Abstract Ongoing climatic changes have altered growing conditions for plants by limiting water availability and inducing unprecedented temperature increases, eliciting plant functional responses that compromise floral trait expression, reduce pollen production and promote early self‐pollination. In a controlled greenhouse study, plants of Collinsia heterophylla, an annual mixed‐mated hermaphrodite, were grown under temperature and water stresses. Floral trait responses and related effects on plant reproductive success were recorded. Plants grown under temperature and water stresses were shorter and had fewer leaves at flowering than control plants. Temperature‐stressed plants flowered earlier and had smaller flowers that produced fewer per capita pollen grains than control and water‐stressed plants. While lifetime flower production in plants experiencing temperature stress alone or water stress alone was similar to control plants, those receiving combined temperature and water stresses had significantly lower lifetime flower production. Abiotic stress did not affect investment in female traits such as ovule number per flower, but impacted male traits seen as fewer pollen grains per flower. In plants experiencing abiotic stress, an increase in autonomous self‐pollination was facilitated through a compromise in herkogamy, a sexual interference mechanism that promotes cross‐pollination. Spatial separation between stigma and anther, a measure of herkogamy was zero or negative in stressed plants. Thus, abiotic stress responses in plants did not compromise plant reproductive success as autonomous selfing was augmented. In nature, such responses will likely reduce available nutrition for pollinators through decreased flower and pollen production.

We document for the first time the larval food plants of Anoncia mosa Hodges (Cosmopterigidae) mining the leaves of Collinsia heterophylla and C. tinctoria (Lamiales: Plantaginaceae) in Madera County, California. We describe and illustrate the mine, larval feeding habits, last instar larva and pupa of Anoncia mosa, then compare the immature stages to other Cosmopterigidae to help identify this species and call attention to some overlooked morphological characters. These include the larval P seta, larval cuticular texture and the genital opening of the pupa in a pit. We partially describe the previously unknown pupa of Euclemensia. We also use characters of the immature stages to briefly discuss the systematic placement of three taxa. The family status of the Chrysopeleiidae and the inclusion of Triclonella in the Cosmopterigidae are supported by the pupa but not the larva. Conversely, the systematic placement of Metharmostis in Cosmopterigidae is better supported by the larva than the pupa. Our study is an example of the need to save larval and pupal exuviae for morphological descriptions.

Even though floral scent is of major importance for pollinator attraction, it is less investigated than other floral traits. Previous studies suggest the importance of joint exploration of olfactory and visual floral cues to understand plant–pollinator interactions. We investigated flower scents inCollinsia heterophylla, a bee‐pollinated, annual herb a with mixed‐mating system combining self‐ and outcross‐pollination. InCollinsia, floral size and development variation is related to mating system, ranging from large‐flowered mixed‐mating species to small‐flowered self‐pollinated species. However, to our knowledge, flower scent has not been described in any species in the genus. We also studied whether flower‐emitted volatiles were coupled to presence versus absence of a coloured band on the upper lip within a population inC. heterophylla, and if these colour morphs affected pollinator visitation. We performed headspace collections of volatiles in the greenhouse from potted flowering plants, and compared these to controls in the bud stage. Flower‐specific volatiles were highly dominated by terpenoid compounds typical of bee‐pollinated plants, such as β‐myrcene, (Z)‐ and (E)‐ocimene and sesquiterpenes (E)‐α‐bergamotene and β‐sesquiphellandrene. The aliphatic ester methyl hexanoate was also prominent, together with additional esters, whereas methyl cinnamate constituted the only aromatic compound. Floral colour morphs showed no qualitative difference in volatiles, but the coloured morph produced significantly higher quantities for seven of the 26 individual flower compounds. A field experiment performed within a natural population, using behavioural observations and florescent dyes dusted on the flowers, could not detect any differences in pollinator visitation between colour morphs. We conclude thatC. heterophyllaflowers emit volatile compounds commonly associated with attraction of their most important pollinators. It would be highly interesting to explore the function of floral scent for pollinator attraction and relate floral scent to mating system variation acrossCollinsiafor a better understanding of pollinator influence on floral evolution.

Mate choice in plants is poorly understood, in particular its indirect genetic benefits, but also the direct benefits of avoiding harmful matings. In the herb Collinsia heterophylla, delayed stigma receptivity has been suggested to enhance pollen competition, potentially functioning as a female mate choice trait. Previous studies show that this trait can mitigate the cost of early fertilization caused by pollen, thus providing a direct benefit. We performed two‐donor pollinations during successive floral stages to assess how this stigma receptivity trait and two pollen traits known to affect siring success influence indirect benefits in terms of offspring performance. We also investigated differential resource allocation by studying the influence of sibling performance in the same capsule. Offspring performance in terms of flower number was mainly affected by parental identities and differential resource allocation. Offspring seed production showed some influence of resource allocation, but was also affected by pollen donor identity and varied positively with late stigma receptivity. However, the effect of late stigma receptivity on offspring seed production was weakened in matings with pollen that advanced stigma receptivity. In conclusion, delayed stigma receptivity may be selected through both direct and indirect fitness effects in C. heterophylla, where pollen‐based delay on stigma receptivity might act as a cue for mate choice. However, selection may also be counteracted by antagonistic selection on pollen to advance stigma receptivity. Our results highlight the challenges of studying indirect genetic benefits and other factors that influence mate choice in plants.

Although sexual selection and sexual conflict are important evolutionary forces in animals, their significance in plants is uncertain. In hermaphroditic organisms, such as many plants, sexual conflict may occur both between mating partners (interlocus conflict) and between male and female sex roles within an individual (intralocus conflict). We performed experimental evolution, involving lines that were crossed with either one or two pollen donors (monogamous or polyandrous lines), in the hermaphroditic plant (Collinsia heterophylla) where early fertilizations are associated with female fitness costs (reduced seed set). Artificial polyandry for four generations resulted in enhanced pollen performance and increased female fitness costs compared to the monogamous and source (starting material) lines. Female fitness was also reduced in the monogamous line, indicating a possible trade-off between sex roles, resulting from early pollination. We performed a second experiment to investigate a potential harming effect of pollen performance on seed set. We found that high siring success of early arriving pollen competing with later-arriving pollen was associated with high female fitness costs, consistent with an interlocus sexual conflict. Our study provides evidence for the importance of sexual selection in shaping evolution of plant reproductive strategies, but also pinpoints the complexity of sexual conflict in hermaphroditic species.

Relatively few studies have investigated how geography, environmental factors, and genetics affect floral trait variation. We used mixed-mating Collinsia heterophylla Buist to explore variation in a suite of floral traits related to mating system in populations representing four geographic regions of California, USA, and relate this variation to geography, climatic factors, and local site characteristics. We evaluated the environmental vs. genetic trait variability in the greenhouse. Stage of anther–stigma contact correlated positively with temperature, stage of stigma receptivity was negatively associated with vegetation cover, and flower size differed among populations without any clear relation to environmental factors. Greenhouse data indicated heritability for stage of anther–stigma contact, flower size, and time to flowering, and positive correlations between field and greenhouse for stage of stigma receptivity and flower size; however, stage of anther–stigma contact showed a high degree of environmental influence. Stage of anther–stigma contact covaried positively with stage of stigma receptivity and flower size across maternal families, indicating genetic correlations between traits. In conclusion, phenotypic floral variation within mixed-mating C. heterophylla is mostly determined by a genetic component. Geography, environment, and genetics affect traits differently, suggesting that ecological and evolutionary processes contribute to shaping variability in mating system-related traits.

While environmental factors strongly influence plant growth and reproduction, less is known about environmental effects on sexual selection and sexual conflict. In this study on mixed‐matingCollinsia heterophyllawe investigated whether soil resource environment affected traits associated with sexual conflict. InC. heterophyllaa sexual conflict over timing of stigma receptivity occurs. Early stigma receptivity benefits pollen parents by securing paternity while late stigma receptivity benefits female fitness in terms of increased seed production. We performed hand‐pollinations combining recipients and donors grown either in high or low resource environments and asked whether these treatments influenced sexual conflict traits – recipient‐ and donor‐based influence on timing of stigma receptivity – and conflict costs related to reduced early seed production. We also asked whether resource environment affected eight traits related to general fitness and mating system. Sexual conflict‐associated traits – timing of stigma receptivity and seed production – were generally unaffected by resource environment. While no universal effect of resources was detected, we did observe donor‐specific responses to environment, suggesting that environment can nonetheless contribute to variation in timing of stigma receptivity. Recipients grown under low resources showed pronounced differences among donors for number of seeds per capsule, indicating that recipients favour some donors over others under resource‐low conditions. Moreover, high resources increased number of flowers but reduced pollen germination rate, while other traits were unaffected, indicating variation in the response to resource environment for fitness‐ and mating system‐traits. Our results suggest that even though soil resource environment had a low impact on the sexual conflict traits and related costs inC. heterophylla, it generated variability in pollen donor‐influence on this trait and in recipient sorting among donors. Thus, it is possible that both sexual conflict and sexual selection is affected by environmental factors not only in animals but also in plants.

Presence of a root neighbor can induce changes in root allocation and pollen traits, but only a limited number of studies have investigated such effects on pollen. To learn more about effects of root contact on pollen competitive ability, we studied plants of the hermaphroditic winter-annual Collinsia heterophylla, native to California. We cultivated plants in two-pot treatments with roots kept either separate or intermingled with the same amount of resources. Pollen-tube growth rate, as an indication of pollen competitive ability, was affected by root treatment but the response varied among competing plant families. The response to root-treatment was not an effect of differential resource uptake of the two competitors. Root biomass was significantly higher when roots were intermingled compared to separate. This finding adds to the number of species with a strategic root response in the presence of competitors, but could also be a consequence of a larger rooting volume. Allocation to pollen performance versus roots in the presence of a competitor was lower in small plants and higher in large plants, potentially implying high costs of producing competitive pollen. We conclude that our study demonstrated that pollen tube growth rate is highly sensitive to the root environment in C. heterophylla.

Sexual conflict and its evolutionary consequences are understudied in plants, but the theory of sexual conflict may help explain how selection generates and maintains variability. Here, we investigated selection on pollen and pistil traits when pollen arrives sequentially to partially receptive pistils in relation to pollen competition and a sexual conflict over timing of stigma receptivity in the mixed-mating annual Collinsia heterophylla (Plantaginaceae). In this species the conflict is generated by early fertilizing pollen that reduces seed production, which is counteracted by delaying receptivity in the recipient. We performed sequential two-donor pollinations at early floral developmental stages involving two pollen deposition schedules (with or without a time lag of 1 day), using only outcross or self and outcross pollen. We investigated pollen and pistil traits in relation to siring success (male fitness) and seed production (female fitness). In contrast to previous findings in receptive pistils in C. heterophylla and in other species, last arriving pollen donors showed highest siring success in partially receptive pistils. The last male advantage was weaker when self pollen was the first arriving donor. Two measures of germination rate (early and late) and pollen tube growth rate of first arriving donors were important for siring success in crosses with a time lag, while only late germination rate had an effect in contemporary crosses. Curiously, late stigma receptivity was negatively related to seed production in our contemporary crosses, which was opposite to expectation. Our results in combination with previous studies suggest that pollen and pistil traits in C. heterophylla are differentially advantageous depending on stage of floral development and varying pollen deposition schedules. Variation in success of these traits over floral development time may result from sexually antagonistic selection.

Although much attention has focused on the diversity of plant mating systems, only a few studies have considered the joint effects of mating system and sexual conflict in plant evolution. In mixed-mating Collinsia heterophylla, a sexual conflict over timing of stigma receptivity is proposed: pollen with a capacity to induce early onset of stigma receptivity secures paternity for early-arriving pollen (at the expense of reduced maternal seed set), whereas late onset of stigma receptivity mitigates the negative effects of early-arriving pollen. Here we investigated whether selection on pollen and pistil traits involved in sexual conflict is affected by the presence of both outcross- and self-pollen (mixed mating) during pollen competition. We conducted two-donor crosses at different floral developmental stages to explore male fitness (siring ability) and female fitness (seed set) in relation to male and female identity, pollen and pistil traits, and type of competitor pollen (outcross vs. self). Late-fertilizing pollen rather than rapidly growing pollen tubes was most successful in terms of siring success, especially in competition with self-pollen after pollination at early floral stages. Late stigma receptivity increased seed set after early-stage pollinations, in agreement with selection against antagonistic pollen. Selection on pollen and pistil traits in C. heterophylla is affected by both sexual conflict and mixed mating, suggesting the importance of jointly considering these factors in plant evolution.

Sexual conflicts and their evolutionary outcomes may be influenced by population-specific features such as mating system and ecological context; however, very few studies have investigated the link between sexual conflict and mating system. The self-compatible, mixed-mating hermaphrodite Collinsia heterophylla (Plantaginaceae) is thought to exhibit a sexual conflict over timing of stigma receptivity. This conflict involves (i) delayed stigma receptivity, which intensifies pollen competition, and (ii) early fertilization forced by pollen, which reduces seed set. We investigated the potential for the conflict to occur under field conditions and performed glasshouse crosses within eight populations to assess its consistency across populations. Flowers were visited, and produced seeds after pollination, at all developmental stages, suggesting that the conflict can be of significance under natural conditions. In the glasshouse, early pollination imposed costs in all populations. Overall, the timing of first seed set was most strongly affected by the maternal parent, denoting stronger female than male ability to influence the onset of stigma receptivity. Crosses also revealed a negative relationship between donor- and recipient-related onset of receptivity within individuals, a novel result hinting at trade-offs in sex allocation or a history of antagonistic selection. Neither timing of stigma receptivity, timing of first seed set, nor pollen competitive ability covaried with population outcrossing rate. In conclusion, these results indicate that sexually antagonistic selection may be present in varying degrees in different populations of C. heterophylla, but this variation does not appear to be directly related to mating system variation.

A plant investing in reproduction partitions resources between flowering and seed production. Under resource limitation, altered allocations may result in floral trait variations, leading to compromised fecundity. Floral longevity and timing of selfing are often the traits most likely to be affected. The duration of corolla retention determines whether fecundity results from outcrossing or by delayed selfing-mediated reproductive assurance. In this study, the role of pollination schedules and soil water availability on floral longevity and seed production is tested in Collinsia heterophylla (Plantaginaceae). Using three different watering regimes and pollination schedules, effects on floral longevity and seed production were studied in this protandrous, flowering annual. The results reveal that soil water status and pollination together influence floral longevity with low soil water and hand-pollinations early in the floral lifespan reducing longevity. However, early pollinations under excess water did not extend longevity, implying that resource surplus does not lengthen the outcrossing period. The results also indicate that pollen receipt, a reliable cue for fecundity, accelerates flower drop. Early corolla abscission under drought stress could potentially exacerbate sexual conflict in this protandrous, hermaphroditic species by ensuring self-pollen paternity and enabling male control of floral longevity. While pollination schedules did not affect fecundity, water stress reduced per-capita seed numbers. Unmanipulated flowers underwent delayed autonomous selfing, producing very few seeds, suggesting that inbreeding depression may limit benefits of selfing. In plants where herkogamy and dichogamy facilitate outcrossing, floral longevity determines reproductive success and mating system. Reduction in longevity under drought suggests a strong environmental effect that could potentially alter the preferred breeding mode in this mixed-mated species. Extrapolating the findings to unpredictable global drought cycles, it is suggested that in addition to reducing yield, water stress may influence the evolutionary trajectory of plant mating system.

Estimating total pollen number and viability is labor intensive and time consuming. Nevertheless the information is crucial for a range of plant biologists from plant breeders to evolutionary biologists. Viability is determined by dye staining and counting colored (viable) and transparent (inviable) grains under a compound microscope. Existing protocols have been standardized to speed up total pollen counts but their success in determining viability is rather limited because they do not incorporate staining techniques. Some of the published protocols that determine viability do so based on statistical methods but focus on just one parameter such as pollen diameter requiring manual standardization to generate two distinct size distributions for viable and inviable pollen. We demonstrate a digital image processing protocol that can count viable and inviable pollen by distinguishing colored and transparent objects in the image space, saving valuable labor and time. Using pollen grains from two plants, Collinsia heterophylla and Brassica napus, we show that differences between viable and inviable pollen are best described by a complex measure, the grain shape. By measuring several parameters such as area, length, width, circularity and elongation, while retaining all the advantages of traditional staining process, our procedure increases the accuracy of viability estimates. The only drawback of our protocol is that it uses the NIS elements a software specific to Nikon instruments.

Costs related to pollen competition have rarely been considered, but are expected in the case of sexual conflict where male and female sexual functions have opposing evolutionary interests. In Collinsia heterophylla, delayed stigma receptivity is beneficial as it enhances pollen competition. A sexual conflict over timing of stigma receptivity has been proposed in this species as early pollination, following one-time pollinations, is advantageous to pollen donors at a cost of reduced maternal seed set (measured as seed number). In this study, we explored whether the maternal cost was still present following an additional pollination. We hypothesized that the cost is caused either by harm related to early pollen presence or by factors unrelated to harm. We performed pollinations at different stages of floral development, either one or two pollinations (24-h time lag), and varied the size of the first pollen load in the latter category. Early pollination reduced seed biomass also after two-time pollinations, suggesting a persistent maternal cost of early pollen presence. Further, pollen load size modified seed production, possibly indicating that dose-dependent harm influences the maternal cost of early fertilization. Our results strongly suggest negative effects of pollen competition on maternal fitness following early pollination, which is consistent with the existence of a sexual conflict over timing of stigma receptivity. In conclusion, we propose that much could be gained if more plant studies considered the potential for fitness costs in relation to sexual conflict, particularly those investigating pollen-pistil interactions.

Heritable genetic variation is crucial for selection to operate, yet there is a paucity of studies quantifying such variation in interactive male/female sexual traits, especially those of plants. Previous work on the annual plant Collinsia heterophylla, a mixed-mating species, suggests that delayed stigma receptivity is involved in a sexual conflict: pollen from certain donors fertilize ovules earlier than others at the expense of reduced maternal seed set and lower levels of pollen competition. Parent-offspring regressions and sib analyses were performed to test for heritable genetic variation and co-variation in male and female interactive traits related to the sexual conflict. SOME heritable variation and evolvability were found for the female trait (delayed stigma receptivity in presence of pollen), but no evidence was found for genetic variation in the male trait (ability to fertilize ovules early). The results further indicated a marginally significant correlation between a male's ability to fertilize early and early stigma receptivity in offspring. However, despite potential indirect selection of these traits, antagonistic co-evolution may not occur given the lack of heritability of the male trait. To our knowledge, this is the first study of a plant or any hermaphrodite that examines patterns of genetic correlation between two interactive sexual traits, and also the first to assess heritabilities of plant traits putatively involved in a sexual conflict. It is concluded that the ability to delay fertilization in presence of pollen can respond to selection, while the pollen trait has lower evolutionary potential.

Even though pollen deposition schedules may have profound effects on the evolutionary outcome of pollen competition, few studies have investigated such effects in relation to pistil traits such as delayed stigma receptivity that enhance pollen competition. In Collinsia heterophylla, a largely outcrossing species with delayed stigma receptivity, we performed a series of controlled crosses involving several donors to understand how timing of pollen deposition influences siring ability, paternal diversity, and offspring fitness. Pollen was applied to fully receptive stigmas either as mixtures or consecutively with or without a time lag to mimic cases with early or delayed stigma receptivity. We used a genetic marker to assess offspring paternity. As expected, siring ability was affected by application order in crosses without a time lag, providing a first-donor advantage for pollen arriving on unreceptive stigmas. However, because pollen donor identity influenced siring ability, delaying stigma receptivity may still favor pollen of high competitive ability. In crosses on fully receptive pistils with a time lag of 24 h, a surprisingly high proportion of seeds (12-47%) were sired by pollen applied last. A novel finding was that pollen applied only once (as a mixture), mimicking delayed stigma receptivity, led to higher paternal diversity within progeny families, which was associated with increased seed production. Our results suggest fitness advantages of enhancing pollen competition by delaying stigma receptivity in C. heterophylla, particularly in relation to increased paternal diversity.

Understanding the genetics of a polymorphic trait is important to predict its likely evolution. In Collinsia heterophylla, the upper petal lip colour can be either be white or white with a purple band, while the lower petal lip colour is invariably purple. Because the corolla is only partly polymorphic, the polymorphism can not have evolved due to a mutation where a pigment was lost in the entire plant, which is common in other polymorphic species. In a previous study, high frequency of the purple band was found in populations with darker flowers, indicating possible selection for this trait. In this study, I determined inheritance of the colour polymorphism using two populations (one with only white morph and other with both morphs). I conducted experimental crosses within and between floral morphs to determine whether patterns of segregation in offspring conform to single-gene predictions. Data from F1, F2, F3 and backcross progeny are consistent with a genetic model of one major locus with presence of the band being completely dominant, as indicated in earlier studies using distantly related populations. A novel finding in this study was that the two morphs did not show a difference in seed germination frequency or seedling survival. This trait can thus be valuable as a genetic marker. Even though more thorough ecological data are needed to understand the potential selection pressures on upper petal lip colour in C. heterophylla, its simple inheritance may indicate the possibility of fast evolutionary response to selective forces acting on this trait.

BackgroundSexual conflict theory predicts sexually antagonistic coevolution of reproductive traits driven by conflicting evolutionary interests of two reproducing individuals. Most studies of the evolutionary consequences of sexual conflicts have, however, to date collectively investigated only a few species. In this study we used the annual herb Collinsia heterophylla to experimentally test the existence and evolutionary consequences of a potential sexual conflict over onset of stigma receptivity.Methodology/Principal FindingsWe conducted crosses within and between four greenhouse-grown populations originating from two regions. Our experimental setup allowed us to investigate male-female interactions at three levels of geographic distances between interacting individuals. Both recipient and pollen donor identity affected onset of stigma receptivity within populations, confirming previous results that some pollen donors can induce stigma receptivity. We also found that donors were generally better at inducing stigma receptivity following pollen deposition on stigmas of recipients from another population than their own, especially within a region. On the other hand, we found that donors did worse at inducing stigma receptivity in crosses between regions. Interestingly, recipient costs in terms of lowered seed number after early fertilisation followed the same pattern: the cost was apparent only if the pollen donor belonged to the same region as the recipient.Conclusion/SignificanceOur results indicate that recipients are released from the cost of interacting with local pollen donors when crossed with donors from a more distant location, a pattern consistent with a history of sexually antagonistic coevolution within populations. Accordingly, sexual conflicts may have important evolutionary consequences also in plants.

Theory predicts that, during pollen competition, selection may favor a pollen trait that increases donor competitive ability at the expense of the female reproductive function. One such pollen trait could be manipulation of the onset of stigma receptivity. We evaluated the potential occurrence of this kind of sexual conflict by testing female control of the timing of stigma receptivity in the self-compatible annual Collinsia heterophylla. By performing one-donor crosses in the greenhouse, we found that differences in both recipients and pollen donors influenced when stigmas became receptive. Because we did not detect an interaction effect, our result suggests that some donors were consistently better than others at germinating pollen and siring seeds earlier. Unexpectedly, self-pollen was able to fertilize seeds earlier during floral development compared with outcross pollen. These results suggest that female control on timing of stigma receptivity is not complete in this species. In addition, fertilizations that occurred early during floral development resulted in fewer seeds than later fertilizations, possibly indicating a cost of lost control over the onset of receptivity. The ability of pollen donors to influence the timing of stigma receptivity might reflect a conflict between the sexual functions in C. heterophylla.

To increase our knowledge about mating-system evolution, we need to understand the relationship between specific floral traits and mating system. Species of Collinsia (Plantaginaceae) vary extensively in mating system; this variation is associated with variation in floral morphology and development and with the timing of self-pollination. Counterintuitively, large-flowered, more outcrossing species tend to have delayed stigma receptivity, reducing the amount of time that the stigma is receptive to cross-pollination before autonomous self-pollination. To understand how the timing of stigma receptivity is related to mating-system evolution, we studied in detail the timing of both stigma receptivity and self-pollination (anther-stigma contact) in two greenhouse-grown populations of large-flowered Collinsia heterophylla. Crosses on emasculated flowers at different stages of floral development always produced seeds, suggesting that cross-fertilization can be effected by pollen arriving prior to physiological receptivity. Phenotypic and genetic variation within populations in the timing of stigma receptivity and anther-stigma contact was substantial, although slightly less for the contact. Despite strong interspecific and interpopulation correlations, we did not find an among-genet phenotypic correlation between the traits. This indicates that each trait may respond independently to selection, and the trait association may be the result of correlational selection.