Transgenerational Genetic Effects Help Explain Latitudinal Variation in Seed Mass and Germination Timing in Plantago lanceolata.

We know little about the underlying genetic control of phenotypic patterns of seed traits across large-scale geographic and environmental gradients. Such knowledge is important for understanding the evolution of populations within species and for improving species conservation. Therefore, to test for genetic variation in Plantago lanceolata, we made reciprocal crosses between northern and southern genotypes that span the species' range in Europe. The results provide evidence of transgenerational genetic effects on seed mass and germination timing. Northern mothers produced larger seeds with delayed germination, in contrast to southern mothers, which produced smaller seeds with accelerated germination. A maternal latitude affected both the seed coat, solely maternal tissue, and embryo/endosperm tissues. Thus, latitudinal variation in seed size and germination timing can be explained, in part, by the direct influence of maternal genotype, independent of zygotic genes that parents pass directly to the embryo and endosperm. Data suggest that researchers exploring the existence and evolution of large-scale geographic variation within species test for transgenerational genetic effects. In addition, data suggest that transgenerational control of seed traits should be considered when developing procedures designed to facilitate species conservation and restoration.

Multiple modes of selection can influence the role of phenotypic plasticity in species' invasions: Evidence from a manipulative field experiment.

In exploring the roles of phenotypic plasticity in the establishment and early evolution of invading species, little empirical attention has been given to the importance of correlational selection acting upon suites of functionally related plastic traits in nature. We illustrate how this lack of attention has limited our ability to evaluate plasticity's role during invasion and also, the costs and benefits of plasticity. We addressed these issues by transplanting clones of European-derived Plantago lanceolata L. genotypes into two temporally variable habitats in the species' introduced range in North America. Phenotypic selection analyses were performed for each habitat to estimate linear, quadratic, and correlational selection on phenotypic trait values and plasticities in the reproductive traits: flowering onset and spike and scape lengths. Also, we measured pairwise genetic correlations for our "colonists." Results showed that (a) correlational selection acted on trait plasticity after transplantation, (b) selection favored certain combinations of genetically correlated and uncorrelated trait values and plasticities, and (c) using signed, instead of absolute, values of plasticity in analyses facilitated the detection of correlational selection on trait value-plasticity combinations and their adaptive value. Based on our results, we urge future studies on species invasions to (a) measure correlational selection and (b) retain signed values of plasticity in order to better discriminate between adaptive and maladaptive plasticity.

Bacterial efflux inhibitors are widely distributed in land plants.

ETHNOPHARMACOLOGICAL RELEVANCE: Secondary metabolites play a critical role in plant defense against disease and are of great importance to ethnomedicine. Bacterial efflux pumps are active transport proteins that bacterial cells use to protect themselves against multiple toxic compounds, including many antimicrobials. Efflux pump inhibitors from plants can block these efflux pumps, increasing the potency of antimicrobial compounds. This study demonstrates that efflux pump inhibition against the Gram-positive bacterial pathogen Staphylococcus aureus is widespread in extracts prepared from individual species throughout the land plant lineage. It therefore suggests a general mechanism by which plants used by indigenous species may be effective as a topical treatment for some bacterial infections. AIM OF THE STUDY: The goal of this research was to evaluate the distribution of efflux pump inhibitors in nine plant extracts with an ethnobotanical use suggestive of an antimicrobial function for the presence of efflux pump inhibitory activity against Staphylococcus aureus. MATERIALS AND METHODS: Plants were collected, dried, extracted, and vouchers submitted to the Herbarium of the University of North Carolina Chapel Hill (NCU). The extracts were analyzed by quantitative mass spectrometry (UPLC-MS) to determine the presence and concentration of flavonoids with known efflux pump inhibitory activity. A mass spectrometry-based assay was employed to measure efflux pump inhibition for all extracts against Staphylococcus aureus. The assay relies on UPLC-MS measurement of changes in ethidium concentration in the spent culture broth when extracts are incubated with bacteria. RESULTS: Eight of these nine plant extracts inhibited toxic compound efflux at concentrations below the MIC (minimum inhibitory concentration) value for the same extract. The most active extracts were those prepared from Osmunda claytoniana L. and Pinus strobes L., which both demonstrated IC50 values for efflux inhibition of 19 ppm. CONCLUSIONS: Our findings indicate that efflux pump inhibitors active against Staphylococcus aureus are common in land plants. By extension, this activity is likely to be important in many plant-derived antimicrobial extracts, including those used in traditional medicine, and evaluation of efflux pump inhibition may often be valuable when studying natural product efficacy.

Two reproductive traits show contrasting genetic architectures in Plantago lanceolata.

In many species, temperature-sensitive phenotypic plasticity (i.e., an individual's phenotypic response to temperature) displays a positive correlation with latitude, a pattern presumed to reflect local adaptation. This geographical pattern raises two general questions: (a) Do a few large-effect genes contribute to latitudinal variation in a trait? (b) Is the thermal plasticity of different traits regulated pleiotropically? To address the questions, we crossed individuals of Plantago lanceolata derived from northern and southern European populations. Individuals naturally exhibited high and low thermal plasticity in floral reflectance and flowering time. We grew parents and offspring in controlled cool- and warm-temperature environments, mimicking what plants would encounter in nature. We obtained genetic markers via genotype-by-sequencing, produced the first recombination map for this ecologically important nonmodel species, and performed quantitative trait locus (QTL) mapping of thermal plasticity and single-environment values for both traits. We identified a large-effect QTL that largely explained the reflectance plasticity differences between northern and southern populations. We identified multiple smaller-effect QTLs affecting aspects of flowering time, one of which affected flowering time plasticity. The results indicate that the genetic architecture of thermal plasticity in flowering is more complex than for reflectance. One flowering time QTL showed strong cytonuclear interactions under cool temperatures. Reflectance and flowering plasticity QTLs did not colocalize, suggesting little pleiotropic genetic control and freedom for independent trait evolution. Such genetic information about the architecture of plasticity is environmentally important because it informs us about the potential for plasticity to offset negative effects of climate change.

Natural selection contributes to geographic patterns of thermal plasticity in Plantago lanceolata.

A long-standing debate in evolutionary biology concerns the relative importance of different evolutionary forces in explaining phenotypic diversification at large geographic scales. For example, natural selection is typically assumed to underlie divergence along environmental gradients. However, neutral evolutionary processes can produce similar patterns. We collected molecular genetic data from 14 European populations of Plantago lanceolata to test the contributions of natural selection versus neutral evolution to population divergence in temperature-sensitive phenotypic plasticity of floral reflectance. In P. lanceolata, reflectance plasticity is positively correlated with latitude/altitude. We used population pairwise comparisons between neutral genetic differentiation (F ST and Jost's D) and phenotypic differentiation (P ST) to assess the contributions of geographic distance and environmental parameters of the reproductive season in driving population divergence. Data are consistent with selection having shaped large-scale geographic patterns in thermal plasticity. The aggregate pattern of P ST versus F ST was consistent with divergent selection. F ST explained thermal plasticity differences only when geographic distance was not included in the model. Differences in the extent of cool reproductive season temperatures, and not overall temperature variation, explained plasticity differences independent of distance. Results are consistent with the hypothesis that thermal plasticity is adaptive where growing seasons are shorter and cooler, that is, at high latitude/altitude.

Multiple Plantago species (Plantaginaceae) modify floral reflectance and color in response to thermal change.

PREMISE OF THE STUDY: Understanding how plant reproduction responds to temperature has become increasingly important because of global climate change. Temperature-sensitive plasticity in floral reflectance is likely involved in some of these responses. Such plasticity, which underlies thermoregulatory ability, affects reproductive success in Plantago lanceolata. To see whether other Plantago species also show thermal plasticity in reflectance, we measured plasticity in P. lagopus, P. coronopus, P. major, P. subulata, P. albicans, P. tomentosa, P. maritima, and P. weldenii. METHODS: We induced plants to flower at two temperatures in growth chambers and recorded floral reflectance (362-800 nm). KEY RESULTS: All species were thermally plastic in visible and near-IR regions. Species and populations differed in response. Some showed greater variation in reflectance at warm temperature, while the reverse was true for others. Plasticity was greatest in the P. lanceolata clade. Cosmopolitan species were not more plastic than were geographically restricted species. CONCLUSIONS: The data suggest that (1) thermal plasticity is an ancestral trait for Plantago, (2) plasticities in visible and near-IR regions have evolved along different pathways within the genus, and (3) phylogenetic history partially explains this evolutionary divergence. Our data combined with those of previous studies suggest that global climate change will modify floral reflectance and color in many plant species. These modifications are likely to affect plant reproductive success.

Fitness effects of floral plasticity and thermoregulation in a thermally changing environment.

To better understand the evolution of phenotypic plasticity and thermoregulation and their potential value for ectotherms in the face of global warming, we conducted field experiments to measure their effects on fitness and their association with reproductive phenology in Plantago lanceolata in a thermally variable environment. We measured the reproductive timing and success of genotypes varying in thermoregulation, as mediated by floral-reflectance plasticity. Results were consistent with the hypothesis that thermoregulation is more adaptive when thermally variable reproductive seasons are shorter and cooler. Strong thermoregulation/plasticity increased reproductive success during the cool portion of the reproductive season but not during the warm portion. Directional selection that favored strongly thermoregulating genotypes early in the season shifted to stabilizing selection that favored genotypes with weaker thermoregulation later in the season. Thermoregulation and reproductive phenology were negatively correlated. Although reproductive onset and duration were similar between genotypes, strong thermoregulators produced more and larger spikes (clutches) early; weak thermoregulators produced more spikes late. Results suggest that with atmospheric warming, the benefit of raising body temperature via thermoregulation when it is cool should decline in extant populations. The negative correlation between thermoregulation and phenology should accelerate the evolutionary shift toward thermoconformity, that is, reduced plasticity.

Floral reflectance, color, and thermoregulation: what really explains geographic variation in thermal acclimation ability of ectotherms?

Adaptive phenotypic plasticity in thermally sensitive traits, that is, thermal acclimation, generally increases with increasing latitude and altitude. The presumed explanation is that high-latitude/altitude organisms have evolved greater acclimation ability because of exposure to greater temperature fluctuations. Using a conceptual model of the thermal environment during the reproductive season, we tested this hypothesis against an alternative that plasticity is greater because of increased exposure to specific temperatures that strongly select for thermal acclimation. We examined geographic variation in floral reflectance/color plasticity among 29 European populations of a widespread perennial herb, Plantago lanceolata. Individuals partially thermoregulate reproduction through temperature-sensitive plasticity in floral reflectance/color. Plasticity was positively correlated with latitude and altitude. Path analyses support the hypothesis that the thermal environment mediates these geographic effects. Plasticity declined as seasonal temperature range increased, and it increased as duration of the growing season shortened and as the proportion of time exposed to temperatures favoring thermoregulation increased. Data provide evidence that floral reflectance/color plasticity is adaptive and that it has evolved in response not to the magnitude of temperature variation during the reproductive season but rather to the relative exposure to low temperatures, which favor thermoregulation.

Temperature-sensitive alternative oxidase protein content and its relationship to floral reflectance in natural Plantago lanceolata populations.

In many plant species, the alternative respiratory pathway consisting of alternative oxidase (AOX) is affected by growth temperature. The adaptive significance of this temperature-sensitivity is unresolved. Here, leaf and spike (flower cluster) AOX protein content and spike/floral reflectance of genotypes from European Plantago lanceolata populations found in regions differing in reproductive season temperatures were measured. Cloned genotypes grown at controlled warm and cool temperatures were used to assess the natural within- and between-population variation in AOX content, temperature-sensitive phenotypic plasticity in content, and the relationship between AOX and temperature-sensitive floral/spike reflectance. AOX content and plasticity were genetically variable. Leaf AOX content, although greater at cool temperature, was relatively low and not statistically different across populations. Spike AOX content was greater than in leaves. Spike AOX plasticity differed significantly among populations and climate-types and showed significant negative correlation with floral reflectance plasticity, which also varied among populations. Genotypes with more AOX at cool than at warm temperature had greater floral reflectance plasticity; genotypes with relatively more AOX at warm temperature had less floral reflectance plasticity. The data support the hypothesis that plasticity of AOX content in reproductive tissues is associated with long-term thermal acclimatization.

Phenotypic plasticity, parental effects, and parental care in plants? I. An examination of spike reflectance in Plantago lanceolata (Plantaginaceae).

We explore the relationships among phenotypic plasticity, parental effects, and parental care in plants by presenting data from four experiments examining reflectance/color patterns in Plantago lanceolata. In three experiments, we measured spike (inflorescence) reflectance between 362 and 850 nm using a spectrophotometer with an integrating sphere. Experiments show that (1) spike reflectance changes seasonally within and outside the visible portion of the spectrum of radiant energy, (2) increasing ambient temperature causes an individual plant to produce flowering and fruiting spikes that reflect more/lighten in color (the greatest changes occur in the regions around 550 nm and between 750 and 850 nm, the visible and near-infrared regions, respectively), (3) responses are reversible, (4) genotypes within populations and populations from different latitudes differ in mean reflectance and degree of phenotypic plasticity. In a fourth experiment, we measured internal spike temperature. Darker spikes, those produced at lower temperature, got hotter than did lighter spikes in full sun. Thus, plants can partially thermoregulate reproduction and the embryonic development of their offspring. In light of a previous experiment, data suggest that thermoregulation produces adaptive parental effects and is a mechanism by which P. lanceolata provides parental care.

PARENTAL EFFECTS IN PLANTAGO LANCEOLATA L. I.: A GROWTH CHAMBER EXPERIMENT TO EXAMINE PRE- AND POSTZYGOTIC TEMPERATURE EFFECTS.

In spite of the potential evolutionary importance of parental effects, many aspects of these effects remain inadequately explained. This paper explores both their causes and potential consequences for the evolution of life-history traits in plants. In a growth chamber experiment, I manipulated the pre- and postzygotic temperatures of both parents of controlled crosses of Plantago lanceolata. All offspring traits were affected by parental temperature. On average, low parental temperature increased seed weight, reduced germination and offspring growth rate, and accelerated onset of reproduction by 7%, 50%, 5%, and 47%, respectively, when compared to the effects of high parental temperature. Both pre- and postzygotic parental temperatures (i.e., prior to fertilization vs. during fertilization and seed set, respectively) influenced offspring traits but not always in the same direction. In all cases, however, the postzygotic effect was stronger. The prezygotic effects were more often transmitted paternally than maternally. Growth and onset of reproduction were influenced both directly by parental temperature as well as indirectly via the effects of parental temperature on seed weight and germination. Significant interactions between parental genotypes and prezygotic temperature treatment (G × E interactions) show that genotypes differ in their intergenerational responses to temperature with respect to germination and growth. The data suggest that temperature is involved in both genetically based and environmentally induced parental effects and that parental temperature may accelerate the rate of evolutionary change in flowering time in natural populations of P. lanceolata. The environmentally induced temperature effects, as mediated through G × (prezygotic) E interactions are not likely to affect the rate or direction of evolutionary change in the traits examined because postzygotic temperature effects greatly exceed prezygotic effects.

CARBON INTEGRATION IN TWO PLANTAGO SPECIES.

We measured the pattern of export of 14 C-assimilate from reproductive spikes and leaves subtending spikes in Plantago aristata and P. virginica. In P. arislata, little 14 carbon was exported to any other reproductive metamer (leaf + associated spike) from the leaf subtending a flowering spike. In P. virginica a large amount was exported. Thus, like clonal species, rosette species can vary in patterns of carbon integration among the repeated morphological subunits comprising an individual. When considered in the context of other studies, these data suggest that comparisons of carbon integration in species differing in morphological complexity would be useful in trying to understand the evolution of patterns of carbon integration in plants.

Effect of parental flowering and dispersal times on offspring fate in Daucus carota (Apiaceae).

Seeds collected from parents that flowered at different times were dispersed onto experimental plots at different times during the normal dispersal season. Parental flowering and dispersal times, which are correlated with each other, independently affected offspring germination, growth, and time of reproduction. Estimated population growth rates were highest for offspring that were dispersed early in the dispersal season and that came from early flowering parents. The data provide evidence that 1) an individual's fate is determined by the environment of the previous generation, and that 2) an individual's fitness should be calculated from life history data that span more than one generation.

The influence of hygroscopic movement on seed dispersal in Daucus carota (Apiaceae).

The influence of hygroscopic movement on seed dispersal in Daucus carota was examined. When relative humidity increases, umbels containing mature fruits close; when relative humidity drops, umbels open. Tests of the effectiveness of smalland large-angled umbels on dispersing seeds under various conditions demonstrate that umbels responding greatly to relative humidity (i.e. opening wide) lose seeds more quickly than do umbels responding little, and do not disperse them as far. As relative humidity increases, number of dispersing seeds drops to near zero. Response to changes in relative humidity within an umbel progressively increases from late August when dispersal begins. Umbels that slowly increase their response retain some seeds that may disperse over snow in winter. Individual variation in response to relative humidity is high among plants beginning dispersal at the same time. This variability is probably maintained by the variable consequences of dispersing seeds at different times.