Evolutionary breakdown of distyly to homostyly is accompanied by reductions of floral scent in Primula oreodoxa

Reproductive traits that function in pollinator attraction may be reduced or lost during evolutionary transitions from outcrossing to selfing. Although floral scent plays an important role in attracting pollinators in outcrossing species, few studies have investigated associations between floral scent variation and intraspecific mating system transitions. The breakdown of distyly to homostyly represents a classic example of a shift from outcrossing to selfing and provides an opportunity to test whether floral fragrances have become reduced and/or changed in composition with increased selfing. Here, we evaluate this hypothesis by quantifying floral volatiles using gas chromatography-mass spectrometry in two distylous and four homostylous populations of Primula oreodoxa Franchet, a perennial herb from SW China. Our analysis revealed significant variation of volatile organic compounds (VOCs) among populations of P. oreodoxa. Although there was no difference in VOCs between floral morphs in distylous populations as predicted, we detected a substantial reduction in VOC emissions and the average number of scent compounds in homostylous compared with distylous populations. A total of 12 compounds, mainly monoterpenoids and sesquiterpenoids, distinguished homostylous and distylous morphs; of these, (E)-β-ocimene was the most important in contributing to the difference in volatiles, with significantly lower emissions in homostyles. Our findings support the hypothesis that the transition from outcrossing to selfing is accompanied by the loss of floral volatiles. The modification to floral fragrances in P. oreodoxa associated with mating system change might occur because high selfing rates in homostylous populations result in relaxed selection for floral attractiveness.     

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.     

The Genomic Selfing Syndrome Accompanies the Evolutionary Breakdown of Heterostyly

The evolutionary transition from outcrossing to selfing can have important genomic consequences. Decreased effective population size and the reduced efficacy of selection are predicted to play an important role in the molecular evolution of the genomes of selfing species. We investigated evidence for molecular signatures of the genomic selfing syndrome using 66 species of Primula including distylous (outcrossing) and derived homostylous (selfing) taxa. We complemented our comparative analysis with a microevolutionary study of P. chungensis, which is polymorphic for mating system and consists of both distylous and homostylous populations. We generated chloroplast and nuclear genomic data sets for distylous, homostylous, and distylous–homostylous species and identified patterns of nonsynonymous to synonymous divergence (d_N/d_S) and polymorphism (π_N/π_S) in species or lineages with contrasting mating systems. Our analysis of coding sequence divergence and polymorphism detected strongly reduced genetic diversity and heterozygosity, decreased efficacy of purifying selection, purging of large-effect deleterious mutations, and lower rates of adaptive evolution in samples from homostylous compared with distylous populations, consistent with theoretical expectations of the genomic selfing syndrome. Our results demonstrate that self-fertilization is a major driver of molecular evolutionary processes with genomic signatures of selfing evident in both old and relatively young homostylous populations.     

Comparative reproductive biology of Primula merrilliana Schltr. and P. cicutariifolia Pax

The reproductive biology of Primula merrilliana Schltr. and P. cicutariifolia Pax that are sister taxa and the only two members of Primula sect. Ranunculoides were studied. The results showed that P. merrilliana is typically a distylous species not only in morphology but also in function; it has an incompatibility system that restricts the legitimate pollination to that between pin and thrum flower morphs, but self-fertilization may occur under certain conditions. P. cicutariifolia is self-compatible. The pin morph of P. merrilliana has longer stigma papillae and smaller pollen than the thrum morph. The results stated above support the view that P. filchnerae should be moved from sect. Pinnatae to sect. Auganthus.     

Primula zhui (Primulaceae) sp. nov. from south Yunnan,southwest China

Primula zhui a new species of Primula endemic to the Yunnan Province, China, is described and illustrated. It is morphologically similar to P. intanoensis from Thailand, and P. calyptrata from southeast Yunnan, affiliated to Primula sect. Carolinella (Hemsl.) Pax. However, it is easily distinguished from the Thailand species by its longer corolla tube and distylous flowers, and it differs from the Chinese species by its leaf ovate to ovate‐elliptic blade and distylous flowers.     

Characterization of 24 microsatellite markers in Primula chungensis (Primulaceae), a distylous homostylous species,using MiSeq sequencing

Primula chungensis is a species with considerable floral and mating-system variation, including distylous (outcrossing), homostylous (selfing) and mixed populations that contain both outcrossing and selfing forms. We isolated 24 microsatellite markers from P.chungensis using Illumina MiSeq sequencing. Polymorphism and genetic diversity were then measured based on a sample of 24 individuals from a natural population in southern Tibet. All loci were polymorphic with the number of alleles per locus ranging from 2 to4. The observed and expected heterozygosity ranged from 0 to 1 and 0.219 to 0.708, respectively. The microsatellite markers we have identified will serve as valuable tools for the investigation of the population genetic structure and phylogeography of P.chungensis and will inform models of the evolutionary history of mating systems in the species.     

Characterization of 24 microsatellite markers in Primula chungensis(Primulaceae),a distylous-homostylous species,using MiSeq sequencing

Primula chungensis is a species with considerable floral and mating-system variation,including distylous(outcrossing),homostylous(selfing) and mixed populations that contain both outcrossing and selfing forms.We isolated 24 microsatellite markers from P.chungensis using Illumina Mi Seq sequencing.Polymorphism and genetic diversity were then measured based on a sample of 24 individuals from a natural population in southern Tibet.All loci were polymorphic with the number of alleles per locus ranging from 2 to 4.The observed and expected heterozygosity ranged from 0 to 1 and 0.219 to 0.708,respectively.The microsatellite markers we have identified will serve as valuable tools for the investigation of the population genetic structure and phylogeography of P.chungensis and will inform models of the evolutionary history of mating systems in the species.     

On the occurrence of unilateral incompatibility in Primula section Aleuritia Duby and the origin of Primula scotica Hook

The occurrence of unilateral incompatibility was tested for a distylous heteromorphic system, using crosses between a self-sterile and three self-fertile species in Primula L. section Aleuritia Duby. The crosses showed non-reciprocity but not in the same direction as would be predicted in the case of unilateral incompatibility. Pollen from the self-compatible (s-c) species was not always inhibited on the style of the self-incompatible (s-i) species, and cross-fertility between s-i and s-c crosses more resembled that between different s-c species, which was also non-reciprocal. Cytoplasmic-nuclear DNA interactions and the possibility of embryo-endosperm imbalance could both explain these results. In crosses between Primula farinosa L. (s-i diploid) and P. scotica Hook. (s-c hexaploid), heterostylous pin tetraploid offspring were produced. This result is discussed in relation to the genome of P. scotica and the possibility that pin morph plants may occur in wild P scotica populations.     

Reproductive ecology in the endemic Primula apennina Widmer (Primulaceae)

Abstract

Disassortative mating in distylous self-incompatible species should result in the equilibrium of morph types in natural populations. Deviation from isoplethy may affect pollen transfer, and in isolated populations it could lead to Allee effect and genetic drift. Pollen limitation has been found to occur in several distylous species, for which mating opportunities are actually reduced to half population. In this study, we investigated the reproductive features and pollination ecology of the narrow endemic Primula apennina. We recorded equilibrium of morph frequencies in the studied population, reflecting the comparable fecundity found in the two morphs. Long-styled flowers produce more pollen grains of smaller size than short-styled ones: we hypothesize that in thrum flowers, pollen is more easily removed by the insect pollinator Macroglossum stellatarum, resulting in equal pollen amounts carried to both short styles and long styles. This lower pollen transfer efficiency from long-styled to short-styled flowers is also reflected in legitimate pollen–ovule ratio values. Despite results show no evidence of imminent threats to population persistence at study site, the strict dependence on one or very few pollinator species, and ecological traits, may increase extinction risks in the long-term period.     

Primula jiugongshanensis sp. nov. (Primulaceae) from China,based on morphological and molecular evidence

Primula jiugongshanensis J. W. Shao, a new species from Mt Jiugongshan in Hubei Province, China, is described and illustrated. This species is a biennial glabrous herb with pinnately compound leaves, and affiliated to P. sect. Ranunculoides C. M. Hu. It is distinguished from other species of the section by outer simple leaves ovate, flowers distylous, corolla lobe apex conspicuously emarginate, and lack of bulblets. Molecular phylogenetic analysis of nrDNA internal transcribed spacer sequences (ITS) was conducted and the results also supported the recognition of P. jiugongshanensis as a new species. The species is known from only three separated unprotected populations and is categorized as ‘Endangered’ (EN).     

VARIATION AND EVOLUTION OF BREEDING SYSTEMS IN THE TURNERA ULMIFOLIA L. COMPLEX (TURNERACEAE)

The evolutionary and functional relationships among breeding systems and floral morphology were investigated in the Turnera ulmifolia complex. Predictions of a model of breeding system evolution among distylous and homostylous varieties were tested. Chromosome counts of 73 accessions revealed an association between breeding system and chromosome number. Diploid and tetraploid populations of five taxonomic varieties are distylous and self-incompatible, whereas hexaploid populations of three varieties are homostylous and self-compatible. The latter occur at different margins of the geographical range of the complex. Crossing studies and analyses of pollen and ovule fertility in F₁'s revealed that the three homostylous varieties are intersterile. To test the prediction that, homostylous varieties are long homostyles that have originated by crossing over within the distyly supergene, a crossing program was undertaken among distylous and homostylous plants. Residual incompatibility was observed in styles and pollen of each homostylous variety with patterns consistent with predictions of the cross-over model. The intersterility of hexaploid varieties suggests that long homostyly has arisen on at least three occasions in the complex by recombination within the supergene controlling distyly. Deviation from expected compatibility behavior occurs in populations of var. angustifolia that have the longest styles. These phenotypes displayed the greatest separation between anthers and stigmas (herkogamy) and set little seed in crosses with long- or short-styled plants. This suggests that they are derived from long homostyles with shorter length styles. It is proposed that selection for increased outcrossing has favored the evolution of herkogamy in long homostyles. Estimates of outcrossing rate in a distylous population using allozyme markers confirmed that dimorphic incompatibility enforces complete outcrossing. Significant genetic variation for floral traits likely to influence the mating system, such as stigma-anther separation, occurs within and among homostylous populations of var. angustifolia on Jamaica. Estimates of the mating system of families from a population with varying degrees of stigma-anther separation, using five isozyme loci, were heterogeneous and ranged from t = 0.04–0.79. Families exhibiting the largest mean stigma-anther separation have higher outcrossing rates than those with little separation.     

Osservazioni sulla polistelia in Primula glaucescens Moretti e Primula longobarda Porta

Abstract

Notes on the polystelic structure in Primula glaucescens Moretti and Primula longobarda Porta. — The rhizome anatomy of Primula glaucescens Moretti and Primula longobarda Porta (two species endemic to Lombardy) have been studied. It has been found that the polystelic structure is present in the rhizome of both species, but with some differences. The taxa show a difference in the number and size of steles and amount of sclerenchymatic tissue within the single stele.

Our investigations confirm the necessity to separate in a specific rank Primula glaucescens Moretti from Primula longobarda Porta, as already proposed by Arietti and Crescini (1976).     

Skewed morph ratios lead to lower genetic diversity of the heterostylous Primula veris in fragmented grasslands

• Populations of heterostylous plant species are ideally composed of equal frequencies of two (distylous) or three (tristylous) morphologically different floral morphs. Intra-morph incompatibility helps to avoid inbreeding and to maintain genetic diversity, supporting plant fitness and long-term viability. Habitat fragmentation can lead to skewed morph ratios and thereby reduce the abundance of compatible mates. This, in turn, can result in a loss of genetic diversity. We tested whether the genetic diversity of heterostylous plants is affected by morph ratio bias using populations of the distylous grassland plant Primula veris in recently fragmented grasslands.
• We recorded morph frequencies and population sizes in 30 study populations of P. veris on two Estonian islands characterised by different degrees of habitat fragmentation. Examining variation of thousands of single nucleotide polymorphisms (SNPs) and heterostyly-specific genetic markers, we quantified overall and morph-specific genetic diversity and differentiation in these populations.
• Morph frequencies deviated more in smaller populations. Skewed morph ratios had a negative effect on the genetic diversity of P. veris in more fragmented grasslands. In the populations of better-connected grassland systems, genetic differentiation among S-morphs was higher than among L-morphs.
• Our study shows that deviations from morph balance are stronger in small populations and have a negative impact on the genetic diversity of the distylous plant P. veris. Together with the direct negative effects of habitat loss and decreased population size on the genetic diversity of plants, morph ratio bias may intensify the process of genetic erosion, thus exacerbating the local extinction of heterostylous species.

     

Distribution of style and pollen polygalacturonases among distylous and homostylous Turnera and Piriqueta spp. (Turneraceae)

We explore the distribution of a style and pollen polygalacturonase in a number of distylous and homostylous species of Turnera, and two species of Piriqueta (Turneraceae). We show, using immunoblotting with antibodies made against these proteins, that the style polygalacturonase is specific to styles of short-styled plants of all the six distylous species of Turnera we have investigated. Styles of a somatic homostylous mutant derived from a short-styled plant do not possess the style polygalacturonase. Distylous P. caroliniana did not appear to possess this protein. We show that the pollen polygalacturonase, while associated with the short-styled morph in three species, is polymorphic among short-styled plants of T. krapovickasii, and absent from T. joelii, T. grandiflora and P. caroliniana. These data support a role for the style polygalacturonase in distyly, possibly in the incompatibility system, but cast doubt on any role for the pollen polygalacturonase. In concert with the predictions for the mode of origin, and the response of styles of homostylous species to pollen from long- and short-styled plants, we find that none of the homostylous species possess the style polygalacturonase. The pollen polygalacturonase does occur in some homostylous species, but not in others. It is not clear that the pollen polygalacturonase, however, provides a marker for the mode of origin of homostyly.     

Temporal variations in the linkages between plants and flower visitors and the pollination success of Primula modesta along the snowmelt gradient

Although pollination networks between plants and flower visitors are diverse and flexible, seed production of many plant species is restricted by pollen limitation. Obligate outcrossers often suffer from low pollinator activity or severe interspecific competition for pollinator acquisition among co-flowering species. This study focused on seasonal changes in plant–flower visitor linkages in an alpine ecosystem and examined whether and how this seasonality affected the seed-set of Primula modesta, a self-incompatible distylous herb having long-tubed flowers. First, we recorded the linkages between plants and flower visitors along the snowmelt gradient. Then, pollination experiment was conducted to estimate the degree of pollen limitation over the course of flowering season of P. modesta. Flower visitors were classified by their tongue length based on the morphological matching with P. modesta flowers. As the season progressed, plant–visitor linkages became more diverse and generalized, and the visitation frequency to P. modesta flowers increased. In the later part of the season, however, the seed set of P. modesta was significantly reduced due to severe pollen limitation, presumably because of increased competition for long-tongued pollinators among co-flowering species. The present study revealed that pollinator availability for specialist species may be restricted even when plant–visitor linkages are diverse and generalized as a whole. In the case of P. modesta, morphological matching and competition for pollinators might be the main factors explaining this discrepancy.     

Heterostyly promotes compatible pollination in buckwheats: Comparisons of intraflower,intraplant, and interplant pollen flow in distylous and homostylous Fagopyrum

Premise of the Study

Heterostyly, the reciprocal positioning of stigmas and anthers in different floral morphs, has long been thought to promote intermorph pollination. However, extensive intramorph pollination occurs commonly in heterostylous species, leading to recurrent questions about the functional and evolutionary significance of heterostyly.

Methods

To identify the sources of stigmatic pollen (autogamous [intraflower], geitonogamous [intraplant], vs. interplant), we emasculated either one flower or entire plants in experimental populations of the two closely related buckwheat species, distylous Fagopyrum esculentum and homostylous F. tataricum. Differences in pollen size allowed unambiguous identification of pollen on stigmas.

Results

Only 2.4% of F. tataricum pollen and 1.5% of F. esculentum pollen arrived successfully on compatible stigmas of other plants. In the former (homostylous) species, 71.3% of the pollen load on stigmas was autogamous, 10.8% was geitonogamous, and 17.9% was interplant. In the latter (distylous) species, 37.45% of the pollen on stigmas was autogamous, 13.8% was geitonogamous, 17.0% was intramorph, and 31.75% was intermorph. The amount of incompatible pollen arriving on stigmas was greatly decreased by both one‐flower and whole‐plant emasculations, and thus, the proportion of compatible pollen deposited increased with one‐flower emasculation and increased even more with whole‐plant emasculation.

Conclusions

Our quantification of pollen‐donor sources in these two species indicated that heterostyly in Fagopyrum esculentum provided a nearly 2‐fold fitness advantage (in terms of compatible pollination) over expected (random) pollen transfers between morphs. Because of reduced herkogamy, the homostylous F. tataricum was highly autogamous.     

Shortened anther-stigma distance reduces compatible pollination in two distylous Primula species

Aims Distyly has been regarded as an adaptation to improve compatible pollination between two floral morphs with reciprocal herkogamy. The hypothesis that the different positions of anthers and stigmas within flowers as well as their reciprocal position between morphs, reduce the probability of self pollination raised by Darwin has been rarely tested. In this study, we measured stigmatic pollen loads in response to reduced reciprocal herkogamy in two Primula species.Methods To see whether reciprocal herkogamy can increase compatible and/or reduce incompatible pollen deposition, thus promoting compatible pollination, we shortened the distance between anthers and stigmas within the flowers by changing the position of the corolla tube, to which the anthers were fused, i.e. reduced herkogamy in natural populations of Primula secundiflora and P. poissonii and quantified stigmatic pollen loads in the field over 2 years.Important findings In both species, stigmatic pollen loads were significantly higher in the long-styled (L-morph) than in the short-styled morph (S-morph) in both control and manipulated flowers, but percentage of compatible pollen in S-morph were higher. Flowers manipulated to halve the anther–stigma distance showed a similar pattern for 2 years: total pollen grain counts on stigmas did not differ significantly but compatible pollen grains in L- and S-morphs were significantly decreased in both species. The percentage of compatible pollen loads was decreased by 68.7% in P. secundiflora and 65.3% in P. poissonii in L-morphs, while it decreased by 30.6% and 2.9% in S-morphs, respectively. Our manipulation of the relative position of anthers and stigmas in the two distylous species indicated that a lower degree of herkogamy reduced compatible but incompatible pollen transfer was likely to increase. The higher proportion of compatible pollen in the S-morph than in the L-morph in the two Primula species could be attributed to the accessibility of two-level sexual organs, floral orientations and pollinator behaviors. This is a first attempt to manipulate intraflower herkogamy for understanding adaptation of heterostyly, shedding insights into how the reciprocal herkogamy promotes compatible pollination.     

EVOLUTIONARY HISTORY OF THE MATING SYSTEM IN AMSINCKIA (BORAGINACEAE)

A survey of restriction site variation in the chloroplast genome of the annual plant genus Amsinckia, together with estimation of outcrossing rates, was conducted to analyze the evolutionary history of the mating system. Species, and in some cases populations within species, differ markedly in their mating system. Five taxa are distylous and predominantly outcrossing, or show mixed mating systems, while the remaining taxa are homostylous and predominantly self-fertilizing. Reconstruction of the molecular phylogeny of the group places different distylous and homostylous taxa at four separate branch tips. When distyly is treated as ancestral in the group, or when the loss of distyly is assumed to be more common than its gain, the results of the phylogenetic analysis support the hypothesis that the self-fertilizing taxa are of recent origin from outcrossing relatives. These findings are discussed with respect to theory for the evolution and breakdown of distyly and the probability of extinction of selfing lineages.     

Assessment of distyly syndrome in Primula palinuri Petagn. a rare species living on maritime vertical cliffs

The distinctive habitat of vertical cliffs provides refuges for many relict plant species. Primula palinuri Petagn. is the only maritime and Mediterranean species of the genus Primula. It only occurs on the cliffs of a narrow area of southern Italy and is classified as being at risk of extinction. Given that only fragmentary information on the biology and ecology of P. palinuri is available, we studied flower morphology, breeding system and other reproductive traits of this species to furnish data for the evaluation of its conservation status. We assessed that plants of P. palinuri develop either long-styled or short-styled flowers and both morphs are equally represented in the studied populations. Reciprocal position of anthers and stigmas is complemented by differences in pollen–ovule ratios, pollen size and exine ornamentation. In both morphs, about 90 % of open-pollinated flowers developed mature capsules and the average number of seeds per capsule was around sixty. Hand pollination experiments showed that reproductive success was high (92.86 and 100 % fruit set) following inter-morph crossings, but no fruit develops after same-morph pollination or selfing. We have ascertained that full distyly syndrome (distinct floral morphs, intra-morph incompatibility and morphs equally represented in the populations) occurs in P. palinuri. However, a further reduction of the severely fragmented range, any rapid change of the environmental conditions due to human impact on coastal areas or stochastic events of plant mortality may alter the proportion of the two morphs, affecting the fragile conservation status of the species.