Apomixis and genetic background affect distinct traits in Hieracium pilosella L. grown under competition.

BACKGROUND: Apomixis, the asexual reproduction through seeds, occurs in over 40 plant families and avoids the hidden cost of sex. Apomictic plants are thought to have an advantage in sparse populations and when colonizing new areas but may have a disadvantage in changing environments because they propagate via fixed genotypes. In this study, we separated the influences of different genetic backgrounds (potentially reflecting local adaptation) from those of the mode of reproduction, i.e., sexual vs. apomictic, on nine fitness-related traits in Hieracium pilosella L. We aimed to test whether apomixis per se may provide a fitness advantage in different competitive environments in a common garden setting. RESULTS: To separate the effects of genetic background from those of reproductive mode, we generated five families of apomictic and sexual full siblings by crossing two paternal with four maternal parents. Under competition, apomictic plants showed reproductive assurance (probability of seeding, fertility), while offspring of sexual plants with the same genetic background had a higher germination rate. Sexual plants grew better (biomass) than apomictic plants in the presence of grass as a competitor but apomictic plants spread further vegetatively (maximum stolon length) when their competitors were sexual plants of the same species. Furthermore, genetic background as represented by the five full-sibling families influenced maximum stolon length, the number of seeds, and total fitness. Under competition with grass, genetic background influenced fecundity, the number of seeds, and germination rate. CONCLUSIONS: Our results suggest that both the mode of reproduction as well as the genetic background affect the success of H. pilosella in competitive environments. Total fitness, the most relevant trait for adaptation, was only affected by the genetic background. However, we also show for the first time that apomixis per se has effects on fitness-related traits that are not confounded by-and thus independent of-the genetic background.

Demography of the giant monocarpic herb Rheum nobile in the Himalayas and the effect of disturbances by grazing.

BACKGROUND: Perennity of giant rosette species in combination with a single 'big bang' reproduction followed by death of the genetic individual is relatively rare among plants. Such long-lived monocarpic plants are usually slow growing and can be found in deserts, bogs or in alpine regions of the tropics or sub-tropics. Due to their longevity, monocarpic perennials risk losing everything before reproduction, which make them particularly susceptible to disturbances. Because of the inherent difficulties in assessing whether long-lived populations are growing or declining, usually neither their demography nor the consequences of increasing grazing pressure are known. METHODS: We used integral projection modelling (IPM) to measure the growth rate and passage time to flowering of Rheum nobile, a monocarpic perennial, and one of the most striking alpine plants from the high Himalayas. Rosettes which were no longer found due to disturbances or grazing by yaks were either treated as missing or as dead in two series of analysis, thereby simulating demography with and without the impact of grazing cattle. Data were collected from plants at 4500 m a.s.l. in Shangri-la County, Yunnan Province, south-west China. In four consecutive years (2011-2014) and in two populations, 372 and 369 individuals were measured, respectively, and size-dependent growth, survival and fecundity parameters were estimated. In addition, germination percentage, seedling survival and establishment probability were assessed. KEY RESULTS: The probability of survival, flowering and fecundity were strongly size dependent. Time to reach flowering size was 33.5 years [95 % confidence interval (CI) 21.9-43.3, stochastic estimate from pooled transitions and populations]. The stochastic population growth rate (λs) of Rheum nobile was 1.013 (95 % CI 1.010-1.017). When disturbance by grazing cattle (yaks) was accounted for in the model, λs dropped to values <1 (0.940, 95 % CI 0.938-0.943). CONCLUSION: We conclude that natural populations of this unique species are viable, but that conservation efforts should be made to minimize disturbances by grazing and to protect this slow-growing flagship plant from the high Himalayas.

Reversible colour change in leaves enhances pollinator attraction and reproductive success in Saururus chinensis (Saururaceae).

Background and Aims: Although there has been much experimental work on leaf colour change associated with selection generated by abiotic environmental factors and antagonists, the role of leaf colour change in pollinator attraction has been largely ignored. We tested whether whitening of the apical leaves subtending the inflorescences of Saururus chinensis during flowering enhances pollinator attraction, and whether re-greening of the white leaves after flowering increases carbon assimilation and promotes seed development. Methods: White leaves were removed or covered, and the effects of these manipulations on pollinator visitation and subsequent reproductive success were assessed. The net photosynthetic rates of leaves of different colour were measured and their photosynthetic contributions to seed development were evaluated. Key Results: Saururus chinensis is able to self-pollinate autonomously, but depends largely on flies for pollination. White leaves had different reflectance spectra from green leaves, and white leaves attracted significantly more pollinators and led to significantly higher fruit and seed set. Although leaf whitening resulted in a reduction in photosynthetic capacity, it translated into only a small decrease in seed mass. When leaves had turned back from white to green after flowering their photosynthetic capacity was similar to that of 'normal' green leaves and promoted seed development. Conclusions: The reversible leaf colour change in S. chinensis appears to be adaptive because it enhances pollination success during flowering, with a small photosynthetic cost, while re-greening of these leaves after flowering helps to meet the carbon requirements for seed development.

Lower plasticity exhibited by high- versus mid-elevation species in their phenological responses to manipulated temperature and drought.

BACKGROUND AND AIMS: Recent global changes, particularly warming and drought, have had worldwide repercussions on the timing of flowering events for many plant species. Phenological shifts have also been reported in alpine environments, where short growing seasons and low temperatures make reproduction particularly challenging, requiring fine-tuning to environmental cues. However, it remains unclear if species from such habitats, with their specific adaptations, harbour the same potential for phenological plasticity as species from less demanding habitats. METHODS: Fourteen congeneric species pairs originating from mid and high elevation were reciprocally transplanted to common gardens at 1050 and 2000 m a.s.l. that mimic prospective climates and natural field conditions. A drought treatment was implemented to assess the combined effects of temperature and precipitation changes on the onset and duration of reproductive phenophases. A phenotypic plasticity index was calculated to evaluate if mid- and high-elevation species harbour the same potential for plasticity in reproductive phenology. KEY RESULTS: Transplantations resulted in considerable shifts in reproductive phenology, with highly advanced initiation and shortened phenophases at the lower (and warmer) site for both mid- and high-elevation species. Drought stress amplified these responses and induced even further advances and shortening of phenophases, a response consistent with an 'escape strategy'. The observed phenological shifts were generally smaller in number of days for high-elevation species and resulted in a smaller phenotypic plasticity index, relative to their mid-elevation congeners. CONCLUSIONS: While mid- and high-elevation species seem to adequately shift their reproductive phenology to track ongoing climate changes, high-elevation species were less capable of doing so and appeared more genetically constrained to their specific adaptations to an extreme environment (i.e. a short, cold growing season).

A new pollinating seed-consuming mutualism between Rheum nobile and a fly fungus gnat, Bradysia sp., involving pollinator attraction by a specific floral compound.

Pollinating seed-consuming mutualisms are regarded as exemplary models for studying coevolution, but they are extremely rare. In these systems, olfactory cues have been thought to play an important role in facilitating encounters between partners. We present a new pollinating seed-consuming mutualism from the high Himalayas between the endemic herb, Rheum nobile, and a fly fungus gnat, Bradysia sp. Seed production resulting from pollination by Bradysia flies and seed consumption by their larvae were measured to determine the outcome of this interaction. Floral scent analyses and behavioural tests were conducted to investigate the role of olfactory cues in pollinator attraction. Rheum nobile is self-compatible, but it depends mainly on Bradysia sp. females for pollination. Seed production resulting from pollination by adult flies is substantially higher than subsequent seed consumption by their larvae. Behavioural tests showed that an unusual floral compound, 2-methyl butyric acid methyl ester, emitted by plants only during anthesis, was attractive to female flies. Our results indicate that the R. nobile-Bradysia sp. interaction represents a new pollinating seed-consuming mutualism, and that a single unusual compound is the specific signal in the floral scent of R. nobile that plays a key role in attracting its pollinator.

Multifunctional bracts enhance plant fitness during flowering and seed development in Rheum nobile (Polygonaceae), a giant herb endemic to the high Himalayas.

Specialized bracts are thought to be important for the successful reproduction of some plants and are regarded as adaptations to diverse driving forces. However, few empirical studies have quantified the adaptive significance of bracts within a cost-benefit framework. We explored the adaptive significance of large and showy bracts for reproduction in Rheum nobile, a giant herb endemic to the high Himalayas. We examined whether the bracts enhance reproductive success during flowering and seed development. Bracts increased flower and fruit temperature on sunny days, greatly decreased the intensity of ultraviolet-B (UV-B) radiation reaching flowers and fruits, and prevented pollen grains being washed away by rain. Experiments indicated that high temperature could promote pollen germination, while pollen grains exposed to rain and UV-B radiation at ambient levels were seriously damaged. Furthermore, bract removal decreased the number of pollinators visiting flowers. When bracts were removed before or after flowering, fecundity and progeny quality were adversely affected, but seed predation by larvae of pollinators decreased. A cost-benefit analysis demonstrated that the cost of bracts, i.e., increased seed predation, is modest. Our results suggest that the bracts of R. nobile promote pollen germination, protect pollen grains from rain and intense UV-B radiation, enhance pollinator visitation during flowering, and facilitate the development of fertilized ovules during seed development. We conclude that multifunctional bracts of R. nobile are an effective adaptive strategy in alpine environments and might have been selected for because of abiotic environmental conditions as well as for enhancing pollination success.

AFLP markers reveal high clonal diversity and extreme longevity in four key arctic-alpine species.

We investigated clonal diversity, genet size structure and genet longevity in populations of four arctic-alpine plants (Carex curvula, Dryas octopetala, Salix herbacea and Vaccinium uliginosum) to evaluate their persistence under past climatic oscillations and their potential resistance to future climate change. The size and number of genets were determined by an analysis of amplified fragment length polymorphisms and a standardized sampling design in several European arctic-alpine populations, where these species are dominant in the vegetation. Genet age was estimated by dividing the size by the annual horizontal size increment from in situ growth measurements. Clonal diversity was generally high but differed among species, and the frequency distribution of genet size was strongly left-skewed. The largest C. curvula genet had an estimated minimum age of c. 4100 years and a maximum age of c. 5000 years, although 84.8% of the genets in this species were <200 years old. The oldest genets of D. octopetala, S. herbacea and V. uliginosum were found to be at least 500, 450 and 1400 years old, respectively. These results indicate that individuals in the studied populations have survived pronounced climatic oscillations, including the Little Ice Age and the postindustrial warming. The presence of genets in all size classes and the dominance of presumably young individuals suggest repeated recruitment over time, a precondition for adaptation to changing environmental conditions. Together, persistence and continuous genet turnover may ensure maximum ecosystem resilience. Thus, our results indicate that long-lived clonal plants in arctic-alpine ecosystems can persist, despite considerable climatic change.

Pollen dispersal and gene flow within and into a population of the alpine monocarpic plant Campanula thyrsoides.

BACKGROUND AND AIMS: Gene flow by seed and pollen largely shapes the genetic structure within and among plant populations. Seed dispersal is often strongly spatially restricted, making gene flow primarily dependent on pollen dispersal within and into populations. To understand distance-dependent pollination success, pollen dispersal and gene flow were studied within and into a population of the alpine monocarpic perennial Campanula thyrsoides. METHODS: A paternity analysis was performed on sampled seed families using microsatellites, genotyping 22 flowering adults and 331 germinated offspring to estimate gene flow, and pollen analogues were used to estimate pollen dispersal. The focal population was situated among 23 genetically differentiated populations on a subalpine mountain plateau (<10 km(2)) in central Switzerland. KEY RESULTS: Paternity analysis assigned 110 offspring (33·2 %) to a specific pollen donor (i.e. 'father') in the focal population. Mean pollination distance was 17·4 m for these offspring, and the pollen dispersal curve based on positive LOD scores of all 331 offspring was strongly decreasing with distance. The paternal contribution from 20-35 offspring (6·0-10·5 %) originated outside the population, probably from nearby populations on the plateau. Multiple potential fathers were assigned to each of 186 offspring (56·2 %). The pollination distance to 'mother' plants was negatively affected by the mothers' degree of spatial isolation in the population. Variability in male mating success was not related to the degree of isolation of father plants. CONCLUSIONS: Pollen dispersal patterns within the C. thyrsoides population are affected by spatial positioning of flowering individuals and pollen dispersal may therefore contribute to the course of evolution of populations of this species. Pollen dispersal into the population was high but apparently not strong enough to prevent the previously described substantial among-population differentiation on the plateau, which may be due to the monocarpic perenniality of this species.

Distribution of bulbil- and seed-producing plants of Poa alpina (Poaceae) and their growth and reproduction in common gardens suggest adaptation to different elevations.

PREMISE OF THE STUDY: The European Alps harbor a spatially heterogeneous environment. Plants can be adapted genetically to this heterogeneity but may also respond to it by phenotypic plasticity. We expected the important fodder grass Poa alpina to be adapted to elevation either genetically or plastically. • METHODS: We investigated in three elevational common gardens whether growth and reproductive allocation of plants reproducing either by seeds or bulbils suggest adaptation to their elevation of origin and to what extent they can respond plastically to different elevations. Additionally, we analyzed genetic diversity using microsatellites and tested whether seeds are of sexual origin. • KEY RESULTS: In the field, bulbil-producing plants occurred more often at higher elevations, whereas seed-producing plants occurred more often at lower elevations, but bulbil-producing plants were generally less vigorous in the common gardens. The response of plants to elevational transplantation was highly plastic, and vigor was always best at the highest location. The small genetic differences were not clinally related to elevation of origin, underlining the importance of phenotypic plasticity. Reproductive allocation was, however, independent of elevational treatments. Seed-producing plants had higher genetic diversity than the bulbil-producing plants even though we found that seed-producing plants were facultative apomicts mostly reproducing asexually. • CONCLUSIONS: Bulbil-producing P. alpina, showing a fitness cost at lower elevations compared with seed-producing plants, seem better adapted to higher elevations. By means of its two reproductive modes and the capacity to adjust plastically, P. alpina is able to occupy a broad ecological niche across a large elevational range.

Global warming pushes the distribution range of the two alpine 'glasshouse' Rheum species north- and upwards in the Eastern Himalayas and the Hengduan Mountains.

Alpine plants' distribution is being pushed higher towards mountaintops due to global warming, finally diminishing their range and thereby increasing the risk of extinction. Plants with specialized 'glasshouse' structures have adapted well to harsh alpine environments, notably to the extremely low temperatures, which makes them vulnerable to global warming. However, their response to global warming is quite unexplored. Therefore, by compiling occurrences and several environmental strata, we utilized multiple ensemble species distribution modeling (eSDM) to estimate the historical, present-day, and future distribution of two alpine 'glasshouse' species Rheum nobile Hook. f. & Thomson and R. alexandrae Batalin. Rheum nobile was predicted to extend its distribution from the Eastern Himalaya (EH) to the Hengduan Mountains (HM), whereas R. alexandrae was restricted exclusively in the HM. Both species witnessed a northward expansion of suitable habitats followed by a southerly retreat in the HM region. Our findings reveal that both species have a considerable range shift under different climate change scenarios, mainly triggered by precipitation rather than temperature. The model predicted northward and upward migration for both species since the last glacial period which is mainly due to expected future climate change scenarios. Further, the observed niche overlap between the two species presented that they are more divergent depending on their habitat, except for certain regions in the HM. However, relocating appropriate habitats to the north and high elevation may not ensure the species' survival, as it needs to adapt to the extreme climatic circumstances in alpine habitats. Therefore, we advocate for more conservation efforts in these biodiversity hotspots.

Longevity of clonal plants: why it matters and how to measure it.

BACKGROUND: Species' life-history and population dynamics are strongly shaped by the longevity of individuals, but life span is one of the least accessible demographic traits, particularly in clonal plants. Continuous vegetative reproduction of genets enables persistence despite low or no sexual reproduction, affecting genet turnover rates and population stability. Therefore, the longevity of clonal plants is of considerable biological interest, but remains relatively poorly known. SCOPE: Here, we critically review the present knowledge on the longevity of clonal plants and discuss its importance for population persistence. Direct life-span measurements such as growth-ring analysis in woody plants are relatively easy to take, although, for many clonal plants, these methods are not adequate due to the variable growth pattern of ramets and difficult genet identification. Recently, indirect methods have been introduced in which genet size and annual shoot increments are used to estimate genet age. These methods, often based on molecular techniques, allow the investigation of genet size and age structure of whole populations, a crucial issue for understanding their viability and persistence. However, indirect estimates of clonal longevity are impeded because the process of ageing in clonal plants is still poorly understood and because their size and age are not always well correlated. Alternative estimators for genet life span such as somatic mutations have recently been suggested. CONCLUSIONS: Empirical knowledge on the longevity of clonal species has increased considerably in the last few years. Maximum age estimates are an indicator of population persistence, but are not sufficient to evaluate turnover rates and the ability of long-lived clonal plants to enhance community stability and ecosystem resilience. In order to understand the dynamics of populations it will be necessary to measure genet size and age structure, not only life spans of single individuals, and to use such data for modelling of genet dynamics.

Genotypic and environmental variation in specific leaf area in a widespread Alpine plant after transplantation to different altitudes.

Specific leaf area (SLA) is an important plant functional trait as it is an indicator of ecophysiological characteristics like relative growth rate, stress tolerance and leaf longevity. Substantial intraspecific variation in SLA is common and usually correlates with environmental conditions. For instance, SLA decreases with increasing altitude, which is understood as adjustment to temperature. It is generally assumed that intraspecific variation is mostly the result of environmentally induced phenotypic plasticity, but genetic effects may also be present, due to local adaptation or genetic drift. In this study, genotypic and environmental effects on SLA were experimentally separated for the widespread Alpine bell flower Campanula thyrsoides by transplanting plants to three common gardens at contrasting altitudes (600, 1,235 and 1,850 m a.s.l.). Seeds were sampled from 18 populations in four phylogeographic regions within the European Alps. A strong plastic response was observed: SLA decreased with increasing altitude of the common gardens (22.0% of variation). The phylogeographic regions were differentiated in SLA in the common gardens (10.1% of variation), indicating that SLA is at least partly genetically determined. Plants from the six easternmost populations experienced a submediterranean climate and showed decreased SLA values in the three common gardens compared to populations to the west, which may be explained as adaptation to drought. Within these submediterranean populations, SLA decreased with altitude of origin in two out of three common gardens. Concluding, SLA shows strong phenotypic plasticity as well as substantial genetic effects, the latter probably being the result of adaptation to local conditions rather than genetic drift.

Dynamics of apomictic and sexual reproduction during primary succession on a glacier forefield in the Swiss Alps.

Apomixis, the asexual reproduction through seeds, is thought to provide reproductive assurance when ploidy is not even and/or when population density is low. Therefore, apomicts are expected to be more abundant, and the frequency of apomictic offspring higher, at early stages of primary succession when mates are rare. To test this hypothesis, we sampled facultative apomictic Hieracium pilosella L. along the successional gradient on a glacier forefield and determined their ploidy, the level of apomixis in their offspring, and the genetic diversity of the entire meta-population and within subpopulations. We found that apomixis is more common in odd- and aneuploid cytotypes, which are more frequent at early stages of primary succession. However, apomixis was uncommon at all successional stages and sexual hexaploids were dominating throughout. Reproductive assurance was reflected in the higher fertility of all odd-ploid apomictic plants (3×, 5×) by avoiding meiosis, illustrating that apomixis provides an escape from sterility, as proposed by Darlington. Odd-ploid plants are supposedly better colonizers (Baker's law), which is supported by their higher occurrence close to the glacier snout. Independent of succession, we found gene flow between apomicts and sexuals, which allows for the continuous creation of new apomictic and sexual genotypes. We conclude that apomixis in H. pilosella does indeed provide an escape from sterility, and therefore reproductive assurance, in aneuploid cytotypes. We further propose that apomixis preserves beneficial combinations of unlinked alleles in every generation for as long as apomictic genotypes persist in the population.

Isolated populations of a rare alpine plant show high genetic diversity and considerable population differentiation.

BACKGROUND AND AIMS: Gene flow and genetic variability within and among alpine plant populations can be greatly influenced by the steep environmental gradients and heterogeneous topography of alpine landscapes. In this study, the effects are examined of natural isolation of alpine habitats on genetic diversity and geographic structure in populations of C. thyrsoides, a rare and isolated European Alpine monocarpic perennial with limited seed dispersal capacity. METHODS: Molecular diversity was analysed for 736 individuals from 32 populations in the Swiss Alps and adjacent Jura mountains using five polymorphic microsatellite loci. Pollen flow was estimated using pollen grain-sized fluorescent powder. In addition, individual-based Bayesian approaches were applied to examine population structure. KEY RESULTS: High within-population genetic diversity (H(E) = 0.76) and a relatively low inbreeding coefficient (F(IS) = 0.022) were found. Genetic differentiation among populations measured with a standardized measure was considerable (G'(ST) = 0.53). A significant isolation-by-distance relationship was found (r = 0.62, P < 0.001) and a significant geographic sub-structure, coinciding with proposed postglacial migration patterns. Altitudinal location and size of populations did not influence molecular variation. Direct measures of pollen flow revealed that insect-mediated pollen dispersal was restricted to short distances within a population. CONCLUSIONS: The natural isolation of suitable habitats for C. thyrsoides restricts gene flow among the populations as expected for a monocarpic species with very limited seed dispersal capacities. The observed high within-population genetic diversity in this rare monocarpic perennial is best explained by its outcrossing behaviour, long-lived individuals and overlapping generations. Despite the high within-population genetic diversity, the considerable genetic differentiation and the clear western-eastern differentiation in this species merits consideration in future conservation efforts.

Microsatellite diversity of the agriculturally important alpine grass Poa alpina in relation to land use and natural environment.

BACKGROUND AND AIMS: The Alpine Meadow Grass Poa alpina is common in subalpine and alpine natural sites and agriculturally used land, where it is an important fodder grass. Natural factors and human land use are supposed to have been shaping its genetic diversity for hundreds of years. The species comprises sexually and vegetatively reproducing plants. The aim of this study was to investigate the effects of agricultural land use, environmental factors and the mode of reproduction on the distribution of its microsatellite diversity within and among populations and to analyse whether its genetic diversity is correlated with plant species diversity in grassland parcels. METHODS: Genetic diversity of P. alpina was assessed with five microsatellite markers for 569 plants originating from 20 natural sites and from 54 grassland parcels of different cultural tradition, land use and altitude in the Swiss Alps. Due to polyploidy and frequent aneuploidy of the species, data analyses were based on the presence of microsatellite bands. KEY RESULTS: A low but significant differentiation was found in microsatellite bands among natural sites and agriculturally used parcels, while their microsatellite band diversity within populations did not differ. An increased differentiation was found in microsatellite bands with increasing geographic distance among parcels, and a differentiation among grazed and mown parcels, and among sexually and vegetatively reproducing populations. Band richness of sampled plants per village was higher for villages where parcels represented more different land-use types. Within populations, microsatellite band diversity was higher in grazed than in mown parcels. CONCLUSIONS: The diversity of human land use in the Alps was associated with genetic diversity of P. alpina. Therefore, the ongoing socio-economically motivated land-use changes, which reduce the number of different land-use types, will affect the genetic diversity of P. alpina negatively.

Host specificity of two pollinating seed-consuming fly species is not related to soil moisture of host plant in the high Himalayas.

Studying the drivers of host specificity can contribute to our understanding of the origin and evolution of obligate pollination mutualisms. The preference-performance hypothesis predicts that host plant choice of female insects is related mainly to the performance of their offspring. Soil moisture is thought to be particularly important for the survival of larvae and pupae that inhabit soil. In the high Himalayas, Rheum nobile and R. alexandrae differ in their distribution in terms of soil moisture; that is, R. nobile typically occurs in scree with well-drained soils, R. alexandrae in wetlands. The two plant species are pollinated by their respective mutualistic seed-consuming flies, Bradysia sp1. and Bradysia sp2. We investigated whether soil moisture is important for regulating host specificity by comparing pupation and adult emergence of the two fly species using field and laboratory experiments. Laboratory experiments revealed soil moisture did have significant effects on larval and pupal performances in both fly species, but the two fly species had similar optimal soil moisture requirements for pupation and adult emergence. Moreover, a field reciprocal transfer experiment showed that there was no significant difference in adult emergence for both fly species between their native and non-native habitats. Nevertheless, Bradysia sp1., associated with R. nobile, was more tolerant to drought stress, while Bradysia sp2., associated with R. alexandrae, was more tolerant to flooding stress. These results indicate that soil moisture is unlikely to play a determining role in regulating host specificity of the two fly species. However, their pupation and adult emergence in response to extremely wet or dry soils are habitat-specific.

Oviposition by mutualistic seed-consuming pollinators reduces fruit abortion in a recently discovered pollination mutualism.

A prerequisite for the evolutionary stability of pollinating seed-consuming mutualisms is that each partner benefits from the association. However, few studies of such mutualism have considered the benefit gained by the pollinators. Here, we determined how the pollinating seed-predators ensure the provisioning of their offspring in the recently discovered mutualism between Rheum nobile and Bradysia flies. The correlation between flower fate and fly oviposition was examined. Floral traits and patterns of variation in fruit abortion and fly oviposition were investigated to determine whether female flies exhibit preferences for particular flowers when laying eggs. Indole-3-acetic acid (IAA) was quantified to determine whether female flies manipulate host physiology. Flowers that flies oviposited on had a significantly lower probability of fruit abortion compared with intact flowers. Females did not exhibit oviposition preference for any of the floral traits examined. There was no significant correlation between fruit abortion and fly oviposition in terms of either flower position or timing of flowering. IAA concentrations in oviposited flowers were significantly higher than in intact flowers. Our results suggest that oviposition by the mutualistic seed-consuming pollinator Bradysia sp., greatly reduces the probability of fruit abortion of its host, R. nobile; this may be attributed to the manipulation of host physiology through regulating IAA levels.

Plants with longer-lived seeds have lower local extinction rates in grassland remnants 1950-1985.

We investigated whether plant species with longer-lived seeds in the soil had lower rates of local extinction between 1950 and 1985 than species with shorter-lived seeds in 26 intact remnants of extensively used calcareous grasslands. In a previous study we had found higher rates of local extinction for smaller populations, for species with shorter life cycles, and for species with higher habitat specificity. We compiled information on seed longevity from recent literature. Seed longevity had a highly significant effect on the rate of population extinction. Rates of local extinction were lower for species with seed longevity >5 years (33.8%; 31 species) compared with species with shorter-lived seeds (1-5 years: 60.7%, 46 species; <1 year: 58.5%, 81 species). The integration of seed longevity into our analysis did not change our previous conclusions on the dependence of local extinction rates on habitat specificity and on life form. Species in our data set with higher habitat specificity tended to have shorter-lived seeds than species with lower habitat specificity. Thus, most characteristic species of calcareous grassland do not persist in the seed bank and can not rely on this mechanism as a buffer against local extinction.

Effects of elevated CO2 and phosphorus addition on productivity and community composition of intact monoliths from calcareous grassland.

We investigated the effects of elevated CO2 (600 µl l-1 vs 350 µl l-1) and phosphorus supply (1 g P m-2 year-1 vs unfertilized) on intact monoliths from species-rich calcareous grassland in a greenhouse. Aboveground community dry mass remained almost unaffected by elevated CO2 in the first year (+6%, n.s.), but was significantly stimulated by CO2 enrichment in year two (+26%, P<0.01). Among functional groups, only graminoids contributed significantly to this increase. The effect of phosphorus alone on community biomass was small in both years and marginally significant only when analyzed with MANOVA (+6% in year one, +9% in year two, 0.1 ≥P > 0.05). Belowground biomass and stubble after two seasons were not different in elevated CO2 and when P was added. The small initial increase in aboveground community biomass under elevated CO2 is explained by the fact that some species, in particular Carex flacca, responded very positively right from the beginning, while others, especially the dominant Bromus erectus, responded negatively to CO2 enrichment. Shifts in community composition towards more responsive species explain the much larger CO2 response in the second year. These shifts, i.e., a decline in xerophytic elements (B. erectus) and an increase in mesophytic grasses and legumes occurred independently of treatments in all monoliths but were accelerated significantly by elevated CO2. The difference in average biomass production at elevated compared to ambient CO2 was higher when P was supplied (at the community level the CO2 response was enhanced from 20% to 33% when P was added, in graminoids from 17% to 27%, in legumes from 4% to 60%, and in C. flacca from 120% to 298% by year two). Based on observations in this and similar studies, we suggest that interactions between CO2 concentration, species presence, and nutrient availability will govern community responses to elevated CO2.

Nutrient relations in calcareous grassland under elevated CO2.

Plant nutrient responses to 4 years of CO2 enrichment were investigated in situ in calcareous grassland. Beginning in year 2, plant aboveground C:N ratios were increased by 9% to 22% at elevated CO2 (P < 0.01), depending on year. Total amounts of N removed in biomass harvests during the first 4 years were not affected by elevated CO2 (19.9 ± 1.3 and 21.1 ± 1.3 g N m-2 at ambient and elevated CO2), indicating that the observed plant biomass increases were solely attained by dilution of nutrients. Total aboveground P and tissue N:P ratios also were not altered by CO2 enrichment (12.5 ± 2 g N g-1 P in both treatments). In contrast to non-legumes (>98% of community aboveground biomass), legume C/N was not reduced at elevated CO2 and legume N:P was slightly increased. We attribute the less reduced N concentration in legumes at elevated CO2 to the fact that virtually all legume N originated from symbiotic N2 fixation (%Ndfa ≈ 90%), and thus legume growth was not limited by soil N. While total plant N was not affected by elevated CO2, microbial N pools increased by +18% under CO2 enrichment (P = 0.04) and plant available soil N decreased. Hence, there was a net increase in the overall biotic N pool, largely due increases in the microbial N pool. In order to assess the effects of legumes for ecosystem CO2 responses and to estimate the degree to which plant growth was P-limited, two greenhouse experiments were conducted, using firstly undisturbed grassland monoliths from the field site, and secondly designed `microcosm' communities on natural soil. Half the microcosms were planted with legumes and half were planted without. Both monoliths and microcosms were exposed to elevated CO2 and P fertilization in a factored design. After two seasons, plant N pools in both unfertilized monoliths and microcosm communities were unaffected by CO2 enrichment, similar to what was found in the field. However, when P was added total plant N pools increased at elevated CO2. This community-level effect originated almost solely from legume stimulation. The results suggest a complex interaction between atmospheric CO2 concentrations, N and P supply. Overall ecosystem productivity is N-limited, whereas CO2 effects on legume growth and their N2 fixation are limited by P.