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.

Plant functional trait change across a warming tundra biome.

The tundra is warming more rapidly than any other biome on Earth, and the potential ramifications are far-reaching because of global feedback effects between vegetation and climate. A better understanding of how environmental factors shape plant structure and function is crucial for predicting the consequences of environmental change for ecosystem functioning. Here we explore the biome-wide relationships between temperature, moisture and seven key plant functional traits both across space and over three decades of warming at 117 tundra locations. Spatial temperature-trait relationships were generally strong but soil moisture had a marked influence on the strength and direction of these relationships, highlighting the potentially important influence of changes in water availability on future trait shifts in tundra plant communities. Community height increased with warming across all sites over the past three decades, but other traits lagged far behind predicted rates of change. Our findings highlight the challenge of using space-for-time substitution to predict the functional consequences of future warming and suggest that functions that are tied closely to plant height will experience the most rapid change. They also reveal the strength with which environmental factors shape biotic communities at the coldest extremes of the planet and will help to improve projections of functional changes in tundra ecosystems with climate warming.

Habitat-based biodiversity responses to macroclimate and edaphic factors in European fen ecosystems.

Understanding large-scale drivers of biodiversity in palustrine wetlands is challenging due to the combined effects of macroclimate and local edaphic conditions. In boreal and temperate fen ecosystems, the influence of macroclimate on biodiversity is modulated by hydrological settings across habitats, making it difficult to assess their vulnerability to climate change. Here, we investigate the influence of macroclimate and edaphic factors on three Essential Biodiversity Variables across eight ecologically defined habitats that align with ecosystem classifications and red lists. We used 27,555 vegetation plot samples from European fens to assess the influence of macroclimate and groundwater pH predictors on the geographic distribution of each habitat type. Additionally, we modeled the relative influence of macroclimate, water pH, and water table depth on community species richness and composition, focusing on 309 plant specialists. Our models reveal strong effects of mean annual temperature, diurnal thermal range, and summer temperature on biodiversity variables, with contrasting differences among habitats. While macroclimatic factors primarily shape geographic distributions and species richness, edaphic factors emerge as the primary drivers of composition for vascular plants and bryophytes. Annual precipitation exhibits non-linear effects on fen biodiversity, with varying impact across habitats with different hydrological characteristics, suggesting a minimum requirement of 600 mm of annual precipitation for the occurrence of fen ecosystems. Our results anticipate potential impacts of climate warming on European fens, with predictable changes among habitat types and geographic regions. Moreover, we provide evidence that the drivers of biodiversity in boreal and temperate fens are closely tied to the ecological characteristics of each habitat type and the dispersal abilities of bryophytes and vascular plants. Given that the influence of macroclimate and edaphic factors on fen ecosystems is habitat specific, climate change research and conservation actions should consider ecological differentiation within functional IUCN ecosystems at continental and regional scales.

The oldest known clones of Salix herbacea growing in the Northern Apennines, Italy are at least 2000 years old.

PREMISE: Dominant in many ecosystems around the world, clonal plants can reach considerable ages and sizes. Due to their modular growth patterns, individual clonal plants (genets) can consist of many subunits (ramets). Since single ramets do not reflect the actual age of genets, the ratio between genet size (radius) and longitudinal annual growth rate (LAGR) of living ramets is often used to approximate the age of clonal plants. However, information on how the LAGR changes along ramets and how LAGR variability may affect age estimates of genets is still limited. METHODS: We assessed the variability of LAGR based on wood-section position along the ramets and on the duration of the growing season on three genetically distinct genets of Salix herbacea growing in the Northern Apennines (Italy). We compared genet ages estimated by dividing genet radius by the LAGRs of its ramets. RESULTS: LAGR increased significantly from the stem apex to the root collar; indicating that ramet growth rate decreased with time. Furthermore, a difference of ca. 2 weeks in the onset of the growing period did not impact LAGR. Considering the high LAGR variability, we estimated that the three genets started to grow between ~2100 and ~7000 years ago, which makes them the oldest known clones of S. herbacea even considering the most conservative age estimate. CONCLUSIONS: Our findings indicate that analyzing ramets at the root collar provides an integrative measurement of their overall LAGR, which is crucial for estimating the age of genets.

Glutamate dehydrogenase in "Liverworld"-A study in selected species to explore a key enzyme of plant primary metabolism in Marchantiophyta.

In plants, glutamate dehydrogenase (GDH) is an ubiquitous enzyme that catalyzes the reversible amination of 2-oxoglutarate in glutamate. It contributes to both the amino acid homeostasis and the management of intracellular ammonium, and it is regarded as a key player at the junction of carbon and nitrogen assimilation pathways. To date, information about the GDH of terrestrial plants refers to a very few species only. We focused on selected species belonging to the division Marchantiophyta, providing the first panoramic overview of biochemical and functional features of GDH in liverworts. Native electrophoretic analyses showed an isoenzymatic profile less complex than what was reported for Arabidposis thaliana and other angiosperms: the presence of a single isoform corresponding to an α-homohexamer, differently prone to thermal inactivation on a species- and organ-basis, was found. Sequence analysis conducted on amino acid sequences confirmed a high similarity of GDH in modern liverworts with the GDH2 protein of A. thaliana, strengthening the hypothesis that the duplication event that gave origin to GDH1-homolog gene from GDH2 occurred after the evolutionary bifurcation that separated bryophytes and tracheophytes. Experiments conducted on Marchantia polymorpha and Calypogeia fissa grown in vitro and compared to A. thaliana demonstrated through in gel activity detection and monodimensional Western Blot that the aminating activity of GDH resulted in strongly enhanced responses to ammonium excess in liverworts as well, even if at a different extent compared to Arabidopsis and other vascular species. The comparative analysis by bi-dimensional Western Blot suggested that the regulation of the enzyme could be, at least partially, untied from the protein post-translational pattern. Finally, immuno-electron microscopy revealed that the GDH enzyme localizes at the subcellular level in both mitochondria and chloroplasts of parenchyma and is specifically associated to the endomembrane system in liverworts.

Directional turnover towards larger-ranged plants over time and across habitats.

Species turnover is ubiquitous. However, it remains unknown whether certain types of species are consistently gained or lost across different habitats. Here, we analysed the trajectories of 1827 plant species over time intervals of up to 78 years at 141 sites across mountain summits, forests, and lowland grasslands in Europe. We found, albeit with relatively small effect sizes, displacements of smaller- by larger-ranged species across habitats. Communities shifted in parallel towards more nutrient-demanding species, with species from nutrient-rich habitats having larger ranges. Because these species are typically strong competitors, declines of smaller-ranged species could reflect not only abiotic drivers of global change, but also biotic pressure from increased competition. The ubiquitous component of turnover based on species range size we found here may partially reconcile findings of no net loss in local diversity with global species loss, and link community-scale turnover to macroecological processes such as biotic homogenisation.

Seed dormancy and germination changes of snowbed species under climate warming: the role of pre- and post-dispersal temperatures.

BACKGROUND AND AIMS: Climate warming has major impacts on seed germination of several alpine species, hence on their regeneration capacity. Most studies have investigated the effects of warming after seed dispersal, and little is known about the effects a warmer parental environment may have on germination and dormancy of the seed progeny. Nevertheless, temperatures during seed development and maturation could alter the state of dormancy, affecting the timing of emergence and seedling survival. Here, the interplay between pre- and post-dispersal temperatures driving seed dormancy release and germination requirements of alpine plants were investigated. METHODS: Three plant species inhabiting alpine snowbeds were exposed to an artificial warming treatment (i.e. +1·5 K) and to natural conditions in the field. Seeds produced were exposed to six different periods of cold stratification (0, 2, 4, 8, 12 and 20 weeks at 0 °C), followed by four incubation temperatures (5, 10, 15 and 20 °C) for germination testing. KEY RESULTS: A warmer parental environment produced either no or a significant increase in germination, depending on the duration of cold stratification, incubation temperatures and their interaction. In contrast, the speed of germination was less sensitive to changes in the parental environment. Moreover, the effects of warming appeared to be linked to the level of (physiological) seed dormancy, with deeper dormant species showing major changes in response to incubation temperatures and less dormant species in response to cold stratification periods. CONCLUSIONS: Plants developed under warmer climates will produce seeds with changed germination responses to temperature and/or cold stratification, but the extent of these changes across species could be driven by seed dormancy traits. Transgenerational plastic adjustments of seed germination and dormancy shown here may result from increased seed viability, reduced primary and secondary dormancy state, or both, and may play a crucial role in future plant adaptation to climate change.

Micro-climatic controls and warming effects on flowering time in alpine snowbeds.

Alpine snowbed communities are among the habitats most threatened by climate change. The warmer temperature predicted, coupled with advanced snowmelt time, will influence flowering phenology, which is a key process in species adaptation to changing environmental conditions and plant population dynamics. However, we know little about the effects of changing micro-climate on flowering time in snowbeds and the mechanisms underlying such phenological responses. The flowering phenology of species inhabiting alpine snowbeds was assessed with weekly observations over five growing seasons. We analysed flowering time in relation to micro-climatic variation in snowmelt date, soil and air temperature, and experimental warming during the snow-free period. This approach allowed us to test hypotheses concerning the processes driving flowering phenology. The plants were finely tuned with inter-annual and intra-seasonal variations of their micro-climate, but species did not track the same micro-climatic feature to flower. At the growing-season time-scale, the air surrounding the plants was the most common trigger of the blooming period. However, at the annual time-scale, the snowmelt date was the main controlling factor for flowering time, even in warmer climate. Moreover, spatial patterns of the snowmelt influenced the developmental rate of the species because in later snowmelt sites the plants needed a lower level of heat accumulation to enter anthesis. Phenological responses to experimental warming differed among species, were proportional to the pre-flowering time-span of plants, and did not show consistent trends of change over time. Finally, warmer temperature produced an overall increase of flowering synchrony both within and among plant species.

A Cd/Fe/Zn-responsive phytochelatin synthase is constitutively present in the ancient liverwort Lunularia cruciata (L.) dumort.

Lunularia cruciata occupies a very basal position in the phylogenetic tree of liverworts, which in turn have been recognized as a very early clade of land plants. It would therefore seem appropriate to take L. cruciata as the startingpoint for investigating character evolution in plants' metal(loid) response. One of the strongest evolutionary pressures for land colonization by plants has come from potential access to much greater amounts of nutritive ions from surface rocks, compared to water. This might have resulted in the need to precisely regulate trace element homeostasis and to minimize the risk of exposure to toxic concentrations of certain metals, prompting the evolution of a number of response mechanisms, such as synthesis of phytochelatins, metal(loid)-binding thiol-peptides. Accordingly, if the ability to synthesize phytochelatins and the occurrence of an active phytochelatin synthase are traits present in a basal liverwort species, and have been even reinforced in 'modern' tracheophytes, e.g. Arabidopsis thaliana, then such traits would presumably have played an essential role in plant fitness over time. Hence, we demonstrated here that: (i) L. cruciata compartmentalizes cadmium in the vacuoles of the phototosynthetic parenchyma by means of a phytochelatin-mediated detoxification strategy, and possesses a phytochelatin synthase that is activated by cadmium and homeostatic concentrations of iron(II) and zinc; and (ii) A. thaliana phytochelatin synthase displays a higher and broader response to several metal(loid)s [namely: cadmium, iron(II), zinc, copper, mercury, lead, arsenic(III)] than L. cruciata phytochelatin synthase.

The capability to synthesize phytochelatins and the presence of constitutive and functional phytochelatin synthases are ancestral (plesiomorphic) characters for basal land plants.

Bryophytes, a paraphyletic group which includes liverworts, mosses, and hornworts, have been stated as land plants that under metal stress (particularly cadmium) do not synthesize metal-binding peptides such as phytochelatins. Moreover, very little information is available to date regarding phytochelatin synthesis in charophytes, postulated to be the direct ancestors of land plants, or in lycophytes, namely very basal tracheophytes. In this study, it was hypothesized that basal land plants and charophytes have the capability to produce phytochelatins and possess constitutive and functional phytochelatin synthases. To verify this hypothesis, twelve bryophyte species (six liverworts, four mosses, and two hornworts), three charophytes, and two lycophyte species were exposed to 0-36 µM cadmium for 72 h, and then assayed for: (i) glutathione and phytochelatin quali-quantitative content by HPLC and mass spectrometry; (ii) the presence of putative phytochelatin synthases by western blotting; and (iii) in vitro activity of phytochelatin synthases. Of all the species tested, ten produced phytochelatins in vivo, while the other seven did not. The presence of a constitutively expressed and functional phytochelatin synthase was demonstrated in all the bryophyte lineages and in the lycophyte Selaginella denticulata, but not in the charophytes. Hence, current knowledge according to phytochelatins have been stated as being absent in bryophytes was therefore confuted by this work. It is argued that the capability to synthesize phytochelatins, as well as the presence of active phytochelatin synthases, are ancestral (plesiomorphic) characters for basal land plants.

Accumulation and Release of Cadmium Ions in the Lichen Evernia prunastri (L.) Ach. and Wood-Derived Biochar: Implication for the Use of Biochar for Environmental Biomonitoring.

Biochar (BC) boasts diverse environmental applications. However, its potential for environmental biomonitoring has, surprisingly, remained largely unexplored. This study presents a preliminary analysis of BC's potential as a biomonitor for the environmental availability of ionic Cd, utilizing the lichen Evernia prunastri (L.) Ach. as a reference organism. For this purpose, the lichen E. prunastri and two types of wood-derived biochar, biochar 1 (BC1) and biochar 2 (BC2), obtained from two anonymous producers, were investigated for their ability to accumulate, or sequester and subsequently release, Cd when exposed to Cd-depleted conditions. Samples of lichen and biochar (fractions between 2 and 4 mm) were soaked for 1 h in a solution containing deionized water (control), 10 µM, and 100 µM Cd2+ (accumulation phase). Then, 50% of the treated samples were soaked for 24 h in deionized water (depuration phase). The lichen showed a very good ability to adsorb ionic Cd, higher than the two biochar samples (more than 46.5%), and a weak ability to release the metal (ca. 6%). As compared to the lichen, BC2 showed a lower capacity for Cd accumulation (-48%) and release (ca. 3%). BC1, on the other hand, showed a slightly higher Cd accumulation capacity than BC2 (+3.6%), but a release capacity similar to that of the lichen (ca. 5%). The surface area and the cation exchange capacity of the organism and the tested materials seem to play a key role in their ability to accumulate and sequester Cd, respectively. This study suggests the potential use of BC as a (bio)monitor for the presence of PTEs in atmospheric depositions and, perhaps, water bodies.

Aridity drives the response of soil total and particulate organic carbon to drought in temperate grasslands and shrublands.

The increasing prevalence of drought events in grasslands and shrublands worldwide potentially has impacts on soil organic carbon (SOC). We leveraged the International Drought Experiment to study how SOC, including particulate organic carbon (POC) and mineral-associated organic carbon (MAOC) concentrations, responds to extreme drought treatments (1-in-100-year) for 1 to 5 years at 19 sites worldwide. In more mesic areas (aridity index > 0.65), SOC and POC concentrations decreased by 7.9% (±3.9) and 15.9% (±6.2) with drought, respectively, but there were no impacts on MAOC concentrations. However, drought had no impact on SOC, POC, or MAOC concentrations in drylands (aridity index < 0.65). The response of SOC to drought varied along an aridity gradient, concomitant with interannual precipitation variability and standing SOC concentration gradients. These findings highlight the differing response magnitudes of POC and MAOC concentrations to drought and the key regulating role of aridity.

Biochar effects on early decomposition of standard litter in a European beech forest (northern Italy).

The release of biochar (BC) on forest soil is a strategy aimed at increasing carbon reserves and forest productivity. The effect of BC amendments on the decomposition of different quality litter is, however, poorly understood. With this study we investigate the effects of wood-derived BC applications on early decomposition in a European beech (Fagus sylvatica L.) forest through the burial of standard material, i.e. green tea and rooibos tea (high- and low-quality litter surrogates, respectively). Two main questions were addressed: 1) Do BC applications influence the decomposition of high- and low-quality standard litter and, if so, in what way? and 2) Does this effect (if measurable) depend on where the sample is placed with respect to the BC application layer? To test BC amendment effects, four application percentages were employed (0, 10, 20 and 100 %), after which standard litter mass loss was recorded. To investigate the effects of sample position, only three BC application percentages were used (0, 10 and 20 %), with teabags buried at three different depths - within the BC amended layer, between this layer and the unamended soil, and below the latter. Results show that early decomposition of high-quality standard litter was not influenced by BC applications, while a significant reduction in mass loss of low-quality standard litter was observed when the percentage of BC application was higher, specifically of litter within the 20 % and 100 % BC amended layers. Decomposition was also affected by sample position relative to the BC layer, exhibiting higher levels of mass loss when samples were placed within the BC amended layer. Overall, BC applications on beech forest soils not only seem to produce negligible effects on the early decomposition rate of high-quality standard litter, but such applications also seem to have the ability to reduce carbon loss following plant material degradation.

Effects of Wood-Derived Biochar on Germination, Physiology, and Growth of European Beech (Fagus sylvatica L.) and Turkey Oak (Quercus cerris L.)

Biochar (BC) soil amendments could partially counteract soil carbon (C) stock decrease in broad-leaved forests in Italy; however, its effects on the growth of representative tree species­Fagus sylvatica L. and Quercus cerris L.­has not yet been addressed. We examine whether seed germination and growth of these species are affected by addition of BC obtained from deciduous broadleaf trees. Seeds were left to germinate in greenhouse conditions under three different BC amendments: 0% (control), 10% and 20% (v/v). Seedlings were then subjected to controlled conditions under the same BC percentage. Biochar effects on seed germination were assessed measuring germination time and percentage, while effects on photosynthesis were assessed using leaf chlorophyll content (mg/m2) and photosynthetic efficiency (FV/FM). Plant growth was estimated by recording leaf number, longest leaf length and plant height. Biochar treatments had no negative effects on germination and early growth stage of the two species. Positive effects were found on the chlorophyll content of both species (ca. +8%) regardless of the treatment and on the leaf number (+30%), leaf length (+14%) and plant height (+48%) of Q. cerris (only with 10% BC). Biochar applications seem, therefore, a suitable method for increasing broad-leaved forest C stock in Italy.

Experimental warming differentially affects vegetative and reproductive phenology of tundra plants.

Rapid climate warming is altering Arctic and alpine tundra ecosystem structure and function, including shifts in plant phenology. While the advancement of green up and flowering are well-documented, it remains unclear whether all phenophases, particularly those later in the season, will shift in unison or respond divergently to warming. Here, we present the largest synthesis to our knowledge of experimental warming effects on tundra plant phenology from the International Tundra Experiment. We examine the effect of warming on a suite of season-wide plant phenophases. Results challenge the expectation that all phenophases will advance in unison to warming. Instead, we find that experimental warming caused: (1) larger phenological shifts in reproductive versus vegetative phenophases and (2) advanced reproductive phenophases and green up but delayed leaf senescence which translated to a lengthening of the growing season by approximately 3%. Patterns were consistent across sites, plant species and over time. The advancement of reproductive seasons and lengthening of growing seasons may have significant consequences for trophic interactions and ecosystem function across the tundra.

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.

The phytochelatin synthase from Nitella mucronata (Charophyta) plays a role in the homeostatic control of iron(II)/(III).

Although some charophytes (sister group to land plants) have been shown to synthesize phytochelatins (PCs) in response to cadmium (Cd), the functional characterization of their phytochelatin synthase (PCS) is still completely lacking. To investigate the metal response and the presence of PCS in charophytes, we focused on the species Nitella mucronata. A 40 kDa immunoreactive PCS band was revealed in mono-dimensional western blot by using a polyclonal antibody against Arabidopsis thaliana PCS1. In two-dimensional western blot, the putative PCS showed various spots with acidic isoelectric points, presumably originated by post-translational modifications. Given the PCS constitutive expression in N. mucronata, we tested its possible involvement in the homeostasis of metallic micronutrients, using physiological concentrations of iron (Fe) and zinc (Zn), and verified its role in the detoxification of a non-essential metal, such as Cd. Neither in vivo nor in vitro exposure to Zn resulted in PCS activation and PC significant biosynthesis, while Fe(II)/(III) and Cd were able to activate the PCS in vitro, as well as to induce PC accumulation in vivo. While Cd toxicity was evident from electron microscopy observations, the normal morphology of cells and organelles following Fe treatments was preserved. The overall results support a function of PCS and PCs in managing Fe homeostasis in the carophyte N. mucronata.