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Recurrent Loss of Sex Is Associated with Accumulation of
Deleterious Mutations in Oenothera
Jesse D. Hollister,*,1,2 Stephan Greiner,3 Wei Wang,1 Jun Wang,4 Yong Zhang,4 Gane Ka-Shu Wong,*,4,5
Stephen I. Wright,y,1 and Marc T.J. Johnsony,2
Department of Ecology and Evolutionary Biology, University of Toronto, Toronto, ON, Canada
Department of Biology, University of Toronto at Mississauga, Mississauga, ON, Canada
Max Planck Institute of Molecular Plant Physiology, Potsdam-Golm, Germany
BGI-Shenzhen, Beishan Industrial Zone, Shenzhen, Yantian District, China
Department of Biological Sciences and Department of Medicine, University of Alberta, Edmonton, AB, Canada
These authors cosupervised the project.
*Corresponding author: E-mail: [email protected]; [email protected]
Associate editor: Michael Purugganan
Sexual reproduction is nearly universal among eukaryotes. Theory predicts that the rarity of asexual eukaryotic species is
in part caused by accumulation of deleterious mutations and heightened extinction risk associated with suppressed
recombination and segregation in asexual species. We tested this prediction with a large data set of 62 transcriptomes
from 29 species in the plant genus Oenothera, spanning ten independent transitions between sexual and a functionally
asexual genetic system called permanent translocation heterozygosity. Illumina short-read sequencing and de novo
transcript assembly yielded an average of 16.4 Mb of sequence per individual. Here, we show that functionally asexual
species accumulate more deleterious mutations than sexual species using both population genomic and phylogenetic
analysis. At an individual level, asexual species exhibited 1.8 higher heterozygosity than sexual species. Within species,
we detected a higher proportion of nonsynonymous polymorphism relative to synonymous variation within asexual
compared with sexual species, indicating reduced efficacy of purifying selection. Asexual species also exhibited a greater
proportion of transcripts with premature stop codons. The increased proportion of nonsynonymous mutations was also
positively correlated with divergence time between sexual and asexual species, consistent with Muller’s ratchet. Between
species, we detected repeated increases in the ratio of nonsynonymous to synonymous divergence in asexual species
compared with sexually reproducing sister taxa, indicating increased accumulation of deleterious mutations. These
results confirm that an important advantage of sex is that it facilitates selection against deleterious alleles, which
might help to explain the dearth of extant asexual species.
Key words: evolution of sex, deleterious mutations, asexual reproduction, purifying selection, Muller’s ratchet.
Sexual reproduction is virtually ubiquitous among higher eukaryotes, despite significant costs in comparison to asexual
reproduction (Williams 1975; Maynard Smith 1978). The
rarity of asexual eukaryotes has spurred much debate on
the advantages of sexual over asexual reproduction
(Weismann 1889; Maynard Smith 1978; Bell 1982; Fisher
1930). Theory predicts that asexuality may reduce the efficacy
of natural selection, leading to accumulation of deleterious
mutations and/or stochastic loss of the fittest genotypic classes (Muller’s ratchet), and reducing genomic rates of adaptive
evolution (Muller 1964; Felsenstein 1974; Keightley and Otto
2006). Asexual lineages may therefore have higher extinction
risk compared with sexual populations (Lynch et al. 1993),
potentially explaining the rarity of asexual species.
Numerous theoretical studies support these predictions,
but empirical investigations have proved more difficult
(Paland and Lynch 2006; Neiman and Taylor 2009; Tucker
et al. 2013).
A potentially informative approach for testing these predictions is to compare the abundance of mildly deleterious
alleles in closely related sexual and asexual populations or
species (Neiman and Taylor 2009). A reduction in the efficacy
of selection is predicted to increase the proportion of mildly
deleterious alleles, such as nonsynonymous single nucleotide
polymorphism (SNPs), segregating within populations or accumulating as fixed differences between species (Gabriel et al.
1993). This approach has been adopted in a handful of systems, including microcrustaceans, mollusks, plants, and insects (Barraclough et al. 2007; Johnson and Howard 2007;
Neiman et al. 2010; Henry et al. 2012; Hersch-Green et al.
2012). However, previous studies have examined only a
small number of genes and/or taxa, and thus the genomewide effects of transitions to asexuality, and the timing over
which these changes occur, remain uncertain (Johnson and
Howard 2007; Neiman et al. 2010).
Ideally, a comparative investigation of this question would
make use of a system in which a loss of sex has occurred
ß The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution. All rights reserved. For permissions, please
e-mail: [email protected]
Mol. Biol. Evol. 32(4):896–905 doi:10.1093/molbev/msu345 Advance Access publication December 21, 2014
Asexuality and deleterious mutations . doi:10.1093/molbev/msu345
repeatedly in independent lineages over a range of timescales,
allowing for an evaluation of the time-dependent effect of
asexuality over multiple comparisons (Muller 1964; Gabriel
et al. 1993). Based on these criteria, the plant genus Oenothera
(Onagraceae) provides a powerful system for investigating the
evolutionary consequences of sex (Ranganath 2008; Johnson
et al. 2009). The ancestral state of the genus is sexual reproduction, and sexual species are typically highly outcrossing
(Stebbins 1950; Raven et al. 1979). However, a significant proportion (29%) (Johnson et al. 2011) of Oenothera species have
evolved to be functionally asexual due to a genetic system
called Permanent Translocation Heterozygosity (PTH). PTH
evolves through reciprocal chromosome translocations, and
is characterized by nearly complete suppression of recombination, and balanced lethal mutations that prevent segregation during meiosis. Combined with self-fertilization, PTH
results in functionally asexual individuals (i.e., progeny are
genetically identical to parents) that transmit two independently evolving haploid genomes (Cleland 1972; Rauwolf et al.
2008). Importantly, closely related PTH (hereafter asexual/
asex) and sexual Oenothera species typically have similar
life-history, morphology, and ecological requirements, and
in many cases they exhibit overlapping ranges. Moreover,
PTH and sexual species are typically diploid, whereas in
other systems asexuality is often associated with polyploidy.
Therefore, the PTH system allows us to test the consequences
of suppressed recombination and segregation specifically,
without confounding effects of ecological differences
(Ranganath 2008).
Here, we present the first genome-wide analysis of polymorphism and divergence at protein-coding genes from 29
species of Oenothera, spanning ten independent sexual/asexual transitions. This data set allows for an unprecedented
opportunity to examine the consequences of loss of sex, replicated across thousands of genes and a phylogeny. We test
the hypothesis that loss of sex is associated with reduced
efficacy of purifying selection, leading to an accumulation of
deleterious mutations in asexual lineages.
Results and Discussion
Sampling of Individuals and Short-Read Sequencing
We generated 62 leaf-tissue transcriptomes from 29
Oenothera species using Illumina short-read sequencing, encompassing at least ten independent transitions from sexual
to functionally asexual reproduction (fig. 1; supplementary
table S1, Supplementary Material online). On average, we sequenced 2.5 billion nucleotides per individual. For all sequenced individuals, we produced de novo assemblies of
mRNA transcripts. Individuals had on average 22,098 transcript assemblies of at least 300 nucleotides in length and 16.4
Mb of total assembled sequence, providing a genome-wide
view of genetic diversity in protein-coding genes. Our sample
included within- and among-species variation, allowing us to
investigate both the short-term (population genomic) and
long-term (phylogenomic) consequences of asexual
FIG. 1. Maximum likelihood phylogenetic tree of the sample of 62
Oenothera individuals. The tree is based on 1.9 Mb of concatenated
sequence from 939 orthologous loci for which outgroup data were
available from Chamerion angustifolium (Onagraceae). The node labels
(large font) 1–7 designate the seven monophyletic clades that are considered separately in subsequent analysis. Blue and red tip labels designate sexual and functionally asexual (PTH) Oenothera species,
respectively. The percentage of bootstrap replicates supporting the
tree topology above the species level (small font) are shown as follows:
*96–100%; 51–95%, listed; 50%, not shown. Further information about
each sample is available in supplementary table S1, Supplementary
Material online.
Patterns of Individual Heterozygosity
We first examined how transitions between sexual and asexual reproduction affected within-individual heterozygosity
and within-species polymorphism. Asexual species are expected to have elevated heterozygosity, both because nucleotide differences inherited from sexual progenitors are fixed in
a heterozygous state, and a lack of segregation prevents coalescence between founding alleles. Asexual Oenothera
Hollister et al. . doi:10.1093/molbev/msu345
species are self-fertilizing—a mating system that typically
leads to near-complete homozygosity because segregation
allows sampling of identical alleles from selfing individuals
(Nordborg 2000). However, if functional asexuality in
Oenothera prevents recombination and segregation across
much of the genome, we expect to observe high levels of
heterozygosity throughout the transcriptome of asexual
To genotype individuals, we selected a single reference
transcriptome from each of seven closely related clades in
our data set, with each clade containing one or more transitions between sexual and asexual reproduction (fig. 1). We
then mapped reads from all individuals within a given clade to
the reference, and called SNPs simultaneously for all individuals within each clade. We implemented a maximum-likelihood method to exclude SNPs resulting from spurious
mapping of reads from multiple transcripts to single reference
loci (See Materials and Methods) (Gayral et al. 2013).
Consistent with the expectation for asexuality, the
genome-wide proportion of heterozygous SNPs in asexual
individuals was on average nearly twice as high as in sexual
individuals (fig. 2a; 22% [n = 34] vs. 12% [n = 28]; t-test
P < 0.002). Our Oenothera sample also included within-species sampling of four independent sexual/asexual species pairs
(table 1), allowing us to examine the contribution of heterozygosity to within-species polymorphism. For each sexual/
asexual pair, we calculated FIT, a measure of the amount of
inbreeding within individuals relative to the total population
(table 1). For all comparisons, FIT was lower among asexual
species, reflecting a greater contribution of heterozygosity to
total polymorphism compared with sexual sister species.
Within-Species Polymorphism
If the evolution of asexuality is associated with an ongoing
reduction in the efficacy of natural selection to purge deleterious mutations, we expect to see an accumulation of segregating
polymorphisms (N) compared with synonymous polymorphisms (S) in asexual species. To test this prediction, we
annotated protein-coding regions within transcripts, and
compared the N/S ratio between four pairs of sexual/asexual sister species for which we had within-species sampling
(n = 3–5 per species; table 1). Transcriptome-wide, there was
a significantly higher N/S ratio in asexual species for three of
four comparisons (fig. 2b; clade 4 nonsignificant; clade
7 P < 103; clade 2 P < 1010; clade 3 P < 107). The single
exception was clade 4, which exhibits very low sexual/asexual
species divergence (0.01%), high inbreeding (FIT = 0.8), and
low effective population size (S = 0.0046 0.0003) in the
sexual species (Oenothera grandiflora). These patterns likely
reflect the fact that the sexual O. grandiflora is geographically
restricted in southeastern United States where it occurs in
small fragmented populations. By contrast, its asexual sister
taxon O. biennis occurs throughout eastern North America
(Dietrich et al. 1997).
Importantly, the observed increase in N/S was positively
correlated with the degree of sequence divergence between
sexual/asexual pairs (fig. 2b), and with levels of individual
heterozygosity as a proportion of segregating sites in the asexual species (supplementary fig. S1, Supplementary Material
online, R2 = 0.85 and 0.80 for divergence and heterozygosity,
respectively). As both measures provide rough estimates of
the age of the transition to asexual reproduction, these patterns are consistent with a time-dependent process in which
asexuality decreases the efficacy of selection against mildly
deleterious alleles (Charlesworth 2012). We acknowledge
that this pattern is based on a small number of comparisons
(N = 4) and additional sampling within and/or between species (see below) would strengthen this conclusion.
Furthermore, higher levels of heterozygosity in some asexual
lineages could in part be due to their hybrid origins (see
To further infer the extent to which higher N/S ratios in
asexual species reflect less efficient purifying selection, we fit a
model of the distribution of fitness effects of deleterious
nonsynonymous variants for the four sexual/asexual pairs,
using polymorphisms at 4-fold degenerate sites as a proxy
for neutral evolution. Consistent with the N/S patterns,
we inferred an increase in the proportion of effectively neutral
(NeS 1) nonsynonymous variants in asexual species compared with their sexual sister species for all comparisons
except clade 4 (supplementary fig. S2, Supplementary
Material online). This indicates that a significant proportion
of sites under efficient purifying selection in sexual clades are
effectively neutral in asexual lineages, consistent with less efficient purifying selection in the latter.
An additional prediction from the hypothesis of reduced
efficacy of selection in asexuals is that deleterious mutations
of large effect, such as premature termination codons, should
increase in abundance following loss of sex. Although such
mutations are under strong purifying selection in sexual populations, a lack of segregation in asexual species ensures that a
functional gene copy is consistently inherited, possibly leading
to relaxed selection against such mutations. To test this, we
quantified the occurrence of premature termination codons
as a proportion of polymorphic sites in sexual and asexual
species; this proportion was significantly higher in asexuals on
average (fig. 2c; P < 0.02). Taken together, these results are
consistent with a genome-wide reduction in the efficacy of
purifying selection and accumulation of deleterious nonsynonymous mutations in asexual species.
Between-Species Divergence
To investigate whether the observed reduction in the efficacy
of purifying selection translates into an accumulation of deleterious mutations across species, we determined orthology
for a core set of 4,081 de novo assembled transcripts across all
individuals in our data set. We aligned orthologs, inferred
locus-specific phylogenetic trees using maximum likelihood,
and estimated the ratio of nonsynonymous to synonymous
divergence between species (dN/dS = !) (Yang and Nielsen
1998). To account for possible heterogeneity in ! across the
Oenothera phylogeny, for example, from different levels of
divergence between asexual species and their sexual
Asexuality and deleterious mutations . doi:10.1093/molbev/msu345
FIG. 2. Heterozygosity, within-species variation, and between species divergence for sexual and functional asexual Oenothera species. (a) Barplot of
observed heterozygosity among sexual (blue; n = 28) and functionally asexual (red, n = 34) individuals. Horizontal lines denote median values; vertical
box lengths denote interquartile distances; whiskers denote 1.5 upper and lower quartile values. (b) The ratio of Watterson’s estimator of the
population mutation rate for nonsynonymous and synonymous sites (N/S) for four sexual/asexual pairs with within-species sampling. Here, clade 4
refers only to the monophyletic O. grandiflora and O. biennis individuals (see fig. 1). Inset: Plot of means and 95% confidence intervals on the N/S
difference as a function of divergence time between sexual/asexual pairs. The black dashed line shows the slope of the regression (R2 = 0.85). (c) Boxplot
showing the proportion of total polymorphic sites represented by premature termination codons in population genomic data from sexual and asexual
species. (d) Means and 95% confidence intervals of average ! ratios across loci for sexual and asexual species within each of seven clades. Clades are
separated by vertical dashed lines.
Table 1. Summary of Within-Species Data for Four Sexual/Asexual Species Pairs.
Species Pair (Sex/Asex)
Oenothera speciosa
Oenothera rosea
Oenothera filiformis
Oenothera gaura
Oenothera grandiflora
Oenothera biennis
Oenothera grandis
Oenothera laciniata
Total Filtered Coding Sequence
NOTE.—n, sample size; S-SNPs, synonymous SNPs; NS-SNPs, nonsynonymous SNPs; FIT, Wright’s Fixation Index; asexuals are given in underline.
progenitors, we fit a “free-ratio” model that estimated ! separately for each locus along each branch. We then compared
the distributions of ! along terminal branches leading to
sexual/asexual species separately for the seven clades.
In all seven clades, average ! across the 4,081 loci was
higher in asexual than sexual lineages, and this difference
was statistically significant for four of the seven comparisons
(fig. 2d; t-test clade 2: P < 1.0 106; clades 1 and 6: P < 0.04;
Hollister et al. . doi:10.1093/molbev/msu345
clade 5: P < 0.05). Although the P value of some of these tests
is not particularly low, the binomial probability of obtaining
four of seven tests with P less than 0.05 by chance is exceedingly unlikely (Binomial expansion test: P = 0.0002; Zar 1984).
Furthermore, when we focus on the three clades for which !
was not significantly elevated, and consider only those loci
that have accumulated a comparatively larger number of
mutations since divergence between asexual and sexual species (i.e., loci with dS 4 median across loci), we find that !
was also significantly elevated within asexual species in clades
3 and 7 (P < 0.05 and P < 0.03, respectively). Therefore, the
hypothesis of greater ! in asexual species is conditionally
supported within these clades as well. Importantly, ! was
never significantly elevated in a sexually reproducing species,
for either small or large divergence levels between species.
Oenothera species are diploid, therefore the evolution of
asexuality involves loss of chromosome segregation and recombination during meiosis. The observed increase in ! can
therefore be interpreted as fixation of heterozygous nonsynonymous changes captured in de novo transcript assemblies
from asexual species. It is important to distinguish this from
the familiar definition of fixation of homozygous mutations in
sexually reproducing diploids. Homozygous fixation in asexuals would require either gene conversion or recurrent mutation between nonrecombining (nonsegregating) gene
copes (Haag and Roze 2007).
Our phylogenetic analysis of ! is consistent with the patterns of within-species polymorphism (see above). This comparative analysis also suggests that the patterns of
polymorphism in asexual species did not result simply from
demographic effects, such as population bottlenecks associated with speciation. If bottlenecks drove our polymorphism
results, we would not expect the observed patterns of !,
because it considers mutations in individual genomes.
It is important to point out that the heterogeneity in !
among sampled Oenothera clades was often greater than the
difference between sexual/asexual sister taxa. This highlights
that our data do not imply a simple relationship between the
! ratio, reproductive system and extinction risk in the genus,
and clearly additional factors are influencing rates of molecular evolution. However, the consistent observation that
shifts to asexuality are associated with increased genomewide ! within a clade provides evidence for the theoretical
prediction of accumulation of deleterious mutations in asexuals (Muller 1964; Felsenstein 1974; Keightley and Otto 2006).
It also supports the corollary hypothesis that asexuality may
increase the risk of extinction relative to closely related sexually reproducing species (Lynch et al. 1993).
The Age of Asexual Lineages
Asexual lineages are hypothesized to be evolutionarily shortlived, which lends support to the hypothesis that fitness costs
of asexuality accrue over time. However, for the costs of asexuality to be apparent in Oenothera, asexual species must have
persisted long enough for such costs to manifest themselves
at the molecular level. For the four species pairs that had
within-species sampling (table 1), we obtained an
approximate age of asexual species by estimating the frequency of fixed differences in synonymous sites between
sister sexual/asexual species and published mutation rates.
Assuming a neutral mutation rate of 7.09 109 from
Arabidopsis (Ossowski et al. 2010), and a generation time
scaled to species’ life-history, point estimates for divergence
time between sexual and asexual lineages are 506,477, 8,906,
5,534, and 12,014 years for clades 2 (O. speciosa/O. rosea; perennial/annual), 3 (O. filiformis/O. gaura; biennial), 4 (O. grandiflora/O. biennis; biennial), and 7 (O. grandis/O. laciniata;
annual), respectively (figs. 1 and 2).
It must be emphasized that there is considerable uncertainty in estimates based on fixed differences. On the one
hand, these are estimates of divergence between sister species,
which is likely greater than the time since the evolution of
asexuality. On the other hand, in the absence of recurrent
mutations or gene conversion events between alleles, new
mutations can no longer fix in asexual lineages due to permanent heterozygosity, which will lead to a downward bias in
estimates of divergence (though mutations can still fix in
sexual species). These estimates also assume a constant generation time for species pairs, an assumption known to be
violated in O. speciosa (perennial)/O. rosea (annual).
Despite these caveats, our estimates do indicate that asexual lineages have likely evolved independently for thousands
to hundreds of thousands of generations, enough time for the
evolutionary consequences of asexual reproduction to
become apparent (Lynch et al. 1993). It is important, however,
to separate the consequences of asexuality in Oenothera from
the evolutionary processes that give rise to asexual species,
and we explore this issue below.
The Impact of Hybrid Origins on Sequence Variation
in Asexual Oenothera
The evolution of asexuality by hybridization of divergent genomes is widespread in both plants and animals (Rieseberg
1997; Simon et al. 2003). A hybrid origin introduces initial
allelic heterozygosity in asexual species. Additional heterozygosity accumulates in asexual lineages over evolutionary time,
leading to high levels of divergence between homologous loci
in ancient asexual lineages (Simon et al. 2003). Asexual
Oenothera species are heterozygous for two haploid
genome complexes that have accumulated reciprocal translocations within or between species (Cleland 1972). Because
recombination between translocated chromosomes is suppressed, they diverge from one another over time. Among
the functionally asexual Oenothera species we sampled, levels
of heterozygosity were nearly twice as high as in sexual individuals. Some proportion of heterozygous sites may have
arisen from allelic divergence after the evolution of asexuality,
but some may have been inherited from historical divergence
between parental genotypes (Levy and Levin 1975). Therefore,
it is important to consider whether hybrid origins, rather than
ongoing reductions in selection efficacy, could explain the
elevated rate of nonsynonymous substitution and polymorphism in asexual Oenothera species.
Asexuality and deleterious mutations . doi:10.1093/molbev/msu345
To explore the effect of hybridization, we took advantage
of the extensive prior knowledge of genome structure and
hybrid origins in subsection Oenothera (fig. 1; clade 4). Species
in this subsection are differentiated by three well-defined genomic complexes, designated A, B, and C (Stubbe 1964;
Dietrich et al. 1997). Sexual species are homozygous for AA
(O. elata, O. jamesii, O. longissima), BB (O. grandiflora), or CC
(O. argillicola). Asexual species are either hybrids of two divergent genomic complexes (intercomplex hybrids; e.g., O.
biennis, AB; O. parviflora, BC; O. oakesiana, AC) or are derived
from a single ancestral complex that has accumulated structural heterozygosity through chromosomal translocations (intracomplex hybrids; e.g., O. wolfii and O. villosa, AA; O. nutans,
BB) (Dietrich et al. 1997). Thus, we were able to explore the
impact of hybrid origin, and divergence between founding
genomes, on the proportion of nonsynonymous (relative to
synonymous) mutations in the sample of transcriptomes in
clade 4.
Among clade 4 asexual individuals, intercomplex hybrids
harbored twice as much genomic heterozygosity (mean
0.006/site/individual) as intracomplex hybrids (mean 0.003/
site/individual) at silent sites (P < 0.002). In contrast, intercomplex hybrids had a lower ratio of nonsynonymous to
synonymous heterozygosity than intracomplex hybrids
(mean 0.40 and 0.48, respectively, P < 0.04). Thus, asexual
species derived from more divergent parental genomes
have lower proportions of nonsynonymous variation compared with asexuals derived from more closely related parental genomes.
One limitation of the above analysis is that asexual species
exhibit patterns of variation that reflect their genetic origins
as well as subsequent sequence evolution. To infer the unique
impact of historical divergence between parental genomes on
the proportion of nonsynonymous variants in a hybrid (asexual) genome, we utilized polymorphism data from sexual
Oenothera species in clade 4. We simulated “hybrids” between
O. elata ssp. hookeri cv. Johansen Standard (JS), which has the
putative ancestral genomic complex (Cleland 1972), and
other sexual Oenothera species in clade 4, each of which
varies in genomic divergence from JS (fig. 1; see Materials
and Methods). In these simulated hybrid genomes, the ratio
of nonsynonymous to synonymous heterozygosity was
strongly negatively correlated with the homozygous divergence between “parental” genomes (r = 0.91, P < 0.001;
supplementary fig. S3, Supplementary Material online).
Taken together, these analyses indicate that hybridization
per se does not increase the ratio of nonsynonymous to synonymous polymorphism, but rather decreases it, particularly
in hybrid crosses of more divergent genotypes.
There is a straightforward explanation for why this should
be the case. Most heterozygous sites in hybrid individuals are
derived from high frequency polymorphisms, or fixed differences, that have accumulated between parental populations
or species. Because purifying selection generally prevents fixation of nonsynonymous variants and constrains them to low
frequencies, hybrid individuals generated from divergent parents are expected to initially have a reduced ratio of nonsynonymous to synonymous polymorphism at heterozygous
sites. Hybrids derived from more closely related parents, conversely, combine more recent mutations (i.e., low-frequency
polymorphisms), and therefore have higher proportions of
nonsynonymous mutations (Mugal et al. 2014). Thus, to
the extent that hybridization drives the relationship between
polymorphism and N/S ratios among clades, we would
expect the opposite pattern to what we actually observed
(fig. 2b and supplementary fig. S1, Supplementary Material
In contrast, a time-dependent increase in the proportion
of deleterious alleles within asexual species is predicted under
Muller’s ratchet or a “mutational meltdown” model (Muller
1964; Lynch et al. 1993). We see such a time-dependent correlation among asexual Oenothera species (fig. 2 and supplementary fig. S1, Supplementary Material online), which
suggests that the increased proportion of nonsynonymous
variation is driven by suppressed recombination and segregation associated with asexuality, rather than by hybridization
or other demographic influences. Genome-wide linkage in
asexuals likely decreases the efficacy of purifying selection
on deleterious mutations due to interference among mutations. However, a reduction in the strength of selection may
also contribute, because asexual species are permanently heterozygous, which could mask the deleterious effects of partially recessive mutations.
Asexuality and Heterozygous Mutation Accumulation
We compared pN/pS ratios spanning four sexual-asexual
transitions. These comparisons indicate increased accumulation of deleterious mutation in asexuals, compared with sister
sexual species. Although we did not sample multiple individuals for all Oenothera species in our data set, we were able to
compare the ratio of heterozygosity at nonsynonymous compared with synonymous sites (hN/hS) for all individuals and
species. Because of a loss of recombination and segregation,
diploid asexual species are predicted to accumulate deleterious heterozygous mutations over a large range of evolutionary parameters. To what extent this leads to decline in mean
fitness in asexuals depends in important ways on the distribution of dominance coefficients, population size, and population structure (Haag and Roze 2007). In addition, as shown
above, the impact of phylogenetic relationships and diversity
at silent sites could also have strong effects on levels of
nonsynonymous diversity (heterozygosity).
To evaluate evidence for accumulation of deleterious heterozygous mutations among all sampled Oenothera individuals, we fit a linear model testing the effects of clade, mode of
reproduction (asexual vs. sexual), and silent site heterozygosity (hS) on nonsynonymous heterozygosity (hN). This model
also took into account the interaction between clade and hS.
Overall, the model explained greater than 99% of the variance
in hN (adjusted R2 = 0.9927; P << 1010) across individuals.
Taking into account the effects and interaction of hS and
clade, sexual reproduction was significantly negatively correlated with hN (P < 0.00005). Thus, patterns of heterozygosity
across the full data set further support the theoretical
Hollister et al. . doi:10.1093/molbev/msu345
prediction that asexuality is associated with accumulation of
deleterious mutations.
Theoretical work predicts that interference among linked mutations may select for increased meiotic recombination and
segregation associated with sexual reproduction (Felsenstein
1974; Barton and Otto 2005; Keightley and Otto 2006;
Agrawal 2009). These studies further indicate that genomewide linkage in asexual populations can cause reduced effective population size, allowing deleterious mutations to rise to
high frequencies and fix within populations (Muller 1964;
Charlesworth 2012). Our analyses confirm the prediction of
accumulation of deleterious mutations following repeated
loss of sex in the genus Oenothera. These results were
robust and consistent at both microevolutionary and macroevolutionary time-scales. Given these patterns, repeated loss
of sex in this genus likely results from short-term benefits of
reproductive assurance (Baker 1955; Barrett 2002), whereas
the ephemerality of functionally asexual reproduction
(Johnson et al. 2011) may be associated with the long-term
costs of accumulating deleterious mutations. This work provides strong genome-wide support for the link between asexuality and mutation accumulation (Muller 1964; Lynch et al.
1993), providing a plausible mechanism underlying the rarity
and ephemerality of asexual eukaryotic lineages due to increased extinction risk.
Materials and Methods
Plant Materials
All plant material used in the experiment originated from
seeds taken from either natural populations or previous experiments. We sampled a total of 62 individual plants from 32
species and subspecies (supplementary table S1,
Supplementary Material online). These species were sampled
to maximize phylogenetic diversity across the Oenothera
genus, where the functionally asexual PTH genetic system is
most prevalent (Raven 1979; Wagner et al. 2007; Johnson et al.
2009). Our sampling included 32 individuals known to have a
PTH genetic system (referred to as “asexual species” hereafter)
and 30 individuals that are non-PTH (referred to as “sexual
species” hereafter). This sampling represented a minimum of
ten independent transitions between sexual and functionally
asexual reproduction (see fig. 1). To gain insight into population genomic patterns of evolution in sexual and asexual
species, we sampled one individual from each of 3–5 populations in each of eight species. These eight species represented four pairs of closely related sexual and asexual
species. The species’ names, genetic system, and the origin
of samples are included in supplementary table S1,
Supplementary Material online. Seed material and vouchers
for all samples studied here are curated by M.T.J.J.
Germination, Plant Growth, and Tissue Harvesting
Seeds were initially soaked at 4 C for 24 h in 1 ml of 0.1%
agarose solution in filtered water. All species except those
with indehiscent fruits (i.e., O. gaura, O. filiformis, O. suffulta)
were then germinated for 1–3 weeks in 55-mm Petri dishes
on wet filter paper left in a windowsill exposed to full sun for
3–4 h per day. Plant species with indehiscent fruits were
planted directly 1 cm below the soil surface and placed
under fluorescent lights at room temperature until seedlings
emerged. Upon germination, seedlings were transplanted individually into plastic plug tray wells containing approximately 150 ml of steam-sterilized potting soil (Ready Earth
Seedling and Plug Soil Mixture, Sungro Horticulture,
Bellevue, WA). We grew all plants in a single reach-in
growth chamber set to a 16 h (25 C):8 h (20 C) light:dark
cycle. Lighting was provided by fluorescent and incandescent
bulbs at an intensity of 450 mol m2 s1. Plants were watered ad libitum with deionized water containing a one-third
strength standard Hoagland’s solution that provided macroand micronutrients. We harvested the fourth true leaf from
each plant, which we excised when it reached one-quarter of
the size of the first true leaf. This criterion insured that all
plants were harvested at the same developmental stage, and
at a time when the leaf was undergoing active growth and
development. Leaves were flash-frozen and stored at 80 C
until extraction of total RNA. For details on Oenothera cultivation, see Greiner and K€ohl (2014).
RNA Extraction
Total RNA was extracted using a hybrid CTAB/acid phenol/
silica membrane method developed specifically for this project. The full details of our extraction protocol are available in
Johnson et al. (2012) (see protocol 9), and we provide a brief
summary here. We extracted total RNA from 50 to 100 mg of
tissue per sample. Tissue was first ground to a powder with a
prechilled mortar and pestle sterilized with RNase Zap
(Ambion, Austin, TX). CTAB extraction buffer (2–5 ml) was
added and mixed with the sample in the mortar. We then
transferred the sample to a 15-ml tube, vortexed the sample
(15 s), and incubated it at 65 C (15 min). The sample was
centrifuged at 14,000 g (3 min) and the supernatant was
transferred to a new 2-ml tube. An equal volume of 24:1
chloroform:isoamyl alcohol was added to each sample, vortexed, and then centrifuged at 14,000 g (3 min). The upper
aqueous phase was transferred to a new 2-ml tube and the
solvent extraction was repeated. After transferring the upper
aqueous phase to a new tube we added an equal volume of
5:1 phenol:chloroform (pH 4.5), followed by vortexing and
centrifugation at 14,000 g (3 min). The upper aqueous
phase was moved to a new 2-ml tube and the phenol:chloroform solvent extraction was repeated multiple times until
we saw a clear boundary layer between the upper and lower
aqueous solutions (usually 2–3 extractions). After transferring
each sample to a new tube we added an equal volume of 24:1
chloroform:isoamyl alcohol, vortexed the sample, and then
centrifuged samples at 14,000 g (3 min). The upper aqueous phase was transferred to a new tube and the extraction
was repeated once. After estimating the volume of each
sample, 0.5 volumes of RLT solution from the Qiagen
RNeasy Plant minikit (Qiagen, Valencia, CA) was added to
each sample and thoroughly mixed by inversion. We
Asexuality and deleterious mutations . doi:10.1093/molbev/msu345
precipitated RNA by adding 0.5 volumes of 95% EtOH followed by inverting tubes. Total RNA was bound to a silica
membrane by transferring the entire sample to a pink Qiagen
miniRNA spin column followed by centrifugation at 8,000 g
(15 s). We added 350 l of RW1 buffer wash to the spin
column and centrifuged the column at 8,000 g (15 s).
DNase solution (80 l) was applied to the membrane and
incubated (15 min), followed by repeating the RW1 wash
step. The column was then washed with 500 l of RPE and
centrifuged at 6,300 g (15 s); this step was repeated once.
After additional centrifugation to remove all remaining liquid
from the column we added 44 l of RNase-free water onto
each column. Samples were incubated for 1 min to allow RNA
to fully dissolve before centrifuging samples at 6,300 g into
a RNase free tube. The purity of total RNA was assessed on a
NanoDrop 8000 spectrophotometer (Thermo Scientific,
Wilmington, DE). The concentration, integrity, and yield of
total RNA were determined by assaying 1 l of diluted total
RNA on an Agilent 2100 Bioanalyzer using the Plant RNA
Nano or Pico chip following the manufacturer’s instructions
(Agilent Technologies, Sanata Clara, CA).
Transcriptome Library Preparation and Sequencing
Library preparation and sequencing were performed by BGIShenzhen (Shenzhen, China) as part of the One Thousand
Plants Consortium project (1KP,, as well as
at North Carolina State University’s (NCSU) Genomic
Sciences Lab (see supplementary table S1, Supplementary
Material online). The methods for library preparation and
sequencing at BGI were previously published and are briefly
described below.
For the BGI samples, we isolated polyA mRNA from 20 mg
of total RNA using the Dynabeads mRNA Purification Kit (Life
Technologies, Grand Island, NY). mRNA was fragmented to a
size of 200–300 nt using the RNA Fragmentation Reagents kit
(Life Technologies). The first and second strands of cDNA
were synthesized using reverse transcriptase and DNA polymerase, respectively. Double-stranded DNA strands were
then isolated using a QIAquick PCR Purification Kit
(Qiagen), followed by end-repair and the addition of a 30
adenosine (A base). The Illumina PE Adapters were ligated
onto the A base and polymerase chain reaction (PCR) amplified for 15 cycles using “indexed” paired-end PCR primers.
Successful library preparation was confirmed by gel electrophoresis and the transcriptome library was further sizeselected by gel-purification to ensure that insert size was
200 bp and total fragment size was 322 bp. The NCSU samples
were prepared in a similar manner except that we used the
Illumina TruSeq RNA protocol to isolate mRNA and we only
attached single-read adapters.
All paired-end sequencing was performed on the Illumina
HiSeq platform. We sequenced samples as paired-ends at a
read length of 90 bp with up to 11 samples multiplexed into
each lane of the flow cell. Single-read sequences at NCSU were
performed on the Illumina GAIIx platform at a read length of
72 bp with one sample per lane. Prior to assembly, sequences
were filtered to minimize contamination and maximize
sequence quality. All sequences met the following four criteria: 1) Contained a unique indexed barcode, 2) less than 15 bp
of continuous adapter sequence, 3) less than 0.5% uncalled
bases on either paired read, and 4) 80% of bases with a Qscore of greater than 10 in both paired reads.
De Novo Assembly of Transcripts
Illumina reads from each individual were assembled using
Velvet Oases (Schulz et al. 2012). VelvetOptimiser version
2.2.4 (Zerbino and Birney 2008) was invoked to choose the
best kmer size (between 27 and 61 nt) for each individual
transcript. To avoid missing low coverage transcripts, the
final total number of bases in each assembly was used to
evaluate the best kmer size. Oases version 0.2.08 was run
under default parameters. For each set of transcript isoforms,
the longest was chosen as the final transcript.
Read-Mapping and Genotyping
To genotype individuals, a single reference transcriptome was
chosen from among the individuals in each clade (1–7) on the
basis of N50 contig length. Reads from all individuals in each
clade were then mapped using Stampy version 1.0.21 (Lunter
and Goodson 2011). Postmapping alignment processing was
performed using Picard Tools version 1.4.8 (http://picard. Regions surrounding insertion/deletion
polymorphisms were realigned, and genotyping was performed using the Genome Analysis Tool-Kit version 2.39
(McKenna et al. 2010). For all read-mapping and genotyping
steps, default program settings were used.
Base-Call Filtering
After genotyping, sites were filtered for genotype quality (genotype likelihood ratios 60 for all individuals) and read coverage (10 for all individuals). We implemented an
additional filter following Gayral et al. (2013). This filter is
based on a likelihood ratio test of a single-locus versus twolocus model, where the former represents the likelihood that
reads from the same orthologous locus in all individuals map
to a single reference locus, and the latter represents the likelihood that reads from multiple loci map to a single reference
locus. The two-locus scenario might take place when, for
example, reads from two closely related duplicate genes are
assembled as a single locus in the reference transcriptome.
This would result in excess heterozygosity (compared with
Hardy–Weinberg expectations) for a given allele frequency,
and uneven representation of segregating bases among reads
mapping to the site. We filtered sites that were a significantly
better fit to the two-locus model, and filtered entire contigs
for which 50% of SNPs were a significantly better fit. Models
were fit using data from the outcrossing samples only, because asexual species are not expected to fit Hardy–Weinberg
expectations even in the absence of paralogous mapping. The
majority of sites filtered using this method were fixed heterozygotes in both sexual and asexual samples. The results presented above were qualitatively equivalent considering data
filtered only by sequencing depth and genotype quality
Hollister et al. . doi:10.1093/molbev/msu345
Genomic Analysis
Open reading frames (ORFs) were identified using the getorf
program from the EMBOSS suite version 6.3.1. For all loci in all
individuals, a custom PERL script was used to identify synonymous and nonsynonymous variants from the read-mapped
data (available upon request). Per-site estimates of the
number of synonymous and nonsynonymous polymorphisms were calculated following Li (1993). For comparison
of within-species polymorphism, we restricted analysis to subsets of within-species samples that were monophyletic with
respect to sister taxa (see fig. 1 and table 1). Statistical analysis
was performed using R version 3.0. Watterson’s was estimated following Tajima (1989) using a custom R function. We
estimated the distribution of fitness effects of nonsynonymous variants using the method of Eyre-Walker and
Keightley (2009), implemented in a PERL script kindly provided by Dan Halligan. For this analysis, we used 4-fold degenerate positions as a proxy for neutrally evolving sites.
Ortholog Identification
We identified orthologous genes within each clade separately
by all-against-all BLASTn searches among individuals within a
clade. We subsequently identified orthologs across clades by
all-against-all BLASTn searches using reference assemblies
from each clade. We filtered loci by BLAST e value 1010
and reciprocal best hits over 80% of sequence length. In
total, we assigned orthology for 4,081 genes.
Sequence Alignment
Orthologous sequences were compiled into single fasta-formatted files. All alignments were done using the program
MUSCLE version 3.8.31 (Edgar 2004). For dN/dS analysis,
ORFs were aligned for each ortholog set. ORF alignments
were then used to guide in-frame alignments of nucleotide
Phylogenetic Analysis
Maximum-likelihood phylogenetic trees were estimated using
RaXML version 7.0.4 (Stamatakis 2006), using default settings
(including substitution model = GTRGAMMA). For the phylogeny presented in figure 1, we identified orthologous transcripts by all-against-all BLASTp of the Oenothera ortholog set
against a de novo assembly of Chamerion angustifolium (produced using the same methods as above). We identified 939
orthologous outgroup loci. Orthologs and outgroup sequences were aligned as above. Alignments were concatenated into a single large alignment 1,976,729 nucleotides in
length. The phylogeny for the sample was estimated based on
the concatenated sequence. For each ORF-guided ortholog
alignment, a separate maximum-likelihood tree was also
! (dN/dS) Analysis
Individual gene trees were used as input for ! analysis in
PAML version 3.4 (
paml.html; last accessed December 26, 2014). For each of
the 4,081 orthologs, we fit a “free-ratio” model, in which a
separate ! value was estimated for every branch on the
phylogeny. For the free-ratio model, branch-specific !
values from the PAML output file were extracted and reformatted to conform to Newick format and read into R using
the “phytools” package (Revell 2012). Terminal branch
values were then extracted in R, and values across loci recorded for each individual. We restricted the analysis to loci
with ! ratios 2, to examine patterns among genes under
selective constraint.
Simulation of Hybrid Genomes
To simulate the initial effect of hybridization on the ratio of
nonsynonymous to synonymous heterozygous mutations, we
simulated hybridization between O. elata (JS) and all other
sexual clade 4 Oenothera species (fig. 1). The proportion of
genotypic differences at nonsynonymous and synonymous
sites was calculated, simulating the proportion of nonsynonymous and synonymous heterozygous sites (hN/hS) that
would initially be present in a hybrid between O. elata (JS)
and other (clade 4) sexual species with varying levels of divergence. The hN/hS ratio was evaluated as a function of hS
across all simulated hybrids (supplementary fig. S3,
Supplementary Material online).
Supplementary Material
Supplementary figures S1–S3 and table S1 are available at
Molecular Biology and Evolution online (http://www.mbe.
The authors thank H. Myburg for performing RNA extractions, the NC State Phytotron staff for providing technical and
infrastructural support, L. Yaneva-Roder and E. Carpenter for
providing technical and logistical support, and M. Deyholos
for outgroup data. A.A. Agrawal, K. Krakos, R. Raguso, and the
Ornamental Plant Germplasm Centre provided seed material.
This work was supported by an NSERC Discovery Grant, NSF
(DEB-0919869), the Early Researcher Award (Ontario
Government), the Ontario Research Fund, the Canadian
Foundation for Innovation to M.T.J.J.; Alberta Enterprise
and Advanced Education, Genome Alberta, Alberta
Innovates Technology Futures iCORE, Musea Ventures, and
BGI-Shenzhen to G.K.S.W.; the Max Planck Society and the
Deutsche Forschungsgemeinschaft to S.G.; and an NSERC
Discovery Grant and an NSERC Accelerator Award to S.W.
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