Institute of Cell, Animal, and Population Biology, University of Edinburgh, Edinburgh, Scotland
École Normale Supérieure, Paris, France
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Abstract |
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Introduction |
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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 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 Y-linked 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 Charlesworth 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).
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Materials and Methods |
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Molecular Methods
Genomic DNA was isolated from leaves of individual Silene plants by a CTAB plant miniprep method (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 x 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 2.3-kb region previously described (Filatov et al. 2000)
. The Y-specific primer ("-8"; 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 "+18": 5'-CCTCTTAACAAGATTCACTACGTCTC-3'). This was used with primer "-10" (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 "+11" and "-10" as in Filatov et al. 2000)
of a 1.7-kb region spanning introns 1013. 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 "+11" and "-10" 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) (5'-AGGGATTCAATGGTGGTCGTGG-3') and 37.1Rb (reverse) (5'-GTGCAAATGAAATTCAGAGGAC-3'). 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 (5'-CTTTGCCACATCATCCATAG-3'), 37.1F6 (5'-TGCCTTCGTTTTTGTCTCTTGA-3'), 37.1R7 (5'-GGTACTGAAATACCAGGCCAAGAG-3'). 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
).
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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 non-sex-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
2-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 statistics 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 (
= 4Neµ) 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 1216 in Wakeley and Hey (1997)
by Newton-Raphson iteration (Press et al. 1992
).
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Results |
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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.
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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 diversity. For S. latifolia, CHud = 0.012 for SlX1 and 0.122 for CCLS37.1, and ZnS = 0.131 for SlX1 and 0.08 for CCLS37.1. For S. dioica, both statistics suggest lower recombination than in S. latifolia (CHud = 0.004 for SlX1 and 0.013 for CCLS37.1; ZnS = 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 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, 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.
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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 polymorphisms 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
).
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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 (µ) 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.
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Discussion |
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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 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 X-linked 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 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 (
) is independent of the migration rate and is given by 4NTµ, 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, 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)
neo-Y 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 selective-sweep 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 Y-linked 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. 3 B) with the observed spectrum for the entire sample (fig. 3 A). Tajima's D remains positive for the subsamples (mean value 0.418). Thus, the SlY1 frequency spectrum shows no bias toward rare polymorphic sites, as would be expected under the selective-sweep hypothesis.
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Acknowledgements |
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Footnotes |
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1 Present address: School of Biosciences, University of Birmingham, Birmingham, England.
1 Keywords: Y chromosomes
sex linkage
selective sweeps
background selection
gene flow
2 Address for correspondence and reprints: Dmitry A. Filatov, School of Biosciences, Diversity of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom. d.filatov{at}bham.ac.uk
.
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