Characterization of the cryptic interspecific hybrid Lemna × mediterranea by an integrated approach provides new insights into duckweed diversity.

Lemnaceae taxonomy is challenged by the particular morphology of these tiny free-floating angiosperms. Although molecular taxonomy has helped clarify the phylogenetic history of this family, some inconsistency with morphological data leads to frequent misclassifications in the genus Lemna. Recently, the finding that Lemna japonica is an interspecific hybrid between Lemna minor and Lemna turionifera, provided a clear explanation to one of such taxonomic questions. Here we demonstrated that L. minor is also capable to hybridize with Lemna gibba, generating a cryptic but widespread taxon in the Mediterranean area. The nothotaxon Lemna × mediterranea is described and compared with clones of the putative parental species L. minor and L. gibba. Genetic analysis by nuclear and plastid markers, as well as genome size measurement, revealed that two different cytotypes, diploid and triploid, originated by at least two independent hybridization events. Despite high overall similarity, morphometrical, physiological and biochemical analyses showed an intermediate position of L. × mediterranea between its parental species in most qualitative and quantitative characters, and also separation of the two hybrid cytotypes by some criteria. These data provide evidence that hybridization and polyploidization, driving forces of terrestrial plant evolution, contribute to the duckweed genetic diversity and may have shaped the phylogenetic history of these mainly asexual, aquatic plants.

Dye-Doped Polymeric Microplastics: Light Tools for Bioimaging in Test Organisms.

Ecosystems around the world are experiencing a major environmental impact from microplastic particles (MPs 0.1 µm-1 mm). Water, sediments, and aquatic biota show the widespread presence of this pollutant. However, MPs are rarely used in laboratory studies as they are scarcely available for purchase or expensive, especially if one wishes to trace the particle with a dye or fluorescent. Furthermore, existing preparation techniques have limited application in biological studies. In this work, we propose a new, easy, and cheap way to prepare fluorescent MPs. The protocol is based on the osmosis method in order to obtain spherical polymeric particles of P(S-co-MMA), with 0.7-9 micron diameter, made fluorescent because dye-doped with rhodamine B isothiocyanate (RITC) or fluorescein isothiocyanate (FITC). The dye loading was studied and optimized, and the MPs-dye conjugates were characterized by UV-vis FTIR and XPS spectrometry and scanning electron microscopy (SEM). Furthermore, preliminary tests on aquatic organisms demonstrated the possible use of these fluorescent MPs in bioimaging studies, showing their absorption/adsorption by duckweeds (Lemna minuta) and insect larvae (Cataclysta lemnata).

Impact of the Invasive Alien Macrophyte Ludwigia hexapetala on Freshwater Ecosystems: Evidence from Field Data.

Biological invasions are a serious threat to biodiversity conservation, especially in freshwater ecosystems. The American macrophyte Ludwigia hexapetala, which colonizes both the aquatic and bank habitats of lakes, rivers, and canals, is invading many waterbodies in Europe, becoming an increasingly worrisome threat in several European countries, including Italy. However, only fragmentary information is available on the actual impact of its invasion in these habitats. This study aims to collect field data from various freshwater habitats in central and northern Italy, to assess the possible impact of L. hexapetala on the environmental parameters and plant biodiversity of the invaded habitats. The results show that in aquatic habitats, dense floating L. hexapetala populations reduce the light levels and oxygen available in the water, consequently limiting the growth of other aquatic plants. Indeed, L. hexapetala populations negatively affect aquatic plant diversity, as an increase in L. hexapetala cover corresponded to a decrease in Simpson's diversity index. In contrast, in bank habitats, L. hexapetala has no significant impact on plant diversity. Evidence suggests that some native species, such as Phragmites australis, which generally form compact populations along the banks, effectively counteract the invasion of L. hexapetala. This information may prove valuable for the environmental managers of those freshwater habitats where L. hexapetala invasion needs to be addressed and controlled.

Trophic transfer of microplastics from producer (Lemna minuta) to primary consumer (Cataclysta lemnata) in a freshwater food chain.

Contamination by microplastics (0.1 µm-5 mm plastic fragments) is currently one of the major threats to the conservation of aquatic and terrestrial ecosystems worldwide. Growing awareness on this issue has led to an increase in studies on the effects of microplastics on freshwater organisms, although there are still few investigations on possible transfer of this contaminant along water trophic chains from producers to primary consumers. In this study, aquatic herbivorous larvae of the moth Cataclysta lemnata were fed on microplastic-free (control) and contaminated (MPs treatment) Lemna minuta fronds. For treatments, Lemna fronds were grown in mineral water enriched with fluorescent microparticles of poly(styrene-co-methyl methacrylate) (MPs, 100 mg/L) and then fed to the larvae as a food source. Microplastics effects on larvae were tested at 0, 7, 14 and 21 days of exposure, corresponding to sensitive phases of the insect life cycle. Contaminant impact was assessed based on some parameters such as viability, larva body size/weight, feeding alterations and regularity of the insect life cycle. Using scanning electron and fluorescence microscopy, the presence of microplastics in the larvae was verified. The finding of fluorescent microplastics in both the intestinal lumen and excrement samples showed that larvae ingested contaminated Lemna fronds. In addition, larvae fed contaminated fronds were strongly affected by the presence of microplastic contaminant over time, showing high mortality (90 %) and total inability to complete the life cycle after 21 days by failing to reach the winged adult phase. In control tests, survival rates were higher than in treatments, and 50 % of the larvae managed to pupate and emerge as moths, reaching the adult phase. The results show that there was a trophic transfer of microplastics from producer to primary consumer along a freshwater food chain, generating negative effects on the life cycle of this aquatic herbivore.

Impact of the Alien Aquatic Plant Ludwigia hexapetala on the Native Utricularia australis: Evidence from an Indoor Experiment.

Ludwigia hexapetala is an alien aquatic plant considered highly invasive in Europe since it alters freshwater habitats by forming dense mats both in water and along banks, outcompeting natives. Ludwigia effects on the native carnivorous plant Utricularia australis were investigated here. A 21-day indoor experiment was performed by setting up some separate tests in which Utricularia was made to grow both alone (control tests) and together with Ludwigia (arena tests). Water chemical and physical parameters and growth and morphological traits of Utricularia and Ludwigia were analysed weekly. Water samples were also analysed by UV-visible spectra to verify allelochemical (quercitrin) production by Ludwigia. In arena tests, oxygen concentration and pH were lower and conductivity higher than in control tests. Utricularia grew significantly less in arena tests in both shoot length and internode number, and its fresh weight, trap number and internode length decreased more than the control. Quercitrin was found only in arena tests as an allelochemical product released by Ludwigia. Overall, this study demonstrated Ludwigia significantly alters water parameters and negatively affects the growth of Utricularia, showing aggressive and competitive behaviour against this native species. Such evidence suggest that the widespread of L. hexapetala can represent a serious threat to the conservation of native plant diversity occurring in the freshwater habitats it invades.

Effects of Microplastic Contamination on the Aquatic Plant Lemna minuta (Least Duckweed).

Microplastics are widely spread in aquatic environments. Although they are considered among the most alarming contaminants, toxic effects on organisms are unclear, particularly on freshwater plants. In this study, the duckweed Lemna minuta was grown on different concentrations (50, 100 mg/L) of poly(styrene-co-methyl methacrylate) microplastics (MP) and exposure times (T0, T7, T14, T28 days). The phytotoxic effects of MP were investigated by analyzing several plant morphological and biochemical parameters (frond and root size, plant growth, chlorophyll, and malondialdehyde content). Observations by scanning electron microscope revealed MP adsorption on plant surfaces. Exposition to MP adversely affected plant growth and chlorophyll content with respect to both MP concentrations and exposure times. Conversely, malondialdehyde measurements did not indicate an alteration of oxidative lipid damage in plant tissue. The presence of MP induced root elongation when compared to the control plants. The effects of MP on L. minuta plants were more evident at T28. These results contribute to a better understanding of MP's impact on aquatic plants and highlight that MP contamination manifests with chronic-type effects, which are thus detectable at longer exposure times of 7 days than those traditionally used in phytotoxicology tests on duckweeds.

Effects of Partial Substitution of Conventional Protein Sources with Duckweed (Lemna minor) Meal in the Feeding of Rainbow Trout (Oncorhynchus mykiss) on Growth Performances and the Quality Product.

Duckweed (Lemna minor) meal was included in the formulation of three experimental feeds (L1, L2, L3) for rainbow trout at 10%, 20%, 28% of the protein source, respectively. Increasing the duckweed inclusion, the other protein sources were adjusted to get isonitrogenous (41%) and isolipidic (20%) diets, as the control diet (LC). 540 fish (mean body weight 124.5 ± 0.7 g) were randomly allocated in 12 tanks divided equally among the four different diets. After 90 days, fish were weighed and the most important productive performances, fillet quality and fatty acid profile were determined. The final body weight in L1 (340.53 g) and L2 (339.42 g) was not different from LC (348.80 g); L3 trout significantly (p < 0.05) exhibited the lowest one (302.16 g). Similar trends were found in final mean length, weight gain, specific growth rate, food conversion rate. Somatic indices were affected by duckweed inclusion. Diets had not significant effects on the proximate composition and fatty acids of the fillet in L1, L2, L3 respect to LC. Based on this study, duckweed meal derived from Lemna minor can be included in the feed for the rainbow trout without negative effects on the growth performances at 20% of the protein substitution.

Plant DNA Barcode as a Tool for Root Identification in Hypogea: The Case of the Etruscan Tombs of Tarquinia (Central Italy).

Roots can produce mechanical and chemical alterations to building structures, especially in the case of underground historical artifacts. In archaeological sites, where vegetation plays the dual role of naturalistic relevance and potential threat, trees and bushes are under supervision. No customized measures can be taken against herbaceous plants lacking fast and reliable root identification methods that are useful to assess their dangerousness. In this study, we aimed to test the efficacy of DNA barcoding in identifying plant rootlets threatening the Etruscan tombs of the Necropolis of Tarquinia. As DNA barcode markers, we selected two sections of the genes rbcL and matK, the nuclear ribosomal internal transcribed spacer (nrITS), and the intergenic spacer psbA-trnH. All fourteen root samples were successfully sequenced and identified at species (92.9%) and genus level (7.01%) by GenBank matching and reference dataset implementation. Some eudicotyledons with taproots, such as Echium italicum L., Foeniculum vulgare Mill., and Reseda lutea L. subsp. lutea, showed a certain recurrence. Further investigations are needed to confirm this promising result, increasing the number of roots and enlarging the reference dataset with attention to meso-Mediterranean perennial herbaceous species. The finding of herbaceous plants roots at more than 3 m deep confirms their potential risk and underlines the importance of vegetation planning, monitoring, and management on archaeological sites.

Aquatic plants and ecotoxicological assessment in freshwater ecosystems: a review.

This paper reviews the current state-of-the-art, limitations, critical issues, and new directions in freshwater plant ecotoxicology. We selected peer-reviewed studies using relevant databases and for each (1) publication year, (2) test plant species, (3) reference plant group (microalgae, macroalgae, bryophytes, pteridophytes, flowering plants), (4) toxicant tested (heavy metal, pharmaceutical product, hydrocarbon, pesticide, surfactant, plastic), (5) experiment site (laboratory, field), and (6) toxicant exposure duration. Although aquatic plant organisms play a key role in the functioning of freshwater ecosystems, mainly linked to their primary productivity, their use as biological models in ecotoxicological tests was limited if compared to animals. Also, toxicant effects on freshwater plants were scarcely investigated and limited to studies on microalgae (80%), or only to a certain number of recurrent species (Pseudokirchneriella subcapitata, Chlorella vulgaris, Lemna minor, Myriophyllum spicatum). The most widely tested toxicants on plants were heavy metals (74%), followed by pharmaceutical products and hydrocarbons (7%), while the most commonly utilized endpoints in tests were plant growth inhibition, variations in dry or fresh weight, morpho-structural alterations, chlorosis, and/or necrosis. The main critical issues emerged from plant-based ecotoxicological tests were the narrow range of species and endpoints considered, the lack of environmental relevance, the excessively short exposure times, and the culture media potentially reacting with toxicants. Proposals to overcome these issues are discussed.

Impact of invasive alien plants on native plant communities and Natura 2000 habitats: State of the art, gap analysis and perspectives in Italy.

Invasive alien plants are a major threat to biodiversity and they contribute to the unfavourable conservation status of habitats of interest to the European Community. In order to favour implementation of European Union Regulation no. 1143/2014 on invasive alien species, the Italian Society of Vegetation Science carried out a large survey led by a task force of 49 contributors with expertise in vegetation across all the Italian administrative regions. The survey summed up the knowledge on impact mechanisms of invasive alien plants in Italy and their outcomes on plant communities and the EU habitats of Community Interest, in accordance with Directive no. 92/43/EEC. The survey covered 241 alien plant species reported as having deleterious ecological impacts. The data collected illustrate the current state of the art, highlight the main gaps in knowledge, and suggest topics to be further investigated. In particular, the survey underlined competition as being the main mechanism of ecological impact on plant communities and Natura 2000 habitats. Of the 241 species, only Ailanthus altissima was found to exert an ecological impact on plant communities and Natura 2000 habitats in all Italian regions; while a further 20 species impact up to ten out of the 20 Italian administrative regions. Our data indicate that 84 out of 132 Natura 2000 Habitats (64%) are subjected to some degree of impact by invasive alien plants. Freshwater habitats and natural and semi-natural grassland formations were impacted by the highest number of alien species, followed by coastal sand dunes and inland dunes, and forests. Although not exhaustive, this research is the first example of nationwide evaluation of the ecological impacts of invasive alien plants on plant communities and Natura 2000 Habitats.

Phytoremediation potential of the duckweeds Lemna minuta and Lemna minor to remove nutrients from treated waters.

Phytoremediation potential of duckweeds (Lemna minuta, Lemna minor) to remove nutrients from simulated wastewater was analyzed. In two separate experiments, the two species were grown for 28 days in waters enriched with nitrate and phosphate to simulate nutrient concentrations of domestic wastewater. Water physical and chemical measurements (temperature, pH, conductivity, oxygen) and plant physiological and biochemical analysis (biomass, relative growth rate-RGR, nutrient and chlorophyll contents, peroxidative damage, bioconcentration factor-BCF) were made to test and compare the phytoremediation capacity of the two Lemna species. L. minuta biomass increased almost tenfold during the time-course of the treatment resulting in a doubling of the mat thickness and a RGR of 0.083 ± 0.001 g/g day. Maximum frond content of phosphate was reached by day 21 (increase over 165%) and nitrate by day 7 (10%). According to the BCF results (BCF > 1000), L. minuta was a hyperaccumulator for both nutrients. On the other hand, L. minor biomass and mat thickness decreased continuously during incubation (RGR = - 0.039 ± 0.004 g/g day). In L. minor fronds, phosphate content increased until day 14, after which there was a decrease until the end of the incubation. Frond nitrate content significantly decreased by day 7, but then remained relatively constant until the end of the experiment. L. minor proved to be hyperaccumulator for phosphates, but not for nitrates. Results indicated L. minuta has a greater potential than L. minor to remove both nutrients by bioaccumulation, especially phosphates, demonstrated also by better physiological and biochemical responses. However, during the incubation, the chlorophyll content of L. minuta mat did continuously decrease and peroxidative damage had increased until day 14, indicating that the system was under some kind of stress. Strategies to avoid this stress were discussed.

The Hydra regeneration assay reveals ecological risks in running waters: a new proposal to detect environmental teratogenic threats.

The regenerative ability of Hydra vulgaris was tested as potential biomarker for the development of a new eco-toxicological index. The test is based on the regeneration rate and the aberration frequency of the columna (body and adhesive foot) after separation from head and tentacles by a bistoury. Particularly, 45 columnae were submerged in the rearing solution (that is Hydra medium) to have control, and 285 in potential contaminated waters to have treatments, collected from 19 sites along 10 rivers in central Italy. ANCOVA and chi-square tests were used to compare values from each site to a laboratory control. Subsequently the values on regeneration rate and aberration frequency were inserted in a double entry matrix, where the match of the two entries in the matrix provides the score of the proposed Teratogenic Risk Index (TRI). Each score corresponded to one of the 5 teratogenic risk classes, to which a risk level was associated: from 1 (no risk) to 5 (very high risk). On the whole, 32% of the studied sites were classified as no teratogenic risk while the remaining showed a variable risk level from low to very high. This study proposed for the first time an early warning system to detect the presence of teratogens in running waters, providing a rapid and cost-effective evaluation method. Therefore, TRI may contribute to initiate adequate measures to manage riverine habitats, and to monitor the running water teratogenic status. Specifically, this index may provide the opportunity to identify the disturbance sources and then to drive the decisions, together with competent authorities, on the catchment and landscape management and on the possible use of waters for urban, agricultural, and industrial activities, since they may show significant effects on the human health.

Response of macrophyte communities to flow regulation in mountain streams.

River macrophytes are widely used in freshwater ecosystem assessment because of their sensitivity to anthropogenic pressures, even if there are only a few studies that investigated how macrophytes respond to water regime alterations. In this study, we analyzed the effects of dams on river macrophyte communities through a comparison between upstream and downstream sides from 18 dams located in Alps and Apennines. A co-inertia analysis and a Mantel test were applied to assess if the analysis of environmental parameters could be effective in predicting macrophyte community structure. We analyzed morphological and physicochemical inter-site differences and tested the influence of dams on various aspects of community structure (composition, richness, diversity, dominance, coverage) using multivariate randomized block permutation procedure. Plant similarity between sites was evaluated at the level of phylum, and indicator species analysis was performed to identify the taxa most sensitive or tolerant to water regulation. We found that the overall environmental setting overwhelms the dam impact and that the influence of hydrological alteration became apparent when comparing upstream and downstream assemblages at the same dam. In particular, we found that most of taxa had a higher affinity with the downstream side and that in general, stream regulation increases plant richness and coverage, but reduces community evenness. Analyses based on higher taxonomic groups (phyla) demonstrated that this community can be effectively used in bioassessment even at phylum level analysis. In particular, we found that bryophytes, strictly linked with changes in substrate stability, show particular sensitivity to water regulation in mountain streams.

The effect of river damming on vegetation: is it always unfavourable? A case study from the River Tiber (Italy).

River damming leads to strong hydromorphological alterations of the watercourse, consequently affecting river vegetation pattern. A multitemporal and spatial analysis of the dam effect on composition, structure and dynamic of the upstream vegetation was performed on Tiber River at Nazzano-dam (Rome). The main research questions were as follows: How does plant landscape vary over time and along the river? Where does the dam effect on vegetation end? How does naturalistic importance of the vegetation affected by damming change over time? Data collection was performed mapping the vegetation in aerial photos related to the period before (1944), during (1954) and after dam construction (1984, 2000). The plant landscape has significantly changed over time and along the river, particularly as a result of the dam construction (1953). The major vegetation changes have involved riparian forests and macrophytes. Dam effect on vegetation is evident up to 3 km, and gradually decreases along an attenuation zone for about another 3 km. Despite the fact that the damming has caused strong local hydromorphological modification of the river ecosystem transforming it into a sub-lacustrine habitat, it has also led to the formation of wetlands of considerable naturalistic importance. Indeed, in these man-made wetlands, optimal hydrological conditions have been created by favouring both the expansion of pre-existing riparian communities and the rooting of new aquatic communities, albeit typical of lacustrine ecosystems. Some of these plant communities have become an important food resource, refuge or nesting habitats for aquatic fauna, while others fall into category of Natura 2000 habitats. Therefore, river damming seems to have indirectly had a "favourable" effect for habitat conservation and local biodiversity.

Concordance between macrophytes and macroinvertebrates in a Mediterranean river of central Apennine region.

The main aim of this study was to improve the knowledge about the concordance among macrophytes and macroinvertebrates to provide complementary information and facilitate the procedures for quality assessment of river ecosystems. Macrophytes and macroinvertebrates were collected in 11 sampling sites along a central Apennine calcareous river in October 2008 and June 2009. The concordance between the two biomonitoring groups was tested according to several environmental parameters. The comparison of data matrix similarities by Mantel test showed differences in the assemblage of macrophytes and macroinvertebrates along the river since correlation values were 0.04, p > 0.05 in October 2008 and 0.39, p > 0.05 in June 2009. The study revealed lack of concordance between the two groups, emphasizing that the information provided by macrophytes and macroinvertebrates does not overlap in terms of response to environmental parameters. Indeed, the two different biological groups resulted useful descriptors of different parameters. Together, they could represent a complementary tool to reflect the river environmental quality.

Multitemporal floristic analysis on a humid area in Rome's archaeological site as indicator for environmental change.

A multitemporal analysis on a humid area in Rome's archaeological site is presented; the floristic data are used as bioindicators for main environmental changes over the last 50 years. By comparing the structural, biogeographical and ecological features of today's florula with the ones of a 1955 survey, and by assessing the new, the rare and the locally disappeared species, it was possible to define and analyse the main floristic changes and their ecological meaning for this site. Our results show that the floristic richness of the area did not differ significantly from 1955. However, the species composition has changed considerably: over 40% of the species have disappeared. More than half of these were rare and typical of humid environments. Approximately the same number of species has appeared but these are mainly ruderal and widespread entities, with the overall result being a progressive and general vulgarization of the local flora. Finally, we analyse the main reasons, linked for the most part to human activities, which led to the reported floristic changes.