Phenotype-fitness relationships and pollen-transfer efficiency of five orchid species with different pollination strategies.

PREMISE: Deceptive pollination, a fascinating mechanism that independently originated in several plant families for benefiting from pollinators without providing any reward, is particularly widespread among orchids. Pollination efficiency is crucial in orchids due to the aggregated pollen in a pollinarium, which facilitates pollen transfer and promotes cross-pollination as pollinators leave after being deceived. METHODS: In this study, we compiled data on reproductive ecology from five orchid species with different pollination strategies: three deceptive-strategy species (shelter imitation, food deception, sexual deception), one nectar-rewarding species, and one shelter-imitation but spontaneously selfing species. We aimed to compare the reproductive success (female fitness: fruit set; male fitness: pollinarium removal) and pollination efficiency of species representing these strategies. We also investigated pollen limitation and inbreeding depression among the pollination strategies. RESULTS: Male and female fitness were strongly correlated in all species but the spontaneously selfing species, which had high fruit set and low pollinarium removal. As expected, pollination efficiency was highest for the rewarding species and the sexually deceptive species. Rewarding species had no pollen limitation but did have high cumulative inbreeding depression; deceptive species had high pollen limitation and moderate inbreeding depression; and spontaneously selfing species did not have pollen limitation or inbreeding depression. CONCLUSIONS: Pollinator response to deception is critical to maintain reproductive success and avoid inbreeding in orchid species with non-rewarding pollination strategies. Our findings contribute to a better understanding of the trade-offs associated with different pollination strategies in orchids and highlight the importance of pollination efficiency in orchids due to the pollinarium.

Pollinators differ in their contribution to the male fitness of a self-incompatible composite.

PREMISE: Reproductive fitness in plants is often determined by the quantity and quality of pollen transferred by pollinators. However, many fitness studies measure only female fitness or rely on proxies for male fitness. Here we assessed how five bee taxon groups affect male fitness in a prairie plant by quantifying pollen removal, visitation, and siring success using paternity assignments and a unique pollinator visitation experiment. METHODS: In Echinacea angustifolia, we measured per-visit pollen removal for each pollinator taxon and estimated the number of pollen grains needed for successful ovule fertilization. Additionally, we directly measured pollinator influence on siring by allowing only one bee taxon to visit each pollen-donor plant, while open-pollinated plants acted as unrestricted pollen recipients. We genotyped the resulting offspring, assigned paternity, and used aster statistical models to quantify siring success. RESULTS: Siring success of pollen-donor plants differed among the five pollinator groups. Nongrooming male bees were associated with increased siring success. Bees from all taxa removed most of the flowering head's pollen in one visit. However, coneflower-specialist bee Andrena helianthiformis removed the most pollen per visit. Female fitness and proxy measures of male fitness, such as pollinator visitation and pollen removal, did not align with our direct quantifications of male fitness. CONCLUSIONS: Our results illustrate the need for more studies to directly quantify male fitness, and we caution against using male fitness proxy measures. In addition, conservation efforts that preserve a diverse pollinator community can benefit plants in fragmented landscapes.

Pollen layering and male-male competition: Quantum dots demonstrate that pollen grains compete for space on pollinators.

PREMISE: Almost nothing is known about what happens to pollen grains once they attach to pollinators, although some have postulated that pollen from different donors may form complex, two- or three-dimensional landscapes (e.g., layers or mosaics) that can facilitate male-male competition. For example, pollen that is already on pollinators may preclude the deposition of subsequent pollen grains. METHODS: Using quantum dots to mark the pollen of individual flowers, we explored the possibilities of layering and preclusion in a fly-pollinated iris, Moraea lurida. RESULTS AND CONCLUSIONS: The proportion of labeled pollen from the last flower visited diminished in sequential pollen samples taken from the top to the bottom of the pollen load, representing the first empirical evidence for pollen layering. However, the consequences in terms of pollen preclusion were equivocal: Although the pre-existing pollen load size was not a good predictor of new pollen receipt, labeled pollen loads from the last flower visited were significantly smaller than pollen loads from the previous flower visited. Thus, pollen from the previous flower may preclude pollen placement from a subsequently visited flower, and pollen from different flowers may compete for space on pollinators.

How do arbuscular mycorrhizas affect reproductive functional fitness of host plants?

Arbuscular mycorrhizal (AM) symbiosis in soil may be directly or indirectly involved in the reproductive process of sexually reproducing plants (seed plants), and affect their reproductive fitness. However, it is not clear how underground AM symbiosis affects plant reproductive function. Here, we reviewed the studies on the effects of AM symbiosis on plant reproductive fitness including both male function (pollen) and female function (seed). AM symbiosis regulates the development and function of plant sexual organs by affecting the nutrient using strategy and participating in the formation of hormone networks and secondary compounds in host plants. The nutrient supply (especially phosphorus supply) of AM symbiosis may be the main factor affecting plant's reproductive function. Moreover, the changes in hormone levels and secondary metabolite content induced by AM symbiosis can also affect host plants reproductive fitness. These effects can occur in pollen formation and transport, pollen tube growth and seed production, and seedling performance. Finally, we discuss other possible effects of AM symbiosis on the male and female functional fitness, and suggest several additional factors that may be involved in the influence of AM symbiosis on the reproductive fitness of host plants. We believe that it is necessary to accurately identify and verify the mechanisms driving the changes of reproductive fitness of host plant in symbiotic networks in the future. A more thorough understanding of the mechanism of AM symbiosis on reproductive function will help to improve our understanding of AM fungus ecological roles and may provide references for improving the productivity of natural and agricultural ecosystems.

Stamen dimorphism in bird-pollinated flowers: Investigating alternative hypotheses on the evolution of heteranthery.

Heteranthery, the presence of distinct stamen types within a flower, is commonly explained as functional adaptation to alleviate the "pollen dilemma," defined as the dual and conflicting function of pollen as pollinator food resource and male reproductive agent. A single primary hypothesis, "division of labor," has been central in studies on heteranthery. This hypothesis postulates that one stamen type functions in rewarding pollen-collecting pollinators and the other in reproduction, thereby minimizing pollen loss. Only recently, alternative functions (i.e., staggered pollen release), were proposed, but comparative and experimental investigations are lagging behind. Here, we used 63 species of the tribe Merianieae (Melastomataceae) to demonstrate that, against theory, heteranthery occurs in flowers offering rewards other than pollen, such as staminal food bodies or nectar. Although shifts in reward type released species from the "pollen dilemma," heteranthery has evolved repeatedly de novo in food-body-rewarding, passerine-pollinated flowers. We used field investigations to show that foraging passerines discriminated between stamen types and removed large stamens more quickly than small stamens. Passerines removed small stamens on separate visits towards the end of flower anthesis. We propose that the staggered increase in nutritive content of small stamens functions to increase chances for outcross-pollen transfer.

Female-limited X-chromosome evolution effects on male pre- and post-copulatory success.

Intralocus sexual conflict arises when the expression of shared alleles at a single locus generates opposite fitness effects in each sex (i.e. sexually antagonistic alleles), preventing each sex from reaching its sex-specific optimum. Despite its importance to reproductive success, the relative contribution of intralocus sexual conflict to male pre- and post-copulatory success is not well-understood. Here, we used a female-limited X-chromosome (FLX) evolution experiment in Drosophila melanogaster to limit the inheritance of the X-chromosome to the matriline, eliminating possible counter-selection in males and allowing the X-chromosome to accumulate female-benefit alleles. After more than 100 generations of FLX evolution, we studied the effect of the evolved X-chromosome on male attractiveness and sperm competitiveness. We found a non-significant increase in attractiveness and decrease in sperm offence ability in males expressing the evolved X-chromosomes, but a significant increase in their ability to avoid displacement by other males' sperm. This is consistent with a trade-off between these traits, perhaps mediated by differences in body size, causing a small net reduction in overall male fitness in the FLX lines. These results indicate that the X-chromosome in D. melanogaster is subject to selection via intralocus sexual conflict in males.

Effects of herbivory and its timing on reproductive success of a tropical deciduous tree.

BACKGROUND AND AIMS: The implications of herbivory for plant reproduction have been widely studied; however, the relationship of defoliation and reproductive success is not linear, as there are many interacting factors that may influence reproductive responses to herbivore damage. In this study we aimed to disentangle how the timing of foliar damage impacts both male and female components of fitness, and to assess when it has greater impacts on plant reproductive success. METHODS: We measured herbivore damage and its effects on floral production, male and female floral attributes as well as fruit yield in three different phenological phases of Casearia nitida (Salicaceae) over the course of two consecutive years. Then we tested two models of multiple causal links among herbivory and reproductive success using piecewise structural equation models. KEY RESULTS: The effects of leaf damage differed between reproductive seasons and between male and female components of fitness. Moreover, the impact of herbivory extended beyond the year when it was exerted. The previous season's cumulated foliar damage had the largest impact on reproductive characters, in particular a negative effect on the numbers of inflorescences, flowers and pollen grains, indirectly affecting the numbers of infructescences and fruits, and a positive one on the amount of foliar damage during flowering. CONCLUSIONS: For perennial and proleptic species, the dynamics of resource acquisition and allocation patterns for reproduction promote and extend the effects of herbivore damage to longer periods than a single reproductive event and growing season, through the interactions among different components of female and male fitness.

Development of Microsatellite Markers for a Dioecious Herpetospermum pedunculosum (Cucurbitaceae).

Understanding the evolution of flower diversity is a central topic in plant evolutionary ecology, and natural selection on floral traits via male fitness could be estimated quantitatively using microsatellites. Here, based on RNA sequencing, we developed simple sequence repeat primers and verified polymorphisms in 2 wild populations of Herpetospermum pedunculosum (Cucurbitaceae), a dioecious annual plants native to the Himalaya Mountains. A total of 131 paired primers were designed; 15 paired primers were found to be polymorphic, with the expected heterozygosity varying between 0.280 and 0.767. We also identified 58 genotypes in 20 plants from the 2 populations. Conclusively, these primers could be effective in examining male fitness and population genetic structure of H pedunculosum in future studies.

Heat shock sensitivity of adult male fertility in the parasitoid wasp Anisopteromalus calandrae (Hymenoptera, Pteromalidae).

In insects, decreased reproduction is a sublethal consequence of high temperatures, with males being more sensitive to this in many species. In hymenoptera, arrhenotokous parthenogenesis means that female offspring are produced using sperm and are thus diploid, while males are haploid. Consequently, sperm stocks in males and females (after copulation) are a key regulator of the sex ratio. Anisopteromalus calandrae is a parasitoid wasp in which males can suffer from subfertility due to a drastic decrease in sperm count after exposure to high temperatures during a critical early pupal stage. However, in this species spermatogenesis continues during adulthood, therefore the heat sensitivity of adult males remains to be studied. Laboratory studies were conducted on virgin and previously mated young adult males under control (30 °C) and heat shock (10 min at 48 °C) conditions to exhaust their initial sperm stock. After heat shock, in both virgin and already mated males, the individual sperm potential was half that of controls. Both groups continuously produced sperm, but sperm stock of heat shocked males' never reached that of the controls. Heat shock reduced survival at 10 days only in previously experienced males but had no impact on the mating ability in competition for a female compared to controls. Despite a reduced sperm count, heat shocked males had fully fertile spermatozoa. Such a physiological response to heat shock in a species with continuous sperm production could be of major interest for both wild populations in a context of temperature variations and parasitoid wasps introduced for agronomical purposes.

Hermaphroditism promotes mate diversity in flowering plants.

PREMISE: Genetically diverse sibships are thought to increase parental fitness through a reduction in the intensity of sib competition, and through increased opportunities for seedling establishment in spatially or temporally heterogeneous environments. Nearly all research on mate diversity in flowering plants has focused on the number of fathers siring seeds within a fruit or on a maternal plant. Yet as hermaphrodites, plants can also accrue mate diversity by siring offspring on several pollen recipients in a population. Here we explore whether mate composition overlaps between the dual sex functions, and discuss the implications for plant reproductive success. METHODS: We established an experimental population of 49 Mimulus ringens (monkeyflower) plants, each trimmed to a single flower. Following pollination by wild bees, we quantified mate composition for each flower through both paternal and maternal function. Parentage was successfully assigned to 240 progeny, 98% of the sampled seeds. RESULTS: Comparison of mate composition between male and female function revealed high mate diversity, with almost no outcross mates shared between the two sexual functions of the same flower. CONCLUSIONS: Dual sex roles contribute to a near doubling of mate diversity in our experimental population of Mimulus ringens. This finding may help explain the maintenance of hermaphroditism under conditions that would otherwise favor the evolution of separate sexes.

Size advantage for male function and size-dependent sex allocation in Ambrosia artemisiifolia, a wind-pollinated plant.

In wind-pollinated plants, male-biased sex allocation is often positively associated with plant size and height. However, effects of size (biomass or reproductive investment) and height were not separated in most previous studies. Here, using experimental populations of monoecious plants, Ambrosia altemisiifolia, we examined (1) how male and female reproductive investments (MRI and FRI) change with biomass and height, (2) how MRI and height affect male reproductive success (MRS) and pollen dispersal, and (3) how height affects seed production. Pollen dispersal kernel and selection gradients on MRS were estimated by 2,102 seeds using six microsatellite markers. First, MRI increased with height, but FRI did not, suggesting that sex allocation is more male-biased with increasing plant height. On the other hand, both MRI and FRI increased with biomass but often more greatly for FRI, and consequently, sex allocation was often female-biased with biomass. Second, MRS increased with both height and MRI, the latter having the same or larger effect on MRS. Estimated pollen dispersal kernel was fat-tailed, with the maximum distance between mates tending to increase with MRI but not with height. Third, the number of seeds did not increase with height. Those findings showed that the male-biased sex allocation in taller plants of A. artemisiifolia is explained by a direct effect of height on MRS.

Temperature fluctuations during development reduce male fitness and may limit adaptive potential in tropical rainforest Drosophila.

Understanding the potential for organisms to tolerate thermal stress through physiological or evolutionary responses is crucial given rapid climate change. Although climate models predict increases in both temperature mean and variance, such tolerances are typically assessed under constant conditions. We tested the effects of temperature variability during development on male fitness in the rainforest fly Drosophila birchii, by simulating thermal variation typical of the warm and cool margins of its elevational distribution, and estimated heritabilities and genetic correlations of fitness traits. Reproductive success was reduced for males reared in warm (mean 24 °C) fluctuating (±3 °C) vs. constant conditions but not in cool fluctuating conditions (mean 17 °C), although fluctuations reduced body size at both temperatures. Male reproductive success under warm fluctuating conditions was similar to that at constant 27 °C, indicating that briefly exceeding critical thermal limits has similar fitness costs to continuously stressful conditions. There was substantial heritable variation in all traits. However, reproductive success traits showed no genetic correlation between treatments reflecting temperature variation at elevational extremes, which may constrain evolutionary responses at these ecological margins. Our data suggest that even small increases in temperature variability will threaten tropical ectotherms living close to their upper thermal limits, both through direct effects on fitness and by limiting their adaptive potential.

Bigger testes increase paternity in a simultaneous hermaphrodite, independently of the sperm competition level.

Hermaphroditic animals face the fundamental evolutionary optimization problem of allocating their resources to their male vs. female reproductive function (e.g. testes and sperm vs. ovaries and eggs), and this optimal sex allocation can be affected by both pre- and post-copulatory sexual selection. For example, local sperm competition (LSC) - the competition between related sperm for the fertilization of a partner's ova - occurs in small mating groups and can favour a female-biased sex allocation, because, under LSC, investment into sperm production is predicted to show diminishing fitness returns. Here, we test whether higher testis investment increases an individual's paternity success under sperm competition, and whether the strength of this effect diminishes when LSC is stronger, as predicted by sex allocation theory. We created two subsets of individuals of the simultaneously hermaphroditic flatworm Macrostomum lignano - by sampling worms from either the highest or lowest quartile of the testis investment distribution - and estimated their paternity success in group sizes of either three (strong LSC) or eight individuals (weak LSC). Specifically, using transgenic focal individuals expressing a dominant green-fluorescent protein marker, we showed that worms with high testis investment sired 22% more offspring relative to those with low investment, corroborating previous findings in M. lignano and other species. However, the strength of this effect was not significantly modulated by the experienced group size, contrasting theoretical expectations of more strongly diminishing fitness returns under strong LSC. We discuss the possible implications for the evolutionary maintenance of hermaphroditism in M. lignano.

The importance of male body size on sperm uptake and usage, and female fecundity in Aedes aegypti and Aedes albopictus.

BACKGROUND: Adult mosquito density is a critical factor in the transmission of arboviruses by container Aedes spp. mosquitoes. Female fecundity drives population growth, and therefore contributes to adult mosquito density. Previous studies have focused on female body size as the major determinant of fecundity, paying little attention to male condition. In this study, we examined the effects of male body size on the abundance of sperm in spermatheca, depletion of sperm over time, and female fecundity. METHODS: We generated males in two size classes using different larval densities, and allowed them to mate with females generated from a moderately dense larval environment. We counted sperm in female spermatheca in a sample of females immediately after mating, then every week for four weeks post-mating. We provided weekly blood meals to females and determined their fecundity over four weeks after the initial blood meal. RESULTS: We found significantly more sperm in Aedes albopictus females than in Aedes aegypti, and detected depletion of sperm in Ae. aegypti, but not in Ae. albopictus. We did not see significant differences in number of sperm in spermathecae in relation to male body size in either species over subsequent gonotrophic cycles. We found a significant effect of male body size on fecundity in Ae. albopictus, but not Ae. aegypti, with a 46 % increase in fecundity for female Ae. albopictus offered four blood meals. CONCLUSIONS: Our results suggest substantial differences in the mating biology of these ecologically similar species and the importance of considering males in understanding female fecundity. The substantial increase in fecundity in Ae. albopictus has implications for population growth, estimating vector density, and modeling the transmission of pathogens.

Toxic Effect of Blood Feeding in Male Mosquitoes.

Blood- and sugar feeding of female mosquitoes has been frequently observed in the laboratory and in the field, but only sugar feeding of males has been reported. Here, we describe for the first time that Culex quinquefasciatus males feed on blood as well. Blood feeding easily happened on a blood-soaked cotton roll and, to a lesser extent, through a thin artificial layer. Mating history of a male specimen does not affect his blood feeding behavior. Male mosquitoes feed on blood even when they have a readily available sugar source. Nevertheless, feeding on blood reduces the survival rate of males to just a few days, as compared to more than a month for mosquitoes fed only on sugar. Comparing survival of male mosquitoes fed on blood only, sugar only, and a combination of both clearly demonstrated that mortality is not affected by malnutrition (reduced sugar levels), but rather due to ingested blood. On average male mosquitoes ingested ca. 0.5 µl of blood, i.e., about 10% of the amount of blood ingested by an engorged female. Although this unexpected observation of blood feeding in the laboratory by male mosquitoes is interesting, structural impairment prevents male feeding on vertebrate blood. In agreement with the literature, male and female proboscises and stylets were in general of similar size, but male mandibles were significantly shorter than female counterparts, thus explaining their inability to pierce through skin layers.

Altitudinal flower size variation correlates with local pollinator size in a bumblebee-pollinated herb, Prunella vulgaris L. (Lamiaceae).

The influence of locally different species interactions on trait evolution is a focus of recent evolutionary studies. However, few studies have demonstrated that geographically different pollinator-mediated selection influences geographic variation in floral traits, especially across a narrow geographic range. Here, we hypothesized that floral size variation in the Japanese herb Prunella vulgaris L. (Lamiaceae) is affected by geographically different pollinator sizes reflecting different pollinator assemblages. To evaluate this hypothesis, we posed two questions. (1) Is there a positive correlation between floral length and the proboscis length of pollinators (bumblebees) across altitude in a mountain range? (2) Does the flower-pollinator size match influence female and male plant fitness? We found geographic variation in the assemblage of pollinators of P. vulgaris along an altitudinal gradient, and, as a consequence, the mean pollinator proboscis length also changed altitudinally. The floral corolla length of P. vulgaris also varied along an altitudinal gradient, and this variation strongly correlated with the local pollinator size but did not correlate with altitude itself. Furthermore, we found that the size match between the floral corolla length and bee proboscis length affected female and male plant fitness and the optimal size match (associated with peak fitness) was similar for the female and male fitness. Collectively, these results suggest that pollinator-mediated selection influences spatial variation in the size of P. vulgaris flowers at a fine spatial scale.

Within-plant variation in reproductive investment: consequences for selection on flowering time.

Variation among the leaves, flowers or fruit produced by a plant is often regarded as a nuisance to the experimenter and an impediment to selection. Here, we suggest that within-plant variation can drive selection on other plant-level traits. We examine within-plant variation in floral sex allocation and in fruit set and predict that such variation generates variation in male success among plants, thereby driving selection on flowering time. We tested this prediction in a simulation model estimating selection on flowering time through male fitness when floral sex allocation and/or fruit set vary directionally among flowers on plants. We parameterized the model through a quantitative literature survey of within-plant change in sex allocation. As predicted, within-plant variation in floral sex allocation and in fruit set probability can generate selection on flowering time through male fitness. Declining fruit set from first to last flowers on plants, as occurs in many species, selected for early flowering onset through male fitness. This result was robust to self-incompatibility and to varying returns on male versus female investment. Selection caused by declining fruit set was strong enough to reverse the selection for late flowering that can be caused by intrafloral protandry. Our model provides testable predictions regarding selection on flowering time through male fitness. The model also establishes the intriguing possibility that within-plant variation may influence selection on other traits, regardless of whether that variation is under selection itself.

Effect of three larval diets on larval development and male sexual performance of Anopheles gambiae s.s.

Population replacement/elimination strategies based on mass-release of sterile or otherwise genetically modified (male) mosquitoes are being considered in order to expand the malaria vector control arsenal on the way to eradication. A challenge in this context, is to produce male mosquitoes that will be able to compete and mate with wild females more efficiently than their wild counterparts, i.e. high fitness males. This study explored the effect of three larval food diets developed by the International Atomic Energy Agency on the overall fitness and mating performance of male Anopheles gambiae s.s. mosquitoes (Kisumu strain). Larval development (pupation and emergence rate, development time) was monitored, and adult wing length and energy reserves at emergence (i.e. lipids, sugars, glycogen and proteins) were measured. Male sexual performance was assessed through an insemination test whereby one male and 10 virgin females were maintained together in the same cage in order to record the number of inseminated females per 24h. Our results show that males reared on Diets 2 and 3 performed best during larval development. Males provided with treatment 2.2 had a shorter development time and performed best in insemination tests. However, these males had the lowest overall lifespan, suggesting a trade-off between longevity and sexual performances which needs to be taken into consideration when planning release. The results from this work were discussed in the context of sterile insect techniques or genetic control methods which is today one of the strategy in the overall mosquito control and elimination efforts.

Selection on Polemonium brandegeei (Polemoniaceae) flowers under hummingbird pollination: in opposition, parallel, or independent of selection by hawkmoths?

Particular floral phenotypes are often associated with specific groups of pollinators. However, flowering plants are often visited, and may be effectively pollinated by more than one type of animal. Therefore, a major outstanding question in floral biology asks: what is the nature of selection on floral traits when pollinators are diverse? This study examined how hummingbirds selected on the floral traits of Polemonium brandegeei, a species pollinated by both hummingbirds and hawkmoths. In array populations of P. brandegeei, we measured pollen movement, and female (seeds set) and male (seeds sired) fitness under hummingbird pollination. We then compared the patterns of selection by hummingbirds with our previous study examining selection by hawkmoths. We documented contrasting selection on sex organ positioning through female function, with hummingbirds selecting for stigmas exserted beyond the anthers and hawkmoths selecting for stigmas recessed below the anthers. Furthermore, hummingbirds selected for longer and wider corolla tubes, and hawkmoths selected for narrower corolla tubes. Therefore, contrasting selection by hawkmoths and hummingbirds may account for variation in sex organ arrangements and corolla dimensions in P. brandegeei. We documented how floral traits under selection by multiple pollinators can result in either an intermediate "compromise" between selective pressures (sex organs) or apparent specialization (corolla tube length) to one pollinator.