Selection of Reliable Reference Genes for Gene Expression Normalization in Sagittaria trifolia.

Real-time quantitative PCR (RT-qPCR) is a method with high sensitivity and convenience that has been extensively used to analyze the expression level of target genes. A reference gene with a highly stable expression is required to ensure the accuracy of experimental results. However, the report on appropriate reference genes in arrowheads (Sagittaria trifolia) is still limited. In this study, eight candidate reference genes (ACT5, UBQ, GAPDH, CYP, NAC, IDH, SLEEPER and PLA) were selected. The candidate genes were employed in a RT-qPCR assay in different tissues at different developmental stages of the same tissue (including corm, leaf and leafstalk) in arrowheads. Five statistical algorithms, GeNorm, NormFinder, BestKeeper, delta cycle threshold (ΔCt) and RefFinder, were used to evaluate the stability of these genes' expressions in order to identify the appropriate reference genes. The results showed that UBQ was the optimum reference gene in leaf, leafstalk, root, stolon and corm, IDH exhibited the most stable expression during the expansion of corm, UBQ and PLA were the most stable reference genes in developmental stages of leaf and leafstalk, respectively. Finally, the reliability of reference genes was further confirmed by the normalization of PDS and EXP1 genes under different arrowhead tissues and developmental stages of corm, respectively. This study constitutes important guidance for the selection of reliable reference genes for analyzing the tissue- and developmental-stage-specific expression of genes in arrowheads.

Mutation at the 197 site and P450-mediated metabolic resistance are involved in bensulfuron-methyl resistance in Sagittaria trifolia.

Sagittaria trifolia control is threatened by the emergence of resistance to acetolactate synthase (ALS)-inhibiting herbicides. Hence, we systematically uncovered the molecular mechanism of resistance to the main herbicide (bensulfuron-methyl) in Liaoning Province from target-site and non-target-site resistance perspectives. The suspected resistant population (TR-1) exhibited high-level resistance. A new amino acid substitution (Pro-197-Ala) in resistant Sagittaria trifolia for ALS was detected, and the molecular docking results showed that the spatial structure of ALS changed significantly after the substitution, manifested by an increase in the number of contacted amino acid residues and the disappearance of hydrogen bonds. Dose-response test of transgenic Arabidopsis thaliana further demonstrated that the Pro-197-Ala substitution conferred bensulfuron-methyl resistance. The assays found that the sensitivity of the ALS enzyme in TR-1 to this herbicide was decreased in vitro; and this population had developed resistance to other types of ALS-inhibiting herbicides. Furthermore, the resistance of TR-1 to bensulfuron-methyl was significantly alleviated after co-treatment with a P450-inhibitor (malathion). TR-1 metabolized bensulfuron-methyl significantly faster than sensitive population (TS-1) did, but this gap was narrowed after malathion treatment. Overall, the resistance of Sagittaria trifolia to bensulfuron-methyl was derived from the mutation of the target-site gene and the enhancement of the P450s-mediated detoxification metabolism.

Contrasting patterns of genetic diversity and differentiation across the continental disjunct range of a sexually polymorphic aquatic plant.

BACKGROUND AND AIMS: Reproductive systems enabling opportunities for self-fertilization influence population genetic structure and play a key role in colonization and genetic differentiation during range expansion. Because of their well-developed powers of dispersal, aquatic plants often have widespread disjunct geographical distributions, providing opportunities to investigate the role of reproductive systems in structuring genetic variation between parts of the range that differ in migration history and ecology. METHODS: We compared reproductive systems and spatial genetic structure of the freshwater aquatic macrophyte Sagittaria latifolia between disjunct western and eastern ranges of North America (NA). Populations of this species are most commonly either monoecious or dioecious. We examined chloroplast DNA haplotype variation to test the hypothesis that the western range of this species represents a secondary colonization from the east, and evaluated the roles of reproductive system differences and geography in structuring contemporary patterns of genetic variation at 11 polymorphic SSR (simple sequence repeat) loci. KEY RESULTS: Chloroplast haplotyping revealed a single haplotype in western NA compared to numerous haplotypes in eastern NA, consistent with a genetic bottleneck during westward migration. Estimates of genetic diversity in eastern NA populations differed significantly between reproductive systems, but this pattern was not evident in the western range. Eastern populations could be reliably assigned to genetic clusters based on their reproductive systems, whereas western populations clustered primarily by geographical location. CONCLUSIONS: The sparser distribution of aquatic habitats in the drier western range of S. latifolia, combined with secondary colonization of this region, probably cause the lower genetic diversity and increased differentiation among populations, possibly overriding the effects of reproductive system evident in the eastern portion of the range. Our findings demonstrate that the complex interplay between migratory history, reproductive systems and habitat availability plays an important role in structuring spatial patterns of genetic variation in disjunct plant populations.

[Sagittaria sagittifolia polysaccharides regulates Nrf2/HO-1 to relieve liver injury caused by multiple heavy metals in vivo and in vitro].

This study explored whether Sagittaria sagittifolia polysaccharides(SSP) activates the nuclear factor erythroid-2-related factor2(Nrf2)/heme oxygenase-1(HO-1) signaling pathway to protect against liver damage jointly induced by multiple heavy metals. First, based on the proportion of dietary intake of six heavy metals in rice available in Beijing market, a heavy metal mixture was prepared for inducing mouse liver injury and HepG2 cell injury. Forty male Kunming mice were divided into five groups: control group, model group, glutathione positive control group, and low-and high-dose SSP groups, with eight mice in each group. After 30 days of intragastric administration, the liver injury in mice was observed by HE staining. In the in vitro experiment, MTT assay was conducted to detect the effects of SSP at 0.25, 0.5, 1, and 2 mg·mL~(-1) on HepG2 cell survival at different time points. The content of alanine transaminase(ALT) and aspartate aminotransferase(AST) in the 48-h cell culture fluid was measured using micro-plate cultivation method, followed by the detection of the change in reactive oxygen species(ROS) content by flow cytometry. The mRNA expression levels of Nrf2 and HO-1 in cells were determined by RT-PCR, and their protein expression by Western blot. HE staining results showed that compared with the model group, the SSP administration groups exhibited significantly alleviated inflammatory cell infiltration and fatty infiltration in the liver, with better outcomes observed in the high-dose SSP group. In the in vitro MTT assay, compared with the model group, SSP at four concentrations all significantly increased the cell survival rate, decreased the ALT, AST, and ROS content(P<0.05), and down-regulated Nrf2 and HO-1 mRNA and protein expression(P<0.05). SSP significantly improves inflammatory infiltration in the liver tissue of mice exposed to a variety of heavy metals and corrects the liver fat degeneration, which may be related to its regulation of the Nrf2/HO-1 signaling pathway, reduction of ROS, and alleviation of oxidative damage.

SMRT and Illumina RNA-Seq Identifies Potential Candidate Genes Related to the Double Flower Phenotype and Unveils SsAP2 as a Key Regulator of the Double-Flower Trait in Sagittaria sagittifolia.

Double flowers are one of the important objectives of ornamental plant breeding. Sagittaria sagittifolia is an aquatic herb in the Alismataceae family that is widely used as an ornamental plant in gardens. However, the reference genome has not been published, and the molecular regulatory mechanism of flower formation remains unclear. In this study, single molecule real-time (SMRT) sequencing technology combined with Illumina RNA-Seq was used to perform a more comprehensive analysis of S. sagittifolia for the first time. We obtained high-quality full-length transcripts, including 53,422 complete open reading frames, and identified 5980 transcription factors that belonged to 67 families, with many MADS-box genes involved in flower formation being obtained. The transcription factors regulated by plant hormone signals played an important role in the development of double flowers. We also identified an AP2 orthologous gene, SsAP2, with a deletion of the binding site for miR172, that overexpressed SsAP2 in S. sagittifolia and exhibited a delayed flowering time and an increased number of petals. This study is the first report of a full-length transcriptome of S. sagittifolia. These reference transcripts will be valuable resources for the analysis of gene structures and sequences, which provide a theoretical basis for the molecular regulatory mechanism governing the formation of double flowers.

Increased spatial-genetic structure in a population of the clonal aquatic plant Sagittaria latifolia (Alismataceae) following disturbance.

The spatial genetic structure (SGS) of plant populations is determined by the outcome of key ecological processes, including pollen and seed dispersal, the intensity of local resource competition among newly recruited plants, and patterns of mortality among established plants. Changes in the magnitude of SGS over time can provide insights into the operation of these processes. We measured SGS in a population of the clonal aquatic plant, Sagittaria latifolia that had been disturbed by flooding, both before and after the flood. Over the four-year interval between measurements, we found substantial changes in the magnitude of SGS. In the first measurement (pre-flood), SGS was weak, even over short distances. By contrast, there was substantial SGS in the second measurement (post-flood), particularly over short distances. This change in SGS was accompanied by near complete turnover in the genotypic composition of the population. The genotypic richness of the population (the number of unique clones scaled by the sample size) was halved over the four-year interval. The clonal subrange-the distances between shoots within clones-also shrank considerably, with more than 5% of shoots having clone-mates at distances >10 m before the flood, but fewer than 5% of shoots having clone-mates at distances beyond 2 m afterwards. Clonal turnover and the re-establishment of SGS in clonal populations are both expected following local extirpation and recruitment. These data reveal the genetic signatures of disturbance and a subsequent flush of seedling recruitment and clonal expansion.

Genetic Diversity and DNA Fingerprints of Three Important Aquatic Vegetables by EST-SSR Markers.

Twenty-two sacred lotus (Nelumbo nucifera), 46 taros (Colocasia esculenta) and 10 arrowheads (Sagittaria trifolia) were used as materials and combined with EST-SSR (expressed sequence tag-simple sequence repeats) primers developed by our laboratory. Core primers were screened from a large number of primers that were able to distinguish all materials with a high frequency of polymorphisms. Six pairs, twenty pairs and three pairs of core primers were screened from sacred lotus, taro, and arrowhead, respectively. The SSR fingerprints of these three important aquatic vegetables, producing 17-, 87- and 14-bit binary molecular identity cards, respectively, were separately determined by using the core primers. Since there were few core primers of sacred lotus and arrowhead, 3 and 9 primer pairs with higher polymorphic information content (PIC), respectively, were selected as candidate primers. These core and candidate primers were used to identify the purities of No.36 space lotus, Shandong 8502 taro and Wuhan arrowhead, which were 93.3% (84/90), 98.9% (89/90) and 100.0% (90/90), respectively. The fingerprints, displayed as binary molecular identification cards of three important aquatic vegetables, were obtained, and their purity was successfully determined with EST-SSR labeling technology. Phylogenetic trees were also constructed to analyze the genetic diversity of 22 sacred lotus, 46 taros and 10 arrowheads. This study classifies and identifies germplasm resources and is an important reference to test the authenticity and variety purity of other aquatic vegetables in the future.

Transcriptome analysis of starch and sucrose metabolism across bulb development in Sagittaria sagittifolia.

Sagittaria sagittifolia L is an important bulb vegetable that has high nutritional and medical value. Bulb formation and development are crucial to Sagittaria sagittifolia; however, its sucrose metabolism is poorly understood and there are a lack of sufficient transcriptomic and genomic data available to fully understand the molecular mechanisms underlying bulb formation and development as well as the bulb transcriptome. Five cDNA libraries were constructed at different developmental stages and sequenced using high-throughput Illumina RNA sequencing. From approximately 63.53 Gb clean reads, a total of 60,884 unigenes, with an average length of 897.34 bp and N50 of 1.368 kb, were obtained. A total of 36,590 unigenes were successfully annotated using five public databases. Across different developmental stages, 4195, 827, 832, 851, and 1494 were differentially expressed in T02, T03, T04, T05, and T06 libraries, respectively. Gene ontology (GO) analysis revealed several differentially-expressed genes (DEGs) associated with catalytic activity, binding, and transporter activity. The Kyoto encyclopedia of genes and genomes (KEGG) revealed that these DEGs are involved in physiological and biochemical processes. RT-qPCR was used to profile the expression of these unigenes and revealed that the expression patterns of the DEGs were consistent with the transcriptome data. In this study, we conducted a comparative gene expression analysis at the transcriptional level using RNA-seq across the different developmental stages of Sagittaria sagittifolia. We identified a set of genes that might contribute to starch and sucrose metabolism, and the genetic mechanisms related to bulblet development were also explored. This study provides important data for future studies of the genetic and molecular mechanisms underlying bulb formation and development in Sagittaria sagittifolia.

The role of hybridization in the evolution of sexual system diversity in a clonal, aquatic plant.

The stable coexistence within populations of females, males, and hermaphrodites (subdioecy) is enigmatic because theoretical models indicate that maintenance of this sexual system involves highly restricted conditions. Subdioecy is more commonly interpreted as a transitory stage along the gynodioecious pathway from hermaphroditism to dioecy. The widespread, North American, aquatic plant Sagittaria latifolia is largely composed of monoecious or dioecious populations; however, subdioecious populations with high frequencies of hermaphrodites (mean frequency = 0.50) characterize the northern range boundary of dioecy in eastern North America. We investigated two hypotheses for the origin of subdioecy in this region. Using polymorphic microsatellite loci, we evaluated whether subdioecy arises through selection on standing genetic variation for male sex inconstancy in dioecious populations, or results from hybridization between monoecious and dioecious populations. We found evidence for both pathways to subdioecy, although hybridization was the more common mechanism, with genetic evidence of admixture in nine of 14 subdioecious populations examined. Hybridization has also played a role in the origin of androdioecious populations in S. latifolia, a mechanism not often considered in the evolution of this rare sexual system. Our study demonstrates how hybridization has the potential to play a role in the diversification of plant sexual systems.

Target-site resistance to bensulfuron-methyl in Sagittaria trifolia L. populations.

Sagittaria trifolia L. is one of the most serious weeds in paddy fields in northeast of China and cannot be controlled effectively by bensulfuron-methyl in recent years. In this study, two suspected resistant S. trifolia populations (R1 and R2) were collected in Liaoning province of China. Whole-plant dose-response studies showed that R1 and R2 were highly resistant to bensulfuron-methyl, with the GR50 R/S ratios of 76.99 and 49.94 respectively. In vitro acetolactate synthase (ALS) assays revealed that resistance was due to reduced sensitivity of the ALS to bensulfuron-methyl inhibition, with I50 R/S ratios of 81.86 and 67.48 for R1 and R2, respectively. Total ALS activity was similar for the S and R2 populations, whereas the R1 population displayed significantly higher ALS activity than did the S population. The mutations Pro-197-Leu and Pro-197-Ser were identified in the ALS gene of the R1 and R2 populations, respectively. This is the first report examining bensulfuron-resistant S. trifolia in Liaoning province, China. The Pro197 mutation is likely responsible for resistance to bensulfuron-methyl in S. trifolia populations.

Clonal genetic structure and diversity in populations of an aquatic plant with combined vs. separate sexes.

Clonality is often implicated in models of the evolution of dioecy, but few studies have explicitly compared clonal structure between plant sexual systems, or between the sexes in dioecious populations. Here, we exploit the occurrence of monoecy and dioecy in clonal Sagittaria latifola (Alismataceae) to evaluate two main hypotheses: (i) clone sizes are smaller in monoecious than dioecious populations, because of constraints imposed on clone size by costs associated with geitonogamy; (ii) in dioecious populations, male clones are larger and flower more often than female clones because of sex-differential reproductive costs. Differences in clone size and flowering could result in discordance between ramet- and genet-based sex ratios. We used spatially explicit sampling to address these hypotheses in 10 monoecious and 11 dioecious populations of S. latifolia at the northern range limit in Eastern North America. In contrast to our predictions, monoecious clones were significantly larger than dioecious clones, probably due to their higher rates of vegetative growth and corm production, and in dioecious populations, there was no difference in clone size between females and males; ramet- and genet-based sex ratios were therefore highly correlated. Genotypic diversity declined with latitude for both sexual systems, but monoecious populations exhibited lower genotypic richness. Differences in life history between the sexual systems of S. latifolia appear to be the most important determinants of clonal structure and diversity.

Identification of differentially expressed genes relevant to corm formation in Sagittaria trifolia.

Sagittaria trifolia is a good model of wetland plants to elucidate the formation of corm. However, few studies have been conducted to uncover the complexity of gene expression involved in corm formation. In this study, high-throughput tag-sequencing based on Solexa Genome Analyzer Platform was applied to monitor the changes in gene expression with three libraries of differentially expressed genes (DEGs) (C1 library: stolon stage, C2 library: initial swelling stage and C3 library: swelling stage) during corm formation in Sagittaria trifolia. Approximately 6.0 million tags were sequenced, and 5854021, 5983454, and 5761079 clean tags including 138319, 116804, and 101739 distinct tags were obtained after removal of low quality tags from each library, respectively. About 46% distinct tags were unambiguous tags mapping to the reference genes, and 33% were unambiguous tag-mapped genes. Totally, 20575, 19807, and 18438 were annotated in C1, C2, and C3 libraries, respectively, after mapping their functions in existing databases. In addition, we found that profiling of gene expression in C1/C2 and C2/C3 libraries were different among most of the selected 20 DEGs. Most DEGs in C1/C2 libraries were relevant to hormone synthesis and response; energy metabolism and stress response, while most of the genes in C2/C3 libraries were involved in carbohydrate metabolism. All up-regulated transcriptional factors and 16 important genes relevant to corm formation in three libraries were also identified. To further analyze the expression of 9 genes, from the results of tag-sequencing, qRT-PCR was applied. In summary, this study provides a comprehensive understanding of gene expression, during the formation of corm in Sagittaria trifolia.

Extra-gynoecial pollen-tube growth in apocarpous angiosperms is phylogenetically widespread and probably adaptive.

• Fusion of floral carpels (syncarpy) in angiosperms is thought to have allowed for significant improvements in offspring quantity and quality in syncarpous species over gymnosperms and apocarpous (free-carpelled) angiosperms. Given the disadvantages of apocarpy, it remains an evolutionary puzzle why many angiosperm lineages with free carpels (apocarpy) have been so successful and why some lineages show reversals to apocarpy. • To investigate whether some advantages of syncarpy may accrue in other ways to apocarpous species, we reviewed previous studies of pollen-tube growth in apocarpous species and also documented pollen-tube growth in nine additional apocarpous species in six families. • Anatomical studies of a scattering of apocarpous paleodicots, monocots, and eudicots show that, after transiting the style, 'extra' pollen tubes exit fully fertilized carpels and grow to other carpels with unfertilized ovules. In many species this occurs via openings in the simple carpels, as we report here for Sagittaria potamogetifolia, Sagittaria pygmaea, Sedum lineare, and Schisandra sphenanthera. • The finding that extra-gynoecial pollen-tube growth is widespread in apocarpous species eliminates the possibility of a major fitness cost of apocarpy relative to syncarpy and may help to explain the persistence of, and multiple reversals to, apocarpy in the evolutionary history of angiosperms.

Development and characterization of microsatellite markers for Sagittaria trifolia var. sinensis (Alismataceae).

PREMISE OF THE STUDY: Microsatellite markers were developed for the aquatic plant Sagittaria trifolia var. sinensis to assess its genetic diversity and population structure. Cross-species transferability was assayed in eight congeneric species. METHODS AND RESULTS: Seventeen microsatellite markers were isolated and characterized in Sagittaria trifolia var. sinensis using Fast Isolation by AFLP of Sequence COntaining Repeats (FIASCO) protocol. Across the evaluated populations, 14 of the markers showed polymorphisms with 3 to 11 alleles per locus; the observed and expected heterozygosity (H(o) and H(E)) ranged from 0.0000 to 0.6364 and from 0.0000 to 0.8386, respectively. Nine of the loci were successfully amplified in the congeneric species. CONCLUSIONS: These markers will be useful for further investigation of population genetics in Sagittaria trifolia var. sinensis and related research in Sagittaria species.

Phylogeography of a marsh herb Sagittaria trifolia (Alismataceae) in China inferred from cpDNA atpB-rbcL intergenic spacers.

Sagittaria trifolia L. is a perennial, erect herb that is confined to ponds, rice fields, ditches, and freshwater wetlands. Using chloroplast DNA (cpDNA) atpB-rbcL intergenic spacer sequences, we studied the phylogeographic pattern and demographic history of S. trifolia with 108 samples from 42 populations representing the entire geographic range in China. Twenty-seven haplotypes were characterized and two of them were widely distributed in the populations. In the minimum-spanning network, all tip haplotypes were unique to a particular population, while the interior nodes represented widespread haplotypes. Nested clade analysis (NCA) of cpDNA haplotypes indicated that long distance dispersal characterized the post-glacial recolonization of S. trifolia in China. No specific refugia areas were suggested because genetic differentiation was low among the sampled regions and among populations within regions although a large number of the haplotypes were unique to a single population. The present data support that the unique haplotypes in individual population most likely represent recent mutational derivatives after long distance dispersal rather than the relics in refugia. These results for S. trifolia represent the first phylogeographic analysis of a widespread marsh herb in China and support the importance of long distance dispersal events in the post-glacial migrations of plants.

Expression of alpha-expansin and xyloglucan endotransglucosylase/hydrolase genes associated with shoot elongation enhanced by anoxia, ethylene and carbon dioxide in arrowhead (Sagittaria pygmaea Miq.) tubers.

BACKGROUND AND AIMS: Shoot elongation of arrowhead tubers (Sagittaria pygmaea Miq.) is stimulated by anoxia, ethylene and CO2. The aim of this study was to characterize anoxic elongation by comparison with elongation stimulated by ethylene and CO2. METHODS: The effects of the inhibitors aminoethoxyvinylglycine (AVG) as an ethylene biosynthesis inhibitor, 1-methylcyclopropene (1-MCP) as a potent inhibitor of ethylene action, and pyrazol, an inhibitor of alcohol dehydrogenase, on shoot elongation were examined. Moreover, the effects of these gaseous factors on expression of genes possibly involved in modification of cell wall architecture were examined by polymerase chain reaction (PCR) methods. KEY RESULTS AND CONCLUSIONS: In air, promotion by 5% CO2 and 5 microL L-1 ethylene of shoot elongation occurred. At 1% O2, ethylene also stimulated shoot elongation but CO2 did not. Pyrazol inhibited shoot elongation in hypoxia but not in normoxia, suggesting that alcohol fermentation contributes to elongation enhanced by hypoxia. AVG and 1-MCP partially prevented shoot elongation both in normoxia and in hypoxia, but they did not have significant effects in anoxia, suggesting that endogenous ethylene acts as a stimulator of shoot elongation in normoxia and in hypoxia but not in anoxia. Ethylene is not involved in anoxia-enhanced elongation. We cloned four cDNAs (SpEXPA1, 2, 3 and 4) encoding alpha-expansin (EXPA) and five cDNAs (SpXTH1, 2, 3, 4 and 5) encoding xyloglucan endotransglucosylase/hydrolase (XTH) from shoots of arrowhead tubers. The transcript levels of SpEXPA1 and 2 were increased by anoxia and those of SpEXPA2 were increased by 5% CO2. Ethylene slightly elevated the level of SpEXPA4 transcripts. Anoxia enhanced the transcript levels of SpXTH1 and 4; neither ethylene nor CO2 had any effect. CO2 enhanced transcript levels of SpXTH3 and depressed those of SpXTH5. Ethylene decreased transcript levels of SpXTH5. These results suggest that four SpEXPA genes and five SpXTH genes are differently responsive to anoxia, CO2 and ethylene. Enhancement of SpEXPA1 and 2, and SpXTH1 and 4 transcript levels suggests that these gene products are involved in anoxic shoot elongation through modification of cell wall architecture.

Chloroplast haplotype variation among monoecious and dioecious populations of Sagittaria latifolia (Alismataceae) in eastern North America.

Aquatic plants commonly have extensive geographical distributions, implying few restrictions to dispersal. Here we investigate the postglacial history of an aquatic plant with contrasting sexual systems (monoecy and dioecy), which are predicted to affect dispersal ability. We examined the distribution of cpDNA haplotypes using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) among 76 populations (32 monoecious, 38 dioecious, two mixed and four undetermined populations) of Sagittaria latifolia sampled throughout eastern North America. We also use these data to investigate the polarity of the evolutionary transition between monoecy and dioecy. Using PCR-RFLP, we identified eight cpDNA haplotypes. All haplotypes were found in unglaciated areas of the species' range, clustered primarily in the southeastern United States, providing evidence that glacial refugia probably occurred in this area. Genetic diversity (hT) was more than six times greater among monoecious compared to dioecious populations. All seven of the haplotypes for which the sexual system could be determined were represented among monoecious populations. In contrast, only four haplotypes were detected in dioecious populations and 94% of individuals from dioecious populations possessed a single haplotype. Monoecious populations possessing this widespread haplotype were restricted to the southern portion of the range, indicating that dioecy probably originated in this region and then spread northwards. The distribution of cpDNA haplotypes in dioecious populations represents a subset of the variation found in monoecious populations, a pattern expected if dioecy has evolved from monoecy in S. latifolia.