How Effective Is Environmental Protection for Ensuring the Vitality of Wild Orchid Species? A Case Study of a Protected Area in Italy.

Orchids are among the plants most threatened by anthropic impact and environmental changes. Therefore, all known orchid species are protected in several countries by regional, national and international legislation. Several studies have cast doubts on the effectiveness of legislation to ensure the protection of wild orchids. We assessed the vitality of four orchid populations in a coastal area in Northern Italy, by monitoring the vegetative and reproductive traits of the orchid populations growing both in the protected sites comprising the Natura 2000 network, and in non-protected sites. We also monitored the level of environmental threat to orchid vitality. The early-flowering deceptive species (Ophrys sphegodes and Anacamptis morio) exhibited high vegetative vitality and experienced similar levels of environmental threat in the protected and non-protected areas. However, their reproductive success was strongly jeopardized, probably by a failed pollination that impeded the fruit set. The late-flowering, partially or totally rewarding species (Anacamptis pyramidalis and Anacamptis coriophora) were more strongly impacted by spring mowing and ungulate herbivory and alien species. Only for A. coriophora were the herbivory and alien species invasions lower at the protected vs. non-protected sites, which ensured a higher population vitality at the protected sites. We conclude that the environmental protection in our study area is ineffective for preserving orchids without targeted actions against specific environmental threats.

Ice holes microrefugia harbor genetically and functionally distinct populations of Vaccinium vitis-idaea (Ericaceae).

In the mountain terrain, ice holes are little depressions between rock boulders that are characterized by the exit of cold air able to cool down the rock surface even in summer. This cold air creates cold microrefugia in warmer surroundings that preserve plant species probably over thousands of years under extra-zonal climatic conditions. We hypothesized that ice hole populations of the model species Vaccinium vitis-idaea (Ericaceae) show genetic differentiation from nearby zonal subalpine populations, and high functional trait distinctiveness, in agreement with genetic patterns. We genotyped almost 30,000 single nucleotide polymorphisms using restriction site-associated DNA sequencing and measured eight functional traits indicative of individual performance and ecological strategies. Genetic results showed high differentiation among the six populations suggesting isolation. On siliceous bedrock, ice hole individuals exhibited higher levels of admixture than those from subalpine populations which could have experienced more bottlenecks during demographic fluctuations related to glacial cycles. Ice hole and subalpine calcareous populations clearly separated from siliceous populations, indicating a possible effect of bedrock in shaping genetic patterns. Trait analysis reflected the bedrock effect on populations' differentiation. The significant correlation between trait and genetic distances suggests the genetic contribution in shaping intraspecific functional differentiation. In conclusion, extra-zonal populations reveal a prominent genetic and phenotypic differentiation determined by history and ecological contingency. Therefore, microrefugia populations can contribute to the overall variability of the species and lead to intraspecific-driven responses to upcoming environmental changes.

Amyotrophic Lateral Sclerosis and Air Pollutants in the Province of Ferrara, Northern Italy: An Ecological Study.

The etiopathogenesis of amyotrophic lateral sclerosis (ALS) is still largely unknown, but likely depends on gene-environment interactions. Among the putative sources of environmental exposure are air pollutants and especially heavy metals. We aimed to investigate the relationship between ALS density and the concentration of air pollution heavy metals in Ferrara, northern Italy. An ecological study was designed to correlate the map of ALS distribution and that of air pollutants. All ALS cases diagnosed between 2000 and 2017 (Ferrara University Hospital administrative data) were plotted by residency in 100 sub-areas, and grouped in 4 sectors: urban, rural, northwestern and along the motorway. The concentrations of silver, aluminium, cadmium, chrome, copper, iron, manganese, lead, and selenium in moss and lichens were measured and monitored in 2006 and 2011. Based on 62 ALS patients, a strong and direct correlation of ALS density was observed only with copper concentrations in all sectors and in both sexes (Pearson coefficient (ρ) = 0.758; p = 0.000002). The correlation was higher in the urban sector (ρ = 0.767; p = 0.000128), in women for the overall population (ρ = 0.782, p = 0.000028) and in the urban (ρ = 0.872, p = 0.000047) population, and for the older cohort of diagnosed patients (2000-2009) the assessment correlated with the first assessment of air pollutants in 2006 (ρ = 0.724, p = 0.008). Our data is, in part, consistent with a hypothesis linking copper pollution to ALS.

Legacy effect of green manure crops fertilized with calcium phosphite on maize production and soil properties.

Recycling phosphorus (P) is crucial to meet future P demand for crop production. We investigated the possibility to use calcium phosphite (Ca-Phi) waste, an industrial by-product, as P fertilizer following the oxidation of phosphite (Phi) to phosphate (Pi) during green manure (GM) cropping in order to target P nutrition of subsequent maize crop. In a greenhouse experiment, four GM crops were fertilized (38 kg P ha-1) with Ca-Phi, triple super phosphate (TSP) or without P (Control) in sandy and clay soils. The harvested GM biomass (containing Phi after Ca-Phi fertilization) was incorporated into the soil before maize sowing. Incorporation of GM residues containing Phi slowed down organic carbon mineralization in clay soil and mass loss of GM residues in sandy soil. Microbial enzymatic activities were affected by Ca-Phi and TSP fertilization at the end of maize crop whereas microbial biomass was similarly influenced by TSP and Ca-Phi in both soils. Compared to Control, Ca-Phi and TSP increased similarly the available P (up to 5 mg P kg-1) in sandy soil, whereas in clay soil available P increased only with Ca-Phi (up to 6 mg P kg-1), indicating that Phi oxidation occurred during GM crops. Accordingly, no Phi was found in maize biomass. However, P fertilization did not enhance aboveground maize productivity and P export, likely because soil available P was not limiting. Overall, our results indicate that Ca-Phi might be used as P source for a subsequent crop since Phi undergoes oxidation during the preliminary GM growth.

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.

Bedrock geology affects foliar nutrient status but has minor influence on leaf carbon isotope discrimination across altitudinal gradients.

Carbon isotope discrimination (Δ13C) in plant leaves generally decreases with increasing altitude in mountains. Lower foliar Δ13C at high elevation usually is associated with higher leaf mass per area (LMA) in thicker leaves. However, it is unclear if lower foliar Δ13C in high-altitude plants is caused by improved photosynthetic capacity as an effect of higher nutrient, especially nitrogen, content in thicker leaves. We investigated trends of foliar Δ13C in four species, each belonging to a different plant functional type (PFT), across two altitudinal gradients, each on a different bedrock type (carbonate and silicate bedrock, respectively) in a region of the southern Alps (Italy) where the foliar Δ13C was not affected by water limitation. Our objective was to assess whether the altitudinal patterns of foliar Δ13C in relation to leaf morphology and foliar nutrients were conditioned by indirect control of bedrock geology on soil nutrient availability. The foliar Δ13C of the four species was mainly affected by LMA and, secondarily, by stomatal density (SD) but the relative importance of these foliar traits varied among species. Area-based nutrient contents had overall minor importance in controlling C discrimination. Relationships among foliar Δ13C, foliar nutrient content and leaf growth rate strongly depended on soil nutrient availability varying differently across the two gradients. In the absence of water limitation, the foliar Δ13C was primarily controlled by irradiance which can shape anatomical leaf traits, especially LMA and/or SD, whose relative importance in determining C isotope discrimination differed among species and/or PFT. Decreasing foliar Δ13C across altitudinal gradients need not be determined by improved photosynthetic capacity deriving from higher nutrient content in thicker leaves.

Monitoring temporal trends of air pollution in an urban area using mosses and lichens as biomonitors.

Monitoring air quality by using living organisms as biomonitors has received increasing attention in recent years. However, rather few studies were based on the concomitant use of passive biomonitoring (based on the different sensitivity of living organisms to air pollution) and active biomonitoring (based on their capacity to accumulate pollutants in the tissues). We carried out a repeated survey of an urban area in Northern Italy, with the objective of comparing temporal trends of different kinds of air pollutants with bioindication (passive biomonitoring) and bioaccumulation (active biomonitoring) techniques. During a five-year interval, temporal patterns of moss metal concentrations underwent significant changes probably due to intercurring variations in the importance of different pollution sources. Nitrogen (N) concentration in moss tissues also decreased and was paralleled by increasing diversity of epiphytic lichens. Increasing δ(15)N in moss tissues suggested a higher contribution of oxidized N species compared with reduced N species.

Mowing regime has different effects on reed stands in relation to habitat.

Reed (Phragmites australis) is widespread in aquatic habitats in Europe where it plays an important ecological role, especially as stabilizer of lake and river shores and as filter against pollutants. Reed is also abundant in ecotones towards terrestrial habitats, especially fen meadows, where its expansion can out-compete rare slowly-growing fen species. Therefore, defining appropriate guidelines for managing reed in wetlands has to consider differences in the ecological roles that reed plays in different wetland habitats. In a small pre-alpine lake in N Italy, we mowed reed stands in three plant communities located along a transect from the lake shore to the periphery. In each community, three areas were subjected to reed mowing in late winter, as traditionally done in the past. Three additional areas were subjected to mowing in winter and summer, while three areas served as un-treated controls. Summer mowing was carried out in August, when the nesting period of birds was concluded. Mowing in winter did not affect reed aboveground biomass (RAB) in any community but enhanced the efficiency of removing nutrients by reducing litter accumulation in the soil. Mowing in winter and summer only slightly decreased RAB in the riparian community, not at all in the intermediate community but significantly diminished RAB in fen meadows. Phosphorus deficiency and/or reduced competition with other species probably accounted for RAB reduction in fen meadows. In conclusion, winter mowing can be overall recommended for preventing eutrophication of littoral habitats while summer mowing is advisable for preventing reed expansion in fen meadows. However, the timing of summer mowing has to be defined considering all requirements needed for optimal management of each individual site.

Heatwave 2003: high summer temperature, rather than experimental fertilization, affects vegetation and CO2 exchange in an alpine bog.

Nitrogen and phosphorus were added experimentally in a bog in the southern Alps. It was hypothesized that alleviating nutrient limitation will increase vascular plant cover. As a consequence, more carbon will be fixed through higher rates of net ecosystem CO(2) exchange (NEE). The vascular cover did increase at the expense of Sphagnum mosses. However, such vegetation changes were largely independent of the treatment and were probably triggered by an exceptional heatwave in summer 2003. Contrary to the tested hypothesis, NEE was unaffected by the nutrient treatments but was strongly influenced by temperature and water-table depth. In particular, ecosystem respiration in the hot summer of 2003 increased dramatically, presumably owing to enhanced heterotrophic respiration in an increased oxic peat layer. At the end of the experiment, the Sphagnum cover decreased significantly in the nitrogen-fertilized treatment at hummock microhabitats. In the long term, this will imply a proportionally greater accumulation of vascular litter, more easily decomposable than the recalcitrant Sphagnum litter. As a result, rates of carbon fixation may decrease because of stimulated respiration.

Global negative vegetation feedback to climate warming responses of leaf litter decomposition rates in cold biomes.

Whether climate change will turn cold biomes from large long-term carbon sinks into sources is hotly debated because of the great potential for ecosystem-mediated feedbacks to global climate. Critical are the direction, magnitude and generality of climate responses of plant litter decomposition. Here, we present the first quantitative analysis of the major climate-change-related drivers of litter decomposition rates in cold northern biomes worldwide. Leaf litters collected from the predominant species in 33 global change manipulation experiments in circum-arctic-alpine ecosystems were incubated simultaneously in two contrasting arctic life zones. We demonstrate that longer-term, large-scale changes to leaf litter decomposition will be driven primarily by both direct warming effects and concomitant shifts in plant growth form composition, with a much smaller role for changes in litter quality within species. Specifically, the ongoing warming-induced expansion of shrubs with recalcitrant leaf litter across cold biomes would constitute a negative feedback to global warming. Depending on the strength of other (previously reported) positive feedbacks of shrub expansion on soil carbon turnover, this may partly counteract direct warming enhancement of litter decomposition.

Atmospheric nitrogen deposition promotes carbon loss from peat bogs.

Peat bogs have historically represented exceptional carbon (C) sinks because of their extremely low decomposition rates and consequent accumulation of plant remnants as peat. Among the factors favoring that peat accumulation, a major role is played by the chemical quality of plant litter itself, which is poor in nutrients and characterized by polyphenols with a strong inhibitory effect on microbial breakdown. Because bogs receive their nutrient supply solely from atmospheric deposition, the global increase of atmospheric nitrogen (N) inputs as a consequence of human activities could potentially alter the litter chemistry with important, but still unknown, effects on their C balance. Here we present data showing the decomposition rates of recently formed litter peat samples collected in nine European countries under a natural gradient of atmospheric N deposition from approximately 0.2 to 2 g.m(-2).yr(-1). We found that enhanced decomposition rates for material accumulated under higher atmospheric N supplies resulted in higher carbon dioxide (CO2) emissions and dissolved organic carbon release. The increased N availability favored microbial decomposition (i) by removing N constraints on microbial metabolism and (ii) through a chemical amelioration of litter peat quality with a positive feedback on microbial enzymatic activity. Although some uncertainty remains about whether decay-resistant Sphagnum will continue to dominate litter peat, our data indicate that, even without such changes, increased N deposition poses a serious risk to our valuable peatland C sinks.

Effects of altitude on element accumulation in alpine moss.

We determined trace metal concentrations in mature living tissues of the moss Hylocomium splendens, at different altitude, in the south-eastern Alps of Italy. We hypothesized that if precipitation does not vary with altitude trace element concentrations in moss tissues will not be affected by altitude. Contrary to our hypothesis, element concentrations did vary with altitude even in the absence of any precipitation pattern related to elevation. In particular, concentrations of anthropogenic pollutants (especially Cd and Pb) peaked at mid altitude (1400-1800 m) where frequency of cloud cover was the highest. We concluded that the trace metal concentrations in alpine mosses can be regarded as a reliable estimate of total atmospheric deposition, i.e., including occult deposition by cloudwater. The latter may account for a significant fraction of the total deposition of anthropogenic trace metals, in close relation to cloud cover frequency.

Testate amoebae (Protista) communities in Hylocomium splendens (Hedw.) B.S.G. (Bryophyta): relationships with altitude, and moss elemental chemistry.

We studied the testate amoebae in the moss Hylocomium splendens along an altitudinal gradient from 1000 to 2200 m asl. in the south-eastern Alps of Italy in relation to micro- and macro-nutrient content of moss plants. Three mountainous areas were chosen, two of them characterised by calcareous bedrock, the third by siliceous bedrock. A total of 25 testate amoebae taxa were recorded, with a mean species richness of 9.3 per sampling plot. In a canonical correspondence analysis, 63.1% of the variation in the amoebae data was explained by moss tissue chemistry, namely by C, P, Ca, Mg, Al, Fe, and Na content and a binary site variable. We interpreted this result as an indirect effect of moss chemistry on testate amoebae through an influence on prey organisms. Although two species responded to altitude, there was no overall significant relationship between testate amoebae diversity or community structure and altitude, presumably because our sampling protocol aimed at minimizing the variability due to vegetation types and soil heterogeneity. This suggests that previous evidence of altitudinal or latitudinal effects on testate amoebae diversity may at least in part be due to a sampling bias, namely differences in soil type or moss species sampled.

Nutritional constraints in ombrotrophic Sphagnum plants under increasing atmospheric nitrogen deposition in Europe.

• We studied the effects of increasing levels of atmospheric nitrogen (N) deposition on nutrient limitation of ombrotrophic Sphagnum plants. • Fifteen mires in 11 European countries were selected across a natural gradient of bulk atmospheric N deposition from 0.1 to 2 g/m2  year-1 . Nutritional constraints were assessed based on nutrient ratios of N, phosphorus (P), and potassium (K) in Sphagnum plants collected in hummocks (i.e. relatively drier microhabitats) and in lawns (i.e. relatively wetter microhabitats). • Nutrient ratios in Sphagnum plants increased steeply at low atmospheric N input, but above a threshold of N deposition of c. 1 g/m2  year-1 the N : P and N : K ratios tended to saturation. Increasing atmospheric N deposition was also accompanied by a reduced retention of Ca and Mg in Sphagnum plants and a decreased stem volumetric density in hummock Sphagnum plants. • We suggest a critical load of N deposition in Europe of 1 g/m2  year-1 above which Sphagnum plants change from being N-limited to be K + P colimited, at N : P > 30 and N : K > 3.

Low-molecular-weight organic acids and hormone-like activity of dissolved organic matter in two forest soils in N Italy.

Concentrations of aliphatic acids, phenolic acid, and inorganic nutrients, as well as hormone-like activity, were determined in soil dissolved organic matter (DOM) from two forest sites in N Italy showing differing degrees of silver fir regeneration. In the site where silver fir recruitment was abundant, humification processes prevailed, and the soil DOM had a high content in aliphatic and phenolic acids. This enhanced the hormone-like activity in the soil, which could in turn promote growth of silver fir seedlings. In the site with poor fir recruitment, the soil DOM underwent rapid mineralization and was richer in inorganic nutrients, but had lower concentrations of aliphatic and phenolic acids, and lower hormone-like activity.

Element concentrations in the forest moss Hylocomium splendens: variation associated with altitude, net primary production and soil chemistry.

Net primary production (NPP) of the forest moss Hylocomium splendens increased significantly along an elevational gradient in the southern Alps of Italy. Extracellularly bound metals (Al, Ca, Co, Cr, Fe, Ni, Mo, Ni, Pb) showed declining concentrations in moss tissue with increasing altitude, presumably because the amount of exchange sites on the cell wall increases less than total biomass. Concentrations of intracellular elements did not vary (Cd, Cu, Mg, Na, Zn), or even increased (K) with altitude. The observed patterns were always independent of precipitation amount and soil concentrations of exchangeable elements. A higher soil nutrient status only enhanced K uptake by the moss. We concluded that variations in moss NPP, associated with elevational gradients, may significantly affect estimates of atmospheric deposition based on moss analysis in mountainous regions.

Nutrient and carbon relations in subalpine dwarf shrubs after neighbour removal or fertilization in northern Italy.

Two subalpine dwarf-shrub heath communities with differing levels of soil nutrient availability were subjected to a 3-year experimental manipulation, including nutrient addition or removal of one of the two co-dominant species from each community. The main objective of our study was to assess the relative importance of interspecific competition versus nutrient limitation in relation to soil fertility. We also aimed to investigate if and to what extent current-year shoot size, leaf-based rates of net photosynthesis and foliar nutrient status accounted for the observed changes in the aboveground biomass of the shrubs. At the end of the experiment, neighbour removal increased the aboveground biomass of all shrubs, especially in the more fertile community, while fertilization did not. We concluded that: (1) competition is more effective than nutrient limitation in structuring the vegetation of subalpine heathlands; and (2) competition intensity is stronger in the more fertile community. The observed patterns of variations in aboveground biomass were not consistently related to net photosynthetic rates, size of individual shoots and foliar nutrient status. Hence, we also concluded that the growth response of dwarf shrubs to altered environmental conditions is primarily determined by developmental plasticity.