Diurnal temperature range as a key predictor of plants' elevation ranges globally.

A prominent hypothesis in ecology is that larger species ranges are found in more variable climates because species develop broader environmental tolerances, predicting a positive range size-temperature variability relationship. However, this overlooks the extreme temperatures that variable climates impose on species, with upper or lower thermal limits more likely to be exceeded. Accordingly, we propose the 'temperature range squeeze' hypothesis, predicting a negative range size-temperature variability relationship. We test these contrasting predictions by relating 88,000 elevation range sizes of vascular plants in 44 mountains to short- and long-term temperature variation. Consistent with our hypothesis, we find that species' range size is negatively correlated with diurnal temperature range. Accurate predictions of short-term temperature variation will become increasingly important for extinction risk assessment in the future.

Chemical Characterization and Antioxidant Activity of Nine Hypericum Species from Greece.

Hypericum L. comprises about 500 species distributed almost worldwide. Research has mainly focused on H. perforatum with confirmed biological activity on the alleviation of depression symptoms, among others. The compounds responsible for such activity are considered naphthodianthrones and acylphloroglucinols. Other Hypericum species are less studied or not studied, and further research is needed to complete the characterization of the genus. In this study we evaluated the qualitative and quantitative phytochemical profile of nine Hypericum species native to Greece, namely H. perforatum, H. tetrapterum, H. perfoliatum, H. rumeliacum subsp. apollinis, H. vesiculosum, H. cycladicum, H. fragile, H. olympicum and H. delphicum. Qualitative analysis was performed using the LC/Q-TOF/HRMS technique, while quantitative data were calculated with the single point external standard method. Additionally, we estimated the antioxidant activity of the extracts using DPPH and ABTS assays. Three species endemic to Greece (H. cycladicum, H. fragile, H. delphicum) were studied for the first time. Our results indicated that all studied species are rich in secondary metabolites, mainly of the flavonoids family, with strong antioxidant activity.

Climate and Land-Cover Change Impacts and Extinction Risk Assessment of Rare and Threatened Endemic Taxa of Chelmos-Vouraikos National Park (Peloponnese, Greece).

Chelmos-Vouraikos National Park is a floristic diversity and endemism hotspot in Greece and one of the main areas where Greek endemic taxa, preliminary assessed as critically endangered and threatened under the IUCN Criteria A and B, are mainly concentrated. The climate and land-cover change impacts on rare and endemic species distributions is more prominent in regional biodiversity hotspots. The main aims of the current study were: (a) to investigate how climate and land-cover change may alter the distribution of four single mountain endemics and three very rare Peloponnesian endemic taxa of the National Park via a species distribution modelling approach, and (b) to estimate the current and future extinction risk of the aforementioned taxa based on the IUCN Criteria A and B, in order to investigate the need for designing an effective plant micro-reserve network and to support decision making on spatial planning efforts and conservation research for a sustainable, integrated management. Most of the taxa analyzed are expected to continue to be considered as critically endangered based on both Criteria A and B under all land-cover/land-use scenarios, GCM/RCP and time-period combinations, while two, namely Alchemilla aroanica and Silene conglomeratica, are projected to become extinct in most future climate change scenarios. When land-cover/land-use data were included in the analyses, these negative effects were less pronounced. However, Silene conglomeratica, the rarest mountain endemic found in the study area, is still expected to face substantial range decline. Our results highlight the urgent need for the establishment of micro-reserves for these taxa.

Cytotoxic Effect of Rosmarinus officinalis Extract on Glioblastoma and Rhabdomyosarcoma Cell Lines.

Rosmarinus officinalis is a well-studied plant, known for its therapeutic properties. However, its biological activity against several diseases is not known in detail. The aim of this study is to present new data regarding the cytotoxic activity of a hydroethanolic extract of Rosmarinus officinalis on glioblastoma (A172) and rhabdomyosarcoma (TE671) cancer cell lines. The chemical composition of the extract is evaluated using liquid chromatography combined with time-of-flight mass spectrometry, alongside its total phenolic content and antioxidant activity. The extract showed a promising time- and dose-dependent cytotoxic activity against both cell lines. The lowest IC50 values for both cell lines were calculated at 72 h after treatment and correspond to 0.249 ± 1.09 mg/mL for TE671 cell line and 0.577 ± 0.98 mg/mL for A172 cell line. The extract presented high phenolic content, equal to 35.65 ± 0.03 mg GAE/g of dry material as well as a strong antioxidant activity. The IC50 values for the antioxidant assays were estimated at 12.8 ± 2.7 µg/mL (DPPH assay) and 6.98 ± 1.9 µg/mL (ABTS assay). The compound detected in abundance was carnosol, a phenolic diterpene, followed by the polyphenol rosmarinic acid, while the presence of phenolic compounds such as rhamnetin glucoside, hesperidin, cirsimaritin was notable. These preliminary results suggest that R. officinalis is a potential, alternative source of bioactive compounds to further examine for abilities against glioblastoma and rhabdomyosarcoma.

GC-MS and LC-DAD-MS Phytochemical Profiling for Characterization of Three Native Salvia Taxa from Eastern Mediterranean with Antiglycation Properties.

Salvia fruticosa and S. pomifera subsp. calycina are native to Eastern Mediterranean and S. pomifera subsp. pomifera is endemic to Greece. The primary aim of this study was to develop an analytical methodology for metabolomic profiling and to study their efficacy in combating glycation, the major biochemical complication of diabetes. After sequential ultrasound-assisted extraction of 2 g of leaves with petroleum ether and 70% methanol, the volatile metabolites in the petroleum ether extracts were studied with GC-MS (Gas Chromatography-Mass Spectrometry), whereas the polar metabolites in the hydroalcoholic extracts were determined and quantified by UHPLC-DAD-ESI-MS (Ultra-High Performance Liquid Chromatography-Diode Array Detector-Mass Spectrometry). This methodology was applied to five populations belonging to the three native taxa. 1,8-Cineole was the predominant volatile (34.8-39.0%) in S. fruticosa, while S. pomifera had a greater content of α-thujone (19.7-41.0%) and ß-thujone (6.0-39.1%). Principal Component Analysis (PCA) analysis of the volatiles could discriminate the different taxa. UHPLC-DAD-ESI-MS demonstrated the presence of 50 compounds, twenty of which were quantified. PCA revealed that not only the taxa but also the populations of S. pomifera subsp. pomifera could be differentiated. All Salvia samples inhibited advanced glycation end-product formation in a bovine serum albumin/2-deoxyribose assay; rosmarinic and carnosic acid shared this activity. This study demonstrates the antiglycation activity of S. fruticosa and S. pomifera extracts for the first time and presents a miniaturized methodology for their metabolomic profiling, which could aid chemotaxonomic studies and serve as a tool for their authentication and quality control.

Conservation of Micromeria browiczii (Lamiaceae), Endemic to Zakynthos Island (Ionian Islands, Greece).

The massive decline in biodiversity due to anthropogenic threats has led to the emergence of conservation as one of the central goals in modern biology. Conservation strategies are urgently needed for addressing the ongoing loss of plant diversity. The Mediterranean basin, and especially the Mediterranean islands, host numerous rare and threatened plants in need of urgent conservation actions. In this study, we assess the current conservation status of Micromeria browiczii, a local endemic to Zakynthos Island (Ionian Islands, Greece), and estimate its future risk of extinction by compiling and assessing scientific information on geographical distribution, population dynamics and reproductive biology. The population size and the geographical distribution of the species were monitored for five years. The current population of the species consists of 15 subpopulations. Considerable annual fluctuation of population size was detected. The species is assessed as Endangered according to the International Union for Conservation of Nature threat categories. According to population viability analysis results, its extinction risk was estimated to be 5.6% over the next 50 years, when six of the fifteen subpopulations (40%) might become extinct. The investigation of certain aspects of the species' biology yielded important data necessary to identify critical aspects for its survival and to propose conservation measures.

Conservation biology of threatened Mediterranean chasmophytes: The case of Asperula naufraga endemic to Zakynthos island (Ionian islands, Greece).

Asperula naufraga is a rare and threatened obligate chasmophyte, endemic to Zakynthos island (Ionian islands, Greece). In this study, we provide a combined approach (including monitoring of demographic and reproductive parameters and study of genetic diversity) to assess the current conservation status of the species and to estimate its future extinction risk. The five subpopulations of A. naufraga were monitored for five years (2014-2018). Population size markedly fluctuated between 68-130 mature individuals during the monitoring period. The extent of occurrence (EOO) was estimated at 28.7 km2 and the area of occupancy (AOO) was 8 km2. Stage-structure recordings were similar for all subpopulations, characterized by high proportions of adult and senescent individuals, following a common pattern, which has been observed in other cliff-dwelling plants. Preliminary genetic analysis with SSRs markers revealed low heterozygosity within subpopulations and significant departure from H-W equilibrium, which combined with small population size suggest increased threat of genetic diversity loss. Our results indicate that the species should be placed in the Critically Endangered (CR) IUCN threat category, while according to Population Viability Analysis results its extinction risk increases to 47.8% in the next 50 years. The small population size combined with large fluctuations in its size, low recruitment and low genetic diversity, indicate the need of undertaking effective in situ and ex situ conservation measures.

Spatial Phylogenetics, Biogeographical Patterns and Conservation Implications of the Endemic Flora of Crete (Aegean, Greece) under Climate Change Scenarios.

Human-induced biodiversity loss has been accelerating since the industrial revolution. The climate change impacts will severely alter the biodiversity and biogeographical patterns at all scales, leading to biotic homogenization. Due to underfunding, a climate smart, conservation-prioritization scheme is needed to optimize species protection. Spatial phylogenetics enable the identification of endemism centers and provide valuable insights regarding the eco-evolutionary and conservation value, as well as the biogeographical origin of a given area. Many studies exist regarding the conservation prioritization of mainland areas, yet none has assessed how climate change might alter the biodiversity and biogeographical patterns of an island biodiversity hotspot. Thus, we conducted a phylogenetically informed, conservation prioritization study dealing with the effects of climate change on Crete's plant diversity and biogeographical patterns. Using several macroecological analyses, we identified the current and future endemism centers and assessed the impact of climate change on the biogeographical patterns in Crete. The highlands of Cretan mountains have served as both diversity cradles and museums, due to their stable climate and high topographical heterogeneity, providing important ecosystem services. Historical processes seem to have driven diversification and endemic species distribution in Crete. Due to the changing climate and the subsequent biotic homogenization, Crete's unique bioregionalization, which strongly reminiscent the spatial configuration of the Pliocene/Pleistocene Cretan paleo-islands, will drastically change. The emergence of the 'Anthropocene' era calls for the prioritization of biodiversity-rich areas, serving as mixed-endemism centers, with high overlaps among protected areas and climatic refugia.

The Use of Essential Oil and Hydrosol Extracted from Satureja hellenica for the Control of Meloidogyne incognita and M. javanica.

Essential oil (EO) and hydrosol (HL) isolated from an indigenous plant species Satureja hellenica were evaluated against Meloidogyne incognita and M. javanica. Particularly, the activity of extracts on a second stage juvenile's (J2s) motility, the hatching of J2s from eggs, egg differentiation and the effect on J2s in soil were tested. A paralysis of 100% of the J2s of both species was recorded after 96 h of immersion in the essential oil, at a dose of 2000 µL/L. At the same dose, the percentage of paralyzed J2s after 48 h of immersion was more than 80%, for both Meloidogyne species. The use of hydrosol has shown encouraging results only in the dilution of 50%, where for both Meloidogyne species tested, the percentage of paralyzed J2s was more than 70% after 48 h of immersion, while the percentage was increased to 90% after 96 h of immersion. Egg differentiation was ceased after immersion, either in EO or HL. However, this decrease in egg differentiation was evident only at higher concentrations of EO and at the highest HL dilution (0.5 v/v). The hatching of M. incognita J2s was decreasing as the dose was increasing. The lowest numbers of hatched J2s were recorded at the doses of 2000 and 4000 µL/L. A clear reduction in M. javanica J2s hatching was observed as the dose was increased to 250 µL/L, a fact constantly observed as the dose was increasing up to 4000 µL/L. Lower numbers of nematodes were recorded in roots grown in infested soil after the application of EO or HL at the highest doses. The EO of S. hellenica is characterized by the presence of p-cymene (27.46%) and carvacrol (23.25%), and in a lesser extent of other constituents, such as borneol (6.79%), carvacrol methylether (6.77%), γ-terpinene (4.63%) and 4-terpineol (3.65%). Carvacrol was the major constituent found in the HL (50.12%), followed by borneol and 4-terpineol (20.42 and 6.72%, respectively).

High Resolution Melting (HRM) Genotyping in the Genus Origanum: Molecular Identification and Discrimination for Authentication Purposes.

Origanum L. (Lamiaceae) is an important genus of medicinal and aromatic plants used since ancient times as culinary herbs and remedies in traditional medicine. Although it is a relatively small genus, intra-generic species delineation, as well as its inter-generic relationships within tribe Mentheae, are still poorly understood. High resolution melting (HRM) analysis, coupled with microsatellite markers (SSRs), could facilitate the molecular identification and characterization of certain genotypes more efficiently and relatively faster when compared to other analytical methods. In this study, 38 Origanum samples corresponding to six Origanum taxa (O. dictamnus, O. majorana, O. onites, O. scabrum, O. sipyleum, and O. vulgare subsp. hirtum) were analyzed, using six microsatellite loci. Our goal was to molecularly identify and discriminate among the selected samples and to evaluate the ability of the HRM technique as an analytical tool for the discrimination of Origanum species from Greece. The temperature-shifted melting curves produced by the HRM analysis, resulted in 98 unique HRM profiles, which enabled the discrimination of the Origanum genotypes studied. According to the similarity dendrogram based on the HRM profiles, six unique clusters were formed, each one corresponding to a single taxon. In conclusion, HRM genotyping provided a fast, cost-effective method, well suited for the molecular characterization and identification of Origanum taxa and for the authentication of the original genetic material.

Population genetic variability and distribution of the endangered Greek endemic Cicer graecum under climate change scenarios.

The Mediterranean hot spot includes numerous endemic and socio-economically important plant species seriously threatened by climate change and habitat loss. In this study, the genetic diversity of five populations of Cicer graecum, an endangered endemic species from northern Peloponnisos, Greece and a wild relative of the cultivated Cicer arietinum, was investigated using inter-simple sequence repeats (ISSRs) and amplified fragment length polymorphism (AFLP) markers in order to determine levels and structure of genetic variability. Nei's gene diversity by ISSR and AFLP markers indicated medium to high genetic diversity at the population level. Moreover, AMOVA results suggest that most of the variation exists within (93 % for AFLPs and 65 % for ISSRs), rather than among populations. Furthermore, Principal Component Analysis based on ISSRs positively correlated the genetic differentiation among the populations to the geographic distances, suggesting that the gene flow among distant populations is limited. The ecological adaptation of C. graecum populations was also investigated by correlation of their genetic diversity with certain environmental variables. Aridity arose as the dominant factor positively affecting the genetic diversity of C. graecum populations. We modelled the realized climatic niche of C. graecum in an ensemble forecasting scheme under three different global circulation models and two climate change scenarios. In all cases, a severe range contraction for C. graecum is projected, highlighting the high extinction risk that is probably going to face during the coming decades. These results could be a valuable tool towards the implementation of an integrated in situ and ex situ conservation scheme approach for activating management programmes for this endemic and threatened species.

Characterization of the chemical composition of Drimia numidica plant parts using high-resolution mass spectrometry: study of their total phenolic content and antioxidant activity.

Drimia species have been used since ancient times for their medicinal properties. Their bulbs are considered as the main source of secondary metabolites with biological activity but the chemical composition of the other plant parts has not yet been considered. The aim of this study is to contribute to the existing knowledge with new data on the total phenolic content, the antioxidant activity and the chemical profile of different parts of Drimia numidica. The total phenolic content was estimated by the Folin-Ciocalteu assay and the antioxidant activity with DPPH· and ABTS·+ reagents. The separation and the identification of the compounds were performed with liquid chromatography combined with time-of-flight high-resolution mass spectrometry (LC/Q-TOF/HRMS). The extract of leaves presented the highest phenolic content while the highest antioxidant activity was presented by the extract of flowers. Results of the chemical analysis verify the presence of bufadienolides and phenolic compounds. Graphical abstract.

Elevational gradient of vascular plant species richness and endemism in Crete--the effect of post-isolation mountain uplift on a continental island system.

Understanding diversity patterns along environmental gradients and their underlying mechanisms is a major topic in current biodiversity research. In this study, we investigate for the first time elevational patterns of vascular plant species richness and endemism on a long-isolated continental island (Crete) that has experienced extensive post-isolation mountain uplift. We used all available data on distribution and elevational ranges of the Cretan plants to interpolate their presence between minimum and maximum elevations in 100-m elevational intervals, along the entire elevational gradient of Crete (0-2400 m). We evaluate the influence of elevation, area, mid-domain effect, elevational Rapoport effect and the post-isolation mountain uplift on plant species richness and endemism elevational patterns. Furthermore, we test the influence of the island condition and the post-isolation mountain uplift to the elevational range sizes of the Cretan plants, using the Peloponnese as a continental control area. Total species richness monotonically decreases with increasing elevation, while endemic species richness has a unimodal response to elevation showing a peak at mid-elevation intervals. Area alone explains a significant amount of variation in species richness along the elevational gradient. Mid-domain effect is not the underlying mechanism of the elevational gradient of plant species richness in Crete, and Rapoport's rule only partly explains the observed patterns. Our results are largely congruent with the post-isolation uplift of the Cretan mountains and their colonization mainly by the available lowland vascular plant species, as high-elevation specialists are almost lacking from the Cretan flora. The increase in the proportion of Cretan endemics with increasing elevation can only be regarded as a result of diversification processes towards Cretan mountains (especially mid-elevation areas), supported by elevation-driven ecological isolation. Cretan plants have experienced elevational range expansion compared to the continental control area, as a result of ecological release triggered by increased species impoverishment with increasing elevation.