Copyright ? 2006 by the Genetics Society of America
Trans-specificity at Loci Near the Self-Incompatibility Loci in Arabidopsis
Deborah Charlesworth,1Esther Kamau, Jenny Hagenblad2and Chunlao Tang3
Institute of Evolutionary Biology, School of Biological Sciences, University of Edinburgh, Edinburgh EH9 3JT, United Kingdom
Manuscript received October 3, 2005
Accepted for publication February 1, 2006
We compared allele sequences of two loci near the Arabidopsis lyrata self-incompatibility (S) loci with
sequences of A. thaliana orthologs and found high numbers of shared polymorphisms, even excluding
singletons and sites likely to be highly mutable. This suggests maintenance of entire S-haplotypes for long
evolutionary times and extreme recombination suppression in the region.
stated that the times can exceed the ages of related
species, i.e., that variants arose before the split of re-
lated species (e.g., Ioerger et al. 1990; Klein et al. 1993;
Richman et al. 1996; Clark 1997; Wu et al. 1998; Adams
et al. 2000; Muirhead et al. 2002). Trans-specific poly-
morphism can provide strong evidence of long-term
balancing selection, because it is highly unlikely to exist
under neutrality, except between very closely related
species that can share variants present in their common
ancestor (Wiuf et al. 2004), and is expected only when
the same alleles persist for long times, and not when
alleles are regularly replaced by new alleles (‘‘turnover’’;
see Muirhead et al. 2002).
Recently,byexamininghumanand chimpanzee gene
sequences for trans-specific polymorphism, a search for
evidence of long-term balancing selection concluded
that it is infrequent in humans (Asthana et al. 2005).
The principle of such tests depends on the fact that
long-term balancing selection not only affects diversity
at the sites that are under selection, but also leads to
species, a gene under balancing selection maintains dif-
ferent functional classes of alleles, and each allele class
will acquire its own unique set of neutral mutations,
ALANCING selection can sometimes maintain vari-
ants for very long evolutionary times, and it is often
causing variants to be associated with the allele in which
they arose until recombination allows ‘‘migration’’ into a
different allele (reviewed in Charlesworth et al. 2003a).
Thus functionally different alleles will be differentiated
at the amino acids that define those types and also at
other sites within the region (linkage disequilibrium);
i.e., there will be higher polymorphism over a region
whose size depends on the local recombination fre-
quency than in unlinked genome regions (Wiuf et al.
2004). When a species with such a balanced polymor-
phism splits into two, multiple different haplotypes will
often pass to the daughter species (Figure 1 shows a
morphism will initially maintain the same associations
of variants as in the ancestor, but over evolutionary time
this signal will become indistinct, as the sequences in
each daughter species’s copies of the functionally same
allele recombine with other haplotypes of the locus,
acquire new mutations, and evolve new, functionally
different alleles, leading to allele turnover. Low recom-
bination will lead to trans-specific polymorphism for
longer evolutionary times.
Here we show that the expected effect of balancing
selection in leading to trans-specific polymorphism is
detectable at loci near the self-incompatibility (SI) loci
(S-loci) in species of the plant genus Arabidopsis. Be-
cause the formal population genetics theory shows that
the region affected by a locus under balancing selection
will be very small, in terms of the recombination dis-
tance (Wiuf et al. 2004), this result suggests very low
recombination in the region. With recombination, the
region of high diversity within the ancestral species
is small (Takahata and Satta 1998), and only this
region is likely to yield trans-specific polymorphisms.
The results also suggest recent maintenance of multiple
1Corresponding author: Institute of Evolutionary Biology, School of Bio-
logical Sciences, University of Edinburgh, Ashworth Lab, King’s Bldgs.,
W. Mains Rd., Edinburgh EH9 3JT, United Kingdom.
2Present address: Department of Biology, IFM, Linko ¨ping University,
SE-581 83 Linko ¨ping, Sweden.
3Present address: Department of Molecular and Computational Biology,
University ofSouthernCalifornia,835W. 37thSt.,SHS172, LosAngeles,
Genetics 172: 2699–2704 (April 2006)
S-haplotypes in Arabidopsis thaliana, even though this
species is now highly self-compatible. Balancing selec-
plant species (Richman et al. 1996; Takebayashi et al.
2003), with large numbers of functionally different
S-alleles maintained for very long evolutionary times
by the advantage of rare incompatibility alleles, i.e., very
slow turnover, as theoretically predicted (Takahata
1990; Vekemans and Slatkin 1994). This should pro-
duce extremely high polymorphism at linked neutral
Takebayashi et al. 2004), and the considerable data
now available document the expected high variability
throughout the S-locus gene sequences. In all species
where multiple S-alleles have been studied, their se-
quences differ greatly. Nucleotide diversity is extremely
high in pistil recognition genes of gametophytic SI
systems(e.g.,Richman et al.1996; Lu2001, 2002) andin
the pistil and pollen S-loci of species with sporophytic
SI (Sato et al. 2002; Charlesworth et al. 2003c). Con-
sistent with this evidence for long-term maintenance of
S-alleles, several alleles with the same specificity are
shared between Brassica oleracea and B. rapa (¼campestris)
(Kimura et al. 2002; Sato et al. 2003).
Our previous work on diversity at loci linked to the
A.lyrata S-locussuggests low recombinationintheregion
on the basis of two kinds of evidence. First, we find high
nucleotide diversity in the sequences of at least three
of five such loci studied, even though they are not in-
volved in incompatibility functions and show no evi-
dence of themselves being under balancing selection;
two of them have extremely high diversity (Kamau and
Charlesworth 2005). Applying a recently developed
et al. 2004), we roughly estimated the recombination
rate for the region to be ,1 cM/6 Mb (Kamau and
Charlesworth 2005), a value lower than most, if not
all, other estimates for noncentromeric regions of plant
genescarried in haplotypes with the same SRK sequence;
J. Bechsgaard, E. Kamau and D. Charlesworth, un-
published results). Here we add an independent type of
evidence from trans-specific polymorphism between A.
lyrata and its self-compatible relative, A. thaliana, in the S-
locus region, also suggesting low recombination in the
thus with an even lower recombination than our rough
estimates from polymorphism within A. lyrata. Sequence
diversity data from these species have recently become
comparisons to be made. Shared polymorphisms were
found in both genes, particularly in B80. These plants are
far too distantly related for trans-specific polymorphism to
be expected, unless very long-term balancing selection
has acted at a locus very closely linked to the ones
variants of these loci over an extremely long timescale.
Sequence diversity in the A. thaliana genome region
pollen S-locus, SCR1, but high at the pistil S-locus, SRK
(Shimizu et al. 2004), and also at the linked orthologs
of B80 (the U-box gene in Shimizu et al. 2004). Thus,
despite having lost self-incompatibility, A. thaliana re-
This might happen if self-compatibility was lost in this
species’s ancestor by selection for a loss-of-function al-
lele at a locus not in the S-locus region or one that
If this occurred recently enough, there might not have
been enough time for genetic drift to lead to fixation of
one of the haplotypes present (see below). By compar-
ing our B80 sequences with sequences of the ortholo-
gous gene of A. thaliana, we obtained evidence that
recombination is indeed very infrequent.
We studied two loci, B80 and ARK3 (called Aly8 in
A. lyrata; see Charlesworth et al. 2003c), for which
multiple sequences are available from A. thaliana and
A. lyrata. These genes are located physically close to
the functional self-incompatibility loci, SRK and SCR
sequences of the A. thaliana ortholog and with a se-
quence from the inbreeding species Arabis glabra (syn-
onymous site divergence from A. thaliana averages 0.2;
A. Kawabe, unpublished data). The B80 gene contains
no introns and is a single exon of 1125 bp in A. thaliana;
Figure 1.—Lineages at an S-locus under long-term balanc-
ing selection and the effects of speciation. As an example, two
haplotypes are shown with different S-alleles, x and y, which
diverged before the common ancestor of two species (1
and 2). The different alleles at the pistil and pollen S-loci,
SRK and SCR, are denoted by solid and shaded boxes. Muta-
tions in the regions in and around the S-locus (thin horizontal
lines in the tree) before the species became isolated remain
associated with the haplotype in which they arose (shown as
thin vertical lines in the haplotypes) until recombination oc-
curs with a different haplotype. Species-specific differences
(dotted lines) will also accumulate.
2700D. Charlesworth et al.
no alignment gaps were required. SRK diversity is very
low in A. glabra, and the sequences from three different
populations are all similar to SRK allele 31 of A. lyrata.
We used the alignment of 577 nucleotides to infer the
lineages in which the variants originated and to exam-
ine the data for polymorphisms shared between the 21
A. thaliana sequences obtained by Shimizu et al. (2004)
and the sequencesof 54A.lyrataalleles, representing 25
different S-haplotypes (there are 6 different A. thaliana
sequences, or 4 if singleton sites are ignored, and 28
different A. lyrata sequences, or 25 if ignoring single-
fixed differences between those two species. Of the 19
sites polymorphic within A. thaliana, 9 are apparently
trans-specific polymorphisms, one of them a nonsynon-
ymous variant (Figure 2). None of the shared variants in-
volves CpNpG or CpG-prone sites (because of the known
high mutation rate of such sites, these sites should be
excluded, together with sites that might recently have
mutated from such sites). None of the sites with shared
polymorphisms is a singleton variant in A. lyrata, but 3
are singletons in A. thaliana, all of these being variants
in the Cvi strain that were not seen in sequences of
ces of this gene between these two species is close to the
mean for other loci so there is no evidence for an un-
usually high mutation rate at this locus (Kamau and
et al. 2001; Schierup et al. 2001), encodes an S-domain
protein, resembling SRK. The results are less straight-
forward, because Aly8 is duplicated in at least some A.
lyrata haplotypes (J. Hagenblad, J. Bechsgaard and
D. Charlesworth, unpublished data); nevertheless,
we can test for shared variants in the two species. Our
sequences are from the first exon, slightly 59 of the re-
gion where high polymorphism was found in A. thaliana
(Shimizu et al. 2004) and just 39 of the region where high
et al. 2003c). With a larger number of sequences (93
from A. thaliana and 34 from A. lyrata) in a sequence
alignment (including both species) of only 318 bp after
excluding gaps, three nonsingleton shared polymor-
phisms were found, plus four shared polymorphisms
that are singletons in the A. thaliana sample; one other
site is polymorphic in both species at a CpG-prone site,
but not in the identical variants. There were 13 sites
with fixed differences between the two species (4.1%), a
strikingly low proportion for these species.
Trans-specific polymorphisms can arise in several ways.
Onepossibility ischanceoccurrenceofmutationsat the
same site since two species split. We calculated the prob-
ability that a polymorphism observed in one species will
be found in a related species, using a recently derived
analytical formula (equation16 of Charlesworth et al.
2005). The split between A. thaliana and A. lyrata is esti-
mated to have been ?5 MYA on the basis of net silent-
site divergence values of ?12% (Wright et al. 2002;
Figure 2.—Variants in the B80 (U-box) gene within A. thaliana and A. lyrata, excluding fixed differences from A. glabra and
singleton variants other than in the A. thaliana Cvi-0 strain. For A. thaliana and A. lyrata, a single sequence of each type seen within
the species is shown. For A. thaliana, the haplogroups of the strains (defined by SRK sequences; see the U-box gene in Shimizu
et al. 2004) are shown; for A. lyrata, the SRK allele, when known, is shown for each haplotype whose B80 sequence was used in the
analysis (the numbers of each A. lyrata S-haplotype sequenced are also shown in the left column). The nine shared variants be-
tween the two species are boxed with thin double lines; the nonsynonymous one is at position 245.
Schmid et al. 2005); this divergence estimates 2mT,
where m is the neutral mutation rate and T is the time
of the speciation event. Within either species, silent-site
diversity values are ?10-fold lower than this, and these
values are estimates of 4Nem, where Neis the species ef-
fective population size. Thus the observed divergence/
diversity values provide an estimate of (2mT/4Nem) ¼ T,
in units of 2Ne, of ?10. With T ¼ 10 we obtain a proba-
bility value of ?4 3 10?5. A sequence of 577 bp, such as
B80, is thus not expected to include as many as a single
shared polymorphism. With T ¼ 5, allowing for a gen-
eration time of .1 year, and only 318 bp, as for ARK3,
two are expected. Moreover, if recurrent mutation caused
the polymorphisms at the same sites in both Arabidopsis
species, most of these sites should have polymorphisms
of different nucleotides, whereas trans-specific polymor-
phisms due to long-term associations must be identical
nucleotides. In the B80 gene, there are only two such
polymorphic sites shared between A. thaliana and A.
lyrata [at sites 343 and 367 (see Figure 2); these are, of
course, not included in the count of shared polymor-
phisms]. T is also large enough that ancestral poly-
morphisms would be very unlikely to be retained in A.
thaliana (Clark 1997), assuming that since this species
lost functionalself-incompatibility, the S-locusregionwas
not maintained polymorphic by balancing selection.
Another possibility is thus ancestral polymorphism. If
we take as our null hypothesis that in A. thaliana no
balancing selection has recently affected this region,
because this species has lost self-incompatibility, A. tha-
liana alleles should have a common ancestor consid-
erably more recent than the time of the split between A.
thaliana and A. lyrata. We tested this null hypothesis
further by calculating the probability of finding in A.
thaliana nine or more shared polymorphisms at sites at
which polymorphisms areobserved in A. lyrata (81/577,
or 14%, of sites in the B80 gene). We used the binomial
theorem to approximate the hypergeometric probabil-
ities of each possible number of A. thaliana polymor-
of the chances of a variant occurring at different sites in
in A. lyrata is not close to zero). The approximation is
valid since the number of sites examined is large (see
Keeping 1962). The probability of finding nineor more
shared polymorphic sites is 0.021. The probability that
these will each have the same variants in both species is
considerably lower, since if mutation occurs randomly,
mutation, assuming the same ancestral nucleotide for
both species. For the ARK3/Aly8 genes, the chance of
three or more shared polymorphic sites is high (53%),
given the polymorphism level in A. lyrata (12.6% of all
sites), but the chance that all three will have identical
variants inbothspeciesislower.Thus thislocusalsomay
have shared polymorphisms, although the conclusion is
weaker than that for B80.
Our analysis underestimates the number of trans-
specific polymorphisms, because our samples might not
include rare variants, and the A. lyrata sample is from a
limited sampling of populations (Schierup et al. 2001).
However, many different alleles were included in the
samples from both species, so this is not likely to be a
Because of their large divergence times, shared poly-
morphisms between A. thaliana and A. lyrata seem a
priori highly unlikely. To check this, we examined loci
that are probably not close to genes under balancing
selection and whose alleles are thus not expected to be
maintained for long evolutionary times. We found six
reference loci for which sequence samples are available
from both these species; the loci are Adh (Savolainen
et al. 2000), Cauliflower (Purugganan and Suddith
1998; Wright et al. 2003), and Chi, FAH1, F3H, and
of sequences of these six loci, with a total of 5172 bp, we
found no shared polymorphisms, as expected for spe-
cies that have diverged for a long evolutionary time [the
expected life span of neutral alleles is 4Ne, although the
range of values is wide, so that much longer times may
occasionally be observed (Clark 1997)]. Thus the ob-
served trans-specific polymorphisms in the S-locus re-
gion seem to require a selective explanation.
The shared polymorphisms are probably variants that
differed between S-haplotypes in a self-incompatible
ancestral species. Analysis of the A. lyrata B80 sequences
does not suggest balancing selection acting at this
locus itself, despite its high diversity, which seems to
be attributable to linkage to the SRK locus (Kamau and
Charlesworth 2005). Our results thus suggest that re-
combination between different S-alleles is rare enough
across the region including the flanking genes (B80
and perhaps also ARK3) that entire haplotype sequences
have been preserved between the species studied since
their common ancestor. In the absence of recombina-
tion, each functionally distinct haplotype is expected to
be almost uniform in sequence within the ancestral self-
in A. lyrata (Charlesworth et al. 2003b). When daugh-
ter species become reproductively isolated, they will
often have ‘‘trans-specific’’ allelic lineages with the same
incompatibility type and, initially, similar sequences. If
the regions flanking the S-locus also recombine very
S-allele in related species, differ only by mutations that
have substituted in one lineage or the other since the
species split (Figure 1). There should thus be unusually
few fixed differences between species, as observed,
whereas raw divergence (uncorrected for polymorphism
within the species) will be high, due to the long times
to the common ancestors of the sequences, whether
compared within or between species. It therefore ap-
pears that the species studied here must have shared
S-allele lineages recently.
2702D. Charlesworth et al.
As already mentioned, A. thaliana is self-compatible.
Using A. thaliana in our comparisons of sequence var-
iants therefore does not correspond precisely to the
situation in which the same alleles are maintained by
balancing selection in two related self-incompatible
species. If loss of SI in A. thaliana was due to a selective
sweep at one of the S-loci, it should have caused low
diversity across this region, so at most a few variants that
arose after the event are expected in the species, since
loss of SI was probably recent (Shimizu et al. 2004).
Trans-specific polymorphisms would then be highly
unlikely. Even if self-compatibility evolved through a
mutation at an unlinked locus (which would not cause
such rapid diversity loss), there has probably been
enough time for genetic drift to have led to loss of all
but one lineage at the S-locus. Applying the standard
population genetics formula, a reduction in diversity to
40% as reported for the SRK pseudogene in a popula-
tion with the Nevalue of ?400,000 estimated for A.
generations (Charlesworth and Vekemans 2005). Yet
in A. thaliana diversity is very high in both SRK and the
closely linked ortholog of B80 (Shimizu et al. 2004). A.
thaliana therefore probably retained incompatibility
of A. lyrata during most of the much longer time (see
above) since the species’s common ancestor, and balanc-
in both species until quite recently. This is supported by
our finding of trans-specific variants, which should not
be present if A. thaliana evolved self-compatibility very
long ago. If so, our comparison is essentially equivalent
to one between two self-incompatible species (the self-
incompatible ancestor of A. thaliana and A. lyrata). Un-
less there is another nearby locus at which alleles are
maintained by long-term balancing selection in A. tha-
liana and A. lyrata, we can account for our findings
at the B80 locus only if A. thaliana lost functional self-
incompatibility recently enough that multiple haplo-
types have remained present.
The results suggest that the region, including at least
the S-loci and B80, has recombined very rarely since
the species split. Low recombination is predicted in the
S-locus region, because recombination generates self-
patibility loci (Casselman et al. 2000). This conclusion
is similar to that for a part of the human MHC region
containing three class II genes, where very high nucle-
otide diversity and strong linkage disequilibrium were
found for sites in the intergenic regions, suggesting
that entire haplotypes across the region have been
maintained since before humans evolved and that re-
combination may have been rare since before the com-
mon ancestor with other species such as chimpanzee
(Raymond et al. 2005). However, as explained above,
balancing selection maintaining many alleles can af-
fect diversity at nearby neutral sites, even without the
evolution of a low recombination rate (Takahata and
Satta 1998). Given the generally low recombination
rate in humans, it is thus unclear whether the findings
for MHC could be explained by a model in which many
alleles are maintained by balancing selection under
average recombination rates.
The size of the region of low recombination around
the Arabidopsis S-locus cannot yet be accurately esti-
mated, because different S-haplotypes are rearranged
Physical distance information is currently available for
three haplotypes, two functional S-haplotypes in A. ly-
rata, and one nonfunctional haplotype from A. thaliana
(Kusaba et al. 2001). The gene order is the same in all
three haplotypes, with B80 and ARK3 (or Aly8 in A.
lyrata) on opposite sides of the S-loci, spanning a region
of several tens of kilobases. The low recombination
suggested by our results thus seems to extend into the
nonrearranged regions flanking the S-loci at least as
far as B80. Given the apparent extreme suppression of
recombination, and likelihood that a quite large ge-
nome region is affected, family studies may be able to
(which could potentially be estimated with data from
the planned A. lyrata genome sequence). Such inde-
pendent tests for recombination suppression would be
very valuable, particularly as there is a puzzle about the
results from A. thaliana. Shimizu et al. (2004) suggested
that the loss-of-function mutation that causes loss of
incompatibility in A. thaliana occurred at the SCR1
locus. This requires the assumption that recombination
occurs frequently enough that the resulting selective
sweep as the compatible haplotype spread affected only
theSCR1locus(which suffered aseverelossofsequence
diversity), but not the flanking ARK3 and U-box genes.
If recombination occurred very infrequently, this in-
terpretation would be less plausible (Charlesworth
and Vekemans 2005). Thus, either the low SCR1 diver-
sity must be due to some cause other than the proposed
selective sweep or this region of the genome must have
had much higher recombination in A. thaliana’s ances-
tor at the time when self-compatibility evolved. This
puzzle should be resolved in the future.
We thank Brian Charlesworth, Magnus Nordborg (University of
Southern California), and Xavier Vekemans (University of Lille I) for
helpful discussions. This work was funded by a grant to D.C. from the
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