Genetic Causes and Genomic Consequences of Breakdown of Distyly in Linum trigynum.

Distyly is an iconic floral polymorphism governed by a supergene, which promotes efficient pollen transfer and outcrossing through reciprocal differences in the position of sexual organs in flowers, often coupled with heteromorphic self-incompatibility. Distyly has evolved convergently in multiple flowering plant lineages, but has also broken down repeatedly, often resulting in homostylous, self-compatible populations with elevated rates of self-fertilization. Here, we aimed to study the genetic causes and genomic consequences of the shift to homostyly in Linum trigynum, which is closely related to distylous Linum tenue. Building on a high-quality genome assembly, we show that L. trigynum harbors a genomic region homologous to the dominant haplotype of the distyly supergene conferring long stamens and short styles in L. tenue, suggesting that loss of distyly first occurred in a short-styled individual. In contrast to homostylous Primula and Fagopyrum, L. trigynum harbors no fixed loss-of-function mutations in coding sequences of S-linked distyly candidate genes. Instead, floral gene expression analyses and controlled crosses suggest that mutations downregulating the S-linked LtWDR-44 candidate gene for male self-incompatibility and/or anther height could underlie homostyly and self-compatibility in L. trigynum. Population genomic analyses of 224 whole-genome sequences further demonstrate that L. trigynum is highly self-fertilizing, exhibits significantly lower genetic diversity genome-wide, and is experiencing relaxed purifying selection and less frequent positive selection on nonsynonymous mutations relative to L. tenue. Our analyses shed light on the loss of distyly in L. trigynum, and advance our understanding of a common evolutionary transition in flowering plants.

Parallel evolution of morphological and genomic selfing syndromes accompany the breakdown of heterostyly.

Evolutionary transitions from outcrossing to selfing in flowering plants have convergent morphological and genomic signatures and can involve parallel evolution within related lineages. Adaptive evolution of morphological traits is often assumed to evolve faster than nonadaptive features of the genomic selfing syndrome. We investigated phenotypic and genomic changes associated with transitions from distyly to homostyly in the Primula oreodoxa complex. We determined whether the transition to selfing occurred more than once and investigated stages in the evolution of morphological and genomic selfing syndromes using 22 floral traits and both nuclear and plastid genomic data from 25 populations. Two independent transitions were detected representing an earlier and a more recently derived selfing lineage. The older lineage exhibited classic features of the morphological and genomic selfing syndrome. Although features of both selfing syndromes were less developed in the younger selfing lineage, they exhibited parallel development with the older selfing lineage. This finding contrasts with the prediction that some genomic changes should lag behind adaptive changes to morphological traits. Our findings highlight the value of comparative studies on the timing and extent of transitions from outcrossing to selfing between related lineages for investigating the tempo of morphological and molecular evolution.

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.

Phylogenomic analysis reveals multiple evolutionary origins of selfing from outcrossing in a lineage of heterostylous plants.

Evolutionary transitions from outcrossing to selfing often occur in heterostylous plants. Selfing homostyles originate within distylous populations and frequently evolve to become reproductively isolated species. We investigated this process in 10 species of Primula section Obconicolisteri using phylogenomic approaches and inferred how often homostyly originated from distyly and its consequences for population genetic diversity and floral trait evolution. We estimated phylogenetic relationships and reconstructed character evolution using the whole plastome comprised of 76 protein-coding genes. To investigate mating patterns and genetic diversity we screened 15 microsatellite loci in 40 populations. We compared floral traits among distylous and homostylous populations to determine how phenotypically differentiated homostyles were from their distylous ancestors. Section Obconicolisteri was monophyletic and we estimated multiple independent transitions from distyly to homostyly. High selfing rates characterised homostylous populations and this was associated with reduced genetic diversity. Flower size and pollen production were reduced in homostylous populations, but pollen size was significantly larger in some homostyles than in distylous morphs. Repeated transitions to selfing in section Obconicolisteri are likely to have been fostered by the complex montane environments that species occupy. Unsatisfactory pollinator service is likely to have promoted reproductive assurance in homostyles leading to subsequent population divergence through isolation.

'A most complex marriage arrangement': recent advances on heterostyly and unresolved questions.

Heterostylous genetic polymorphisms provide paradigmatic systems for investigating adaptation and natural selection. Populations are usually comprised of two (distyly) or three (tristyly) mating types, maintained by negative frequency-dependent selection resulting from disassortative mating. Theory predicts this mating system should result in equal style-morph ratios (isoplethy) at equilibrium. Here, I review recent advances on heterostyly, focusing on examples challenging stereotypical depictions of the polymorphism and unresolved questions. Comparative analyses indicate multiple origins of heterostyly, often within lineages. Ecological studies demonstrate that structural components of heterostyly are adaptations improving the proficiency of animal-mediated cross-pollination and reducing pollen wastage. Both neutral and selective processes cause deviations from isoplethy in heterostylous populations, and, under some ecological and demographic conditions, cause breakdown of the polymorphism, resulting in either the evolution of autogamy and mixed mating, or transitions to alternative outcrossing systems, including dioecy. Earlier ideas on the genetic architecture of the S-locus supergene governing distyly have recently been overturned by discovery that the dominant S-haplotype is a hemizygous region absent from the s-haplotype. Ecological, phylogenetic and molecular genetic data have validated some features of theoretical models on the selection of the polymorphism. Although heterostyly is the best-understood floral polymorphism in angiosperms, many unanswered questions remain.

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.

Outbreeding depression and breeding system evolution in small, remnant populations of Primula vulgaris: consequences for genetic rescue.

Many species suffer from anthropogenic habitat fragmentation. The resulting small and isolated populations are more prone to extinction due to, amongst others, genetic erosion, inbreeding depression and Allee-effects. Genetic rescue can help mitigate such problems, but might result in outbreeding depression. We evaluated offspring fitness after selfing and outcrossing within and among three very small and isolated remnant populations of the heterostylous plant Primula vulgaris. We used greenhouse-grown offspring from these populations to test several fitness components. One population was fixed for the pin-morph, and was outcrossed with another population in the field to obtain seeds. Genetic diversity of parent and offspring populations was studied using microsatellites. Morph and population-specific heterosis, inbreeding and outbreeding depression were observed for fruit and seed set, seed weight and cumulative fitness. Highest fitness was observed in the field-outcrossed F1-population, which also showed outbreeding depression following subsequent between-population (back)crossing. Despite outbreeding depression, fitness was still relatively high. Inbreeding coefficients indicated that the offspring were more inbred than their parent populations. Offspring heterozygosity and inbreeding coefficients correlated with observed fitness. One population is evolving homostyly, showing a thrum morph with an elongated style and high autonomous fruit and seed set. This has important implications for conservation strategies such as genetic rescue, as the mating system will be altered by the introduction of homostyles.

Phylogeographic insights on the evolutionary breakdown of heterostyly.

The breakdown of heterostyly to homostyly is a classic system for the investigation of evolutionary transitions from outcrossing to selfing. Loss of sexual polymorphism is characterized by changes to population morph structure and floral morphology. Here, we used molecular phylogeography to investigate the geographical context for the breakdown process in Primula chungensis, a species with distylous and homostylous populations. We genotyped plants from 20 populations throughout the entire range in south-west China using the chloroplast intergenic spacer (trnL-trnF), nuclear internal transcribed spacer (ITS) and 10 nuclear microsatellite loci, and determined the genetic relationships among populations and the variation in floral traits associated with homostyle evolution. The marker data identified two multi-population lineages (Tibet and Sichuan) and one single-population lineage (Yunnan), a pattern consistent with at least two independent origins of homostyly. Evidence from flower and pollen size variation is consistent with the hypothesis that transitions to selfing have arisen by the same genetic mechanism involving recombination and/or mutation at the distyly linkage group. Nevertheless, flowers of homostylous lineages have followed divergent evolutionary trajectories following their origin, resulting in populations with both approach and reverse herkogamy. Our study illustrates a rare example of the near-complete replacement of sexual polymorphism by floral monomorphism in a heterostylous species.

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.

Transitions from distyly to homostyly are associated with floral evolution in the buckwheat genus (Fagopyrum).

PREMISE OF THE STUDY: Documenting trait transitions among species with dimorphic flowers can help to test whether similar patterns of selection are responsible for divergence in floral traits among different species. Heterostyly is thought to promote outcrossing. Theory suggests that the evolutionary transition from heterostylous to homostylous flowers should be accompanied by a reduction in floral size in which pollen size and style length are expected to covary. Patterns of such correlated floral trait evolution have not, however, been widely examined. METHODS: The evolutionary history of heterostyly and homostyly was reconstructed from a molecular phylogeny of 13 species of Fagopyrum and two outgroups, based on one nuclear gene (nrITS) and three chloroplast regions (matK, atpB-rbcL, and psbA-trnH spacer). Floral traits of nine Fagopyrum species including pollen number and size, as well as stigma depth (length of the capitate stigma), were measured and ancestral characters of the herkogamic condition were reconstructed. KEY RESULTS: Three transitions from distyly to homostyly were observed. In two transitions, flower size (corolla diameter, pedicel length), herkogamy (stigma-anther distance) and pollen production decreased, but stigma depth and pollen size did not. Changes of anther height and style length were inconsistent. The predicted positive relationship between style length and pollen size in the two transitions to homostyly was not observed. CONCLUSIONS: Floral evolution accompanying transitions to homostyly in Fagopyrum were found to be consistent with predictions of mating system evolution theory, and the correlation of traits in distylous vs. homostylous species revealed that pollen size generally correlates with stigma depth rather than style length.

Development of polymorphic microsatellite markers for distylous-homostylous Primula secundiflora (Primulaceae) using HiSeq sequencing.

Primula secundiflora is an insect-pollinated, perennial herb belonging to the section Proliferae (Primulaceae) that exhibits considerable variation in its mating system, with predominantly outcrossing populations comprising long-styled and short-styled floral morphs and selfing populations comprising only homostyles. To facilitate future investigations of the population genetics and mating patterns of this species, we developed 25 microsatellite markers from P. secundiflora using next-generation sequencing and measured polymorphism and genetic diversity in a sample of 30 individuals from three natural populations. The markers displayed high polymorphism, with the number of observed alleles per locus ranging from three to 16 (mean = 8.36). The observed and expected heterozygosities ranged from 0.100 to 1.000 and 0.145 to 0.843, respectively. Twenty-one of the loci were also successfully amplified in P. denticulata. These microsatellite markers should provide powerful tools for investigating patterns of population genetic diversity and the evolutionary relationships between distyly and homostyly in P. secundiflora.

Floral morph variation mediated by clonal growth and pollinator functional groups of Limonium otolepis in a heterostylous fragmented population.

Abstract. Heterostyly, a genetic style polymorphism, is linked to symmetric pollen transfer, vital for its maintenance. Clonal growth typically impacts sexual reproduction by influencing pollen transfer. However, the floral morph variation remains poorly understood under the combined effects of pollinators and clonal growth in heterostyly characterized by negative frequency-dependent selection and disassortative mating. We estimated morph ratios, ramets per genet and heterostylous syndrome and quantified legitimate pollen transfer via clonal growth, pollinators and reciprocal herkogamy between floral morphs in Limonium otolepis, a fragmented population composed of five subpopulations in the desert environment of northwestern China, with small flower and large floral morph variation. All subpopulations but one exhibited pollen-stigma morphology dimorphism. The compatibility between mating types with different pollen-stigma morphologies remained consistent regardless of reciprocal herkogamy. Biased ratios and ramets per genet of the two mating types with distinct pollen-stigma morphologies caused asymmetric pollen flow and varying fruit sets in all subpopulations. Short-tongued insects were the primary pollinators due to small flower sizes. However, pollen-feeding Syrphidae sp. triggered asymmetry in pollen flow between high and low sex organs, with short-styled morphs having lower stigma pollen depositions and greater variation. Clonal growth amplified this variation by reducing intermorph pollen transfer. All in all, pollinators and clonal growth jointly drive floral morph variation. H-morphs with the same stigma-anther position and self-incompatibility, which mitigate the disadvantages of sunken low sex organs with differing from the classical homostyly, might arise from long- and short-styled morphs through a 'relaxed selection'. This study is the first to uncover the occurrence of the H-morph and its associated influencing factors in a distylous plant featuring clonal growth, small flowers and a fragmented population.

The rapid appearance of homostyly in a cultivated distylous population of Primula forbesii.

Evolutionary breakdown from rigorous outbreeding to self-fertilization frequently occurs in angiosperms. Since the pollinators are not necessary, self-compatible populations often reduce investment in floral display characteristics and pollination reward. Primula forbesii is a biennial herb with distribution restricted to southwest China; it was initially a self-incompatible distylous species, but after 20 years of artificial domestication, homostyly appeared. This change in style provides an ideal material to explore the time required for plant mating systems to adapt to new environmental changes and test whether flower attraction has reduced following transitions to selfing. We did a survey in wild populations of P. forbesii where its seeds were originally collected 20 years ago and recorded the floral morph frequencies and morphologies. The floral morphologies, self-incompatibility, floral scent, and pollinator visitation between distyly and homostyly were compared in greenhouse. Floral morph frequencies of wild populations did not change, while the cultivated population was inclined to L-morph and produced homostyly. Evidence from stigma papillae and pollen size supports the hypothesis that the homostyly possibly originated from mutations of large effect genes in distylous linkage region. Transitions to self-compatible homostyly are accompanied by smaller corolla size, lower amounts of terpenoids, especially linalool and higher amounts of fatty acid derivatives. The main pollinators in the greenhouse were short-tongued Apis cerana. However, homostyly had reduced visiting frequency. The mating system of P. forbesii changed rapidly in just about 20 years of domestication, and our findings confirm the hypothesis that the transition to selfing is accompanied by decreased flower attraction.

 Primulaxinningensis (Primulaceae), a new species from karst caves in Hunan, China.

Primulaxinningensis Wei Zhang bis & J.W.Shao, a new species from Hunan Province, China, is described. Its leaf morphology is similar to the P.merrilliana complex and flower morphology similar to P.cicutariifolia, but it can be distinguished from the former by the black pollen sac, corolla lobes apex obviously emarginate and can be differed from the latter by cotyledon triangular obovate, plants densely covered with glandular hairs and special habitat (karst caves). The whole plastid genome of this new species is 151, 601-151, 630 bp in length. Based on the whole plastid genome sequences, phylogenetic trees revealed that the new species did not genetically relate to the above two mentioned morphologically similar species, but it was closely related to P.hubeiensis. Currently, only three populations were discovered within a small distribution area, thus, it is preliminarily considered as Vulnerable (VU) according to criteria of the IUCN Red List.

Evolution of Autonomous Selfing in Marginal Habitats: Spatiotemporal Variation in the Floral Traits of the Distylous Primula wannanensis.

Outcrossing plant species are more likely to exhibit autonomous selfing in marginal habitats to ensure reproduction under conditions of limited pollinator and/or mate availability. Distyly is a classical paradigm that promotes outcrossing; however, little is known about the variation in floral traits associated with distylous syndrome in marginal populations. In this study, we compared the variation in floral traits including stigma and anther height, corolla tube length, herkogamy, and corolla diameter between the central and peripheral populations of the distylous Primula wannanensis, and assessed the variation of floral traits at early and late florescence stages for each population. To evaluate the potential consequences of the variation in floral traits on the mating system, we investigated seed set in each population under both open-pollinated and pollinator-excluded conditions. The flower size of both short- and long-styled morphs was significantly reduced in late-opening flowers compared with early opening flowers in both central and peripheral populations. Sex-organ reciprocity was perfect in early opening flowers; however, it was largely weakened in the late-opening flowers of peripheral populations compared with central populations. Of these flowers, disproportionate change in stigma height (elongated in S-morph and shortened in L-morph) was the main cause of reduced herkogamy, and seed set was fairly high under pollinator-excluded condition. Our results provide empirical support for the hypothesis on the evolution of delayed autonomous selfing in marginal populations of distylous species. Unsatisfactory pollinator service is likely to have promoted reproductive assurance of distylous plants with largely reduced herkogamy mimicking "homostyles."

Intraspecific variation of self-incompatibility in the distylous plant Primula merrilliana.

Heteromorphic self-incompatibility can prevent self- and intramorph fertilization while favouring intermorph mating and the maintenance of morph-ratio stability in heterostylous populations. However, variation in the expression of self-incompatibility intraspecies has seldom been assessed. Through hand pollinations and microsatellite markers, the variation in the expression of self-incompatibility and genetic diversity were studied in distylous plant Primula merrilliana. We discovered that the strength of self-incompatibility varied extensively among individuals and populations, from pronounced to weaker self-incompatibility in distylous populations, all the way to strong self-compatibility in homostylous populations. Each distylous population included self-incompatible (SI), partly self-compatible (PSC) and self-compatible (SC) individuals, with the index of self-compatibility (ISC) ranging from 0.07 to 0.68 across populations. Self-compatible populations (ISC > 0.25) were not genetically clustered but were more closely related to populations with high SI and SC individuals were mixed with SI individuals within populations. The ISC and the proportions of SC and PSC individuals were higher in peripheral than in central populations, but no decrease of genetic diversity and no deviations of floral morph ratio from isoplethy were detected in peripheral populations. Additionally, the expression of self-incompatibility was stronger in long-styled flowers than in short-styled flowers. The variation in the strength of self-incompatibility documented in P. merrilliana cautions against the estimation of ISC from a few individuals or populations in distylous species and provides a more complex and nuanced understanding of the role of self-incompatibility in heterostyly.

Characterization of 30 microsatellite markers in distylous Primula sinolisteri (Primulaceae) using HiSeq sequencing.

PREMISE OF THE STUDY: Microsatellite markers were developed for Primula sinolisteri, a perennial distylous herb belonging to section Obconicolisteri (Primulaceae), to facilitate future investigations of the population genetics and mating patterns of populations in this species. METHODS AND RESULTS: We developed 30 microsatellite markers for P. sinolisteri using HiSeq X-Ten sequencing and measured polymorphism and genetic diversity in a sample of 36 individuals from three natural populations. The markers displayed relatively high polymorphism, with the number of observed alleles per locus ranging from one to 19 (mean = 4.42). The observed and expected heterozygosity ranged from 0-1.000 and 0.083-0.882, respectively. Twenty-nine of the loci were also successfully amplified in homostylous P. sinolisteri var. aspera. CONCLUSIONS: The microsatellite markers we have identified in P. sinolisteri provide powerful tools for investigating patterns of population genetic diversity and the evolutionary relationships between heterostyly and homostyly in this species.

Variation in the degree of reciprocal herkogamy affects the degree of legitimate pollination in a distylous species.

Distyly is a widespread floral polymorphism characterized by the flowers within a population showing reciprocal placement of the anthers and stigma. Darwin hypothesizes that distyly evolves to promote precise pollen transfer between morphs. Primula chungensis exhibits two types of anther heights, and these two types of anthers show pollen of two different size classes. To understand whether the stigma could capture more pollen grains from the anthers of the pollen donor as the separation between the stigma of pollen receiver and the anther of pollen donor decreased, the present research assessed the source of the pollen load in a series of open-pollinated flowers with continuous variation of style lengths. Individuals with continuous variation of style length were tagged, and the selected flowers in the tagged plants were emasculated the day before dehiscence. The stigma of the emasculated flowers was fixed in fuchsin gel at the end of blooming. We assessed the pollen sources on each stigma by taking photos under a microscope and measured the diameter of each conspecific pollen grain with ImageJ. We found that a shorter distance from the stigmas to the anthers of a pollen donor gave the flower a higher capacity to receive pollen from those anthers. Our result provides a new evidence that distyly could promote the pollen transfer between morphs, which is consistent with Darwin's hypothesis of disassortative pollination. An alternative hypothesis for the evolution of distyly (e.g. selfing avoidance) might also be true, but less likely, because self-incompatibility would greatly avoid self-fertilization for many distylous species.

Morphological and molecular data confirm the transfer of homostylous species in the typically distylous genus Galianthe (Rubiaceae), and the description of the new species Galianthe vasquezii from Peru and Colombia.

Galianthe (Rubiaceae) is a neotropical genus comprising 50 species divided into two subgenera, Galianthe subgen. Galianthe, with 39 species and Galianthe subgen. Ebelia, with 11 species. The diagnostic features of the genus are: usually erect habit with xylopodium, distylous flowers arranged in lax thyrsoid inflorescences, bifid stigmas, 2-carpellate and longitudinally dehiscent fruits, with dehiscent valves or indehiscent mericarps, plump seeds or complanate with a wing-like strophiole, and pollen with double reticulum, rarely with a simple reticulum. This study focused on two species that were originally described under Diodia due to the occurrence of fruits indehiscent mericarps: Diodia palustris and D. spicata. In the present study, classical taxonomy is combined with molecular analyses. As a result, we propose that both Diodia species belong to Galianthe subgen. Ebelia. The molecular position within Galianthe, based on ITS and ETS sequences, has been supported by the following morphological characters: thyrsoid, spiciform or cymoidal inflorescences, bifid stigmas, pollen grains with a double reticulum, and indehiscent mericarps. However, both species, unlike the remainder of the genus Galianthe, have homostylous flowers, so the presence of this type of flower significantly modifies the generic concept. In this framework, a third homostylous species, Galianthe vasquezii, from the Andean region is also described. Until now, this species remained cryptic under specimens of Galianthe palustris It differs however from the latter by having longer calyx lobes, the presence of dispersed trichomes inside the corolla lobes (vs. glabrous), fruits that are acropetally dehiscent (vs. basipetally dehiscent), and its Andean geographical distribution (vs. Paranaense). Additionally, a lectotype has been chosen for Diodia palustris, Borreria pterophora has been placed under synonymy of Galianthe palustris, and Galianthe boliviana is reported for the first time from Peru. A key of all Galianthe species with indehiscent mericarps is also provided.

Pollination biology of the hexaploid self-compatible species Turnera velutina (Passifloraceae).

The evolution of monomorphisms from heterostylous ancestors has been related to the presence of homostyly and the loss of self-incompatibility, allowing the occurrence of selfing, which could be advantageous under pollinator limitation. However, flowers of some monomorphic species show herkogamy, attraction and rewarding traits that presumably favour cross-pollination and/or a mixed mating system. This study evaluated the contributions of pollinators, breeding system and floral traits to the reproduction of Turnera velutina, a herkogamous monomorphic species. Floral visitors and frequency of visits were recorded, controlled hand cross-pollinations were conducted under greenhouse and natural conditions, and individual variation in floral traits was characterised to determine their contribution to seed production. Apis mellifera was the most frequent floral visitor. Flowers presented approach herkogamy, high variation in nectar features, and a positive correlation of floral length with nectar volume and sugar concentration. Seed production did not differ between manual self- and cross-pollinations, controls or open cross-pollinations, but autonomous self-pollination produced, on average, 82.74% fewer seeds than the other forms, irrespective of the level of herkogamy. Differences in seed production among autonomous self-pollination and other treatments showed that T. velutina flowers depend on insect pollination for reproduction, and that approach herkogamy drastically reduced seed production in the absence of pollen vectors. The lack of differences in seed production from manual cross- and self-pollinations suggests the possible presence of a mixed mating system in the studied population. Overall, this species was possibly derived from a distylous ancestor but appears fully capable of outcrossing despite being monomorphic.