Sexual dimorphism in floral longevity and flowering synchrony in relation to pollination and mating success in three dioecious Ilex species.

UNLABELLED: • PREMISE OF THE STUDY: Sexual floral dimorphism may have evolved under pollinator-mediated natural selection by which female and male functions are differently influenced. We hypothesized that mating success is differently influenced by display size between sexes, under which sexual dimorphism in flowering schedule has evolved in dioecious species.• METHODS: We examined sexual dimorphism in the total number of flowers per shoot, maximum daily display size, longevity of individual flowers, and flowering synchrony (maximum proportion of open flowers) in three dioecious Ilex species (I. pedunculosa, I. serrata, and I. crenata) whose female flowers are not smaller than male flowers. We compared pollinator response to natural variation of daily display size between sexes. We also examined the effect of display size on female and male success (fruit set and pollen deposition and removal).• KEY RESULTS: In the three species, male shoots produced significantly more flowers than female shoots did. Although female flowers lasted longer and opened more synchronously than male flowers, maximum daily display size was larger on male shoots than on female shoots. Fruit set was significantly pollen-limited in the field in all species. Pollen deposition and/or fruit set increased with female display size, whereas pollen removal decreased or was approximately constant with male display size in the three species.• CONCLUSIONS: We suggest that sexual dimorphism in floral longevity and flowering synchrony might enhance both female and male success in relation to the display size-mating success (pollinator attraction) association irrespective of flower size dimorphism.

Functional adjustments of xylem anatomy to climatic variability: insights from long-term Ilex aquifolium tree-ring series.

The present study assessed the effects of climatic conditions on radial growth and functional anatomical traits, including ring width, vessel size, vessel frequency and derived variables, i.e., potential hydraulic conductivity and xylem vulnerability to cavitation in Ilex aquifolium L. trees using long-term tree-ring time series obtained at two climatically contrasting sites, one mesic site in Switzerland (CH) and one drought-prone site in Italy (ITA). Relationships were explored by examining different xylem traits, and point pattern analysis was applied to investigate vessel clustering. We also used generalized additive models and bootstrap correlation functions to describe temperature and precipitation effects. Results indicated modified radial growth and xylem anatomy in trees over the last century; in particular, vessel frequency increased markedly at both sites in recent years, and all xylem traits examined, with the exception of xylem cavitation vulnerability, were higher at the CH mesic compared with the ITA drought site. A significant vessel clustering was observed at the ITA site, which could contribute to an enhanced tolerance to drought-induced embolism. Flat and negative relationships between vessel size and ring width were observed, suggesting carbon was not allocated to radial growth under conditions which favored stem water conduction. Finally, in most cases results indicated that climatic conditions influenced functional anatomical traits more substantially than tree radial growth, suggesting a crucial role of functional xylem anatomy in plant acclimation to future climatic conditions.

Where do seeds go when they go far? Distance and directionality of avian seed dispersal in heterogeneous landscapes.

Seed dispersal at large scales strongly influences plant population dynamics. Still, ecologists have rarely measured seed dispersal at relevant scales, and the role of habitat types in affecting seed dispersal at long distances remains unexplored. We studied seed dispersal of Ilex aquifolium and Crataegus monogyna in northern Spain, hypothesizing that seeds would be recovered at higher rates and at longer distances (LDD) at habitats with fleshy-fruited trees, compared to habitats with other tree types or at open habitats. We tracked seeds in eight landscapes by enriching trees with 15N isotopes at the center of landscapes, and then detected 15N-marked seeds by sampling at distances of up to 700 m. We found that seeds arrive in greater densities and at longer distances in habitats with trees, particularly fleshy-fruited types, producing different LDD probabilities for each habitat. Results also show a disproportional arrival of seeds in habitats similar to those of mother plants, which should affect seed establishment and the genetic diversity of plant neighborhoods. Findings reveal the strong dependence of seed dispersal on the existing templates that guide the movements of avian dispersers in heterogeneous landscapes and also suggest that LDD above tree lines and beyond hard habitat edges can be difficult.

[Responses of Ilex integra Thunb. seedlings to elevated air ozone concentration].

One-year-old Ilex integra seedlings were exposed to charcoal-filtered (CF) and elevated ozone (E-O3, approximately 150 microL x L(-1)) for 84 days in six open-top chambers. Visible injury, growth parameters, pigments content, gas exchange, chlorophyll a fluorescence and antioxidant system were investigated during the growing season. At the end of experiment, foliage showed remarkable visible symptoms with dark-brown necrotic spots and patches which were concaved on the upper surface of the current-year leaves. Although relative height and diameter increment, total biomass and specific leaf weight (SLW) remained unaffected, E-O3 significantly decreased the percentage of stem biomass in total biomass. E-O3 induced significant decrease in net photosynthetic rate, chlorophyll a/b ratio and total phenolic compound content by 19%, 9% and 36%, respectively. However, stomatal conductance, intercellular CO2 concentration, chlorophyll a fluorescence parameters, pigment contents, MDA contents, total antioxidant capacity and total ascorbate content remained unaffected by E-O3. The results suggested that E-O3-induced change in components of chlorophyll contributed to the reduction of photosynthesis in Ilex integra seedlings. In addition, although visible symptom was found during the experiment, antioxidant system, most of the physiological parameters and growth were not significantly affected by E-O3.

Foliar methyl salicylate emissions indicate prolonged aphid infestation on silver birch and black alder.

It is well documented that when plants are damaged by insects they respond by emitting a range of volatile organic compounds (VOCs). While there have been numerous reports concerning VOCs induced by chewing herbivores, there are relatively few studies detailing the VOCs induced by aphid feeding. The effects of aphid feeding on VOCs emitted by boreal forest trees have been particularly neglected. Herbivore-induced VOCs have relevance to direct and indirect plant defence and atmospheric chemistry. In this study, we analysed the VOCs emitted by Betula pendula (Roth) and Alnus glutinosa (L.) (Gaertn.) infested by specialist aphid species under laboratory conditions. We also complemented this by collecting VOCs from leaf beetle-damaged saplings under field conditions. In addition to induction of some inducible terpenes, we detected substantial aphid-induced emissions of methyl salicylate (MeSA) in both B. pendula and A. glutinosa. MeSA emission intensity depended on the length of aphid infestation. Feeding by beetles induced emission of (E)-DMNT in both tree species and (E)-beta-ocimene in A. glutinosa but had no effect on MeSA emissions. MeSA has been shown to have aphid-repellent qualities and has been shown recently to have impact on formation of secondary organic aerosols in the atmosphere. We discuss our results in relation to these two phenomena.

A cryopreservation protocol for immature zygotic embryos of species of Ilex (Aquifoliaceae).

Tropical Ilex species have recalcitrant seeds. This work describes experiments demonstrating the feasibility of long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. intergerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos were aseptically removed from the seeds and precultured (7 days) in the dark, at 27 +/- 2 degrees C on solidified (0.8% agar) 1/4MS medium, [consisting of quarter-strength salts and vitamins of Murashige and Skoog (1962) medium] with 3% sucrose and 0.1 mg/l Zeatin. The embryos were then encapsulated in 3% calcium alginate beads and pretreated at 24 h intervals in liquid medium supplemented with progressively increasing sucrose concentrations (0.5, 0.75 and 1 M). Beads were dehydrated for 5 h with silicagel to 25% water content (fresh weight basis) and then placed in sterile 5 ml cryovials. Then the beads were either plunged rapidly in liquid nitrogen were they were kept for 1 h (rapid cooling) or cooled at 1 degrees C min(-1) to -30 degrees C. Then the beads were immersed in liquid nitrogen for 1 h (slow cooling). The beads were rewarmed by immersion of the cryovials for 1 min in a water bath thermostated at 30 degrees C. Finally, beads were transferred onto culture medium (1/4MS, 3% sucrose, 0.1 mg/l zeatin, solidified with 0.8% agar) and incubated in a growth room at 27 +/- 2 degrees C under a 14 h light (116 micromol. m(-2) x s(-1))/ 10 h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on de the species and the treatment) were obtained with the cryopreserved embryos.

A cryopreservation protocol for immature zygotic embryos of species of Ilex (Aquifoliaceae)

Tropical Ilex species have recalcitrant seeds. This work describes experiments demonstrating the feasibility of long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. intergerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos were aseptically removed from the seeds and precultured (7 days) in the dark, at 27 +/- 2 degrees C on solidified (0.8% agar) 1/4MS medium, [consisting of quarter-strength salts and vitamins of Murashige and Skoog (1962) medium] with 3% sucrose and 0.1 mg/l Zeatin.The embryos were then encapsulated in 3% calcium alginate beads and pretreated at 24 h intervals in liquid medium supplemented with progressively increasing sucrose concentrations (0.5, 0.7 5 and 1 M). Beads were dehydrated for 5 h with silicagel to 25% water content (fresh weight basis) and then placed in sterile 5 ml cryovials. Then the beads were either plunged rapidly in liquid nitrogen were they were kept for 1 h (rapid cooling) or cooled at 1 degrees C min(-1) to -30 degrees C. Then the beads were immersed in liquid nitrogen for 1 h (slow cooling). The beads were rewarmed by immersion of the cryovials for 1 min in a water bath thermostated at 30 degrees C. Finally, beads were transferred onto culture medium (1/4MS, 3% sucrose, 0.1 mg/l zeatin, solidified with 0.8% agar) and incubated in a growth room at 27 +/- 2 degrees C under a 14 h light (116 micromol. m(-2) x s(-1))/ 10 h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on de the species and the treatment) were obtained with the cryopreserved embryos.

A cryopreservation protocol for immature zygotic embryos of species of Ilex (Aquifoliaceae)

Tropical Ilex species have recalcitrant seeds. This work describes experiments demonstrating the feasibility of long-term conservation of Ilex brasiliensis, I. brevicuspis, I. dumosa, I. intergerrima, I. paraguariensis, I. pseudoboxus, I. taubertiana, and I. theezans through cryopreservation of zygotic rudimentary embryos at the heart developmental stage. The embryos were aseptically removed from the seeds and precultured (7 days) in the dark, at 27 +/- 2 degrees C on solidified (0.8% agar) 1/4MS medium, [consisting of quarter-strength salts and vitamins of Murashige and Skoog (1962) medium] with 3% sucrose and 0.1 mg/l Zeatin.The embryos were then encapsulated in 3% calcium alginate beads and pretreated at 24 h intervals in liquid medium supplemented with progressively increasing sucrose concentrations (0.5, 0.7 5 and 1 M). Beads were dehydrated for 5 h with silicagel to 25% water content (fresh weight basis) and then placed in sterile 5 ml cryovials. Then the beads were either plunged rapidly in liquid nitrogen were they were kept for 1 h (rapid cooling) or cooled at 1 degrees C min(-1) to -30 degrees C. Then the beads were immersed in liquid nitrogen for 1 h (slow cooling). The beads were rewarmed by immersion of the cryovials for 1 min in a water bath thermostated at 30 degrees C. Finally, beads were transferred onto culture medium (1/4MS, 3% sucrose, 0.1 mg/l zeatin, solidified with 0.8% agar) and incubated in a growth room at 27 +/- 2 degrees C under a 14 h light (116 micromol. m(-2) x s(-1))/ 10 h dark photoperiod. Maximum recovery percentages between 15 and 83% (depending on de the species and the treatment) were obtained with the cryopreserved embryos.(AU)

Effects of kin-structured seed dispersal on the genetic structure of the clonal dioecious shrub ilex leucoclada.

Nonrandom patterns of gene dispersal have been identified as possible causes of genetic structuring within populations. Attempts to model these patterns have generally focused solely on the effects of isolation by distance, but the processes involved are more complex than such modeling suggests. Here, we extend considerations of gene dispersal processes beyond simple isolation by distance effects by directly evaluating the effects of kin-structured gene dispersal mediated by the group dispersal of related seeds within fruits (i.e., kin-structured seed dispersal) by birds on genetic structure in Ilex leucoclada, a clonal dioecious shrub. To examine the genetic structure patterns, we established two 30x30 m plots (one with immature soils in old-growth forest and one in secondary forest, designated IM and SC, respectively) with different I. leucoclada stem densities. In these two plots 145 and 510 stems were found, representing 78 and 85 genets, respectively, identified by analyzing their genotypes at eight microsatellite loci. The clonal structure was stronger in the SC plot than in the IM plot. Correlograms of coancestry for genets in both plots exhibited significant, positive, high values in the shortest distance class, indicating the presence of strong genetic structure. However, Sp statistics revealed that the pattern of the genetic structure differed between the plots. In addition, to estimate the family structure within fruits, we sampled forty fruits, in total, from 15 randomly selected plants in the area around the IM and SC plots, and found that 80% of the fruits were multiseeded and 42-100% of the multiseeded fruits contained at least one pair of full sibs. Simulations based on these estimates demonstrated that the group dispersal of related seeds produced through correlated mating both within and across fruits, but not unstructured half-sib dispersal, could generate the observed magnitude and trends of genetic structure found in the IM plot. Furthermore, in addition to kin-structured seed dispersal, isolation by distance processes is also likely to promote genetic substructuring in the SC plot. After discussing possible ecological factors that may have contributed to the observed genetic structure, we contrast our results with those predicted by general isolation by distance models, and propose that kin-structured seed dispersal should promote some evolutionary phenomena, and thus should be incorporated, where appropriate, in models of gene dispersal in natural plant populations.

The effect of winter stress on Ilex aquifolium L.previously fumigated with ozone.

European Holly (Ilex aquifolium) received either charcoal-filtered air (CFA) or CFA with 70 nl l(-1) ozone added for 7 h day(-1) over a 28 day period. Plants were then transferred into cooling incubators for hardening (4 degrees C day/2 degrees C night; day length 12 h) for 7 days and then to the frosting stage (2 degrees C day and -5, -10 or -15 degrees C night) for 4 days. The plants were then placed in ambient conditions. Treatment produced significant differences in chlorophyll fluorescence data. Stomatal conductance was significantly higher for the ozone treatments though both showed a general decline over all temperature regimes. Ozone also significantly increased electrolyte leakage and reduced winter survival. These results show that ambient concentrations of ozone can reduce the tolerance of I. aquifolium to freezing stress, which may have serious implications for its establishment and survival.

Ozone induced leaf loss and decreased leaf production of European Holly (Ilex aquifolium L.) over multiple seasons.

European Holly (Ilex aquifolium L.) was used to study the impact of one short (28 day) ozone fumigation episode on leaf production, leaf loss and stomatal conductance (g(s)), in order to explore potential longer term effects over 3 growing seasons. Young I. aquifolium plants received an episode of either charcoal-filtered air or charcoal-filtered air with 70 nl l(-1) O(3) added for 7 h d(-1) over a 28 day period from June 15th 1996, then placed into ambient environment, Stoke-on-Trent, U.K. Data were collected per leaf cohort over the next three growing seasons. Ozone exposure significantly increased leaf loss and stomatal conductance and reduced leaf production over all subsequent seasons. Impact of the initial ozone stress was still detected in leaves that had no direct experimental ozone exposure. This study has shown the potential of ozone to introduce long-term phenological perturbations into ecosystems by influencing productivity over a number of seasons.

Daily transpiration rates of woody species on drying soil.

Among annual plants, daily transpiration rates, expressed as a fraction of volumetric soil water content available for transpiration, show a common pattern in response to soil drying. Initially, as soil dries, there is little decrease in transpiration rate until water availability has fallen to about one third that at field capacity. With further soil drying, relative transpiration rate decreases in a more-or-less linear fashion until all available water has been used. Data previously obtained for perennial woody species have often been confounded by different methods for determining available soil water. In this study, we investigated the daily transpiration response to soil drying in five woody perennial species: Thuja plicata Donn ex D. Don, Acer rubrum L., Robinia pseudoacacia L., Hibiscus sp. and Ibex aquifolium L. Transpiration was unaffected by soil drying until the initial estimated transpirable soil water fraction had decreased to between 0.23 and 0.32 of that at field capacity. Beyond this point, transpiration rate declined linearly with available soil water fraction until reaching one fifth the rate observed in well-watered plants. With further soil drying, the relative transpiration rates remained between 10 and 20% of that observed in well-watered plants. Maintenance of transpiration at these rates with further soil drying was hypothesized to result from contributions to transpiration of water stored in plant tissues. After taking tissue water storage into account, it was estimated that transpiration was curtailed as the available soil water fraction fell to between 0.26 and 0.37 of that at field capacity, which is comparable to values reported for annual crop plants.

An ecological 'footprint' of climate change.

Recently, there has been increasing evidence of species' range shifts due to changes in climate. Whereas most of these shifts relate ground truth biogeographic data to a general warming trend in regional or global climate data, we here present a reanalysis of both biogeographic and bioclimatic data of equal spatio-temporal resolution, covering a time span of more than 50 years. Our results reveal a coherent and synchronous shift in both species' distribution and climate. They show not only a shift in the northern margin of a species, which is in concert with gradually increasing winter temperatures in the area, they also confirm the simulated species' distribution changes expected from a bioclimatic model under the recent, relatively moderate climate change.

Shade tolerance, photoinhibition sensitivity and phenotypic plasticity of Ilex aquifolium in continental Mediterranean sites.

Shade tolerance, plastic phenotypic response to light and sensitivity to photoinhibition were studied in holly (Ilex aquifolium L.) seedlings transported from the field to a greenhouse and in adult trees in the field. All plants were growing in, or originated from, continental Mediterranean sites in central Spain. Seedlings tolerated moderate but not deep shade. Mortality was high and growth reduced in 1% sunlight. Survival was maximal in 12% sunlight and minimal in full sunlight, although the relative growth rate of the seedlings surviving in high light was similar to that of plants in moderate shade. Maximum photochemical efficiency at predawn was significantly lower in sun plants than in shade plants in the field, revealing chronic photoinhibition that was most pronounced in winter. Plasticity in response to available light varied according to the variable studied, being low for photosynthetic capacity and stomatal conductance, and high for specific leaf area, root:shoot ratio and leaf area ratio, particularly in seedlings. Differences in water relations and hydraulic features between sun and shade plants in the field were marginal. High water potential at the turgor loss point of field-grown plants suggested that holly is sensitive to drought during both the seedling and the adult stage. Low relative growth rates in both high and low light with low physiological plasticity in response to light indicate the existence of a stress-tolerance mechanism. We conclude that holly is a facultative understory plant in areas of oceanic and relatively mild climate, but an obligate understory plant in dry continental areas such as the study site. The impact of abandonment of traditional management practices and climate change on these Mediterranean populations is discussed.

Hydraulic conductivity, photosynthesis and leaf water balance in six evergreen woody species from fall to winter.

To confirm that freeze-thaw embolism is a primary stress for evergreen woody species in winter, hydraulic conductivity, photosynthesis and leaf water potential were measured during fall and winter in trees growing in a cool temperate zone (Nikko) and in a warm temperate zone (Tokyo). We examined two evergreen conifers that naturally occur in the cool temperate zone (Abies firma Siebold & Zucc. and Abies homolepis Siebold & Zucc.), and four evergreen broad-leaved woody species that are restricted to the warm temperate zone (Camellia japonica L., Cinnamomum camphora (L.) J. Presl, Ilex crenata Thunb. and Quercus myrsinaefolia Blume). In Tokyo, where no freeze-thaw cycles of xylem sap occurred, hydraulic conductivity, photosynthesis and water balance remained constant during the experimental period. In Nikko, where there were 38 daily freeze-thaw cycles by February, neither of the tracheid-bearing evergreen conifers showed xylem embolism or leaf water deficits. Similarly, the broad-leaved evergreen trees with small-diameter vessels did not exhibit severe embolism or water deficits and maintained CO(2) assimilation even in January. In contrast, the two broad-leaved evergreen trees with large-diameter vessels showed significantly reduced hydraulic conductivity and shoot die-back in winter. We conclude that freeze-thaw embolism restricts evergreen woody species with large-diameter vessels to the warm temperate zone, whereas other stresses limit the distribution of broad-leaved trees, that have small-diameter vessels, but which are restricted to the warm temperate zone.

Two types of matter economy for the wintering of evergreen shrubs in regions of heavy snowfall.

Plant adaptation to an environment subject to heavy snowfalls was investigated in four species of evergreen shrubs growing in a Fagus crenata forest in an area of Honshu on the Sea of Japan. These shrubs stored carbohydrates in some organs before the snowy season and were covered with snow for 4-5 months. Aucuba japonica var. borealis, Camellia rusticana, and Ilex crenata var. paludosa maintained a reserve of carbohydrates during the snowy season. In Daphniphyllum macropodum var. humile, the reserve of carbohydrates decreased during winter. The respiration rates in the first three species decreased from autumn to winter, whereas the decrease in D. macropodum was slight. It was found that the first three species could use reserve carbohydrates for the growth of new shoots after the thaw, whereas in the last species the growth of new shoots depends on high photosynthetic activity in late spring. Our findings suggest some types of matter economy in evergreen shrubs for wintering in an environment of heavy snow.