Intraspecific variability of leaf form and function across habitat types.

Trait-based ecology has already revealed main independent axes of trait variation defining trait spaces that summarize plant adaptive strategies, but often ignoring intraspecific trait variability (ITV). By using empirical ITV-level data for two independent dimensions of leaf form and function and 167 species across five habitat types (coastal dunes, forests, grasslands, heathlands, wetlands) in the Italian peninsula, we found that ITV: (i) rotated the axes of trait variation that define the trait space; (ii) increased the variance explained by these axes and (iii) affected the functional structure of the target trait space. However, the magnitude of these effects was rather small and depended on the trait and habitat type. Our results reinforce the idea that ITV is context-dependent, calling for careful extrapolations of ITV patterns across traits and spatial scales. Importantly, our study provides a framework that can be used to start integrating ITV into trait space analyses.

Functional responses of understory plants to natural disturbance-based management in eastern and western Canada.

Natural disturbance-based management (NDBM) is hypothesized to maintain managed forest ecosystem integrity by reducing differences between natural and managed forests. The effectiveness of this approach often entails local comparisons of species composition or diversity for a variety of biota from managed and unmanaged forests. Understory vegetation is regularly the focus of such comparison because of its importance in nutrient cycling, forest regeneration, and for wildlife. However, larger scale comparisons between regions with distinct species assemblages may require a trait-based approach to better understand understory responses to disturbance. We compared the long-term effects of retention harvesting on understory vegetation in two large experimental study sites located in eastern and western regions of the Canadian boreal forest. These sites included the Sylviculture en Aménagement Forestier Ecosystémique (SAFE) experiment and the Ecosystem Management Emulating Natural Disturbance (EMEND) experiment, located in the eastern and western regions of Canada, respectively. EMEND and SAFE share common boreal understory species but have distinct tree communities, soils, and climate. Both experiments were designed to evaluate how increasing tree retention after harvest affects biodiversity. Here, we examined taxonomic richness, functional diversity, and functional composition (using community trait mean values) of understory plant communities, and also examine intraspecific trait variability (ITV) for five species common and abundant in both experiments. We observed the limited impacts of retention level on richness, functional diversity, and functional composition of understory plants 20 years postharvest. However, ITV of leaf morphological traits varied between retention levels within each experiment, depending on the species identity. Common species had different functional responses to retention level, showing species-specific reactions to environmental variation. Our result suggests that understory plant communities in the boreal forest achieve resilience to disturbance both in terms of interspecific and intraspecific functional trait diversity. Such diversity may be key to maintaining understory biodiversity in the face of future disturbances and environmental change. Our results reveal the significance of ITV in plant communities for understanding responses to forest harvesting and the importance of choosing appropriate traits when studying species responses to the environment.

Trait-dependent importance of intraspecific variation relative to species turnover in determining community functional composition following nutrient enrichment.

Community weighted mean trait, i.e., functional composition, has been extensively used for upscaling of individual traits to the community functional attributes and ecosystem functioning in recent years. Yet, the importance of intraspecific trait variation relative to species turnover in determining changes in CWM still remains unclear, especially under nutrient enrichment scenarios. In this study, we conducted a global data synthesis analysis and three nutrient addition experiments in two sites of alpine grassland to reveal the extent to which species turnover and ITV contribute to shift in CWM in response to nutrient enrichment. The results consistently show that the importance of ITV relative to species turnover in regulating CWM in response to nutrient enrichment strongly depends on trait attributes rather than on environmental factors (fertilization type, climatic factors, soil properties, and light transmittance). For whole plant traits (height) and leaf morphological traits, species turnover is generally more important than ITV in determining CWM following most treatments of nutrient addition. However, for leaf nutrient traits, ITV outweighed species turnover in determining shifts in CWM in response to almost all treatments of nutrient addition, regardless of types and gradients of the nutrient addition. Thus, our study not only provides robust evidence for trait-dependent importance of ITV in mediating community functional composition, but also highlights the need to consider the nature of functional traits in linking ITV to community assembly and ecosystem functioning under global nutrient enrichment scenarios.

Higher local intra- than interspecific variability in water- and carbon-related leaf traits among Neotropical tree species.

BACKGROUND AND AIMS: Intraspecific variability in leaf water-related traits remains little explored despite its potential importance in the context of increasing drought frequency and severity. Studies comparing intra- and interspecific variability of leaf traits often rely on inappropriate sampling designs that result in non-robust estimates, mainly owing to an excess of the species/individual ratio in community ecology or, on the contrary, to an excess of the individual/species ratio in population ecology. METHODS: We carried out virtual testing of three strategies to compare intra- and interspecific trait variability. Guided by the results of our simulations, we carried out field sampling. We measured nine traits related to leaf water and carbon acquisition in 100 individuals from ten Neotropical tree species. We also assessed trait variation among leaves within individuals and among measurements within leaves to control for sources of intraspecific trait variability. KEY RESULTS: The most robust sampling, based on the same number of species and individuals per species, revealed higher intraspecific variability than previously recognized, higher for carbon-related traits (47-92 and 4-33 % of relative and absolute variation, respectively) than for water-related traits (47-60 and 14-44 % of relative and absolute variation, respectively), which remained non-negligible. Nevertheless, part of the intraspecific trait variability was explained by variation of leaves within individuals (12-100 % of relative variation) or measurement variations within leaf (0-19 % of relative variation) and not only by individual ontogenetic stages and environmental conditions. CONCLUSIONS: We conclude that robust sampling, based on the same number of species and individuals per species, is needed to explore global or local variation in leaf water- and carbon-related traits within and among tree species, because our study revealed higher intraspecific variation than previously recognized.

Extent of intraspecific trait variability in ecologically central and marginal populations of a dominant alpine plant across European mountains.

BACKGROUND AND AIMS: Studying trait variability and restricted gene flow between populations of species can reveal species dynamics. Peripheral populations commonly exhibit lower genetic diversity and trait variability due to isolation and ecological marginality, unlike central populations experiencing gene flow and optimal conditions. This study focused on Carex curvula, the dominant species in alpine acidic meadows of European mountain regions. The species is sparser in dry areas such as the Pyrenees and Balkans, compared to the Central-Eastern Alps and Carpathians. We hypothesized that distinct population groups could be identified based on their mean functional trait values and their correlation with the environment; we predicted that ecologically marginal populations would have stronger trait correlations, lower within-population trait variability (intraspecific trait variability, ITV) and lower genetic diversity than populations of optimal habitats. METHODS: Sampling was conducted in 34 populations that spanned the entire distribution range of C. curvula. We used hierarchical clustering to identify emergent functional groups of populations, defined by combinations of multiple traits associated with nutrient economy and drought tolerance (e.g. specific leaf area, anatomy). We contrasted the geographical distribution of these groups in relation to environment and genetic structure. We compared pairwise trait relationships, within-population trait variation (ITV) and neutral genetic diversity between groups. KEY RESULTS: Our study identified emergent functional groups of populations. Those in the southernmost ranges, specifically the Pyrenees and Balkan region, showed drought-tolerant trait syndromes and correlated with indicators of limited water availability. While we noted a decline in population genetic diversity, we did not observe any significant changes in ITV in ecologically marginal (peripheral) populations. CONCLUSIONS: Our research exemplifies the relationship between ecological marginality and geographical peripherality, which in this case study is linked to genetic depauperation but not to reduced ITV. Understanding these relationships is crucial for understanding the biogeographical factors shaping trait variation.

Low resource availability drives feeding niche partitioning between wild bees and honeybees in a European city.

Cities are socioecological systems that filter and select species, therefore establishing unique species assemblages and biotic interactions. Urban ecosystems can host richer wild bee communities than highly intensified agricultural areas, specifically in resource-rich urban green spaces such as allotments and family gardens. At the same time, urban beekeeping has boomed in many European cities, raising concerns that the fast addition of a large number of managed bees could deplete the existing floral resources, triggering competition between wild bees and honeybees. Here, we studied the interplay between resource availability and the number of honeybees at local and landscape scales and how this relationship influences wild bee diversity. We collected wild bees and honeybees in a pollination experiment using four standardized plant species with distinct floral morphologies. We performed the experiment in 23 urban gardens in the city of Zurich (Switzerland), distributed along gradients of urban and local management intensity, and measured functional traits related to resource use. At each site, we quantified the feeding niche partitioning (calculated as the average distance in the multidimensional trait space) between the wild bee community and the honeybee population. Using multilevel structural equation models (SEM), we tested direct and indirect effects of resource availability, urban beekeeping, and wild bees on the community feeding niche partitioning. We found an increase in feeding niche partitioning with increasing wild bee species richness. Moreover, feeding niche partitioning tended to increase in experimental sites with lower resource availability at the landscape scale, which had lower abundances of honeybees. However, beekeeping intensity at the local and landscape scales did not directly influence community feeding niche partitioning or wild bee species richness. In addition, wild bee species richness was positively influenced by local resource availability, whereas local honeybee abundance was positively affected by landscape resource availability. Overall, these results suggest that direct competition for resources was not a main driver of the wild bee community. Due to the key role of resource availability in maintaining a diverse bee community, our study encourages cities to monitor floral resources to better manage urban beekeeping and help support urban pollinators.

Microanatomical traits track climate gradients for a dominant C4 grass species across the Great Plains, USA.

BACKGROUND AND AIMS: Andropogon gerardii is a highly productive C4 grass species with a large geographic range throughout the North American Great Plains, a biome characterized by a variable temperate climate. Plant traits are often invoked to explain growth rates and competitive abilities within broad climate gradients. For example, plant competition models typically predict that species with large geographic ranges benefit from variation in traits underlying high growth potential. Here, we examined the relationship between climate variability and leaf-level traits in A. gerardii, emphasizing how leaf-level microanatomical traits serve as a mechanism that may underlie variation in commonly measured traits, such as specific leaf area (SLA). METHODS: Andropogon gerardii leaves were collected in August 2017 from Cedar Creek Ecosystem Science Reserve (MN), Konza Prairie Biological Station (KS), Platte River Prairie (NE) and Rocky Mountain Research Station (SD). Leaves from ten individuals from each site were trimmed, stained and prepared for fluorescent confocal microscopy to analyse internal leaf anatomy. Leaf microanatomical data were compared with historical and growing season climate data extracted from PRISM spatial climate models. KEY RESULTS: Microanatomical traits displayed large variation within and across sites. According to AICc (Akaike's information criterion adjusted for small sample sizes) selection scores, the interaction of mean precipitation and temperature for the 2017 growing season was the best predictor of variability for the anatomical and morphological traits measured here. Mesophyll area and bundle sheath thickness were directly correlated with mean temperature (annual and growing season). Tissues related to water-use strategies, such as bulliform cell and xylem area, were significantly correlated with one another. CONCLUSIONS: The results indicate that (1) microanatomical trait variation exists within this broadly distributed grass species, (2) microanatomical trait variability appears likely to impact leaf-level carbon and water use strategies, and (3) microanatomical trait values vary across climate gradients, and may underlie variation in traits measured at larger ecological scales.

Within-individual trophic variability drives short-term intraspecific trait variation in natural populations.

Intraspecific trait variability (ITV) maintains functional diversity in populations and communities, and plays a crucial role in ecological and evolutionary processes such as trophic cascades or speciation. Furthermore, functional variation within a species and its populations can help buffer against harmful environmental changes. Trait variability within species can be observed from differences among populations, and between- and within individuals. In animals, ITV can be driven by ontogeny, the environment in which populations live and by within-individual specialization or variation unrelated to growth. However, we still know little about the relative strength of these drivers in determining ITV variation in natural populations. Here, we aimed to (a) measure the relative strength of between- and within-individual effects of body size on ITV over time, and (b) disentangle the trophic changes due to ontogeny from other sources of variability, such as the environment experienced by populations and individual preferences at varying temporal and spatial scales. We used as a model system the endangered marble trout Salmo marmoratus, a freshwater fish living in a restricted geographical area (<900 km2 ) that shows marked changes in diet through ontogeny. We investigated two trophic traits, trophic position and resource use, with stable isotopes (δ15 N and δ13 C), and followed over time 238 individually tagged marble trout from six populations to estimate the trophic changes between and within individuals through ontogeny at three different time-scales (short term: 3 months, medium term: 1 year and long term: 2 years). We found that the relative strength of between- and within-individual effects of body size on trophic position and resource use change strongly over time. Both effects played a similar role in ITV over medium- and long-term time-scales, but within-individual effects were significantly driving trophic variability over short-term scales. Apart from ontogenetic shifts, individuals showed variability in trophic traits as big as the variability estimated between populations. Overall, our results show how the relative strengths of ITV drivers change over time. This study evidences the crucial importance of considering effects of time-scales on functional variability at individual, population and species levels.

Effects of extreme drought on specific leaf area of grassland species: A meta-analysis of experimental studies in temperate and sub-Mediterranean systems.

Here, we conducted a meta-analysis of experimental drought manipulation studies using rainout shelters in five sites of natural grassland ecosystems of Europe. The single studies assess the effects of extreme drought on the intraspecific variation of the specific leaf area (SLA), a proxy of plant growth. We evaluate and compare the effect size of the SLA response for the functional groups of forbs and grasses in temperate and sub-Mediterranean systems. We hypothesized that the functional groups of grasses and forbs from temperate grassland systems have different strategies in short-term drought response, measured as adjustment of SLA, with SLA-reduction in grasses and SLA-maintenance in forbs. Second, we hypothesized that grasses and forbs from sub-Mediterranean systems do not differ in their drought response as both groups maintain their SLA. We found a significant decrease of SLA in grasses of the temperate systems in response to drought while SLA of forbs showed no significant response. Lower SLA is associated with enhanced water-use efficiency under water stress and thus can be seen as a strategy of phenotypic adjustment. By contrast, in the sub-Mediterranean systems, grasses significantly increased their SLA in the drought treatment. This result points towards a better growth performance of these grasses, which is most likely related to their strategy to allocate resources to belowground parts. The observed SLA reduction of forbs is most likely a direct drought response given that competitive effect of grasses is unlikely due to the scanty vegetation cover. We point out that phenotypic adjustment is an important driver of short-term functional plant response to climatic extremes such as drought. Differential reactions of functional groups have to be interpreted against the background of the group's evolutionary configuration that can differ between climatic zones.

Variable prey development time suppresses predator-prey cycles and enhances stability.

Although theoretical models have demonstrated that predator-prey population dynamics can depend critically on age (stage) structure and the duration and variability in development times of different life stages, experimental support for this theory is non-existent. We conducted an experiment with a host-parasitoid system to test the prediction that increased variability in the development time of the vulnerable host stage can promote interaction stability. Host-parasitoid microcosms were subjected to two treatments: Normal and High variance in the duration of the vulnerable host stage. In control and Normal-variance microcosms, hosts and parasitoids exhibited distinct population cycles. In contrast, insect abundances were 18-24% less variable in High- than Normal-variance microcosms. More significantly, periodicity in host-parasitoid population dynamics disappeared in the High-variance microcosms. Simulation models confirmed that stability in High-variance microcosms was sufficient to prevent extinction. We conclude that developmental variability is critical to predator-prey population dynamics and could be exploited in pest-management programs.

Ecoregion and community structure influences on the foliar elemental niche of balsam fir (Abies balsamea (L.) Mill.) and white birch (Betula papyrifera Marshall).

Changes in foliar elemental niche properties, defined by axes of carbon (C), nitrogen (N), and phosphorus (P) concentrations, reflect how species allocate resources under different environmental conditions. For instance, elemental niches may differ in response to large-scale latitudinal temperature and precipitation regimes that occur between ecoregions and small-scale differences in nutrient dynamics based on species co-occurrences at a community level. At a species level, we compared foliar elemental niche hypervolumes for balsam fir (Abies balsamea (L.) Mill.) and white birch (Betula papyrifera Marshall) between a northern and southern ecoregion. At a community level, we grouped our focal species using plot data into conspecific (i.e., only one focal species is present) and heterospecific groups (i.e., both focal species are present) and compared their foliar elemental concentrations under these community conditions across, within, and between these ecoregions. Between ecoregions at the species and community level, we expected niche hypervolumes to be different and driven by regional biophysical effects on foliar N and P concentrations. At the community level, we expected niche hypervolume displacement and expansion patterns for fir and birch, respectively-patterns that reflect their resource strategy. At the species level, foliar elemental niche hypervolumes between ecoregions differed significantly for fir (F = 14.591, p-value = .001) and birch (F = 75.998, p-value = .001) with higher foliar N and P in the northern ecoregion. At the community level, across ecoregions, the foliar elemental niche hypervolume of birch differed significantly between heterospecific and conspecific groups (F = 4.075, p-value = .021) but not for fir. However, both species displayed niche expansion patterns, indicated by niche hypervolume increases of 35.49% for fir and 68.92% for birch. Within the northern ecoregion, heterospecific conditions elicited niche expansion responses, indicated by niche hypervolume increases for fir of 29.04% and birch of 66.48%. In the southern ecoregion, we observed a contraction response for birch (niche hypervolume decreased by 3.66%) and no changes for fir niche hypervolume. Conspecific niche hypervolume comparisons between ecoregions yielded significant differences for fir and birch (F = 7.581, p-value = .005 and F = 8.038, p-value = .001) as did heterospecific comparisons (F = 6.943, p-value = .004, and F = 68.702, p-value = .001, respectively). Our results suggest species may exhibit biogeographical specific elemental niches-driven by biophysical differences such as those used to describe ecoregion characteristics. We also demonstrate how a species resource strategy may inform niche shift patterns in response to different community settings. Our study highlights how biogeographical differences may influence foliar elemental traits and how this may link to concepts of ecosystem and landscape functionality.

Foliar optical traits capture physiological and phenological leaf plasticity in Tilia×euchlora in the urban environment.

Knowledge on the response of trees to the urban heat island (UHI) effect and soil sealing is currently limited, yet of vital importance in an era characterized by both climate change and urbanization. We investigated the physiological and phenological leaf plasticity of Tilia×euchlora trees to the UHI effect and soil sealing and explored the potential of leaf optical traits to quantify the magnitude of leaf plasticity. Temporal changes of leaf water content (LWC), specific leaf area (SLA), total chlorophyll (Chl) and carotenoids (Car) content, Car:Chl ratio and leaf reflectance for 46 Tilia×euchlora trees were measured along a soil sealing and urbanization gradient. The leaf functional traits displayed trait-specific temporal patterns during the growing season. We observed higher LWC and SLA but lower Chl and Car contents in the coolest zones. We found earlier autumn downregulation in Chl and Car content at paved sites compared to unsealed sites (maximum difference = 13 days). The magnitude of plasticity in relation to the UHI and soil sealing varied in leaf functional traits with largest variation observed in Chl (38%), followed by Car:Chl (31%), Car (29%), SLA (26%) and LWC (8%). The proposed spectral indices calculated using leaf reflectance measurements were able to track the spatiotemporal variations and phenology in the leaf functional traits. Our results clearly demonstrate the leaf plasticity of Tilia×euchlora trees, which provides Tilia×euchlora trees the necessary capacity to adapt to rapid changes in the urban environment. More importantly, we demonstrated the suitability of leaf optical traits to serve as a proxy of leaf functional traits for studying the spatiotemporal response of urban trees to environmental factors, which opens up new possibilities for large scale ecological studies using remote sensing.

Phenotypic dissimilarity index: Correcting for intra- and interindividual variability when quantifying phenotypic variation.

In trait-based ecology, phenotypic variation (PVar) is often quantified with measures expressing average differences between populations standardized in the range 0-1. However, these measures disregard the within-population trait variability. In addition, some of them cannot be partitioned between populations. These aspects can either alter their interpretation or limit their applicability. To overcome these problems, we propose a new measure, the phenotypic dissimilarity (PhD) index, to quantify PVar between populations in scenarios of varying within-population interindividual trait variability. PhD can also quantify within-population PVar while accounting for intraindividual trait variability. Using simulated and real data, we show that using the PhD index becomes important when the within-population trait variability is not negligible, as in all ecological studies. By accounting for within-population trait variability, the PhD index does not overestimate PVar across an environmental gradient compared to other estimators. Traits inherently vary within species. Accounting for such variability is essential to understanding species' phenotypic responses to environmental cues. The proposed PhD index will provide ecologists with a tool for quantifying PVar within species and compare it between species at different levels of biological organization. We provide an R function to calculate the PhD index.

Grazing effects on intraspecific trait variability vary with changing precipitation patterns in Mongolian rangelands.

Functional traits are proxies for plant physiology and performance, which do not only differ between species but also within species. In this work, we hypothesized that (a) with increasing precipitation, the percentage of focal species which significantly respond to changes in grazing intensity increases, while under dry conditions, climate-induced stress is so high that plant species hardly respond to any changes in grazing intensity and that (b) the magnitude with which species change their trait values in response to grazing, reflected by coefficients of variation (CVs), increases with increasing precipitation. Chosen plant traits were canopy height, plant width, specific leaf area (SLA), chlorophyll fluorescence, performance index, stomatal pore area index (SPI), and individual aboveground biomass of 15 species along a precipitation gradient with different grazing intensities in Mongolian rangelands. We used linear models for each trait to assess whether the percentage of species that respond to grazing changes along the precipitation gradient. To test the second hypothesis, we assessed the magnitude of intraspecific trait variability (ITV) response to grazing, per species, trait, and precipitation level by calculating CVs across the different grazing intensities. ITV was most prominent for SLA and SPI under highest precipitation, confirming our first hypothesis. Accordingly, CVs of canopy height, SPI, and SLA increased with increasing precipitation, partly confirming our second hypothesis. CVs of the species over all traits increased with increasing precipitation only for three species. This study shows that it remains challenging to predict how plant performance will shift under changing environmental conditions based on their traits alone. In this context, the implications for the use of community-weighted mean trait values are discussed, as not only species abundances change in response to changing environmental conditions, but also values of traits considerably change. Including this aspect in further studies will improve our understanding of processes acting within and among communities.

Intraspecific variability in fluctuating environments: mechanisms of impact on species diversity.

Recent studies have found considerable trait variations within species. The effect of such intraspecific trait variability (ITV) on the stability, coexistence, and diversity of ecological communities received considerable attention and in many models it was shown to impede coexistence and decrease species diversity. Here we present a numerical study of the effect of genetically inherited ITV on species persistence and diversity in a temporally fluctuating environment. Two mechanisms are identified. First, ITV buffers populations against varying environmental conditions (portfolio effect) and reduces variation in abundances. Second, the interplay between ITV and environmental variations tends to increase the mean fitness of diverse populations. The first mechanism promotes persistence and tends to increase species richness, while the second reduces the chance of a rare species population (which is usually homogeneous) to invade, thus decreasing species richness. We show that for large communities the portfolio effect is dominant, leading to ITV promoting species persistence and richness.

Intraspecific Functional Trait Response to Advanced Snowmelt Suggests Increase of Growth Potential but Decrease of Seed Production in Snowbed Plant Species.

In ecological theory, it is currently unclear if intraspecific trait responses to environmental variation are shared across plant species. We use one of the strongest environmental variations in alpine ecosystems, i.e., advanced snowmelt due to climate warming, to answer this question for alpine snowbed plants. Snowbeds are extreme habitats where long-lasting snow cover represents the key environmental factor affecting plant life. Intraspecific variation in plant functional traits is a key to understanding the performance and vulnerability of species in a rapidly changing environment. We sampled snowbed species after an above-average warm winter to assess their phenotypic adjustment to advanced snowmelt, based on differences in the natural snowmelt dynamics with magnitudes reflecting predicted future warming. We measured nine functional traits related to plant growth and reproduction in seven vascular species, comparing snowbeds of early and late snowmelt across four snowbed sites in the southern Alps in Italy. The early snowbeds provide a proxy for the advanced snowmelt caused by climatic warming. Seed production was reduced under advanced snowmelt in all seed-forming snowbed species. Higher specific leaf area (SLA) and lower leaf dry matter content (LDMC) were indicative of improved growth potential in most seed-forming species under advanced snowmelt. We conclude, first, that in the short term, advanced snowmelt can improve snowbed species' growth potential. However, in the long term, results from other studies hint at increasing competition in case of ongoing improvement of conditions for plant growth under continued future climate warming, representing a risk for snowbed species. Second, a lower seed production can negatively affect the seed rain. A reduction of propagule pressure can be crucial in a context of loss of the present snowbed sites and the formation of new ones at higher altitudes along with climate warming. Finally, our findings encourage using plant functional traits at the intraspecific level across species as a tool to understand the future ecological challenges of plants in changing environments.

Seed release by a serotinous pine in the absence of fire: implications for invasion into temperate regions.

In pines, the release of seeds from serotinous cones is primarily considered a response to the high temperatures of a fire. However, the naturalization of serotinous pines in regions where fires are rare highlights the need to quantify environmental conditions that determine seed release to allow accurate prediction of dispersal and spread risk. We investigated the conditions that break cone serotiny in Pinus radiata, a widely planted forestry species that has naturalized in temperate regions worldwide. We quantified the cone temperatures at which cones open in this species, while also assessing potential confounding effects of cone moisture and age on these temperature requirements. We compared our laboratory results with cone opening behaviour under typical field conditions during summer in Canterbury, New Zealand. Cones opened at a mean temperature of 45 °C, much higher than maximum ambient air temperatures recorded in New Zealand. We found no influence of cone age or moisture content on opening temperature. Under field conditions, cones opened upon reaching similar temperatures to those determined in the laboratory; however, passive solar heating caused cones to reach temperatures up to 15 °C higher than ambient conditions. This resulted in 50 % of cones opening in field conditions where maximum air temperatures never exceeded 30 °C. Our results highlight the need for complementary laboratory and field experiments for understanding seed release from serotinous cones. Our findings have important implications for weed risk assessments, showing that serotinous pines can release seed in temperate climates without fire.

Single-Cell Growth Rates in Photoautotrophic Populations Measured by Stable Isotope Probing and Resonance Raman Microspectrometry.

A new method to measure growth rates of individual photoautotrophic cells by combining stable isotope probing (SIP) and single-cell resonance Raman microspectrometry is introduced. This report explores optimal experimental design and the theoretical underpinnings for quantitative responses of Raman spectra to cellular isotopic composition. Resonance Raman spectra of isogenic cultures of the cyanobacterium, Synechococcus sp., grown in 13C-bicarbonate revealed linear covariance between wavenumber (cm-1) shifts in dominant carotenoid Raman peaks and a broad range of cellular 13C fractional isotopic abundance. Single-cell growth rates were calculated from spectra-derived isotopic content and empirical relationships. Growth rates among any 25 cells in a sample varied considerably; mean coefficient of variation, CV, was 29 ± 3% (σ/[Formula: see text]), of which only ~2% was propagated analytical error. Instantaneous population growth rates measured independently by in vivo fluorescence also varied daily (CV ≈ 53%) and were statistically indistinguishable from single-cell growth rates at all but the lowest levels of cell labeling. SCRR censuses of mixtures prepared from Synechococcus sp. and T. pseudonana (a diatom) populations with varying 13C-content and growth rates closely approximated predicted spectral responses and fractional labeling of cells added to the sample. This approach enables direct microspectrometric interrogation of isotopically- and phylogenetically-labeled cells and detects as little as 3% changes in cellular fractional labeling. This is the first description of a non-destructive technique to measure single-cell photoautotrophic growth rates based on Raman spectroscopy and well-constrained assumptions, while requiring few ancillary measurements.