Genome copy number predicts extreme evolutionary rate variation in plant mitochondrial DNA.

Nuclear and organellar genomes can evolve at vastly different rates despite occupying the same cell. In most bilaterian animals, mitochondrial DNA (mtDNA) evolves faster than nuclear DNA, whereas this trend is generally reversed in plants. However, in some exceptional angiosperm clades, mtDNA substitution rates have increased up to 5,000-fold compared with closely related lineages. The mechanisms responsible for this acceleration are generally unknown. Because plants rely on homologous recombination to repair mtDNA damage, we hypothesized that mtDNA copy numbers may predict evolutionary rates, as lower copy numbers may provide fewer templates for such repair mechanisms. In support of this hypothesis, we found that copy number explains 47% of the variation in synonymous substitution rates of mtDNA across 60 diverse seed plant species representing ~300 million years of evolution. Copy number was also negatively correlated with mitogenome size, which may be a cause or consequence of mutation rate variation. Both relationships were unique to mtDNA and not observed in plastid DNA. These results suggest that homologous recombinational repair plays a role in driving mtDNA substitution rates in plants and may explain variation in mtDNA evolution more broadly across eukaryotes. Our findings also contribute to broader questions about the relationships between mutation rates, genome size, selection efficiency, and the drift-barrier hypothesis.

Sorting of mitochondrial and plastid heteroplasmy in Arabidopsis is extremely rapid and depends on MSH1 activity.

The fate of new mitochondrial and plastid mutations depends on their ability to persist and spread among the numerous organellar genome copies within a cell (heteroplasmy). The extent to which heteroplasmies are transmitted across generations or eliminated through genetic bottlenecks is not well understood in plants, in part because their low mutation rates make these variants so infrequent. Disruption of MutS Homolog 1 (MSH1), a gene involved in plant organellar DNA repair, results in numerous de novo point mutations, which we used to quantitatively track the inheritance of single nucleotide variants in mitochondrial and plastid genomes in Arabidopsis. We found that heteroplasmic sorting (the fixation or loss of a variant) was rapid for both organelles, greatly exceeding rates observed in animals. In msh1 mutants, plastid variants sorted faster than those in mitochondria and were typically fixed or lost within a single generation. Effective transmission bottleneck sizes (N) for plastids and mitochondria were N ∼ 1 and 4, respectively. Restoring MSH1 function further increased the rate of heteroplasmic sorting in mitochondria (N ∼ 1.3), potentially because of its hypothesized role in promoting gene conversion as a mechanism of DNA repair, which is expected to homogenize genome copies within a cell. Heteroplasmic sorting also favored GC base pairs. Therefore, recombinational repair and gene conversion in plant organellar genomes can potentially accelerate the elimination of heteroplasmies and bias the outcome of this sorting process.

Rewiring of Aminoacyl-tRNA Synthetase Localization and Interactions in Plants With Extensive Mitochondrial tRNA Gene Loss.

The number of tRNAs encoded in plant mitochondrial genomes varies considerably. Ongoing loss of bacterial-like mitochondrial tRNA genes in many lineages necessitates the import of nuclear-encoded counterparts that share little sequence similarity. Because tRNAs are involved in highly specific molecular interactions, this replacement process raises questions about the identity and trafficking of enzymes necessary for the maturation and function of newly imported tRNAs. In particular, the aminoacyl-tRNA synthetases (aaRSs) that charge tRNAs are usually divided into distinct classes that specialize on either organellar (mitochondrial and plastid) or nuclear-encoded (cytosolic) tRNAs. Here, we investigate the evolution of aaRS subcellular localization in a plant lineage (Sileneae) that has experienced extensive and rapid mitochondrial tRNA loss. By analyzing full-length mRNA transcripts (PacBio Iso-Seq), we found predicted retargeting of many ancestrally cytosolic aaRSs to the mitochondrion and confirmed these results with colocalization microscopy assays. However, we also found cases where aaRS localization does not appear to change despite functional tRNA replacement, suggesting evolution of novel interactions and charging relationships. Therefore, the history of repeated tRNA replacement in Sileneae mitochondria reveals that differing constraints on tRNA/aaRS interactions may determine which of these alternative coevolutionary paths is used to maintain organellar translation in plant cells.

Stochastic organelle genome segregation through Arabidopsis development and reproduction.

Organelle DNA (oDNA) in mitochondria and plastids is vital for plant (and eukaryotic) life. Selection against damaged oDNA is mediated in part by segregation - sorting different oDNA types into different cells in the germline. Plants segregate oDNA very rapidly, with oDNA recombination protein MSH1 a key driver of this segregation, but we have limited knowledge of the dynamics of this segregation within plants and between generations. Here, we reveal how oDNA evolves through Arabidopsis thaliana development and reproduction. We combine stochastic modelling, Bayesian inference, and model selection with new and existing tissue-specific oDNA measurements from heteroplasmic Arabidopsis plant lines through development and between generations. Segregation proceeds gradually but continually during plant development, with a more rapid increase between inflorescence formation and the next generation. When MSH1 is compromised, the majority of observed segregation can be achieved through partitioning at cell divisions. When MSH1 is functional, mtDNA segregation is far more rapid; we show that increased oDNA gene conversion is a plausible mechanism quantitatively explaining this acceleration. These findings reveal the quantitative, time-dependent details of oDNA segregation in Arabidopsis. We also discuss the support for different models of the plant germline provided by these observations.

MSH1 is required for maintenance of the low mutation rates in plant mitochondrial and plastid genomes.

Mitochondrial and plastid genomes in land plants exhibit some of the slowest rates of sequence evolution observed in any eukaryotic genome, suggesting an exceptional ability to prevent or correct mutations. However, the mechanisms responsible for this extreme fidelity remain unclear. We tested seven candidate genes involved in cytoplasmic DNA replication, recombination, and repair (POLIA, POLIB, MSH1, RECA3, UNG, FPG, and OGG1) for effects on mutation rates in the model angiosperm Arabidopsis thaliana by applying a highly accurate DNA sequencing technique (duplex sequencing) that can detect newly arisen mitochondrial and plastid mutations even at low heteroplasmic frequencies. We find that disrupting MSH1 (but not the other candidate genes) leads to massive increases in the frequency of point mutations and small indels and changes to the mutation spectrum in mitochondrial and plastid DNA. We also used droplet digital PCR to show transmission of de novo heteroplasmies across generations in msh1 mutants, confirming a contribution to heritable mutation rates. This dual-targeted gene is part of an enigmatic lineage within the mutS mismatch repair family that we find is also present outside of green plants in multiple eukaryotic groups (stramenopiles, alveolates, haptophytes, and cryptomonads), as well as certain bacteria and viruses. MSH1 has previously been shown to limit ectopic recombination in plant cytoplasmic genomes. Our results point to a broader role in recognition and correction of errors in plant mitochondrial and plastid DNA sequence, leading to greatly suppressed mutation rates perhaps via initiation of double-stranded breaks and repair pathways based on faithful homologous recombination.

Migration through a Major Andean Ecogeographic Disruption as a Driver of Genetic and Phenotypic Diversity in a Wild Tomato Species.

Evolutionary dynamics at the population level play a central role in creating the diversity of life on our planet. In this study, we sought to understand the origins of such population-level variation in mating systems and defensive acylsugar chemistry in Solanum habrochaites-a wild tomato species found in diverse Andean habitats in Ecuador and Peru. Using Restriction-site-Associated-DNA-Sequencing (RAD-seq) of 50 S. habrochaites accessions, we identified eight population clusters generated via isolation and hybridization dynamics of 4-6 ancestral populations. Detailed characterization of mating systems of these clusters revealed emergence of multiple self-compatible (SC) groups from progenitor self-incompatible populations in the northern part of the species range. Emergence of these SC groups was also associated with fixation of deleterious alleles inactivating acylsugar acetylation. The Amotape-Huancabamba Zone-a geographical landmark in the Andes with high endemism and isolated microhabitats-was identified as a major driver of differentiation in the northern species range, whereas large geographical distances contributed to population structure and evolution of a novel SC group in the central and southern parts of the range, where the species was also inferred to have originated. Findings presented here highlight the role of the diverse ecogeography of Peru and Ecuador in generating population differentiation, and enhance our understanding of the microevolutionary processes that create biological diversity.

Spread of self-compatibility constrained by an intrapopulation crossing barrier.

Mating system transitions from self-incompatibility (SI) to self-compatibility (SC) are common in plants. In the absence of high levels of inbreeding depression, SC alleles are predicted to spread due to transmission advantage and reproductive assurance. We characterized mating system and pistil-expressed SI factors in 20 populations of the wild tomato species Solanum habrochaites from the southern half of the species range. We found that a single SI to SC transition is fixed in populations south of the Rio Chillon valley in central Peru. In these populations, SC correlated with the presence of the hab-6 S-haplotype that encodes a low activity S-RNase protein. We identified a single population segregating for SI/SC and hab-6. Intrapopulation crosses showed that hab-6 typically acts in the expected codominant fashion to confer SC. However, we found one specific S-haplotype (hab-10) that consistently rejects pollen of the hab-6 haplotype, and results in SI hab-6/hab-10 heterozygotes. We suggest that the hab-10 haplotype could act as a genetic mechanism to stabilize mixed mating in this population by presenting a disadvantage for the hab-6 haplotype. This barrier may represent a mechanism allowing for the persistence of SI when an SC haplotype appears in or invades a population.

Transcriptomic analysis links gene expression to unilateral pollen-pistil reproductive barriers.

BACKGROUND: Unilateral incompatibility (UI) is an asymmetric reproductive barrier that unidirectionally prevents gene flow between species and/or populations. UI is characterized by a compatible interaction between partners in one direction, but in the reciprocal cross fertilization fails, generally due to pollen tube rejection by the pistil. Although UI has long been observed in crosses between different species, the underlying molecular mechanisms are only beginning to be characterized. The wild tomato relative Solanum habrochaites provides a unique study system to investigate the molecular basis of this reproductive barrier, as populations within the species exhibit both interspecific and interpopulation UI. Here we utilized a transcriptomic approach to identify genes in both pollen and pistil tissues that may be key players in UI. RESULTS: We confirmed UI at the pollen-pistil level between a self-incompatible population and a self-compatible population of S. habrochaites. A comparison of gene expression between pollinated styles exhibiting the incompatibility response and unpollinated controls revealed only a small number of differentially expressed transcripts. Many more differences in transcript profiles were identified between UI-competent versus UI-compromised reproductive tissues. A number of intriguing candidate genes were highly differentially expressed, including a putative pollen arabinogalactan protein, a stylar Kunitz family protease inhibitor, and a stylar peptide hormone Rapid ALkalinization Factor. Our data also provide transcriptomic evidence that fundamental processes including reactive oxygen species (ROS) signaling are likely key in UI pollen-pistil interactions between both populations and species. CONCLUSIONS: Gene expression analysis of reproductive tissues allowed us to better understand the molecular basis of interpopulation incompatibility at the level of pollen-pistil interactions. Our transcriptomic analysis highlighted specific genes, including those in ROS signaling pathways that warrant further study in investigations of UI. To our knowledge, this is the first report to identify candidate genes involved in unilateral barriers between populations within a species.

Mating system transitions in Solanum habrochaites impact interactions between populations and species.

In plants, transitions in mating system from outcrossing to self-fertilization are common; however, the impact of these transitions on interspecific and interpopulation reproductive barriers is not fully understood. We examined the consequences of mating system transition for reproductive barriers in 19 populations of the wild tomato species Solanum habrochaites. We identified S. habrochaites populations with self-incompatible (SI), self-compatible (SC) and mixed population (MP) mating systems, and characterized pollen-pistil interactions among S. habrochaites populations and between S. habrochaites and other tomato species. We examined the relationship between mating system, floral morphology, interspecific and interpopulation compatibility and pistil SI factors. We documented five distinct phenotypic groups by combining reproductive behavior with molecular data. Transitions from SI to MP were not associated with weakened interspecific reproductive barriers or loss of known pistil SI factors. However, transitions to SC at the northern range margin were accompanied by loss of S-RNase, smaller flowers, and weakened (or absent) interspecific pollen-pistil barriers. Finally, we identified a subset of SC populations that exhibited a partial interpopulation reproductive barrier with central SI populations. Our results support the hypothesis that shifts in mating system, followed by additional loss-of-function mutations, impact reproductive barriers within and between species.

Interspecific reproductive barriers between sympatric populations of wild tomato species (Solanum section Lycopersicon).

PREMISE OF THE STUDY: Interspecific reproductive barriers (IRBs) often prevent hybridization between closely related species in sympatry. In the tomato clade (Solanum section Lycopersicon), interspecific interactions between natural sympatric populations have not been evaluated previously. In this study, we assessed IRBs between members of the tomato clade from nine sympatric sites in Peru. METHODS: Coflowering was assessed at sympatric sites in Peru. Using previously collected seeds from sympatric sites in Peru, we evaluated premating prezygotic (floral morphology), postmating prezygotic (pollen-tube growth), and postzygotic barriers (fruit and seed development) between sympatric species in common gardens. Pollen-tube growth and seed development were examined in reciprocal crosses between sympatric species. KEY RESULTS: We confirmed coflowering of sympatric species at five sites in Peru. We found three types of postmating prezygotic IRBs during pollen-pistil interactions: (1) unilateral pollen-tube rejection between pistils of self-incompatible species and pollen of self-compatible species; (2) potential conspecific pollen precedence in a cross between two self-incompatible species; and (3) failure of pollen tubes to target ovules. In addition, we found strong postzygotic IRBs that prevented normal seed development in 11 interspecific crosses, resulting in seed-like structures containing globular embryos and aborted endosperm and, in some cases, overgrown endothelium. Viable seed and F1 hybrid plants were recovered from three of 19 interspecific crosses. CONCLUSIONS: We have identified diverse prezygotic and postzygotic IRBs that would prevent hybridization between sympatric wild tomato species, but interspecific hybridization is possible in a few cases.

Disruption of recombination machinery alters the mutational landscape in plant organellar genomes.

Land plant organellar genomes have extremely low rates of point mutation yet also experience high rates of recombination and genome instability. Characterizing the molecular machinery responsible for these patterns is critical for understanding the evolution of these genomes. While much progress has been made towards understanding recombination activity in land plant organellar genomes, the relationship between recombination pathways and point mutation rates remains uncertain. The organellar targeted mutS homolog MSH1 has previously been shown to suppress point mutations as well as non-allelic recombination between short repeats in Arabidopsis thaliana. We therefore implemented high-fidelity Duplex Sequencing to test if other genes that function in recombination and maintenance of genome stability also affect point mutation rates. We found small to moderate increases in the frequency of single nucleotide variants (SNVs) and indels in mitochondrial and/or plastid genomes of A. thaliana mutant lines lacking radA, recA1, or recA3. In contrast, osb2 and why2 mutants did not exhibit an increase in point mutations compared to wild type (WT) controls. In addition, we analyzed the distribution of SNVs in previously generated Duplex Sequencing data from A. thaliana organellar genomes and found unexpected strand asymmetries and large effects of flanking nucleotides on mutation rates in WT plants and msh1 mutants. Finally, using long-read Oxford Nanopore sequencing, we characterized structural variants in organellar genomes of the mutant lines and show that different short repeat sequences become recombinationally active in different mutant backgrounds. Together, these complementary sequencing approaches shed light on how recombination may impact the extraordinarily low point mutation rates in plant organellar genomes.

Expansion of the MutS Gene Family in Plants.

The MutS gene family is distributed across the tree of life and is involved in recombination, DNA repair, and protein translation. Multiple evolutionary processes have expanded the set of MutS genes in plants relative to other eukaryotes. Here, we investigate the origins and functions of these plant-specific genes. Land plants, green algae, red algae, and glaucophytes share cyanobacterial-like MutS1 and MutS2 genes that presumably were gained via plastid endosymbiotic gene transfer. MutS1 was subsequently lost in some taxa, including seed plants, whereas MutS2 was duplicated in Viridiplantae (i.e., land plants and green algae) with widespread retention of both resulting paralogs. Viridiplantae also have two anciently duplicated copies of the eukaryotic MSH6 gene (i.e., MSH6 and MSH7) and acquired MSH1 via horizontal gene transfer - potentially from a nucleocytovirus. Despite sharing the same name, "plant MSH1" is not directly related to the gene known as MSH1 in some fungi and animals, which may be an ancestral eukaryotic gene acquired via mitochondrial endosymbiosis and subsequently lost in most eukaryotic lineages. There has been substantial progress in understanding the functions of MSH1 and MSH6/MSH7 in plants, but the roles of the cyanobacterial-like MutS1 and MutS2 genes remain uncharacterized. Known functions of bacterial homologs and predicted protein structures, including fusions to diverse nuclease domains, provide hypotheses about potential molecular mechanisms. Because most plant-specific MutS proteins are targeted to the mitochondria and/or plastids, the expansion of this family appears to have played a large role in shaping plant organelle genetics.

Avoiding misleading estimates using mtDNA heteroplasmy statistics to study bottleneck size and selection.

Mitochondrial DNA heteroplasmy samples can shed light on vital developmental and genetic processes shaping mitochondrial DNA populations. The sample means and sample variance of a set of heteroplasmy observations are typically used both to estimate bottleneck sizes and to perform fits to the theoretical "Kimura" distribution in seeking evidence for mitochondrial DNA selection. However, each of these applications raises problems. Sample statistics do not generally provide optimal fits to the Kimura distribution and so can give misleading results in hypothesis testing, including false positive signals of selection. Using sample variance can give misleading results for bottleneck size estimates, particularly for small samples. These issues can and do lead to false positive results for mitochondrial DNA mechanisms-all published experimental datasets we re-analyzed, reported as displaying departures from the Kimura model, do not in fact give evidence for such departures. Here we outline a maximum likelihood approach that is simple to implement computationally and addresses all of these issues. We advocate the use of maximum likelihood fits and explicit hypothesis tests, not fits and Kolmogorov-Smirnov tests via summary statistics, for ongoing work with mitochondrial DNA heteroplasmy.

Chromosome-Level Genome Assembly for the Angiosperm Silene conica.

The angiosperm genus Silene has been the subject of extensive study in the field of ecology and evolution, but the availability of high-quality reference genome sequences has been limited for this group. Here, we report a chromosome-level assembly for the genome of Silene conica based on Pacific Bioscience HiFi, Hi-C, and Bionano technologies. The assembly produced 10 scaffolds (1 per chromosome) with a total length of 862 Mb and only ∼1% gap content. These results confirm previous observations that S. conica and its relatives have a reduced base chromosome number relative to the genus's ancestral state of 12. Silene conica has an exceptionally large mitochondrial genome (>11 Mb), predominantly consisting of sequence of unknown origins. Analysis of shared sequence content suggests that it is unlikely that transfer of nuclear DNA is the primary driver of this mitochondrial genome expansion. More generally, this assembly should provide a valuable resource for future genomic studies in Silene, including comparative analyses with related species that recently evolved sex chromosomes.

Chromosome-level genome assembly for the angiosperm Silene conica.

The angiosperm genus Silene has been the subject of extensive study in the field of ecology and evolution, but the availability of high-quality reference genome sequences has been limited for this group. Here, we report a chromosome-level assembly for the genome of Silene conica based on PacBio HiFi, Hi-C and Bionano technologies. The assembly produced 10 scaffolds (one per chromosome) with a total length of 862 Mb and only ~1% gap content. These results confirm previous observations that S. conica and its relatives have a reduced base chromosome number relative to the genus's ancestral state of 12. Silene conica has an exceptionally large mitochondrial genome (>11 Mb), predominantly consisting of sequence of unknown origins. Analysis of shared sequence content suggests that it is unlikely that transfer of nuclear DNA is the primary driver of this mitochondrial genome expansion. More generally, this assembly should provide a valuable resource for future genomic studies in Silene, including comparative analyses with related species that recently evolved sex chromosomes. Significance: Whole-genome sequences have been largely lacking for species in the genus Silene even though these flowering plants have been used for studying ecology, evolution, and genetics for over a century. Here, we address this gap by providing a high-quality nuclear genome assembly for S. conica, a species known to have greatly accelerated rates of sequence and structural divergence in its mitochondrial and plastid genomes. This resource will be valuable in understanding the coevolutionary interactions between nuclear and cytoplasmic genomes and in comparative analyses across this highly diverse genus.

Pollen-Pistil Interactions as Reproductive Barriers.

Pollen-pistil interactions serve as important prezygotic reproductive barriers that play a critical role in mate selection in plants. Here, we highlight recent progress toward understanding the molecular basis of pollen-pistil interactions as reproductive isolating barriers. These barriers can be active systems of pollen rejection, or they can result from a mismatch of required male and female factors. In some cases, the barriers are mechanistically linked to self-incompatibility systems, while others represent completely independent processes. Pollen-pistil reproductive barriers can act as soon as pollen is deposited on a stigma, where penetration of heterospecific pollen tubes is blocked by the stigma papillae. As pollen tubes extend, the female transmitting tissue can selectively limit growth by producing cell wall-modifying enzymes and cytotoxins that interact with the growing pollen tube. At ovules, differential pollen tube attraction and inhibition of sperm cell release can act as barriers to heterospecific pollen tubes.

Long-read transcriptome and other genomic resources for the angiosperm Silene noctiflora.

The angiosperm genus Silene is a model system for several traits of ecological and evolutionary significance in plants, including breeding system and sex chromosome evolution, host-pathogen interactions, invasive species biology, heavy metal tolerance, and cytonuclear interactions. Despite its importance, genomic resources for this large genus of approximately 850 species are scarce, with only one published whole-genome sequence (from the dioecious species Silene latifolia). Here, we provide genomic and transcriptomic resources for a hermaphroditic representative of this genus (S. noctiflora), including a PacBio Iso-Seq transcriptome, which uses long-read, single-molecule sequencing technology to analyze full-length mRNA transcripts. Using these data, we have assembled and annotated high-quality full-length cDNA sequences for approximately 14,126 S. noctiflora genes and 25,317 isoforms. We demonstrated the utility of these data to distinguish between recent and highly similar gene duplicates by identifying novel paralogous genes in an essential protease complex. Furthermore, we provide a draft assembly for the approximately 2.7-Gb genome of this species, which is near the upper range of genome-size values reported for diploids in this genus and threefold larger than the 0.9-Gb genome of Silene conica, another species in the same subgenus. Karyotyping confirmed that S. noctiflora is a diploid, indicating that its large genome size is not due to polyploidization. These resources should facilitate further study and development of this genus as a model in plant ecology and evolution.

Detecting de novo mitochondrial mutations in angiosperms with highly divergent evolutionary rates.

Although plant mitochondrial genomes typically show low rates of sequence evolution, levels of divergence in certain angiosperm lineages suggest anomalously high mitochondrial mutation rates. However, de novo mutations have never been directly analyzed in such lineages. Recent advances in high-fidelity DNA sequencing technologies have enabled detection of mitochondrial mutations when still present at low heteroplasmic frequencies. To date, these approaches have only been performed on a single plant species (Arabidopsis thaliana). Here, we apply a high-fidelity technique (Duplex Sequencing) to multiple angiosperms from the genus Silene, which exhibits extreme heterogeneity in rates of mitochondrial sequence evolution among close relatives. Consistent with phylogenetic evidence, we found that Silene latifolia maintains low mitochondrial variant frequencies that are comparable with previous measurements in Arabidopsis. Silene noctiflora also exhibited low variant frequencies despite high levels of historical sequence divergence, which supports other lines of evidence that this species has reverted to lower mitochondrial mutation rates after a past episode of acceleration. In contrast, S. conica showed much higher variant frequencies in mitochondrial (but not in plastid) DNA, consistent with an ongoing bout of elevated mitochondrial mutation rates. Moreover, we found an altered mutational spectrum in S. conica heavily biased towards AT→GC transitions. We also observed an unusually low number of mitochondrial genome copies per cell in S. conica, potentially pointing to reduced opportunities for homologous recombination to accurately repair mismatches in this species. Overall, these results suggest that historical fluctuations in mutation rates are driving extreme variation in rates of plant mitochondrial sequence evolution.

S-RNase Alleles Associated With Self-Compatibility in the Tomato Clade: Structure, Origins, and Expression Plasticity.

The self-incompatibility (SI) system in the Solanaceae is comprised of cytotoxic pistil S-RNases which are countered by S-locus F-box (SLF) resistance factors found in pollen. Under this barrier-resistance architecture, mating system transitions from SI to self-compatibility (SC) typically result from loss-of-function mutations in genes encoding pistil SI factors such as S-RNase. However, the nature of these mutations is often not well characterized. Here we use a combination of S-RNase sequence analysis, transcript profiling, protein expression and reproductive phenotyping to better understand different mechanisms that result in loss of S-RNase function. Our analysis focuses on 12 S-RNase alleles identified in SC species and populations across the tomato clade. In six cases, the reason for gene dysfunction due to mutations is evident. The six other alleles potentially encode functional S-RNase proteins but are typically transcriptionally silenced. We identified three S-RNase alleles which are transcriptionally silenced under some conditions but actively expressed in others. In one case, expression of the S-RNase is associated with SI. In another case, S-RNase expression does not lead to SI, but instead confers a reproductive barrier against pollen tubes from other tomato species. In the third case, expression of S-RNase does not affect self, interspecific or inter-population reproductive barriers. Our results indicate that S-RNase expression is more dynamic than previously thought, and that changes in expression can impact different reproductive barriers within or between natural populations.