Development of polymorphic microsatellite markers for Fagus pashanica (Fagaceae) using next-generation sequencing.

Fagus pashanica is an endangered and endemic tree species in China. To understand its genetic diversity and structure for effective conservation, we used next-generation sequencing data to develop a set of microsatellite markers. Twenty-three of the 68 designed loci were successfully amplified. Fifteen polymorphic loci with clear peaks were selected for further analyses in three F. pashanica populations sampled from Nanjiang, Wangcang and Pingwu counties in Sichuan Province, China. The number of alleles per locus ranged from two to 11. The levels of observed and expected heterozygosity ranged from 0.033-0.852 and 0.033-0.787, respectively. All 23 loci were also successfully amplified in F. longipetiolata and F. lucida, and 19 were successfully amplified in F. engleriana. These microsatellite markers will be useful for population genetic studies of F. pashanica and other Fagus species.

Host insect specificity and interspecific competition drive parasitoid diversification in a plant-insect community.

Ecological interactions among plants, insect herbivores, and parasitoids are pervasive in nature and play important roles in community assembling, but the codiversification of tri-trophic interactions has received less attention. Here we compare pairwise codiversification patterns between a set of 22 fig species, their herbivorous pollinating and galling wasps, and their parasitoids. The parasitoid phylogeny showed significant congruence and more cospeciation events with host insects phylogeny than with host plants. These results suggest that parasitoid phylogeny and speciation is more closely related to their host insects than to their host plants. The pollinating wasps hosted more parasitoid species than gallers and indicated a more intense interspecific competition among parasitoids associated with pollinators. Closer matching and fewer evolutionary host shifts were found between parasitoids and galler hosts than between parasitoids and pollinator hosts. These results suggest that interspecific competition among parasitoids, rather than resource availability of host wasps, is the main driver of the codiversification pattern in this community. Therefore, our study highlights the important role of interspecific competition among high trophic level insects in plant-insect tri-trophic community assembling.

Habitat effects on reproductive phenotype, pollinator behavior, fecundity, and mating outcomes of a bumble bee-pollinated herb.

PREMISE: Fecundity and mating outcomes commonly differ among plant populations occupying contrasting environments. If self-pollination occurs primarily among flowers within plants, contrasting reproductive outcomes among populations must reflect environmental effects on plant-pollinator interactions. Specifically, local conditions could affect features of plant phenotypes that influence pollinator behavior, in turn modifying plant reproductive outcomes. METHODS: We compared phenotypes, pollinator abundance and behavior, and female fecundity and mating in two meadow populations and two forest populations of Aconitum kusnezoffii within 3 km of each other. Mating outcomes were assessed using microsatellites. RESULTS: Meadow plants generally produced more, shorter ramets with more, larger flowers, but less nectar per flower than forest plants. These differences likely largely represent phenotypic plasticity. Individual bumble bees visited more flowers on forest plants, likely because the more abundant bees in the meadows depleted nectar availability, as indicated by briefer visits to individual flowers. Despite similar fruit set in both habitats, forest plants set more seeds per fruit. Nevertheless, meadow plants produced more seeds overall, owing to sevenfold greater flower production. Consistent with individual bees visiting fewer flowers on meadow plants, more of their seeds were outcrossed. However, the outcrossed seeds of forest plants included more male mates. CONCLUSIONS: Reproductive outcomes can vary among populations of animal-pollinated plants as a result of differences in the availability of effective pollinators and environmental effects on plant phenotypes, and their functional consequences for pollinator behavior that governs pollen dispersal.

The nature of interspecific interactions and co-diversification patterns, as illustrated by the fig microcosm.

Interactions between mutualists, competitors, and antagonists have contrasting ecological effects that, sustained over generations, can influence micro- and macroevolution. Dissimilar benefits and costs for these interactions should cause contrasting co-diversification patterns between interacting clades, with prevalent co-speciation by mutualists, association loss by competitors, and host switching by antagonists. We assessed these expectations for a local assemblage of 26 fig species (Moraceae: Ficus), 26 species of mutualistic (pollinating), and 33 species of parasitic (galling) wasps (Chalcidoidea). Using newly acquired gene sequences, we inferred the phylogenies for all three clades. We then compared the three possible pairs of phylogenies to assess phylogenetic congruence and the relative frequencies of co-speciation, association duplication, switching, and loss. The paired phylogenies of pollinators with their mutualists and competitors were significantly congruent, unlike that of figs and their parasites. The distributions of macroevolutionary events largely agreed with expectations for mutualists and antagonists. By contrast, that for competitors involved relatively frequent association switching, as expected, but also unexpectedly frequent co-speciation. The latter result likely reflects the heterogeneous nature of competition among fig wasps. These results illustrate the influence of different interspecific interactions on co-diversification, while also revealing its dependence on specific characteristics of those interactions.

A comparison of reproductive isolation between two closely related oak species in zones of recent and ancient secondary contact.

BACKGROUND: Much of the debate over the evolutionary consequences of hybridization on genetic divergence and speciation results from the breakdown or reinforcement of reproductive barriers in secondary hybrid zones. Among hybrid populations established for different lengths of time following secondary contact, stronger reproductive barriers are generally expected to occur in zones with longer contact. However, in plants no detailed investigation of recent and ancient zones of secondary contact has been conducted despite the importance of such a comparative study. Here, we compare pre- and postzygotic reproductive barriers between two closely related oak species, Quercus mongolica and Q. liaotungensis, in such a situation. RESULTS: The recorded flowering times of both species overlapped in both contact zones. The fruit set at 10 and 30 days after interspecific hand pollination was not significantly lower than that after intraspecific pollination whenever Q. mongolica or Q. liaotungensis comprised the maternal parents in both populations. These results indicated that neither prezygotic phenological barriers nor interspecific incompatibility could have resulted in the reproductive isolation between the two species in both hybrid zones. However, the proportion of hybrid seeds produced by both species in the ancient zone was significantly lower than that recorded in the recent zone of secondary contact. In addition, the proportion of hybrid seeds simulated to form, assuming both random mating and an absence of postpollination barriers, was significantly higher than that detected in the ancient contact zone but not in the recent contact zone. These results suggest stronger early-acting postzygotic isolation between the two oak species in the ancient relative to the recent contact zone. CONCLUSIONS: Our comparative study demonstrated that postzygotic barriers during seed maturity were the main contributing factor to total reproductive isolation, particularly in the ancient contact zone, which aided species delimitation. In the recently formed secondary contact zone, pre- and postzygotic barriers were not well developed, and a high frequency of natural hybridization was evident. To our knowledge this study provides the first comparison of reproductive isolation between the ancient and recent secondary contact zones in plants and helps to clarify the evolutionary consequences of hybridization in a temporal context.

Identification of Cronobacter species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry with an optimized analysis method.

Rapid and precise identification of Cronobacter species is important for foodborne pathogen detection, however, commercial biochemical methods can only identify Cronobacter strains to genus level in most cases. To evaluate the power of mass spectrometry based on matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF MS) for Cronobacter species identification, 51 Cronobacter strains (eight reference and 43 wild strains) were identified by both MALDI-TOF MS and 16S rRNA gene sequencing. Biotyper RTC provided by Bruker identified all eight reference and 43 wild strains as Cronobacter species, which demonstrated the power of MALDI-TOF MS to identify Cronobacter strains to genus level. However, using the Bruker's database (6903 main spectra products) and Biotyper software, the MALDI-TOF MS analysis could not identify the investigated strains to species level. When MALDI-TOF MS analysis was performed using the combined in-house Cronobacter database and Bruker's database, bin setting, and unweighted pair group method with arithmetic mean (UPGMA) clustering, all the 51 strains were clearly identified into six Cronobacter species and the identification accuracy increased from 60% to 100%. We demonstrated that MALDI-TOF MS was reliable and easy-to-use for Cronobacter species identification and highlighted the importance of establishing a reliable database and improving the current data analysis methods by integrating the bin setting and UPGMA clustering.

Characterization of 19 microsatellite loci in the clonal monkshood Aconitum kusnezoffii (Ranunculaceae).

PREMISE OF THE STUDY: Microsatellite loci were isolated and characterized from Aconitum kusnezoffii (Ranunculaceae) to estimate male and female reproductive success and evaluate the effects of clonal growth on sexual reproduction. METHODS AND RESULTS: A genomic enrichment approach was used to develop microsatellite markers. In three investigated A. kusnezoffii populations, a total of 19 microsatellite loci were successfully amplified, and 13 of these loci were polymorphic. Most of the primer pairs designed for the identified loci also amplified corresponding microsatellite loci in A. barbatum var. puberulum and A. alboviolaceum. CONCLUSIONS: The identified microsatellite loci will be useful for quantifying male and female fitness in A. kusnezoffii and evaluating the effects of clonal growth on sexual reproduction.

Multiple glacial refugia for cool-temperate deciduous trees in northern East Asia: the Mongolian oak as a case study.

In East Asia, temperate forests are predicted to have retracted southward to c. 30° N during the last glacial maximum (LGM) based on fossil pollen data, whereas phylogeographic studies have often suggested glacial in situ survival of cool-temperate deciduous trees in their modern northern ranges. Here we report a study of the genetic diversity and structure of 29 natural Mongolian oak (Quercus mongolica) populations using 19 nuclear simple sequence repeat (nSSR) loci and four chloroplast DNA fragments. Bayesian clustering analysis with nSSRs revealed five groups, which were inferred by approximate Bayesian computation (ABC) to have diverged in multiple refugia through multiple glacial-interglacial cycles. Analysis of chloroplast DNA variation revealed four lineages that were largely but incompletely geographically disjunct. Ecological niche modelling (ENMs) indicated a southward range shift of the oak's distribution at the LGM, although high suitability scores were also evident in the Changbai Mts. (Northeast China), the Korean Peninsula, areas surrounding the Bohai Sea, and along the coast of the Russian Far East. In addition, endemic chloroplast DNA haplotypes and nuclear lineages occurred in high-latitude northern areas where the ENM predicted no suitable habitat. The combined evidence from nuclear and chloroplast DNA, and the results of the ENM clearly demonstrate that multiple northern refugia, including cryptic ones, were maintained across the current distributional range of the Mongolian oak during the LGM or earlier glacial periods. Though spatially limited, postglacial expansions from these refugia have led to a pattern of decreased genetic diversity with increasing latitude.

Experimental analysis of mating patterns in a clonal plant reveals contrasting modes of self-pollination.

Hermaphrodite plants commonly practice self-fertilization (selfing), but the mechanisms responsible vary depending on the mode of self-pollination, pollinator behavior, and degree of clonality. Whether selfing occurs within (autogamy) or between flowers (geitonogamy) is of evolutionary significance because their fitness consequences differ. We used floral manipulations and genetic markers to determine the relative contribution of autogamy and within- versus between-ramet geitonogamy to the selfing rate of the bumblebee-pollinated, clonal herb Aconitum kusnezoffii. Data on flowering phenology and bumblebee foraging were also collected to determine opportunities for different modes of self-pollination. Autogamy accounted for only 12% of the selfing rate with the remainder resulting from geitonogamy. Whole-ramet emasculation of clones with multiple ramets reduced selfing by 78%, indicating that within-ramet geitonogamy contributed significantly (68%) to total selfing. Selfing of single-ramet plants was 44% less than multiple-ramet plants, indicating that the contribution of between-ramet geitonogamy was substantially less (20%) than within-ramet geitonogamy, probably because of bumblebee foraging behavior. Our results demonstrate for the first time in a clonal plant that within-ramet geitonogamy is substantially greater than between-ramet geitonogamy and highlight the importance of considering the influence of clonal architecture and pollinator foraging on modes of self-pollination.

Life history trait differentiation and local adaptation in invasive populations of Ambrosia artemisiifolia in China.

Local adaptation has been suggested to play an important role in range expansion, particularly among invasive species. However, the extent to which local adaptation affects the success of an invasive species and the factors that contribute to local adaptation are still unclear. This study aimed to investigate a case of population divergence that may have contributed to the local adaptation of invasive populations of Ambrosia artemisiifolia in China. Common garden experiments in seven populations indicated clinal variations along latitudinal gradients, with plants from higher latitudes exhibiting earlier flowering and smaller sizes at flowering. In reciprocal transplant experiments, plants of a northern Beijing origin produced more seeds at their home site than plants of a southern Wuhan origin, and the Wuhan-origin plants had grown taller at flowering than the Beijing-origin plants in Wuhan, which is believed to facilitate pollen dispersal. These results suggest that plants of Beijing origin may be locally adapted through female fitness and plants from Wuhan possibly locally adapted through male fitness. Selection and path analysis suggested that the phenological and growth traits of both populations have been influenced by natural selection and that flowering time has played an important role through its direct and indirect effects on the relative fitness of each individual. This study evidences the life history trait differentiation and local adaptation during range expansion of invasive A. artemisiifolia in China.

The incidence and pattern of copollinator diversification in dioecious and monoecious figs.

Differences in breeding system are associated with correlated ecological and morphological changes in plants. In Ficus, dioecy and monoecy are strongly associated with different suites of traits (tree height, population density, fruiting frequency, pollinator dispersal ecology). Although approximately 30% of fig species are pollinated by multiple species of fig-pollinating wasps, it has been suggested that copollinators are rare in dioecious figs. Here, we test whether there is a connection between the fig breeding system and copollinator incidence and diversification by conducting a meta-analysis of molecular data from pollinators of 119 fig species that includes new data from 15 Asian fig species. We find that the incidence of copollinators is not significantly different between monoecious and dioecious Ficus. Surprisingly, while all copollinators in dioecious figs are sister taxa, only 32.1% in monoecious figs are sister taxa. We present hypotheses to explain those patterns and discuss their consequences on the evolution of this mutualism.

Consequences of multiple inflorescences and clonality for pollinator behavior and plant mating.

Angiosperms engage in distributed reproduction, producing sex organs in multiple flowers on one or more inflorescences, including on different physical individuals of clonal plants. We investigated the effects of alternative deployments of artificial flowers for pollinator behavior and simulated pollen dispersal. Plants presented 18 flowers on either one inflorescence (1-I plants) or three inflorescences (3-I plants) spaced either closely or widely. Bees often skipped inflorescences on 3-I plants, visiting an average of 1.5 fewer flowers overall than on 1-I plants. In simulations with all flowers receiving and donating pollen, this behavior caused 7% less geitonogamy for 3-I plants, contradicting a common supposition that clonality increases geitonogamy. Bees generally moved upward within inflorescences and downward between inflorescences. Consequently, in simulations, segregation of pollen receipt to lower flowers and pollen donation to upper flowers reduced self-pollination and enhanced pollen export much more for 1-I plants. Nectar volume per flower had little relevant influence on bee behavior. The observed bee responses and simulated mating results suggest that production of multiple inflorescences and clonality promote pollination quality when flowers simultaneously receive and donate pollen, whereas a single large inflorescence is advantageous when segregation of sex roles among flowers reduces geitonogamy effectively.

The role of late-acting self-incompatibility and early-acting inbreeding depression in governing female fertility in monkshood, Aconitum kusnezoffii.

Reduced seed yields following self-pollination have repeatedly been observed, but the underlying mechanisms remain elusive when self-pollen tubes can readily grow into ovaries, because pre-, post-zygotic late-acting self-incompatibility (LSI), or early-acting inbreeding depression (ID) can induce self-sterility. The main objective of this study was to differentiate these processes in Aconitum kusnezoffii, a plant lacking stigmatic or stylar inhibition of self-pollination. We performed a hand-pollination experiment in a natural population of A. kusnezoffii, compared seed set among five pollination treatments, and evaluated the distribution of seed size and seed set. Embryonic development suggested fertilization following self-pollination. A partial pre-zygotic LSI was suggested to account for the reduced seed set by two lines of evidence. The seed set of chase-pollination treatment significantly exceeded that of self-pollination treatment, and the proportion of unfertilized ovules was the highest following self-pollination. Meanwhile, early-acting ID, rather than post-zygotic LSI, was suggested by the findings that the size of aborted selfed seeds varied continuously and widely; and the selfed seed set both exhibited a continuous distribution and positively correlated with the crossed seed set. These results indicated that the embryos were aborted at different stages due to the expression of many deleterious alleles throughout the genome during seed maturation. No signature of post-zygotic LSI was found. Both partial pre-zygotic LSI and early-acting ID contribute to the reduction in selfed seed set in A. kusnezoffii, with pre-zygotic LSI rejecting part of the self-pollen and early-acting ID aborting part of the self-fertilized seeds.

No evolutionary shift in the mating system of north American Ambrosia artemisiifolia (Asteraceae) following its introduction to China.

The mating system plays a key role during the process of plant invasion. Contemporary evolution of uniparental reproduction (selfing or asexuality) can relieve the challenges of mate limitation in colonizing populations by providing reproductive assurance. Here we examined aspects of the genetics of colonization in Ambrosia artemisiifolia, a North American native that is invasive in China. This species has been found to possess a strong self-incompatibility system and have high outcrossing rates in North America and we examined whether there has been an evolutionary shift towards the dependence on selfing in the introduced range. Specifically, we estimated outcrossing rates in one native and five invasive populations and compared levels of genetic diversity between North America and China. Based on six microsatellite loci we found that, like the native North American population, all five Chinese populations possessed a completely outcrossing mating system. The estimates of paternity correlations were low, ranging from 0.028-0.122, which suggests that populations possessed ~8-36 pollen donor parents contributing to each maternal plant in the invasive populations. High levels of genetic diversity for both native and invasive populations were found with the unbiased estimate of gene diversity ranging from 0.262-0.289 for both geographic ranges based on AFLP markers. Our results demonstrate that there has been no evolutionary shift from outcrossing to selfing during A. artemisiifolia's invasion of China. Furthermore, high levels of genetic variation in North America and China indicate that there has been no erosion of genetic variance due to a bottleneck during the introduction process. We suggest that the successful invasion of A. artemisiifolia into Asia was facilitated by repeated introductions from multiple source populations in the native range creating a diverse gene pool within Chinese populations.

Geographic variation in the structure of oak hybrid zones provides insights into the dynamics of speciation.

Studying geographic variation in the rate of hybridization between closely related species could provide a useful window on the evolution of reproductive isolation. Reinforcement theory predicts greater prezygotic isolation in areas of prolonged contact between recently diverged species than in areas of recent contact, which implies that old contact zones would be dominated by parental phenotypes with few hybrids (bimodal hybrid zones), whereas recent contact zones would be characterized by hybrid swarms (unimodal hybrid zones). Here, we investigate how the hybrid zones of two closely related Chinese oaks, Quercus mongolica and Q. liaotungensis, are structured geographically using both nuclear and chloroplast markers. We found that populations of Q. liaotungensis located around the Changbai Mountains in Northeast China, an inferred glacial refugium, were introgressed by genes from Q. mongolica, suggesting historical contact between the two species in this region. However, these introgressed populations form sharp bimodal hybrid zones with Q. mongolica. In contrast, populations of Q. liaotungensis located in North China, which show no sign of ancient introgression with Q. mongolica, form unimodal hybrid zones with Q. mongolica. These results are consistent with the hypothesis that selection against hybrids has had sufficient time to reinforce the reproductive barriers between Q. liaotungensis and Q. mongolica in Northeast China but not in North China.

Exploring species limits in two closely related Chinese oaks.

BACKGROUND: The species status of two closely related Chinese oaks, Quercus liaotungensis and Q. mongolica, has been called into question. The objective of this study was to investigate the species status and to estimate the degree of introgression between the two taxa using different approaches. METHODOLOGY/PRINCIPAL FINDINGS: Using SSR (simple sequence repeat) and AFLP (amplified fragment length polymorphism) markers, we found that interspecific genetic differentiation is significant and higher than the differentiation among populations within taxa. Bayesian clusters, principal coordinate analysis and population genetic distance trees all classified the oaks into two main groups consistent with the morphological differentiation of the two taxa rather than with geographic locations using both types of markers. Nevertheless, a few individuals in Northeast China and many individuals in North China have hybrid ancestry according to Bayesian assignment. One SSR locus and five AFLPs are significant outliers against neutral expectations in the interspecific FST simulation analysis, suggesting a role for divergent selection in differentiating species. MAIN CONCLUSIONS/SIGNIFICANCE: All results based on SSRS and AFLPS reached the same conclusion: Q. liaotungensis and Q. mongolica maintain distinct gene pools in most areas of sympatry. They should therefore be considered as discrete taxonomic units. Yet, the degree of introgression varies between the two species in different contact zones, which might be caused by different population history or by local environmental factors.

Nuclear and chloroplast DNA phylogeography reveal two refuge areas with asymmetrical gene flow in a temperate walnut tree from East Asia.

• Recently, there has been a debate about whether the temperate forests of East Asia merged or fragmented during glacial periods in the Pleistocene. Here, we tested these two opposing views through phylogeographical studies of the temperate-deciduous walnut tree, Juglans mandshurica (Juglandaceae) in northern and northeastern China, as well as Japan and Korea. • We assessed the genetic structure of 33 natural populations using 10 nuclear microsatellite loci and seven chloroplast DNA (cpDNA) fragments. • The cpDNA data showed the complete fixation of two different haplotype lineages in northeastern vs northern populations. This pronounced phylogeographic break was also indicated by nuclear microsatellite data, but there were disparities regarding individual populations. Among those populations fixed for haplotype A (the northeastern group), three were clustered in the northern group and four showed evidence of mixed ancestry based on microsatellite data. • Our results support the hypothesis that two independent refugia were maintained across the range of J. mandshurica in the north of China during the last glacial maximum, contrary to the inference that all temperate forests migrated to the south (25-30°N). The discordance between the patterns revealed by cpDNA and microsatellite data indicate that asymmetrical gene flow has occurred between the two refugia.

Identification of 12 animal species meat by T-RFLP on the 12S rRNA gene.

The verification of authenticity of meat products is relevant for economical, religious or public health concerning reasons. A molecular approach using terminal restriction fragment length polymorphism (T-RFLP) was developed to distinguish 12 common economically important meat species. The partial 12S rRNA gene was amplified with double-fluorescently labeled primers. The amplified fragments were digested with two endonucleases and only the terminal restriction fragment containing labeled primer was detected on capillary electrophoresis system ABI3100. AluI and Tru9I generated differently-sized terminal fragments in different species. Pig and buffalo can be separated by 3'-terminal fragment of AluI digestion. Horse, turkey, goat, sheep, deer, and cattle can be further separated by 5'-terminal fragment of Tru9I digestion. Dog and chicken, sturgeon and salmon can finally be separated by 5'-terminal fragment of AluI digestion and 3'-terminal fragment of Tru9I digestion. Our results demonstrated the potential feasibility and applicability of T-RFLP method for rapid and accurate identification of animal species.

Female reproductive success decreases with display size in monkshood, Aconitum kusnezoffii (Ranunculaceae).

BACKGROUND AND AIMS: Reduction in female fitness in large clones can occur as a result of increased geitonogamous self-fertilization and its influence through inbreeding depression. This possibility was investigated in the self-compatible, bee-pollinated perennial herb Aconitum kusnezoffii which varies in clone size. METHODS: Field investigations were conducted on pollinator behaviour, flowering phenology and variation in seed set. The effects of self-pollination following controlled self- and cross-pollination were also examined. Selfing rates of differently sized clones were assessed using allozyme markers. KEY RESULTS: High rates of geitonogamous pollination were associated with large display size. Female fitness at the ramet level decreased with clone size. Fruit and seed set under cross-pollination were significantly higher than those under self-pollination. The pre-dispersal inbreeding depression was estimated as 0.502 based on the difference in seed set per flower between self- and cross-pollinated flowers. Selfing rates of differently sized clones did not differ. CONCLUSIONS: It is concluded that in A. kusnezoffii the negative effects of self-pollination causing reduced female fertility with clone size arise primarily from a strong early-acting inbreeding depression leading to the abortion of selfed embryos prior to seed maturation.

Increased maleness at flowering stage and femaleness at fruiting stage with size in an andromonoecious perennial, Veratrum nigrum.

Theory predicts that cosexual plants should adjust their resource investment in male and female functions according to their size if female and male fitness are differentially affected by size. However, few empirical studies have been carried out at both the flowering and fruiting stages to adequately address size-dependent sex allocation in cosexual plants. In this paper, we investigated resource investment between female and male reproduction, and their size-dependence in a perennial andromonoecious herb, Veratrum nigrum L. We sampled 192 flowering plants, estimated their standardized phenotypic gender, and assessed the resource investment in male and female functions in terms of absolute dry biomass. At the flowering stage, male investment increased with plant size more rapidly than female investment, and the standardized phenotypic femaleness (ranging from 0.267 to 0.776) was negatively correlated with plant size. By contrast, female biased allocation was found at the fruiting stage, although both flower biomass and fruit biomass were positively correlated with plant size. We propose that increased maleness with plant size at the flowering stage may represent an adaptive strategy for andromonoecious plants, because male flowers promote both male and female fertility by increasing pollinator attraction without aggravating pollen discounting.