BACKGROUND
abiotic stresses, such as drought, high salinity and low soil nutrient levels, negatively impact crop production worldwide and are predicted to increase in coming decades due to climate change  <cit> .
much work has focused on identifying molecular mechanisms of abiotic stress tolerance in plants with the goal of breeding more tolerant crops  <cit> .
however, the genetic basis of plant adaptation to abiotic stress remains poorly understood, even in genomic model species such as rice.
identification of stress tolerance genes is often limited to mutant lines or a relatively few individuals used in mapping populations, and wider analysis of natural allelic variants in cultivated and wild populations is rare .
one factor that may shape the evolution of abiotic stress adaptation is fitness trade-offs in contrasting environments, where genotypes that can tolerate abiotic stress show reduced competitive ability or yields when grown in more favorable conditions.
at the genetic level, antagonistic pleiotropy describes genotypes that are adaptive in one environment but that negatively impact fitness in another  <cit> .
fitness trade-offs for stress tolerance may be especially important for crops such as rice , where cultivated varieties show tremendous variation in their agroecological adaptations.
in rice this range includes upland, drought-tolerant varieties; deep-water varieties that grow in monsoon-flooded fields; short-season, cold-adapted varieties grown at high elevations and latitudes; and varieties with high tolerance to salinity and nutrient-poor soils  <cit> .
rice is the primary staple food for over one-third of the world’s population  <cit> , and rice cultivation extends over  <dig> nations spanning six continents  <cit> .
soil phosphorus  availability varies widely across rice production areas.
a large proportion of crop varieties are grown in p-depleted soils, particularly in upland regions of tropical asia  <cit> .
at the same time, rice is also widely cultivated in p-rich or p-supplemented soils, and the varieties grown in these conditions are typically high yielding but intolerant of nutrient deprivation.
this pattern suggests potential fitness trade-offs associated with tolerance of nutrient-poor soils.
at the genetic level, if spatial heterogeneity in soil p availability has selected for both low-p-tolerant and -intolerant genotypes, then genes underlying this adaptive variation would be expected to evolve under balancing selection to maintain both allele classes.
to the extent that soil nutrient heterogeneity also affects populations of rice’s wild and weedy relatives, non-cultivated oryza populations could also be subject to balancing selection for low-p tolerance.
asian cultivated rice  consists of two major subspecies, japonica and indica, which were domesticated from wild rice, o. rufipogon, starting about 8000 years ago  <cit> .
the indica subspecies consists of indica and aus varieties, and the japonica subspecies consists of tropical japonica, temperate japonica, and aromatic varieties.
all five cultivated variety groups can be distinguished genetically  <cit> .
in addition to domestication, asian rice has also undergone de-domestication, leading to conspecific weedy strains that infest crop fields.
several genetically distinct weedy rice populations have been described, including straw-hulled u.s. weeds derived from indica crop progenitors  <cit> , black-hulled u.s. weeds from aus progenitors  <cit> , korean weeds from indica progenitors  <cit> , korean weeds from japonica progenitors  <cit> , and malaysian weeds from indica progenitors  <cit> .
in africa, an independent domestication event starting about 3500 years ago led to african cultivated rice , which is derived from the wild african species oryza barthii  <cit> .
both asian and african domesticated rice belong to the aa genome oryza species clade, which, in addition to their wild progenitor species, includes the south american species o. glumaepatula, the australian species o. meridionalis, and the african species o. longistaminata  <cit> .
phosphorus starvation tolerance  <dig>  has recently been identified as a major genetic determinant of low-p tolerance in rice  <cit> .
this gene, which encodes a protein kinase conferring p starvation tolerance, was identified in a low-p tolerant aus rice cultivar, kasalath  <cit> .
interestingly, pstol <dig> was found to be absent from the rice reference genome , where it falls within a ~90 kb insertion-deletion polymorphism  on chromosome  <dig>  prior to the functional characterization of pstol <dig> by gamuyao et al., this ~90 kb indel was identified as widely polymorphic across asian rice varieties; insertions are associated with upland rice grown in poor soil conditions, and deletions are associated with lowland rice grown in favorable soil conditions  <cit> .
given that the ~90 kb indel likely corresponds to pstol <dig> presence/absence, this pattern suggests that the pstol <dig> presence/absence variation could potentially reflect geographically heterogeneous selection for low-p-tolerant and -intolerant genotypes.
although pstol <dig> appears to be an important gene underlying adaptation of cultivated rice to poor soil environments, sequence variation at this locus has only been directly examined in a handful of cultivars  <cit> .
in this study we report on pstol <dig> molecular evolution and distribution of the gene presence/absence polymorphism across wild, cultivated and weedy rice.
using these data we address the following questions: 1) what does the distribution of pstol <dig> presence/absence variation across cultivated, weedy and wild rice tell us about long-term evolution of adaptation to low-p soil conditions?
2) for plants that carry pstol <dig>  what does the molecular evolution of this gene reveal about mechanisms of selection on functional variation?
3) how does pstol <dig> variation correspond to phenotypic variation for low-p tolerance in rice plants grown under controlled greenhouse conditions?
our results suggest long-term balancing selection at the pstol <dig> locus that predates and transcends rice domestication, with strong and persistent selection to maintain both functional and nonfunctional alleles at this agronomically important gene.
RESULTS
phylogenetic distribution of the pstol <dig> presence/absence polymorphism
of the  <dig> screened plants,  <dig>  carried pstol <dig> and  <dig>  were negative for all pcr-genotyping reactions .
across the six aa genome oryza species sampled, all accessions of o. barthii, o. glaberrima, o. glumaepatula and o. meridionalis were found to carry the gene.
in contrast, the gene was absent in 30 % of o. rufipogon samples and 52 % of o. sativa samples.
these patterns suggest that the pstol <dig> presence/absence polymorphism is restricted to asian cultivated rice and its wild progenitor.
within o. sativa, the presence/absence polymorphism spans both the indica and japonica subspecies.
pstol <dig> was present in all aus and aromatic crop samples , as well as all us weed accessions , while it was absent from all temperate japonica samples.
indica and tropical japonica crop varieties were polymorphic, as were malaysian and korean weeds .fig.
<dig> distribution of pstol <dig> presence absence polymorphism and clade type.
absence of pstol <dig> was recorded when no pcr product was produced for any of three sets of primers.
early stop mutations are included in the clade type percentages
additional loss-of-function mutations at pstol1
although the pstol <dig> presence/absence polymorphism is restricted to o. sativa and o. rufipogon in the sampled accessions, the gene appears to have independently evolved loss-of-function alleles across multiple species through other mutational events .
among the subset of samples that carry the gene, a 2-bp frameshift deletion at nucleotide positions 381– <dig> was detected in all six o. meridionalis accessions; this deletion was also detected in one of the five o. glumaepatula accessions.
within o. sativa, a g to a substitution at position  <dig>  which results in a premature stop codon at amino acid position  <dig>  was detected in an indica crop variety and four korean indica-like weeds.
interestingly, the same codon is altered through an independent premature stop detected in five aus varieties and one o. rufipogon accession; these samples carry a g to a substitution at nucleotide position  <dig>  thus, the occurrence of both functional and nonfunctional pstol <dig> alleles spans multiple aa genome oryza species, and the variation in pstol <dig> functionality within o. rufipogon and o. sativa includes both presence/absence variation and loss-of-function mutations.
this pattern is consistent with taxonomically- and geographically-widespread selection to maintain polymorphism in pstol <dig> functionality.fig.
<dig> location of early stop mutations in pstol <dig> gene.
pstol <dig> consists of a single 975 bp exon.
two different nonsense mutations are located in the same codon .
the 410 g to a mutation results in early stop in five aus accessions and the 411 g to a mutation results in early stop in one indica and four korean weed accessions.
a two basepair deletion at  <dig> leads to an early stop in a single o. glumaepatula accession and six o. meridionalis accessions
evolutionarily diverged pstol <dig> clades and signatures of balancing selection
phylogenetic analysis of pstol <dig> sequences revealed divergence between o. meridionalis, the most distantly related of the sampled aa genome species  <cit> , and the other sampled taxa .
for the remaining samples, sequences fell into two well supported and highly diverged clades , which are separated by  <dig> mutational steps including  <dig> nonsynonymous substitutions.
clade distributions among the sampled accessions are shown in table  <dig> and in figs.
<dig> and  <dig>  haplotypes of o. glumaepatula and african rice  are grouped by species, with o. glumaepatula sequences in clade a and african rice sequences in clade b .
in contrast, haplotypes of both wild and domesticated asian rice are present in both clades.
within o. sativa, three of the four cultivated variety groups that carry pstol <dig> are also present in both clades , with no apparent correlation between clade divergence and indica/japonica subspecies designation.
given the deep divergence between the two clades and the incongruence of o. rufipogon/sativa haplotypes with known species relationships, these distributions suggest long-term selective maintenance of both the a and b allele classes in asian rice.
consistent with this inference, frequency spectrum-based tests of neutral equilibrium revealed statistically significant signatures of balancing selection for tropical japonica sequences , although deviations from neutrality were not significant for aus, aromatic or o. rufipogon samples .
a statistically significant signature of directional selection was found in indica samples , likely due to only  <dig> of  <dig> accessions possessing the b clade allele.fig.
<dig> maximum likelihood phylogeny of pstol <dig>  parentheses contain numbers of accessions within a group.
bootstrap values are percentages from  <dig>  replicates.
the red x symbol indicates locations of early stop mutations
o. sativa
- indica
- aus
- tropical japonica
- aromatic
o. rufipogon
o. barthii
o. glaberrima
o. meridionalis
o. glumaepatula
pi and theta are per base pair at silent sites
<dig>  coalescent simulations for significance values
*p-value <  <dig>
**p-value <  <dig>
it is important to note that blast searches using pstol <dig> sequences from each of the major clades revealed no evidence for additional pstol <dig> gene copies in published databases, nor did we ever amplify both clade a and b haplotypes in the same accession.
thus, the a and b clades appear to be highly diverged alleles of the same gene rather than paralogous and/or pseudogenized gene copies.
interestingly, o. sativa and o. rufipogon sequences with premature stop mutations occur exclusively within clade a , which might further point to some difference in functionality between the a and b allele classes.
comparisons of nucleotide diversity between asian domesticated rice and its wild progenitor further suggest non-neutral evolution at pstol <dig>  whereas neutrally evolving genes across the rice genome show evidence of a domestication bottleneck, with a lower diversity in the crop compared to o. rufipogon  <cit> , pstol <dig> nucleotide diversity is instead higher in cultivated rice .
this pattern suggests that balancing selection has maintained elevated diversity at this locus through the domestication event.
consistent with this finding, we observed a high, albeit non-significant, value for tajima’s d for all cultivated rice samples pooled .
in contrast to o. sativa, african domesticated rice  showed no evidence of balancing selection at pstol <dig> during domestication; diversity is lower in the crop than its wild progenitor, consistent with neutral evolution during the african rice domestication bottleneck  <cit> .
for weedy rice strains, patterns of nucleotide diversity at pstol <dig> are consistent with demographic bottlenecks that occurred as these weeds evolved from their domesticated progenitors .
us weeds are fixed for a single, common clade a haplotype, which is also present in both indica and aus cultivated variety groups  .
all malaysian weeds also share this common clade a haplotype, consistent with their close relationship to indica crop varieties  <cit> .
the four indica-like korean weed accessions that carry pstol <dig> are fixed for a different clade a haplotype; as with the other weedy rice haplotypes, this haplotype is shared with an indica crop accession .
greenhouse growth experiments
in contrast to the findings of gamuyao et al.
<cit> , comparisons of genotype performance in low- vs. high-p conditions did not reveal an obvious fitness advantage for plants with functional pstol <dig> gene copies under low-p conditions.
although plants with functional gene copies showed a non-significant trend towards better growth under low-p conditions than those with premature stop mutations, the plants that performed best of all were those that lack the gene altogether; these results were consistent for both root and shoot growth .
we also found no major difference in root or shoot growth between the two major allele classes  when plants were grown under low-p conditions.
under high-p conditions, plants grew larger shoots than plants grown in low-p conditions regardless of pstol <dig> genotype .
root growth did not show significant differences between high- and low-p conditions for plants other than those with premature stop mutations, where root weight was greater under high-p conditions , and for those that lack pstol <dig>  where roots were longer in low-p conditions .
while our greenhouse experiment was modeled on the methods of gamuyao et al., our failure to detect the phenotypic associations with pstol <dig> variation observed in that study may reflect differences in experimental design, including differences in the developmental stages assayed.
two other recent studies have also reported no clear correlation between pstol <dig> genotypic variation and plant performance in high- vs. low-p conditions  <cit> .fig.
<dig> performance of pstol <dig> genotypes in low and high phosphorus conditions.
plants were measured after 21 days in high phosphorus  and low phosphorus  media.
a dry root weight; b dry shoot weight
discussion
pstol <dig> is a highly polymorphic gene in aa genome oryza species.
we find evidence for the selective maintenance of the gene presence/absence polymorphism across domesticated asian rice and its wild progenitor , independent evolution of loss-of-function alleles though nonsense and frameshift mutations , and maintenance of two distinct clades of functional alleles in asian rice that bear clear signatures of balancing selection .
the divergence of these two pstol <dig> clades likely predates asian rice domestication, as alleles from both clades are found in wild rice  accessions .
there is also evidence of long-term balancing selection to maintain the two separate clades across several cultivated rice groups .
these findings raise intriguing questions on the selective factors that have maintained this functional variation and its implications for adaptation to high- and low-p environments.
functional implications of pstol <dig> variation
there is abundant evidence that pstol <dig> confers increased uptake of phosphorus in plants that possess functional copies of the gene.
this gene, which encodes a receptor-like cytoplasmic kinase  <cit> , was identified through fine mapping of a major qtl for p uptake, pup <dig>  which had been found to explain nearly 80 % of the variation in this trait between kasalath and the nipponbare  <cit> .
pstol <dig> overexpression in two transgenic lines increased grain yield by more than 60 % under low-p conditions, with improved uptake of p and other nutrients arising through inreased root length and surface area  <cit> .
in addition, a survey of  <dig> cultivated asian rice accessions indicated that the pup <dig> qtl was differentially represented in varieties adapted to unfavorable, stress-prone growing conditions  <cit> .
homologs of pstol <dig> have also been implicated in increased p-uptake efficiency in sorghum  <cit>  and maize  <cit> .
all of these findings would suggest that selection should strongly favor pstol <dig> functionality rice plants growing in stressful, low-p soil conditions.
correspondingly, the phylogenetically and geographically widespread occurrence of loss-of function and gene-deletion alleles can most plausibly be explained as an instance of antagonistic pleiotropy, with energetic tradeoffs or other costs disfavoring pstol <dig> expression in environments with greater p availability.
whereas pstol <dig> has been completely lost in temperate japonica rice, functional copies are present in some indica, aus, tropical japonica and african cultivated rices, indicating that phosphorus starvation tolerance could be beneficial in parts of the regions where the latter varieties are cultivated.
transgenic expression experiments provide additional evidence that pstol <dig> likely has pleiotropic effects.
for example, 35s:pstol <dig> overexpression lines cause changes in expression of genes related to root growth and stress response when compared pstol <dig> null lines  <cit> ; affected genes include those involved in drought response and leaf senescence, two traits that are likely under strong selective constraints of their own.
examples of antagonistic pleiotropy are rare in plants, and further experiments with the pstol <dig> polymorphism will be required to definitively confirm that functional alleles are selected against in some environmental conditions.
just as the polymorphism in pstol <dig> functionality is likely to reflect an adaptive polymorphism, the occurrence of two evolutionarily diverged clades within asian rice also suggests balancing selection, in this case to maintain two functionally distinct allele classes.
clade a and b haplotypes differ by  <dig> nonsynonyous subsititutions , and a subset of these amino acid differences could underlie functional differences in the protein kinase activity.
however, the functional differences between clade a and b alleles, if any, remain to be characterized.
the well characterized kasalath pstol <dig> allele falls within clade a haplotypes , and studies to date on the phenotypic effects of pstol <dig> variation have either focused specifically on that allele  <cit>  or have assessed the pup1/pstol <dig> polymorphism without information on underlying sequence variation  <cit> .
thus, as with the gene presence/absence polymorphism, further work is needed to assess functional differences between the major pstol <dig> haplotype clades.
pariasca-tanaka et al.
<cit>  recently described an african rice pstol <dig> sequence that matches the clade b haplotype we found in the majority of our african rice samples .
they note that while differing at 6 % of amino acid sites from the kasalath allele, the sequence is conserved in the kinase catalytic domain.
similarly, all but three accessions in our clade b haplotypes also have a completely conserved kinase catalytic domain.
the exceptions include two o. rufipogon accessions and one indica accession which each contain a single amino acid replacement in that domain.
these findings suggest that if there are functional differences in the clade a and b alleles, they most likely do not arise through alterations of the protein’s catalytic domain.
given the previously documented evidence that pstol <dig> enhances p uptake under low-p conditions  <cit>  and our evidence of balancing selection at this locus , it is somewhat surprising that we did not detect any obvious effects of pstol <dig> variation on plant growth in the greenhouse .
on the other hand, at least two other recent studies have also found no clear correlation between pstol <dig> genotypes and plant performance in high- and low-p environments.
in a survey of  <dig> indica rice varieties, sarkar et al.
<cit>  reported that the pup <dig> locus was only present in three of the nine varieties showing greatest efficiency in p uptake in low-and high-p soils.
similarly, mukherjee et al.
<cit>  found no association between pstol <dig> presence and p-deficiency tolerance in a study of  <dig> diverse indian varieties.
the latter study also assessed the effects of pstol <dig> presence on low-p tolerance using a panel of  <dig> recombinant inbred lines and again found no evidence that the gene confers increased p-uptake efficiency.
like all physiological responses to abiotic stress, p uptake efficiency is undoubtedly a complex trait, and as such the phenotypic effects of pstol <dig> would be expected to vary across genetic backgrounds and environments.
it seems likely that the variable effects reported for pstol <dig> in different studies are a manifestation of these gene-by-gene and gene-by-environment interactions.
balancing selection
the phylogenetic distribution of putatively functional and non-functional pstol <dig> alleles suggests that selection has maintained this functional polymorphism across multiple oryza species .
trans-specific polymorphisms are typically established when balancing selection has been acting on a gene for very long periods of time  <cit> , indicating that the same differential selective pressures may have been acting in this genus long before the domestication of asian rice.
examples of trans-specific polymorphism are rare; self-incompatibility loci provide one of the best examples of long term balancing selection in plants .
however, the selective pressures acting on self-incompatibility are due to negative frequency dependent selection, where rare alleles have a selective advantage over common alleles.
balancing selection on abiotic stress genes is instead likely due to differential selection based on variable environmental conditions.
one example of long term balancing selection on abiotic stress genes was found in two species of wild tomato solanum peruvianum and s. chilense  <cit> .
the c-repeat binding factor  is involved in plant responses to cold and drought, environmental conditions that vary across solanum species ranges.
mboup et al.
found trans-species polymorphism at cbf <dig> that maintains signatures of balancing selection.
environmental heterogeneity is likely driving balancing selection in both cbf <dig> and pstol <dig>  and is likely not limited to these two examples of abiotic stress tolerance genes.
genome-wide studies of polymorphism in plants have identified several genomic regions that have signatures of balancing selection , but few studies have looked across species for trans-species polymorphisms in closely related plants.
CONCLUSIONS
agricultural environments exhibit a range of abiotic stresses including drought, low nutrient availability and high salinity.
geographical variation in these stresses can lead to balancing selection at loci that underlie adaptation to these stresses.
our findings for pstol <dig> suggest that adaptive variation at this gene has been a target of environmentally heterogeneous selection not only within cultivated rice, but also on a larger time frame and geographical scale that encompasses multiple aa genome oryza species.
while we did not observe clear phenotypic associations with pstol <dig> molecular variation in the greenhouse, the molecular signature of balancing selection we detect at this locus suggests that functional variation is important in the field — both in cultivated rice and wild oryza species.
it is likely that other genes involved in abiotic stress response will also show signatures of balancing selection in crop plants and their wild relatives.

 abiotic stresses, such as drought, high salinity and low soil nutrient levels, negatively impact crop production worldwide and are predicted to increase in coming decades due to climate change. much work has focused on identifying molecular mechanisms of abiotic stress tolerance in plants with the goal of breeding more tolerant crops. however, the genetic basis of plant adaptation to abiotic stress remains poorly understood, even in genomic model species such as rice. identification of stress tolerance genes is often limited to mutant lines or a relatively few individuals used in mapping populations, and wider analysis of natural allelic variants in cultivated and wild populations is rare. one factor that may shape the evolution of abiotic stress adaptation is fitness trade-offs in contrasting environments, where genotypes that can tolerate abiotic stress show reduced competitive ability or yields when grown in more favorable conditions. at the genetic level, antagonistic pleiotropy describes genotypes that are adaptive in one environment but that negatively impact fitness in another. fitness trade-offs for stress tolerance may be especially important for crops such as rice, where cultivated varieties show tremendous variation in their agroecological adaptations. in rice this range includes upland, drought-tolerant varieties; deep-water varieties that grow in monsoon-flooded fields; short-season, cold-adapted varieties grown at high elevations and latitudes; and varieties with high tolerance to salinity and nutrient-poor soils. rice is the primary staple food for over one-third of the world’s population, and rice cultivation extends over nations spanning six continents. soil phosphorus availability varies widely across rice production areas. a large proportion of crop varieties are grown in p-depleted soils, particularly in upland regions of tropical asia. at the same time, rice is also widely cultivated in p-rich or p-supplemented soils, and the varieties grown in these conditions are typically high yielding but intolerant of nutrient deprivation. this pattern suggests potential fitness trade-offs associated with tolerance of nutrient-poor soils. at the genetic level, if spatial heterogeneity in soil p availability has selected for both low-p-tolerant and -intolerant genotypes, then genes underlying this adaptive variation would be expected to evolve under balancing selection to maintain both allele classes. to the extent that soil nutrient heterogeneity also affects populations of rice’s wild and weedy relatives, non-cultivated oryza populations could also be subject to balancing selection for low-p tolerance. asian cultivated rice consists of two major subspecies, japonica and indica, which were domesticated from wild rice, o. rufipogon, starting about 8000 years ago. the indica subspecies consists of indica and aus varieties, and the japonica subspecies consists of tropical japonica, temperate japonica, and aromatic varieties. all five cultivated variety groups can be distinguished genetically. in addition to domestication, asian rice has also undergone de-domestication, leading to conspecific weedy strains that infest crop fields. several genetically distinct weedy rice populations have been described, including straw-hulled u.s. weeds derived from indica crop progenitors, black-hulled u.s. weeds from aus progenitors, korean weeds from indica progenitors, korean weeds from japonica progenitors, and malaysian weeds from indica progenitors. in africa, an independent domestication event starting about 3500 years ago led to african cultivated rice, which is derived from the wild african species oryza barthii. both asian and african domesticated rice belong to the aa genome oryza species clade, which, in addition to their wild progenitor species, includes the south american species o. glumaepatula, the australian species o. meridionalis, and the african species o. longistaminata. phosphorus starvation tolerance has recently been identified as a major genetic determinant of low-p tolerance in rice. this gene, which encodes a protein kinase conferring p starvation tolerance, was identified in a low-p tolerant aus rice cultivar, kasalath. interestingly, pstol was found to be absent from the rice reference genome, where it falls within a ~90 kb insertion-deletion polymorphism on chromosome prior to the functional characterization of pstol by gamuyao et al., this ~90 kb indel was identified as widely polymorphic across asian rice varieties; insertions are associated with upland rice grown in poor soil conditions, and deletions are associated with lowland rice grown in favorable soil conditions. given that the ~90 kb indel likely corresponds to pstol presence/absence, this pattern suggests that the pstol presence/absence variation could potentially reflect geographically heterogeneous selection for low-p-tolerant and -intolerant genotypes. although pstol appears to be an important gene underlying adaptation of cultivated rice to poor soil environments, sequence variation at this locus has only been directly examined in a handful of cultivars. in this study we report on pstol molecular evolution and distribution of the gene presence/absence polymorphism across wild, cultivated and weedy rice. using these data we address the following questions: 1) what does the distribution of pstol presence/absence variation across cultivated, weedy and wild rice tell us about long-term evolution of adaptation to low-p soil conditions? 2) for plants that carry pstol what does the molecular evolution of this gene reveal about mechanisms of selection on functional variation? 3) how does pstol variation correspond to phenotypic variation for low-p tolerance in rice plants grown under controlled greenhouse conditions? our results suggest long-term balancing selection at the pstol locus that predates and transcends rice domestication, with strong and persistent selection to maintain both functional and nonfunctional alleles at this agronomically important gene.  phylogenetic distribution of the pstol presence/absence polymorphism of the screened plants, carried pstol and were negative for all pcr-genotyping reactions. across the six aa genome oryza species sampled, all accessions of o. barthii, o. glaberrima, o. glumaepatula and o. meridionalis were found to carry the gene. in contrast, the gene was absent in 30 % of o. rufipogon samples and 52 % of o. sativa samples. these patterns suggest that the pstol presence/absence polymorphism is restricted to asian cultivated rice and its wild progenitor. within o. sativa, the presence/absence polymorphism spans both the indica and japonica subspecies. pstol was present in all aus and aromatic crop samples, as well as all us weed accessions, while it was absent from all temperate japonica samples. indica and tropical japonica crop varieties were polymorphic, as were malaysian and korean weeds.fig. distribution of pstol presence absence polymorphism and clade type. absence of pstol was recorded when no pcr product was produced for any of three sets of primers. early stop mutations are included in the clade type percentages additional loss-of-function mutations at pstol1 although the pstol presence/absence polymorphism is restricted to o. sativa and o. rufipogon in the sampled accessions, the gene appears to have independently evolved loss-of-function alleles across multiple species through other mutational events. among the subset of samples that carry the gene, a 2-bp frameshift deletion at nucleotide positions 381– was detected in all six o. meridionalis accessions; this deletion was also detected in one of the five o. glumaepatula accessions. within o. sativa, a g to a substitution at position which results in a premature stop codon at amino acid position was detected in an indica crop variety and four korean indica-like weeds. interestingly, the same codon is altered through an independent premature stop detected in five aus varieties and one o. rufipogon accession; these samples carry a g to a substitution at nucleotide position thus, the occurrence of both functional and nonfunctional pstol alleles spans multiple aa genome oryza species, and the variation in pstol functionality within o. rufipogon and o. sativa includes both presence/absence variation and loss-of-function mutations. this pattern is consistent with taxonomically- and geographically-widespread selection to maintain polymorphism in pstol functionality.fig. location of early stop mutations in pstol gene. pstol consists of a single 975 bp exon. two different nonsense mutations are located in the same codon. the 410 g to a mutation results in early stop in five aus accessions and the 411 g to a mutation results in early stop in one indica and four korean weed accessions. a two basepair deletion at leads to an early stop in a single o. glumaepatula accession and six o. meridionalis accessions evolutionarily diverged pstol clades and signatures of balancing selection phylogenetic analysis of pstol sequences revealed divergence between o. meridionalis, the most distantly related of the sampled aa genome species, and the other sampled taxa. for the remaining samples, sequences fell into two well supported and highly diverged clades, which are separated by mutational steps including nonsynonymous substitutions. clade distributions among the sampled accessions are shown in table  and in figs.  and haplotypes of o. glumaepatula and african rice are grouped by species, with o. glumaepatula sequences in clade a and african rice sequences in clade b. in contrast, haplotypes of both wild and domesticated asian rice are present in both clades. within o. sativa, three of the four cultivated variety groups that carry pstol are also present in both clades, with no apparent correlation between clade divergence and indica/japonica subspecies designation. given the deep divergence between the two clades and the incongruence of o. rufipogon/sativa haplotypes with known species relationships, these distributions suggest long-term selective maintenance of both the a and b allele classes in asian rice. consistent with this inference, frequency spectrum-based tests of neutral equilibrium revealed statistically significant signatures of balancing selection for tropical japonica sequences, although deviations from neutrality were not significant for aus, aromatic or o. rufipogon samples. a statistically significant signature of directional selection was found in indica samples, likely due to only of accessions possessing the b clade allele.fig. maximum likelihood phylogeny of pstol parentheses contain numbers of accessions within a group. bootstrap values are percentages from replicates. the red x symbol indicates locations of early stop mutations o. sativa    - indica    - aus    - tropical japonica    - aromatic o. rufipogon o. barthii o. glaberrima o. meridionalis o. glumaepatula pi and theta are per base pair at silent sites coalescent simulations for significance values *p-value <  **p-value <  it is important to note that blast searches using pstol sequences from each of the major clades revealed no evidence for additional pstol gene copies in published databases, nor did we ever amplify both clade a and b haplotypes in the same accession. thus, the a and b clades appear to be highly diverged alleles of the same gene rather than paralogous and/or pseudogenized gene copies. interestingly, o. sativa and o. rufipogon sequences with premature stop mutations occur exclusively within clade a, which might further point to some difference in functionality between the a and b allele classes. comparisons of nucleotide diversity between asian domesticated rice and its wild progenitor further suggest non-neutral evolution at pstol whereas neutrally evolving genes across the rice genome show evidence of a domestication bottleneck, with a lower diversity in the crop compared to o. rufipogon, pstol nucleotide diversity is instead higher in cultivated rice. this pattern suggests that balancing selection has maintained elevated diversity at this locus through the domestication event. consistent with this finding, we observed a high, albeit non-significant, value for tajima’s d for all cultivated rice samples pooled. in contrast to o. sativa, african domesticated rice showed no evidence of balancing selection at pstol during domestication; diversity is lower in the crop than its wild progenitor, consistent with neutral evolution during the african rice domestication bottleneck. for weedy rice strains, patterns of nucleotide diversity at pstol are consistent with demographic bottlenecks that occurred as these weeds evolved from their domesticated progenitors. us weeds are fixed for a single, common clade a haplotype, which is also present in both indica and aus cultivated variety groups. all malaysian weeds also share this common clade a haplotype, consistent with their close relationship to indica crop varieties. the four indica-like korean weed accessions that carry pstol are fixed for a different clade a haplotype; as with the other weedy rice haplotypes, this haplotype is shared with an indica crop accession. greenhouse growth experiments in contrast to the findings of gamuyao et al., comparisons of genotype performance in low- vs. high-p conditions did not reveal an obvious fitness advantage for plants with functional pstol gene copies under low-p conditions. although plants with functional gene copies showed a non-significant trend towards better growth under low-p conditions than those with premature stop mutations, the plants that performed best of all were those that lack the gene altogether; these results were consistent for both root and shoot growth. we also found no major difference in root or shoot growth between the two major allele classes when plants were grown under low-p conditions. under high-p conditions, plants grew larger shoots than plants grown in low-p conditions regardless of pstol genotype. root growth did not show significant differences between high- and low-p conditions for plants other than those with premature stop mutations, where root weight was greater under high-p conditions, and for those that lack pstol where roots were longer in low-p conditions. while our greenhouse experiment was modeled on the methods of gamuyao et al., our failure to detect the phenotypic associations with pstol variation observed in that study may reflect differences in experimental design, including differences in the developmental stages assayed. two other recent studies have also reported no clear correlation between pstol genotypic variation and plant performance in high- vs. low-p conditions.fig. performance of pstol genotypes in low and high phosphorus conditions. plants were measured after 21 days in high phosphorus and low phosphorus media. a dry root weight; b dry shoot weight discussion pstol is a highly polymorphic gene in aa genome oryza species. we find evidence for the selective maintenance of the gene presence/absence polymorphism across domesticated asian rice and its wild progenitor, independent evolution of loss-of-function alleles though nonsense and frameshift mutations, and maintenance of two distinct clades of functional alleles in asian rice that bear clear signatures of balancing selection. the divergence of these two pstol clades likely predates asian rice domestication, as alleles from both clades are found in wild rice accessions. there is also evidence of long-term balancing selection to maintain the two separate clades across several cultivated rice groups. these findings raise intriguing questions on the selective factors that have maintained this functional variation and its implications for adaptation to high- and low-p environments. functional implications of pstol variation there is abundant evidence that pstol confers increased uptake of phosphorus in plants that possess functional copies of the gene. this gene, which encodes a receptor-like cytoplasmic kinase, was identified through fine mapping of a major qtl for p uptake, pup which had been found to explain nearly 80 % of the variation in this trait between kasalath and the nipponbare. pstol overexpression in two transgenic lines increased grain yield by more than 60 % under low-p conditions, with improved uptake of p and other nutrients arising through inreased root length and surface area. in addition, a survey of cultivated asian rice accessions indicated that the pup qtl was differentially represented in varieties adapted to unfavorable, stress-prone growing conditions. homologs of pstol have also been implicated in increased p-uptake efficiency in sorghum and maize. all of these findings would suggest that selection should strongly favor pstol functionality rice plants growing in stressful, low-p soil conditions. correspondingly, the phylogenetically and geographically widespread occurrence of loss-of function and gene-deletion alleles can most plausibly be explained as an instance of antagonistic pleiotropy, with energetic tradeoffs or other costs disfavoring pstol expression in environments with greater p availability. whereas pstol has been completely lost in temperate japonica rice, functional copies are present in some indica, aus, tropical japonica and african cultivated rices, indicating that phosphorus starvation tolerance could be beneficial in parts of the regions where the latter varieties are cultivated. transgenic expression experiments provide additional evidence that pstol likely has pleiotropic effects. for example, 35s:pstol overexpression lines cause changes in expression of genes related to root growth and stress response when compared pstol null lines; affected genes include those involved in drought response and leaf senescence, two traits that are likely under strong selective constraints of their own. examples of antagonistic pleiotropy are rare in plants, and further experiments with the pstol polymorphism will be required to definitively confirm that functional alleles are selected against in some environmental conditions. just as the polymorphism in pstol functionality is likely to reflect an adaptive polymorphism, the occurrence of two evolutionarily diverged clades within asian rice also suggests balancing selection, in this case to maintain two functionally distinct allele classes. clade a and b haplotypes differ by nonsynonyous subsititutions, and a subset of these amino acid differences could underlie functional differences in the protein kinase activity. however, the functional differences between clade a and b alleles, if any, remain to be characterized. the well characterized kasalath pstol allele falls within clade a haplotypes, and studies to date on the phenotypic effects of pstol variation have either focused specifically on that allele or have assessed the pup1/pstol polymorphism without information on underlying sequence variation. thus, as with the gene presence/absence polymorphism, further work is needed to assess functional differences between the major pstol haplotype clades. pariasca-tanaka et al. recently described an african rice pstol sequence that matches the clade b haplotype we found in the majority of our african rice samples. they note that while differing at 6 % of amino acid sites from the kasalath allele, the sequence is conserved in the kinase catalytic domain. similarly, all but three accessions in our clade b haplotypes also have a completely conserved kinase catalytic domain. the exceptions include two o. rufipogon accessions and one indica accession which each contain a single amino acid replacement in that domain. these findings suggest that if there are functional differences in the clade a and b alleles, they most likely do not arise through alterations of the protein’s catalytic domain. given the previously documented evidence that pstol enhances p uptake under low-p conditions and our evidence of balancing selection at this locus, it is somewhat surprising that we did not detect any obvious effects of pstol variation on plant growth in the greenhouse. on the other hand, at least two other recent studies have also found no clear correlation between pstol genotypes and plant performance in high- and low-p environments. in a survey of indica rice varieties, sarkar et al. reported that the pup locus was only present in three of the nine varieties showing greatest efficiency in p uptake in low-and high-p soils. similarly, mukherjee et al. found no association between pstol presence and p-deficiency tolerance in a study of diverse indian varieties. the latter study also assessed the effects of pstol presence on low-p tolerance using a panel of recombinant inbred lines and again found no evidence that the gene confers increased p-uptake efficiency. like all physiological responses to abiotic stress, p uptake efficiency is undoubtedly a complex trait, and as such the phenotypic effects of pstol would be expected to vary across genetic backgrounds and environments. it seems likely that the variable effects reported for pstol in different studies are a manifestation of these gene-by-gene and gene-by-environment interactions. balancing selection the phylogenetic distribution of putatively functional and non-functional pstol alleles suggests that selection has maintained this functional polymorphism across multiple oryza species. trans-specific polymorphisms are typically established when balancing selection has been acting on a gene for very long periods of time, indicating that the same differential selective pressures may have been acting in this genus long before the domestication of asian rice. examples of trans-specific polymorphism are rare; self-incompatibility loci provide one of the best examples of long term balancing selection in plants. however, the selective pressures acting on self-incompatibility are due to negative frequency dependent selection, where rare alleles have a selective advantage over common alleles. balancing selection on abiotic stress genes is instead likely due to differential selection based on variable environmental conditions. one example of long term balancing selection on abiotic stress genes was found in two species of wild tomato solanum peruvianum and s. chilense. the c-repeat binding factor is involved in plant responses to cold and drought, environmental conditions that vary across solanum species ranges. mboup et al. found trans-species polymorphism at cbf that maintains signatures of balancing selection. environmental heterogeneity is likely driving balancing selection in both cbf and pstol and is likely not limited to these two examples of abiotic stress tolerance genes. genome-wide studies of polymorphism in plants have identified several genomic regions that have signatures of balancing selection, but few studies have looked across species for trans-species polymorphisms in closely related plants.  agricultural environments exhibit a range of abiotic stresses including drought, low nutrient availability and high salinity. geographical variation in these stresses can lead to balancing selection at loci that underlie adaptation to these stresses. our findings for pstol suggest that adaptive variation at this gene has been a target of environmentally heterogeneous selection not only within cultivated rice, but also on a larger time frame and geographical scale that encompasses multiple aa genome oryza species. while we did not observe clear phenotypic associations with pstol molecular variation in the greenhouse, the molecular signature of balancing selection we detect at this locus suggests that functional variation is important in the field — both in cultivated rice and wild oryza species. it is likely that other genes involved in abiotic stress response will also show signatures of balancing selection in crop plants and their wild relatives. 
for the subset of samples that carry pstol haplotype sequences suggest long-term selective maintenance of functional alleles, but with repeated evolution of loss-of-function alleles through premature stops and frameshift mutations. the loss-of-function alleles have evolved convergently in multiple rice species and cultivated rice varieties. we find that the presence/absence polymorphism spans cultivated, weedy and wild asian rice groups. greenhouse assessments of plant growth under low- and high-phosphorus conditions did not reveal significant associations with pstol genotype variation; however, the striking signature of balancing selection at this locus suggests that further phenotypic characterizations of pstol allelic variants is warranted and may be useful for crop improvement. the ability to grow in phosphorus-depleted soils is an important trait for rice cultivation in many world regions, especially in the tropics. these findings suggest balancing selection for both functional and non-functional pstol alleles that predates and transcends asian rice domestication, a pattern that may reflect fitness tradeoffs associated with geographical variation in soil phosphorus content. we assessed pstol presence/absence in a geographically diverse sample of wild, domesticated and weedy rice and sequenced the gene in samples where it is present. balancing selectioncrop domesticationgene presence/absence variation phosphorus starvation tolerance weedy riceabiotic stresshttp://dx.doi.org/ /100000076directorate for biological sciences1032023olsen kenneth m. issue-copyright-statement© the author 2016
 the ability to grow in phosphorus-depleted soils is an important trait for rice cultivation in many world regions, especially in the tropics. the phosphorus starvation tolerance gene has been identified as underlying the ability of some cultivated rice varieties to grow under low-phosphorus conditions; however, the gene is absent from other varieties. we assessed pstol presence/absence in a geographically diverse sample of wild, domesticated and weedy rice and sequenced the gene in samples where it is present.  we find that the presence/absence polymorphism spans cultivated, weedy and wild asian rice groups. for the subset of samples that carry pstol haplotype sequences suggest long-term selective maintenance of functional alleles, but with repeated evolution of loss-of-function alleles through premature stops and frameshift mutations. the loss-of-function alleles have evolved convergently in multiple rice species and cultivated rice varieties. greenhouse assessments of plant growth under low- and high-phosphorus conditions did not reveal significant associations with pstol genotype variation; however, the striking signature of balancing selection at this locus suggests that further phenotypic characterizations of pstol allelic variants is warranted and may be useful for crop improvement.  these findings suggest balancing selection for both functional and non-functional pstol alleles that predates and transcends asian rice domestication, a pattern that may reflect fitness tradeoffs associated with geographical variation in soil phosphorus content. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords balancing selectioncrop domesticationgene presence/absence variation phosphorus starvation tolerance weedy riceabiotic stresshttp://dx.doi.org/ /100000076directorate for biological sciences1032023olsen kenneth m. issue-copyright-statement© the author 2016 