Pollen limitation and Allee effect related to population size and sex ratio in the endangered Ottelia acuminata (Hydrocharitaceae): implications for conservation and reintroduction

Small populations may suffer more severe pollen limitation and result in Allee effects. Sex ratio may also affect pollination and reproduction success in dioecious species, which is always overlooked when performing conservation and reintroduction tasks. In this study, we investigated whether and how population size and sex ratio affected pollen limitation and reproduction in the endangered Ottelia acuminata, a dioecious submerged species. We established experimental plots with increasing population size and male sex ratio. We observed insect visitation, estimated pollen limitation by hand‐pollinations and counted fruit set and seed production per fruit. Fruit set and seed production decreased significantly in small populations due to pollinator scarcity and thus suffered more severe pollen limitation. Although frequently visited, female‐biased larger populations also suffered severe pollen limitation due to few effective visits and insufficient pollen availability. Rising male ratio enhanced pollination service and hence reproduction. Unexpectedly, pollinator preferences did not cause reduced reproduction in male‐biased populations because of high pollen availability. However, reproductive outputs showed more variability in severe male‐biased populations. Our results revealed two component Allee effects in fruit set and seed production, mediated by pollen limitation in O. acuminata. Moreover, reproduction decreased significantly in larger female‐biased populations, increasing the risk of an Allee effect.     

Diversity of S-alleles and mate availability in 3 populations of self-incompatible wild pear (Pyrus pyraster)

Small populations of self-incompatible plants may be expected to be threatened by the limitation of compatible mating partners (i.e., S-Allee effect). However, few empirical studies have explicitly tested the hypothesis of mate limitation in small populations of self-incompatible plants. To do so, we studied wild pear (Pyrus pyraster), which possesses a gametophytic self-incompatibility system. We determined the S-genotypes in complete samplings of all adult trees from 3 populations using a PCR-RFLP approach. We identified a total of 26 different S-alleles, homologous to S-alleles of other woody Rosaceae. The functionality of S-alleles and their Mendelian inheritance were verified in artificial pollination experiments and investigations of pollen tube growth. The smallest population (N = 8) harbored 9 different S-alleles and showed a mate availability of 92.9%, whereas the 2 larger populations harbored 18 and 25 S-alleles and exhibited mate availabilities of 98.4% and 99.2%, respectively. Therefore, we conclude that even small populations of gametophytic self-incompatible plants may exhibit high diversity at the S-locus and are not immediately threatened owing to reduced mate availability.     

S-allele diversity suggests no mate limitation in small populations of a self-incompatible plant

Small populations of self-incompatible plants are assumed to be threatened by a limitation of compatible mating partners due to low genetic diversity at the self-incompatibility (S) locus. In contrast, we show by using a PCR-RFLP approach for S-genotype identification that 15 small populations (N = 8-88) of the rare wild pear (Pyrus pyraster) displayed no mate limitation. S-allele diversity within populations was high (N = 9-21) as was mate availability (92.9-100%). Although population size and S-allele diversity were strongly related, no relationship was found between population size and mate availability, gene diversity (He), or fixation index (F(IS)), based on five neutral microsatellite loci. As we determined the principal mate availability within populations based on the S-genotypes observed, the realized mate availability under natural conditions may differ from our estimates, for example, due to spatially limited pollen dispersal. We therefore urge studies on self-incompatible plants to proceed from the simple assessment of principal mate availability to the determination of realized mate availability in natural populations.     

Loss of gametophytic self-incompatibility with evolution of inbreeding depression

Gametophytic self-incompatibility (SI) in plants is a widespread mechanism preventing self-fertilization and the ensuing inbreeding depression, but it often evolves to self-compatibility. We analyze genetic mechanisms for the breakdown of gametophytic SI, incorporating a dynamic model for the evolution of inbreeding depression allowing for partial purging of nearly recessive lethal mutations by selfing, and accounting for pollen limitation and sheltered load linked to the S-locus. We consider two mechanisms for the breakdown of gametophytic SI: a nonfunctional S-allele and an unlinked modifier locus that inactivates the S-locus. We show that, under a wide range of conditions, self-compatible alleles can invade a self-incompatible population. Conditions for invasion are always less stringent for a nonfunctional S-allele than for a modifier locus. The spread of self-compatible genotypes is favored by extremely high or low selfing rates, a small number of S-alleles, and pollen limitation. Observed parameter values suggest that the maintenance of gametophytic SI is caused by a combination of high inbreeding depression in self-incompatible populations coupled with intermediate selfing rates of the self-compatible genotypes and sheltered load linked to the S-locus.     

The evolution of self-incompatibility when mates are limiting

Self-incompatibility (SI) is a genetic barrier to inbreeding that is broadly distributed in angiosperms. In finite populations of SI plants, the loss of S-allele diversity can limit plant reproduction by reducing the availability of compatible mates. Many studies have shown that small or fragmented plant populations suffer from mate limitation. The advent of molecular typing of S-alleles in many species has paved the way to address quantitatively the importance of mate limitation, and to provide greater insight into why and how SI systems breakdown frequently in nature. In this review, we highlight the ecological factors that contribute to mate limitation in SI taxa, discuss their consequences for the evolution and functioning of SI, and propose new empirical research directions.     

Pollen quality limitation in the Iberian critically endangered genus <Emphasis Type="Italic">Pseudomisopates</Emphasis> (Antirrhinaceae)

Pollen limitation occurs when sexual reproduction is decreased due to inadequate pollen receipt. Limitation is usually associated with the quality and the quantity of pollen; still, most studies do not discriminate between the two. We used hand-pollination experiments and observations of floral visitors to determine the mating system and limitations to seed production of the Spanish endangered species Pseudomisopates rivas-martinezii. We tested for agamospermy, autogamy, obligated autogamy, cross-pollination and supplementation. The response variables considered were fruit set, seed set and viable seed set. Previous studies indicated that (i) the species exhibits extensive clonal growth, (ii) plants flower profusely in summer, (iii) seeds showed to be highly unviable, and (iv) no seedlings were observed in the field. We found that P. rivas-martinezii is predominantly self-incompatible, has an unexpected generalized pollination system considering its occluded corolla, and is limited by qualitative rather than quantitative pollen limitation. These results indicate that, in addition to other presumed environmental factors, the low seed viability of this self-incompatible species is due to pollen quality limitation as a result of reduced mate availability.     

文冠果花粉供体的多样性对果实产出的影响

很多植物的花,其柱头上接受的花粉供体多样性数量通常与果实成熟和种子产出有关。关于提高花粉多样性能否增加植物的生殖产出问题,文献中时常有相互矛盾的结论。本研究通过对自交不亲和性植物文冠果(Xanthoceras sorbifolium)进行1、2、3、4种花粉供体传粉实验,调查了不同花粉供体数量对文冠果雌性生殖的影响。结果表明,增加亲和花粉供体的多样性,一定程度上提高了花序上果实的坐果数量,同时也增加了果实中种子的数量及单粒种子重量。对文冠果授以4种亲和性花粉供体后,花序上成熟果实的平均数最多;3种亲和性花粉供体传粉后,每个果实中的种子数最多,单粒种子也最重。如果混合花粉中含有自交花粉,那么果实将败育。除花粉多样性影响雌性生殖产出外,母本基因型也具有重要影响,即增加文冠果种植品系的多样性,将有助于提高果实和种子产量。本研究还讨论了花粉遗传多样性效应、母本交配选择及果实选择性败育假说。     

Relative impact of mate versus pollinator availability on pollen limitation and outcrossing rates in a mass‐flowering species

Plant mating systems are driven by several pre‐pollination factors, including pollinator availability, mate availability and reproductive traits. We investigated the relative contributions of these factors to pollination and to realized outcrossing rates in the patchily distributed mass‐flowering shrub Rhododendron ferrugineum. We jointly monitored pollen limitation (comparing seed set from intact and pollen‐supplemented flowers), reproductive traits (herkogamy, flower size and autofertility) and mating patterns (progeny array analysis) in 28 natural patches varying in the level of pollinator availability (flower visitation rates) and of mate availability (patch floral display estimated as the total number of inflorescences per patch). Our results showed that patch floral display was the strongest determinant of pollination and of the realized outcrossing rates in this mass‐flowering species. We found an increase in pollen limitation and in outcrossing rates with increasing patch floral display. Reproductive traits were not significantly related to patch floral display, while autofertility was negatively correlated to outcrossing rates. These findings suggest that mate limitation, arising from high flower visitation rates in small plant patches, resulted in low pollen limitation and high selfing rates, while pollinator limitation, arising from low flower visitation rates in large plant patches, resulted in higher pollen limitation and outcrossing rates. Pollinator‐mediated selfing and geitonogamy likely alleviates pollen limitation in the case of reduced mate availability, while reduced pollinator availability (intraspecific competition for pollinator services) may result in the maintenance of high outcrossing rates despite reduced seed production.     

MATE AVAILABILITY AND FECUNDITY SELECTION IN MULTI-ALLELIC SELF-INCOMPATIBILITY SYSTEMS IN PLANTS

We investigate mate availability in different models of multiallelic self-incompatibility systems in mutation-selection-drift balance in finite populations. Substantial differences among self-incompatibility systems occur in average mate availability, and in variances of mate availability among individual plants. These differences are most pronounced in small populations in which low mate availability may reduce seed set in some types of sporophytic self-incompatibility. In cases where the pollination system causes a restriction in the number of pollen genotypes available to an individual plant, the fecundity of that plant depends on the availability of compatible pollen, which is determined by its genotype at the incompatibility locus. This leads to an additional component of selection acting on self-incompatibility systems, which we term “fecundity selection.” Fecundity selection increases the number of alleles maintained in finite populations and increases mate availability in small populations. The strength of fecundity selection is dependent on the type of self-incompatibility. In some cases, fecundity selection markedly alters the equilibrium dynamics of self-incompatibility alleles. We discuss the population genetic consequences of mate availability and fecundity selection in the contexts of conservation management of self-incompatible plant species and experimental investigations on self-incompatibility in natural populations.     

SRK-SCR转基因拟南芥自交不亲和性的研究进展

十字花科植物自交不亲和性（SI）受墨位点（S-locus）编码的sRK和sCR控制,它们分别是柱头和花粉中的sI特异识别因子。野生型拟南芥不具有sI,而近来通过转基因手段将外源艘K—scR基因转入野生型拟南芥可以使其表现sI,由此建立了一个可用于十字花科sI研究的新型模式植物。本文综述了利用这种转基因拟南芥在SI机制及进化方面取得的进展,包括sI新基因的挖掘、候选基因功能分析和拟南芥生殖模式的转变等。     

Pollen limitation or mate search need not induce an Allee effect

When a process modelling the availability of gametes is included explicitly in population models a critical depensation or Allee effect usually results. Non-spatial models cannot describe clumping and so small populations must be assumed very diffuse. Consequently individuals in small populations experience low contact rates and so reproduction is limited. In Nature invasions into new territory are unlikely to be as diffuse as those described by non-spatial models. We develop pair approximations to a probabilistic cellular automata model with independent pollination and seed setting processes (equivalently mate search and reproduction processes). Each process can be either global (population-wide) or local (within a small neighbourhood) or a mixture of the two. When either process is global the resulting model recaptures the Allee effect found in non-spatial models. However, if both processes are at least partially local we obtain a model in which Allee effects can be avoided altogether if individuals are suitably strong pollinators and colonisers. The Allee effect disappears because small populations are dramatically more clumped when colonisation is local and less wasteful of pollen when pollination is local.     

Microevolution of S-allele frequencies in wild cherry populations: respective impacts of negative frequency dependent selection and genetic drift

Negative frequency dependent selection (NFDS) is supposed to be the main force controlling allele evolution at the gametophytic self-incompatibility locus (S-locus) in strictly outcrossing species. Genetic drift also influences S-allele evolution. In perennial sessile organisms, evolution of allelic frequencies over two generations is mainly shaped by individual fecundities and spatial processes. Using wild cherry populations between two successive generations, we tested whether S-alleles evolved following NFDS qualitative and quantitative predictions. We showed that allelic variation was negatively correlated with parental allelic frequency as expected under NFDS. However, NFDS predictions in finite population failed to predict more than half S-allele quantitative evolution. We developed a spatially explicit mating model that included the S-locus. We studied the effects of self-incompatibility and local drift within populations due to pollen dispersal in spatially distributed individuals, and variation in male fecundity on male mating success and allelic frequency evolution. Male mating success was negatively related to male allelic frequency as expected under NFDS. Spatial genetic structure combined with self-incompatibility resulted in higher effective pollen dispersal. Limited pollen dispersal in structured distributions of individuals and genotypes and unequal pollen production significantly contributed to S-allele frequency evolution by creating local drift effects strong enough to counteract the NFDS effect on some alleles.     

Patterns of variation within self-incompatibility loci

Diverse self-incompatibility (SI) mechanisms permit flowering plants to inhibit fertilization by pollen that express specificities in common with the pistil. Characteristic of at least two model systems is greatly reduced recombination across large genomic tracts surrounding the S-locus, which regulates SI. In three angiosperm families, including the Solanaceae, the gene that controls the expression of gametophytic SI in the pistil encodes a ribonuclease (S-RNase). The gene that controls pollen SI expression is currently unknown, although several candidates have recently been proposed. Although each candidate shows a high level of polymorphism and complete allelic disequilibrium with the S-RNase gene, such properties may merely reflect tight linkage to the S-locus, irrespective of any functional role in SI. We analyzed the magnitude and nature of nucleotide variation, with the objective of distinguishing likely candidates for regulators of SI from other genes embedded in the S-locus region. We studied the S-RNase gene of the Solanaceae and 48A, a candidate for the pollen gene in this system, and we also conducted a parallel analysis of the regulators of sporophytic SI in Brassica, a system in which both the pistil and pollen genes are known. Although the pattern of variation shown by the pollen gene of the Brassica system is consistent with its role as a determinant of pollen specificity, that of 48A departs from expectation. Our analysis further suggests that recombination between 48A and S-RNase may have occurred during the interval spanned by the gene genealogy, another indication that 48A may not regulate SI expression in pollen.     

CLONAL STRUCTURE OF ARCTIC DWARF BIRCH (BETULA GLANDULOSA) AT ITS NORTHERN LIMIT

Betula glandulosa Michx. (Betulaceae) at Tarr Inlet, Baffin Island, Northwest Territories reproduces primarily by vegetative layering with little, if any, viable seed production or seedling recruitment. Information from polymorphic enzyme loci revealed 15 three-locus genotypes with a single genotype usually dominating each of the nine sampled sites. Clonal reproduction combined with self-incompatibility may be one factor contributing to a decreased seed set by reducing the probability of xenogamy. However, multiple genotypes were found at some sites providing an opportunity for cross compatible pollen to effect fertilization. The lack of seed set in these areas appears to be due to additional factors inhibiting sexual reproduction. Based on pollen records, the present limited distribution of Betula glandulosa on Baffin Island represents the remnants of once more widely distributed populations, and the failure of sexual reproduction at the northern edge of its range may be responsible for the decline in abundance. Genotypic diversity observed in the Tarr Inlet area appears to be residual from a once sexually reproducing population. No evidence was found for differential selection of genotypes indicating that random processes are probably eroding the genotypic diversity during the decrease of population size in this species.     

基于S- 核酸酶的自交不亲和性的分子机制

自交不亲和性是一种广泛存在于显花植物中的种内生殖障碍, 可以抑制近亲繁殖而促进异交。其中, 以茄科、玄参科和蔷薇科为代表的配子体自交不亲和性是最常见的类型。这类自交不亲和性是由单一的多态性S-位点所控制。目前的研究发现这一位点至少包含两个自交不亲和反应特异性决定因子: 花柱中的S-核酸酶和花粉中的SLF(S-Locus F-box)蛋白。该文将主要介绍并讨论基于S-核酸酶的自交不亲和性分子机制的研究进展。     

基于S-核酸酶的自交不亲和性的分子机制

自交不亲和性是一种广泛存在于显花植物中的种内生殖障碍,可以抑制近亲繁殖而促进异交。其中,以茄科、玄参科和蔷薇科为代表的配子体自交不亲和性是最常见的类型。这类自交不亲和性是由单一的多态性S-位点所控制。目前的研究发现这一位点至少包含两个自交不亲和反应特异性决定因子：花柱中的S-核酸酶和花粉中的SLF（S-Locus F-box）蛋白。该文将主要介绍并讨论基于S-核酸酶的自交不亲和性分子机制的研究进展。     

Small populations are mate-poor but pollinator-rich in a rare, self-incompatible plant, Hymenoxys herbacea (Asteraceae)

If pollinators or compatible mates are scarce, plants in small populations may be pollen-limited and consequently produce fewer offspring. However, determining the relative importance of the genetic vs ecological mechanisms limiting successful pollination is challenging. We explored the relationships among population size, population connectivity, pollinator visitation, allozyme diversity, mate availability (measured as percent compatible crosses among plants within a population), and pollen limitation in 12 populations (N = 39-885,274) of the self-incompatible plant Hymenoxys herbacea in Ontario, Canada. Unexpectedly, small populations had more insect flower visitors per capitulum than large populations. Consistent with the effects of genetic drift, both allozyme polymorphism and mate availability decreased with decreasing population size. Pollen limitation was low and significant in only one population and could not be predicted based on knowledge of population size, connectivity, compatible mate availability, or pollinator visitation. Population size had detectable effects on both pollinator activity and mate availability. However, because the effect of population size was complementary on these two potentially limiting mechanisms, this threatened plant was rarely pollen-limited.     

Mating system,life-history,and reproduction in Canada thistle (Cirsium arvense; Asteraceae)

Canada thistle (Cirsium arvense) (L.) Scop, is an almost perfectly dioecious, perennial plant that can express strong vegetative reproduction by means of its extensive root system. We explore some of the consequences of this type of reproductive strategy on the plant's pollination success, its ability to allocate resources to individual achenes, its ability to abort excess achenes, and on how pollen limitation affects the primary sex ratio of its offspring. Seed set in females is constrained by the availability of pollen. Clumps of female thistle isolated from males by at least 50 m set far fewer achenes per head than females that are interspersed with males. Even when such interspersion occurs, distance to nearest effective pollen donor correlates negatively with fertilization. Achene mass is significantly higher in plants that set fewer seed due to pollen limitation; however, the proportion of achenes that abort is not affected by the availability of pollen. Plants partially compensate for pollen limitation by maintaining stigmas in a receptive stage longer when pollination is sparse. Primary sex ratio is not affected by the availability of pollen; both high- and low-pollen availability treatments produced highly female-biased sex ratios.     

Non-additive effects of pollen limitation and self-incompatibility reduce plant reproductive success and population viability

Background and Aims

Mating system is a primary determinant of the ecological and evolutionary dynamics of wild plant populations. Pollen limitation and loss of self-incompatibility genotypes can both act independently to reduce seed set and these effects are commonly observed in fragmented landscapes. This study used a simulation modelling approach to assess the interacting effects of these two processes on plant reproductive performance and population viability for a range of pollination likelihood, self-incompatibility systems and S-allele richness conditions.

Methods

A spatially explicit, individual-based, genetic and demographic simulation model parameterized to represent a generic self-incompatible, short-lived perennial herb was used to conduct simulation experiments in which pollination probability, self-incompatibility type (gametophytic and sporophytic) and S-allele richness were systematically varied in combination to assess their independent and interacting effects on the demographic response variables of mate availability, seed set, population size and population persistence.

Key Results

Joint effects of reduced pollination probability and low S-allele richness were greater than independent effects for all demographic response variables except population persistence under high pollinator service (>50 %). At intermediate values of 15–25 % pollination probability, non-linear interactions with S-allele richness generated significant reductions in population performance beyond those expected by the simple additive effect of each independently. This was due to the impacts of reduced effective population size on the ability of populations to retain S alleles and maintain mate availability. Across a limited set of pollination and S-allele conditions (P = 0·15 and S = 20) populations with gametophytic SI showed reduced S-allele erosion relative to those with sporophytic SI, but this had limited effects on individual fecundity and translated into only modest increases in population persistence.

Conclusions

Interactions between pollen limitation and loss of S alleles have the potential to significantly reduce the viability of populations of a few hundred plants. Population decline may occur more rapidly than expected when pollination probabilities drop below 25 % and S alleles are fewer than 20 due to non-additive interactions. These are likely to be common conditions experienced by plants in small populations in fragmented landscapes and are also those under which differences in response between gameptophytic and sporophtyic systems are observed.     

Comparison of Petunia inflata S-Locus F-box protein (Pi SLF) with Pi SLF like proteins reveals its unique function in S-RNase based self-incompatibility

Petunia inflata possesses S-RNase-based self-incompatibility (SI), which prevents inbreeding and promotes outcrossing. Two polymorphic genes at the S-locus, S-RNase and P. inflata S-locus F-box (Pi SLF), determine the pistil and pollen specificity, respectively. To understand how the interactions between Pi SLF and S-RNase result in SI responses, we identified four Pi SLF-like (Pi SLFL) genes and used them, along with two previously identified Pi SLFLs, for comparative studies with Pi SLF(2). We examined the in vivo functions of three of these Pi SLFLs and found that none functions in SI. These three Pi SLFLs and two other Pi SLFs either failed to interact with S(3)-RNase (a non-self S-RNase for all of them) or interacted much more weakly than did Pi SLF(2) in vitro. We divided Pi SLF(2) into FD1 (for Functional Domain1), FD2, and FD3, each containing one of the Pi SLF-specific regions, and used truncated Pi SLF(2), chimeric proteins between Pi SLF(2) and one of the Pi SLFLs that did not interact with S(3)-RNase, and chimeric proteins between Pi SLF(1) and Pi SLF(2) to address the biochemical roles of these three domains. The results suggest that FD2, conserved among three allelic variants of Pi SLF, plays a major role in the strong interaction with S-RNase; additionally, FD1 and FD3 (each containing one of the two variable regions of Pi SLF) together negatively modulate this interaction, with a greater effect on interactions with self S-RNase than with non-self S-RNases. A model for how an allelic product of Pi SLF determines the fate of its self and non-self S-RNases in the pollen tube is presented.