Jack of all nectars, master of most: DNA methylation and the epigenetic basis of niche width in a flower-living yeast.

In addition to genetic differences between individuals as a result of nucleotide sequence variation, epigenetic changes that occur as a result of DNA methylation may also contribute to population niche width by enhancing phenotypic plasticity, although this intriguing possibility remains essentially untested. Using the nectar-living yeast Metschnikowia reukaufii as study subject, we examine the hypothesis that changes in genome-wide DNA methylation patterns underlie the ability of this fugitive species to exploit a broad resource range in its heterogeneous and patchy environment. Data on floral nectar characteristics and their use by M. reukaufii in the wild were combined with laboratory experiments and methylation-sensitive amplified polymorphism (MSAP) analyses designed to detect epigenetic responses of single genotypes to variations in sugar environment that mimicked those occurring naturally in nectar. M. reukaufii exploited a broad range of resources, occurring in nectar of 48% of species and 52% of families surveyed, and its host plants exhibited broad intra- and interspecific variation in sugar-related nectar features. Under experimental conditions, sugar composition, sugar concentration and their interaction significantly influenced the mean probability of MSAP markers experiencing a transition from unmethylated to methylated state. Alterations in methylation status were not random but predictably associated with certain markers. The methylation inhibitor 5-azacytidine (5-AzaC) had strong inhibitory effects on M. reukaufii proliferation in sugar-containing media, and a direct relationship existed across sugar × concentration experimental levels linking inhibitory effect of 5-AzaC and mean per-marker probability of genome-wide methylation. Environmentally induced DNA methylation polymorphisms allowed genotypes to grow successfully in extreme sugar environments, and the broad population niche width of M. reukaufii was largely made possible by epigenetic changes enabling genotype plasticity in resource use.

Micro-organisms behind the pollination scenes: microbial imprint on floral nectar sugar variation in a tropical plant community.

BACKGROUND AND AIMS: Variation in the composition of floral nectar reflects intrinsic plant characteristics as well as the action of extrinsic factors. Micro-organisms, particularly yeasts, represent one extrinsic factor that inhabit the nectar of animal-pollinated flowers worldwide. In this study a 'microbial imprint hypothesis' is formulated and tested, in which it is proposed that natural community-wide variation in nectar sugar composition will partly depend on the presence of yeasts in flowers. METHODS: Occurrence and density of yeasts were studied microscopically in single-flower nectar samples of 22 animal-pollinated species from coastal xeric and sub-humid tropical habitats of the Yucatán Peninsula, Mexico. Nectar sugar concentration and composition were concurrently determined on the same samples using high-performance liquid chromatography (HPLC) methods. KEY RESULTS: Microscopical examination of nectar samples revealed the presence of yeasts in nearly all plant species (21 out of 22 species) and in about half of the samples examined (51·8 % of total, all species combined). Plant species and individuals differed significantly in nectar sugar concentration and composition, and also in the incidence of nectar yeasts. After statistically controlling for differences between plant species and individuals, nectar yeasts still accounted for a significant fraction of community-wide variance in all nectar sugar parameters considered. Significant yeast × species interactions on sugar parameters revealed that plant species differed in the nectar sugar correlates of variation in yeast incidence. CONCLUSIONS: The results support the hypothesis that nectar yeasts impose a detectable imprint on community-wide variation in nectar sugar composition and concentration. Since nectar sugar features influence pollinator attraction and plant reproduction, future nectar studies should control for yeast presence and examine the extent to which microbial signatures on nectar characteristics ultimately have some influence on pollination services in plant communities.

Untangling individual variation in natural populations: ecological, genetic and epigenetic correlates of long-term inequality in herbivory.

Individual variation in ecologically important features of organisms is a crucial element in ecology and evolution, yet disentangling its underlying causes is difficult in natural populations. We applied a genomic scan approach using amplified fragment length polymorphism (AFLP) markers to quantify the genetic basis of long-term individual differences in herbivory by mammals at a wild population of the violet Viola cazorlensis monitored for two decades. In addition, methylation-sensitive amplified polymorphism (MSAP) analyses were used to investigate the association between browsing damage and epigenetic characteristics of individuals, an aspect that has been not previously explored for any wild plant. Structural equation modelling was used to identify likely causal structures linking genotypes, epigenotypes and herbivory. Individuals of V. cazorlensis differed widely in the incidence of browsing mammals over the 20-year study period. Six AFLP markers (1.6% of total) were significantly related to herbivory, accounting altogether for 44% of population-wide variance in herbivory levels. MSAP analyses revealed considerable epigenetic variation among individuals, and differential browsing damage was significantly related to variation in multilocus epigenotypes. In addition, variation across plants in epigenetic characteristics was related to variation in several herbivory-related AFLP markers. Statistical comparison of alternative causal models suggested that individual differences in herbivory are the outcome of a complex causal structure where genotypes and epigenotypes are interconnected and have direct and indirect effects on herbivory. Insofar as methylation states of MSAP markers influential on herbivory are transgenerationally heritable, herbivore-driven evolutionary changes at the study population will involve correlated changes in genotypic and epigenotypic distributions.

Clonality, genetic diversity and support for the diversifying selection hypothesis in natural populations of a flower-living yeast.

Vast amounts of effort have been devoted to investigate patterns of genetic diversity and structuring in plants and animals, but similar information is scarce for organisms of other kingdoms. The study of the genetic structure of natural populations of wild yeasts can provide insights into the ecological and genetic correlates of clonality, and into the generality of recent hypotheses postulating that microbial populations lack the potential for genetic divergence and allopatric speciation. Ninety-one isolates of the flower-living yeast Metschnikowia gruessii from southeastern Spain were DNA fingerprinted using amplified fragment length polymorphism (AFLP) markers. Genetic diversity and structuring was investigated with band-based methods and model- and nonmodel-based clustering. Linkage disequilibrium tests were used to assess reproduction mode. Microsite-dependent, diversifying selection was tested by comparing genetic characteristics of isolates from bumble bee vectors and different floral microsites. AFLP polymorphism (91%) and genotypic diversity were very high. Genetic diversity was spatially structured, as shown by amova (Φ(st) = 0.155) and clustering. The null hypothesis of random mating was rejected, clonality seeming the prevailing reproductive mode in the populations studied. Genetic diversity of isolates declined from bumble bee mouthparts to floral microsites, and frequency of five AFLP markers varied significantly across floral microsites, thus supporting the hypothesis of diversifying selection on clonal lineages. Wild populations of clonal fungal microbes can exhibit levels of genetic diversity and spatial structuring that are not singularly different from those shown by sexually reproducing plants or animals. Microsite-dependent, divergent selection can maintain high local and regional genetic diversity in microbial populations despite extensive clonality.

A comment on García et al. (2005, 2007) and related papers on mating patterns and gene dispersal in Prunus mahaleb.

In two studies on mating patterns and spatial components of pollen and seed dispersal of Prunus mahaleb based on parentage analysis, García et al. (2005, 2007) depicted their 196 focal trees as a spatially isolated population where all reproductive trees had been genotyped. Additional distributional data for P. mahaleb trees in their study area, however, revealed that García and colleagues' depiction of their study system bears little resemblance to reality. The trees these authors studied did not form a discrete, geographically isolated population. Around 300 ungenotyped reproductive trees occurred within the 1.5-km distributional gap to the nearest population proclaimed by García and colleagues. Since exhaustive sampling of potential parental genotypes is essential in parentage analyses, the occurrence of a large number of ungenotyped trees in the immediate neighbourhood of focal trees can severely affect the main conclusions of García et al. (2005, 2007) as well as of several related publications on gene dispersal and mating patterns of P. mahaleb conducted on the same trees and relying on the same false premises of spatial isolation and exhaustive sampling.

Quantifying the genetic component of phenotypic variation in unpedigreed wild plants: tailoring genomic scan for within-population use.

The study of adaptive genetic variation in natural populations is central to evolutionary biology. Quantitative genetics methods, however, are hardly applicable to long-lived organisms, and current knowledge on adaptive genetic variation in wild plants mostly refers to annuals and short-lived perennials. Studies on long-lived species are essential to explore possible life-history correlates of genetic variation, selection, and trait heritability. In this paper, we propose a method based on molecular markers to quantify the genetic basis of individual phenotypic differences in wild plants under natural conditions. Rather than focusing on inferring individual relatedness to estimate the heritability of phenotypic traits, we directly estimate the proportion of observed phenotypic variance that is statistically accounted for by genotypic differences between individuals. This is achieved by (i) identifying loci that are correlated across individuals with the phenotypic trait of interest by means of an amplified fragment length polymorphism (AFLP)-based explorative genomic scan, and (ii) fitting multiple regression and linear random effect models to estimate the effects of genotype, environment and genotype x environment on phenotypes. We apply this method to estimate genotypic and environmental effects on cumulative maternal fecundity in a wild population of the long-lived Viola cazorlensis monitored for 20 years. Results show that between 56-63% (depending on estimation method) of phenotypic variance in fecundity is accounted for by genotypic differences in 11 AFLP loci that are significantly related to fecundity. Genotype x environment effects accounted for 38% of fecundity variance, which may help to explain the unexpectedly high levels of genetic variance for fecundity found.

Local adaptation of Ruellia nudiflora (Acanthaceae) to biotic counterparts: complex scenarios revealed when two herbivore guilds are considered.

This study evaluated whether the herb Ruellia nudiflora is locally adapted to a specialist insect seed predator (SP) and insect folivores, and if plant local adaptation (LA) to the former is more likely. A reciprocal transplant experiment was conducted using three sites in Yucatan (Mexico) (n = 864 plants). A third of the plants of each origin were placed at each site, and we recorded the following during a 9-month period: fruit number, leaf damage, and fruits attacked by SP. Results indicated lack of plant LA for all the variables measured. Instead, seed predation was c. 100% greater for native plants at one study site, suggesting insect LA or plant maladaptation; folivory was homogeneous across sites/origins. Based on these results, we discuss differences in the potential each herbivore guild has to promote plant LA, as well as divergent evolutionary outcomes of plant-herbivore interactions across sites.

Population-genomic approach reveals adaptive floral divergence in discrete populations of a hawk moth-pollinated violet.

Local adaptation to contrasting biotic or abiotic environments is an important evolutionary step that presumably precedes floral diversification at the species level, yet few studies have demonstrated the adaptive nature of intraspecific floral divergence in wild plant populations. We combine a population-genomic approach with phenotypic information on floral traits to examine whether the differentiation in metric floral traits exhibited by 14 populations of the southern Spanish hawk moth-pollinated violet Viola cazorlensis reflects adaptive divergence. Screening of many amplified fragment length polymorphism (AFLP) loci using a multiple-marker-based neutrality test identified nine outlier loci (2.6% of the total) that departed from neutral expectations and were potentially under selection. Generalized analysis of molecular variance revealed significant relationships between genetic distance and population divergence in three floral traits when genetic distance was based on outlier loci, but not when it was based on neutral ones. Population means of floral traits were closely correlated with population scores on the first principal coordinate axis of the genetic distance matrix using outlier loci, and with the allelic frequencies of four of the outlier loci. Results strongly support the adaptive nature of intraspecific floral divergence exhibited by V. cazorlensis and illustrate the potential of genome scans to identify instances of adaptive divergence when used in combination with phenotypic information.

Adding a third dimension to the edge of a species' range: altitude and genetic structuring in mountainous landscapes.

In addition to the topographical and ecological barriers, other landscape features may also subtly influence the patterns of gene flow and spatial genetic structuring at species' borders. This paper focuses on the role played by altitudinal gradients that characterize mountainous landscapes. We formulate and test the hypothesis that when the distribution boundaries of plant species intersect mountainous landscapes, altitudinal gradients in ecological conditions may considerably enhance population subdivision and genetic structuring at the regional level. Using amplified fragment length polymorphism markers, we studied genetic diversity and differentiation in a set of 21 peripheral populations of the evergreen shrub Lavandula latifolia Med. (Labiatae) at its southernmost distribution limit in the Betic mountain ranges of southern Spain. Population size and abundance, and within-population genetic diversity, varied predictably with altitude, being highest at middle elevations and declining steadily towards both the upper and lower altitudinal distribution margins. Genetic differentiation tended to follow the opposite trend. These altitudinal patterns result from variation with elevation in the relative influence of gene flow and drift on the distribution of genetic variation. Genetic drift prevails around the upper and lower altitudinal limits, whereas a situation closer to a drift-gene flow equilibrium exists at the center of the altitudinal distribution. Altitudinal variation in the relative influences of gene flow and drift appears as an essential element in the interpretation of regional genetic structuring of L. latifolia at its mountainous distribution edge, and a factor which may influence the evolutionary potential of peripheral populations and the likelihood of local adaptation.

Sex-specific phenotypic selection and geographic variation in gender divergence in a gynodioecious shrub.

In sexually polymorphic plant species the extent of gender divergence in floral morphology and phenology may be influenced by gender-specific selection patterns imposed by pollinators, which may change geographically. Distribution margins are areas where changes in the pollinator fauna, and thus variation in gender divergence of floral traits, are expected. We tested for pollination-driven geographic variation in the gender divergence in floral and phenological traits in the gynodioecious shrub Daphne laureola, in core and marginal areas differing in the identity of the main pollinator. Pollinators selected for longer corolla tubes in hermaphrodite individuals only in core populations, which in turn recorded higher fruit set. Consistent with these phenotypic selection patterns, gender divergence in flower corolla length was higher in core populations. Moreover, pollinators selected towards delayed flowering on hermaphrodite individuals only in marginal populations, where the two sexes differed more in flowering time. Our results support that a shift in main pollinators is able to contribute to geographic variation in the gender divergence of sexually polymorphic plant species.

Permanent genetic resources added to molecular ecology resources database 1 April 2013-31 May 2013.

This article documents the addition of 234 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acipenser sinensis, Aleochara bilineata, Aleochara bipustulata, Barbus meridionalis, Colossoma macropomum, Delia radicum, Drosophila nigrosparsa, Fontainea picrosperma, Helianthemum cinereum, Liomys pictus, Megabalanus azoricus, Pelteobagrus vachelli, Pleuragramma antarcticum, Podarcis hispanica type 1A, Sardinella brasiliensis and Sclerotinia homoeocarpa. These loci were cross-tested on the following species: Acipenser dabryanus, Barbus balcanicus, Barbus barbus, Barbus cyclolepis, Drosophila hydei, Drosophila melanogaster, Drosophila obscura, Drosophila subobscura, Fontainea australis, Fontainea fugax, Fontainea oraria, Fontainea rostrata, Fontainea venosa, Podarcis bocagei, Podarcis carbonelli, Podarcis liolepis, Podarcis muralis and Podarcis vaucheri.

Herkogamy and mate diversity in the wild daffodil Narcissus longispathus: beyond the selfing-outcrossing paradigm in the evolution of mixed mating.

Spatial separation of male and female reproductive structures (herkogamy) is a widespread floral trait that has traditionally been viewed as an adaptation that reduces the likelihood of self-pollination. Here we propose that increased herkogamy may also influence another important aspect of plant mating: the diversity of pollen donors siring seeds within fruits. We test this hypothesis in Narcissus longispathus, a wild daffodil species with extensive variation in anther-stigma separation. To study the morphological basis of variation in herkogamy, floral measurements were undertaken in 16 populations of N. longispathus. We then quantified multilocus outcrossing rates and the correlation of outcrossed paternity in three of these populations sampled over several years. Mating system estimates were calculated for each population and year, and also separately for groups of plants that differed markedly in herkogamy within each population and year. In N. longispathus herkogamy was much more variable than other floral traits, and was more closely related to style length than to anther position. Averaged across populations and years, plants with high herkogamy had similar outcrossing rates (0.683) to plants with intermediate (0.648) or low herkogamy (0.590). However, a significant linear trend was found for correlation of outcrossed paternity, which increased monotonically from high herkogamy (0.221), through intermediate herkogamy (0.303) to low herkogamy (0.463) plants. The diversity of pollen donors siring seeds of high herkogamy Narcissus flowers was thus consistently greater than the diversity of pollen donors siring seeds of low herkogamy flowers. Results of this study contribute to the emerging consensus that floral traits can simultaneously influence several aspects of plant mating system in complex ways, thus extending the traditional focus centred exclusively on patterns and relative importance of self- and cross-fertilisation.

Genetic structure of the shrub Daphne laureola across the Baetic Ranges, a Mediterranean glacial refugium and biodiversity hotspot.

Distribution margins constitute areas particularly prone to random and/or adaptive intraspecific differentiation in plants. This trend may be particularly marked in species discontinuously distributed across mountain ranges, where sharp geographic isolation gradients and habitat boundaries will enhance genetic isolation among populations. In this study, we analysed the level of neutral genetic differentiation among populations of the long-lived shrub Daphne laureola (Thymelaeaceae) across the Baetic Ranges, a glacial refugium and biodiversity hotspot in the western Mediterranean Basin. Within this area, core and marginal populations of D. laureola were compared with regard to their spatial isolation, size, genetic diversity and differentiation. A spatially explicit analysis conducted on the vast majority of the species' known populations in the study area (N = 111) showed that marginal populations (western and eastern) present larger spatial isolation than core populations, but are not smaller. We compared genetic diversity and differentiation between core and marginal populations using a subsample of 15 populations and 225 amplified fragment length polymorphism (AFLP) markers. Core and marginal populations did not differ in genetic diversity, probably because of the occurrence of large populations on the local margins. Western populations were strongly differentiated from the other populations. In addition, spatial and genetic differentiation among populations was larger on the western margin. Eastern populations constituted a genetically homogeneous group closely related to core populations, despite their greater spatial isolation. Results suggest that studies on phenotypic differentiation between core and marginal populations of D. laureola, and presumably other species having discontinuous distributions across the Baetic ranges, should take into account geographical differences in levels of genetic differentiation between the different distribution borders.

Mechanical behaviour and rupture of normal and pathological human ascending aortic wall.

The mechanical properties of aortic wall, both healthy and pathological, are needed in order to develop and improve diagnostic and interventional criteria, and for the development of mechanical models to assess arterial integrity. This study focuses on the mechanical behaviour and rupture conditions of the human ascending aorta and its relationship with age and pathologies. Fresh ascending aortic specimens harvested from 23 healthy donors, 12 patients with bicuspid aortic valve (BAV) and 14 with aneurysm were tensile-tested in vitro under physiological conditions. Tensile strength, stretch at failure and elbow stress were measured. The obtained results showed that age causes a major reduction in the mechanical parameters of healthy ascending aortic tissue, and that no significant differences are found between the mechanical strength of aneurysmal or BAV aortic specimens and the corresponding age-matched control group. The physiological level of the stress in the circumferential direction was also computed to assess the physiological operation range of healthy and diseased ascending aortas. The mean physiological wall stress acting on pathologic aortas was found to be far from rupture, with factors of safety (defined as the ratio of tensile strength to the mean wall stress) larger than six. In contrast, the physiological operation of pathologic vessels lays in the stiff part of the response curve, losing part of its function of damping the pressure waves from the heart.

The geographic mosaic in predispersal interactions and selection on Helleborus foetidus (Ranunculaceae).

We examine the hierarchical geographic structure of the interaction between a plant, Helleborus foetidus, and its floral herbivores and pollinators (interactors). Six populations from three distant regions of the Iberian Peninsula were used to examine intra- and inter-regional variation in plant traits, interactors and plant fecundity, and to compare, through selection gradient and path analyses, which traits were under selection, and which interactors were responsible for differential selection. Geographic and temporal congruency in interactor-mediated selection was further tested using a recent analytical approach based on multi-group comparison in Structural Equation Models. Most plant traits, interactors and fecundity differed among regions but not between populations. Similarly, the identity of the traits under selection, the selection gradients (strength and/or the direction of the selection) and the path coefficients (identifying the ecological basis for selection) varied inter- but not intra-regionally. Results show a selection mosaic at the broad scale and, for some traits, a link of differential selection to trait differentiation.

Mating system, sex ratio, and persistence of females in the gynodioecious shrub Daphne laureola L. (Thymelaeaceae).

Although in gynodioecious populations male steriles require a fecundity advantage to compensate for their gametic disadvantage, southern Spanish populations of the long-lived shrub Daphne laureola do not show any fecundity advantage over hermaphrodites in terms of seed production and early seedling establishment. By using allozyme markers, we assess the mating system of this species in five populations differing in sex ratio, and infer levels of inbreeding depression over the whole life cycle by comparing the inbreeding coefficients at the seed and adult plant stages. Extremely low outcrossing rates (0.001

Neither vegetative nor reproductive advantages account for high frequency of male-steriles in southern Spanish gynodioecious Daphne laureola (Thymelaeaceae).

In gynodioecious species, male-steriles (termed "females" hereafter) usually exhibit some reproductive advantage over hermaphrodites that allow them to compensate for the loss of male reproductive function. This compensation can result from higher fecundity, vegetative outperformance, and/or lower inbreeding depression. In this study we compared vegetative and reproductive parameters of female and hermaphrodite Daphne laureola individuals in two southeastern Spanish populations and estimated the magnitude of inbreeding depression up to the seedling emergence stage by conducting controlled pollinations and experimental sowings of seed progenies in the field. Reproductive shrubs of both sexes did not differ significantly in size, leaf production, leaf size, leaf growth, nutrient allocation to leaves, and production of flowers and fruits. Seed set and seed size of cross- and self-pollinated flowers of hermaphrodites and cross-pollinated females were also similar. Seedling emergence rates of self- and cross-pollinated seeds from hermaphrodites under natural field conditions were similar, suggesting a minor role of inbreeding depression up to that reproductive stage. Seeds from females produced more seedlings than selfed seeds from hermaphrodites. In 21 populations surveyed in the study region over a broad geographical and elevational gradient, the proportion of females ranged between 20.6 and 56.1% and was inversely related to elevation. The establishment and maintenance of females in southeastern Spanish populations of D. laureola seem to be mainly mediated by ecological factors.

Annual variability in seed production by woody plants and the masting concept: reassessment of principles and relationship to pollination and seed dispersal.

By analyzing 296 published and unpublished data sets describing annual variation in seed output by 144 species of woody plants, this article addresses the following questions. Do plant species naturally fall into distinct groups corresponding to masting and nonmasting habits? Do plant populations generally exhibit significant bimodality in annual seed output? Are there significant relationships between annual variability in seed production and pollination and seed dispersal modes, as predicted from economy of scale considerations? We failed to identify distinct groups of species with contrasting levels of annual variability in seed output but did find evidence that most polycarpic woody plants seem to adhere to alternating supra-annual schedules consisting of either high or low reproduction years. Seed production was weakly more variable among wind-pollinated taxa than animal-pollinated ones. Plants dispersed by mutualistic frugivores were less variable than those dispersed by either inanimate means or animals that predominantly behave as seed predators. We conclude that there are no objective reasons to perpetuate the concept of mast fruiting in the ecological literature as a shorthand to designate a distinct biological phenomenon. Associations between supra-annual variability in seed output and pollination and seed dispersal methods suggest the existence of important reproductive correlates that demand further investigation.

Mating patterns and genetic diversity in the wild daffodil Narcissus longispathus (Amaryllidaceae).

Despite the importance of Narcissus to ornamental horticulture, there have been no population genetic studies of wild species, many of which have narrow distributions. Here, we measure selfing rates and levels of genetic diversity at allozyme loci in six populations of Narcissus longispathus, a self-compatible daffodil endemic to a few mountain ranges in southeastern Spain. The populations were distributed among four distinct river valleys encompassing two main watersheds in the Sierra de Cazorla mountains. Selfing rates averaged 0.37 (range 0.23-0.46), resulting in significant inbreeding coefficients for the progeny (f = 0.324). In contrast, estimates of inbreeding in parental genotypes were not significantly different from zero (f = 0.001), indicating that few selfed offspring survive to maturity because of inbreeding depression. Species-wide estimates of genetic diversity for the six populations were P(s) = 0.38, H(es) = 0.119 and A(s) = 1.27 with significant genetic differentiation among populations theta = 0.15. The observed patterns of genetic differentiation among populations are likely influenced by the mating system, and a combination of local topography, watershed affinities and gene flow.

Geographical variation in autonomous self-pollination levels unrelated to pollinator service in Helleborus foetidus (Ranunculaceae).

Autonomous self-pollination may be considered as a mechanism enhancing plant reproductive success when plant access to pollen sources may limit seed production. We have studied the relationship between geographical patterns of variation in pollinator service to Helleborus foetidus and self-pollination ability in three widely spaced regions in the Iberian Peninsula. As could be expected from its early flowering period, pollinator visitation rates to both plants and flowers of H. foetidus were very low at all sites. Pollinator composition remained consistent among regions, but there was significant variation among regions in pollinator service. Despite the low visitation rates, fruit set did not appear to be pollen limited in any of the study areas, which may be explained by the long duration of flowers (up to 20 d). When pollinators were excluded experimentally, fruit set decreased significantly, but substantial levels of self-pollination occurred at all regions. Autonomous self-pollination levels were lowest in the two regions with lowest pollinator service and highest in the region with highest pollinator service. This disagreement between our results and the expectations derived from the reproductive assurance hypothesis may reflect a nonequilibrium situation of the northern H. foetidus populations in relation to their current pollinating environment.