Autonomous Selfing Research Articles

Breeding system and pollination ecology of a potentially invasive alien Clematis vitalba L. in Ireland

Invasive alien plants can greatly affect native communities and ecosystem processes but only a small fraction of alien plant species become invasive. Barriers to establishment and invasion include reproductive limitations. Clematis vitalba L. has been a popular horticultural species for the past century and is widely distributed and can be highly invasive. In Ireland, it is considered naturalized and potentially invasive. Despite this, little is known about its reproductive biology. We carried out manipulative field experiments in Ireland and compared fruit and seed set from a number of pollination treatments, namely cross-pollination, geitonogamy, autogamy and natural pollination. We also recorded floral visitation to C. vitalba through a series of timed observations. We found that C. vitalba is capable of uniparental reproduction via geitonogamy and autonomous selfing, albeit at a reduced rate compared with outcrossing treatments. Clematis vitalba was visited by at least 10 native pollinator taxa, with hoverflies dominating visitation. Neither fruit set nor seed set in our study population was pollen limited. Given the lack of reproductive constraint, C. vitalba may easily spread in suitable habitats. This is of concern in Ireland, given its prevalence in some of the country’s most floristically diverse regions.

Small and surrounded: population size and land use intensity interact to determine reliance on autonomous selfing in a monocarpic plant.

Habitat fragmentation has transformed landscapes globally, leaving remnants embedded within a complex matrix that is rapidly becoming more developed. For many plant populations, the associated factors of decreased size and intensification of land use surrounding them are expected to increase pollen limitation ('PL'), unless autonomous self-pollination provides reproductive assurance ('RA'). Decreased pollinator visitation is often assumed to drive these patterns, but other, less studied mechanisms might include increased heterospecific pollen transfer or decreased conspecific pollen availability via florivory. I investigate how PL and RA and their potential underlying mechanisms vary with population size and land use intensity surrounding populations in the biennial Sabatia angularis (Gentianaceae). I estimated the capacity for seed production via autonomous self-pollination (i.e. autofertility). Over 2 years in 22 S. angularis populations across a fragmented landscape, I performed emasculation and pollen supplementation experiments measuring RA and PL, and quantified visitation rates of potential pollinators and a pollen consumer, conspecific pollen loads and rates of heterospecific pollen deposition. Autofertility based on fruit mass was 93 % under PL but only 51.6 % relative to maximal conditions. PL and RA were significant on average across populations in the first year of study. Variation in RA was significantly influenced by the interaction between population size and land use intensity, which in turn rendered PL independent of these factors. Visitation and heterospecific pollen deposition rates were greatest in small populations and declined with population size, while conspecific pollen loads were greatest in intermediate sized populations. Increased reliance on RA is predicted in small S. angularis populations surrounded by intense development, which can explain elevated selfing rates in fragmented populations of plant species more generally. Results from this study point toward forces such as heterospecific pollen transfer, self-pollen limitation or resource availability influencing the need and ability to rely on RA.

Does stigma curvature promote delayed selfing? An experimental investigation in Triodanis perfoliata (Campanulaceae).

Self-fertilisation that is delayed until after opportunities for outcrossing have ceased has been argued to provide both the reproductive assurance benefits of selfing and the genetic advantages of outcrossing. In the Campanulaceae, presentation of pollen on stylar hairs and progressive stigma curvature have been hypothesised to facilitate delayed selfing, but experimental tests are lacking. Stigma curvature is common in Campanula, a genus largely characterised by self-incompatibility, and therefore is unlikely to have initially evolved to promote self-fertilisation. In derived self-compatible species, however, stigma curvature might serve the secondary function of delayed selfing. We investigated delayed selfing in Triodanis perfoliata, a self-compatible relative of Campanula. Using floral manipulation experiments and pollen tube observations, we quantified the extent and timing of self-pollination. Further, we hypothesised that, if stigma curvature provides the benefit of delayed selfing in Triodanis, selection should have favoured retention of self-pollen through the loss of a stylar hair retraction mechanism. Results of a stigma removal experiment indicated that autonomous selfing produces partial seed set, but only some selfing was delayed. Pollen tube observations and a flower senescence assay also supported the finding of partial delayed selfing. Scanning electron microscopy revealed that pollen-collecting hairs retract during anthesis, which may limit the extent of delayed selfing. Delayed selfing appeared to be only partially effective in T.perfoliata. The stylar hair retraction in this species would seem to contradict selection for selfing. We suggest that caution and rigour are needed in interpreting floral traits as adaptive mechanisms for delayed selfing.

Late-acting self-incompatibility: a barrier to self-fertilization in sunnhemp (Crotalaria juncea L.)

In search of causal factors underlying perceived allogamy in sunnhemp (Crotalaria juncea L.), we investigated the breeding system and pollen–pistil interaction. Floral biology study in the species established Fabaceae type hermaphrodite zygomorphic flower with dimorphic anthers. Study of the breeding system revealed preferential success of allogamy despite delayed autonomous selfing mechanism characteristically prevailing in the species. Pollen–pistil interaction study in selfed and crossed pistils revealed significant differences in pollen tube growth rates inside the pistil. Further, in self-pollinated pistils callose formation observed at and around nucellus cells suggesting the late-acting type of self-incompatibility mechanism in the species. To the best of our knowledge, this is the first scientific communication underscoring the prevalence of self-incompatibility mechanism in sunnhemp.

Differences in pollination syndromes and the frequency of autonomous delayed selfing between co-flowering <i>Hibiscus aponeurus</i> (Sprague and Hutch) and <i>H. flavifolius</i> (Ulbr) from Kenya

Delayed autonomous selfing offers a mechanism for seed production when pollination levels are low or unpredictable. At Mpala Research Centre (MRC) in Kenya, we examined the relationships between floral attraction, insect visitation, and delayed autonomous selfing through backwards stylar curvature in the co-flowering Hibiscus aponeurus and H. flavifolius. Despite producing similar pollen and nectar rewards, visitation rates and the composition of floral visitor guilds varied significantly between these species. Across four years of observations, floral visitation in H. flavifolius was dominated by bees, and in H. aponeurus by a mixture of bees, butterflies and beetles. Visitation rates to H. flavifolius flowers (range 0.17 - 2.1 visits flr-1hr-1) were two times greater than to H. aponeurus flowers (range 0 - 2.7 visits flr-1hr-1), which resulted in significantly higher pollen deposition and removal rates in H. flavifolius than in H. aponeurus. Field crosses demonstrated little pollen limitation in either species. In open-pollinated flowers, H. aponeurus displayed significantly greater stylar curvature and apparent self-pollination than did H. flavifolius. Floral attributes in H. aponeurus, such as a smaller corolla size and a downwards orientation of the stylar column, also suggest that delayed selfing is a more important mechanism of reproductive assurance in this species than in H. flavifolius. Determining whether these differences in insect visitation and stylar curvature are characteristic for these species or are unique to MRC will require comparison with populations located in other parts of the ranges, genetic tests of selfing rates, and chemical analyses of nectar, pollen, and floral volatiles.

Aggregate enantiostyly: Floral visitor interactions with a previously unreported form of floral display

Floral herkogamy, or the spatial separation of reproductive structures, plays an important role in the evolution of plant mating systems. One form of herkogamy is enantiostyly, or the alternate presentation of stigmas to the left and right of the floral axis. Theoretically, enantiostyly increases outcrossing rate by promoting reciprocal pollen deposition and export, but empirical evidence of this is lacking. Understanding how pollinators interact with enantiostylous flowers will illuminate the mechanisms by which enantiostyly encourages outcross pollination. We quantified style deployment within and among plants, of Allionia incarnata L. (Nyctaginaceae), a species with a new kind of enantiostyly we call “aggregate enantiostyly”. A. incarnata is the first species known to have “compound blossoms”, with different flowers each containing a left- or right-handed style. We quantify the autonomous selfing rate and describe floral visitor interactions with this unique floral structure. We found equal frequency of left- and right-handed blossoms within and among plants. Common floral visitors, Hemiargus ceraunus and Halictus sp., interacted with either anthers or stigmas, but not both, indicating effective outcrossing at the flower level. Outcrossing produced more fruits than selfing. The order in which floral visitors interacted with reproductive structures indicates that enantiostyly may increase pollen export. However, we did not find evidence that enantiostyly affected pollen deposition. Our study provides an intriguing first look at the reproductive ecology of a species with a new unique form of herkogamy, and provides evidence on the adaptive significance of enantiostyly.

SECONDARY POLLEN PRESENTATION, PSYCHOPHILY AND ANEMOCHORY IN Lagascea mollis Cav. (ASTERACEAE)

Information on pollination ecology and seed dispersal aspects is essential to understand sexual reproduction in Lagascea mollis Cav. The study was aimed at providing details of pollen presentation system, pollination syndrome, pollinators and seed dispersal mode for L. mollis based mostly on field study. Paper chromatography technique was used for recording sugar and amino acid types in the nectar, since they are important to evaluate the pollination syndrome. The study indicated that L. mollis flowers during August-November. The plant produces synflorescences consisting of several capitula. Each capitulum produces a single disc floret. The plant is protandrous, temporally dioecious and exhibits secondary pollen presentation using brush mechanism to prevent autonomous selfing. The floral characteristics such as having synflorescence, narrow tubular corolla, production of sucrose-rich nectar with essential and non-essential amino acids and pollen charactersistics such as having spherical shape, tri-colpate apertures and strongly spinulose exine suggested that the plant is adapted for insect pollination. The plant is principally psychophilous. Other insects also visited the florets opportunistically and acted as supplementary pollinators. Further, thrips used this plant as breeding and feeding sites; their effect mostly geitonogamy. The fruit is an achene, anemochorous and also anthropochorous. Regeneration occurred from seeds and perennial root stock during rainy season.

Plant reproductive ecology and evolution in the Mediterranean islands: state of the art.

The ca. 12,000 islands and islets that encompass the Mediterranean Basin represent a biodiversity hotspot. They have been disconnected from each other and from the continent for hundreds of thousands to millions of years and encompass a high incidence of endemic plant species, with values that can exceed 20% of the local flora. Despite this, relatively few studies have been carried out to unravel ecological and evolutionary aspects of plant reproduction. We synthesise here the available information on the breeding systems, pollination and seed dispersal mode of the Mediterranean island flora. The main objective is to identify general patterns as well as to detect the main gaps in information on reproductive ecology in these particular and vulnerable systems in the face of global change. We also briefly review the information on impacts of invasive species on plant reproduction and dispersal, as these are some of the main threats to island biodiversity in general and Mediterranean island plant diversity in particular. The review has revealed that most available information is very geographically biased towards the western Mediterranean islands, especially the Balearic Islands, although a good fraction of studies have also been carried out on the eastern islands in the Aegean archipelago. Moreover, the majority of data come from species-focused studies, mainly endemic species of restricted range, whereas only a small fraction of studies have been performed at a community level. Relatively little work has involved genetic analyses, mainly focused on assessing the genetic differentiation and variability on narrow endemics. Contrary to our expectations, most island species do not rely on autonomous selfing, which might be related to the relatively high diversity of pollinators. The small, uninhabited, islands might be the last refuges of peculiar interactions that evolved in them in ancient times; they thus should be considered as sanctuaries of extraordinary biodiversity. Finally, we point out the main gaps of information and formulate a set of hypotheses that we believe are worth testing in future studies if we are to advance knowledge on the reproductive biology of Mediterranean island plants.

<b>Pollination ecology of <I>Rhynchosia cana</I> (Willd.) DC. (Fabaceae), an erect sub-shrub, in peninsular India</b>

Rhynchosia cana is a perennial erect sub-shrub. It flowers during November-January with peak flowering in December. The flowers are hermaphroditic, nectariferous, self-compatible and display explosive pollination mechanism adapted for pollination by bees. They do not fruit through autonomous selfing, but rather through manipulated selfing, geitonogamy and xenogamy mediated principally by bees and occasionally by lycaenid butterflies. In the localities of this herb, weeds such as Hyptis suaveolens (Lamiaceae) and Chromolaena odorata (Asteraceae) show luxuriant growth and flower simultaneously, and their intense flowering and high flower density ensures that most flower-visiting insects visit their flowers. In this situation only two bee species, Nomia and Anthidium exhibited fidelity to R. cana flowers. Un-tripped flowers fall off while tripped ones set fruit. In open-pollinations, fruit set was 81% and seed set was 54%. Seed dispersal occurs by explosive pod dehiscence. Perennial root stock resurrects back to life and restarts its reproductive cycle during the rainy season. Seeds also germinate at the same time but their continued growth is subject to the availability of soil moisture content. This study suggests that R. cana is unable to compete with the co-flowering weed species for pollinators, and also has regeneration constraints due to nutrient-deficient rocky habitats with prolific growth of weeds.

Ginger and the beetle: Evidence of primitive pollination system in a Himalayan endemic alpine ginger (Roscoea alpina, Zingiberaceae).

The Himalayan endemic alpine genus Roscoea, like other members of ginger family, exhibits the combination of floral traits that would fit pollination by long distant foragers such as bees, birds or flies. We studied the pollination biology of Roscoea alpina, observed potential floral visitors and determined their foraging behaviour, visitation frequency and pollination efficiency, to seek evidence in support of the pollination syndrome hypothesis. We also measured the floral spectra of R. alpina flowers to evaluate if signals fit with the currently known framework for observed floral visitors. We found that R. alpina have autonomous selfing and pollinator-mediated crossing, but lack apomixis. We observed that a beetle (Mylabris sp.), and a moth (Macroglossum nycteris) visit the flowers of R. alpina for pollen and nectar feeding respectively. Our field observations, the stigmatic pollen count and fruit set data indicated that the visit by the beetle was legitimate, while that of the moth was illegitimate. Emasculated flowers visited by beetles set as many fruits and seeds/fruit as auto-selfed and naturally pollinated flowers, while emasculated flowers excluded from beetle visits did not set fruit and seed; indicating that a single visit of a beetle to the flowers of R. alpina can facilitate pollination. We found that flower spectral signal of R. alpina does not fit typical spectra previously reported for beetle or bee-visited flowers. Our results suggest that, to ensure reproductive success in alpine habitat, R. alpina has evolved autonomous selfing as a predominant mode of reproduction, while beetle pollination would promote genetic diversity of this plant species. The visitation of beetles to the flowers of R. alpina, despite floral signal mismatch with the classically associated beetle vision, suggests that a different visual processing may operate in this plant-pollinator interaction at high altitudes.

Flower colour polymorphism in the Mediterranean Basin: occurrence, maintenance and implications for speciation

Flower colour polymorphism (FCP) is the occurrence of at least two discrete flower colour variants in the same population. Despite a vast body of research concerning the maintenance and evolutionary consequences of FCP, only recently has the spatial variation in morph frequencies among populations been explored. Here we summarise the biochemical and genetic basis of FCP, the factors that have been proposed to explain their maintenance, and the importance of FCP and its geographic variation in the speciation process. We also review the incidence of FCP in the environmentally heterogeneous Mediterranean Basin. Nearly 88% of Mediterranean FCP species showed anthocyanin-based polymorphisms. Concerning the evolutionary mechanisms that contribute to maintain FCP, selection by pollinators is suggested in some species, but in others, selection by non-pollinator agents, genetic drift or gene flow are also found; in some cases different processes interact in the maintenance of FCP. We emphasise the role of both autonomous selfing and clonal reproduction in FCP maintenance. Mediterranean polymorphic species show mainly monomorphic populations with only a few polymorphic ones, which generate clinal or mosaic patterns of variation in FCP. No cases of species with only polymorphic populations were found. We posit that different evolutionary processes maintaining polymorphism the Mediterranean Basin will result in a continuum of geographic patterns in morph compositions and relative frequencies of FCP species.

Geographic variation in floral traits and the capacity of autonomous selfing across allopatric and sympatric populations of two closely related Centaurium species

Floral traits and the relative contribution of autonomous selfing to total seed set varies geographically and is often driven by the availability and abundance of suitable pollinators and/or the presence of co-flowering relatives. In the latter case, competition for pollinator services and costs of hybridization can select for floral traits that reduce interspecific gene flow and contribute to prezygotic isolation, potentially leading to geographic variation in floral divergence between allopatric and sympatric populations. In this study, we investigated variation in floral traits and its implications on the capacity of autonomous selfing in both allopatric and sympatric populations of two closely related Centaurium species(Gentianaceae) across two distinct geographic regions(UK and mainland Europe). Although the magnitude and direction of floral differentiation varied between regions, sympatric populations were always significantly more divergent in floral traits and the capacity to self autonomously than allopatric populations. These results indicate that mating systems can vary substantially within a species and that the joint occurrence of plant species can have a major impact on floral morphology and capacity of autonomous selfing, most likely as a way to reduce the probability of interspecific interference.

High autonomous selfing capacity and low flower visitation rates in a subalpine population of Prunella vulgaris (Lamiaceae)

Background and aims - Autonomous selfing is a potential strategy to overcome pollinator scarcity or unreliability, but the specific mechanisms of autonomous selfing need further study to clarify our understanding of this phenomenon. This study investigated the mechanism of autonomous selling in a subalpine population of Prunella vulgaris (Lamiaceae), a species with didynamous stamens.

Mating System Evolution under Strong Pollen Limitation: Evidence of Disruptive Selection through Male and Female Fitness in Clarkia xantiana.

Selection on floral traits in hermaphroditic plants is determined by both male and female reproductive success. However, predictions regarding floral trait and mating system evolution are often based solely on female fitness. Selection via male fitness has the potential to affect the outcomes of floral evolution. In this study, we used paternity analysis to assess individual selfing rates and selection on floral traits via male and female fitness in an experimental population of Clarkia xantiana where pollen limitation of seed set was strong. We detected selection through both female and male fitness with reinforcing or noninterfering patterns of selection through the two sex functions. For female fitness, selection favored reduced herkogamy and protandry, traits that promote increased autonomous selfing. For male fitness, selection on petal area was disruptive, with higher trait values conferring greater pollinator attraction and outcross siring success and smaller trait values leading to higher selfed siring success. Combining both female and male fitness, selection on petal area and protandry was disruptive because intermediate phenotypes were less successful as both males and females. Finally, functional relationships among male and female fertility components indicated that selfing resulted in seed discounting and pollen discounting. Under these functional relationships, the evolutionarily stable selfing rate can be intermediate or predominantly selfing or outcrossing, depending on the segregating load of deleterious mutations.

Evolutionary consequences of ecological factors: pollinator reliability predicts mating-system traits of a perennial plant.

The reproductive-assurance hypothesis predicts that mating-system traits will evolve towards increased autonomous self-pollination in plant populations experiencing unreliable pollinator service. We tested this long-standing hypothesis by assessing geographic covariation among pollinator reliability, outcrossing rates, heterozygosity and relevant floral traits across populations of Dalechampia scandens in Costa Rica. Mean outcrossing rates ranged from 0.16 to 0.49 across four populations, and covaried with the average rates of pollen arrival on stigmas, a measure of pollinator reliability. Across populations, genetically based differences in herkogamy (anther-stigma distance) were associated with variation in stigmatic pollen loads, outcrossing rates and heterozygosity. These observations are consistent with the hypothesis that, when pollinators are unreliable, floral traits promoting autonomous selfing evolve as a mechanism of reproductive assurance. Extensive covariation between floral traits and mating system among closely related populations further suggests that floral traits influencing mating systems track variation in adaptive optima generated by variation in pollinator reliability.

Floral biology and pollination mechanisms of four Mexico-endemicFuchsiaspecies with contrasting reproductive systems

The evolution of the outstanding variation of reproductive systems in angiosperms has been considered an important driver of lineage diversification. Closely related hermaphroditic and dioecious species with biotic pollination provide the opportunity to study and compare traits related to pollinator attraction and their consequences on reproductive components. A higher predictability of pollination syndromes is expected in dioecious species, which are dependent on pollinators, than in self-compatible hermaphroditic taxa. Dioecious species may suffer pollen limitation depending on the quality of floral rewards and the kind and abundance of pollinators, whereas no pollen limitation is expected in hermaphroditic species with autonomous self-pollination. Additionally, in the absence of pollen limitation, more or better seeds are expected in dioecious species, according to the sexual specialization hypothesis. In natural populations of the hermaphroditic Fuchsia fulgens and Fuchsia arborescens and dioecious Fuchsia parviflora and Fuchsia obconica, all endemic to Mexico, we first described flower phenology, flower production and longevity and nectar volume and concentration. Then, we evaluated the correspondence between floral visitors and pollination syndromes. In hermaphrodite plants, we determined the level of herkogamy and the potential for autonomous self-pollination. Finally, we evaluated the effect of pollen limitation on fruit set and seed number and assessed seed germination for all species. In contrast to our prediction, dioecious species did not show a higher correspondence between pollination syndromes and floral visitors than did hermaphrodites; however, male flowers exhibited a higher correspondence than female flowers. No pollen limitation was detected in dioecious species, for which visitation rate did not differ between male and female flowers. The hermaphroditic F. fulgens showed pollen limitation for seed number, despite the presence of autonomous selfing. Fruit set from autonomous pollination was higher in F. arborescens, which showed a lower level of herkogamy compared with F. fulgens. Finally, dioecious species produced fewer but heavier seeds compared with hermaphrodite species. Although Fuchsia is classified as an outcrossing genus, both hermaphroditic species showed autonomous self-pollination. The heavier but lower number of seeds per fruit in dioecious species may be related to the more efficient resource allocation expected from sexual specialization. This could play an important role in the evolution of dioecy; however, a comparative phylogenetic approach is required to confirm this hypothesis.

Spatio-temporal patterns in pollination of deceptive Aristolochia rotunda L. (Aristolochiaceae).

Pollination success of highly specialised flowers is susceptible to fluctuations of the pollinator fauna. Mediterranean Aristolochia rotunda has deceptive trap flowers exhibiting a highly specialised pollination system. The sole pollinators are kleptoparasitic flies in search of food. This study investigates these pollinators on a spatio-temporal scale and the impact of weather conditions on their availability. Two potential strategies of the plants to cope with pollinator limitation, i.e. autonomous selfing and an increased floral life span, were tested. A total of 6156 flowers were investigated for entrapped pollinators in 10 Croatian populations. Availability of the main pollinator was correlated to meteorological data. Artificial pollination experiments were conducted and the floral life span was recorded in two populations according to pollinator availability. Trachysiphonella ruficeps (Chloropidae) was identified as dominant pollinator, along with less abundant species of Chloropidae, Ceratopogonidae and Milichiidae. Pollinator compositions varied among populations. Weather conditions 15-30days before pollination had a significant effect on availability of the main pollinator. Flowers were not autonomously selfing, and the floral life span exhibited considerable plasticity depending on pollinator availability. A.rotunda flowers rely on insect pollen vectors. Plants are specialised on a guild of kleptoparasitic flies, rather than on a single species. Pollinator variability may result in differing selection pressures among populations. The availability/abundance of pollinators depends on weather conditions during their larval development. Flowers show a prolonged trapping flower stage that likely increases outcrossing success during periods of pollinator limitation.

Pollination ecology of Clerodendrum inerme (L.) Gaertn. (Lamiaceae) in Coringa mangrove ecosystem, Andhra Pradesh, India

Clerodendrum inerme (L.) Gaertn. (Lamiaceae) is bisexual, self-compatible and has a vector-dependent mixed breeding system. They are dichogamous and herkogamous; the day 1 flowers are staminate while the day 2 and 3 flowers are pistillate. The plant blooms in the evening, possesses a white long corolla with a hairy interior to exclude other insects and strong fragrance are adaptations for pollination by the hawk-moth Macroglossum gyrans. The 2nd and 3rd day flowers are nectar-rich and attract hawk-moths during the dawn and dusk hours. The plant is also visited by bees and butterflies. The bees Xylocopa and Anthophora are primary nectar robbers which collect nectar without effecting pollination. In C. inerme, three forms of flowers can be distinguished based on the position of sex organs. The first form is characterized by elongated stamens and a style which occur in close proximity to each other just after anthesis facilitating contact between the stamens and stigma. The second form is characterized by the scattered position of stamens and style. In the third form, the stamens are fully extended while the style is curved away from them, either to the left or to the right; subsequently the stamens curl inward and the style elongates. Interestingly, the three flower forms can be found within a cyme also. These forms of flowers with strong protandry prevent autonomous selfing but not geitonogamy. The fruit is a capsule and breaks open to disperse nutlets. Birds such as Acridotheres tristis, Corvus splendens, Corvus macrorhynchos and Turdoides caudatus disperse nutlets during the early winter season. Seeds germinate in June and seedlings grow gradually to produce new plants.

The importance of autonomous selfing in preventing hybridization in three closely related plant species

Summary In plants that are able to self‐fertilize, autonomous selfing has been hypothesized to function as an effective mechanism that prevents heterospecific mating. However, there have only been few studies that have assessed the relative and absolute contribution of different modes of selfing to total reproductive isolation acting between species. Because selfing can be expected to increase fitness costs when offspring are sired from more outcrossing sister taxa, it can be hypothesized that the contribution of post‐zygotic barriers to total reproductive isolation becomes stronger in selfing than in more outcrossing relatives. To assess the efficiency of different modes of selfing against heterospecific mating, we conducted mixed‐pollination experiments in three Centaurium species showing strong differences in mating system (i.e. delayed selfing in Centaurium erythraea, competing selfing in C. littorale and prior selfing in C. pulchellum). The contribution of selfing to total reproductive isolation was further examined by quantifying the strength of three pre‐zygotic (flowering phenology, pollinator fidelity and pollen production) and four intrinsic post‐zygotic (seed set and germination, survival and flowering capacity) barriers for each of the six species pair combinations. Although autonomous selfing as a reproductive barrier was unable to completely impede gene flow, its efficiency as a mechanism to prevents heterospecific pollination strongly depended on the mode of selfing, with prior selfing being by far the most efficient mechanism and delayed selfing only offering a limited protection against hybrid fertilization at the end of a flower's lifetime. Apart from the prevention effect of selfing, differences in pollinator fidelity and pollen production also contributed substantially to total reproductive isolation. Post‐zygotic hybrid costs were high and most prevalent for seed production and germination success. Reductions in hybrid vigour were significantly associated with the mating system of the maternal species, with the highest reductions in the most selfing C. pulchellum and the lowest reductions in predominant outcrossing C. erythraea. Synthesis. Our findings indicate that selfing may prevent species from interspecific gene flow and hybridization. The association between the capacity to self‐autonomously and hybrid vigour further suggests that selfing may foster post‐zygotic costs following hybridization.

Severe outbreeding and inbreeding depression maintain mating system differentiation in Epipactis (Orchidaceae).

In hermaphroditic plants, theory for mating system evolution predicts that populations will evolve to either complete autonomous selfing (AS) or complete outcrossing, depending on the balance between automatic selection favouring self-fertilization and costs resulting from inbreeding depression (ID). Theory also predicts that selection for selfing can occur rapidly and is driven by purging of genetic load and the loss of ID. Therefore, selfing species are predicted to have low levels of ID or even to suffer from outbreeding depression (OD), whereas predominantly outcrossing species are expected to have high levels of ID. To test these predictions, we related the capacity of AS to the magnitude of early-acting inbreeding or OD in both allogamous and autogamous species of the orchid genus Epipactis. For each species, the level of AS was assessed under controlled greenhouse conditions, whereas hand-pollinations were performed to quantify early costs of inbreeding or OD acting at the level of fruit and seed production. In the autogamous species, the capacity of AS was high (>0.72), whereas in the allogamous species AS was virtually absent (<0.10). Consistent with our hypothesis, allogamous Epipactis species had significantly higher total ID (average: 0.46) than autogamous species, which showed severe costs of OD (average: -0.45). Overall, our findings indicate that strong early-acting ID represents an important mechanism that contributes to allogamy in Epipactis, whereas OD may maintain selfing in species that have evolved to complete selfing.