BACKGROUND
plant-pollinator interactions are increasingly affected by human disturbances  <cit> , probably resulting in strong selective pressure on plant mating systems  <cit> .
pollination failure may promote the evolution of selfing, which can provide reproductive assurance by increasing seed production when pollinators and/or mates are scarce  <cit> .
reproductive assurance is one of the most longstanding and widely accepted explanations for the evolution of selfing and the maintenance of mixed mating systems .
if both pollinators and mates are limiting, reproductive assurance can occur through autonomous selfing  <cit> .
by contrast, if only mates are limiting, reproductive assurance can also result from pollinator-mediated selfing, such as facilitated selfing  <cit> .
however, inbreeding depression  reduces the fitness of selfed progenies  <cit>  and counteracts the advantages of selfing  /  <dig>  where e is the fraction of ovules remaining unfertilized  <cit> .
id is typically caused either by the expression of lethal deleterious recessive alleles formerly masked by the heterozygous state or by the loss of heterozygote advantage  <cit> .
it may evolve with an increase in selfing rate, when deleterious recessive alleles are purged by selection, although this trend is not general in plant species  <cit> .
experimental studies have shown that the expression of id depends on the environment .
however, the variability of id in natural environments and, more specifically, along population size gradients, remains unclear, despite its acknowledged importance  <cit> .
furthermore, id estimates are based principally on experimental studies in herbaceous annual species, and the fitness of selfed progenies in long-lived perennials has been little considered  <cit>  but see  <cit> .
perennial species experience multiplicative effects of id over many years  <cit>  and are exposed to many biotic and abiotic changes.
given that selfing may be a by-product of outcrossing or provide additional benefits in terms of reproductive assurance in cases of pollination failure, strong id may be associated with high or intermediate rates of selfing .
selfing provides reproductive assurance only if seed production is pollen-limited, if selfing increases seed set and if id does not completely counteract the benefits of selfing over the lifetime of the adult plant  <cit> .
however, few studies have empirically estimated the actual long-term benefits of selfing by investigating whether the negative impact of id counteracts the positive effects of the greater seed set due to reproductive assurance in natural populations of contrasting sizes.
given the scarcity of studies simultaneously estimating reproductive assurance, selfing rate and lifetime id along natural population size gradients, predicting the potential of selfing to provide actual reproductive assurance in various ecological contexts remains a major challenge in evolutionary biology.
we focused on a patchily distributed mass-flowering shrub, rhododendron ferrugineum .
we showed in a previous study that insect visitation rates and floral availability are inversely related, resulting in similar pollinator-mediated pollen transfer in small and large r. ferrugineum patches  <cit> .
in this study system, pollen transfer is probably constrained by pollinator limitation in large patches and by mate limitation in small patches, decreasing seed production by 34% on average  <cit> .
here, we investigated the lifetime reproductive assurance benefit of selfing by estimating reproductive success, reproductive assurance, selfing rate and lifetime id along a continuum of patch floral display , used as a proxy for patch size.
we predicted reproductive assurance and selfing rate to be higher in small sparse patches, in which pollen transfer has been shown to be limited principally by the availability of conspecific plants  <cit> .
lifetime id  would be expected to be strong in long-lived perennial species and, if associated with early-acting id, it may counteract the reproductive assurance benefit of selfing in small plant patches.
we also characterized the r. ferrugineum mating system by estimating fruit set  and self-compatibility.
we estimated the magnitude of id in natural populations by using a marker-based approach comparing  inbreeding in adult plants , including any episodes of mortality in natural populations over the long lifetime of individuals and  multilocus selfing rate  in progeny arrays, reflecting selfing before selection in natural populations  <cit> .
we also estimated the following, along the patch size gradient:  pollination parameters, including reproductive assurance, by comparing seed production by emasculated and intact flowers, and self-compatibility, by comparing seed production by hand-selfed and hand-outcrossed flowers and open fruit set;  selfing rate, estimated by progeny array analysis and  id in natural populations, as assessed with neutral genetic markers.
RESULTS
reproductive success and pollination parameters along a natural patch size gradient
the mean fruit set per patch was  <dig> %  and increased significantly with increasing r. ferrugineum floral display per patch .
mean open seed set  decreased significantly with increasing floral display  whereas mean pollinator-mediated seed set  was not significantly related to patch floral display .
open seed set from intact flowers was significantly greater than that of emasculated flowers, consistent with significant reproductive assurance .
reproductive assurance increased seed production by 27%, on average , and declined significantly with increasing patch floral display .
reproductive assurance index could not be calculated for one of the  <dig> patches, for which en seed set could not be determined.table  <dig>
experimental manipulation for estimating pollination parameters: reproductive assurance  and self-compatibility  estimated with seed sets from four different pollination treatments
abbreviation and calculation
definitions
patch mean seed sets/indices
range
tukey-kramer test
reproductive assurance
in
en
ra = <dig> –
self-compatibility
ix
isb
sc =
isbm
/
ix
m
patch means, ranges and tukey-kramer tests are presented to compare in vs. en  and ix vs. isb .
see the method section for model description and additional file 1: table s <dig> for raw data per patch.
effects of
r. ferrugineum
patch floral display on  mean seed set per patch for control flower
s  and emasculated flowers  and  mean reproductive assurance per patch .
see table  <dig> for methods and statistical comparisons of treatments and additional file 1: table s <dig> for seed sets and pollination parameters per patch.
the mean seed set for hand-outcrossed flowers was significantly higher than that for hand self-pollinated bagged flowers , indicating partial self-compatibility.
neither of these seed set values was significantly related to patch floral display .
self-compatibility  was not significantly related to floral display .
patch selfing rate estimates
significant amounts of selfing were observed in all patches .
selfing rates not significantly different from  <dig> were found in two patches , as indicated by the 95% confidence limits of the bootstrap distribution.
overall, r. ferrugineum has a mixed mating system, with a mean multilocus selfing rate per patch  of sm = <dig>  .
selfing rate decreased significantly with increasing r. ferrugineum floral display .table  <dig>
mating system estimates , wright’s fixation index  and lifetime inbreeding depression  for 28
rhododendron ferrugineum
patches from the pyrenees  surveyed in 2009
mating system estimates
s
m
wright’s fixation index
lifetime id
n
f
mean
se
n
i
mean
95% ci  <dig> bootstraps
mean
95% ci  <dig> bootstraps
sample sizes , means and standard errors  or 95% confidence intervals  are presented.
patches are classified from the smallest  to the largest patch floral display .
four patches had a small number of individuals , resulting in an irregular bootstrap distribution of f estimates.
the estimates of f and id for these four patches are, therefore, not presented .
see the method section for lifetime inbreeding depression calculation.
abbreviations:
t
m: multilocus patch outcrossing rates, s
m: multilocus patch selfing rates ; nf: number of families; se: standard errors; ni: number of individuals; ci: confidence interval; id: inbreeding depression; a: patch selfing rate not significantly different from  <dig> based on 95% confidence intervals.
b: f significantly higher than zero  based on 95% confidence limits.
c: bootstrap distribution of id values significantly different from  <dig>  d: bootstrap distribution of id values significantly lower than  <dig>  all other patches had id values significantly higher than  <dig>
lifetime inbreeding depression
estimates of id reached  <dig>  ±  <dig>   on average .
based on the distribution of id bootstrap values, only four patches had values significantly lower than  <dig> .
values above  <dig> were due to negative f values .
in one patch, the rate of selfing for the mother plants exceeded that of the progeny , resulting in a negative value for id .
we found no evidence for a relationship between the strength of id and patch floral display .
the mean observed inbreeding coefficient of mature plants  was  <dig>  .
f was not significantly different from zero in  <dig> of the  <dig> patches, indicating that the selfed progeny contributed little to the adult population .
the observed inbreeding coefficients  of mature plants were almost always lower than expected given the level of self-fertilization  in natural conditions ; figure 2).
finally, there was no evidence of a relationship between f estimates and patch floral display .figure  <dig>
the relationship between estimated levels of self-fertilization in progeny  and the inbreeding coefficient  of adult
r. ferrugineum
individuals in heathland patches.
n = <dig> patches .
the solid line shows the expected relationship between s and f in patches at equilibrium  with no id .
in the case of id, data points are below this solid line.
discussion
the level of selfing  <cit>  and reproductive assurance  <cit>  have been shown to be influenced by the availability of pollinators and/or mates.
however, the benefits of selfing in terms of the reproductive assurance provided when pollinators and/or mates are scarce is dependent on selfed-progeny fitness in natura.
our results demonstrate that seed production and reproductive assurance increase with decreasing patch size.
furthermore, pollinator-mediated seed set was not affected by patch size.
almost half the progeny  were produced by selfing, on average, and the rate of selfing increased with decreasing patch size.
however, the reduced fruit set in small patches and the strong mean lifetime id of  <dig>  strongly counteract the reproductive assurance benefit of selfing.
reproductive assurance and selfing rate estimates for patches of different sizes
we found that autogamy significantly increased seed set, as previously shown for other systems .
we showed in a previous study that flower visitation rates increase  <cit>  and pollen limitation decreases  <cit>  with decreasing patch size, as a result of pollinator monopolization by the mass-flowering plant r. ferrugineum in small patches and intraspecific competition for pollinator services in larger patches.
reproductive assurance  enhanced r. ferrugineum reproductive success in small patches in response to the low availability of conspecific plants with no decrease in pollinator availability.
mate limitation has been shown to be an important driver of plant mating systems  <cit> , potentially resulting in the evolution of simultaneous autogamy  with moderate pollen and seed discounting  <cit> .
the level of self-compatibility was not affected by patch size  and cannot, therefore, be responsible for the variation in reproductive assurance along the patch size gradient.
the greater reproductive assurance in small patches is more likely to result from facilitated self-pollination, as suggested in the case of mate limitation in a previous study  <cit> .
the perennial mass-flowering species studied has a mixed mating system .
the selfing rate in r. ferrugineum is higher than that in other species from the ericaceae .
this indicates that, in addition to reproductive assurance , geitonogamy was an important additional source of self-pollen.
reproductive assurance is particularly costly when associated with ovule usurpation by selfing in situations in which outcrossing could otherwise occur   <cit> .
pollinator-mediated pollen transfer decreased seed production by 34% on average in this system .
early-acting and lifetime inbreeding depression in a mass-flowering shrub
the reduced fruit set in small patches, the mean self-compatibility index of  <dig>  and its variability between individuals  suggest that id acted early on fruit set in small patches  and was probably mediated by a small number of few mutations of major effect.
a similar situation has already been reported in the ericaceae  <cit> , resulting in higher mortality between fertilization and seed maturation for selfed zygotes than for outcrossed zygotes.
lifetime estimates of id in natura were very high , not significantly affected by patch size, and well above the threshold value of  <dig>  below which selfing is favored due to its transmission advantage  <cit> .
this threshold, which was proposed for annuals  <cit> , applies to perennials only for lifetime estimates of id, such as those obtained here  <cit> .
our results suggest that there is strong selective pressure against selfed progenies, as already reported for other perennials  <cit> .
an association of strong id with high levels of self-incompatibility and a predominance of outcrossing is commonly observed in long-lived species , due to the accumulation of somatic mutations over the lifetime of the organism  have been little studied  <cit> .
mixed mating systems may be favored by the ecological context  <cit> , as the benefits of reproductive assurance can outweigh the disadvantages of id if the success of outcrossing is limited by low pollinator and/or mate availability .
reproductive assurance may, therefore, be essential for species forming small populations in subalpine environments, in which there may sometimes be a shortage of pollinators  <cit> .
however, reproductive assurance may be less advantageous in long-lived perennials, in which a failure to reproduce in some years does not compromise lifetime fitness  <cit> .
we found that reproductive assurance clearly increased seed production in small patches in which mates were scarce, but the gain was almost entirely cancelled out by id .
by contrast, stochastic environments, such as high-altitude habitats, may select for outcrossing, leading to high levels of genetic variability and favoring population persistence  <cit> .
the commonly reported low abundance and activity of pollinators in such systems may lead to the evolution of stronger interactions with pollinators via large floral displays  <cit> .
the mass-flowering habit of r. ferrugineum clearly enhances pollinator attraction  <cit> , but it also illustrates the conflict between self- and cross-fertilization  <cit> .
selfing in trees and shrubs may be an unavoidable consequence of insect visitation  <cit>  and may be autonomous or geitonogamous and facilitated .
visitation rates tend to be higher in small patches  <cit> , probably increasing both facilitated self-pollination and geitonogamy  <cit>  and, possibly, pollen  <cit>  or seed discounting  <cit> .
if not opposed by early-acting and lifetime id, the establishment of seedlings from seeds produced via facilitated selfing or geitonogamy may have an impact on the genetic structure of populations  <cit> .
in r. ferrugineum, massive flowering and buzz pollination have positive effects on pollinator attraction and pollen delivery, favoring either outcrossing or selfing .
however, strong lifetime id preserves heterozygosity along the patch size gradient in mature populations.
CONCLUSIONS
the abundances of pollinators and plant species are currently decreasing in parallel  <cit> , and the adaptive evolution of plant mating systems towards self-pollination in response to these changes has been predicted  <cit> .
however, our results suggest that, in species like r. ferrugineum, id strongly counteracts the reproductive assurance benefit of selfing.
in the case of outcross pollen limitation in the presence of such id, small and sparse populations may therefore be at risk of extinction  <cit> .
the higher selfing rates found in small patches appeared to be non-adaptive with respect to lifetime id and to be a by-product of the pollination context, which may reflect recent patch fragmentation.
the intriguing paradox of a high selfing rate despite high lifetime id should be considered in the context of species’ range scale estimates, because both selfing rate and id may vary with the environment.
this study highlights the need to estimate lifetime id along with the actual mating system and pollination parameters if we are to understand the real benefit of selfing and avoid overestimating the benefits of reproductive assurance.

 plant-pollinator interactions are increasingly affected by human disturbances, probably resulting in strong selective pressure on plant mating systems. pollination failure may promote the evolution of selfing, which can provide reproductive assurance by increasing seed production when pollinators and/or mates are scarce. reproductive assurance is one of the most longstanding and widely accepted explanations for the evolution of selfing and the maintenance of mixed mating systems. if both pollinators and mates are limiting, reproductive assurance can occur through autonomous selfing. by contrast, if only mates are limiting, reproductive assurance can also result from pollinator-mediated selfing, such as facilitated selfing. however, inbreeding depression reduces the fitness of selfed progenies and counteracts the advantages of selfing / where e is the fraction of ovules remaining unfertilized. id is typically caused either by the expression of lethal deleterious recessive alleles formerly masked by the heterozygous state or by the loss of heterozygote advantage. it may evolve with an increase in selfing rate, when deleterious recessive alleles are purged by selection, although this trend is not general in plant species. experimental studies have shown that the expression of id depends on the environment. however, the variability of id in natural environments and, more specifically, along population size gradients, remains unclear, despite its acknowledged importance. furthermore, id estimates are based principally on experimental studies in herbaceous annual species, and the fitness of selfed progenies in long-lived perennials has been little considered but see. perennial species experience multiplicative effects of id over many years and are exposed to many biotic and abiotic changes. given that selfing may be a by-product of outcrossing or provide additional benefits in terms of reproductive assurance in cases of pollination failure, strong id may be associated with high or intermediate rates of selfing. selfing provides reproductive assurance only if seed production is pollen-limited, if selfing increases seed set and if id does not completely counteract the benefits of selfing over the lifetime of the adult plant. however, few studies have empirically estimated the actual long-term benefits of selfing by investigating whether the negative impact of id counteracts the positive effects of the greater seed set due to reproductive assurance in natural populations of contrasting sizes. given the scarcity of studies simultaneously estimating reproductive assurance, selfing rate and lifetime id along natural population size gradients, predicting the potential of selfing to provide actual reproductive assurance in various ecological contexts remains a major challenge in evolutionary biology. we focused on a patchily distributed mass-flowering shrub, rhododendron ferrugineum. we showed in a previous study that insect visitation rates and floral availability are inversely related, resulting in similar pollinator-mediated pollen transfer in small and large r. ferrugineum patches. in this study system, pollen transfer is probably constrained by pollinator limitation in large patches and by mate limitation in small patches, decreasing seed production by 34% on average. here, we investigated the lifetime reproductive assurance benefit of selfing by estimating reproductive success, reproductive assurance, selfing rate and lifetime id along a continuum of patch floral display, used as a proxy for patch size. we predicted reproductive assurance and selfing rate to be higher in small sparse patches, in which pollen transfer has been shown to be limited principally by the availability of conspecific plants. lifetime id would be expected to be strong in long-lived perennial species and, if associated with early-acting id, it may counteract the reproductive assurance benefit of selfing in small plant patches. we also characterized the r. ferrugineum mating system by estimating fruit set and self-compatibility. we estimated the magnitude of id in natural populations by using a marker-based approach comparing inbreeding in adult plants, including any episodes of mortality in natural populations over the long lifetime of individuals and multilocus selfing rate in progeny arrays, reflecting selfing before selection in natural populations. we also estimated the following, along the patch size gradient: pollination parameters, including reproductive assurance, by comparing seed production by emasculated and intact flowers, and self-compatibility, by comparing seed production by hand-selfed and hand-outcrossed flowers and open fruit set; selfing rate, estimated by progeny array analysis and id in natural populations, as assessed with neutral genetic markers.  reproductive success and pollination parameters along a natural patch size gradient the mean fruit set per patch was % and increased significantly with increasing r. ferrugineum floral display per patch. mean open seed set decreased significantly with increasing floral display whereas mean pollinator-mediated seed set was not significantly related to patch floral display. open seed set from intact flowers was significantly greater than that of emasculated flowers, consistent with significant reproductive assurance. reproductive assurance increased seed production by 27%, on average, and declined significantly with increasing patch floral display. reproductive assurance index could not be calculated for one of the patches, for which en seed set could not be determined.table experimental manipulation for estimating pollination parameters: reproductive assurance and self-compatibility estimated with seed sets from four different pollination treatments abbreviation and calculation definitions patch mean seed sets/indices range tukey-kramer test reproductive assurance   in     en     ra = –   self-compatibility   ix     isb   sc =  isbm / ix m   patch means, ranges and tukey-kramer tests are presented to compare in vs. en and ix vs. isb. see the method section for model description and additional file 1: table s for raw data per patch. effects of r. ferrugineum patch floral display on mean seed set per patch for control flower s and emasculated flowers and mean reproductive assurance per patch. see table  for methods and statistical comparisons of treatments and additional file 1: table s for seed sets and pollination parameters per patch. the mean seed set for hand-outcrossed flowers was significantly higher than that for hand self-pollinated bagged flowers, indicating partial self-compatibility. neither of these seed set values was significantly related to patch floral display. self-compatibility was not significantly related to floral display. patch selfing rate estimates significant amounts of selfing were observed in all patches. selfing rates not significantly different from were found in two patches, as indicated by the 95% confidence limits of the bootstrap distribution. overall, r. ferrugineum has a mixed mating system, with a mean multilocus selfing rate per patch of sm =. selfing rate decreased significantly with increasing r. ferrugineum floral display.table mating system estimates, wright’s fixation index and lifetime inbreeding depression for 28 rhododendron ferrugineum patches from the pyrenees surveyed in 2009 mating system estimates s m wright’s fixation index lifetime id n f mean se n i mean 95% ci bootstraps mean 95% ci bootstraps sample sizes, means and standard errors or 95% confidence intervals are presented. patches are classified from the smallest to the largest patch floral display. four patches had a small number of individuals, resulting in an irregular bootstrap distribution of f estimates. the estimates of f and id for these four patches are, therefore, not presented. see the method section for lifetime inbreeding depression calculation. abbreviations: t m: multilocus patch outcrossing rates, s m: multilocus patch selfing rates; nf: number of families; se: standard errors; ni: number of individuals; ci: confidence interval; id: inbreeding depression; a: patch selfing rate not significantly different from based on 95% confidence intervals. b: f significantly higher than zero based on 95% confidence limits. c: bootstrap distribution of id values significantly different from d: bootstrap distribution of id values significantly lower than all other patches had id values significantly higher than lifetime inbreeding depression estimates of id reached  ±  on average. based on the distribution of id bootstrap values, only four patches had values significantly lower than. values above were due to negative f values. in one patch, the rate of selfing for the mother plants exceeded that of the progeny, resulting in a negative value for id. we found no evidence for a relationship between the strength of id and patch floral display. the mean observed inbreeding coefficient of mature plants was. f was not significantly different from zero in of the patches, indicating that the selfed progeny contributed little to the adult population. the observed inbreeding coefficients of mature plants were almost always lower than expected given the level of self-fertilization in natural conditions; figure 2). finally, there was no evidence of a relationship between f estimates and patch floral display.figure the relationship between estimated levels of self-fertilization in progeny and the inbreeding coefficient of adult r. ferrugineum individuals in heathland patches. n = patches. the solid line shows the expected relationship between s and f in patches at equilibrium with no id. in the case of id, data points are below this solid line. discussion the level of selfing and reproductive assurance have been shown to be influenced by the availability of pollinators and/or mates. however, the benefits of selfing in terms of the reproductive assurance provided when pollinators and/or mates are scarce is dependent on selfed-progeny fitness in natura. our results demonstrate that seed production and reproductive assurance increase with decreasing patch size. furthermore, pollinator-mediated seed set was not affected by patch size. almost half the progeny were produced by selfing, on average, and the rate of selfing increased with decreasing patch size. however, the reduced fruit set in small patches and the strong mean lifetime id of strongly counteract the reproductive assurance benefit of selfing. reproductive assurance and selfing rate estimates for patches of different sizes we found that autogamy significantly increased seed set, as previously shown for other systems. we showed in a previous study that flower visitation rates increase and pollen limitation decreases with decreasing patch size, as a result of pollinator monopolization by the mass-flowering plant r. ferrugineum in small patches and intraspecific competition for pollinator services in larger patches. reproductive assurance enhanced r. ferrugineum reproductive success in small patches in response to the low availability of conspecific plants with no decrease in pollinator availability. mate limitation has been shown to be an important driver of plant mating systems, potentially resulting in the evolution of simultaneous autogamy with moderate pollen and seed discounting. the level of self-compatibility was not affected by patch size and cannot, therefore, be responsible for the variation in reproductive assurance along the patch size gradient. the greater reproductive assurance in small patches is more likely to result from facilitated self-pollination, as suggested in the case of mate limitation in a previous study. the perennial mass-flowering species studied has a mixed mating system. the selfing rate in r. ferrugineum is higher than that in other species from the ericaceae. this indicates that, in addition to reproductive assurance, geitonogamy was an important additional source of self-pollen. reproductive assurance is particularly costly when associated with ovule usurpation by selfing in situations in which outcrossing could otherwise occur. pollinator-mediated pollen transfer decreased seed production by 34% on average in this system. early-acting and lifetime inbreeding depression in a mass-flowering shrub the reduced fruit set in small patches, the mean self-compatibility index of and its variability between individuals suggest that id acted early on fruit set in small patches and was probably mediated by a small number of few mutations of major effect. a similar situation has already been reported in the ericaceae, resulting in higher mortality between fertilization and seed maturation for selfed zygotes than for outcrossed zygotes. lifetime estimates of id in natura were very high, not significantly affected by patch size, and well above the threshold value of below which selfing is favored due to its transmission advantage. this threshold, which was proposed for annuals, applies to perennials only for lifetime estimates of id, such as those obtained here. our results suggest that there is strong selective pressure against selfed progenies, as already reported for other perennials. an association of strong id with high levels of self-incompatibility and a predominance of outcrossing is commonly observed in long-lived species, due to the accumulation of somatic mutations over the lifetime of the organism have been little studied. mixed mating systems may be favored by the ecological context, as the benefits of reproductive assurance can outweigh the disadvantages of id if the success of outcrossing is limited by low pollinator and/or mate availability. reproductive assurance may, therefore, be essential for species forming small populations in subalpine environments, in which there may sometimes be a shortage of pollinators. however, reproductive assurance may be less advantageous in long-lived perennials, in which a failure to reproduce in some years does not compromise lifetime fitness. we found that reproductive assurance clearly increased seed production in small patches in which mates were scarce, but the gain was almost entirely cancelled out by id. by contrast, stochastic environments, such as high-altitude habitats, may select for outcrossing, leading to high levels of genetic variability and favoring population persistence. the commonly reported low abundance and activity of pollinators in such systems may lead to the evolution of stronger interactions with pollinators via large floral displays. the mass-flowering habit of r. ferrugineum clearly enhances pollinator attraction, but it also illustrates the conflict between self- and cross-fertilization. selfing in trees and shrubs may be an unavoidable consequence of insect visitation and may be autonomous or geitonogamous and facilitated. visitation rates tend to be higher in small patches, probably increasing both facilitated self-pollination and geitonogamy and, possibly, pollen or seed discounting. if not opposed by early-acting and lifetime id, the establishment of seedlings from seeds produced via facilitated selfing or geitonogamy may have an impact on the genetic structure of populations. in r. ferrugineum, massive flowering and buzz pollination have positive effects on pollinator attraction and pollen delivery, favoring either outcrossing or selfing. however, strong lifetime id preserves heterozygosity along the patch size gradient in mature populations.  the abundances of pollinators and plant species are currently decreasing in parallel, and the adaptive evolution of plant mating systems towards self-pollination in response to these changes has been predicted. however, our results suggest that, in species like r. ferrugineum, id strongly counteracts the reproductive assurance benefit of selfing. in the case of outcross pollen limitation in the presence of such id, small and sparse populations may therefore be at risk of extinction. the higher selfing rates found in small patches appeared to be non-adaptive with respect to lifetime id and to be a by-product of the pollination context, which may reflect recent patch fragmentation. the intriguing paradox of a high selfing rate despite high lifetime id should be considered in the context of species’ range scale estimates, because both selfing rate and id may vary with the environment. this study highlights the need to estimate lifetime id along with the actual mating system and pollination parameters if we are to understand the real benefit of selfing and avoid overestimating the benefits of reproductive assurance. 
patch size is likely to affect mate and pollinator availabilities, thereby affecting pollination success and the rate of selfing. we estimated fruit and seed set, reproductive assurance, self-compatibility, the multilocus patch selfing rate and lifetime inbreeding depression in natural patches of rhododendron ferrugineum, a mass-flowering species characterized by considerable patch size variation. open seed set declined linearly with increasing patch size, whereas pollinator-mediated seed set was not significantly affected. progeny array analysis indicated that the selfing rate declined with increasing patch size, consistent with greater reproductive assurance in small sparse patches than in large, dense patches. however, fruit set and adult fitness decreased with decreasing patch size, with an estimated mean lifetime inbreeding depression of. this study highlights the need to estimate lifetime inbreeding depression, together with mating system and pollination parameters, if we are to understand the actual benefit of selfing and avoid the overestimation of reproductive assurance. we investigated the potential for selfing to provide reproductive assurance over the lifetime of a long-lived perennial species and its variation between plant patches of various sizes. inbreeding depressionmixed matingperennial speciesself-compatibilityselfing ratereproductive assurancerhododendron ferrugineumissue-copyright-statement© the author 2014 lifetime inbreeding depression strongly counteracts the advantage of reproductive assurance due to selfing in this long-lived species. an increase in self-fertilization may evolve to compensate for the negative effects of pollination failure.
 decreases in mate and/or pollinator availability would be expected to affect the selective pressure on plant mating systems. an increase in self-fertilization may evolve to compensate for the negative effects of pollination failure. however, the benefit of selfing in variable pollination environments depends on the relative fitnesses of selfed and outcrossed progeny. we investigated the potential for selfing to provide reproductive assurance over the lifetime of a long-lived perennial species and its variation between plant patches of various sizes. patch size is likely to affect mate and pollinator availabilities, thereby affecting pollination success and the rate of selfing. we estimated fruit and seed set, reproductive assurance, self-compatibility, the multilocus patch selfing rate and lifetime inbreeding depression in natural patches of rhododendron ferrugineum, a mass-flowering species characterized by considerable patch size variation.  open seed set declined linearly with increasing patch size, whereas pollinator-mediated seed set was not significantly affected. progeny array analysis indicated that the selfing rate declined with increasing patch size, consistent with greater reproductive assurance in small sparse patches than in large, dense patches. however, fruit set and adult fitness decreased with decreasing patch size, with an estimated mean lifetime inbreeding depression of.  lifetime inbreeding depression strongly counteracts the advantage of reproductive assurance due to selfing in this long-lived species. the poor fitness of selfed offspring should counteract any evolution towards selfing, despite its potential to alleviate the negative consequences of pollen limitation. this study highlights the need to estimate lifetime inbreeding depression, together with mating system and pollination parameters, if we are to understand the actual benefit of selfing and avoid the overestimation of reproductive assurance. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords inbreeding depressionmixed matingperennial speciesself-compatibilityselfing ratereproductive assurancerhododendron ferrugineumissue-copyright-statement© the author 2014 