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 invasion risk inside and outside protected areas: Propagule pressure, abiotic and biotic factors definitively matter.

Invasive alien species are among the main global drivers of biodiversity loss posing major challenges to nature conservation and to managers of protected areas. The present study applied a methodological framework that combined invasive Species Distribution Models, based on propagule pressure, abiotic and biotic factors for 14 invasive alien plants of Union concern in Italy, with the local interpretable model-agnostic explanation analysis aiming to map, evaluate and analyse the risk of plant invasions across the country, inside and outside the network of protected areas. Using a hierarchical invasive Species Distribution Model, we explored the combined effect of propagule pressure, abiotic and biotic factors on shaping invasive alien plant occurrence across three biogeographic regions (Alpine, Continental, and Mediterranean) and realms (terrestrial and aquatic) in Italy. We disentangled the role of propagule pressure, abiotic and biotic factors on invasive alien plant distribution and projected invasion risk maps. We compared the risk posed by invasive alien plants inside and outside protected areas. Invasive alien plant distribution varied across biogeographic regions and realms and unevenly threatens protected areas. As an alien's occurrence and risk on a national scale are linked with abiotic factors followed by propagule pressure, their local distribution in protected areas is shaped by propagule pressure and biotic filters. The proposed modelling framework for the assessment of the risk posed by invasive alien plants across spatial scales and under different protection regimes represents an attempt to fill the gap between theory and practice in conservation planning helping to identify scale, site, and species-specific priorities of management, monitoring and control actions. Based on solid theory and on free geographic information, it has great potential for application to wider networks of protected areas in the world and to any invasive alien plant, aiding improved management strategies claimed by the environmental legislation and national and global strategies.

Improving plant-based genotoxicity bioassay through AFLP technique for trace metal-contaminated water: insights from Myriophyllum aquaticum (Vell.) Verdc. and Cd.

In this work, we evaluated whether the species Myriophyllum aquaticum (Vell.) Verdc. can be a promising material for devising reliable eco-toxicological tests for Cd-contaminated waters. Plants of M. aquaticum were exposed to Cd, using different concentrations (1 mg L-1, 2.5 mg L-1, 5 mg L-1, and 10 mg L-1; experiment 1) and exposure times (2.5 mg L-1 for 3 days, 7 days, 14 days, and 21 days; experiment 2). Plant growth and Cd accumulation were monitored during the treatment period, and Cd genotoxicity was assessed by analyzing Cd-induced changes in the AFLP fingerprinting profiles using famEcoRI(TAC)/MseI(ATG) and hexEcoRI(ACG)/MseI(ATG) pairs of primers. Root and shoot growth was reduced already at the lowest Cd concentration used (about 20% reduction for roots and 60% for shoots at 1 mg L-1; experiment 1) and after 7 days (about 50% reduction for roots and 70% for shoots; experiment 2). The primer combinations produced 154 and 191 polymorphic loci for experiments 1 and 2, respectively. Mean genetic diversity (He) reduction among the treatment groups was observed starting from 2.5 mg L-1 (He 0.211 treated vs 0.236 control; experiment 1) and after 3 days (He 0.169 treated vs 0.261 control; experiment 2), indicating that results obtained from AFLP profiles did not match with plant growth measurements. Therefore, our results showed that M. aquaticum proved to be a suitable model system for the investigation of Cd genotoxicity through AFLP fingerprinting profile, whereas the more classic eco-toxicological tests based only on biometric parameters could not correctly estimate the risk associated with undetected Cd genotoxicity.

Persistence of the Strictly Endemic Plants of Forest Margins: The Case of Cirsium alpis-lunae in the Northern Apennines (Italy).

Narrow endemic plants constitute a pivotal group for conservation, being often reduced to a small contingent of individuals and frequently threatened. However, effective conservation actions require reliable basic information about distribution range, ecological requirements, and population traits. Nevertheless, such knowledge results are incomplete or even completely missing for some neglected or recently described plants, such as Cirsium alpis-lunae, a thistle exclusive to the N-Apennines (Italy). To fill this gap, all sites where C. alpis-lunae grow were monitored, and data on the site and population traits were collected. Our results indicated that this plant is restricted to 16 scattered sites, varied in surface area and number of individuals. Reproductive and juvenile plants showed to be affected by roughly the same variables, in particular the surface of the site, the slope aspect, and the canopy cover. The narrow ecological niche of C. alpis-lunae was mainly determined by the canopy cover, and where coverage increases, the number of individuals decreases. The individuals only grow at forest edges, where the peculiar ecological conditions are limiting factors for the development of forestry cover; some other factors (i.e., high inclination and instability of the substrate) contribute to limiting the development of forestry vegetation and guarantee the persistence of these ecotones. Despite the great difficulties in accessing the sites where this species grows, this study presents, for the first time, a complete picture of the C. alpis-lunae population and yielded important data to identify effective conservation measures.

Improving the efficiency of wastewater treatment plants: Bio-removal of heavy-metals and pharmaceuticals by Azolla filiculoides and Lemna minuta.

In this study, we investigated the removal of Fe(III), Cr(VI), Al(III), diclofenac, and levofloxacin from treated wastewater in the presence of the free-floating plants Azolla filiculoides and Lemna minuta, to understand whether these species can be effectively used in a surface flow constructed wetland as wastewater refining treatment. Fe and Al were selected owing to their wide use as coagulant agents in wastewater treatment plants for promoting clariflocculation processes, whilst Cr was chosen due to its common use in industry. Diclofenac and levofloxacin, two molecules belonging to the most widely used pharmaceutical classes in the world, were studied as representative anti-inflammatory drugs and antibiotics, respectively. The study was performed at laboratory scale, exposing the plants separately to each individual contaminant at the concentrations of 5 mg L-1 for the metals (i.e. 2.5-5 times higher than the European limits concerning discharge into surface water), and 1 µg L-1 for the pharmaceuticals (concentration levels commonly found in wastewater). Depending on the plant species and contaminant tested, the range of different effects observed included low toxicity (i.e. Cr, Fe and diclofenac in L. minuta) and even a stimulatory effect on plant growth (i.e. for A. filiculoides with Al and for L. minuta with Al and levofloxacin). Moreover, both species proved to be very effective in the removal of Fe, Al and levofloxacin, with A. filiculoides showing the best performance (removal efficiency of 92%, 96%, and 60%, respectively), whereas for Cr and diclofenac the removal was always less than 10%. The higher removal capacity of A. filiculoides compared to L. minuta can be attributed to its superior tolerance of the contaminants, probably in turn related to the presence of nitrogen-fixing microorganism in its fronds.

Patterns of change in α and ß taxonomic and phylogenetic diversity in the secondary succession of semi-natural grasslands in the Northern Apennines.

We studied the secondary succession in semi-natural grasslands (dry grasslands and hay meadows) located in the eastern side of the Tuscan Apennines (Tuscany, Central Italy). We compared these habitats, investigating: (i) the changes in species richness, composition and phylogenetic diversity during the succession; (ii) whether the trends in species loss and species turnover in taxonomic diversity matched those in phylogenetic diversity. We performed a stratified random sampling, in a full factorial design between habitat type and succession stage (60 sampled plots, 10 × 2 types of habitat × 3 stages of succession). We constructed a phylogenetic tree of the plant communities and compared the differences in taxonomic/phylogenetic α- and ß-diversity between these two habitats and during their succession. We identified indicator species for each succession stage and habitat. Looking at α-diversity, both habitats displayed a decrease in species richness, with a random process of species selection in the earlier succession stages from the species regional pool. Nevertheless, in the latter stage of dry grasslands we recorded a shift towards phylogenetic overdispersion at the higher-level groups in the phylogenetic tree. In both habitats, while the richness decreased with succession stage, most species were replaced during the succession. However, the hay meadows were characterized by a higher rate of new species' ingression whereas the dry grasslands became dominated with Juniperus communis. Accordingly, the two habitats showed similar features in phylogenetic ß-diversity. The main component was true phylogenetic turnover, due to replacement of unique lineages along the succession. Nevertheless, in dry grasslands this trend is slightly higher than expected considering the major importance of difference in species richness of dry grasslands sites and this is due to the presence of a phylogenetically very distant species (J. communis).

Applying predictive models to decipher rhizobacterial modifications in common reed die-back affected populations.

The microbiota inhabiting the soil, as well as the rhizosphere, represents a key determinant of several plant functions. Like for humans, dysbiosis of the plant-associated microbiota may be a co-causal agent in disease with still obscure eziology. In the last decades, the common reed Phragmites australis has been deeply studied for its disappearance from natural stands, but no clear causative agents have been identified and no laboratory models of such "reed die-back syndrome" (RDBS) have been developed. In this study, we try to shed light on the RDBS, by comparing the rhizosphere microbiota of five Italian P. australis populations with different degrees of decline. Results obtained showed a biogeographical meaningful pattern of rhizosphere microbiota, coupled with an impact of RDBS. Obtained data allowed to construct a two-steps predictive model which enabled the prediction of the plant health status from the microbiota taxonomic composition, independently from their geographic location. In conclusion, this study represents one of the first overviews that statistically links RDBS to alteration of rhizosphere microbiota and suggests a model for the analysis of plant-bacteria relationships in nature.

AFLP Approach Reveals Variability in Phragmites australis: Implications for Its Die-Back and Evidence for Genotoxic Effects.

Phragmites australis is a subcosmopolitan species typical of wetlands being studied in Europe for its disappearance from natural stands, a phenomenon called reed die-back syndrome (RDBS). Although it is conjectured that low genetic variability contributes to RDBS, this aspect remains neglected to this day. Using a molecular fingerprinting approach and a sequence analysis of the trnT-trnL/rbcL-psaI regions of cpDNA, this study aimed to compare the genetic structure of stable vs. RDBS-affected P. australis stands from five wetlands of central Italy. Beforehand, in order to characterize the health condition of reed populations, the occurrence of the main macromorphological descriptors for RDBS was considered on 40 reed stands. Soil samples were also collected to examine the total content of heavy metals. The current study analyzed cpDNA in 19 samples and AFLP profiles in 381 samples to investigate the genetic structure of Phragmites populations. Based on the multinomial-Dirichlet model, an analysis of candidate loci under selective pressure was also performed. The relationships among AFLP data, RDBS descriptors and chemicals were evaluated with the use of Linear Mixed Models. The analysis of the cpDNA shows the occurrence of the haplotypes M (the most widespread), and K here recorded for the first time in Italy. Three new haplotypes were also described. The DNA fingerprinting analysis has produced a total of 322 loci (98% polymorphic) and shows the medium-to-high amount of genetic diversity. The significant genetic differentiation among wetlands (Fst = 0.337) suggests either low gene flow or small effective population size. Moreover, the low amount of outlier loci (only 5; l.5% of the total), seems to indicate the scarce occurrence of selective pressure upon the reed's genome. Genetic diversity increased in relationship to the decrease in diameter and of flowering buds of the reed, two of the trends associated with the die-back. The current study rejects the hypothesis that genetic diversity massively contributed to RDBS. Moreover, significant relationships between genetic diversity and the total concentration of some heavy metals (Cr, Cu, and Zn) were highlighted, indicating possible genotoxic effects on P. australis. The current study represents a fact-finding background useful for the conservation of common reed.

Using Myriophyllum aquaticum (Vell.) Verdc. to remove heavy metals from contaminated water: Better dead or alive?

This study aimed to investigate the potential of the invasive macrophyte Myriophyllum aquaticum to remove heavy metals. The elements tested were Cd, Cr, Ni, and Zn, in single-metal trials, and experiments were performed with both the living and dead biomass of the plant. In respect of metal removal by living plants, the element that was removed the most was Zn, though Cd showed the highest concentration in plant shoots. The metal negative effect on plant growth was, therefore, more important than the level of metal concentration in plant tissue in determining the removal percentages. All the metals were mostly accumulated in the roots, where a considerable fraction of the element was simply adsorbed to root cell wall, except in the case of Cr. In shoots, the fraction of the adsorbed metal was extremely low in respect to roots, thereby implying a lower apoplastic binding capacity. As regards a possible use of the dead biomass for metal removal, we proposed the generation of a hybrid biosorbent enclosing the dried and grounded plant biomass in cotton bags to improve its handling and its adsorption capacity, in view of a valid alternative to reduce the problems of packed beds. Cadmium-and especially Zn-were the elements removed most efficiently with respect to the other metals. On comparing the removal percentages of the living biomass and the hybrid biosorbent, our data deposed in favour of the use of M. aquaticum as dead biomass for a possible application of this invasive macrophyte in the biological treatment of metal-contaminated water. Our findings may be beneficial to metal removal application accompanying wetland management, devising a possible use of M. aquaticum waste material after its removal from the invaded habitats.

How ecosystems change following invasion by Robinia pseudoacacia: Insights from soil chemical properties and soil microbial, nematode, microarthropod and plant communities.

Biological invasions are a global threat to biodiversity. Since the spread of invasive alien plants may have many impacts, an integrated approach, assessing effects across various ecosystem components, is needed for a correct understanding of the invasion process and its consequences. The nitrogen-fixing tree Robinia pseudoacacia (black locust) is a major invasive species worldwide and is used in forestry production. While its effects on plant communities and soils are well known, there have been few studies on soil fauna and microbes. We investigated the impacts of the tree on several ecosystem components, using a multi-trophic approach to combine evidence of soil chemical properties and soil microbial, nematode, microarthropod and plant communities. We sampled soil and vegetation in managed forests, comparing those dominated by black locust with native deciduous oak stands. We found qualitative and quantitative changes in all components analysed, such as the well-known soil nitrification and acidification in stands invaded by black locust. Bacterial richness was the only component favoured by the invasion. On the contrary, abundance and richness of microarthropods, richness of nematodes, and richness and diversity of plant communities decreased significantly in invaded stands. The invasion process caused a compositional shift in all studied biotic communities and in relationships between the different ecosystem components. We obtained clear insights into the effects of invasion of managed native forests by black locust. Our data confirms that the alien species transforms several ecosystem components, modifying the plant-soil community and affecting biodiversity at different levels. Correct management of this aggressive invader in temperate forests is urgently required.

Oomycete Communities Associated with Reed Die-Back Syndrome.

Phragmites australis (Cav.) Trin. ex Steud. die-back is a widely-studied phenomenon that was first discovered in northern Europe and that, until recently, was almost unknown in the Mediterranean basin. It has been described as a complex syndrome affecting reed populations leading to their retreat and decline, with significant impacts on valuable ecosystem services. Among the factors that cause the decline, soil-living microorganisms can be crucial. The aims of this study were to analyze the diversity of oomycetes communities associated with reed stands, and to understand whether they could play a key role in the decline. Variations in the structure of oomycetes communities were studied by metabarcoding of the internal transcribed spacer (ITS) 1 region of ribosomal DNA, from the sediments of five Italian freshwater ecosystems. They were chosen to cover a large variability in terms of surface area, water depth, microclimate, and presence of documented reed retreat. From 96 samples collected from reed roots, rhizosphere, and bulk soil, we assembled 207661 ITS1 reads into 523 OTUs. We demonstrated that oomycete communities were structured by several factors, among which the most important was die-back occurrence. Our study also indicates that Pythiogeton spp. could be potentially involved in the development of die-back. The role of heavy metals in the soil was also explored, and cadmium concentration was shown to affect oomycetes distribution. This study represents a significant step forward for the characterization of microbial communities associated with reed die-back syndrome and helps to gain knowledge of the complexity of these important wet ecosystems.

Review of Hydrometra gracilenta Horváth, 1899 (Hemiptera: Heteroptera: Hydrometridae) in Italy, with notes on its general distribution.

An annotated review of the known Italian distribution of Hydrometra gracilenta Horváth, 1899 is provided, plus unpublished data including a new record which increases the previously known range of the species in Italy. An updated map shows the currently known distribution of the taxon in Italy. An overlooked nomenclatural change is discussed, and the date of description for the genus Hydrometra Lateille is corrected from 1796 to 1797. The presence of H. gracilenta is excluded from Egypt, Sudan, and Rwanda.

New occurrence of reed bed decline in southern Europe: do permanent flooding and chemical parameters play a role?

Based on the experimental design proposed in similar studies, macromorphological and ecological traits of common reed beds were analysed at Lake Chiusi (Central Italy), together with selected chemical parameters in sediments and interstitial waters and aerial images of the site, in order to investigate reed decline and search for possible correlations among data. Typical symptoms of the reed dieback syndrome were detected, thus enlarging the occurrence of this phenomenon in southern Europe. Permanently dry, permanently flooded and partially flooded stands show different levels of decline, with the permanent flooding always co-occurring with reed dieback. Only few of the considered chemical parameters seem to play a role in reed decline (nitrates, rubidium, nickel, barium, manganese), although no clear pattern was identified. Data suggest that the co-occurrence of some chemicals with stressing conditions might affect the growth even of an efficient metal accumulator, as reed is generally considered.

Human disturbance threats the red-listed macrolichen Seirophora villosa (Ach.) Frödén in coastal Juniperus habitats: evidence from western peninsular Italy.

In Europe, coastal dune systems with Juniperus spp. (Natura 2000 habitat code 2250) are a priority habitat for conservation according to the Natura 2000 policies. Currently, anthropogenic pressure is threatening the biodiversity of this habitat. While the impact of human pressure on animals and vascular plants is already documented, information is still scanty for other organisms such as epiphytic lichens. The main aim of this study is to test the effect of human disturbance on the occurrence and abundance of the red-listed macrolichen Seirophora villosa. We also tested the effect of human disturbance on the whole community of epiphytic lichens in terms of species richness and composition. The study was performed along the coast of Tuscany by comparing both disturbed and undisturbed Juniperus stands according to a stratified random sampling design. Our results provided evidence that in coastal systems the long-term conservation of the red-listed macrolichen S. villosa and its characteristic community composed by several Mediterranean species of conservation concern depends on the maintenance of undisturbed Juniperus habitats. Results also support the possibility of using S. villosa as an indicator species of habitat conservation importance and habitat integrity since its occurrence is predicted on nestedness in term of species composition, assemblages of species poor disturbed stands being subsets of those of richer undisturbed stands.

CORINE land cover and floristic variation in a Mediterranean wetland.

The aims of the present study were to: (1) investigate whether CORINE land cover classes reflect significant differences in floristic composition, using a very detailed CORINE land cover map (scale 1:5000); (2) decompose the relationships between floristic assemblages and three groups of explanatory variables (CORINE land cover classes, environmental characteristics and spatial structure) into unique and interactive components. Stratified sampling was used to select a set of 100-m(2) plots in each land cover class identified in the semi-natural wetland surrounding a lake in central Italy. The following six classes were considered: stable meadows, deciduous oak dominated woods, hygrophilous broadleaf dominated woods, heaths and shrublands, inland swamps, canals or watercourses. The relationship between land cover classes and floristic composition was tested using several statistical techniques in order to determine whether the results remained consistent with different procedures. The variation partitioning approach was applied to identify the relative importance of three groups of explanatory variables in relation to floristic variation. The most important predictor was land cover, which explained 20.7% of the variation in plant distribution, although the hypothesis that each land cover class could be associated with a particular floristic pattern was not verified. Multi Response Permutation Analysis did not indicate a strong floristic separability between land cover classes and only 9.5% of species showed a significant indicator value for a specific land cover class. We suggest that land cover classes linked with hygrophilous and herbaceous communities in a wetland may have floristic patterns that vary with fine scale and are not compatible with a land cover map.