrid pollinations.
The difference between pollination success of hybrid and purebred crosses
varied from instances of significantly lower pollination success in hybrid crosses
to significantly greater pollination success in hybrid crosses (Table 4.2). For
example, crosses between the Dunes 1 population and the Inland 1 population
were significantly less successful than crosses within these populations, but
crosses between Inland population 2 and Dunes population 2 were more likely to
be successful than purebred crosses.
Like pollination success, fruit production of F2 progeny of crosses
depended on interactions between pollen donor and pollen recipient population,
but there were environmental effects on fruit production as well. Earlier planting
resulted in greater fruit production (linear regression: β = -0.32 p<0.0001) and
seedlings that were larger at the time of planting (linear regression: β = 4.79
58
p<0.0001) produced more fruits (Table 4.3). I also detected a significant effect of
experimental block nested within planting site (Table 4.3), indicating that smallscale environmental variation influenced fruit number.
I found a marginally significant effect of the pollen donor by pollen
recipient population interaction (Table 4.3), indicating that progeny derived from
different combinations of parental populations did not set similar numbers of
fruits, evidence of the possibility of postzygotic isolation. A significant planting
site by pollen donor population interaction suggests that fruit production
depended on the planting site and the source population of the pollen donor
parent. The lack of a significant planting site by donor population by pollen
recipient population interaction suggests that the fruit set of progeny of crosses
between specific combinations of pollen donor and pollen recipient populations
was similar among planting sites.
Postzygotic reproductive isolation occurs when hybrids between a pair of
populations set fewer fruits than native purebred progeny at a planting site. I
found that hybrid fruit production was sometimes greater and sometimes less
than native purebred fruit production (Table 4.4). For example, all hybrids of
Inland population 1 set significantly more fruits than the purebreds at the Inland 1
planting site, and generally set more fruits than the purebreds at the other
parental sites as well. In contrast, hybrids between Inland population 2 and
Dunes population 1 set significantly fewer fruits than purebreds of Inland site 2,
but not purebreds of Dunes population 1. With the exception of Inland population
1, most hybrids set fewer fruits than the native purebred plants at each site, but
few of these differences were significant (Table 4.4).
Habitat variation and reproductive isolation
If reproductive isolation evolves as a by-product of adaptation to different
habitats and if a barrier to reproduction is an effective isolating mechanism,
plants belonging to different populations of the same habitat type will successfully
interbreed at rates similar to purebred plants, and pollinations between plants
from different habitat types will be less successful than purebred pollinations.
Consistent with this expectation, I found that intra-habitat crosses were not
59
significantly less successful than purebred crosses (logistic regression β = -0.25
p = 0.21; Fig. 4.2) but crosses between plants from different habitats were less
likely to be successful than purebred crosses (logistic regression β = -0.40, p <
0.04). In contrast to the results for pollination success, there was evidence that
purebred progeny were the least successful at setting fruits in the field. Fruit set
of progeny from purebred crosses was 31% less than inter-habitat hybrid fruit set
(Probit model β = -0.48 p < 0.02; Fig. 3.3), and 37% less than intra-habitat
hybrids (Probit model β = -0.57 p < 0.001). Fruit production was similar between
intra-habitat hybrids and inter-habitat hybrids.
Estimates of pairwise postmating/prezygotic reproductive isolation were
mostly positive (5 negative and 10 positive; Table 4.5), indicating reproductive
isolation between the majority of population pairs. The magnitude of isolation
was greater for pairs of populations belonging to different habitat types, and most
negative values were associated with the two Sandhills populations for which the
success rates of purebred crosses were low (Table 4.2). Estimates of
postzygotic reproductive isolation were slight to moderate for some population
pairs, but many pairs displayed strong hybrid advantage, especially those
involving Inland population 1. I was not able to estimate postzygotic isolation
between the Sandhills populations, or between Sandhills population 1 and Dunes
population 1 because only one F1 hybrid between these populations produced
F2 seeds that germinated. Overall postmating/prezygotic isolation was stronger
than postzygotic isolation (mean of 0.08 vs. -0.14: paired t-test, t=2.137,d.f. = 12,
p = 0.0538). Despite variation among population pairs in both measures, my
estimates of postmating prezygotic and postzygotic reproductive isolation were
not significantly correlated (rank correlation = -0.03, p>0.90; Fig. 3.4).
Discussion
Variation among populations and planting sites in cross pollination success and
hybrid fruit set
I found that the degree of postmating/prezygotic isolation varied among
pairs of populations. The probability of a successful hand pollination depended
on the population of the pollen donor and pollen recipient, and there were
60
significant differences between populations when acting as pollen donors and as
pollen recipients (Table 4.1). The variation in a population's ability to receive
pollen may in part reflect differences in population tolerance for emasculation, but
the interaction between pollen donor and pollen recipient also affects the success
rate of crosses. For example, Sandhills 1 plants generally were not good pollen
donors or pollen recipients even in purebred crosses, yet the average pollination
success of crosses between Sandhills1 and Dunes population 2 was greater than
the grand mean pollination success (Table 4.2) suggesting, that Sandhills 1
plants were highly receptive to Dunes 2 pollen. A number of studies have shown
that pollination success in hybrid crosses depends on pollen stigma interactions
(Levin 1976; Cruzan 1990; Carney et al. 1996), which may explain these results.
Some of the larger differences between rates of hand pollination success of
purebred and hybrid crosses were not significant, which suggests low power to
detect differences in pollination success rate, and lack of precision in estimates
of pollination success. Though I attempted each cross five times, the number of
target plants for each cross was low. The small sample sizes probably lead to
more error in estimates of postmating/prezygotic reproductive isolation, because
I measured isolation between 6 pairs of populations, the overall patterns of
isolation are probably robust
Fruit production of hybrid progeny was variable, and was often greater
than purebred fruit production (Table 4.4). Reduced fruit set in hybrids can be
explained by a variety of causes such as the break-up of beneficial coadapted
gene complexes, or the inability of hybrids to fit into the ecological niche of their
parents (Coyne and Orr 2004). Other studies have also shown that hybrids can
have greater fitness than purebreds (Langor 1990; Wang et al. 1996; Rieseberg
et al. 2003; Gross et al. 2004). Hybrids in this study may have greater fitness in
some instances because populations carry an inbreeding load and outcrossing
between populations decreases this load (e.g. Barrett and Charlesworth 1991).
High homozygosity within my study populations suggests that they are highly
inbred (Table 5.2). The heterosis that results from outcrossing between
populations may counterbalance the negative effects of hybrids’ lack of
61
adaptation to parental environments. The combined effects of inbreeding load of
the parents and hybrid maladaptation may contribute to the low and variable
estimates of postzygotic isolation.
Habitat variation and reproductive isolation
Inter-habitat hand pollinations were less successful than purebred hand
pollinations and intra-habitat pollinations, though the latter difference was not
significant (Fig. 4.2). These results suggest that postmating/prezygotic isolation
may arise as a by-product of adaptation to different habitats. The pattern of
pollination success I observed in D. teres is in agreement with the pattern of
prezygotic reproductive isolation reported in other studies of isolation between
populations that occur in similar and dissimilar habitats. Populations of threespine sticklebacks adapted to benthic environments are more reproductively
isolated from populations of the same lakes that are adapted to limnetic
environments than they are to benthic populations that occur in different lakes,
(Rundle et al. 2000). The same pattern of reproductive isolation in association
with habitat or environment differences between populations has been reported
in a variety of other taxa including birds and insects (Schluter 2000; Funk 1998;
Nosil et al. 2002). Similar results have been reported in Leopard frogs, where it
has been shown that adaptation to temperature at different latitudes leads to
postzygotic isolation in the form of hybrid inviability (Moore 1946; 1949).
Unlike rates of hand pollination, hybrid fruit set was not lower for interthan for intra-habitat hybrids. Inter-habitat hybrids set similar numbers of fruits to
intra-habitat hybrids, and both set significantly more fruits than purebred plants
(Fig. 4.3). This result is surprising given evidence for local adaptation at the level
of habitat type for this set of populations, and that most studies report that
hybrids between different habitats or distant populations have lower fitness than
purebreds or hybrids between populations separated by intermediate distances
(Waser and Price 1994; Edmands 2002; but see Fenster and Galloway 2000).
Waser and Price (1994) showed that in Delphinium nelsonii there was an
optimum distance between parents at which cross pollinations between them
yielded the most fit offspring. Crosses between parents separated by greater
62
distances and possibly under different selective pressures (Waser and Price
1985) resulted in offspring with lower fitness. It was assumed that genetic
relatedness was inversely related to the physical distance between parents. In
this study, I assumed that individuals from different habitats were less closely
related than individuals from the same habitat type, and did not find a similar
relationship between relatedness and progeny fruit production (Fig. 4.3). Peer
and Taborskyi (2005) showed that in hybrid bark beetles, the hatching success
rate of progeny of within and between population crosses was similar. Their
results and the results in this study suggest that postzygotic isolation may not be
as commonly associated with habitat differences between populations as
prezygotic or postmating/prezygotic isolation in the early stages of divergence.
The strength of postmating/prezygotic reproductive isolation and postzygotic
reproductive isolation
Postzygotic isolation in D. teres was not as strong as
postmating/prezygotic isolation (Table 4.5), and there was no significant
relationship between the magnitudes of postmating/prezygotic and postzygotic
isolation between populations(Fig. 4.4). It is possible that postzygotic isolation
was underestimated because hybrid disadvantage in the F1 generation was not
accounted for. On the other hand, it has been shown that hybrid disadvantage
may not become apparent until later generations (Burton 1990; Fenster and
Galloway 2000), and thus it is not likely that growing the F1 generation in the
greenhouse resulted in an underestimate of reproductive isolation. In their
review of modes of reproductive isolation, Nosil et al. (2005) found that
prezygotic mechanisms were stronger than postzygotic barriers, and other
studies have shown that hybrids can have equal or higher fitness than parents in
the field (Emms and Arnold 1997; Wang et al. 1997).
Although other studies have found evidence that modes of reproductive
isolation are positively related (Levitan et al. 2003; Ramsey et al. 2003), I found
no correlation between postmating/prezygotic reproductive isolation and
postzygotic isolation, though there was substantial variation in both measures.
Modes of isolation in this study may be uncorrelated because postzygotic
63
isolation is not evolving, while postmating/prezygotic isolation is becoming
stronger with divergence. Proteins that influence fertilization after mating and
before zygote formation evolve fast relative to neutral expectations (Swanson
and Vacquier 2002), and such a mechanisms could cause postmating/prezygotic
isolation in D. teres to evolve faster than postzygotic isolation.
Given that prezygotic isolation is more effective an isolating mechanism
than postzygotic isolation, the genetics and ecology of prezygotic isolation should
be studied more closely. Though small sample sizes of individual estimates of
isolation between specific pairs of populations make it difficult to obtain precise
estimates of isolation, the overall pattern of isolation suggests that
postmating/prezygotic isolation is more extensive in D. teres than postzygotic
isolation. There is a long record of studies of the genetics of postzygotic
isolation (Wu and Palopoli 1994; Coyne and Orr 2004), and recent studies of the
ecology of speciation have shed more light on the role of the ecological niche in
postzygotic isolation (Schluter 1998; 2000; Via 2001; Rundle and Nosil 2005).
Despite its importance as the primary barrier to reproduction, we know little about
the genetics and ecology of prezygotic mechanisms. Because so many traits
such as dispersal distance, mating preferences, or pollen-stigma interactions can
influence the expression of prezygotic isolation, we may not arrive at general
rules for the expression of prezygotic isolation that are analogous to rules for the
expression of postzygotic isolation (e.g. Coyne and Orr 2004). However, we may
come to a more complete understanding of the early stages of speciation, by
focusing on earlier acting isolation barriers which are most effective.
64
Table 4.1. Generalized Linear Model of effects of pollen donor source
population, pollen recipient population and their interaction on probability of
successful hand pollination. Denominator degrees of freedom for all sources of
variation is 98.
χ2
Source of variation
d.f.
Pollen donor source population
5
20.96
0.0008
Pollen recipient source population
5
24.34
0.0002
Pollen donor X recipient source population
21
51.37
0.0002
65
p-value
Table 4.2. Mean (standard deviation, number of pollen recipients) rate of
successful hand pollination between populations as pollen donors and pollen
recipients. Rates in bold are means of purebred crosses. Marginal values are
mean pollination success of populations when acting as pollen donors or pollen
recipients. Marginals that share the same letter superscript are not significantly
different. Asterisks indicate significance of comparisons between a specific
hybrid pollination and the pooled hand pollination success rate of the purebred
crosses of its parental populations.
Pollen recipient
Pollen donor
IN1
IN2
SA1
SA2
DU1
DU2
IN1
0.66
(0.14)
9
0.60
(0.23)
7
0.40
(0.37)
5
0.40
(0.28)
2
0.27*
(0.23)
3
0.44
(0.43)
5
Recipient
mean
0.51B
(0.29)
31
IN2
0.32
(0.41)
9
0.42
(0.41)
10
0.40
(0.49)
4
0.20
(0.20)
4
0.09*
(0.12)
5
0.47
(0.31)
5
0.33C
(0.36)
37
SA1
0.28
(0.30)
3
0.05*
(0.10)
5
0.08
(0.11)
9
0.11
(0.20)
7
0.07**
(0.12)
3
0.52
(0.41)
4
0.17D
(0.26)
28
SA2
0.70*
(0.42)
4
0.34
(0.32)
4
0.44
(0.43)
6
0.23
(0.29)
9
0.20
(0.28)
4
0.20
(0.28)
4
0.33C
(0.34)
31
DU1
0.70
(0.12)
4
0.60
(0.28)
4
0.45**
(0.44)
4
0.73*
(0.23)
3
0.87
(0.12)
9
0.70
(0.29)
7
0.71A
(0.25)
31
DU2
0.55
(0.19)
4
0.75*
(0.19)
4
0.33*
(0.58)
4
0.27
(0.31)
3
0.51
(0.25)
7
0.59
(0.25)
9
0.53B
(0.30)
31
0.44
0.54A
0.44A
0.27B
0.33B
0.47A
0.52A
(0.35)
(0.32)
(0.37)
(0.30)
(0.38)
(0.34)
(0.31)
174
33
31
28
32
34
33
** p < 0.0017 (significance level after correction for multiple comparisons)
* p < 0.05
Donor
mean
66
Table 4.3. Generalized Linear Model of effects of date of planting, initial size at
planting, block nested within planting site, planting site and their interactions on
number of fruit produced by F2 progeny from hand pollinations within and
between populations. Denominator degrees of freedom for all sources of
variation is 2882.
χ2
p-value
1
48.89
0.0001
Initial size at planting
1
166.69
0.0001
Block within planting site
44
1002.89
0.0001
Planting site
5
486.14
0.0001
Pollen donor source population
5
5.14
0.3991
Pollen recipient source population
5
11.58
0.0410
Planting site X pollen donor source population
16
46.68
0.0001
Planting site X pollen recipient source population
16
27.83
0.0331
Pollen donor population X pollen recipient population
14
23.72
0.0495
Planting site X pollen donor X pollen recipient
11
11.99
0.3642
Source of variation
d.f.
Date of planting
67
Table 4.4. Mean (standard deviation, number of fullsib families) number of fruits
produced by fullsib families of hybrid and purebred progeny. Means in bold
indicate purebred progeny at their native planting site. Marginal row totals are
means of all hybrids and the native purebreds at each planting site, and column
marginal totals are means of each population as a hybrid parent across planting
sites. Marginal means that share the same letter superscript are not significantly
different. Asterisks indicate significance of comparisons of the fruit set of hybrids
with the fruit set of the native parental purebred at each planting site.
Planting site and hybrid parent population
Hybrid parent population
IN1
IN2
SA1
SA2
DU1
IN1
25.29
(14.23)
8
66.79**
(34.98)
9
58.34**
(40.90)
4
73.20**
(30.06)
4
82.74**
(55.28)
5
Planting
site
mean
105.39** 65.48A
(86.03)
(53.22)
6
36
IN2
24.96
(10.13)
9
22.30
(5.32)
4
30.00a
(10.20)
2
16.92
(0.94)
2
15.00*
(1.78)
3
16.27*
(7.31)
9
20.99B
(8.89)
29
SA1
17.56a
(7.17)
4
25.08
(8.39)
2
29.56
(10.89)
3
24.00
(-)
1
42.10
(-)
1
32.20
(6.23)
3
24.69B
(11.23)
14
SA2
9.89a
(3.53)
7
6.24
(4.44)
2
6.40
(-)
1
15.28
(1.62)
2
11.33a
(2.78)
4
8.17a
(3.60)
4
10.26C
(4.15)
20
DU1
16.89
(3.42)
5
16.17
(1.51)
3
17.64
(-)
1
15.99
(4.44)
4
23.83
(9.46)
7
23.05
(4.70)
11
20.53B
(6.36)
32
DU2
9.69a
(4.81)
6
5.95
(7.24)
7
0.78
(0.75)
2
4.27
(6.50)
4
8.88
(7.54)
11
4.22
(5.20)
8
6.51C
(6.68)
38
Hybrid
parent
mean
36.97
(28.73)
40
25.16
(19.87)
28
31.49
(14.51)
15
19.06
(15.32)
20
22.31
(17.76)
32
20.52
(23.19)
40
25.35
(21.87)
169
DU2
** p < 0.0017 (significance after correction for multiple comparisons)
* p < 0.05
68
Table 4.5. Matrix of pairwise estimates of postmating / prezygotic reproductive
isolation (above) and postzygotic reproductive isolation (below) between
populations. Negative estimates of postmating/prezygotic isolation indicate that
hybrids crosses were more successful than purebred crosses, and positive
values indicate the opposite. Values of postzygotic isolation less than one
indicate that hybrids had lower relative fitness than purebreds, and values
greater than one indicate that hybrids had greater relative fitness. Only one
hybrid family between the Sandhills populations, and between Dunes population
1 and Sandhills population 1 survived into the F2 generation. Thus no estimate of
postzygotic reproductive isolation is given for these populations.
IN1
IN2
SA1
SA2
DU1
IN1
IN2
SA1
SA2
DU1
IN2
SA1
SA2
DU1
DU2
0.15
0.08
0.10
-0.24
0.17
-0.77
0.37
0.47
0.45
0.15
0.21
-0.21
-0.29
0.43
0.17
IN2
SA1
SA2
DU1
DU2
-0.35
-0.15
0.07
-0.53
0.23
------
-0.37
0.08
-----0.04
-1.05
0.12
0.09
0.07
-0.02
69
Table 4.6. Generalized Linear Model of the effects of date of planting, initial size
at planting, planting site, experimental block within planting site, planting site,
cross combination, and genotype nested within cross combination on hybrid fruit
number. The model included the fruit production of hybrids only. Denominator
degrees of freedom for all sources of variation is 1154.
Date of planting
1
12.60
0.0004
Initial size at planting
1
50.65
0.0001
Block within planting site
44
471.80
0.0001
Planting site
5
190.56
0.0001
Cross combination
13
21.52
0.0633
Planting site X cross combination
11
10.13
0.5185
Genotype within cross combination
54
93.25
0.0007
70
Figure 4.1. A diagram of a flower of D. teres (taken from Zomlefer 1994).
71
Figure 4.2. Meanpollination success of purebred crosses, intra-habitat crosses,
and inter habitat crosses Error bars are one standard error of the mean. Points
that share the same letter superscript are not significantly different.
72
Figure 4.3. Meanfruit number of purebred, intra-habitat, and inter habitat
progeny. Points that share the same letter superscript are not significantly
different. Error bars are one standard error of the mean.
73
Figure 4.4. The relationship between pairwise postmating/prezygotic
reproductive isolation and postzygotic isolation for all pairs of populations. The
rank correlation between measures is -0.03, p>0.90.
74
CHAPTER 5
ARE THERE POSITIVE RELATIONSHIPS BETWEEN DIVERGENCE AND
REPRODUCTIVE ISOLATION WITHIN A SPECIES?
Abstract
Many studies have shown that the strength of reproductive isolation
increases with the strength of nonadaptive divergence, but few studies have
tested the hypothesis that reproductive isolation increases with adaptive
divergence as well. I quantified the relationships between adaptive and
nonadaptive divergence and two modes of reproductive isolation among six
populations of the annual plant Diodia teres. Using estimates of reproductive
isolation and adaptive divergence obtained from previous studies, as well as
estimates of genetic distance obtained from six polymorphic allozyme loci, I
measured the correlations between measures of divergence and reproductive
isolation. Despite significant population genetic structure, and variation in
genetic distance, adaptive divergence, and reproductive isolation, there were no
significant relationships between either measure of divergence and reproductive
isolation. The results suggest that processes that give rise to reproductive
isolation early in divergence may not follow a simple pattern and that the degree
of reproductive isolation between a pair of populations may depend on additional
factors besides the level of divergence between them.
Introduction
Recent empirical and theoretical studies have demonstrated that adaptive
divergence can facilitate speciation. Empirical studies have shown that
reproductive isolation can evolve as a by-product of divergent selection in
different habitats (Macnair and Christie 1983; Nagel and Schluter 1998;
Schemske and Bradshaw 1999; Hawthorne and Via 2001), and mathematical
models have shown that the waiting time to speciation is greatly decreased when
the effects of divergent selection are added to those of neutral divergence
(Gavrilets 2003). Empirical evidence for a role of neutral divergence is less
75
extensive, but there have been fewer attempts to study it in natural populations.
In one example, Burton (1990) showed that F2 hybrids between different
populations of a marine copepod had longer development times relative to
purebreds, in the absence of obvious environmental differences between
parental habitats. Thus, empirical and theoretical evidence suggests that
reproductive isolation can evolve as a by-product of adaptive or neutral
divergence, though these mechanisms are not expected to be mutually
exclusive.
Studies that have quantified the relationship between divergence and
reproductive isolation have typically involved fairly divergent taxa. These studies,
which are typically conducted between different species, have shown that
reproductive isolation increases with divergence, prezygotic isolation evolves
faster than postzygotic isolation, and that sympatric species are more strongly
isolated than allopatric species (Coyne and Orr 1989; Sasa 1998; Presgraves
2002; Mendelson 2003; Moyle et al. 2004). They have also shown that
reproductive isolation tends to become stronger over time, but do not quantify
how reproductive isolation evolves early in divergence. There is evidence that
prezygotic isolation can become stronger after secondary contact (Coyne and
Orr 1989), but we do not know if reproductive isolation is positively related to
divergence at the initial stages of speciation. Effects of inbreeding depression
and heterosis could slow the evolution of reproductive isolation by providing an
advantage to disassortative mating. Thus, the relationship between divergence
and reproductive isolation may become established later in divergence, when
outbreeding depression overwhelms inbreeding depression.
Nonadaptive divergence has been shown to be positively associated with
reproductive isolation (Coyne and Orr 1989; Sasa 1998; Presgraves 2002;
Mendelson 2003; Moyle et al. 2004). Given emerging evidence for the role of
adaptive divergence in speciation and the evolution of reproductive isolation
(Schluter 2000; Coyne and Orr 2004), the relationship between adaptive
divergence and reproductive isolation should be quantified as well. Because
populations adapted to different ecological niches or different environments have
76
been shown to have evolved reproductive isolation (Howard and Berlocher 1998;
Levin 2000; Coyne and Orr 2004), it is expected that reproductive isolation
should increase with adaptive divergence. There have been few attempts to
quantitatively test the predicted relationship between adaptive divergence and
reproductive isolation, and there is no widely accepted method of measuring
adaptive divergence analogous to methods of measuring nonadaptive
divergence such as genetic distance. Finally, any measure of adaptive
divergence will not be a pure measure of divergent selection because other
forces in addition to adaptive divergence such as genetic drift and gene flow will
influence the strength of adaptive divergence.
Prezygotic isolation may evolve faster due to reinforcement after
secondary contact or as a result of differential sexual selection within species.
We do not know if these relationships hold in organisms in which there is not
strong sexual selection, or where there has been little chance of secondary
contact. In organisms with where there is a strong possibility of sexual selection,
such as fruit flies or guppies (Coyne and Orr 1989; Mendelson 2003), there is
evidence that prezygotic isolation evolves faster than postzygotic isolation. In
plants where there is less opportunity for selection based on mate choice, there
is no evidence that prezygotic isolation evolves faster than postzygotic isolation
(Moyle et al 2004).
An appropriate system to test the hypothesis that there is a relationship
between nonadaptive or adaptive divergence and reproductive isolation is a set
of populations that vary in the degree of pairwise divergence and reproductive
isolation. Differentiated populations of the same species will most likely meet
these criteria because they often show variation in the degree of divergence and
are not completely reproductively isolated (Levin 1976b; Schmidt and Levin
1985; Avise 1993; Harrison and Hastings 1996; Hollocher et al. 1997). Study of
the evolution of reproductive isolation between differentiated populations can
also provide insight into the earliest stages of the process of speciation, where
the evolution of reproductive isolation is expected to occur and where most
models of speciation are focused (Gavrilets 2003). At later stages of speciation,
77
it will be difficult to study the effects of adaptive and neutral divergence on
reproductive isolation, because once a system reaches the limit of complete
assortative mating, there will be no variation in reproductive isolation.
Here I test the hypothesis that there are positive relationships between
modes of population divergence and modes of reproductive isolation among six
populations of the annual herb Diodia teres. To measure the degree of
nonadaptive divergence, I estimated hierarchical levels of genetic structure within
and among populations, and pairwise genetic distances between them. I used
two previously quantified measures of reproductive isolation and a metric of
divergence in fitness between the six populations, as well as estimates of genetic
distance to quantify the relationships between all measures of divergence and
reproductive isolation. In this study, I address two questions; (1) what is the
pattern of nonadaptive divergence between all population pairs? (2) What are the
relationships between adaptive divergence, nonadaptive divergence,
postmating/prezygotic isolation, and postzygotic isolation of reproductive
isolation.
Materials and Methods
Diodia teres (Rubiaceae) is a self-compatible annual plant with a range
extending from Panama, north to Connecticut, USA, and west to Michigan, USA
(Kearney and Peebles 1964). In northern Florida and southern Georgia it occurs
in a range of habitats including coastal sand dunes, the margins of inland forests,
and disturbances in sandhill forest. These three habitats differ in soil texture and
in percent cover of both herbaceous vegetation and overhead canopy. Previous
work has demonstrated local adaptation of some populations of D. teres to these
habitats. The present study involves six populations that have been the focus of
previous investigations of adaptive divergence and reproductive isolation. Two
populations are located in each of three habitat types and are designated Dunes
1 and 2, Sandhills 1 and 2, and Inland 1 and 2. Seeds of D. teres disperse by
gravity, and there is overlap in flowering time among populations (Hereford pers.
Obs.), but the study populations occur in discrete patches separated by at least
20km, making frequent gene flow between them by pollen or seeds unlikely.
78
Allozyme markers
To screen for variation at allozyme loci, I collected tissue from the six
study populations and ground young leaf material in a modified buffer solution
from Soltis et al. (1983). The buffer was titrated with monobasic potassium
phosphate to bring the pH to 7.00, and the concentration of PVP was double that
in Soltis et al. (1983). Samples were stored at -80ºC until analysis. I screened
17 enzyme systems for polymorphism within and among populations and
detected polymorphism in four systems using the staining recipes in Soltis et al.
(1983) and 11.5% starch (Starch Art Inc.) in gel preparations. These systems
yielded six polymorphic loci, Phosphoglumutase (Pgm), Phosphoglucoisomerase
1 and 2 (Pgi1, Pgi2), Glutamate dehydrogenase (Gdh), and Acid Phosphatase 1
and 2 (Acp1, Acp2). I used gel and electrode buffer system 4 from Soltis et al.
(1983) to resolve Pgm and Gdh, and a modification of Soltis et al. (1983) system
6 to resolve Pgi and Acp enzymes. A representative gel shows the
polymorphism at the locus PGM (Fig. 5.1)
In June of 2004, I randomly sampled ten individuals from each population
by collecting whole plants at randomly determined points along a transect. Leaf
samples from these plants were genotyped for the six polymorphic loci for
analysis of population genetic structure.
Data analysis
I tested for Hardy-Weinberg equilibrium at each locus within each
populations using Fisher’s Exact Test. This test was carried out by randomly
permuting alleles at each locus within populations, and calculating the proportion
of resulting data sets with allele frequencies more extreme than the observed
data. To quantify genetic diversity within loci and populations, I calculated
observed and expected heterozygosities.
I quantified genetic structure for individuals within populations and among
populations with Wright's F-statistics. I estimated fIS, the correlation of alleles
within individuals within populations of all loci and populations, a measure of
inbreeding within populations, and FIT, the correlation of alleles within individuals
in the total sample, a measure of total inbreeding. I also calculated θ, the
79
unbiased multilocus estimator of FST (Weir 1996), to estimate the correlation of
alleles among individuals within populations.
In addition to estimating θ among populations, I also estimated θ between
populations of the same habitat type to determine if populations from the same
habitat type had diverged less than the overall level of population divergence.
The correlation of alleles among individuals within habitats is θhab. Including the
term for genetic structure at the habitat level, I estimated four F-statistics across
loci. fIS FIT, θhab, and θ.
To determine the statistical significance of multilocus values of fIS, FIT, θhab,
and θ, I used the bootstrap to calculate confidence intervals. With six loci, there
are fewer than 1000 possible unique combinations of loci, therefore I calculated
the confidence intervals with 700 replicates. To test the hypothesis that the
estimates of FST for each individual locus were different from zero, I used a
permutation method to determine statistical significance of differentiation among
populations at individual loci. I used the method described by Raymond and
Rousset (1995a) that simulates a Fishers Exact Test of differentiation among
populations for each locus.
To quantify the degree of neutral divergence between populations, I
estimated Nei's (1978) genetic distance between all pairs of populations based
on allozyme frequencies. These distances reflect divergence in assumed neutral
or nearly neutral characters, and this measure is commonly used as a measure
of the time since divergence. To provide a qualitative description of similarity in
allele frequency of the study populations, I constructed a phenogram using the
standard clustering method of unweighted pair-group method using arithmetic
averages (UPGMA).
I estimated correlations between adaptive and nonadaptive divergence
and two measures of reproductive isolation. Estimates of pairwise reproductive
isolation and adaptive divergence were available from previous studies.
Postmating/prezygotic isolation was calculated as one minus the ratio of the rate
of successful hand pollination of hybrid crosses and the rate of successful hand
pollination of purebred crosses (Coyne and Orr 1989). Positive values of this
80
metric indicate isolation and negative values indicate disassortative mating. A
value of zero indicates random pollination success and therefore no
postmating/prezygotic isolation. Postzygotic isolation was measured similarly as
one minus the ratio of the average fitness of hybrids and purebred progeny
grown in the field. Hybrid disadvantage relative to purebred progeny is indicated
by positive values, and negative values indicate hybrid advantage.
Fitness differences between populations in each planting site were
measured using Fitness Divergence. Fitness Divergence provides a single,
quantitative measure of the degree of divergence in relative fitness between a
pair of populations when reciprocally transplanted. Fitness Divergence is
calculated by summing the absolute value of the difference in relative fitness
between two populations in each environment. Relative fitness is standardized
by the fitness of the population with greater fitness in an environment. Fitness
Divergence was based on results of a reciprocal transplant experiment carried
out in 2002. I did not use data from a second experiment conducted in 2003
because those data are not independent of the data used to measure postzygotic
reproductive isolation. The 2003 experiment included progeny that were
genetically related to some of the plants used to quantify reproductive isolation in
the previous study.
I also estimated the correlation between both measures of reproductive
isolation and geographic distances between populations, calculated from
mapping software in the SAS statistical package (SAS v9 SAS Institute, Cary
NC). The software package GDA (Lewis and Zaykin 1998) was used to test for
Hardy-Weinberg equilibrium, to calculate heterozygosities, F-statistics, and
genetic distances, and to construct the population phenogram. The simulation of
the Exact Test of population differentiation was performed in Genepop v3.3
(Raymond and Rousset 1995b). I used the SAS statistical package (SAS
Institute, Cary NC) to estimate correlations within the procedure CORR.
Results
Population genetic structure
81
The results of Fisher's Exact Test within populations show that many
combinations of loci and populations violated assumptions of Hardy-Weinberg
equilibrium because of a deficiency of heterozygotes (Table 5.1). Consequently I
used genotype frequencies instead of allele frequencies to estimate θ throughout
(Weir 1996b).
I detected few alleles and little genetic diversity within populations for 5 of
the 6 loci. Most loci were segregating for two or three alleles (Table 5.1), and
levels of observed heterozygosity were low (Table 5.2). The average expected
heterozygosity was 0.21 and the average observed heterozygosity was 0.05, with
the exception of gdh for which observed heterozygosity was 0.21. Most loci were
characterized by a high frequency of one allele (Table 5.2). Large fIS and FIT
values for individual loci (Table 5.1) indicate that most individuals within
populations and in the overall sample were homozygous. The θ values for
individual loci were high for all loci except gdh. The average θ of the five other
loci was 0.48, and the value for gdh was 0.09. The lower θ of gdh resulted from
similar allele frequencies among all populations, whereas populations were fixed
for alternate alleles at other loci.
Just as there was little variation within loci, I detected little variation within
populations. Only Inland population 1 was polymorphic at all loci (Table 5.2).
Observed and expected heterozygosities were low, and fIS was high, indicating
that there was little genetic variation within populations, and that most individuals
were homozygous at most loci.
In contrast to limited variation within populations, there was variation
among populations as indicated by the high, significant θ estimated across all loci
(Table 5.1). When the analysis included habitat-level structure, the θ value
between populations from the same habitat was 0.14 (95% confidence interval
0.25, -0.03) and the overall value was 0.45 (95% confidence interval 0.59, 0.22).
Thus, populations from the same habitat tended to be more similar in allele
frequency than populations from different habitats.
Estimates of Nei's (1978) genetic distance varied between 0.03 and 0.57,
and populations from the same habitat type were not always separated by the
82
smallest genetic distances (Table 5.3). The smallest genetic distance (D = 0.03)
was between Sandhills population 2 and Dunes population 2, and the largest
distance (D = 0. 57) was between Sandhills population 1 and Dunes population
1. Given this pattern, it is not surprising that only the Inland populations were
ordered according to habitat type on the phenogram (Fig. 5.2).
Relationships between modes of divergence and modes of reproductive isolation
Despite variation in pairwise genetic distance, adaptive divergence, and
reproductive isolation, there was no significant relationship between either
measure of reproductive isolation and the two measures of divergence (Fig. 5.3).
Values of both modes of reproductive isolation varied from large negative values
to large positive values in the case of postmating/prezygotic isolation and Fitness
Divergence varied from 0.21 to 1.03. I also detected no significant relationship
between the geographic distance separating populations and
postmating/prezygotic isolation (ρ = 0.09, p = 0.75) or postzygotic isolation (ρ =
0.12, p = 0.68). The correlation between adaptive divergence and genetic
distance was weak and not significant (ρ = 0.04, p = 0.87), indicating that
adaptive and nonadaptive divergence were not correlated, and there was a trend
for a positive relationship between geographic distance and genetic distance (ρ =
0.46, p = 0.09).
Discussion
I detected little variation within the six populations of D. teres at allozyme
loci, but greater variation among populations. The small numbers of alleles
segregating within populations, low proportions of polymorphic loci within
populations, low expected heterozygosities, and high fIS, all suggest limited
genetic diversity within populations (Table 5.1). The high overall θ, in addition to
these measures indicates that populations are fixed for alternate alleles and that
there is little gene flow between them. The measures of genetic structure within
and among populations found here are in agreement with other published values
of other autogamous, annual, early successional, wide-ranging plants (Loveless
and Hamrick 1984).
83
The population structure I observed is favorable for evolution by natural
selection and genetic drift because there is little chance that gene flow between
populations will dampen the effects of either neutral divergence or divergent
selection. In addition, a selfing species will be able to found a population with
few individuals (Jain 1976), allowing the effects of founder events on subsequent
evolution. Levin (1976a) found similar structure among populations of Phlox
drummondii, a predominantly outcrossing species, but because the populations
had gone through many generations of biparental inbreeding, these cultivars
were likely to be highly inbred and populations may have been founded by a few
individuals.
My estimates of genetic differentiation among populations for individual
loci were generally in agreement inter se. The only locus for which population
structure was not significant was gdh, and in this test, differentiation was
marginally significant (p = 0.07). These results suggest that the number of loci
used to estimate population structure and genetic distance was adequate and
that adding additional loci would not fundamentally change the results. In
addition, the absence of a relationship between Fitness Divergence and genetic
distance suggests that differentiation among populations at allozyme loci was not
influenced by selection.
There was little structure between populations from the same habitat type
indicated by low genetic distances between them (Table 5.3), but populations
from the same habitat type were not always the most similar (Fig. 5.2). These
results along with the analysis of population structure (Table 5.2) and evidence of
local adaptation at the habitat level (Fig. 3.2), suggest that the founding of new
populations in different habitats is characterized by the effects of both genetic
drift and divergent selection. Populations of D. teres have been shown to be
adapted to different habitats (Jordan 1992), but given the variation between
populations of the same habitat type, the ability of some populations to maintain
high fitness at foreign sites as well as in their home sites, and the heterosis of
between population crosses, it is clear that adaptation in this species is
influenced by genetic drift as well as divergent selection.
84
Genetic drift through founder events or population bottle-necks could
influence evolution of these populations by limiting variation within populations,
and by fixing populations for alternate alleles. Some populations may lack the
genetic variation to adapt to their local habitat, while in other populations,
adaptation to the local environment could confer adaptation to other habitats as a
pleiotropic effect. Differential adaptation under this scenario would require the
effects of drift and divergent selection. Populations could adapt to different
habitats by divergent selection, while populations within a habitat type may
diverge as a result of founder events or population bottle-necks.
Thus
populations in similar habitats could evolve under a shifting balance process
where chance differences in frequencies of alleles that affect fitness lead to
differences in fitness between populations within a habitat. Effects of drift are
probably less important in divergence between populations from different
habitats, where selection will drive different alleles to fixation in populations
adapting to different habitats. There will be fitness peaks in the adaptive
landscape of population in similar habitats, but no peaks between populations
from different habitats. If it is shown that many systems are characterized by
population genetic structure similar to that shown here in which there is little
evidence of gene flow between populations regardless of the similarity of their
environments, and populations are also adapted to different habitats, the
controversy over the importance of selection and genetic drift in evolution (Coyne
et al. 1997; Wade and Goodnight 1998) may come to a logical compromise that
both influence adaptation to new environments.
Although I found evidence of reproduction isolation between some
population pairs, the relationships I observed between adaptive and nonadaptive
divergence and both postmating/prezygotic isolation and postzygotic isolation
were not significant. This result is surprising given the range of variation in
reproductive isolation and divergence, the lower pollination success in crosses
between populations from different habitats relative to populations from the same
habitats, and that a relationship between environmental variation and
reproductive isolation has been reported in another plant species (Montalvo and
85
Ellstrand 2001). Unlike some experimental studies that have not found
reproductive isolation between artificially selected lines or lines subjected to
founder events (Galiana et al. 1993; Rundle 2003), I found evidence of
reproductive isolation, but no measure of divergence was correlated with it. The
lack of a relationship between postzygotic isolation and either measure of
divergence is to be expected because postzygotic isolation was generally weak
relative to postmating/prezygotic isolation.
Given that I detected variation in divergence and reproductive isolation,
the lack of a relationship between divergence and isolation suggests that the
evolution of reproductive isolation between a pair of populations may not follow
an easily predictable path. One pair of populations that showed reciprocal
adaptation to their native sites, Dunes population 2 and Inland population 2
(Hereford and Winn 2005) had low rates of successful hand pollination when
crossed. Other pairs with evidence of postzygotic isolation such as Dunes
population 1 and Sandhills population 2, had only slight genetic distances
between them (Table 5.3). Sister species of plants span a range from strong
ecological similarity and little reproductive isolation, to weak ecological similarity
and strong isolation (Levin 2000). The processes that allow for variation between
sister species may also operate between populations of a single species,
resulting in the absence of a single consistent relationship between divergence
and isolation.
This study demonstrates that in its initial stages, the evolution of
reproductive isolation may not evolve as an inevitable direct consequence of
either adaptive or neutral divergence. Positive values of both measures of
reproductive isolation suggest that reproductive isolation can evolve, but this
isolation is not necessarily driven directly by adaptive or nonadaptive divergence.
Instead it seems that heterosis and inbreeding depression may overwhelm the
expression of postzygotic isolation Populations separated by the greatest
genetic distances like Dunes population 2 and Inland population 1 produced
hybrids that set more that twice the number of fruits as the parental populations
in both parental planting sites, despite reciprocal local adaptation between these
86
populations in one year and low fruit set on Inland population 1 plants at Dunes
population 2 in another year. Despite evidence for a negative associated
between habitat similarity and postzygotic isolation, the evolution of
postmating/prezygotic isolation seems have a positive association with
adaptation to different habitats (Fig 4.2). In more diverged taxa, it has been
shown that reproductive isolation increases with time (Edmands 2002) and that
reproductive isolation commonly evolves as a consequence of selection in
different environments (Schluter 1998; Rundle and Nosil 2005). Coyne and Orr
(2004) concluded that evolution of reproductive isolation was most often
associated with differences in selection, and that there was little evidence for an
effect of genetic drift, a conclusion in direct contrast to widely held views twenty
years earlier (Carson and Templeton 1984). My results show that the early
development of reproductive isolation may not be directly associated with either
form of divergence, and that the influences of adaptive and neutral divergence on
the evolution of reproductive isolation probably differ depending on the attributes
of the study populations and their native environments.
87
Table 5.1. Descriptive statistics of population genetic structure by locus and
population. "Allele/loci" column gives the number of alleles segregating at each
locus, or the proportion of loci that are polymorphic within each population.
Lower bound and upper bound refer to 95% confidence intervals of overall
estimates of F-statistics calculated by bootstrapping 700 replicates.
Locus/population
Obs.
het.
Exp.
het.
fIS
FIT
θ
pgm*
0.00
0.07
1.00
1.00
0.19
pgi1*
0.08
0.23
0.68
0.86
0.58
pgi2*
0.03
0.33
0.91
0.95
0.46
gdh
0.21
0.42
0.52
0.55
0.09
acp1*
0.00
0.08
1.00
1.00
0.69
acp2*
0.00
0.11
1.00
1.00
0.47
Inland 1
0.09
0.34
0.75
Inland 2
0.08
0.19
0.58
Sandhills 1
0.05
0.22
0.79
Sandhills 2
0.05
0.15
0.68
Dunes 1
0.02
0.21
0.92
Dunes 2
0.03
0.14
0.77
Lower bound of C.I.
0.60
0.70
0.22
Upper bound of C.I.
0.96
0.98
0.59
0.21
0.75
0.86
Multilocus
0.53
0.05
0.43
* indicates significant differentiation at the locus according to simulation of exact
test.
88
Table 5.2. Allele frequencies at each locus within each population.
Locus/Allele
ACP_1
1
2
3
ACP_2
1
2
3
GDH
1
2
PGI_1
1
2
3
PGI_2
1
2
3
PGM
1
2
IN 1
IN 2
SA 1
SA 2
DU 1
DU 2
0.90
0.00
0.10
1.00
0.00
0.00
1.00
0.00
0.00
1.00
0.00
0.00
0.20
0.80
0.00
1.00
0.00
0.00
0.90
0.00
0.10
0.00
1.00
0.00
0.60
0.40
0.00
0.00
1.00
0.00
0.00
1.00
0.00
0.00
1.00
0.00
0.65
0.35
0.75
0.25
0.25
0.75
0.75
0.25
0.75
0.25
0.60
0.40
0.00
0.60
0.40
0.30
0.70
0.00
0.30
0.70
0.00
1.00
0.00
0.00
0.40
0.00
0.60
0.20
0.00
0.80
0.20
0.80
0.00
0.85
0.15
0.00
1.00
0.00
0.00
0.60
0.00
0.40
0.40
0.00
0.60
0.20
0.00
0.80
0.70
0.30
1.00
0.00
1.00
0.00
1.00
0.00
1.00
0.00
1.00
0.00
89
Table 5.3. Estimates of Nei's (1978) genetic distance between all pairs of
populations. Habitat abbreviations are, IN = Inland, SA = Sandhills, DU = Dunes.
Distances in bold are between populations from the same habitat type.
IN1
IN2
SA1
SA2
DU1
IN2
SA1
SA2
DU1
DU2
0.13
0.33
0.12
0.28
0.10
0.28
0.55
0.31
0.57
0.14
0.36
0.21
0.38
0.03
0.14
90
Figure 5.1 A representative gel of the locus PGM. The lane that is marked
shows the "fast" allele in an individual native to Inland population 1. All other
lanes contain the “slow” allele.
91
Figure 5.2. Phenogram of the six study populations using allozyme loci and
obtained by cluster analysis (UPGMA).
92
Figure 5.3. Relationships between each of the two modes of reproductive
isolation, and the two measures of divergence. The Pearson correlation
coefficients are A = -0.05, B = 0.05,C = 0.20, and D = -0.47. Only the correlation
between postzygotic isolation and genetic distance (section D) was at least
marginally significant (p = 0.10).
93
CHAPTER 6
CONCLUSIONS
The finding that local adaptation did not always lead to reproductive
isolation between the study populations is not surprising when considering that
populations of the same species often occupy different environments yet all
populations of a species are usually not reproductively isolated. If there was a
stronger relationship between local adaptation and reproductive isolation every
population of a species that occurred in a different environment would be
reproductively isolated. Obviously, other forces besides selection are involved in
local adaptation and the evolution of reproductive isolation. Other forces such as
inbreeding or genetic drift could also influence the degree of local adaptation or
reproductive isolation, weakening the relationship between adaptation and
isolation. For example, the variation in the degree of local adaptation between
populations from the same habitat types and the high fitness of plants from the
Sandhills 1 source population at all planting sites (Table 3.5 and 3.6) suggests
that other forces such as founder effects or populations bottle-necks also
influence the degree of local adaptation. These forces may also influence how
the degree of local adaptation influences reproductive isolation. Other studies
have shown that local adaptation and reproductive isolation are not tightly
correlated. Galloway and Fenster (2000) and Fenster and Galloway (2000)
showed that in Chamaecrista fasciculata there was little evidence of local
adaptation, and the pattern of reproductive isolation was not related to the
pattern of local adaptation or the geographic distance between parents. They did
not find such wide-spread heterosis as I found in this study, though they did find
more heterosis in the F2 generation than in the F3 generation crosses. I did not
plant F3 crosses, thus I may not have detected as much isolation because
parental genomes had not recombined in the hybrids.
94
Heterosis in crosses between populations adapted to different
environments may be a means of maintaining the cohesion of a species in the
face of divergence due to selection or drift. If hybrids between a pair of
populations outperform purebreds, dissasortative mating will be favored and
populations may even coalesce into a single population. This type of mechanism
may explain why so many species are locally adapted (Linhart and Grant 1996;
Mopper and Strauss 1998; Schluter 2000) but not differentiated into different
species. Alternatively, populations may harbor genetic variation for adaptations
to new environments, but there may be little variation in traits that confer
reproductive isolation. Thus, populations could be locally adapted yet there
would be little reproductive isolation between them. It has been argued that
there should not be any variation in traits that confer reproductive isolation
because these traits will be selected against (Dobzhansky 1951). I did not have
the statistical power to quantify variation within populations in reproductive
isolation of hybrid crosses, but I found substantial variation among populations in
reproductive isolation. Though it will be difficult to study why populations have
not speciated, future studies should address the question of mechanisms of
species cohesion.
With the exception of studies of 'ring species' (Wake et al. 1996) most
studies of reproductive isolation within a single species focus on a pair of distinct
populations from extremely different environments (Coyne and Orr 2004). I
performed crosses between populations at varying degrees of divergence. In
this study I found that some pairs of populations were more locally adapted than
others and that some pairs of populations were reproductively isolated while
others were not. I also found that population pairs varied in nonadaptive
divergence. Surprisingly, there were no consistent relationships between local
adaptation and either mode of reproductive isolation or between reproductive
isolation and nonadaptive divergence. In a review of differences between sister
species of plants, Levin (2000) showed that species pairs were highly variable in
the degree of ecological divergence, neutral divergence and reproductive
isolation, where some pairs were very different ecologically, but with little
95
evidence of reproductive isolation, and other pairs were ecologically similar with
evidence of strong reproductive isolation. This pattern of variation may also exist
at the population level. There may not be distinct rules of speciation where with
a certain level of ecological adaptation or neutral divergence, we can expect a
given amount of reproductive isolation. Instead the degree of reproductive
isolation that evolves between a pair of populations probably depends on the
variation within the populations, and the attributes of their native habitat types.
96
REFERENCES
Aigner, P. A. 2004. Floral specialization without trade-offs: Optimal corolla flare in
contrasting pollination environments. Ecology 85:2560-2569.
Allard, R. W., G. R. Babbel, A. L. Kahler, and M. T. Clegg. 1972. Evidence for
Coadaptation in Avena-Barbata. Proceedings of the National Academy of
Sciences of the United States of America 69:3043
Antonovics, J., and R. B. Primack. 1982. Experimental Ecological Genetics in
Plantago .6. The Demography of Seedling Transplants of P Lanceolata.
Journal of Ecology 70:55-75.
Avise, J. C. 1993. Molecular markers, natural history & evolution. Chapman &
Hall, New York.
Barbour, M. G., J. H. Burk, and W. D. Pitts. 1987. Terrestrial Plant Ecology. The
Benjamin/Cummings Publishing Company Inc., Menlo Park, CA.
Barrett, S. C. H., and D. Charlesworth. 1991. Effects of a Change in the Level of
Inbreeding on the Genetic Load. Nature 352:522-524.
Belsley, D. A., E. Kuh, and R. E. Welsch. 1980. Regression diagnostics :
identifying influential data and sources of collinearity. Wiley, New York.
Bennington, C. C., and J. B. Mcgraw. 1995. Natural-Selection and Ecotypic
Differentiation in Impatiens-Pallida. Ecological Monographs 65:303-323.
Berglund, A. B. N., S. Dahlgren, and A. Westerbergh. 2004. Evidence for parallel
evolution and site-specific selection of serpentine tolerance in Cerastium
alpinum during the colonization of Scandinavia. New Phytologist 161:199209.
Bernatchez, L., J. A. Vuorinen, R. A. Bodaly, and J. J. Dodson. 1996. Genetic
evidence for reproductive isolation and multiple origins of sympatric
trophic ecotypes of whitefish (Coregonus). Evolution 50:624-635.
Bocher, T. W. 1949. Racial divergences in Prunella vulgaris in relation to habitat
and climate. New Phytologist 48:285-314.
Bone, E., and A. Farres. 2001. Trends and rates of microevolution in plants.
Genetica 112:165-182.
97
Burton, R. S. 1990. Hybrid Breakdown in Developmental Time in the Copepod
Tigriopus-Californicus. Evolution 44:1814-1822.
Carney, S. E., S. A. Hodges, and M. L. Arnold. 1996. Effects of differential pollentube growth on hybridization in the Louisiana irises. Evolution 50:18711878.
Carson, H. L., and A. R. Templeton. 1984. Genetic Revolutions in Relation to
Speciation Phenomena - the Founding of New Populations. Annual
Review of Ecology and Systematics 15:97-131.
Clausen, J., D. D. Keck, and W. M. Hiesey. 1940. Experimental studies on the
nature of species. I. Effect of varied environments on western North
America plants. Carnegie Institute of Washington, Washington, DC.
Cohan, F. M. 1984. Genetic-Divergence under Uniform Selection .1. Similarity
among Populations of Drosophila-Melanogaster in Their Responses to
Artificial Selection for Modifiers of Cid. Evolution 38:55-71.
Cox, G. W. 1978. Laboratory Manual of General Ecology. Wm. C. Brown
Company Publishers, Dubuque, IA.
Coyne, J. A., N. H. Barton, and M. Turelli. 1997. Perspective: A critique of Sewall
Wright's shifting balance theory of evolution. Evolution 51:643-671.
Coyne, J. A., and H. A. Orr. 1989. Patterns of Speciation in Drosophila. Evolution
43:362-381.
Coyne, J. A., and H. A. Orr. 2004. Speciation. Sinauer Associates, Sunderland,
Mass.
Crawford, T. J. 1984. What is a population? Pp. 135-173 in B. Shorrocks, ed.
Evolutionary Ecology. Blackwell Scientific, Oxford.
Cruzan, M. B. 1990. Variation in Pollen Size, Fertilization Ability, and
Postfertilization Siring Ability in Erythronium-Grandiflorum. Evolution
44:843-856.
Dobzhansky, T. G. 1937. Genetics and the origin of species. Columbia University
Press, New York,.
Doebeli, M., and U. Dieckmann. 2003. Speciation along environmental gradients.
Nature 421:259-264.
98
Dudley, S. A., and J. Schmitt. 1996. Testing the adaptive plasticity hypothesis:
density-dependent selection on manipulated stem length in Impatiens
capensis. American Naturalist 147:445-465.
Edmands, S. 1999. Heterosis and outbreeding depression in interpopulation
crosses spanning a wide range of divergence. Evolution 53:1757-1768.
Edmands, S. 2002. Does parental divergence predict reproductive compatibility?
Trends in Ecology & Evolution 17:520-527.
Effron, B. 1981. Censored data and the bootstrap. Journal of the American
Statistical Association 76:312-319.
Ehrlich, P. R., and P. H. Raven. 1969. Differentiation of Populations. Science
165:1228-&.
Emms, S. K., and M. L. Arnold. 1997. The effect of habitat on parental and hybrid
fitness: Transplant experiments with Louisiana irises. Evolution 51:11121119.
Etterson, J. R. 2004a. Evolutionary potential of Chamaecrista fasciculata in
relation to climate change. 1. Clinal patterns of selection along an
environmental gradient in the great plains. Evolution 58:1446-1458.
Etterson, J. R. 2004b. Evolutionary potential of Chamaecrista fasciculata in
relation to climate change. II. Genetic architecture of three populations
reciprocally planted along an environmental gradient in the great plains.
Evolution 58:1459-1471.
Falconer, D. S. 1952. The problem of environment and selection. American
Naturalist 86:293-298.
Felsenstein, J. 1981. Skepticism towards Santa Rosalia, or why are there so few
kinds of animals. Evolution 35:124-138.
Fenster, C. B., and L. F. Galloway. 2000. Population differentiation in an annual
legume: Genetic architecture. Evolution 54:1157-1172.
Fitzpatrick, B. M. 2002. Molecular correlates of reproductive isolation. Evolution
56:191-198.
Fry, J. D. 1993. The General Vigor Problem - Can Antagonistic Pleiotropy Be
Detected When Genetic Covariances Are Positive. Evolution 47:327-333.
Fry, J. D. 1996. The evolution of host specialization: Are trade-offs overrated?
American Naturalist 148:S84-S107.
99
Funk, D. J. 1998. Isolating a role for natural selection in speciation: Host
adaptation and sexual isolation in Neochlamisus bebbianae leaf beetles.
Evolution 52:1744-1759.
Futuyma, D. J., and T. E. Philippii. 1987. Genetic variation and covariation in
responses to host plants by Alsophila pometaria (Lepidoptera:
Geometridae). Evolution 41:269-279.
Galiana, A., A. Moya, and F. J. Ayala. 1993. Founder-Flush Speciation in
Drosophila-Pseudoobscura - a Large-Scale Experiment. Evolution 47:432444.
Galloway, L. F., and C. B. Fenster. 2000. Population differentiation in an annual
legume: Local adaptation. Evolution 54:1173-1181.
Gavrilets, S. 1999. A dynamical theory of speciation on holey adaptive
landscapes. American Naturalist 154:1-22.
Gavrilets, S. 2003. Perspective: Models of speciation: What have we learned in
40 years? Evolution 57:2197-2215.
Gross, B. L., N. C. Kane, C. Lexer, F. Ludwig, D. M. Rosenthal, L. A. Donovan,
and L. H. Rieseberg. 2004. Reconstructing the origin of Helianthus
deserticola: Survival and selection on the desert floor. American Naturalist
164:145-156.
Hare, J. D., and G. G. Kennedy. 1986. Genetic-variation in plant-insect
associations - survival of leptinotarsa-decemlineata populations on
solanum-carolinense. Evolution 40:1031-1043.
Hatfield, T. 1997. Genetic divergence in adaptive characters between sympatric
species of stickleback. American Naturalist 149:1009-1029.
Hawthorne, D. J., and S. Via. 2001. Genetic linkage of ecological specialization
and reproductive isolation in pea aphids. Nature 412:904-907.
Hereford, J., T. F. Hansen, and D. Houle. 2004. Comparing strengths of
directional selection: How strong is strong? Evolution 58:2133-2143.
Hereford, J., and K. S. Moriuchi. 2005. Variation among populations of Diodia
teres (Rubiaceae) in environmental maternal effects. Journal of
Evolutionary Biology 18:124-131.
Howard, D. J., and S. H. Berlocher. 1998. Endless forms : species and
speciation. Oxford University Press, New York ; Oxford.
100
Jain, S. K., and A. D. Bradshaw. 1966. Evolutionary Divergence among Adjacent
Plant Populations .1. Evidence and Its Theoretical Analysis. Heredity
21:407-&.
Johnston, M. O. 1991. Natural selection on floral traits in two species of lobelia
with different pollinators. Evolution 45:1468-1479.
Jordan, N. 1991. Multivariate-Analysis of Selection in Experimental Populations
Derived from Hybridization of 2 Ecotypes of the Annual Plant Diodia-Teres
W (Rubiaceae). Evolution 45:1760-1772.
Jordan, N. 1992. Path analysis of local adaptation in two ecotypes of the annual
plant Diodia teres Walt. (Rubiaceae). American Naturalist 140:149-165.
Kawecki, T. J. 1997. Sympatric speciation via habitat specialization driven by
deleterious mutations. Evolution 51:1751-1763.
Kawecki, T. J., and D. Ebert. 2004. Conceptual issues in local adaptation.
Ecology Letters 7:1225-1241.
Kearney, T. H., and R. H. Peebles. 1964. Arizona Flora. University of California
Press, Los Angeles.
Kindell, C. E., A. A. Winn, and T. E. Miller. 1996. The effects of surrounding
vegetation and transplant age on the detection of local adaptation in the
perennial grass Aristida stricta. Journal of Ecology 84:745-754.
Kruckeberg, A. R. 1951. Intraspecific variability in the response of certain native
plant species to serpintine soil. American Journal of Botany 38:408-419.
Lande, R. 1976. Natural-Selection and Random Genetic Drift in Phenotypic
Evolution. Evolution 30:314-334.
Lande, R., and S. J. Arnold. 1983. The Measurement of Selection on Correlated
Characters. Evolution 37:1210-1226.
Langerhans, R. B., and T. J. DeWitt. 2004. Shared and unique features of
evolutionary diversification. American Naturalist 164:335-349.
Langor, D. W., J. R. Spence, and G. R. Pohl. 1990. Host Effects on Fertility and
Reproductive Success of Dendroctonus-Ponderosae Hopkins (Coleoptera,
Scolytidae). Evolution 44:609-618.
101
Levene, H. 1960. Robust tests for equality of variances. Pp. 278-292 in I. Olkin,
ed. Contributions to Probability and Statistics. Stanford University Press,
Palo Alto, CA.
Levin, D. A. 1976. Consequences of Long-Term Artificial Selection, Inbreeding,
and Isolation in Phlox .1. Evolution of Cross-Incompatibility. Evolution
30:335-344.
Levin, D. A. 2000. The Origin, Expansion, and Demise of Plant Species. Oxford
University Press, New York Oxford.
Levitan, D. R. 2002. The relationship between conspecific fertilization success
and reproductive isolation among three congeneric sea urchins. Evolution
56:1599-1609.
Linhart, Y. B., and M. C. Grant. 1996. Evolutionary significance of local genetic
differentiation in plants. Annual Review of Ecology and Systematics
27:237-277.
Lorch, P. D., and M. R. Servedio. 2005. Postmating-prezygotic isolation is not an
important source of selection for reinforcement within and between
species in Drosophila pseudoobscura and D-persimilis. Evolution 59:10391045.
Lortie, C. J., and L. W. Aarssen. 1996. The specialization hypothesis for
phenotypic plasticity in plants. International Journal of Plant Sciences
157:484-487.
Macnair, M. R., and P. Christie. 1983. Reproductive Isolation as a Pleiotropic
Effect of Copper Tolerance in Mimulus-Guttatus. Heredity 50:295-302.
Mazer, S., and L. M. Wolfe. 1992. Planting density influences the expression of
genetic variation in seed mass in wild radish (Raphanus sativus L:
Brassicaceae). American Journal of Botany 79:1185-1193.
McCullagh, P., and J. A. Nelder. 1989. Generalized linear models. Chapman and
Hall.
McGuigan, K., C. E. Franklin, C. Moritz, and M. W. Blows. 2003. Adaptation of
rainbow fish to lake and stream habitats. Evolution 57:104-118.
McLachlan, G. J. 1992. Discriminant analysis and statistical pattern recognition.
Wiley, New York.
102
Mendelson, T. C. 2003. Sexual isolation evolves faster than hybrid inviability in a
diverse and sexually dimorphic genus of fish (Percidae : Etheostoma).
Evolution 57:317-327.
Mendelson, T. C., B. D. Inouye, and M. D. Rausher. 2004. Quantifying patterns
in the evolution of reproductive isolation. Evolution 58:1424-1433.
Mooney, H. A., and W. D. Billings. 1961. Comparative Physiological Ecology of
Arctic and Alpine Populations of Oxyria Digyna. Ecological
Monographs 31:1-29.
Montalvo, A. M., and N. C. Ellstrand. 2001. Nonlocal transplantation and
outbreeding depression in the subshrub Lotus scoparius (Fabaceae).
American Journal of Botany 88:258-269.
Moore, J. A. 1946. Incipient intraspecific isolating mechanisms in Rana pipiens.
Genetics 31:304-326.
Moore, J. A. 1949. Geographic variation of adaptive characters in Rana pipiens
Schreber. Evolution 3:1-24.
Mopper, S., and S. Y. Strauss. 1998. Genetic structure and local adaptation in
natural insect populations : effects of ecology, life history, and behavior.
Chapman & Hall, New York.
Moyle, L. C., M. S. Olson, and P. Tiffin. 2004. Patterns of reproductive isolation in
three angiosperm genera. Evolution 58:1195-1208.
Muller, H. J. 1942. Isolating mechanisms, evolution, and temperature. Biological
Symposia 6:71-125.
Nagel, L., and D. Schluter. 1998. Body size, natural selection, and speciation in
sticklebacks. Evolution 52:209-218.
Nagy, E. S. 1997. Selection for native characters in hybrids between two locally
adapted plant subspecies. Evolution 51:1469-1480.
Nagy, E. S., and K. J. Rice. 1997. Local adaptation in two subspecies of an
annual plant: Implications for migration and gene flow. Evolution 51:10791089.
Nosil, P., and B. J. Crespi. 2004. Does gene flow constrain adaptive divergence
or vice versa? A test using ecomorphology and sexual isolation in Timema
cristinae walking-sticks. Evolution 58:102-112.
103
Nosil, P., B. J. Crespi, and C. P. Sandoval. 2002. Host-plant adaptation drives
the parallel evolution of reproductive isolation. Nature 417:440-443.
Nosil, P., T. H. Vines, and D. J. Funk. 2005. Perspective: Reproductive isolation
caused by natural selection against immigrants from divergent habitats.
Evolution 59:705-719.
Peer, K., and M. Taborskyi. 2005. Outbreeding depression, but no inbreeding
depression in haplodiploid ambrosia beetles with regular sibling mating.
Evolution 59:317-323.
Presgraves, D. C. 2002. Patterns of postzygotic isolation in Lepidoptera.
Evolution 56:1168-1183.
Ramsey, J., H. D. Bradshaw, and D. W. Schemske. 2003. Components of
reproductive isolation between the monkeyflowers Mimulus lewisii and Mcardinalis (Phrymaceae). Evolution 57:1520-1534.
Rausher, M. D. 1992. The Measurement of Selection on Quantitative Traits Biases Due to Environmental Covariances between Traits and Fitness.
Evolution 46:616-626.
Raymond, M., and F. Rousset. 1995a. An exact test for population differentiation.
Evolution 49:1280-1283.
Raymond, M., and F. Rousset. 1995b. Genepop (Version-1.2) - PopulationGenetics Software for Exact Tests and Ecumenicism. Journal of Heredity
86:248-249.
Reznick, D. N., and C. K. Ghalambor. 2001. The population ecology of
contemporary adaptations: what empirical studies reveal about the
conditions that promote adaptive evolution. Genetica 112:183-198.
Rice, K. J., and R. N. Mack. 1991. Ecological Genetics of Bromus-Tectorum .3.
The Demography of Reciprocally Sown Populations. Oecologia 88:91-101.
Rice, W. R. 1984. Disruptive Selection on Habitat Preference and the Evolution
of Reproductive Isolation - a Simulation Study. Evolution 38:1251-1260.
Rundle, H. D., L. Nagel, J. W. Boughman, and D. Schluter. 2000. Natural
selection and parallel speciation in sympatric sticklebacks. Science
287:306-308.
Rundle, H. D., and P. Nosil. 2005. Ecological speciation. Ecology Letters 8:336352.
104
Sasa, M. M., P. T. Chippindale, and N. A. Johnson. 1998. Patterns of postzygotic
isolation in frogs. Evolution 52:1811-1820.
Schemske, D. W., and H. D. Bradshaw. 1999. Pollinator preference and the
evolution of floral traits in monkeyflowers (Mimulus). Proceedings of the
National Academy of Sciences of the United States of America 96:1191011915.
Schluter, D. 1995. Adaptive Radiation in Sticklebacks - Trade-Offs in Feeding
Performance and Growth. Ecology 76:82-90.
Schluter, D. 1998. Ecological causes of speciation. Pp. 114-129 in D. J. Howard
and S. H. Berlocher, eds. Endless forms. Oxford University Press, New
York; Oxford.
Schluter, D. 2000. The Ecology of Adaptive Radiation. Oxford University Press,
New York, NY.
Schmidt, K. P., and D. A. Levin. 1985. The comparative demography of
reciprocally sown populations of Phlox drummondii Hook. I. survivorships,
fecundities, and finite rates of increase. Evolution 39:396-404.
Sokal, R. R., and F. J. Rohlf. 1995. Biomery. W. H. Freeman and Company, New
York, NY.
Soltis, D. E., C. H. Haufler, D. C. Darrow, and G. J. Gastony. 1983. Starch-Gel
Electrophoresis of Ferns - a Compilation of Grinding Buffers, Gel and
Electrode Buffers, and Staining Schedules. American Fern Journal 73:927.
Stanton, M. L., and C. Galen. 1997. Life on the edge: Adaptation versus
environmentally mediated gene flow in the snow buttercup, Ranunculus
adoneus. American Naturalist 150:143-178.
Stanton, M. L., D. A. Thiede, and B. A. Roy. 2004. Consequences of intraspecific
competition and environmental variation for selection in the mustard
Sinapsis arvensis: Contrasting ecological and evolutionary perspectives.
American Naturalist 164:736-752.
Stinchcombe, J. R., M. T. Rutter, D. S. Burdick, P. Tiffin, M. D. Rausher, and R.
Mauricio. 2002. Testing for environmentally induced bias in phenotypic
estimates of natural selection: Theory and practice. American Naturalist
160:511-523.
Sumner, F. B. 1932. Genetic, distributional, and evolutionary studies of the
subspecies of deer mice (Peromyscus). Bibliographia Genetica 9
105
Swanson, W. J., and V. D. Vacquier. 2002. Reproductive protein evolution.
Annual Review of Ecology and Systematics 33:161-179.
Tilley, S. G., P. a. Verrell, and S. J. Arnold. 1990. Correspondence between
Sexual Isolation and Allozyme Differentiation - a Test in the Salamander
Desmognathus-Ochrophaeus. Proceedings of the National Academy of
Sciences of the United States of America 87:2715-2719.
Travisano, M., J. A. Mongold, A. F. Bennett, and R. E. Lenski. 1995.
Experimental Tests of the Roles of Adaptation, Chance, and History in
Evolution. Science 267:87-90.
van Tienderen, P. H. 2000. Elasticities and the link between demographic and
evolutionary dynamics. Ecology 81:666-679.
Verhoeven, K. J. F., T. K. Vanhala, A. Biere, E. Nevo, and J. M. M. Van Damme.
2004. The genetic basis of adaptive population differentiation: A
quantitative trait locus analysis of fitness traits in two wild barley
populations from contrasting habitats. Evolution 58:270-283.
Via, S. 1984a. The Quantitative Genetics of Polyphagy in an Insect Herbivore .1.
Genotype-Environment Interaction in Larval Performance on Different
Host Plant-Species. Evolution 38:881-895.
Via, S. 1984b. The Quantitative Genetics of Polyphagy in an Insect Herbivore .2.
Genetic Correlations in Larval Performance within and among Host Plants.
Evolution 38:896-905.
Via, S. 1991. The Genetic-Structure of Host Plant Adaptation in a Spatial
Patchwork - Demographic Variability among Reciprocally Transplanted
Pea Aphid Clones. Evolution 45:827-852.
Via, S., and R. Lande. 1985. Genotype-Environment Interaction and the
Evolution of Phenotypic Plasticity. Evolution 39:505-522.
Wade, M. J., and C. J. Goodnight. 1998. Perspective: The theories of Fisher and
Wright in the context of metapopulations: When nature does many small
experiments. Evolution 52:1537-1553.
Wake, D. B., and K. P. Yanev. 1986. Geographic-Variation in Allozymes in a
Ring Species, the Plethodontid Salamander Ensatina-Eschscholtzii of
Western North-America. Evolution 40:702-715.
Wang, H., E. D. McArthur, S. C. Sanderson, J. H. Graham, and D. C. Freeman.
1997. Narrow hybrid zone between two subspecies of big sagebrush
106
(Artemisia tridentata: Asteraceae) .4. Reciprocal transplant experiments.
Evolution 51:95-102.
Waser, N. M., and M. V. Price. 1985. Reciprocal Transplant Experiments with
Delphinium-Nelsonii (Ranunculaceae) - Evidence for Local
Adaptation. American Journal of Botany 72:1726-1732.
Waser, N. M., and M. V. Price. 1994. Crossing-distance effects in Delphinium
nelsonii: outbreeding and inbreeding depression in progeny fitness.
Evolution 48:842-852.
Weinig, C. 2000. Differing selection in alternative competitive environments:
Shade-avoidance responses and germination timing. Evolution 54:124136.
Weir, B. S. 1996a. Genetic Data Analysis II. Sinauer Associates, Sunderland,
Massachusetts.
Weir, B. S. 1996b. Intraspecific variation. Pp. 385-405 in D. M. Hillis, C. Moritz
and B. K. Mable, eds. Molecular Systemaitcs. Sinauer Associates, Inc.,
Sunderland, MA.
Welch, M. E., and L. H. Rieseberg. 2002. Patterns of genetic variation suggest a
single, ancient origin for the diploid hybrid species Helianthus paradoxus.
Evolution 56:2126-2137.
Willis, J. H. 1996. Measures of phenotypic selection are biased by partial
inbreeding. Evolution 50:1501-1511.
Winn, A. A. 2004. Natural selection, evolvability and bias due to environmental
covariance in the field in an annual plant. Journal of Evolutionary Biology
17:1073-1083.
Wolf, J. B., E. D. Brodie, and M. J. Wade. 2000. Epistasis and the evolutionary
process. Oxford University Press, Oxford ; New York.
Wright, S. 1931. Evolution in Mendelian populations. Genetics 16:97-159.
107
BIOGRAPHICAL SKETCH
Joe Hereford
Department of Biological Science
Florida State University
Tallahassee, Florida 32306-1100
[email protected]
A. Professional Preparation
University of New Orleans
Florida State University
Biology
Biology
B.S. 1997
Ph.D. 2005 (expected)
B. Publications
2004 Hereford, J., T. F. Hansen, and D. Houle. Comparing strengths of
directional selection: how strong is strong. Evolution 58:2133-2143.
2005 Hereford, J., and K. S. Moriuchi. Variation among populations of Diodia
teres in environmental maternal effects. Journal of Evolutionary Biology 18:124131.
C. Presentations at Professional Meetings
1998 “Population genetic structure in the seagrass Thalassia testudinum."
Florida Ecological and Evolutionary Symposium, Lake Placid, FL.
2002 "Environmental dependence of environmental maternal effects in an
annual plant, Diodia teres." Society for the Study of Evolution Annual Meeting,
Urbana, IL.
2003 "The contributions of adaptive divergence and genetic distance to
reproductive isolation between populations of an annual plant". Society for the
Study of Evolution Annual Meeting, Chico, CA.
2004 "Evolution of postmating/prezygotic isolation and postzygotic isolation in
the annual plant Diodia teres". Society for the Study of Evolution Annual Meeting,
Fort Collins, CO.
2005 "The relationship between population divergence and reproductive isolation
among six populations of the annual plant Diodia teres. Society for the Study of
Evolution Annual Meeting, Fairbanks AK.
108
D. Grants and Awards
2004 National Science Foundation Dissertation Improvement Grant
"Contributions of adaptation and genetic drift to reproductive isolation in an
annual plant". Award from the National Science Foundation to support
dissertation research $5430.00.
2003 Robert F. Godfrey award in support of graduate research in Botany at
Florida State University $500.00.
2003 Dissertation Research Award in support of graduate research at Florida
State University. $500.00.
E. Collaborators
Alice A. Winn, Florida State University Department of Biological Science,
Tallahassee, FL.
Ken S. Moriuchi, Florida State University Department of Biological Science,
Tallahassee, FL.
David Houle, Florida State University Department of Biological Science,
Tallahassee, FL
Thomas F. Hansen, Florida State University Department of Biological Science,
Tallahassee, FL.
F. Synergistic activities
1999 Organized the "Florida Ecological and Evolutionary Symposium", Lake
Placid, FL.
109