Context-dependent concordance between physiological divergence and phenotypic selection in sister taxa with contrasting phenology and mating systems.

PREMISE: The study of phenotypic divergence of, and selection on, functional traits in closely related taxa provides the opportunity to detect the role of natural selection in driving diversification. If the strength or direction of selection in field populations differs between taxa in a pattern that is consistent with the phenotypic difference between them, then natural selection reinforces the divergence. Few studies have sought evidence for such concordance for physiological traits. METHODS: Herbarium specimen records were used to detect phenological differences between sister taxa independent of the effects on flowering time of long-term variation in the climate across collection sites. In the field, physiological divergence in photosynthetic rate, transpiration rate, and instantaneous water-use efficiency were recorded during vegetative growth and flowering in 13 field populations of two taxon pairs of Clarkia, each comprising a self-pollinating and a outcrossing taxon. RESULTS: Historically, each selfing taxon flowered earlier than its outcrossing sister taxon, independent of the effects of local long-term climatic conditions. Sister taxa differed in all focal traits, but the degree and (in one case) the direction of divergence depended on life stage. In general, self-pollinating taxa had higher gas exchange rates, consistent with their earlier maturation. In 6 of 18 comparisons, patterns of selection were concordant with the phenotypic divergence (or lack thereof) between sister taxa. CONCLUSIONS: Patterns of selection on physiological traits measured in heterogeneous conditions do not reliably reflect divergence between sister taxa, underscoring the need for replicated studies of the direction of selection within and among taxa.

Maladaptation beyond a geographic range limit driven by antagonistic and mutualistic biotic interactions across an abiotic gradient.

Species' geographic range limits often result from maladaptation to the novel environments beyond the range margin. However, we rarely know which aspects of the n-dimensional environment are driving this maladaptation. Especially of interest is the influence of abiotic versus biotic factors in delimiting species' distributions. We conducted a 2-year reciprocal transplant experiment involving manipulations of the biotic environment to explore how spatiotemporal gradients in precipitation, fatal mammalian herbivory, and pollination affected lifetime fitness within and beyond the range of the California annual plant, Clarkia xantiana ssp. xantiana. In the first, drier year of the experiment, fitness outside the range edge was limited mainly by low precipitation, and there was some evidence for local adaptation within the range. In the second, wetter year, we did not observe abiotic limitations to plant fitness outside the range; instead biotic interactions, especially herbivory, limited fitness outside the range. Together, protection from herbivory and supplementation of pollen resulted in three- to sevenfold increases in lifetime fitness outside the range margin in the abiotically benign year. Overall, our work demonstrates the importance of biotic interactions, particularly as they interact with the abiotic environment, in determining fitness beyond geographic range boundaries.

Floral traits influence the opportunity for selection among male gametophytes: independent and combined effects of style length and petal area.

PREMISE: Strong correlations between traits can obscure their independent effects on components of reproduction. Style length (SL) and petal area (PA) vary within species, for example, but their independent effects on the opportunity for selection among pollen genotypes are poorly understood. Previous work in Clarkia detected a positive effect of SL on pollen receipt, potentially intensifying selection. However, this apparent effect of SL may be influenced by a correlated trait, such as PA. Here, we examine the independent effects of these two traits on pollen receipt and performance. METHODS: We collected petals and styles from wild populations of two insect-pollinated Clarkia taxa and estimated the independent and combined effects of SL and PA on pollen receipt and performance. RESULTS: In both taxa, SL and PA are positively correlated. In C. unguiculata, both traits positively and independently affect pollen receipt, but in C. xantiana ssp. xantiana, the two traits act only in combination to affect pollen receipt. In both taxa, pollen receipt positively affects the numbers of pollen tubes entering and penetrating the style, as well as pollen tube attrition. CONCLUSIONS: The effects of SL and PA on pollen receipt and performance are taxon specific. In C. unguiculata, both traits may be independent targets of selection due to their effects on pollen receipt. In C. xantiana ssp. xantiana, by contrast, the combined (but not independent) effects of SL and PA influence pollen receipt. Ecological differences between these taxa require exploration to understand the mechanisms by which these traits affect pollinator behavior.

Heteranthery in Clarkia: pollen performance of dimorphic anthers contradicts expectations.

PREMISE OF THE STUDY: Wild plant species that require the services of pollen-feeding insects for reliable pollination may evolve features that attract and reward their mutualistic partners. Heterantherous species have been proposed to exhibit a "division of labor" whereby "feeding anthers" (which produce pollen that may be consumed by an insect) are distinguished from "reproductive anthers" (which produce pollen more likely to contribute to reproduction). In some heterantherous species, including Clarkia unguiculata (Onagraceae), these two anther types differ with respect to stamen length, anther size, pollen production, and pollen color. METHODS: The primary goal of this study was to test one component of the "division of labor" hypothesis by comparing the performance of the pollen produced by each type of anther in C. unguiculata. To achieve this goal, under greenhouse conditions, we hand pollinated and assessed pollen performance (using epifluorescence microscopy) within ~228 flowers. KEY RESULTS: The pollen produced by the two anther types differed significantly with respect to both stigma and style penetration. The inner series of anthers produce pollen with higher performance than the outer series of longer, dark red anthers. CONCLUSIONS: These findings contradict previous descriptions of the genus, reporting that the inner diminutive series of anthers in Clarkia produce "abortive and nonfunctional" pollen. We outline the future research required to demonstrate the ecological function of heteranthery in this iconic wildflower group.

Experimental sympatry reveals geographic variation in floral isolation by hawkmoths.

Background and Aims: Under a widely accepted model of pollinator-driven speciation, geographic variation in pollinator assemblage drives floral divergence and automatically causes reproductive isolation. Yet it is unclear whether divergent floral adaptation initially confers strong reproductive isolation, or whether that comes at later stages of speciation and requires other forms of reproductive isolation. This study uses a pair of recently diverged, interfertile and parapatric species in the genus Clarkia to determine whether adaptation to hawkmoths, a novel pollinator functional group, would automatically confer floral isolation upon sympatric contact. Methods: First, genetically based differences in floral traits between C. breweri and C. concinna that would be maintained upon migration are quantified. Then scenarios of experimental sympatry are constructed in which arrays of flowers are exposed to the novel pollinator, the hawkmoth Hyles lineata, and pollinator preference and heterospecific pollen transfer are assessed. Source populations from across the ranges of each species are used to understand how geographic variation in floral traits within species may affect floral isolation. Key Results: Although H. lineata has never been observed visiting C. concinna in the wild, it regularly moves between species in experimental floral arrays. Hawkmoth preference and heterospecific pollen transfer vary both among moths and among geographic source locations of C. concinna. The strength of floral isolation in this system is related to variation in flower size, especially hypanthium tube width, and nectar reward among C. concinna forms. Conclusions: Although C. breweri has adopted a novel hawkmoth pollination system, both ethological and mechanical floral isolation by hawkmoths are incomplete and vary according to the specific phenotype of the C. concinna source population. The results suggest that strong floral isolation is not automatically conferred by a pollinator shift and may require additional evolution of deterrent floral traits and habitat isolation that reduces the immediate spatial co-occurrence of young species.

Ecological sorting and character displacement contribute to the structure of communities of Clarkia species.

Despite long-standing interest in the evolutionary ecology of plants that share pollinators, few studies have explored how these interactions may affect communities during both community assembly (ecological sorting) and through ongoing, in situ evolution (character displacement), and how the effects of these interactions may change with community context. To determine if communities display patterns consistent with ecological sorting, we assessed the frequency of co-occurrence of four species of Clarkia in the southern Sierra foothills (Kern County, CA, USA). To investigate potential character displacement, we measured pollination-related traits on plants grown in a greenhouse common garden from seed collected in communities with one, two or four Clarkia species. Among the four species of Clarkia in this region, the two species that are often found in multi-species communities also co-occur with one another more frequently than expected under a null model. This pattern is consistent with ecological sorting, although further investigation is needed to determine the role of pollinators in shaping community assembly. Patterns of trait variation in a common garden suggest that these two species have diverged in floral traits and converged in flowering time where they co-occur, which is consistent with character displacement. Trait variation across community types also suggests that the process and outcome of character displacement may vary with community context. Because community context appears to affect both the direction and magnitude of character displacement, change in more species-rich communities may not be predictable from patterns of change in simpler communities.

Could seasonally deteriorating environments favour the evolution of autogamous selfing and a drought escape physiology through indirect selection? A test of the time limitation hypothesis using artificial selection in Clarkia.

Background and Aims: The evolution of selfing from outcrossing may be the most common transition in plant reproductive systems and is associated with a variety of ecological circumstances and life history strategies. The most widely discussed explanation for these associations is the reproductive assurance hypothesis - the proposition that selfing is favoured because it increases female fitness when outcross pollen receipt is limited. Here an alternative explanation, the time limitation hypothesis, is addressed, one scenario of which proposes that selfing may evolve as a correlated response to selection for a faster life cycle in seasonally deteriorating environments. Methods: Artificial selection for faster maturation (early flowering) or for low herkogamy was performed on Clarkia unguiculata (Onagraceae), a largely outcrossing species whose closest relative, C. exilis, has evolved higher levels of autogamous selfing. Direct responses to selection and correlated evolutionary changes in these traits were measured under greenhouse conditions. Direct responses to selection on early flowering and correlated evolutionary changes in the node of the first flower, herkogamy, dichogamy, gas exchange rates and water use efficiency (WUE) were measured under field conditions. Key Results: Lines selected for early flowering and for low herkogamy showed consistent, statistically significant responses to direct selection. However, there was little or no evidence of correlated evolutionary changes in flowering date, floral traits, gas exchange rates or WUE. Conclusions: These results suggest that the maturation rate and mating system have evolved independently in Clarkia and that the time limitation hypothesis does not explain the repeated evolution of selfing in this genus, at least through its indirect selection scenario. They also suggest that the life history and physiological components of drought escape are not genetically correlated in Clarkia, and that differences in gas exchange physiology between C. unguiculata and C. exilis have evolved independently of differences in mating system and life history.

Divergence in pollen performance between Clarkia sister species with contrasting mating systems supports predictions of sexual selection.

Animal taxa that differ in the intensity of sperm competition often differ in sperm production or swimming speed, arguably due to sexual selection on postcopulatory male traits affecting siring success. In plants, closely related self- and cross-pollinated taxa similarly differ in the opportunity for sexual selection among male gametophytes after pollination, so traits such as the proportion of pollen on the stigma that rapidly enters the style and mean pollen tube growth rate (PTGR) are predicted to diverge between them. To date, no studies have tested this prediction in multiple plant populations under uniform conditions. We tested for differences in pollen performance in greenhouse-raised populations of two Clarkia sister species: the predominantly outcrossing C. unguiculata and the facultatively self-pollinating C. exilis. Within populations of each taxon, groups of individuals were reciprocally pollinated (n = 1153 pollinations) and their styles examined four hours later. We tested for the effects of species, population, pollen type (self vs. outcross), the number of competing pollen grains, and temperature on pollen performance. Clarkia unguiculata exhibited higher mean PTGR than C. exilis; pollen type had no effect on performance in either taxon. The difference between these species in PTGR is consistent with predictions of sexual selection theory.

Winning in style: Longer styles receive more pollen, but style length does not affect pollen attrition in wild Clarkia populations.

PREMISE OF THE STUDY: One proposed function of long styles is to intensify selection among male gametophytes relative to short styles. If so, given sufficient competition, longer styles will have higher rates of pollen tube attrition (failure to reach the style base) within the style than shorter ones. Alternatively, style length may influence pollen receipt, which itself may affect attrition rates. METHODS: We tested these predictions by collecting senescing styles from wild populations of two insect-pollinated Clarkia species. We examined the number of pollen grains adhering to the stigma, length of styles, and rates of attrition from the stigma surface to the stigma-style junction (SSJ), from the SSJ to the style base, and from the stigma surface to the style base. Multivariate analyses estimated the independent effects of pollen grains per stigma, the number of pollen tubes at the SSJ, and style length on attrition. KEY RESULTS: Style length was generally positively correlated with pollen receipt, and the number of pollen grains per stigma was positively correlated with all three attrition rates. In neither species was any attrition rate affected by style length independent of the number of pollen grains per stigma. CONCLUSIONS: Pollen attrition was mediated by style length, but the function of style length was primarily to increase the number of germinating pollen grains, which affected attrition rates either through stigma clogging or pollen-pollen interactions. Style length may have a direct effect on pollen receipt due to the stigma's position relative to pollinator body parts, but traits correlated with style length may also directly affect pollen receipt.

Effects of range-wide variation in climate and isolation on floral traits and reproductive output of Clarkia pulchella.

PREMISE OF THE STUDY: Plant mating systems and geographic range limits are conceptually linked by shared underlying drivers, including landscape-level heterogeneity in climate and in species' abundance. Studies of how geography and climate interact to affect plant traits that influence mating system and population dynamics can lend insight to ecological and evolutionary processes shaping ranges. Here, we examined how spatiotemporal variation in climate affects reproductive output of a mixed-mating annual, Clarkia pulchella. We also tested the effects of population isolation and climate on mating-system-related floral traits across the range. METHODS: We measured reproductive output and floral traits on herbarium specimens collected across the range of C. pulchella. We extracted climate data associated with specimens and derived a population isolation metric from a species distribution model. We then examined how predictors of reproductive output and floral traits vary among populations of increasing distance from the range center. Finally, we tested whether reproductive output and floral traits vary with increasing distance from the center of the range. KEY RESULTS: Reproductive output decreased as summer precipitation decreased, and low precipitation may contribute to limiting the southern and western range edges of C. pulchella. High spring and summer temperatures are correlated with low herkogamy, but these climatic factors show contrasting spatial patterns in different quadrants of the range. CONCLUSIONS: Limiting factors differ among different parts of the range. Due to the partial decoupling of geography and environment, examining relationships between climate, reproductive output, and mating-system-related floral traits reveals spatial patterns that might be missed when focusing solely on geographic position.

Mating system divergence affects the distribution of sequence diversity within and among populations of recently diverged subspecies of Clarkia xantiana (Onagraceae).

PREMISE OF THE STUDY: The population biology of outcrossing and self-fertilizing taxa is thought to differ because of the advantage that selfers have in colonizing unoccupied sites where mates and pollinators may be limiting (Baker's Law). This reduced tendency for outcrossers to colonize new sites, along with their greater dependence on pollinators to disperse pollen, has the potential to differently influence the genetic diversity and structure of outcrossing and selfing populations. METHODS: We conducted a comparative population genetic study of two sister outcrossing and selfing subspecies of Clarkia xantiana that have very recently diverged. We used DNA sequence variation (>40 kb from eight nuclear loci) from large samples of individuals from 14 populations to assess geographic patterns of genetic diversity and make inferences about the demographic and colonization histories of each subspecies. KEY RESULTS: We show that sequence variation is strongly reduced across all selfing populations. The demographic history of selfing populations exhibits recent colonization bottlenecks, whereas such bottlenecks are rarely observed for the outcrosser. The greater effect of genetic drift in the selfer has resulted in strong population genetic structure, but with no pattern of isolation by distance. By contrast, the stronger effect of gene flow in the outcrosser has resulted in considerably less structure, but a significant pattern of isolation by distance. CONCLUSIONS: Taken together, our results suggest that selfing populations are not at migration-drift equilibrium, are affected by strong episodes of genetic drift during colonization, and experience little or no subsequent gene flow from other populations after those founder events.

Experimental studies of adaptation in Clarkia xantiana. III. Phenotypic selection across a subspecies border.

Sister taxa with distinct phenotypes often occupy contrasting environments in parapatric ranges, yet we generally do not know whether trait divergence reflects spatially varying selection. We conducted a reciprocal transplant experiment to test whether selection favors "native phenotypes" in two subspecies of Clarkia xantiana (Onagraceae), an annual plant in California. For four quantitative traits that differ between subspecies, we estimated phenotypic selection in subspecies' exclusive ranges and their contact zone in two consecutive years. We predicted that in the arid, pollinator-scarce eastern region, selection favors phenotypes of the native subspecies parviflora: small leaves, slow leaf growth, early flowering, and diminutive flowers. In the wetter, pollinator-rich, western range of subspecies xantiana, we expected selection for opposite phenotypes. We investigated pollinator contributions to selection by comparing naturally pollinated and pollen-supplemented individuals. For reproductive traits and for subspecies xantiana, selection generally matched expectations. The contact zone sometimes showed distinctive selection, and in ssp. parviflora selection sometimes favored nonnative phenotypes. Pollinators influenced selection on flowering time but not on flower size. Little temporal variation in selection occurred, possibly because of plastic trait responses across years. Though there were exceptions and some causes of selection remain obscure, phenotypic differentiation between subspecies appears to reflect spatially variable selection.

Seasonal changes in physiological performance in wild Clarkia xantiana populations: Implications for the evolution of a compressed life cycle and self-fertilization.

PREMISE OF THE STUDY: One explanation for the evolution of selfing, the drought escape hypothesis, proposes that self-fertilization may evolve under conditions of intensifying seasonal drought as part of a suite of traits that enable plants to accelerate the completion of their life cycle, thereby escaping late-season drought. Here, we test two fundamental assumptions of this hypothesis in Clarkia xantiana: (1) that a seasonal decline in precipitation causes an increase in drought stress and (2) that this results in changes in physiological performance, reflecting these deteriorating conditions. METHODS: We examined seasonal and interannual variation in abiotic environmental conditions (estimated by ambient temperature, relative humidity, predawn leaf water potentials, and carbon isotope ratios) and physiological traits (photosynthesis, conductance, transpiration, instantaneous water-use efficiency, ascorbate peroxidase and glutathione reductase activities, quantum yield of photosystem II, PSII potential efficiency) in field populations of C. xantiana in 2009 and 2010. KEY RESULTS: In both years, plants experienced intensifying drought across the growing season. Gas exchange rates decreased over the growing season and were lower in 2009 (a relatively dry year) than in 2010, suggesting that the temporal changes from early to late spring were directly linked to the deteriorating environmental conditions. CONCLUSIONS: Seasonal declines in transpiration rate may have increased survival by protecting plants from desiccation. Concomitant declines in photosynthetic rate likely reduced the availability of resources for seed production late in the season. Thus, the physiological patterns observed are consistent with the conditions required for the drought escape hypothesis.

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.

Floral and mating system divergence in secondary sympatry: testing an alternative hypothesis to reinforcement in Clarkia.

BACKGROUND AND AIMS: Reproductive character displacement (RCD) is often an important signature of reinforcement when partially cross-compatible taxa meet in secondary sympatry. In this study, floral evolution is examined during the Holocene range expansion of Clarkia xantiana subsp. parviflora from eastern Pleistocene refugia to a western zone of sympatry with its sister taxon, subsp. xantiana. Floral divergence between the two taxa is greater in sympatry than allopatry. The goal was to test an alternative hypothesis to reinforcement - that floral divergence of sympatric genotypes is simply a by-product of adaptation to pollination environments that differ between the allopatric and sympatric portions of the subspecies' range. METHODS: Floral trait data from two common garden studies were used to examine floral divergence between sympatric and allopatric regions and among phylogeographically defined lineages. In natural populations of C. x. parviflora, the magnitude of pollen limitation and reproductive assurance were quantified across its west-to-east range. Potted sympatric and allopatric genotypes were also reciprocally translocated between geographical regions to distinguish between the effects of floral phenotype versus contrasting pollinator environments on reproductive ecology. KEY RESULTS: Sympatric populations are considerably smaller flowered with reduced herkogamy. Pollen limitation and the reproductive assurance value of selfing are greater in sympatric than in allopatric populations. Most significantly, reciprocal translocation experiments showed these differences in reproductive ecology cannot be attributed to contrasting pollinator environments between the sympatric and allopatric regions, but instead reflect the effects of flower size on pollinator attraction. CONCLUSIONS: Floral evolution occurred during the westward range expansion of parviflora, particularly in the zone of sympatry with xantiana. No evidence was found that strongly reduced flower size in sympatric parviflora (and RCD between parviflora and xantiana) is due to adaptation to limited pollinator availability. Rather, floral divergence appears to have been driven by other factors, such as interactions with congenerics in secondary sympatry.

Novel adaptation to hawkmoth pollinators in Clarkia reduces efficiency, not attraction of diurnal visitors.

BACKGROUND AND AIMS: Plant populations experiencing divergent pollination environments may be under selection to modify floral traits in ways that increase both attractiveness to and efficiency of novel pollinators. These changes may come at the cost of reducing overall effectiveness of other pollinators. The goal of this study was to examine differences in attractiveness and efficiency between Clarkia concinna and C. breweri, sister species of annual plants with parapatric distributions. METHODS: An assessment was made as to whether observed differences in visitors between natural populations are driven by differences in floral traits or differences in the local pollination environment. Differences in floral attractiveness were quantified by setting out arrays of both species in the geographical range of each species and exposing both species to nocturnal hawkmoths (Hyles lineata) in flight cages. Differences in visitor efficiency were estimated by measuring stigma-visitor contact frequency and pollen loads for diurnal visitors, and pollen deposition on stigmas for hawkmoths. KEY RESULTS: The composition of visitors to arrayed plants was similar between plant species at any particular site, but highly divergent among sites, and reflected differences in visitors to natural populations. Diurnal insects visited both species, but were more common at C. concinna populations. Hummingbirds and hawkmoths were only observed visiting within the range of C. breweri. Despite attracting similar species when artificially presented together, C. concinna and C. breweri showed large differences in pollinator efficiency. All visitors except hawkmoths pollinated C. concinna more efficiently. CONCLUSIONS: Differences in the available pollinator community may play a larger role than differences in floral traits in determining visitors to natural populations of C. concinna and C. breweri. However, floral traits mediate differences in pollinator efficiency. Increased effectiveness of the novel hawkmoth pollinator on C. breweri comes at relatively little cost in attractiveness to other visitors, but at large cost in their efficiency as pollinators.

Resource reallocation does not influence estimates of pollen limitation or reproductive assurance in Clarkia xantiana subsp. parviflora (Onagraceae).

PREMISE OF STUDY: Studies of pollen limitation and the reproductive assurance value of selfing are important for examining the process of floral and mating system evolution in flowering plants. Recent meta-analyses have shown that common methods for measuring pollen limitation may often lead to biased estimates. Specifically, experiments involving single- or few-flower manipulations per plant tend to overestimate pollen limitation compared to those involving manipulations on most or all flowers per plant. Little previous work has explicitly tested for reallocation within individual systems using alternative methods and response variables. • METHODS: We performed single-flower and whole-plant pollen supplementation and emasculation of flowers of Clarkia xantiana subsp. parviflora to estimate pollen limitation (PL) and reproductive assurance (RA). We compared levels of PL and RA using the following response variables: fruit set, seeds/flower, and seeds/plant. We also assessed the germination and viability of seeds to evaluate potential variation in pollen quality among treatments. • KEY RESULTS: Autonomous selfing in Clarkia xantiana subsp. parviflora eliminates pollen limitation and provides reproductive assurance. Estimates from single-flower manipulations were not biased, closely resembling those from whole-plant manipulations. All three response variables followed the same pattern, but treatments were only significantly different for seeds/flower. Pollen quality, as indicated by seed viability, did not differ among treatments. • CONCLUSIONS: Partial plant manipulations provided reliable estimates of pollen limitation and reproductive assurance. These estimates were also unaffected by accounting for pollen quality. Although whole plant manipulations are desirable, this experiment demonstrates that in some systems partial plant manipulations can be used in studies where whole-plant manipulations are not feasible.

Indirect competition for pollinators is weak compared to direct resource competition: pollination and performance in the face of an invader.

Invasive plants have the potential to reduce native plant abundance through both direct and indirect interactions. Direct interactions, such as competition for soil resources, and indirect interactions, such as competition for shared pollinators, have been shown to influence native plant performance; however, we know much less about how these interactions influence native plant abundance in the field. While direct competitive interactions are often assumed to drive declines in native abundance, an evaluation of their influence relative to indirect mechanisms is needed to more fully understand invasive plant impacts. We quantified the direct effects of resource competition by the invasive perennial forb, Euphorbia esula (Euphorbiaceae), on the recruitment, subsequent performance, and ultimate adult abundance of the native annual, Clarkia pulchella (Onagraceae). We contrast these direct effects with those that indirectly resulted from competition for shared pollinators. Although E. esula dramatically reduced pollinator visitation to C. pulchella, plants were only weakly pollen-limited. Pollen supplementation increased the number of seeds per fruit from 41.28 to 46.38. Seed addition experiments revealed that the impacts of ameliorating pollen limitation only increased potential recruitment by 12.3 %. In contrast, seed addition experiments that ameliorated direct competition with E. esula resulted in an increase in potential future recruitment of 574 %. Our results show that, while the indirect effects of competition for pollinators can influence plant abundance, its effects are dwarfed by the magnitude of direct effects of competition for resources.

Reduced pollinator service and elevated pollen limitation at the geographic range limit of an annual plant.

Mutualisms are well known to influence individual fitness and the population dynamics of partner species, but little is known about whether they influence species distributions and the location of geographic range limits. Here, we examine the contribution of plant-pollinator interactions to the geographic range limit of the California endemic plant Clarkia xantiana ssp. xantiana. We show that pollinator availability declined from the center to the margin of the geographic range consistently across four years of study. This decline in pollinator availability was caused to a greater extent by variation in the abundance of generalist rather than specialist bee pollinators. Climate data suggest that patterns of precipitation in the current and previous year drove variation in bee abundance because of its effects on cues for bee emergence in the current year and the abundance of floral resources in the previous year. Experimental floral manipulations showed that marginal populations had greater outcross pollen limitation of reproduction, in parallel with the decline in pollinator abundance. Although plants are self-compatible, we found no evidence that autonomous selfing contributes to reproduction, and thus no evidence that it alleviates outcross pollen limitation in marginal populations. Furthermore, we found no association between the distance to the range edge and selfing rate, as estimated from sequence and microsatellite variation, indicating that the mating system has not evolved in response to the pollination environment at the range periphery. Overall, our results suggest that dependence on pollinators for reproduction may be an important constraint limiting range expansion in this system.

Tempo and mode of mating system evolution between incipient Clarkia species.

Mating systems are among the most labile characteristics of flowering plants, with transitions frequently occurring among populations or in association with speciation. The frequency of mating system shifts has made it difficult to reconstruct historical evolutionary dynamics unless transitions have been very recent. Here, we examine molecular and phenotypic variation to determine the polarity, timescale, and causes of a transition between outcrossing and self-fertilization in sister subspecies of Clarkia xantiana. Phylogenetic analyses and coalescent-based estimates of the time to most recent common ancestor indicated that outcrossing is ancestral to selfing and that there has been a single origin of selfing. Estimates of divergence time between outcrossing and selfing subspecies were 10,000 (95% CI [credible interval]: 3169-66,889) and 65,000 years ago (95% CI: 33,035-151,448) based on two different methods, suggesting a recent and rapid evolutionary transition. Population genetic data indicated that the transition to selfing was associated with a 80% reduction in molecular diversity, which is much greater than the 50% reduction expected under a shift from obligate outcrossing to obligate self-fertilization alone. Our data also suggest that this severe loss of diversity was caused by colonization bottlenecks. Together with previous studies, evidence for reproductive assurance in C. xantiana now connects variation in plant-pollinator interactions in the field to phenotypic and molecular evolution.