Integrative taxonomy in a rapid speciation group associated with mating system transition: A case study in the Primula cicutariifolia complex.

Accurate species delimitation is the key to biodiversity conservation and is fundamental to most branches of biology. However, species delimitation remains challenging in those evolutionary radiations associated with mating system transition from outcrossing to self-fertilization, which have frequently occurred in angiosperms and are usually accompanied by rapid speciation. Here, using the Primula cicutariifolia complex as a case, we integrated molecular, morphological and reproductive isolation evidence to test and verify whether its outcrossing (distylous) and selfing (homostylous) populations have developed into independent evolutionary lineages. Phylogenetic trees based on whole plastomes and SNPs of the nuclear genome both indicated that the distylous and homostylous populations grouped into two different clades. Multispecies coalescent, gene flow and genetic structure analyses all supported such two clades as two different genetic entities. In morphology, as expected changes in selfing syndrome, homostylous populations have significantly fewer umbel layers and smaller flower and leaf sizes compared to distylous populations, and the variation range of some floral traits, such as corolla diameter and umbel layers, show obvious discontinuity. Furthermore, hand-pollinated hybridization between the two clades produced almost no seeds, indicating that well post-pollination reproductive isolation has been established between them. Therefore, the distylous and homostylous populations in this studied complex are two independent evolutionary lineages, and thus these distylous populations should be treated as a distinct species, here named Primula qiandaoensis W. Zhang & J.W. Shao sp. nov.. Our empirical study of the P. cicutariifolia complex highlights the importance of applying multiple lines of evidence, in particular genomic data, to delimit species in pervasive evolutionary plant radiations associated with mating system transition.

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.

Soil water and nutrient availability interactively modify pollinator-mediated directional and correlational selection on floral display.

The individual and combined effects of abiotic factors on pollinator-mediated selection on floral traits are not well documented. To examine potential interactive effects of water and nutrient availability on pollinator-mediated selection on three floral display traits of Primula tibetica, we manipulated pollination and nutrient availability in a factorial experiment, conducted at two common garden sites with different soil water content (natural vs addition). We found that both water and nutrient availability affected floral trait expression in P. tibetica and that hand pollination increased seed production most when both nutrient content and water content were high, indicating joint pollen and resource limitation. We documented selection on all floral traits, and pollinators contributed to directional and correlational selection on plant height and number of flowers. Soil water and nutrient availability interactively influenced the strength of both pollinator-mediated directional and correlational selection, with significant selection observed when nutrient or water availability was high, but not when none or both were added. The results suggest that resource limitation constrains the response of P. tibetica to among-individual variation in pollen receipt, that addition of nutrients or water leads to pollinator-mediated selection and that effects of the two abiotic factors are nonadditive.

The Complete Chloroplast Genomes of Primula obconica Provide Insight That Neither Species nor Natural Section Represent Monophyletic Taxa in Primula (Primulaceae).

The genus Primula (Primulaceae) comprises more than 500 species, with 300 species distributed in China. The contradictory results between systematic analyses and morphology-based taxonomy make taxonomy studies difficult. Furthermore, frequent introgression between closely related species of Primula can result in non-monophyletic species. In this study, the complete chloroplast genome of sixteen Primula obconica subsp. obconica individuals were assembled and compared with 84 accessions of 74 species from 21 sections of the 24 sections of the genus in China. The plastome sizes of P. obconica subsp. obconica range from 153,584 bp to 154,028 bp. Genome-wide variations were detected, and 1915 high-quality SNPs and 346 InDels were found. Most SNPs were detected in downstream and upstream gene regions (45.549% and 41.91%). Two cultivated accessions, ZP1 and ZP2, were abundant with SSRs. Moreover, 12 SSRs shared by 9 accessions showed variations that may be used as molecular markers for population genetic studies. The phylogenetic tree showed that P. obconica subsp. obconica cluster into two independent clades. Two subspecies have highly recognizable morphological characteristics, isolated geographical distribution areas, and distinct phylogenetic relationships compared with P. obconica subsp. obconica. We elevate the two subspecies of P. obconica to separate species. Our phylogenetic tree is largely inconsistent with morphology-based taxonomy. Twenty-one sections of Primula were mainly divided into three clades. The monophyly of Sect. Auganthus, Sect. Minutissimae, Sect. Sikkimensis, Sect. Petiolares, and Sect. Ranunculoides are well supported in the phylogenetic tree. The Sect. Obconicolisteri, Sect. Monocarpicae, Sect. Carolinella, Sect. Cortusoides, Sect. Aleuritia, Sect. Denticulata, Sect. Proliferae Pax, and Sect. Crystallophlomis are not a monophyletic group. The possible explanations for non-monophyly may be hybridization, polyploidization, recent introgression, incorrect taxonomy, or chloroplast capture. Multiple genomic data and population genetic studies are therefore needed to reveal the evolutionary history of Primula. Our results provided valuable information for intraspecific variation and phylogenetic relationships within Primula.

Female self-incompatibility type in heterostylous Primula is determined by the brassinosteroid-inactivating cytochrome P450 CYP734A50.

Most flowering plants are hermaphrodites, with flowers having both male and female reproductive organs. One widespread adaptation to limit self-fertilization is self-incompatibility (SI), where self-pollen fails to fertilize ovules.1,2 In homomorphic SI, many morphologically indistinguishable mating types are found, although in heteromorphic SI, the two or three mating types are associated with different floral morphologies.3-6 In heterostylous Primula, a hemizygous supergene determines a short-styled S-morph and a long-styled L-morph, corresponding to two different mating types, and full seed set only results from intermorph crosses.7-9 Style length is controlled by the brassinosteroid (BR)-inactivating cytochrome P450 CYP734A50,10 yet it remains unclear what defines the male and female incompatibility types. Here, we show that CYP734A50 also determines the female incompatibility type. Inactivating CYP734A50 converts short S-morph styles into long styles with the same incompatibility behavior as L-morph styles, and this effect can be mimicked by exogenous BR treatment. In vitro responses of S- and L-morph pollen grains and pollen tubes to increasing BR levels could only partly explain their different in vivo behavior, suggesting both direct and indirect effects of the different BR levels in S- versus L-morph stigmas and styles in controlling pollen performance. This BR-mediated SI provides a novel mechanism for preventing self-fertilization. The joint control of morphology and SI by CYP734A50 has important implications for the evolutionary buildup of the heterostylous syndrome and provides a straightforward explanation for why essentially all of the derived self-compatible homostylous Primula species are long homostyles.11.

The complete chloroplast genome sequences of five pinnate-leaved Primula species and phylogenetic analyses.

The six pinnate-leaved species are a very particular group in the genus Primula. In the present paper, we sequenced, assembled and annotated the chloroplast genomes of five of them (P. cicutarrifolia, P. hubeiensis, P. jiugongshanensis, P. merrilliana, P. ranunculoides). The five chloroplast genomes ranged from ~ 150 to 152 kb, containing 113 genes (four ribosomal RNA genes, 29 tRNA genes and 80 protein-coding genes). The six pinnate-leaved species exhibited synteny of gene order and possessed similar IR boundary regions in chloroplast genomes. The gene accD was pseudogenized in P. filchnerae. In the chloroplast genomes of the six pinnate-leaved Primula species, SSRs, repeating sequences and divergence hotspots were identified; ycf1 and trnH-psbA were the most variable markers among CDSs and noncoding sequences, respectively. Phylogenetic analyses showed that the six Primula species were separated into two distant clades: one was formed by P. filchnerae and P. sinensis and the other clade was consisting of two subclades, one formed by P. hubeiensis and P. ranunculoides, the other by P. merrilliana, P. cicutarrifolia and P. jiugongshanensis. P. hubeiensis was closely related with P. ranunculoides and therefore it should be placed into Sect. Ranunculoides. P. cicutarrifolia did not group first with P. ranunculoides but with P. merrilliana, although the former two were once united in one species, our results supported the separation of P. ranunculoides from P. cicutarrifolia as one distinct species.

Nectar robbers influence the trait-fitness relationship of Primula secundiflora.

The trait-fitness relationship influences the strength and direction of floral evolution. To fully understand and predict the evolutionary trajectories of floral traits, it is critical to disentangle the direct and indirect effects of floral traits on plant fitness in natural populations. We experimentally quantified phenotypic selection on floral traits through female fitness and estimated the casual effects of nectar robbing with different nectar robbing intensities on trait-fitness relationships in both the L- (long-style and short-anther phenotype) and S-morph (short-style and long-anther phenotype) flowers among Primula secundiflora populations. A larger number of flowers and wider corolla tubes had both direct and indirect positive effects on female fitness in the P. secundiflora populations. The indirect effects of these two traits on female fitness were mediated by nectar robbers. The indirect effect of the number of flowers on female fitness increased with increasing nectar robbing intensity. In most populations, the direct and/or indirect effects of floral traits on female fitness were stronger in the S-morph flowers than in the L-morph flowers. In addition, nectar robbers had a direct positive effect on female fitness, but this effect varied between the L- and S-morph flowers. These results show the potential role of nectar robbers in influencing the trait-fitness relationships in this primrose species.

Comparative intra- and interspecific sexual organ reciprocity in four distylous Primula species in the Himalaya-Hengduan Mountains.

Distyly is a mechanism promoting cross-pollination within a balanced polymorphism. Numerous studies show that the degree of inter-morph sexual organ reciprocity (SOR) within species relates to its pollen-mediated gene flow. Similarly, a lower interspecific SOR should promote interspecific isolation when congeners are sympatric, co-blooming and share pollinators. In this comparative study, we address the significance of SOR at both intra- and interspecific levels. Seventeen allopatric and eight sympatric populations representing four Primula species (P. anisodora, P. beesiana, P. bulleyana and P. poissonii) native to the Himalaya-Hengduan Mountains were measured for eight floral traits in both long- and short-styled morphs. GLMM and spatial overlap methods were used to compare intra- and interspecific SOR. While floral morphology differed among four Primula species, SOR within species was generally higher than between species, but in species pairs P. poissonii/P. anisodora and P. beesiana/P. bulleyana, the SOR was high at both intra- and interspecific levels. We did not detect a significant variation in intraspecific SOR or interspecific SOR when comparing allopatric versus sympatric populations for all species studied. As intraspecific SOR increased, disassortative mating may be promoted. As interspecific SOR decreased, interspecific isolation between co-flowering species pairs also may increase. Hybridisation between congeners occurred when interspecific SOR increased in sympatric populations, as confirmed in two species pairs, P. poissonii/P. anisodora and P. beesiana/P. bulleyana.

Ecological correlates and genetic consequences of evolutionary transitions from distyly to homostyly.

Background and Aims: The outbreeding floral polymorphism heterostyly frequently breaks down, resulting in the evolution of self-fertilization as a result of homostyle formation. Here, the loss of floral polymorphism in distylous Primula oreodoxa, a sub-alpine species restricted to western Sichuan, China, was examined by investigating the ecological correlates and genetic consequences of mating system transitions. Several key questions were addressed. (1) What are the frequencies, geographical distribution and reproductive characteristics of floral morphs in distylous and homostylous populations? (2) Does increased elevation influence pollinator service and the likelihood of inbreeding in populations? (3) How often has homostyly originated and what are the consequences of the breakdown of distyly for the amounts and distribution of genetic diversity in populations? Methods: Fourteen populations throughout the range of P. oreodoxa were sampled, and morph frequencies and floral characteristics were recorded. Polymorphism at microsatellite loci and chloroplast DNA (cpDNA) variation were used to quantify population genetic structure and genetic relationships among populations. Controlled pollinations and studies of pollen tube growth and fertility were conducted to determine the compatibility status of populations and their facility for autonomous self-pollination. Finally, visitation rates of long- and short-tongued pollinators to distylous and homostylous populations at different elevations were compared to determine if increased elevation was associated with deterioration in pollinator service. Key Results: In contrast to most heterostylous species, both distylous and homostylous morphs of P. oreodoxa are highly self-compatible, but only homostyles have the facility for autonomous self-pollination. Homostyles set significantly more fruit and seeds following open pollination than the distylous morphs. Visitation by long-tongued pollinators was significantly lower in homostylous populations, and overall rates of insect visitation decreased with elevation. Genetic diversity was significantly lower in homostylous populations, with evidence of increased inbreeding at higher elevation. Patterns of cpDNA variation were consistent with multiple transitions from distyly to homostyly and limited gene flow among populations. Conclusions: The results of this study support the hypothesis that the multiple loss of floral polymorphism in distylous P. oreodoxa is associated with unsatisfactory pollinator service, with homostyles benefiting from reproductive assurance as a result of autonomous self-pollination.

Genetic architecture and evolution of the S locus supergene in Primula vulgaris.

Darwin's studies on heterostyly in Primula described two floral morphs, pin and thrum, with reciprocal anther and stigma heights that promote insect-mediated cross-pollination. This key innovation evolved independently in several angiosperm families. Subsequent studies on heterostyly in Primula contributed to the foundation of modern genetic theory and the neo-Darwinian synthesis. The established genetic model for Primula heterostyly involves a diallelic S locus comprising several genes, with rare recombination events that result in self-fertile homostyle flowers with anthers and stigma at the same height. Here we reveal the S locus supergene as a tightly linked cluster of thrum-specific genes that are absent in pins. We show that thrums are hemizygous not heterozygous for the S locus, which suggests that homostyles do not arise by recombination between S locus haplotypes as previously proposed. Duplication of a floral homeotic gene 51.7 million years (Myr) ago, followed by its neofunctionalization, created the current S locus assemblage which led to floral heteromorphy in Primula. Our findings provide new insights into the structure, function and evolution of this archetypal supergene.

Presence versus absence of CYP734A50 underlies the style-length dimorphism in primroses.

Heterostyly is a wide-spread floral adaptation to promote outbreeding, yet its genetic basis and evolutionary origin remain poorly understood. In Primula (primroses), heterostyly is controlled by the S-locus supergene that determines the reciprocal arrangement of reproductive organs and incompatibility between the two morphs. However, the identities of the component genes remain unknown. Here, we identify the Primula CYP734A50 gene, encoding a putative brassinosteroid-degrading enzyme, as the G locus that determines the style-length dimorphism. CYP734A50 is only present on the short-styled S-morph haplotype, it is specifically expressed in S-morph styles, and its loss or inactivation leads to long styles. The gene arose by a duplication specific to the Primulaceae lineage and shows an accelerated rate of molecular evolution. Thus, our results provide a mechanistic explanation for the Primula style-length dimorphism and begin to shed light on the evolution of the S-locus as a prime model for a complex plant supergene.

Morphological and Molecular Phylogenetic Data Reveal a New Species of Primula (Primulaceae) from Hunan, China.

A new species of Primulaceae, Primula undulifolia, is described from the hilly area of Hunan province in south-central China. Its morphology and distributional range suggest that it is allied to P. kwangtungensis, both adapted to subtropical climate, having contiguous distribution and similar habitat, growing on shady and moist cliffs. Petioles, scapes and pedicels of them are densely covered with rusty multicellular hairs, but the new species can be easily distinguished by its smaller flowers and narrowly oblong leaves with undulate margins. Molecular phylogenetic analysis based on four DNA markers (ITS, matK, trnL-F and rps16) confirmed the new species as an independent lineage and constitutes a main clade together with P. kwangtungensis, P. kweichouensis, P. wangii and P. hunanensis of Primula sect. Carolinella.

The first genetic linkage map of Primulina eburnea (Gesneriaceae) based on EST-derived SNP markers.

Primulina eburnea is a promising candidate for domestication and floriculture, since it is easy to culture and has beautiful flowers. An F2 population of 189 individuals was established for the construction of first-generation linkage maps based on expressed sequence tags-derived single-nucleotide polymorphism markers using the massARRAY genotyping platform. Of the 232 screened markers, 215 were assigned to 18 LG according to the haploid number of chromosomes in the species. The linkage map spanned a total of 3774.7 cM with an average distance of 17.6 cM between adjacent markers. This linkage map provides a framework for identification of important genes in breeding programmes.

On the origins of observations of heterostyly in Primula.

In 1862, Charles Darwin published his landmark study on the different forms of flower in Primula; he coined the term distyly and subsequently expanded his studies to other species, including those with tristyly. Darwin is widely recognized as the first to study pin and thrum flowers in Primula, and to provide an explanation for the functional significance of the two floral morphs. Our laboratory is pursuing the genes that underpin floral heteromorphy in Primula, work influenced by Darwin's observations. One day, while appreciating a print of Primula vulgaris from William Curtis' Flora Londinensis, I was struck by the fact that I was looking at images of dimorphic Primula flowers captured in a late-1700s copper-plate engraving that predated Darwin's observations by over 70 yr. This realization triggered a journey into archives of botanical texts, herbals and florilegia from the 16(th) to 19(th) Centuries, and correspondence archives, in search of earlier documents that could have influenced Darwin and the origins of an idea. Darwin was not the first to observe floral heteromorphy in Primula, but he was the first to realize the significance of the two floral morphs. Darwin's insight and exposition of purpose have underpinned all consequent work on the subject.

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.

Unidirectional hybridization and reproductive barriers between two heterostylous primrose species in north-west Yunnan, China.

BACKGROUND AND AIMS: Heteromorphy in flowers has a profound effect on breeding patterns within a species, but little is known about how it affects reproductive barriers between species. The heterostylous genus Primula is very diverse in the Himalaya region, but hybrids there have been little researched. This study examines in detail a natural hybrid zone between P. beesiana and P. bulleyana. METHODS: Chloroplast sequencing, AFLP (amplified fragment length polymorphism) markers and morphological comparisons were employed to characterize putative hybrids in the field, using synthetic F1s from hand pollination as controls. Pollinator visits to parent species and hybrids were observed in the field. Hand pollinations were conducted to compare pollen tube growth, seed production and seed viability for crosses involving different morphs, species and directions of crossing. KEY RESULTS: Molecular data revealed all hybrid derivatives examined to be backcrosses of first or later generations towards P. bulleyana: all had the chloroplast DNA (cpDNA) of this species. Some individuals had morphological traits suggesting they were hybrids, but they were genetically similar to P. bulleyana; they might have been advanced generation backcrosses. Viable F1s could not be produced with P. bulleyana pollen on P. beesiana females, irrespective of the flower morphs used. Within-morph crosses for each species had very low (<10 %) seed viability, whereas crosses between pin P. bulleyana (female) and pin P. beesiana had a higher seed viability of 30 %. Thus genetic incompatibility mechanisms back up mechanical barriers to within-morph crosses in each species, but are not the same between the two species. The two species share their main pollinators, and pollinators were observed to fly between P. bulleyana and hybrids, suggesting that pollinator behaviour may not be an important isolating factor. CONCLUSIONS: Hybridization is strongly asymmetric, with P. bulleyana the only possible mother and all detected hybrids being backcrosses in this direction. Partial ecological isolation and inhibition of heterospecific pollen, and possibly complete barriers to F1 formation on P. beesiana, may be enough to make F1 formation very rare in these species. Therefore, with no F1 detected, this hybrid zone may have a finite life span as successive generations become more similar to P. bulleyana.

Pollination ecology in the narrow endemic winter-flowering Primula allionii (Primulaceae).

Reduction of pollen flow can affect plant abundance and population viability and cause selection on plant mating system and floral traits. Little is known on the effect of this phenomenon in species naturally restricted to small and isolated habitats, that may have developed strategies to cope with long-term isolation and small population size. We investigated the pollination ecology of the endemic distylous winter-flowering P. allionii to verify the possible limitation of female fitness due to reduced pollinator visits. We recorded a higher production of pollen grains in long-styled morph, and a higher seed set in short-styled morph. The high intra-morph variability of sexual organ position may explain the hybridization phenomena allowing and easier intra-morph pollination. The fruit set is constant, although its winter-flowering period might decrease pollen transfer. Nevertheless, the lower competition for pollinators with neighbouring plants and the long-lasting anthesis may offset its reproductive success. Even if our results show no evidence of imminent threats, changes in plant-pollinator interactions might increase inbreeding, resulting in an increased extinction risk.

Decrease of sexual organ reciprocity between heterostylous primrose species, with possible functional and evolutionary implications.

BACKGROUND AND AIMS: Heterostyly is a floral polymorphism that has fascinated evolutionary biologists since Darwin's seminal studies on primroses. The main morphological characteristic of heterostyly is the reciprocal placement of anthers and stigmas in two distinct (distyly) floral morphs. Variation in the degree of intermorph sexual reciprocity is relatively common and known to affect patterns of pollen transfer within species. However, the partitioning of sexual organ reciprocity within and between closely related species remains unknown. This study aimed at testing whether intermorph sexual reciprocity differs within vs. between primrose species that hybridize in nature and whether the positions of sexual organs are correlated with other floral traits. METHODS: Six floral traits were measured in both floral morphs of 15 allopatric populations of Primula elatior, P. veris and P. vulgaris, and anther-stigma reciprocity was estimated within and between species. A combination of univariate and multivariate approaches was used to test whether positions of reproductive organs were less reciprocal between than within species, to assess correlations between sexual organ positions and other corolla traits, and to quantify differences between morphs and species. KEY RESULTS: The three species were morphologically well differentiated in most floral traits, except that P. veris and P. vulgaris did not differ significantly in sexual organ positions. Overall, lower interspecific than intraspecific sexual organ reciprocity was detected. This decrease was marked between P. elatior and P. vulgaris, intermediate and variable between P. elatior and P. veris, but negligible between P. veris and P. vulgaris. CONCLUSIONS: Differences in anther and stigma heights between the analysed primrose species were of the same magnitude or larger than intraspecific differences that altered pollen flow within other heterostylous systems. Therefore, it is possible to suggest that considerable reductions of sexual organ reciprocity between species may lower interspecific pollen flow, with potential effects on reproductive isolation.

Flavonoids in selected Primula spp.: bridging micromorphology with chemodiversity.

A combined study was carried out on the micromorphology and chemistry of glandular trichomes with focus on Primula vialii and P. vulgaris, respectively. Epifluorescence microscopy was applied to study the auto-fluorescent properties of flavonoids and their localization in glandular trichomes. Both species differed in the morphology of the glandular trichomes and in the exudate flavonoid composition. Leaf glands from P. vialii and from some species of subgen. Primula exhibited uniform glandular fluorescence, but notable differentiation was observed within a single leaf of P. vulgaris. Our observations indicate that exudate flavonoids are not transported from the tissue to the glandular hairs. Conversely, only the newly isolated glycoside 1 (kaempferol 3-O-(2"'-rhamnosyl)-robinobioside) was obtained from leaf tissue of P. vialii after removal of the exudate. Its structure was confirmed by NMR and mass spectrometry. This glycoside was not detected in tissue extracts of P. vulgaris after similar treatment. The observed chemical diversity is discussed, with focus on possible correlation with glandular structures and tissue differentiation in Primula, and also against other studied species. Aspects of biosynthesis in relation to tissue-specific flavonoid diversification are shortly addressed.