Download DNA Diversity in Sex-Linked and Autosomal Genes of the Plant

DNA Diversity in Sex-Linked and Autosomal Genes of the Plant Species
Silene latifolia and Silene dioica
Dmitry A. Filatov,*1 Valerie Laporte,* Clementine Vitte,† and Deborah Charlesworth*
*Institute of Cell, Animal, and Population Biology, University of Edinburgh, Edinburgh, Scotland; and †École Normale
Supérieure, Paris, France
The relatively recent origin of sex chromosomes in the plant genus Silene provides an opportunity to study the
early stages of sex chromosome evolution and, potentially, to test between the different population genetic processes
likely to operate in nonrecombining chromosomes such as Y chromosomes. We previously reported much lower
nucleotide polymorphism in a Y-linked gene (SlY1) of the plant Silene latifolia than in the homologous X-linked
gene (SlX1). Here, we report a more extensive study of nucleotide diversity in these sex-linked genes, including a
larger S. latifolia sample and a sample from the closely related species Silene dioica, and we also study the diversity
of an autosomal gene, CCLS37.1. We demonstrate that nucleotide diversity in the Y-linked genes of both S. latifolia
and S. dioica is very low compared with that of the X-linked gene. However, the autosomal gene also has low
DNA polymorphism, which may be due to a selective sweep. We use a single individual of the related hermaphrodite
species Silene conica, as an outgroup to show that the low SlY1 diversity is not due to a lower mutation rate than
that for the X-linked gene. We also investigate several other possibilities for the low SlY1 diversity, including
differential gene flow between the two species for Y-linked, X-linked, and autosomal genes. The frequency spectrum
of nucleotide polymorphism on the Y chromosome deviates significantly from that expected under a selective-sweep
model. However, we detect population subdivision in both S. latifolia and S. dioica, so it is not simple to test for
selective sweeps. We also discuss the possibility that Y-linked diversity is reduced due to highly variable male
reproductive success, and we conclude that this explanation is unlikely.
Introduction
Heteromorphic sex chromosomes have evolved independently many times (Bull 1983), and the properties
of sex chromosomes are very similar, suggesting that
similar evolutionary processes have operated in the evolution of sex chromosomes in different groups of organisms. The ancient sex chromosome systems of mammals
and Drosophila species are characterized by the absence
of recombination and exhibit genetic degeneration (loss
of functional copies of most Y-chromosome loci; see
Lucchesi 1978). The chief reason for Y-chromosome genetic degeneration is thought to be low efficacy of selection in nonrecombining regions. This could be manifested as slow adaptive evolution of Y-linked genes
compared with genes on the X chromosome (Orr and
Kim 1998) and as reduced effectiveness of purifying
selection, causing accumulation of deleterious mutations
and gradual degeneration of the Y chromosome. It is
thought that accumulation of deleterious mutations occurs due to three population genetic processes. One process is hitchhiking (fixation of deleterious mutations due
to linkage to favorable mutations spreading in the population; Rice 1987). A second possibility is background
selection (selection against deleterious mutations at
linked genes, resulting in reduced effective population
size and stochastic accumulation of mildly deleterious
mutations; Charlesworth, Morgan, and Charlesworth
1993; Charlesworth, Charlesworth, and Morgan 1995).
1 Present address: School of Biosciences, University of Birmingham, Birmingham, England.
Key words: Y chromosomes, sex linkage, selective sweeps, background selection, gene flow.
Address for correspondence and reprints: Dmitry A. Filatov,
School of Biosciences, Diversity of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. E-mail: [email protected].
Mol. Biol. Evol. 18(8):1442–1454. 2001
q 2001 by the Society for Molecular Biology and Evolution. ISSN: 0737-4038
1442
These two processes should considerably reduce the effective population size of a nonrecombining region, facilitating the operation of the third process, Muller’s
ratchet (stochastic loss of chromosomes with the fewest
mutations; Charlesworth 1978), which is slow unless the
size of the population is small (Charlesworth and Charlesworth 1997; Gordo and Charlesworth 2000). Any or
all of these processes may have led to Y chromosomes
gradually accumulating deleterious mutations, such that
Y-linked genes have become less and less functional. A
further consequence of these processes is reduction of
the effective population size of Y-linked genes, which
should therefore have reduced neutral variability. In
chromosome regions with no recombination, such as Y
chromosomes, these effects on diversity should be detectable in any loci, not just at loci that are involved in
the hypothesized selective processes.
To test the theories for Y-chromosome degeneration, it will therefore be helpful to study neutral diversity
of genes in the nonrecombining regions of Y chromosomes to see whether diversity at Y-linked loci is lower
than expected from the ploidy differences between Y
chromosomes and other chromosomes. Effective population sizes of Y-linked genes are expected to be lower
than values for autosomal and X-linked loci, even without the effects of selection just outlined. Ne for Y-linked
genes is, in theory, one fourth of that for autosomal
genes and one third of that for X-linked genes (Caballero 1995). Under neutrality, the nucleotide polymorphism maintained in a population is proportional to the
product of the neutral mutation rate and the effective
population size of the population (Kimura 1983). Thus,
Y-linked genes should have approximately one third of
the nucleotide variation observed for X-linked genes and
one fourth of that for autosomal loci, assuming equal
neutral mutation rates for all genes. These differences
DNA Diversity in Silene
should lead to lower Y-chromosomal diversity even in
sex chromosome systems that are not actively
degenerating.
Some data on the diversity of Y-linked loci are
available for the neo-Y chromosomes of Drosophila
americana (McAllister and Charlesworth 1999) and
Drosophila miranda (Yi and Charlesworth 2000). The
first of these species has a recent X-autosome fusion,
and the second has a somewhat older translocation onto
the Y chromosome. In both, nucleotide polymorphism
of Y-linked genes was decreased relative to the homologous genes on the X chromosome and autosomes, taking into account the ploidy differences. Here, we extend
our previous study of plant sex-linked genes, which also
demonstrated very low variability at a Y-linked locus
(Filatov et al. 2000).
The genus Silene contains about 700 species (Mabberley 1997), most of them hermaphroditic or gynodioecious. Two groups of dioecious species apparently
evolved independently (Desfeux et al. 1996). One group
includes the white-flowered Silene latifolia and its close
relative, Silene dioica (pink flowered). These two species are closely related and form viable and fertile hybrids in nature (Baker 1948; Goulson and Jerrim 1997).
They are estimated to have diverged from a nondioecious ancestor about 20 MYA based on divergence of
ITS sequences (Desfeux et al. 1996). Silene latifolia is
an agricultural weed and commonly grows in open sunny fields, particularly along the edges of paths and
roads, while S. dioica generally grows in more shaded
sites, such as woodlands and shady hedgerows, and has
a rather more northerly distribution in Europe; S. dioica
is absent from North America, while S. latifolia is an
introduced weedy species there. The two species have
similar chromosomal sex-determination systems (XX female and XY male; see Westergaard 1958). In S. latifolia, a small proportion of dihaploid plants, with a Y
chromosome but no X chromosome, are viable (Vagera,
Paulikova, and Dolezel 1994), although haploids with Y
alone are not. In contrast, haploids carrying an X chromosome are viable (Ye et al. 1990). This suggests that
the S. latifolia Y chromosome is at least partly degenerated, although it is not heterochromatic (Vyskot et al.
1993).
Searches for genes involved in sex determination
have yielded several S. latifolia genes (Matsunaga et al.
1996; Barbacar et al. 1997; Delichère et al. 1999), some
of them sex-linked (Guttman and Charlesworth 1998).
The genes studied here are the X- and Y-linked genes
SlX1 and SlY1 (Delichère et al. 1999; Filatov et al.
2000). These genes were isolated in screens for male
organ-specific genes, although SlX1 is expressed in both
sexes (Delichère et al. 1999). We previously found that
nucleotide sequence diversity in the SlY1 gene of S. latifolia is only about one twentieth that of the X-linked
ortholog, SlX1 (Filatov et al. 2000). To test whether Ylinked diversity in S. latifolia and S. dioica is reduced,
versus the alternative that X-linked variability is unusually high, diversity data for autosomal genes are also
needed. Here, we add diversity data on the autosomal
gene CCLS37.1 (Barbacar et al. 1997; Laporte and Char-
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lesworth 2001) In addition, we used outgroup sequences
from Silene conica, a hermaphroditic species closely related to S. latifolia and S. dioica (Desfeux et al. 1996)
to compare divergence in the X-linked, Y-linked, and
autosomal genes and to test whether the Y-chromosomal
mutation rate is low. We also present more detailed studies to test whether the pattern of nucleotide diversity in
the SlY1 gene can be explained by a selective-sweep
model, which our initial study seemed to rule out (Filatov et al. 2000). Finally, we examine possible effects
of the population structure within the species (which
could obscure evidence of selective sweeps) and gene
flow between them (which could increase diversity at
some loci).
Materials and Methods
Species and Populations
We studied nucleotide diversity in two closely related dioecious Silene species: S. latifolia and S. dioica.
White-flowered plants were taken to be S. latifolia, and
ones with deep pink flowers were taken to be S. dioica,
following Baker (1948) and Goulson and Jerrim (1997).
The S. latifolia sample consisted of three males from
laboratory strains and 24 from 10 natural populations,
12 of them used in our previous study (Filatov et al.
2000), while the S. dioica sample consisted of 18 males
from four natural populations (table 1). In one Scottish
population (North Berwick [NB]), S. latifolia, S. dioica,
and hybrid plants with pale pink flowers were growing
together. From this population, two S. latifolia, three S.
dioica and two hybrid males (numbers 270 and 271)
were collected (hybrids were oversampled relative to
their abundance in the population). In addition, one individual initially classified as S. dioica was reclassified
as a hybrid based on sequence data (see Results). A
single S. conica plant was used to provide an outgroup.
Genes
Exon-intron structures for the regions studied in the
three genes were inferred by comparing genomic DNA
sequences with cDNA. In the region sequenced, the intron-exon structures of the sex-linked genes SlX1 and
SlY1 (Delichère et al. 1999; GenBank accession numbers SLA18517 and SLA18519) were identical, with 15
exons and introns (Filatov et al. 2000). The sequences
analyzed here spanned intron 10 to exon 15. The exonintron structure of the autosomal CCLS37.1 gene (Barbacar et al. 1997; accession number SLZ93048) is
known only for the portion we sequenced from genomic
DNA. This contains one intron about 1 kb long, between
nucleotides 308 and 309 of the GenBank CCLS37.1
cDNA sequence. All three genes were sequenced for
most plants (table 1). However, CCLS37.1 was not studied in the Denmark S. latifolia population. For S. conica,
we sequenced the CCLS37.1 homolog and a gene corresponding to SlX1 and SlY1 (denoted Sc).
Molecular Methods
Genomic DNA was isolated from leaves of individual Silene plants by a CTAB plant miniprep method
1444
Filatov et al.
Table 1
Species and Populations Sampled in this Study
Species
Silene latifolia . . .
Hybrids . . . . . . . . .
Silene dioica . . . .
Silene conica . . . .
Country
Population
Denmark
Portugal
Portugal
United States
United States
United States
England
Scotland
Scotland
Scotland
Scotland
Scotland
France
Scotland
France
Scotland
Ebeltoft (DM)
Segier-Chaves (SC)
Sierra de Nogere (SD)
Vandy (VA)
Virginia (VR)
Lab strains (LS)
Sussex (SX)
Dalkeith (DK)
Dunbar (DB)
Edinburgh, Arthur’s seat (EA)
North Berwick (NB)
North Berwick (NB)
Corrèze (CR)
North Berwick (NB)
Corrèze (CR)
Edinburgh, Salisbury Crags (ES)
Scotland
Edinburgh, Blackford Glen (EB)
France
Canche
Plant No.
525 (2, 1, 1), 539 (2, 1, 1), 540 (2, 1, 1)
120a (1, 1, 1), 388 (1, 1, 1), 399 (1, 1, 1)
106a (1, 1, 1)
52a (1, 1, 1), 328 (1, 1, 1), 333 (1, 1, 1)
69a (1, 1, 1), 367 (1, 1, 2), 373 (1, 1, 2)
26a (1, 1, 1), 71a (1, 1, 1), 184a (1, 1, 1)
516 (1, 1, 1)
99a (1, 1, 1), 205a (1, 1, 1)
236a (1, 1, 1)
225a (1, 1, 1), 243a (1, 1, 1)
266 (1, 1, 1), 267 (1, 1, 1)
270 (1, 1, 2), 271 (1, 1, 1)
484 (1, 1, 1)
268 (1, 1, 1), 269 (1, 2, 2), 272 (1, 2, 2)
571 (1, 1, 1), 576 (1, 1, 2), 579 (1, 1, 2)
260 (1, 1, 2), 261 (1, 1, 1), 262 (1, 1, 2),
263 (2, 1, 2), 264 (1, 1, 1), 265 (1, 1, 1)
86 (1, 1, 1), 251 (2, 2, 1), 252 (2, 2, 1)
253 (1, 2, 2), 254 (1, 2, 2), 258 (1, 2, 1)
632
NOTE.—The plant numbers are indicated in the right-hand column, and whether or not each of the genes was sequenced is indicated for each plant by ‘‘1’’
or ‘‘2’’; three genes are indicated in the following order: CCLS37.1, SIX1, SIY1.
(described in Filatov and Charlesworth 1999). For PCR
amplification, Taq polymerase (Promega) or Expand
long template PCR (Boehringer Mannheim; for amplification of a 5-kb region of the S. dioica SlY1 gene) was
used. The PCR products were run on 1% agarose gels
with standard 1 3 TBE buffer (pH 8) and extracted from
gels using the QIAquick gel extraction kit (Qiagen). The
PCR products of Sc1 from S. conica and CCLS37.1 from
all three species were cloned into the pCR4-TOPO vector using the TA-TOPO cloning kit (Invitrogen). Sequencing of clones and direct sequencing of PCR products were performed on an ABI Prism 377 automatic
sequencer (Perkin Elmer) using ABI Prism BigDye terminator cycle sequencing kit (PE Applied Biosystems).
For the S. latifolia SlX1 and SlY1 genes and S. dioica SlX1, we used the same primers and studied the 2.3kb region previously described (Filatov et al. 2000). The
Y-specific primer (‘‘28’’; see Filatov et al. 2000) did
not anneal to the S. dioica SlY1 gene, so a new primer
specific to SlY1 was designed based on a sequence within intron 1 (primer ‘‘118’’: 59-CCTCTTAACAAGATTCACTACGTCTC-39). This was used with primer
‘‘210’’ (see Filatov et al. 2000) for long-range PCR
amplification of a region of about 5 kb spanning introns
1 to 13 of the S. dioica SlY1 gene. This PCR product
was used as a template for reamplification (with primers
‘‘111’’ and ‘‘210’’ as in Filatov et al. 2000) of a 1.7kb region spanning introns 10–13. Thus, the SlY1 region
studied in S. dioica was about 0.5 kb shorter than that
in S. latifolia. SlX1 and SlY1 PCR products of both dioecious species were sequenced directly. For S. conica,
primers ‘‘111’’ and ‘‘210’’ were used to amplify a part
of the homologous Sc1 gene for cloning and sequencing.
The silent-site divergence of this gene from the SlX1
and SlY1 genes was about 7%, and the Sc1 sequence
aligned with SlX1 and SlY1 even in the introns. However, most (about 0.5 kb) of intron 12 was deleted from
the S. conica Sc1 gene, which reduced the number of
nucleotides analyzed to about 1 kb.
For CCLS37.1, we used the primers 37.1F (forward)
(59-AGGGATTCAATGGTGGTCGTGG-39) and 37.1Rb
(reverse) (59-GTGCAAATGAAATTCAGAGGAC-39).
These amplify a single band of about 1.2 kb from both S.
latifolia and S. dioica and a band of about 1 kb from S.
conica. The PCR products of CCLS37.1 from all species
were cloned and sequenced using 37.1F, 37.1Rb, and three
internal primers, 37.1Fb (59-CTTTGCCACATCATCCATAG-39), 37.1F6 (59-TGCCTTCGTTTTTGTCTCTTGA39), 37.1R7 (59-GGTACTGAAATACCAGGCCAAGAG39). Both strands were sequenced for most of the region
studied, but for 11 of the 21 sequences, the first 400 bp
were sequenced twice in the same direction. Since cloning
of PCR products (unlike direct sequencing) may result in
incorporation of Taq polymerase errors, which will inflate
the number of singletons in the sample, we checked all
the singleton polymorphic sites by additional direct sequencing of PCR products.
Sequence Alignment and Analysis
Sequences were aligned using ClustalX, version
1.64 (Thomson et al. 1997), followed by manual adjustment using ProSeq, version 2.71 (Filatov 2001). Estimates of nucleotide diversity, population subdivision,
and gene flow statistics, as well as permutation-based
tests of significance (Hudson, Boos, and Kaplan 1992),
were performed using ProSeq, version 2.71. The neighbor-joining tree (fig. 2) was created using MEGA, version 1.01 (Kumar, Tamura, and Nei 1993).
Within each species, nucleotide diversities were
compared between genes using HKA tests (Hudson,
Kreitman, and Aguadé 1987), taking ploidy differences
into account using DNAsp, version 3.5 (Rozas and Rozas 1999). Interspecific divergence values were estimat-
DNA Diversity in Silene
ed using a single S. conica individual. Because the S.
conica sequence contains deletions in introns relative to
those of S. latifolia and S. dioica from the SlX1 and SlY1
sequences, the total number of sites in this analysis was
1,012 nt. To estimate the recombination statistic, CHud
(Hudson 1987), and for the FS neutrality tests (Fu 1997),
we also used DNAsp, version 3.5, and P values for the
FS neutrality test were estimated by coalescent simulations without recombination.
The possibility of different evolutionary rates of the
X and Y chromosomes was tested by a likelihood ratio
test using the local-molecular-clock method (Yoder and
Yang 2000). To compare mutation rates in the SlX1 and
SlY1 genes, we rooted the branches of the SlX1 and SlY1
sequences using the homologous S. conica sequence,
Sc1. A maximum-likelihood tree for all the SlX1 and
SlY1 sequences of either S. latifolia or S. dioica, plus
the S. conica sequence, was constructed by the PHYLIP
dnaml program (Felsenstein 1993). A model with three
different evolutionary rates (for SlX1, SlY1, and Sc1) in
the ancestry of the sequences was then tested against
one with just two evolutionary rates, one for the nonsex-linked homolog Sc1 and another common to both
the SlX1 and the SlY1 genes. This was done using the
baseml program in the PAML package (Yang 2000) to
calculate likelihoods for the two models. As the log likelihood ratio of these values is x2-distributed (Muse and
Weir 1992), the significance of the differences between
the two models can be evaluated. Since a separate evolutionary rate for Sc1 was allowed in both models, the
rate on the S. conica branch does not affect the results
of the analysis.
Tests for Gene Flow Between S. latifolia and S. dioica
Two approaches were used to test whether our data
from S. latifolia and S. dioica differed significantly from
the predictions of a model with no gene flow. First, we
used coalescent simulations (Hudson 1990) to model a
split into two populations of the same size without subsequent gene flow, but with recombination within populations (using ProSeq, version 2.71; Filatov 2001). The
simulations were conditioned on the actual number of
segregating sites in the pooled sample of the two species
and were run with a recombination rate equal to the
average estimate for the two species. Until the time of
the split (Ta) is reached (going back in time), coalescence and recombination events occur within each of
the two populations. At time Ta, the two populations are
united, and the process continues until the most recent
common ancestor is reached. We obtained bounds for
the values of several descriptive statistics by comparing
the values estimated from the data with those obtained
from simulations with different divergence times (Ta)
between the two species (scaled in terms of Ne values).
The statistics calculated for each run were the net divergence Da (Nei 1987), the population subdivision statistics Fst (Hudson, Boos, and Kaplan 1992), and the
numbers of polymorphic sites that were fixed and shared
between the two populations. After 1,000 runs, the 5%
and 95% percentiles for the distributions of all the sta-
1445
tistics were calculated and used as confidence intervals
for the values of the statistics given the speciation time
Ta.
Second, we used Wakeley and Hey’s (1997) model
of population divergence in a two-island model without
gene flow, taking into account differences in effective
population sizes between the two modern and the ancestral species. The approximate age of the split between
the extant populations and the scaled mutation rates (u
5 4Nem) of all three populations were estimated using
ProSeq, version 2.71 (Filatov 2001). The program uses
the numbers of shared polymorphic sites, fixed sites, and
polymorphic sites exclusive to each of the two populations sampled to obtain numerical solutions of equations
12–16 in Wakeley and Hey (1997) by Newton-Raphson
iteration (Press et al. 1992).
Results
Polymorphism and Population Structure Within
Species in the SlY1, SlX1, and CCLS37.1 Genes
Nucleotide diversity in the three S. latifolia and S.
dioica genes is summarized in table 2. The lengths of
coding and noncoding regions differ between the genes
(for instance, our CCLS37.1 sequences contain only
18% nonintron sites, compared with 25% for SlX1). One
cannot, therefore, directly compare their diversity. At
least in Drosophila, silent sites in exons typically have
higher diversity than intron sites (Moriyama and Powell
1995). We therefore estimated nucleotide diversity separately in noncoding and coding sites of the three genes.
In both species, X-linked and autosomal sequences are
less variable at replacement sites than at silent or intron
sites. For SlX1, diversity values (p) range from 1% to
2%, with no consistent difference in diversity between
synonymous and intron sites (table 2). For all types of
sites, the SlY1 gene of both species had lower diversity
than SlX1 or CCLS37.1. Among the nine S. dioica SlY1
1.5-kb sequences, we found only one nucleotide variant
(in intron 11 of sequences from the Corrèze population;
fig. 1). In the 22 S. latifolia SlY1 sequences for which
the longer sequences (2 kb) were analyzed, we found
five nucleotide variants (four in introns, and a Pro/Leu
amino acid replacement; fig. 1).
In addition, indel polymorphisms were more abundant in the X-linked and autosomal genes than in the Ylinked gene. Treating regions with overlapping indels as
single-indel sites, there are at least 28 insertion/deletion
(indel) polymorphisms in the 24 S. latifolia SlX1 sequences and 17 in the 11 SlX1 S. dioica sequences.
Among the CCLS37.1 sequences, there are seven indels
in the 21 S. latifolia sequences and four in the 15 sequences from S. dioica. In the sample of S. latifolia SlY1
sequences, however, there are only two indels, and there
is one in the S. dioica SlY1 sequences. Indel regions
were excluded from most of the further analyses, but
they were included in analyses of shared and fixed variants in S. latifolia and S. dioica (see below). The S.
latifolia SlY1 sequences fall into four haplotypes, which
differ by several nucleotide substitutions and indels (fig.
1).
1446
Filatov et al.
Table 2
DNA Polymorphism in Coding and Noncoding Regions of SlY1, SlX1, and CCLS37.1 Genes
INTRONS
SPECIES
GENE
Silene latifolia . . SlY1
SlX1
CCLS371
Silene dioica . . . SlY1
SlX1
CCLS37.1
N
L
S
22
24
21
9
11
15
1,518
1,119
905
1,104
1,489
903
4
87
31
1
48
8
EXONS,
p
0.001
0.019
0.009
0.0003
0.012
0.0021
6
6
6
6
6
6
u
0.0003
0.0101
0.0047
0.0001
0.0055
0.0014
0.0007
0.020
0.009
0.0003
0.011
0.003
6
6
6
6
6
6
0.0004
0.0069
0.0035
0.0002
0.0043
0.0013
SYNONYMOUS
L
S
p
106
106
51
76
109
51
0
11
1
0
9
1
—
0.023 6 0.0137
0.0019 6 0.0028
—
0.031 6 0.0146
0.005 6 0.0032
u
0.027
0.005
0.028
0.006
—
6 0.0119
6 0.0054
—
6 0.0138
6 0.0061
NOTE.—N 5 sample size; L 5 length of sequence analyzed; S 5 number of segregating sites.
All the genes studied show evidence of population
subdivision in both S. latifolia and S. dioica, and all Fst
estimates except that for the S. latifolia CCLS37.1 gene
are significantly greater than zero (P , 0.05). The S.
latifolia SlY1 haplotypes are associated with the geographic locations from which the samples originated, although the association is not complete, and several populations have two haplotypes, even with our small samples (fig. 1). The Fst estimates for the SlY1, SlX1, and
CCLS37.1 genes in S. latifolia were 0.76, 0.46, and 0.36,
respectively. For the SlX1 and CCLS37.1 genes of S.
dioica, Fst estimates were 0.79 and 0.18, respectively
(Fst was not calculated for the S. dioica SlY1 because
only one polymorphic site was found in the sample).
HKA Tests and Tests for Mutation Rate Differences in
the SlX1 and SlY1 Genes
To compare nucleotide diversity in the SlY1, SlX1,
and CCLS37.1 genes, we used the HKA test (Hudson,
Kreitman, and Aguadé 1987). This test assumes that sequences follow a neutral coalescent process in which
polymorphism is proportional to divergence, which may
not be true for a subdivided population such as that from
which our samples come (see previous section). However, Wakeley (1999) has shown that the coalescent approach may still be useful in subdivided populations.
The genealogy of such samples can be considered as
having two phases: a very short recent ‘‘scattering
phase’’ and a much longer ‘‘collecting phase,’’ which
starts (going backward in time) when each lineage ancestral to the sample is in a separate deme. Wakeley
(1999) demonstrated that the genealogy of ancestral lineages during the collecting phase is a coalescent. Because the collecting phase lasts much longer than the
scattering phase, the scattering phase can be ignored,
provided that a large enough number of populations are
sampled. Since our S. latifolia sample included 10 natural populations, the genealogy may thus be well approximated by a coalescent process, and the HKA test
may be used. This may not, however, be legitimate for
the S. dioica sample from only four populations.
The HKA test results were similar for both species
(table 3). The SlY1 gene has significantly (P , 0.05)
less diversity than SlX1, taking ploidy into account.
However, for both species, SlX1 has significantly (P ,
0.05) higher nucleotide polymorphism than CCLS37.1,
while the HKA tests for the CCLS37.1/SlY1 comparison
are nonsignificant.
The HKA test takes into account possible mutation
rate differences between genes, so the reduced SlY1 diversity is not likely to be due to a lower neutral mutation
rate of this gene. Moreover, using likelihood ratio tests,
we did not detect significant evolutionary rate differences in the X- and Y-linked genes: a model with two
rates (one for Sc1 and another for the SlX1 and SlY1
genes) accounts for the data, as does one with three rates
(for SlX1, SlY1, and Sc1); x2 5 2.48 for S. latifolia and
1.33 for S. dioica (df 5 1, P . 0.05 for both). The
evolutionary rates in the SlX1 and SlY1 genes are thus
not significantly different. Differences in evolutionary
rates cannot therefore explain the low SlY1
polymorphism.
We can also eliminate the possibility that the lower
diversity of CCLS37.1 compared with SlX1 could simply
be due to different amounts of coding and noncoding
sequence from the two genes. Our data show no sign of
lower intron diversity (table 2). Moreover, the HKA test
remains significant for SlX1 and CCLS37.1 introns only
(table 3).
Possible Causes of Low Diversity in the CCLS37.1 Gene
The much lower diversity in the autosomal gene
than in the X-linked gene is surprising. As noted above,
the effective population size for autosomal genes is expected to be four thirds that of X-linked ones, and thus
neutral diversity of autosomal genes should be somewhat higher than that for X-linked loci. We tested several other possibilities for this difference, including differences in recombination rates, and effects of either balancing selection (which could inflate SlX1 diversity) or
selective sweeps (which could have reduced diversity in
CCLS37.1).
Estimates of Recombination Rates for the X-Linked
and Autosomal Genes
One possible cause of the low CCLS37.1 diversity
is the well-known effect of reduced diversity in regions
of low recombination (e.g., Begun and Aquadro 1992;
Stephan and Langley 1998). To examine this possibility,
we estimated recombination rates using Hudson’s (1987)
measure of recombination rate per nucleotide, CHud, and
calculated Kelly’s (1997) ZnS statistic, a summary of
linkage disequilibrium for all sites. In both species, SlX1
appears to experience less recombination than
CCLS37.1, the opposite of the difference in DNA di-
DNA Diversity in Silene
1447
Table 2
Extended
EXONS,
TOTAL
REPLACEMENTS
L
S
p
u
L
S
381
382
176
289
381
176
1
5
5
0
0
0
0.006 6 0.0004
0.002 6 0.0017
0.0036 6 0.0039
—
—
—
0.0007 6 0.0007
0.004 6 0.0018
0008 6 0.0042
—
—
—
2,006
1,607
1,132
1,469
1,976
1,130
5
102
37
1
57
9
versity. For S. latifolia, CHud 5 0.012 for SlX1 and 0.122
for CCLS37.1, and ZnS 5 0.131 for SlX1 and 0.08 for
CCLS37.1. For S. dioica, both statistics suggest lower
recombination than in S. latifolia (CHud 5 0.004 for SlX1
and 0.013 for CCLS37.1; ZnS 5 0.291 for SlX1 and
0.098 for CCLS37.1). These estimates assume that the
populations are at mutation-drift equilibrium, an assumption which may be violated for the loci and populations studied here. However, subject to this caveat,
there is no evidence in either species that lower recombination causes lower diversity in CCLS37.1 than in
SlX1. Since this approach often appears to underestimate
recombination frequencies (Andolfatto and Przeworski
2000), this conclusion is conservative.
Tests for Selection
If diversity in CCLS37.1 has been reduced by a
recent selective sweep (Kaplan, Hudson, and Langley
1989), the frequency spectrum of polymorphic sites
should exhibit excess rare variants (Langley 1990; Braverman et al. 1995). If, however, SlX1 diversity is inflated
due to balancing selection at a linked site, which can
p
0.0009
0.015
0.007
0.0002
0.011
0.002
6
6
6
6
6
6
u
0.0003
0.0084
0.0045
0.0001
0.0049
0.0012
0.0007
0.017
0.009
0.0002
0.009
0.002
6
6
6
6
6
6
0.0004
0.0056
0.0033
0.0002
0.0038
0.0012
maintain diversity for a very long time (e.g., Strobeck
1983; Nordborg, Charlesworth, and Charlesworth 1996;
Charlesworth, Nordborg, and Charlesworth 1997; Takahata and Satta 1998), the site frequency spectrum
should have a bias toward frequent variants. Population
subdivision affects the frequency spectrum similarly to
balancing selection but should affect all the genes
similarly.
The SlX1 spectrum does not deviate significantly
from the distribution expected for neutral variants. Neither Tajima’s (1989) D nor Fu and Li’s (1993) D* statistic differs significantly from zero for either species
studied (table 4). Thus, it is unlikely that SlX1 has unusually high diversity caused by balancing selection.
These tests are also nonsignificant for CCLS37.1. However, Fu’s (1997) FS statistic, which is very sensitive to
the frequency spectrum bias toward rare polymorphisms, detects a significant deviation from neutrality
for the CCLS37.1 gene in both species, despite the population subdivision, which would tend to bias the frequency spectrum in the opposite direction, masking the
effect of a selective sweep (Strobeck 1983; Nordborg,
FIG. 1.—Polymorphic sites in the SlY1 region studied. Sequences are referred to by the species names, followed by the population abbreviation (see table 1) and the number of the individual plant. The types of sites are indicated by letters (i 5 intron; s 5 synonymous; r 5
replacement). Dots indicate nucleotides or indel sequences identical to the first sequence. The S. latifolia SlY1 sequences fall into four haplotypes,
shown at the right-hand side of the figure. Indels: a 5 AC; b 5 CTG; c 5 AA; d 5 AT; e 5 37 bp; f 5 CCC; g 5 CTG; h1 5 186 bp; h2 5
161 bp; h3 5 44 bp; h4 5 43 bp.
1448
Filatov et al.
FIG. 2.—Neighbor-joining tree of Silene latifolia and Silene dioica SlX1 and SlY1 sequences, rooted by a single Silene conica sequence of
a non-sex-linked gene, Sc1, homologous to SlX1 and SlY1 genes.
Charlesworth, and Charlesworth 1996). This suggests a
recent selective sweep in CCLS37.1. The frequency
spectrum bias toward rare variants is significant for this
gene. We therefore tentatively conclude that the
CCLS37.1 has atypically low diversity for an autosomal
locus and has perhaps experienced a selective sweep. In
what follows, we therefore assume that the lower diversity at SlY1 than SlX1 requires further explanation, rather
than seeking to explain why SlX1 diversity is high.
Gene Flow Between S. latifolia and S. dioica
A possible reason for the difference in diversity
between the SlY1 and SlX1 genes is a difference in gene
flow of these loci between S. latifolia and S. dioica.
Hybridization clearly occurs between the two species.
Both plants with an intermediate flower phenotype (pale
pink; plants 270 and 271 from the North Berwick population in Scotland) have CCLS37.1 and SlY1 sequences
(SlY1 sequenced only for plant 271) that cluster with the
corresponding S. dioica sequences, while their SlX1 sequences cluster with those of S. latifolia SlX1 (fig. 2).
In addition, one S. dioica plant (plant 484) out of four
from the Corrèze population (France) had a CCLS37.1
sequence that clustered with those from S. latifolia, al-
though its SlX1 and SlY1 sequences fell among those of
S. dioica (fig. 2). On the basis of these sequence data,
this plant was reclassified as a hybrid.
Differences in selective pressures against introgressed alleles for X-linked, Y-linked and non-sex-linked
genes could, in theory (Barton and Bengtsson 1986),
result in different rates of gene flow for the SlX1, SlY1,
and CCLS37.1 genes. Does gene flow occur, does it occur at different rates for the loci studied, and could it
account for the diversity differences observed? Table 5
compares nucleotide site and indel differences between
the two species. In SlY1, no polymorphisms of either
kind are shared between the species (excluding all three
hybrids). Most of the variable sites are fixed differences,
and Fst between the species is high. Gene flow between
S. latifolia and S. dioica must therefore be low for this
locus. For SlX1, there are few fixed differences and several shared polymorphic sites, such that net silent-site
divergence and Fst are lower than for SlY1 (table 5). The
autosomal gene, CCLS37.1, is even less diverged than
SlX1, with only two fixed indels and no shared sites (out
of 48 variable sites). The Fst estimates for CCLS37.1
are, however, similar to those for SlX1. Finally, even
after removing from the data the 11 SlX1 sites with poly-
DNA Diversity in Silene
1449
Table 3
Results of Pairwise HKA Tests for the SlX1, SlY1, and CCLS37.1 Genes
INTRONS
GENES COMPARED
SPECIES
x2
SlY1/SlX1 . . . . . . . . . .
SlY1/CCLS37.1 . . . . . .
SlX1/CCLS37.1 . . . . . .
SlY1/SlX1 . . . . . . . . . .
SlY1/CCLS37.1 . . . . . .
SlX1/CCLS37.1 . . . . . .
Silene latifolia
S. latifolia
S. latifolia
Silene dioica
S. dioica
S. dioica
10.489
1.58
4.52
3.96
0.09
8.85
TOTAL SEQUENCE
EXCLUDING SHARED SITES
P
x2
P
x2
P
0.001
0.208
0.033
0.046
0.761
0.003
10.03
1.89
3.42
4.4
0.19
8.97
0.002
0.168
0.065
0.036
0.662
0.003
8.87
1.89
2.94
3.89
0.19
7.368
0.003
0.168
0.086
0.049
0.662
0.007
morphisms that are shared between the two species and
could be caused by gene flow, the diversity differences
between SlX1 and SlY1 remain significant for both species (HKA test; P , 0.05; see table 3).
Polymorphic sites shared between S. latifolia and
S. dioica genes, such as those in SlX1, however, may
not indicate gene flow, but could have persisted since
the time of common ancestry, especially if the time
since speciation is short. To test whether a split without
further gene flow was compatible with our data from
these loci or whether subsequent introgression was required to explain the results, we ran coalescent simulations assuming no gene flow (see Materials and Methods) and estimated divergence times between the two
species (Ta). The Ta values, scaled in terms of Ne values,
are consistent for the SlX1 and CCLS37.1 genes and
suggest speciation between 2Ne and 4Ne generations
ago. If there has been gene flow, this is an
underestimate.
A model of population divergence without gene
flow (Wakeley and Hey 1997) is also compatible with
our data. Estimated numbers of generations since the
split between the two populations, based on the SlX1
and CCLS37.1 data, are shown in table 6. The parameter
values estimated for S. latifolia and S. dioica by this
model are similar to those estimated above within each
species individually. With the same mutation rate (m) in
the ancestral and the modern populations, the estimated
ancestral population size is close to the modern S. latifolia population size. Assuming no gene flow between
the two species, the speciation event is estimated to have
occurred about 2Ne generations ago (table 6), consistent
with the time estimated from the coalescent simulations.
Discussion
Reduced Diversity on the Y Chromosome
Genetic degeneration of nonrecombining Y chromosomes by the processes mentioned above should be
accompanied by considerable loss of nucleotide diversity (reviewed by Charlesworth and Charlesworth 2000).
In the early stages of Y-chromosome evolution, when
recombination has recently ceased, many functional
genes will still be present, and there should thus be a
high rate of both advantageous and deleterious mutations. Since the genes will be linked in a nonrecombining block, the reduction of diversity in the region should
be severe. The S. latifolia Y chromosome has probably
not yet become fully genetically degenerate, as active
genes not thought to be involved in sex determination
or male function, such as SlY1, are present. We previously reported very low SlY1 polymorphism (Filatov et
al. 2000), and this is verified with the present larger
sample. Although it is likely that diversity is reduced
for the Y-linked SlY1 locus, rather than being inflated
for the X-linked one, the low CCLS37.1 diversity makes
this uncertain. There is, however, no evidence for balancing selection acting at or near the X-linked SlX1 locus, and we found some evidence for a selective sweep
at CCLS37.1.
Possible Factors Reducing Nucleotide Diversity on the
Y Chromosome
Our results enable us to eliminate several possible
explanations for the low SlY1 diversity in S. latifolia and
S. dioica. A lower mutation rate on the Y chromosome
is one possibility. There is currently little information
about mutation rates of genes on different chromosomes,
other than for mammals (McVean and Hurst 1997;
Smith and Hurst 1999) and Drosophila (Baur and
Aquadro 1997). No data are available from plants. However, the assumption of equal mutation rates in the three
genes can be tested by comparing their sequences with
those of an outgroup species, since neutral divergence
depends only on the mutation rate (Kimura 1983). Our
results give no evidence of different mutation rates of
Table 4
Results of Neutrality Tests for the SlY1, SlX1, and CCLS37.1 Genes of Silene dioica and
Silene latifolia
Species
S.
S.
S.
S.
S.
latifolia . . .
dioica . . . .
latifolia . . .
dioica . . . .
latifolia . . .
Gene
SlX1
SlX1
CCLS37.1
CCLS37.1
SlY1
Tajima’s D
20.398,
0.176,
21.054,
20.808,
0.743,
NS
NS
NS
NS
NS
Fu and Li’s D*
20.289,
20.045,
21.602,
0.453,
1.176,
NS
NS
NS
NS
NS
Fu’s FS
23.117,
20.591,
23.666,
25.715,
2.842,
NS
NS
P , 005
P , 0.01
NS
NOTE.—Results are not shown for the S. dioica SlY1 gene because only one segregating site was observed. The
significance values for the statistics were calculated from 1,000 coalescent simulations without recombination.
1450
Filatov et al.
Table 5
Differences Between Sequences from Silene latifolia and Silene dioica
GENE
SlX1 . . . . . . .
SlY1 . . . . . . .
CCLS37.1 . .
SAMPLE
(S. latifolia/ SEQUENCE
S. dioica)
LENGTH
24/11
22/9
21/15
SINGLE-NUCLEOTIDE POLYMORPHISMS
1,526, NS
1,405, NS
1,122, NS
INSERTIONS/DELETIONS
S
Da (%)
Fst
Shared
Fixed
S
Da (%)
Fst
Shared
Fixed
127
42
43
1.11
2.7
0.2
0.48
0.97
0.35
11
0
0
6
37
0
17
12
5
0.84
0.98
0.14
0.39
0.91
0.63
7
0
0
2
11
1
NOTE.—The table shows net silent site divergence, Da (Nei 1987), Fst (Hudson, Boos, and Kaplan 1992), and the numbers of shared polymorphic sites and
sites fixed between the two species for single-nucleotide and indel polymorphisms. Hybrids are excluded. Sites with more than two variants were excluded from
the analysis.
the homologous X- and Y-chromosome genes. This conclusion is supported by HKA tests showing significantly
reduced diversity in SlY1 compared with SlX1, taking
into account their relative rates of divergence from the
S. conica sequence.
Another possibility is a high variance in male mating success (including sexual selection) or a female-biased sex ratio. These situations will reduce the effective
population size of Y-linked genes compared with that
for X-chromosomal and autosomal loci (Caballero 1995;
Charlesworth 1996) and can reduce (or even reverse) the
difference in effective population sizes between X chromosomes and autosomes. X-chromosomal diversity can
then reach values similar to, or slightly higher than,
those of autosomal loci (Caballero 1995). It is not
known whether plants are likely to have a high variance
in male mating success, but this possible cause of the
low SlY1 diversity should be testable by comparing nucleotide diversity in X-linked and autosomal genes. Our
results do not support this explanation because nucleotide diversity is considerably higher in the X-linked
gene, SlX1, than in the autosomal CCLS37.1.
Unlike the situation for animal male gametes, a
high proportion of genes are expressed in at least one
pollen grain nucleus (Tanksley, Zamir, and Rick 1981;
Stinson et al. 1987; Vielle-Calzada et al. 2000). If Xlinked genes expressed in pollen are important for mating success, pollen grains carrying Y chromosomes will
be somewhat defective compared with X-bearing pollen.
Indeed, a female-biased sex ratio is often observed in
natural populations of S. latifolia and S. dioica (Correns
1928; Lloyd 1974) and some, but not all, S. latifolia
families (Taylor 1994a, 1994b). However, gene expression in the haploid pollen also allows the possibility of
selection against deleterious mutations in pollen-expressed Y-linked genes (Haldane 1927). On the one
hand, this may lead to background selection, reducing
silent diversity in sequences on plant Y chromosomes,
but on the other hand, it may slow down genetic degeneration, at least at loci under purifying selection during
the pollination process.
Many other factors, including a gene’s local recombination frequency, and selection within the locus, or at
very closely linked loci, affect its diversity. The excess
of singleton polymorphisms in CCLS37.1 indeed suggests that diversity in this gene is unusually low and has
probably been reduced by a recent selective sweep. This
gene may therefore not be a suitable reference locus for
asking whether Y-linked genes have lower diversity than
their X-linked homologs. Studies of diversity levels of
further autosomal and X-linked loci are required to resolve this question.
Gene Flow as a Possible Cause of Elevated Diversity
of X-Linked Genes
Yet another possibility is that hybridization between S. latifolia and S. dioica may contribute to the
high SlX1 diversity in both species. Although hybridization certainly occurs, the S. latifolia or S. dioica sequences form separate clusters, and all sequences of the
X-linked and autosomal genes cluster either with S. latifolia or S. dioica sequences. This might suggest that
only very recent hybrids occur, and not older gene introgressions (which should have generated recombinant
alleles), perhaps indicating some form of selection
against hybrids. Consistent with this, the divergence
time between the two Silene species for SlX1 and
CCLS37.1, estimated by two methods that assume no
gene flow since the time of speciation, is similar to that
estimated directly from the SlY1 nucleotide divergence
of the S. latifolia and S. dioica alleles. These analyses
do not, however, conclusively rule out gene flow. The
evidence of hybridization in our sequence data must underestimate its frequency; if data were available from
more loci, more plants would probably be classified as
Table 6
Estimates of Population Parameters for Silene latifolia and Silene dioica
Gene
Length
Sshared
Sfixed
Sx2lat
Sx2dio
E (ulatifolia)
E (udioica)
E (uancestral)
E (t)
E (T2Ne)
SlX1 . . . . . . . . . . .
CCLS37.1 . . . . . . .
1,532
1,122
11
0
6
0
82
35
28
8
20.2 (0.0013)
9.7 (0.008)
8.1 (0.005)
2.5 (0.002)
31.9 (0.021)
0 (0)
10.9
5.1
1.2–2.5
1.1–4.1
NOTE.—The estimated time of the split between the two species is given in generations (t), scaled in mutational units (t 5 2ut) and in effective population
size (T2Ne 5 2t/u). The values of T2Ne are calculated using the u values estimated for S. latifolia and S. dioica. Estimates of u are given per sequence and per
nucleotide (in parentheses). Sites with more than two variants were excluded from the analysis. S values indicate numbers of variable sites, with subscripts to
indicate those shared between the two species, fixed differences between the species, and exclusive (x) to S. latifolia (lat) or S. dioica (dio).
DNA Diversity in Silene
hybrids. However, we found no evidence of markedly
different rates of gene flow between S. latifolia and S.
dioica for the different loci, although these tests did detect such differences between Drosophila pseudoobscura and its close relatives (Wang, Wakeley, and Hey
1997).
To be as conservative as possible in testing whether
diversity is higher in SlX1 than in SlY1, we could assume that introgression is so frequent between the two
species that diversity has reached equilibrium under
gene flow. Since the Fst values between the two species
for SlX1 are not much higher than those between different populations within each species, the two species
may be viewed as a single subdivided population. Assuming conservative migration (Nagylaki 1998), the expected equilibrium within-species diversity (p) is independent of the migration rate and is given by 4NTm,
where NT is the total population size (Maruyama 1971,
1972; Slatkin 1987; Strobeck 1987). The SlX1 diversity
would then be, at most, doubled as a result of gene flow
(on the most conservative assumption, that migration
occurs in both directions between the two species and
that these two species have the same population size).
We should thus halve the observed SlX1 diversity value;
this gives 51 and 28 polymorphic sites in the S. latifolia
and S. dioica SlX1 sequences, respectively, still considerably higher than the observed SlY1 diversity within
either species. The HKA test remains significant (P ,
0.05) for S. latifolia, but not for S. dioica. Thus, at least
for S. latifolia, correction both for ploidy differences and
for gene flow does not remove the diversity difference
between SlX1 and SlY1.
The diversity difference between X- and Y-linked
genes cannot, therefore, be explained by a low Y-chromosome mutation rate or by a high variance in male
mating success, and probably not by different gene flow
between the two species. This suggests that differences
in effective population sizes of genes on the X and Y
chromosomes are involved.
Testing Between Background Selection and Selective
Sweeps
The different population genetic models to explain
reduced genetic diversity in nonrecombining regions
are, in principle, distinguishable because they lead to
different predicted site frequency spectra of polymorphic variants. The selective-sweep model (Rice 1987)
predicts a frequency spectrum biased toward rare variants (Langley 1990; Braverman et al. 1995). Under the
background selection model, however, the site frequency
spectrum should be close to that expected under neutrality, unless the deleterious mutations driving the process have very small selection coefficients (Charlesworth, Charlesworth, and Morgan 1995). No detailed
study has yet been published of the effects of Muller’s
ratchet on diversity at neutral sites in a nonrecombining
chromosome, but simulations show effects intermediate
between the above two models, with a frequency spectrum less biased than that caused by selective sweeps but
still potentially distinguishable from the neutral spectrum,
1451
at least for the population sizes studied so far (I. Gordo
and B. Charlesworth, personal communication).
No bias in the frequency spectrum was detected for
loci on the D. americana (McAllister and Charlesworth
1999) and D. miranda (Yi and Charlesworth 2000) neoY chromosomes, which tends to support the background
selection model for these sex chromosome systems in
which functional genes are present on the Y chromosome. On the other hand, Zurovcova and Eanes (1999)
report excess singleton polymorphisms in the D. melanogaster Y-linked dynein gene, supporting the selectivesweep model (perhaps caused by selection within this
very large gene).
There are problems in testing site frequency spectra
in subdivided populations such as those of Silene. Seed
migration between populations separated by shorter geographic distances than those studied here is limited in
these species based on studies using genetic markers
(McCauley 1994; Giles, Lundqvist, and Goudet 1998;
Ingvarsson and Giles 1999; Richards, Church, and
McCauley 1999), and gene flow between populations
appears to be mostly via pollen movement (Richards,
Church, and McCauley 1999). Subdivision may therefore be less extensive for Y chromosomes than for autosomes and X chromosomes. On the other hand, the
lower effective population size for Y-linked genes implies that genetic drift will affect these genes more than
autosomal loci, causing them to have lower within-population variability and greater differentiation between
populations. The frequency spectrum of variants in Ylinked loci might thus be particularly affected by subdivision. Further theoretical work is, however, needed to
elucidate the net effects on sex-linked and autosomal
loci of multiple sampling from demes in a subdivided
population.
It is nevertheless clear that if deme sizes are small
and gene flow between populations is low, mutations on
the Y chromosome may quickly be fixed in local populations by drift and/or local selective sweeps, such that
different variants will be present in different populations. Thus, if these species have a subdivided population structure, the site-frequency spectra will be affected.
Sampling of multiple individuals per population generates samples in which all of the polymorphic sites on
the Y chromosomes are non-singletons, such that a negative Tajima’s D might become nonsignificant, and selective sweeps would be wrongly rejected. The only case
in which we obtained a positive D statistic was that of
the S. latifolia SlY1 (table 4), but it may nevertheless be
more appropriate to sample a single sequence per population. We therefore tested whether such a sample
changes the outcome of the Tajima’s tests. Subsamples
of S. latifolia SlY1 sequences, one from each natural
population and one from the laboratory strain, were randomly generated. Figure 3 compares the average frequency spectrum in 1,000 such subsamples of 11 SlY1
sequences (fig. 3B) with the observed spectrum for the
entire sample (fig. 3A). Tajima’s D remains positive for
the subsamples (mean value 0.418). Thus, the SlY1 frequency spectrum shows no bias toward rare polymor-
1452
Filatov et al.
LITERATURE CITED
FIG. 3.—Silene latifolia SlY1 site-frequency spectra. White bars
show the expected relative neutral frequencies. The short horizontal
lines indicate one standard deviation from the mean values, calculated
from 1,000 coalescent simulations without recombination, conditioning
on the actual number of segregating sites observed in the sample. A,
The gray bars show the observed frequency spectrum for the pooled
sample of 22 sequences. B, The gray bars show the average frequency
spectrum for 1,000 random subsamples of 11 S. latifolia SlY1 sequences, one sequence from each population listed in table 1.
phic sites, as would be expected under the selectivesweep hypothesis.
This test is, however, based only on intuitive ideas
about the expected behavior of subdivided populations.
Ideally, we should argue based on results of models that
include population subdivision. Assuming a high selection coefficient and a low migration rate between subpopulations (Nm , 1), a process of selective sweeps in
a subdivided population is dominated by the restricted
migration, and intrasubpopulation fixation can be ignored (Slatkin and Wiehe 1998). For a nonrecombining
chromosome such as the Y chromosome, hitchhiking in
Slatkin and Wiehe’s (1998) model leads to fixation of
one allele in the whole population, eliminating diversity
in the entire linked region. This model is probably reasonable for Silene populations, since Fst is high for all
loci. Moreover, our finding of four S. latifolia SlY1 haplotypes which differ by two or more substitutions, one
of which is found in several different geographic locations (fig. 1), suggests that some gene flow of Y chromosomes between populations must occur. These data
thus appear inconsistent with a simple advantageous
hitchhiking model, and our conclusion that selective
sweeps do not seem to be responsible for low diversity
at the SlY1 locus is therefore probably valid if we take
into account the subdivided population structure.
Acknowledgments
We thank G. A. T. McVean, B. Charlesworth, and
I. Gordo for discussions and advice on analyses, R.
Goodwin for help with S. dioica sequences, Y. Piquot
for providing S. conica material, and the University of
Edinburgh greenhouse staff for plant care. D.A.F. was
supported by a grant to D.C. from the Leverhulme Trust,
V.L. by a grant from the European Science Foundation
and a grant to D.C. from BBSRC, C.V. by the programme Magistère Universitaire, École Normale Supérieure, Paris, and D.C. by the Natural Environment Research Council of Great Britain.
ANDOLFATTO, P., and M. PRZEWORSKI. 2000. A genome-wide
departure from the standard neutral model in natural populations of Drosophila. Genetics 156:257–268.
BAKER, H. G. 1948. Stages in invasion and replacement demonstrated by species of Melandrium. J. Ecol. 36:96–119.
BARBACAR, N., S. HINNISDAELS, I. FARBOS, F. MONEGER, A.
LARDON, C. DELICHÈRE, A. MOURAS, and I. NEGRUTIU.
1997. Isolation of early genes expressed in reproductive organs of the dioecious white campion (Silene latifolia) by
subtraction cloning using an asexual mutant. Plant J. 12:
805–817.
BARTON, N., and B. O. BENGTSSON. 1986. The barrier to genetic exchange between hybridizing populations. Heredity
57:357–376.
BAUR, V. L., and C. F. AQUADRO. 1997. Rates of DNA sequence evolution are not sex-biased in Drosophila melanogaster and D. simulans. Mol. Biol. Evol. 14:1252–1257.
BEGUN, D. J., and C. F. AQUADRO. 1992. Levels of naturally
occurring DNA polymorphism correlate with recombination
rates in D. melanogaster. Nature 356:519–520.
BRAVERMAN, J. M., R. R. HUDSON, N. L. KAPLAN, C. H.
LANGLEY, and W. STEPHAN. 1995. The hitchhiking effect
on the site frequency spectrum of DNA polymorphism. Genetics 140:783–796.
BULL, J. J. 1983. Evolution of sex determining mechanisms.
Benjamin/Cummings, Menlo Park, Calif.
CABALLERO, A. 1995. On the effective size of populations with
separate sexes, with particular reference to sex-linked
genes. Genetics 139:1007–1011.
CHARLESWORTH, B. 1978. Model for evolution of Y chromosomes and dosage compensation. Proc. Natl. Acad. Sci.
USA 75:5618–5622.
———. 1996. Background selection and patterns of genetic
diversity in Drosophila melanogaster. Genet. Res. Camb.
68:131–150.
CHARLESWORTH, B., and D. CHARLESWORTH. 1997. Rapid fixation of deleterious alleles can be caused by Muller’s ratchet. Genet. Res. Camb. 70:63–73.
———. 2000. The degeneration of Y chromosomes. Philos.
Trans. R. Soc. Lond. B Biol. Sci. 355:1563–1572.
CHARLESWORTH, B., M. T. MORGAN, and D. CHARLESWORTH.
1993. The effect of deleterious mutations on neutral molecular variation. Genetics 134:1289–1303.
CHARLESWORTH, B., M. NORDBORG, and D. CHARLESWORTH.
1997. The effects of local selection, balanced polymorphism
and background selection on equilibrium patterns of genetic
diversity in subdivided populations. Genet. Res. Camb. 70:
155–174.
CHARLESWORTH, D., B. CHARLESWORTH, and M. T. MORGAN.
1995. The pattern of neutral molecular variation under the
background selection model. Genetics 141:1619–1632.
CORRENS, C. 1928. Bestimmung, Vererbung und Verteilung des
Geschlechtes bei den höheren Pflanzen. Handb. Vererbungswiss. 2:1–138.
DELICHÈRE, C., J. VEUSKENS, M. HERNOULD, N. BAARBACAR,
A. MOURAS, I. NEGRUTIU, and F. MONÉGER. 1999. SlY1, the
first active gene cloned from a plant Y chromosome, encodes a WD-repeat protein. EMBO J. 18:4169–4179.
DESFEUX, C., S. MAURICE, J. P. HENRY, B. LEJEUNE, and P. H.
GOUYON. 1996. Evolution of reproductive systems in the
genus Silene. Proc. R. Soc. Lond. B Biol. Sci. 263:409–
414.
FELSENSTEIN, J. 1993. PHYLIP (phylogeny inference package).
Distributed by the author, Department of Genetics, University of Washington, Seattle.
DNA Diversity in Silene
FILATOV, D. A. 2001. Processor of sequences manual. University of Edinburgh, Edinburgh, Scotland (http://helios.bto.
ed.ac.uk/evolgen/filatov/proseq.html).
FILATOV, D. A., and D. CHARLESWORTH. 1999. DNA polymorphism, haplotype structure and balancing selection in
the Leavenworthia PgiC locus. Genetics 153:1423–1434.
FILATOV, D. A., F. MONÉGER, I. NEGRUTIU, and D. CHARLESWORTH. 2000. Evolution of a plant Y-chromosome: variability in a Y-linked gene of Silene latifolia. Nature 404:
388–390.
FU, Y.-X. 1997. Statistical tests of neutrality of mutations
against population growth, hitchhiking and background selection. Genetics 147:915–925.
FU, Y.-X., and W.-H. LI. 1993. Statistical tests of neutrality of
mutations. Genetics 133:693–709.
GILES, B. E., E. LUNDQVIST, and J. GOUDET. 1998. Restricted
gene flow and subpopulation differentiation in Silene dioica. Heredity 80:715–723.
GORDO, I., and B. CHARLESWORTH. 2000. The degeneration of
asexual haploid populations and the speed of Muller’s ratchet. Genetics 154:1379–1387.
GOULSON, D., and K. JERRIM. 1997. Maintenance of the species
boundary between Silene dioica and S. latifolia (red and
white campion). Oikos 79:115–126.
GUTTMAN, D. S., and D. CHARLESWORTH. 1998. An X-linked
gene with a degenerate Y-linked homologue in a dioecious
plant. Nature 393:263–265.
HALDANE, J. B. S. 1927. A mathematical theory of natural and
artificial selection. V. Selection and mutation. Proc. Camb.
Philos. Soc. 26:838–844.
HUDSON, R. R. 1987. Estimating the recombination parameter
of a finite population model without selection. Genet. Res.
Camb. 50:245–250.
———. 1990. Gene genealogies and the coalescent process.
Oxf. Surv. Evol. Biol. 7:1–45.
HUDSON, R. R., D. D. BOOS, and N. L. KAPLAN. 1992. A statistical test for detecting geographic subdivision. Mol. Biol.
Evol. 9:138–151.
HUDSON, R. R., M. KREITMAN, and M. AGUADÉ. 1987. A test
of neutral molecular evolution based on nucleotide data.
Genetics 116:153–159.
INGVARSSON, P. K., and B. E. GILES. 1999. Kin-structured colonization and small-scale genetic differentiation in Silene
dioica. Evolution 53:605–611.
KAPLAN, N. L., R. R. HUDSON, and C. LANGLEY. 1989. The
hitchhiking effect revisited. Genetics 123:887–899.
KELLY, J. K. 1997. A test of neutrality based on interlocus
associations. Genetics 146:1197–1206.
KIMURA, M. 1983. The neutral theory of molecular evolution.
Cambridge University Press, Cambridge, England.
KUMAR, S., K. TAMURA, and M. NEI. 1993. MEGA: molecular
evolutionary genetics analysis. Version 1.0. Pennsylvania
State University, University Park.
LANGLEY, C. H. 1990. The molecular genetics of Drosophila.
Pp. 75–91 in N. TAKAHATA and J. F. CROW, eds. Population
biology of genes and molecules. Baifukan, Tokyo.
LAPORTE, V., and D. CHARLESWORTH. 2001. Non sex-linked,
nuclear cleaved amplified polymorphic sequences in Silene
latifolia. J. Hered. (in press).
LLOYD, D. G. 1974. Female-predominant sex ratios in angiosperms. Heredity 32:35–44.
LUCCHESI, J. C. 1978. Gene dosage compensation and the evolution of sex chromosomes. Science 202:711–716.
MABBERLEY, D. 1997. The plant book. 2nd edition. Cambridge
University Press, Cambridge, England.
1453
MCALLISTER, B., and B. CHARLESWORTH. 1999. Reduced sequence variability on the neo-Y chromosome of Drosophila
americana americana. Genetics 153:221–233.
MCCAULEY, D. E. 1994. Contrasting the distribution of chloroplast DNA and allozyme polymorphism among local populations of Silene alba: implications for studies of gene flow
in plants. Proc. Natl. Acad. Sci. USA 91:8127–8131.
MCVEAN, G. T., and L. D. HURST. 1997. Evidence for a selectively favorable reduction in the mutation rate of the X
chromosome. Nature 386:388–392.
MARUYAMA, T. 1971. An invariant property of a structured
population. Genet. Res. Camb. 18:81–84.
———. 1972. Some invariant properties of a geographically
structured finite population: distribution of heterozygotes
under irreversible mutation. Genet. Res. Camb. 20:141–149.
MATSUNAGA, S., S. KAWANO, H. TAKANO, H. UCHIDA, A. SAKAI, and T. KUROIWA. 1996. Isolation and developmental
expression of male reproductive organ-specific genes in a
dioecious campion, Melandrium album (Silene latifolia).
Plant J. 10:679–689.
MORIYAMA, E. N., and J. R. POWELL. 1995. Intraspecific nuclear DNA variation in Drosophila. Mol. Biol. Evol. 13:
261–277.
MUSE, S. V., and B. S. WEIR. 1992. Testing for equality of
evolutionary rates. Genetics 132:269–276.
NAGYLAKI, T. 1998. Fixation indices in subdivided populations.
Genetics 148:1325–1332.
NEI, M. 1987. Molecular evolutionary genetics. Columbia University Press, New York.
NORDBORG, M., B. CHARLESWORTH, and D. CHARLESWORTH.
1996. Increased levels of polymorphism surrounding selectively maintained sites in highly selfing species. Proc. R.
Soc. Lond. B Biol. Sci. 163:1033–1039.
ORR, H. A., and Y. KIM. 1998. An adaptive hypothesis for the
evolution of the Y chromosome. Genetics 150:1693–1698.
PRESS, W. H., S. A. TEUKOLSKY, W. T. VETTERLING, and B. P.
FLANNERY. 1992. Numerical recipes in Fortran. The art of
scientific computing. Cambridge University Press, Cambridge, England.
RICE, W. R. 1987. Genetic hitchhiking and the evolution of
reduced genetic activity of the Y sex chromosome. Genetics
116:161–167.
RICHARDS, C. M., S. CHURCH, and D. E. MCCAULEY. 1999.
The influence of population size and isolation on gene flow
by pollen in Silene alba. Evolution 53:63–73.
ROZAS, J., and R. ROZAS. 1999. DnaSP version 3.0: an integrated program for molecular population genetics and molecular evolution analysis. Bioinformatics 15:174–175.
SLATKIN, M. 1987. The average number of sites separating
(DNA) sequences drawn from a subdivided population.
Theor. Popul. Biol. 32:42–49.
SLATKIN, M., and T. WIEHE. 1998. Genetic hitch-hiking in a
subdivided population. Genet. Res. Camb. 17:155–160.
SMITH, N. G. C., and L. D. HURST. 1999. The causes of synonymous rate variation in the rodent genome: can substitution rates be used to estimate the sex bias in mutation
rate? Genetics 152:661–673.
STEPHAN, W., and C. H. LANGLEY. 1998. DNA polymorphism
in Lycopersicon and crossing-over per physical length. Genetics 150:1585–1593.
STINSON, J. R., A. J. EISENBERG, R. P. WILLING, M. E. PE, D.
D. HANSON, and J. P. MASCARENHAS. 1987. Genes expressed in the male gametophyte of flowering plants and
their isolation. Plant Physiol. 83:442–447.
STROBECK, C. 1983. Expected linkage disequilibrium for a
neutral locus linked to a chromosomal arrangement. Genetics 103:545–555.
1454
Filatov et al.
———. 1987. Average number of nucleotide differences in a
sample from a single population: a test for population subdivision. Genetics 117:149–153.
TAJIMA, F. 1989. Statistical method for testing the neutral mutation hypothesis by DNA polymorphism. Genetics 123:
585–595.
TAKAHATA, N., and Y. SATTA. 1998. Footprints of intragenic
recombination at HLA loci. Immunogenetics 47:430–441.
TANKSLEY, S. D., D. ZAMIR, and C. M. RICK. 1981. Evidence
for extensive overlap of sporophytic and gametophytic gene
expression in Lycopersicon esculentum. Science 213:453–
455.
TAYLOR, D. R. 1994a. The genetic-basis of sex-ratio in Silene
alba (5S. latifolia). Genetics 136:641–651.
———. 1994b. Sex-ratio in hybrids between Silene alba and
Silene dioica—evidence for Y-linked restorers. Heredity 73:
518–526.
THOMSON, J. D., T. J. GIBSON, F. PLEWNIAK, F. JEANMOUGIN,
and D. G. HIGGINS. 1997. The ClustalX windows interface:
strategies for multiple sequences alignment aided by quality
analysis tools. Nucleic Acids Res. 25:4876–4882.
VAGERA, J., D. PAULIKOVA, and J. DOLEZEL. 1994. The development of male and female regenerants by in-vitro androgenesis in dioecious plant Melandrium album. Ann. Bot.
73:455–459.
VIELLE-CALZADA, J.-P., R. BASKAR, and U. GROSSNIKLAUS.
2000. Delayed activation of the paternal genome during
seed development. Nature 404:91–94.
VYSKOT, B., A. ARAYA, J. VEUSKENS, I. NEGRUTIU, and A.
MOURAS. 1993. DNA methylation of sex chromosomes in
a dioecious plant, Melandrium album. Mol. Gen. Genet.
239:219–224.
WAKELEY, J. 1999. Nonequilibrium migration in human history. Genetics 153:1863–1871.
WAKELEY, J., and J. HEY. 1997. Estimating ancestral population parameters. Genetics 145:847–855.
WANG, R. L., J. WAKELEY, and J. HEY. 1997. Gene flow and
natural selection in the origin of Drosophila pseudoobscura
and close relatives. Genetics 147:1091–1106.
WESTERGAARD, M. 1958. The mechanism of sex determination
in dioecious flowering plants. Adv. Genet. 9:217–281.
YANG, Z. 2000. Phylogenetic analysis by maximum likelihood
(PAML). Version 3.0. University College London, London.
YE, D., P. INSTALLE, D. CIUPERCESCU, J. VEUSKENS, Y. WU,
G. SALESSES, M. JACOBS, and I. NEGRUTIU. 1990. Sex determination in the dioecious Melandrium. I. First lessons
from androgenic haploids. Sex. Plant Reprod. 3:179–186.
YI, S., and B. CHARLESWORTH. 2000. Contrasting patterns of
molecular evolution of the genes on the new and old sex
chromosomes of Drosophila miranda. Mol. Biol. Evol. 17:
703–717.
YODER, A., and Z. YANG. 2000. Estimation of primate speciation dates using local molecular clocks. Mol. Biol. Evol.
17:1081–1090.
ZUROVCOVA, M., and W. F. EANES. 1999. Lack of nucleotide
polymorphism in the Y-linked sperm flagellar dynein gene
Dhc-Yh3 of Drosophila melanogaster and D. simulans. Genetics 153:1709–1715.
WOLFGANG STEPHAN, reviewing editor
Accepted April 9, 2001