We present the first isotopic dataset for two shallow, slightly brackish Mediterranean lakes in Sicily colonised by Chara baltica (Lago Preola) and Najas marina (Lago Murana). Seasonal stable isotopes (δ13C, δ15N) and elements concentration (C%, N%) were measured in macrophytes and sediments over one year. Lago Murana sediments were more 13C-depleted than Lago Preola. Both lakes showed similar seasonality: 13C-enriched and 15N-depleted values in spring–summer, lower in autumn–winter. Greater seasonal variability in Preola indicates macrophyte-derived organic matter input to sediments, whereas Murana reflects run-off from surrounding pastures and vineyards. These results offer a preliminary framework for future work.

This study aimed to evaluate the effects of ultrasound treatment (UA), pasteurization (P), and their combination on juices from seven Citrus aurantium cultivars: Consolei, Fasciata, Listata, Crispifolia, Foetifera, Bizzarria, and Canaliculata. Both untreated and processed juices were analysed using high-performance liquid chromatography (HPLC) and physicochemical methods. The health-promoting properties were assessed by testing the inhibition of carbohydrate hydrolysing enzymes, and pancreatic lipase. Antioxidant activity was also measured using the 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 '-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and Ferric Reducing Antioxidant Power (FRAP) assays. The combined use of ultrasound followed by pasteurization proved effective in enhancing the extraction and stabilization of bioactive compounds, thus improving the antioxidant and nutritional profiles of the juices. Hesperidin was identified as the predominant compound, with concentrations ranging from 176.4 to 1804.6 mg/L in untreated C. aurantium cv Foetifera and Consolei, respectively, followed by naringenin. Although the impact of pasteurization alone differed among cultivars, it generally led to a decrease in antioxidant activity and the ability to inhibit alpha-amylase, alpha-glucosidase and pancreatic lipase. Across all cultivars, combined ultrasound and pasteurization significantly increased (p < 0.01) phenolic and flavonoid levels compared to untreated juice, demonstrating a consistent treatment effect. The application of pasteurization alone reduced the antioxidant activity as well as the enzyme inhibitory activity but combining it with ultrasound assisted extraction led to significant recovery, with an increase of + 100.0 % in alpha-amylase inhibition and + 74.5 % in pancreatic lipase inhibition. Overall, the obtained results showed that ultrasound pre-treatment of juice is an effective method to improve the extraction of bioactive compounds and led to maintain high levels of these molecules even after the pasteurization process.

Conventional thermal pasteurization is widely applied to ensure the safety of fruit juices, although its impact on bioactive compounds and functional properties may vary according to cultivar. This study evaluated the effects of conventional pasteurization on physicochemical parameters, bioactive composition, antioxidant capacity, and enzyme inhibitory activities of juices obtained from five Sicilian Citrus grandis cultivars (Todarii, Maxima, Pyriformis, Chadock, and Terracciani). Total polyphenols, flavonoids, and carotenoids were quantified, while flavanone profiles were characterized by means of HPLC analysis. Antioxidant activity was assessed using DPPH, ABTS, FRAP, and β-carotene bleaching assays, and in vitro inhibitory activities against α-amylase, α-glucosidase, and pancreatic lipase were determined. Pasteurization led to cultivar-dependent reductions in total polyphenols (up to ~40%), flavonoids (up to ~45%), and carotenoids (up to ~25%), accompanied by decreased radical scavenging capacity and reducing power. Naringin was identified as the predominant flavanone, with thermal processing inducing both degradation and release phenomena depending on the cultivar. Fresh juices exhibited stronger enzyme inhibitory activities, particularly against α-amylase and α-glucosidase. Multivariate analysis discriminated against fresh and pasteurized juices, identifying phenolics as the main contributors to antioxidant capacity. Despite bioactive reductions, functional quality was partially preserved, supporting targeted cultivar selection for optimized industrial processing.

Plants as animals are associated with a cortege of microbes influencing their health, fitness and evolution. Scientists refer to all living organisms as holobionts, complex genetic units that coevolve simultaneously. This is what has been recently proposed as the hologenome theory. This exciting theory has important implications on animal and plant health; however, it still needs consistent proof to be validated. Indeed, holobionts are still poorly studied in their natural habitats where coevolution processes occur. Compared to animals, wild plant populations are an excellent model to explore the hologenome theory. These sessile holobionts have coevolved with their microbiota for decades, and natural selection and adaptive processes acting on wild plants are likely to regulate the plant-microbe interactions. Here, we conducted a microbiota survey, plant genome sequencing and genome-environmental analysis (GEA) of 26 natural populations of the plant species Brassica rapa. We collected plants over two seasons in Italy and France and analysed the root and rhizosphere microbiota. When conducting GEA, we evidenced neat peaks of association correlating with both fungal and bacterial microbiota. Surprisingly, we found 13 common genes between fungal and bacterial diversity descriptors that we referred to under the name of holobiont generalist genes (HGGs).

In the present study, the chemical composition of the essential oil from aerial parts of a very rare Centaurea species, not previously investigated, Centaurea erycina Raimondo and Bancheva, collected in Sicily, was evaluated by GC-MS. The new species, classified just twenty years ago, belongs to Centaurea cineraria group (sect. Pannophyllum Hayek, Asteraceae) and grows in an extremely limited area in the NW part of Sicily. The oil was shown to be rich of aldehydes (41.4%) and sesquiterpenes (33.4%). The main components of the essential oil were β-caryophyllene (13.4%), caryophyllene oxide (12.6%), hexanal (11.9%), and trans-2-hexenal (10.0%). Furthermore, a complete literature review on the composition of the essential oils of all the other taxa of Centaurea, belonging to sections Pannophyllum, studied so far, was carried out.

Mandarin, one of the winter fruits commonly used in the preparation of foods and juices, is a fruit native to China and Southeast Asia. In this work, essential oils (EOs) obtained from by-products of the Citrus reticulata Blanco flavedo of five cultivars present and cultivated within the Botanical Garden of Palermo were chemically and biologically studied: C. reticulata ‘Avana’ (C1), C. reticulata ‘Tardivo di Ciaculli’ (C2), C. reticulata ‘Bombajensis’ (C3), C. reticulata ‘Aurantifolia’ (C4), and C. reticulata ‘Padre Bernardino’ (C5). The GC and GC-MS analysis performed on all the extracted samples clearly highlighted the notable presence of limonene, a characteristic hydrocarbon monoterpene of EOs of the Citrus genus. C1, C2, C3, C4, and C5 were tested in relation to their possible antibacterial and allelopathic activity, also highlighting the activity of limonene, the main compound. For the antibacterial activity, eight different bacterial strains were used, both Gram-positive and Gram-negative (Staphylococcus aureus, Proteus vulgaris, Klebsiella pneumoniae, Enterobacter cloacae, Pseudomonas aeruginosa, Escherichia coli, Salmonella typhi, and Enterobacter aerogens). For the allelopathic effect, two model systems were chosen: the germination of radish seeds (Raphanus sativus L.) and of spores in the moss Tortula muralis (Hedw.). The EOs from all cultivars showed pronounced antibacterial effects against all strains with an MIC comprised in the range of 16–256 μg/mL. Limonene showed the highest activity with an MIC between 4 and 16. The allelopathic effects showed a decrease in the percentage of seed germination, root, and epicotyl growth on Raphanus and a strong reduction in the germination of Tortula spores with an alteration in the development of the protonema. Limonene showed the same but more intense allelopathic activity.

Agriculture faces great challenges to overcome global warming and improve system sustainability, requiring access to novel genetic diversity. So far, wild populations and local landraces remain poorly explored. This is notably the case for the two diploid species, Brassica oleracea L. (CC, 2n=2x=18) and B. rapa L. (AA, 2n=2x=20). In order to explore the genetic diversity in both species, we have collected populations in their centre of origin, the Mediterranean basin, on a large contrasting climatic and soil gradient from northern Europe to southern sub-Saharan regions. In these areas, we also collected 14 populations belonging to five B. oleracea closely related species. Our objective was to ensure the absence of species misidentification at the seedling stage among the populations collected and to describe thereafter their origins. We combined flow cytometry, sequencing of a species-specific chloroplast genomic region, as well as cytogenetic analyses in case of unexpected results for taxonomic verification. Out of the 112 B. oleracea and 154 B. rapa populations collected, 103 and 146, respectively, presented a good germination rate and eighteen populations were misidentified. The most frequent mistake was the confusion of these diploid species with B. napus. Additionally for B. rapa, two autotetraploid populations were observed. Habitats of the collected and confirmed wild populations and landraces are described in this study. The unique plant material described here will serve to investigate the genomic regions involved in adaptation to climate and microbiota within the framework of the H2020 Prima project ‘BrasExplor’.

AbstractNowadays, secondary raw materials (SRM) obtained from plant matrices are of great interest for circular economy, suitable for sustainable measures to reduce environmental impact. This work focused on the extraction, characterization and quantification of compounds obtained from leaves and fruits of the Sicilian sumac, Rhus coriaria L. and their application as natural dyes on textile fibres. Extractions were performed with Extractor Naviglio®, maceration and ultrasound assisted methods and food-grade solvents (aqueous and hydroalcoholic) to evaluate the yields for dye compounds. The presence of colouring molecules was evaluated by UV–Vis spectrophotometer, and the extracts selected for colouring were quantified and characterized by LC–MS. The results showed that Extractor Naviglio® achieved the best extraction yield, and the ethanol–water mixture extracts had a higher amount of total phenolic compounds (TPC) and a higher content of total colouring compounds (TCC). These extracts were selected for subsequent applications as dyes for linen, cotton and wool. The chemical profile of selected extracts was rich in compounds such as gallotannin and anthocyanin class. Fibre dyeing was verified by recording CIELAB colouring coordinates. The results suggest that the dyes obtained from R. coriaria can be of great interest for artisanal and industrial processes, in accordance with environmental sustainability.

Jacobaea is a small genus of plants belonging to Asteraceae family, formerly joint to Senecio genus, that includes more than 60 species and sub-species. Several taxa of this genus have been extensively studied both for their non-volatile and volatile metabolites. In the present study the chemical composition of the essential oil (EO) from aerial parts of Jacobaea delphiniifolia (Vahl) Pelser & Veldkamp, collected in Sicily, was analyzed by GC-MS. No one report has been previously published on the EO of this species. The result showed the presence of a large quantity of two metabolites, namely 1-undecene (63.57%) and thymol methyl ether (13.65%). Chemotaxonomic considerations were carried out by comparing the other oils of Jacobaea taxa studied so far.

Plants as animals are strictly associated with a cortege of microbial communities influencing their health, fitness and evolution. Therefore, scientists refer to all living organisms as holobionts; complex genetic units that coevolve simultaneously. This is what has been recently proposed as the hologenome theory of evolution. This exciting and attractive theory has important implications on animal and plant health; however, it still needs consistent proof to be validated. Indeed, holobionts are still poorly studied in their natural habitats where coevolution and natural selective processes occur. Compared to animals and crops, wild plant populations are an excellent and unique model to explore the hologenome theory. These sessile holobionts have strictly coevolved with their microbiota for decades and natural selection and adaptive processes acting on wild plants are likely to regulate the plant-microbe interactions. Here we conducted for the first time a microbiota survey, plant genome sequencing and Genome-Environmental Analysis (GEA) of 26 natural populations of the non-model plant species Brassica rapa . We collected plants over two seasons in Italy and France, and analyzed the microbiota on two plant compartments (root and rhizosphere). We identified that plant compartment and season modulate B. rapa microbiota. More importantly, when conducting GEA we evidenced neat peaks of association correlating with both fungal and bacterial microbiota. Surprisingly, we found 13 common genes between fungal and bacterial diversity descriptors that we referred under the name of Holobiont Generalist Genes (HGG). These genes might strongly regulate the diversity and composition of plant microbiota at the inter-kingdom level.Significance Statement The novel hologenome concept claims that hosts and their associated microbes (considered as holobionts) are a unique evolutionary unit on which natural selection acts. Thus, the hologenome theory assumes that hosts and microbiomes simultaneously coevolve. This novel vision of universal evolutionary entities is promising for both animal and plant health purposes. However, it is still quietly controversial as it suffers from a lack of tangible evidence. How can we enrich the debate on holobionts? How can we translate this concept in discoveries that can change farming practices? Our study is filling the gaps of the hologenome theory by showing that certain genes under natural selection and regulating plant microbiota are generalist in response to fungal and bacterial communities.### Competing Interest StatementThe authors have declared no competing interest.

Agriculture faces great challenges to overcome global warming and to improve system sustainability, requiring access to novel genetic diversity. So far, wild populations and local landraces remain poorly explored. This is notably the case for the two diploid species, Brassica oleracea L. (CC, 2n=2x=18) and B. rapa L. (AA, 2n=2x=20). In order to explore genetic diversity in both species, we have collected numerous populations in their center of origin, the Mediterranean basin, on a large contrasting climatic and soil gradient from northern Europe to southern sub-Saharan regions. In these areas, we also collected 14 populations belonging to five B. oleracea closely related species. Before further genetic and agronomic investigations, we controlled the absence of species misidentification using flow cytometry, sequencing of species specific chloroplast genomic region, as well as cytogenetic analyses in case of unexpected results. Looking at the 102 B. oleracea and 146 B. rapa populations showing a good germination among the 112 and 154 populations collected, seventeen populations were misidentified. The most frequent mistake was a confusion of these diploid species with B. napus . Additionally for B. rapa , 2 autotetraploid populations were observed. Habitats of the collected wild populations and landraces are described in our work. This provides a unique plant material characterization that will pave the way for further analyses investigating the genomic regions involved in climatic and microbiota adaptation. This research is supported by the H2020 Prima project ‘BrasExplor’.

Viscum album L., (Common Mistletoe), is a hemiparasitic shrub that lives on a wide range of woody plant species, known since Neolithic period for its ritual and for pharmacological properties. Up to now, no investigation has been reported on the chemical composition of the essential oils of V. album aerial parts in relation with the diverse host trees. Consequently, the essential oils compositions of three Sicilian accession of V. album, growing on Quercus suber L., Crataegus monogyna Jacq. and Olea europaea L. var. europaea cv. 'Santagatese', respectively, were evaluated by GC-MS. The three populations showed quite different profiles indicating that the nature of the host considerably influences the chemical composition of the guest plant, V. album.

The valorisation and conservation of plant genetic resources (PGRs) and wild fruit PGRs are critical to ensure the maintenance of genetic and cultural heritage and to promote new perspectives on resource use. New strategies to characterize PGRs are needed, and the omics approach can provide information that is still largely unknown. The Strawberry tree (Arbutus unedo L.) is an underutilized, drought and fire-resistant species distributed in the Mediterranean area and its berries have large ethnobotanical use. Although their phenolic profile and antioxidant capacity are known, they are not well characterised, particularly from a proteomic perspective. The aim of this work is the characterisation of two ecotypes of A. unedo (Campania and Sicily) from a molecular viewpoint to valorise and encourage the preservation of this wild fruit. Samples were collected from two different geographical areas to assess whether different geographical conditions could influence the characteristics of leaves and fruits at the three stages of ripening (green, veraison, red). Proteomic analysis identified 904 proteins, of which 122 showed significance along the ripening. Some of these differentially abundant proteins, such as chalcone synthase, show a marked increase during ripening. The protein functional classes with the highest representation are involved in protein and amino acid metabolism, glycolysis and in secondary metabolism. From a proteomic perspective, there are no differences between the fruits from the two regions compared by the ripening stage. However, the pedoclimatic metabolic imprinting allowed the observation of good diversity in the metabolomic profiles between the two ecotypes, especially for anthocyanins, 4 times more abundant in the Sicilian veraisoned fruit than in the Campania one, and catechins, with double the abundance in the Campania ecotype compared to the Sicilian ecotype in the green phase, but more abundant (3x) in the Sicilian veraisoned fruit. Phenolic compounds show a 20% greater abundance in the Campania green arbutus fruit than in the Sicilian one, values that then equalise as ripening progresses. Multi-omic characterisation enhanced the knowledge on a wild fruit plant species which shows specific adaptations and responses to the environment to be considered when addressing the issue of local agrobiodiversity.

This work focused on the extraction, quantification, and characterization of bioactive compounds of Arbutus unedo L. fruits, comparing the results obtained from the different ripening states. Extractions were performed by different methods (such as maceration extraction and ultrasonic extraction) and food grade solvents (aqueous and hydroalcoholic solvents) in each of the all ripening states (four states considered, associated with four different colors, i.e., green, yellow, orange, and red). The presence of (poly)phenols was quantified and characterized, and scavenging activity was determined by the Folin–Ciocâlteu reagent and the DPPH method, respectively. The content of bioactive compounds was characterized by LC-MS/MS, such as multiple reaction monitoring (MRM) mass spectrometry. The results showed that ultrasound-assisted extraction (UAE) performed better than maceration extraction; ethanol–water mixture extracts showed a more positive effect than the use of aqueous extracts regarding the content of total phenolic compounds. Overall, the total phenolic compounds in the EtOH:H 2 O mixture at a ratio of 7:3 (v:v) were higher than that of the other solvents for both extraction methods. Some bioactive molecules were characterized for the first time in the extracts of A. unedo . The chemical profile of the strawberry tree extracts depended on the degree of fruit ripeness. The results suggest that A. unedo fruits may be of great interest for food and nutraceutical applications.

In this work, seven Citrus aurantium essential oils (EOs) derived from flavedo of cultivars ‘Canaliculata’, ‘Consolei’, ‘Crispifolia’, ‘Fasciata’, ‘Foetifera’, ‘Listata’, and ‘Bizzaria’ were investigated. EOs were also combined in 1:1 (v/v) ratio to identify possible synergism or antagonism of actions. GC-MS analysis was done to investigate Eos’ phytochemical profiles. The antioxidant activity was studied by using a multi-target approach based on FRAP, DPPH, ABTS, and β-carotene bleaching tests. A great difference was observed in EOs’ phytochemical profiles. d-limonene (33.35–89.17%) was the main monoterpene hydrocarbon, and α-Pinene, β-myrcene, and β-linalool were identified in almost all samples. Among EOs, only C3 showed high quantitative and qualitative variability in its chemical composition. The chemical diversity of EOs was also demonstrated by PCA and HCA statistical analysis. Samples C2, C4, C5, C6, and C7 were statistically similar to each other, while C1 and C3 were characterized as having a different amount of other compounds and oxygenated monoterpenes, respectively, with respect to the other EOs mentioned. The global antioxidant score (GAS) revealed that among the tested EOs, C. aurantium ‘Fasciata’ EO had the highest antioxidant potential, with a GAS value of −0.47, whereas among combinations, the EO obtained by mixing ‘Canaliculata’ + ‘Bizzaria’ was the most active. Comparison by theoretical and real data on inhibitory concentration (IC50) and FRAP values did not reveal any significant effect of synergism or antagonism of actions to be valid in all biological applied tests. These findings, considered together, represent an important starting point to understand which compounds are responsible for the activities and their future possible industrial application.

The aim of the present study is to investigate the chemical profile, antioxidant activity, carbohydrate-hydrolysing enzyme inhibition, and hypolipidemic effect of essential oils (EOs) extracted from Sicilian Citrus maxima (pomelo) flavedo. Using gas-chromatography-mass spectrometry analysis (GC-MS) we analysed the Eos of five cultivars of C. maxima, namely, ‘Chadock’, ‘Maxima’, ‘Pyriformis’, ‘Terracciani’, and ‘Todarii’, and their blends. The antioxidant activity was performed by using a multi-target approach using 2,2′-Azino-Bis-3-Ethylbenzothiazoline-6-Sulfonic acid (ABTS), 2,2-Diphenyl-1-picrylhydrazyl (DPPH), ferric reducing ability power (FRAP), and β-carotene bleaching tests. The α-amylase, α-glucosidase, and lipase-inhibitory activities were also assessed. GC-MS analyses revealed D-limonene as the main monoterpene hydrocarbon in all cultivars, albeit with different percentages in the range of 21.72–71.13%. A good content of oxygenated monoterpenes was detected for all cultivars, especially for ‘Todarii’. The analysis of the principal components (PCA), and related clusters (HCA), was performed to find chemo-diversity among the analysed samples. EOs from ‘Chadock’ and ‘Maxima’ were statistically similar to each other, and they differed from P3 in the smaller amount of sesquiterpene hydrocarbons, while the oils from ‘Terracciani’ and ‘Todarii’ were found to be chemically and statistically different. ‘Chadock’ EO was the most active to scavenge radicals (IC50 values of 22.24 and 27.23 µg/mL in ABTS and DPPH tests, respectively). ‘Terracciani’ EO was the most active against both lipase and α-amylase, whereas the blends obtained by the combination (1:1 v/v) of C. maxima ‘Maxima’ + ‘Todarii’ were the most active against α-glucosidase. Generally, the blends did not exert a unique behaviour in potentiating or reducing the bioactivity of the pomelo EOs.

Abies nebrodensis (Lojac.) Mattei is an endemic species of the north-west of Sicily located in an 84 ha area in the Madonie Regional park. The current population is limited to 30 relic adult trees and a fluctuating number of juveniles of natural regeneration. The species is defined as “Critically Endangered” in the Italian list of threatened plants and is classified as CR-D in the 2000 IUCN Red List of Threatened Species. This article reports the key action undertaken by the LIFE4FIR project aimed at preserving A. nebrodensis, and the results obtained so far in three years of activity. OpenArrays SNPs genotyping revealed a high rate of inbreeding in the natural population and that the adult trees are genetically related. Controlled cross-pollination was consequently performed to increase the genetic variability of the progeny. Outbred offspring are currently being grown in the nursery. Reforestation has been planned by using 4000 selected outbred seedlings in 10 areas within Madonie Park to create re-diffusion cores. Support and protection of the relic population have been implemented through regular phytosanitary surveys, as well as new fencing and video surveillance systems against grazing and wild herbivores. A seedbank and cryobank for the long-term germplasm conservation have been established.

Sumac (Rhus coriaria L.), a spice obtained by grinding whole sumac berries, is a complex natural product with a plethora of biological activities that can be favorably explored in nutraceutical and pharmaceutical fields. Sicilian sumac is herein chemically investigated by means of a combined NMR/HR MS-based profiling. A hydroalcoholic extract was prepared and its complexity unraveled following fractionation in an alcoholic and an aqueous fraction. The 1H-NMR spectrum of this latter fraction was dominated by the signals of gallic acid and its derivatives, whereas HR MS and HR MS/MS analyses highlighted the diversity in malic acid derivatives. Gallotannins and galloyl flavonol glycosides with quercetin and myricetin as the main aglycones were highly represented in the alcoholic fraction. To improve bioavailability of chemicals in sumac extract, the feasibility of a microencapsulation process by spray drying was investigated. In particular, the addition of maltodextrin, cyclodextrin and gum arabic as covering agents was evaluated. The tests were carried out at different temperatures and concentrations. The addition of maltodextrin was found to be suitable as a carrier for the spray drying of sumac extract, having the highest yields, always > 82%. The highest values of a* (red index) and Chroma were recorded with cyclodextrin and maltodextrin at the lowest concentrations and temperatures of spray drying (120 °C). The correlations between the characteristics of the studied microcapsules (yields, moisture, total and surface polyphenols and colour) have been represented by Principal Component Analysis (PCA).

BACKGROUND: This study investigated the chemical profile and biological activity of Diplotaxis erucoides subsp. erucoides (L.) DC. (Brassicaceae) collected in Sicily (Italy). RESULTS: Liquid chromatography coupled with electrospray ionization and high-resolution mass spectrometry (LC-ESI/HRMS) analysis of the ethanol extract revealed the presence of 42 compounds - glucosinolates, hydroxycinnamic acids, flavonoids, and oxylipins. The extract was tested for its antioxidant activity using 2,2-diphenyl-1-picrylhydrazyl (DPPH), 2,2 '-azinobis(3-ethylbenzothiazoline-6-sulfonic) acid (ABTS), ferric reducing ability power (FRAP), and beta-carotene bleaching tests. Promising protection from lipid peroxidation was observed after 30 min of incubation in a beta-carotene bleaching test (IC50 of 3.32 mu g mL(-1)). The inhibition of carbohydrates-hydrolyzing enzymes resulted in IC50 values of 85.18 and 92.36 mu g mL(-1) for alpha-amylase and alpha-glucosidase, respectively. Significant inhibition against lipase enzyme was observed (IC50 of 61.27 mu g mL(-1)). CONCLUSION: Diplotaxis erucoides can be considered a potential source of antioxidant, hypoglycemic, and hypolipidemic bioactives. (c) 2021 Society of Chemical Industry.

To elucidate the taxonomic status of Ruppia drepanensis Tineo ex Guss. (Alismatales, Ruppiaceae), we performed morphological analysis and DNA barcoding of historical materials (including the lectotype) and fresh samples (including those from a recently discovered population near the locus classicus in Sicily, Italy). We conclude that R. drepanensis is a separate species, closely related to R. spiralis L. ex Dumort., that occurs in temporary inland waters from the western to central sectors of the Mediterranean region. We also highlight the importance of vouchers and the need to link molecular investigations to field, ecological, and morphological investigations.