Global transcriptome and gene co-expression network analyses on the development of distyly in Primula oreodoxa.

Distyly is a genetically controlled flower polymorphism that has intrigued both botanists and evolutionary biologists ever since Darwin's time. Despite extensive reports on the pollination and evolution of distylous systems, the genetic basis and mechanism of molecular regulation remain unclear. In the present study, comparative transcriptome profiling was conducted in primrose (Primula oreodoxa), the prime research model for heterostyly. Thirty-six transcriptomes were sequenced for styles at different stages and corolla tube in the three morphs of P. oreodoxa. Large numbers of differentially expressed genes (DEGs) were detected in the transcriptomes of styles across different morphs. Several transcription factors (TFs) and phytohormone metabolism-related genes were highlighted in S-morphs. A growing number of genes showed differential expression patterns along with the development of styles, suggesting that the genetic control of distyly may be more complicated than ever expected. Analysis of co-expression networks and module-trait relationships identified modules significantly associated with style development. CYP734A50, a key S-locus gene whose products degrade brassinosteroids, was co-expressed with many genes in the module and showed significant negative association with style length. In addition, crucial TFs involved in phytohormone signaling pathways were found to be connected with CYP734A50 in the co-expression module. Our global transcriptomic analysis has identified DEGs that are potentially involved in regulation of style length in P. oreodoxa, and may shed light on the evolution and broad biological processes of heterostyly.

Genetics of distyly and homostyly in a self-compatible Primula.

The transition from outcrossing to selfing through the breakdown of distyly to homostyly has occurred repeatedly among families of flowering plants. Homostyles can originate by major gene changes at the S-locus linkage group, or by unlinked polygenic modifiers. Here, we investigate the inheritance of distyly and homostyly in Primula oreodoxa, a subalpine herb endemic to Sichuan, China. Controlled self- and cross-pollinations confirmed that P. oreodoxa unlike most heterostylous species is fully self-compatible. Segregation patterns indicated that the inheritance of distyly is governed by a single Mendelian locus with the short-styled morph carrying at least one dominant S-allele (S-) and long-styled plants homozygous recessive (ss). Crossing data were consistent with a model in which homostyly results from genetic changes at the distylous linkage group, with the homostylous allele (Sh) dominant to the long-styled allele (s), but recessive to the short-styled allele (S). Progeny tests of open-pollinated seed families revealed high rates of intermorph mating in the L-morph but considerable selfing and possibly intramorph mating in the S-morph and in homostyles. S-morph plants homozygous at the S-locus (SS) occurred in several populations but may experience viability selection. The crossing data from distylous and homostylous plants are consistent with either recombination at the S-locus governing distyly, or mutation at gene(s) controlling sex-organ height; both models predict the same patterns of segregation. Recent studies on the molecular genetics of distyly in Primula demonstrating the hemizygous nature of genes at the S-locus make it more likely that homostyles have resulted from mutation rather than recombination.

Genomic Signature of Adaptive Divergence despite Strong Nonadaptive Forces on Edaphic Islands: A Case Study of Primulina juliae.

Both genetic drift and divergent selection are expected to be strong evolutionary forces driving population differentiation on edaphic habitat islands. However, the relative contribution of genetic drift and divergent selection to population divergence has rarely been tested simultaneously. In this study, restriction-site associated DNA-based population genomic analyses were applied to assess the relative importance of drift and divergent selection on population divergence of Primulina juliae, an edaphic specialist from southern China. All populations were found with low standing genetic variation, small effective population size (NE), and signatures of bottlenecks. Populations with the lowest genetic variation were most genetically differentiated from other populations and the extent of genetic drift increased with geographic distance from other populations. Together with evidence of isolation by distance, these results support neutral drift as a critical evolutionary driver. Nonetheless, redundancy analysis revealed that genomic variation is significantly associated with both edaphic habitats and climatic factors independently of spatial effects. Moreover, more genomic variation was explained by environmental factors than by geographic variables, suggesting that local adaptation might have played an important role in driving population divergence. Finally, outlier tests and environment association analyses identified 31 single-nucleotide polymorphisms as candidates for adaptive divergence. Among these candidates, 26 single-nucleotide polymorphisms occur in/near genes that potentially play a role in adaptation to edaphic specialization. This study has important implications that improve our understanding of the joint roles of genetic drift and adaptation in generating population divergence and diversity of edaphic specialists.

Integration of genetic and physical maps of the Primula vulgaris S locus and localization by chromosome in situ hybridization.

Heteromorphic flower development in Primula is controlled by the S locus. The S locus genes, which control anther position, pistil length and pollen size in pin and thrum flowers, have not yet been characterized. We have integrated S-linked genes, marker sequences and mutant phenotypes to create a map of the P. vulgaris S locus region that will facilitate the identification of key S locus genes. We have generated, sequenced and annotated BAC sequences spanning the S locus, and identified its chromosomal location. We have employed a combination of classical genetics and three-point crosses with molecular genetic analysis of recombinants to generate the map. We have characterized this region by Illumina sequencing and bioinformatic analysis, together with chromosome in situ hybridization. We present an integrated genetic and physical map across the P. vulgaris S locus flanked by phenotypic and DNA sequence markers. BAC contigs encompass a 1.5-Mb genomic region with 1 Mb of sequence containing 82 S-linked genes anchored to overlapping BACs. The S locus is located close to the centromere of the largest metacentric chromosome pair. These data will facilitate the identification of the genes that orchestrate heterostyly in Primula and enable evolutionary analyses of the S locus.

Oakleaf: an S locus-linked mutation of Primula vulgaris that affects leaf and flower development.

In Primula vulgaris outcrossing is promoted through reciprocal herkogamy with insect-mediated cross-pollination between pin and thrum form flowers. Development of heteromorphic flowers is coordinated by genes at the S locus. To underpin construction of a genetic map facilitating isolation of these S locus genes, we have characterised Oakleaf, a novel S locus-linked mutant phenotype. We combine phenotypic observation of flower and leaf development, with classical genetic analysis and next-generation sequencing to address the molecular basis of Oakleaf. Oakleaf is a dominant mutation that affects both leaf and flower development; plants produce distinctive lobed leaves, with occasional ectopic meristems on the veins. This phenotype is reminiscent of overexpression of Class I KNOX-homeodomain transcription factors. We describe the structure and expression of all eight P. vulgaris PvKNOX genes in both wild-type and Oakleaf plants, and present comparative transcriptome analysis of leaves and flowers from Oakleaf and wild-type plants. Oakleaf provides a new phenotypic marker for genetic analysis of the Primula S locus. We show that none of the Class I PvKNOX genes are strongly upregulated in Oakleaf leaves and flowers, and identify cohorts of 507 upregulated and 314 downregulated genes in the Oakleaf mutant.

Disruption of the distylous syndrome in Primula veris.

BACKGROUND AND AIMS: Distyly is a floral polymorphism characterized by the presence of two discrete morphs with reciprocal positioning of anthers and stigmas in flowers on different plants within the same population. Although reciprocal herkogamy and associated floral traits are generally thought to be discrete and strict polymorphisms, little is known about variation in floral traits related to the distylous syndrome within and among populations of a single species. In this study, variation in floral morphology and reciprocal positioning of the sexual organs in the distylous Primula veris (cowslip) is quantified. METHODS: Data were collected in ten populations occurring in two contrasting habitat types (grasslands and forests), and for each population the average level of reciprocity was assessed, the strength of the self-incompatibility system was determined, and seed production under natural conditions was quantified. RESULTS: In grassland populations, flowers showed clear distyly with low and symmetric reciprocity indices at both the lower and upper level. In forests, P. veris produced larger flowers that showed strong deviations in stigma-anther separation, especially in the L-morph. This deviation was mainly driven by variation in stigma height, resulting in high and asymmetric reciprocity indices and the occurrence of several short-styled homostylous plants. Self-incompatibility was, however, strict in both habitats, and morph ratios did not differ significantly from isoplethy. The observed shift in reciprocity in forest populations was associated with a significant reduction in seed production in the L-morph. CONCLUSIONS: The results indicate that populations of P. veris show habitat-specific variation in flower morphology. Deviations from perfect reciprocal positioning of stigmas and anthers also translate into reduced seed production, suggesting that small changes in sexual organ reciprocity can have far-reaching ecological and evolutionary implications.

Occurrence of morphological and anatomical adaptive traits in young and adult plants of the rare Mediterranean cliff species Primula palinuri Petagna.

Cliffs worldwide are known to be reservoirs of relict biodiversity. Despite the presence of harsh abiotic conditions, large endemic floras live in such environments. Primula palinuri Petagna is a rare endemic plant species, surviving on cliff sites along a few kilometres of the Tyrrhenian coast in southern Italy. This species is declared at risk of extinction due to human impact on the coastal areas in question. Population surveys have shown that most of the plants are old individuals, while seedlings and plants at early stages of development are rare. We followed the growth of P. palinuri plants from seed germination to the adult phase and analysed the morphoanatomical traits of plants at all stages of development. Our results showed that the pressure of cliff environmental factors has been selected for seasonal habitus and structural adaptive traits in this species. The main morphoanatomical modifications are suberized cell layers and accumulation of phenolic compounds in cell structures. These features are strictly related to regulation of water uptake and storage as well as defence from predation. However, we found them well established only in adult plants and not in juvenile individuals. These findings contribute to explain the rare recruitment of the present relict populations, identifying some of the biological traits which result in species vulnerability.

Production of interspecific hybrids in ornamental plants.

In breeding of ornamental plants, interspecific hybridization and polyploidization have successfully been used to produce novel cultivars with blended traits of both parents and to introgress useful traits of one species to another. Embryo rescue techniques and molecular cytogenetic methods have successfully been used to produce and characterize interspecific hybrids in various genera. In this paper, recent advances in interspecific hybridization are described based on the results obtained in Primula, Cosmos, and Kalanchoe with special references to the use of embryo culture techniques for rescuing the abortive hybrid embryos. The methods for production and characterization of interspecific hybrids are categorized into three steps, i.e., (1) pollination, (2) rescue culture of immature embryo, and (3) confirmation of hybridity and ploidy level of the plants obtained. For interspecific crosses, emasculation step is usually needed to avoid self-pollination even in the genera with self-incompatibility system, such as Primula and Cosmos, since self-incompatibility is not always complete. Since interspecific crosses are usually hindered by various cross-incompatibility mechanisms, successful production of interspecific hybrids could be achieved only from limited crosses among those using many cultivars/strains of both parents, suggesting the importance of the selection of the compatible genotypes. Unilateral cross incompatibility is commonly observed in interspecific cross combinations, so reciprocal crosses should be conducted as an indispensable step. At the rescue culture step, addition of plant hormones, e.g., auxin cytokinin and gibberellin, to the culture medium at the appropriate concentrations is proved to be effective and necessary. The hybridity of the plants is efficiently confirmed at the seedling stage by DNA analysis in addition to the observation of morphological characters. The analysis of relative DNA contents by flow cytometry is an easy and rapid means to confirm hybridity and to estimate ploidy level and genomic combination.

Impact of herbivory on flowering behaviour and life history trade-offs in a polycarpic herb: a 10-year experiment.

Herbivores can have strong deleterious effects on plant growth, reproduction, and even survival. Because these effects might be strongly interrelated, the direct consumptive effects of herbivores and a variety of indirect effects are difficult to untangle. Reductions in growth, for example, may strongly impact the flowering behaviour of plant species in the current season, but at the same time incur costs to survival, growth and reproduction in the next growing season(s). To get better insights in the effects of herbivory on the flowering behaviour of the long-lived polycarpic grassland herb Primula veris L., flowering patterns were monitored over ten consecutive years under two treatments (grazing and control mowing regimes). We tested the hypothesis that the size at flowering was affected by the presence of herbivores, and whether this translated into costs to future reproduction and survival. Overall, grazed plants were significantly smaller than control plants, and the size at which plants flowered was also significantly smaller when herbivores were present. The transition probability of flowering and of surviving into the next year was significantly smaller for all plants in the current year if they had been grazed than if they had been mown, indicating that herbivory incurred costs to both flowering and survival. Grazed plants also needed longer to start flowering, had fewer flowers and flowered less frequently, causing a significantly lower proportion of flowering adults in the population. These results suggest that the observed regression in plant size due to herbivory does not allow plants to capture enough resources to guarantee regular flowering in the longer run.

Plasticity in response to phosphorus and light availability in four forest herbs.

The differential ability of forest herbs to colonize secondary forests on former agricultural land is generally attributed to different rates of dispersal. After propagule arrival, however, establishing individuals still have to cope with abiotic soil legacies from former agricultural land use. We focused on the plastic responses of forest herbs to increased phosphorus availability, as phosphorus is commonly found to be persistently bioavailable in post-agricultural forest soils. In a pot experiment performed under field conditions, we applied three P levels to four forest herbs with contrasting colonization capacities: Anemone nemorosa, Primula elatior, Circaea lutetiana and Geum urbanum. To test interactions with light availability, half of the replicas were covered with shade cloths. After two growing seasons, we measured aboveground P uptake as well as vegetative and regenerative performance. We hypothesized that fast-colonizing species respond the most opportunistically to increased P availability, and that a low light availability can mask the effects of P on performance. All species showed a significant increase in P uptake in the aboveground biomass. The addition of P had a positive effect on the vegetative performances of two of the species, although this was unrelated to their colonization capacities. The regenerative performance was affected by light availability (not by P addition) and was related to the species' phenology. Forest herbs can obviously benefit from the increased availability of P in post-agricultural forests, but not all species respond in the same way. Such differential patterns of plasticity may be important in community dynamics, as they affect the interactions among species.

Hose in Hose, an S locus-linked mutant of Primula vulgaris, is caused by an unstable mutation at the Globosa locus.

Hose in Hose mutants of primrose and cowslip have been cultivated since the early 17th century and show dominant homeotic conversion of sepals to petals. The phenotype shows variable penetrance and expressivity and is linked to the S locus, which controls floral heteromorphy in Primula species. Here we demonstrate that the homeotic conversion of sepals to petals in Hose in Hose is associated with up-regulation of both Primula B-function MADS box genes PvDef and PvGlo in the first floral whorl. We have defined a restriction fragment length polymorphism associated with PvGlo that cosegregates with the Hose in Hose phenotype and have also identified and characterized a retrotransposon insertion in the PvGlo promoter which is associated with the up-regulated expression of PvGlo. Excision of this retrotransposon, associated with epigenetic changes at the locus, causes reversion toward normal calyces and restores wild-type flower development. These data define the molecular basis of the Hose in Hose mutation and provide an explanation for its long-documented phenotypic instability.

Linking environmental and demographic data to predict future population viability of a perennial herb.

Recent advances in stochastic demography provide tools to examine the importance of random and periodic variation in vital rates for population dynamics. In this study, we explore with simulations the effect of disturbance regime on population dynamics and viability. We collected 7 years of demographic data in three populations of the perennial herb Primula farinosa, and used these data to examine how variation in vital rates affected population viability parameters (stochastic growth rate, lambda(S)), and how vital rates were related to weather conditions. Elasticity analysis indicated that the stochastic growth rate was very sensitive to changes in regeneration, quantified as the production, survival, and germination of seeds. In one of the study years, all seedlings and mature plants in the demography plots died. This extinction coincided with the driest summer during the study period. Simulations suggested that a future increase in the frequency of high-mortality years due to climate change would result in reduced population growth rate, and an increased importance of survival in the seed bank for population viability. The results illustrate how the limited demographic data typically available for many natural systems can be used in simulation models to assess how environmental change will affect population viability.

Reconsideration for conservation units of wild Primula sieboldii in Japan based on adaptive diversity and molecular genetic diversity.

Primula sieboldii E. Morren is a perennial clonal herb that is widely distributed in Japan, but in danger of extinction in the wild. In a previous study, we revealed the genetic diversity of the species using chloroplast and nuclear DNA and used this information to define conservation units. However, we lacked information on adaptive genetic diversity, which is important for long-term survival and, thus, for the definition of conservation units. In order to identify adaptive traits that showed adaptive differentiation among populations, we studied the genetic variation in six quantitative traits within and among populations for 3 years in a common garden using 110 genets from five natural populations from three regions of Japan. The number of days to bud initiation was adaptive quantitative trait for which the degree of genetic differentiation among populations (QST) was considerably larger than that in eight microsatellite markers (FST). The relationship between this trait and environmental factors revealed that the number of days to bud initiation was negatively correlated, with the mean temperature during the growing period at each habitat. This suggests that adaptive differentiation in the delay before bud initiation was caused by selective pressure resulting from temperature differences among habitats. Our results suggest that based on adaptive diversity and neutral genetic diversity, the Saitama population represents a new conservation unit.

Why didn't Darwin discover Mendel's laws?

Darwin's focus on small quantitative variations as the raw material of evolution may have prevented him from discovering the laws of inheritance.

Spatial variability in seed predation in Primula farinosa: local population legacy versus patch selection.

Spatio-temporal variation in seed predation may strongly influence both plant population dynamics and selection on plant traits. The intensity of seed predation may depend on a number of factors, but the relative importance of previous predator abundance ("local legacy"), spatial distribution of the host plant, environmental factors and plant characteristics has been explored in few species. We monitored seed predation in the perennial herb Primula farinosa, which is dimorphic for scape length, during 5 consecutive years, in a 10-km x 4-km area comprising 79 P. farinosa populations. A transplant experiment showed that the seed predator, the oligophagous tortricid moth Falseuncaria ruficiliana, was not dispersal limited at the spatial scale corresponding to typical distances between P. farinosa populations. Correlations between population characteristics and incidence and intensity of seed predation varied among years. The incidence of the seed predator was positively correlated with host population size and mean number of flowers, while intensity of seed predation in occupied patches was positively related to the frequency of the long-scaped morph in 2 years and negatively related to host population size in 1 year. In both scape morphs, predation tended to increase with increasing frequency of the long morph. There was no evidence of a local legacy; incidence and intensity of seed predation were not related to the abundance of the seed predator in the population in the previous year. Taken together, the results indicate that among-population variation in seed predation intensity is determined largely by patch selection and that the seed predator's preference for tall and many-flowered inflorescences may not only affect selection on plant traits within host plant populations, but also the overall intensity of seed predation.

Effects of population size on reproductive success of the endangered and endemic species Primula merrilliana.

The reproductive ability related to the population size of the endangered and endemic species Primula merrilliana Schltr. was investigated. In 26 populations observed, only four contain more than 500 flowering individuals, whereas most of them (53.8%) consist of less than 100 flowering individuals. Though the ratio of pin and thrum plants keeps its balance at 1:1 for all populations, the frequency of pin and thrum flowers was significantly biased in most small populations. Population size strongly affected reproductive success; plants in small populations produced significantly fewer flowers and fruits per plant and fewer seeds per fruit, and therefore fewer seeds per plant. The floral density was another important factor that influenced the reproductive success of P. merrilliana, because four main reproductive success parameters (fruits per plant, seeds per fruit, seeds per plant, and the proportion of flowers setting fruit) were all positively correlated with floral density. The size of plants and the number of leaves per plant (measure of habitat quality) were not influenced by the variation of population size, suggesting that the reduced fecundity in small populations may not be a consequence of lower habitat quality. Inbreeding depression and pollen limitation as a result of less attractiveness in small populations are therefore likely explanations for these patterns.

Mutualists and antagonists mediate frequency-dependent selection on floral display.

Theory predicts that, with conflicting selection pressures mediated by mutualists and antagonists, alternative reproductive strategies can be maintained by negative frequency-dependent selection if it results in rare-morph advantage. We combined field experiments and surveys of natural populations to determine whether selection on floral display is frequency dependent in the self-incompatible herb Primula farinosa, which is polymorphic for inflorescence height and occurs in a short-scaped and a long-scaped morph. Among short-scaped plants, both pollination success, quantified as initiation of fruits and seeds, and seed predation were positively correlated with the relative frequency of the long-scaped morph. The relative strength of these effects and the direction of the resulting frequency-dependent selection on scape morph varied among years and populations. The results suggest that both mutualists and antagonists may mediate frequency-dependent selection and that frequency dependence may vary from positive to negative with rare-morph advantage, depending on the relative strength of these interactions.

Effects of stand age on the demography of a temperate forest herb in post-agricultural forests.

Changes in land use have been shown to have profound effects on forest plant community structure and diversity. Dispersal limitation has been invoked as a major factor hampering colonization of forest plant species, while seed-sowing experiments and performance observations have provided some evidence for recruitment limitation determining forest plant distribution in post-agricultural forests. However, most of these studies were relatively short-term, and very few studies have investigated long-term growth rates of populations occurring in recent and ancient forests. In this study, matrix models using demographic data collected for four consecutive years were used to study the effect of forest age on population dynamics of the temperate forest herb Primula elatior. A life table response experiment (LTRE) and elasticity analysis were used to analyze the effect of forest age on population growth rate (lambda) and to decompose the effect of forest age on lambda into contributions from each matrix element. Population growth increased logarithmically with increasing forest age. Bootstrap analyses showed that populations located in very recent forests (< 50-years-old) had growth rates that were significantly < 1, whereas populations located in forests > 150-years-old had growth rates that were significantly > 1. Summed elasticities for individual growth significantly decreased with increasing forest age, whereas summed elasticities for survival and fertility significantly increased with increasing forest age. The LTRE analysis showed that the increase in lambda with increasing forest age was mainly due to increased seedling and juvenile growth and increased juvenile and adult survival. Our results indicate that past agricultural land use has long-lasting effects on the demography of forest herbs and may provide an additional mechanistic explanation for the poor colonization capacity of many forest herbs in post-agricultural forests.

Fine-scale spatial genetic structure of the distylous Primula veris in fragmented habitats.

In Flanders (northern Belgium), the distylous self-incompatible perennial herb Primula veris is common, but mainly occurs in fragmented habitats. Distyly, which favours disassortative mating, is characterized in P. veris by two genetically determined floral morph types (pin or thrum). Using 18 polymorphic loci, we investigated fine-scale spatial genetic structure (SGS) and spatial distribution of the morphs within four populations from two regions that differ in degree of habitat fragmentation. We studied the contributions made by sexual reproduction and clonal propagation and compared the SGS patterns between pin and thrum morph types. Clonal growth was very restricted to a few individuals and to short distances. One population showed a non-random spatial distribution of the morphs. Pin and thrum individuals differed in SGS patterns at a small scale, suggesting intrapin biparental inbreeding, also related to high plant densities. This may be explained by partial self-compatibility of the pin morph combined with restricted seed dispersal and pollinator behaviour. There is an indication of more pronounced SGS when populations occur in highly fragmented habitats. From our findings, we may hypothesize disruption of the gene flow processes if these large populations evolve into patchworks of small remnants, but also a possible risk for long-term population survival if higher intrapin biparental inbreeding leads to inbreeding depression. Our study emphasizes the need for investigating the interactions between the heterostylous breeding system, population demographic and genetic structure for understanding population dynamics in fragmented habitats and for developing sustainable conservation strategies.