Pollinator response to yellow UV-patterned versus white UV-patternless flower dimorphism in Anemone palmata.

Flower colour polymorphisms are uncommon but widespread among angiosperms and can be maintained by a variety of balancing selection mechanisms. Anemone palmata is mostly yellow-flowered, but white-flowered plants coexist in some populations. We analysed the distribution of colour morphs of A. palmata across its range. We also characterised their colours and compared their vegetative and sexual reproductive traits, pollinator attention and fitness. The range of A. palmata is limited to the Western Mediterranean, while white-flowered plants are restricted to Portugal and SW Spain, where they occur at low proportions. Yellow flowers have a characteristic UV pattern, with a UV-absorbing centre and UV-reflecting periphery, which is absent in the white morph. Colour features of both morphs were highly delineated, making it easy for pollinators to distinguish them. Both morphs were protogynous, with the same duration of sexual stages, and the main floral traits related to pollinator attraction, apart from flower colour, were similar. Hymenoptera and Diptera were the main pollinators, showing preference for the yellow morph, clear partitioning of pollinator groups between the two colour morphs and a marked constancy to flower colour during foraging. Both morphs combined clonal propagation with sexual reproduction, but sexual reproductive potential was lower in white-flowered plants. Finally, female fitness was higher in the yellow morph. Pollinator partitioning and colour constancy could maintain this polymorphism, despite the lower visitation rate and fitness of white-flowered plants, which could facilitate their clonal propagation.

The effects of pollination, herbivory and autonomous selfing on the maintenance of flower colour variation in Silenelittorea.

Intraspecific flower colour variation has been generally proposed to evolve as a result of selection driven by biotic or abiotic agents. In a polymorphic population of Silene littorea with pink- and white-flowered plants, we studied pollinators, analysed flower colour perception and tested for differences in pollinator visitation. We also experimentally analysed pollinator limitation in fruit and seed set, and the degree of autonomous selfing. The incidence of florivory and leaf herbivory was compared over 3-4 years. Silene littorea is mainly pollinated by bees and butterflies. Pollinators preferred pink flowers, which did not show pollinator limitation. On the contrary, white flowers showed pollinator limitation in fruit set. White-flowered plants had less floral display and higher levels of florivory than pink plants. Flower colour morphs of S. littorea can reproduce in the absence of pollinators by autonomous selfing, setting 20% and 12% of fruit and seeds in the pink morph and 27% and 20% in the white morph, respectively. Fruit set of white flowers produced by autonomous selfing did not differ from open-pollinated flowers. In conclusion, S. littorea is pollinated by insects of different orders that more frequently visit pink flowers, which is reflected in pollinator limitation of fruit set in white flowers. Moreover, this species has a mixed mating system in which both colour morphs can reproduce in the absence of pollinators by autonomous selfing, although white flowers mainly produce fruits by autogamy. We suggest that reproductive assurance by autonomous selfing helps to maintain flower colour polymorphism in this population.

Whole-plant reddening in Silene germana is due to anthocyanin accumulation in response to visible light.

The phenology of anthocyanin accumulation in leaves has been widely studied in perennial plants; several hypotheses have been proposed to explain their adaptive significance. Here, we explored the photoprotection hypothesis in Silene germana, a Mediterranean annual plant with late-spring/summer flowering. We analysed the temporal patterns of anthocyanin accumulation in photosynthetic calyces, leaves and stems and throughout the reproductive season, and their relationship with flower abortion, florivory and plant mortality due to drought. In addition, the flavonoid production and the photoinhibitory response were measured in a shading experiment. The whole plant becomes red at the end of the flowering and remains red until fruiting and senescence. Calyces were redder on the side with more sun exposition. Aborted flowers showed redder calyces than those of fruiting flowers. No effect of plant redness on florivory or plant mortality was found. The shading experiment showed a positive relationship between anthocyanin accumulation and intensity of solar radiation, but plants growing in absence of UV showed similar redness than full sunlight plants. Plants growing in natural shade lack anthocyanins but produced the same amount of non-anthocyanin flavonoids. Anthocyanic and non-anthocyanic plants showed similar photochemical efficiency (Fv /Fm ) after sun exposition, but in early morning, the former showed lower Fv /Fm values. Plants growing in full sunlight produced more fruits than those of natural shade plants. Whole-plant reddening during fruiting and senescence appears to be a property of S. germana. Our results suggest that anthocyanin accumulation depends on sunlight intensity, but non-anthocyanin flavonoids are produced constitutively.

Narrow endemics on coastal plains: Miocene divergence of the critically endangered genus Avellara (Compositae).

Critically endangered species representing ancient, evolutionarily isolated lineages must be given priority when allocating resources for conservation projects. Sound phylogenetic analyses and divergence time estimations are required to detect them, and studies on their population genetics, ecological requirements and breeding system are needed to understand their evolutionary history and to design efficient conservation strategies. Here we present the paradigmatic case of Avellara, a critically endangered monotypic genus of Compositae inhabiting a few swamps in the west-southwest Iberian coastal plains. Our phylogenetic and dating analyses based on nuclear (ITS) and plastid (matK) DNA sequences support a Miocene (>8.6 Ma) divergence between Avellara and closely related genera, resulting in marked morphological and ecological differentiation. We found alarmingly low levels of genetic diversity, based on AFLPs and plastid DNA sequences, and confirmed the prevalence of clonal reproduction. Species distribution modelling suggested a large macroclimatically suitable area for Avellara in the western Iberian Peninsula, but its apparently narrow microecological requirements restrict its distribution to peatlands with low-mineralised waters. Although five populations have been recorded from Spain and Portugal in the past, its current distribution may be reduced to only one population, recurrently found in the last decade but threatened by herbivory and habitat degradation. All this confirms the consideration of Avellara as a threatened species with high phylogenetic singularity, and makes it a flagship species for plant conservation in both Spain and Portugal that should be given priority in the design of in situ and ex situ conservation programmes.

Transcriptome and Biochemical Analysis of a Flower Color Polymorphism in Silene littorea (Caryophyllaceae).

Flower color polymorphisms are widely used as model traits from genetics to ecology, yet determining the biochemical and molecular basis can be challenging. Anthocyanin-based flower color variations can be caused by at least 12 structural and three regulatory genes in the anthocyanin biosynthetic pathway (ABP). We use mRNA-Seq to simultaneously sequence and estimate expression of these candidate genes in nine samples of Silene littorea representing three color morphs (dark pink, light pink and white) across three developmental stages in hopes of identifying the cause of flower color variation. We identified 29 putative paralogs for the 15 candidate genes in the ABP. We assembled complete coding sequences for 16 structural loci and nine of ten regulatory loci. Among these 29 putative paralogs, we identified 622 SNPs, yet only nine synonymous SNPs in Ans had allele frequencies that differentiated pigmented petals (dark pink and light pink) from white petals. These Ans allele frequency differences were further investigated with an expanded sequencing survey of 38 individuals, yet no SNPs consistently differentiated the color morphs. We also found one locus, F3h1, with strong differential expression between pigmented and white samples (>42x). This may be caused by decreased expression of Myb1a in white petal buds. Myb1a in S. littorea is a regulatory locus closely related to Subgroup 7 Mybs known to regulate F3h and other loci in the first half of the ABP in model species. We then compare the mRNA-Seq results with petal biochemistry which revealed cyanidin as the primary anthocyanin and five flavonoid intermediates. Concentrations of three of the flavonoid intermediates were significantly lower in white petals than in pigmented petals (rutin, quercetin and isovitexin). The biochemistry results for rutin, quercetin, luteolin and apigenin are consistent with the transcriptome results suggesting a blockage at F3h, possibly caused by downregulation of Myb1a.

The roles of female and hermaphroditic flowers in the gynodioecious-gynomonoecious Silene littorea: insights into the phenology of sex expression.

Some gynodioecious species have intermediate individuals that bear both female and hermaphroditic flowers. This phenomenon is known as a gynodioecious-gynomonoecious sexual system. Gender expression in such species has received little attention in the past, and the phenologies of male and female functions have also yet to be explored. In this study, we examined variations in gender patterns, their effects on female reproductive success and sex expression in depth throughout the flowering period in two populations. The studied populations of Silene littorea contained mostly gynomonoecious plants and the number of pure females was very low. The gynomonoecious plants showed high variability in the total proportion of female flowers. In addition, the proportion of female flowers in each plant varied widely across the flowering season. Although there was a trend towards maleness, our measures of functional gender suggested that most plants transmit their genes via both pollen and ovules. Fruit set and seed set were not significantly different among populations; in contrast, flower production significantly varied between the two populations - and among plants - with consequent variation in total seed production. Conversely, gender and sex expression were similar in both populations. Plants with higher phenotypic femaleness did not have higher fruit set, seed set or total female fecundity. The mating environment fluctuated little across the flowering period, but fluctuations were higher in the population with low flower production. We therefore conclude that the high proportion of gynomonoecious individuals in our studied populations of S. littorea may be advantageous for the species, providing the benefits of both hermaphroditic and female flowers.

Molecular phylogeny of Scorzoneroides (Asteraceae): evolution of heterocarpy and annual habit in unpredictable environments.

It has been hypothesized that annual life history and heterocarpy are an adaptation to unpredictable habitats. In this paper, we infer a phylogenetic history for Scorzoneroides from the analysis of internal transcribed spacer (ITS) and plastid ndhF-rpl32 and rpl32-trnL sequences, on which we trace the evolution of one ecological (predictable vs unpredictable habitats) and two morphological (annual vs perennial and dimorphic vs homomorphic achenes) characters. Our results confirm the monophyly of Scorzoneroides and reveal the existence of two significant evolutionary lineages that do not coincide with major taxonomic subdivisions traditionally proposed for this genus. The first major group comprises taxa with all types of possible combinations of character states. The second major group includes exclusively homocarpic perennial taxa occurring in predictable habitats. It is strongly inferred that the most recent common ancestor of Scorzoneroides was perennial and homomorphic, and occurred in predictable habitats. The evolution of both annual life history and dimorphic achenes was significantly associated with occurrence in unpredictable habitats, which may suggest an adaptation process.

Breeding system in the dichogamous hermaphrodite Silene acutifolia (Caryophyllaceae).

The breeding system of the dichogamous hermaphrodite species Silene acutifolia, endemic to north-west Spain and north and central Portugal, is examined. Pollen germinability and style-stigma receptivity were analysed to determine whether protandry is a barrier to self-fertilization. By 48 h after anthesis, pollen germinability had declined to approx. 10 %. The short straight styles are not receptive when flowers first open. They gradually elongate and curve outwards, develop stigma papillae and become receptive. There is no clear separation between stigma and style: the stigma papillae appear in a line along the length of the style. Fruit set is high regardless of pollen source; however, seed set is significantly reduced after both spontaneous and facilitated autogamy. Seed set following spontaneous autogamy was 30 % (86 % in controls) in 1998 and 33 % (87 % in controls) in 1999. Seed set following facilitated autogamy was 62 % (86 % in controls) in 1998 and 67 % (89 % in controls) in 1999. Thus, separation of the male and female phases does not prevent production of seeds by self-pollination, although it does reduce the likelihood of this. Furthermore, results of the present experiments indicate that this species has no self-incompatibility mechanisms (self-compatibility index = 0.98). The selfing rate in the study population was 0.41, which is supported by the lack of self-incompatibility systems and by the incomplete protandry.