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.

Characterization of Sideritis clandestina subsp. peloponnesiaca Polar Glycosides and Phytochemical Comparison to Other Mountain Tea Populations.

Sideritis clandestina (Bory & Chaub.) Hayek subsp. peloponnesiaca (Boiss. & Heldr.) Baden (SCP) is endemic to the mountains of the Northern Peloponnese (Greece). This and other Sideritis taxa, collectively known as mountain tea, are widely ingested as beverages for refreshment or medicinal purposes. We describe a methodology for the characterization of SCP. Four iridoid glycosides (monomelittoside, melittoside, ajugoside, and 7-O-acetyl-8-epiloganic acid), two phenolic acid glycosides (vanillic and salicylic acid glycosides), and three caffeoyl ester glycosides (chlorogenic acid, verbascoside, and isoverbascoside) were isolated from SCP for the first time. We used ultrasound-assisted extraction of 3 g of plant material to produce petroleum ether and aqueous extracts, which we then analyzed using GC/MS and LC/MS. This was applied to eight samples from four different taxa. In total, 70 volatile and 27 polar metabolites were determined. The S. clandestina samples had a lower phenolic content and weaker antioxidant properties than S. raeseri and S. scardica. However, S. clandestina ssp. clandestina seemed to be the most aromatic taxon, with almost double the number of volatiles as the others. Τhis study could contribute to authentication and chemotaxonomic studies of Sideritis taxa.

Exploration of genetic, morphological and essential oil variation reveals tools for the authentication and breeding of Salvia pomifera subsp. calycina (Sm.) Hayek.

Salvia pomifera subsp. calycina (Sm.) Hayek (Lamiaceae), is an Eastern Mediterranean element, which is used in traditional medicine and cuisine in the same manner as S. fruticosa Mill. and S. officinalis L.. The essential oil (EO) and the extracts of S. pomifera possess bioactive compounds with anti-proliferative, anticholinesterase, antioxidant, antiviral and antifungal properties. In this study, the chemical (EO), genetic (DNA microsatellites, SSRs) and morphological diversity of forty-nine individuals of Salvia pomifera subsp. calycina, originating from five natural populations of the Peloponnese (Greece) were determined, in order to explore the potential for successful breeding and to reveal tools and biomarkers for identification and authentication. Chemical and genetic analyses revealed high levels of variation both within and among populations, while morphological analysis mainly within populations. Essential oil yield ranged from 1.79 to 5.79 ml 100 g-1 dry wt, among individuals while ß-thujone ranged from 6.04 to 64.75%. Consistency was found in the EO yield and composition of specific individuals, when sampled during the same period, for three consecutive years, while the analysis during spring and summer months showed differentiation that still retained individual's discrimination. Genetic analysis using SSRs showed that the observed population heterozygosity (Ho) ranged from 0.48 to 0.67, while high number of private alleles were revealed in all populations. Considerable genetic differentiation was observed among the three Salvia taxa (S. pomifera subsp. calycina, S. fruticosa, S. officinalis) (Fst values ranged from 0.27 to 0.48) and lower among S. pomifera subsp. calycina populations (Fst values ranged from 0.06 to 0.13). The great variation that was revealed in all measured traits, in combination with the demonstrated, genetically based, consistency of their EO yield and composition, advocates to a successful breeding, whereas SSR genotyping presents a strong identification and authentication tool.

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.

Metabolomic fingerprinting and genetic discrimination of four Hypericum taxa from Greece.

Hypericum perforatum has plenty of uses in traditional medicine and is the source of top-selling herbal drugs and food supplements. The secondary metabolite chemistry for most of the nearly 500 Hypericum taxa is still unknown, even though they are used interchangeably. In the present study, we characterized four Hypericum populations from Achaia, Greece, belonging to H. perforatum ssp. veronense, H. perfoliatum, H. triquetrifolium, and an uninvestigated taxon, H. empetrifolium ssp. empetrifolium, in terms of their essential oils and polar bioactives in methanolic extracts via GC-MS, LC-HRMS, LC-DAD-MS, and HPLC-DAD. We also performed sequence analysis of nrITS to explore the genetic profile of these taxa and to examine whether their genotype is correlated to the metabolome. Sixty-three non-volatile compounds, phloroglucinols in their majority, and over one hundred (113) volatiles, mostly sesqui- and mono- terpenes, were detected. The concentration of the major polar constituents varied greatly among samples. In particular, phloroglucinols' diversity and abundance in H. empetrifolium ssp. empetrifolium was remarkable. The PCA and Biplot analysis revealed the contribution of each compound to the total chemodiversity and also revealed certain compounds that contribute to the discrimination of the samples. Sequence analysis of nrITS revealed different genetic profiles and markers which can be used for the identification of the four Hypericum taxa. The Mantel test showed a relatively strong correlation between the genetic profile and the volatile compounds and low with the main polar metabolites.

Phytochemical and genetic characterization of styles of wild Crocus species from the island of Crete, Greece and comparison to those of cultivated C. sativus.

The aim of this study was to contribute to the characterization of Crocus taxa using morphological, phytochemical and genetic analysis. The styles of C. cartwrightianus, C. oreocreticus and C. laevigatus, collected in the island of Crete were compared to those of C. sativus cultivated at the region of Western Macedonia (Greece). Phytochemical analysis was done using GC-MS and HPLC methods, while ISSR markers were used for their genetic characterization. Safranal was the major volatile component of the styles of C. sativus, 4-hydroxy-2,6,6-trimethyl-1-cyclohexene-1-carboxaldehyde of C. cartwrightianus and C. oreocreticus, and isophorone of C. laevigatus. C. sativus had the highest content of crocins and picrocrocin, while C. laevigatus the lowest (only 5% of C. sativus' quantity) and negligible amount of picrocrocin. According to both the genetic and the chemical analysis, C. cartwrightianus is more related to C. oreocreticus, while C. sativus and C. laevigatus are more distinct. Concordance between the two different types of data was also confirmed by the Mantel test (r = 0.932, P = .68). This is the first thorough screening of secondary metabolites (volatile and non-volatile) and also genetic and morphological characters of wild Crocus styles simultaneously, that contributes to the identification and valorisation of genotypes with similar to C. sativus traits which may be introduced as new cultivars through breeding.

Effect of acidic and enzymatic pretreatment on the analysis of mountain tea (Sideritis spp.) volatiles via distillation and ultrasound-assisted extraction.

A number of beneficial medicinal properties are attributed to the extract and essential oil of the aerial parts of Sideritis species (Lamiaceae). Hydrodistillation of the aerial parts of wild Sideritis clandestina ssp. peloponnesiaca (an endemic taxon in northern Peloponnesus, Greece) gave a low essential oil yield (<0.12%); about 65 components, mainly α-pinene, ß-caryophyllene, ß-pinene, globulol, caryophyllene oxide, were identified via GC-MS. Internal and external standards were used for quantification. For miniaturization of the procedure, we studied side-by-side maceration (MAC) and ultrasound-assisted extraction (UAE) methods, as well as the effect of preincubation in acidic medium (pH 4.8) for 75min at 37°C with or without a mixture of cellulase, hemicellulase and pectinase. Maceration and UAE provide consistent chemoprofiling of the main volatile compounds (about 20); UAE has lower demands on time, solvent, plant material (3g) and results in higher yields. Pretreatment with enzymes can increase the respective yields of hydrodistillation and UAE, but this effect is definitely attributed to the concurrent acidic pretreatment. In conclusion, incubation of plant material prior to hydrodistillation or UAE in citrate buffer, pH 4.8, significantly enhances the overall yield and number of components obtained and is recommended for the analysis of Sideritis volatiles. The acidic pre-treatment method was also successfully applied to analysis of cultivated Sideritis raeseri Boiss. & Heldr. in Boiss. ssp. raeseri; α-pinene, α- and γ-terpinene and ß-thujene were predominant albeit in different percentages in flowers and leaves.

Quantitation of Crocins and picrocrocin in saffron by HPLC: application to quality control and phytochemical differentiation from other crocus taxa.

A chromatographic method was developed and fully validated for the determination of the major saffron constituents, i.e., picrocrocin and five major crocins. Dried samples (styles of Crocus sativus and other Crocus taxa) were extracted with MeOH : water (1 : 1, v/v), and chromatographic separation of the analytes was achieved by reversed-phase chromatography using a gradient elution. Full validation was performed using spiked samples with analytes, which were isolated, purified, and characterized by MS due to a lack of commercial standards. The method showed a good fit (r2 > 0.999) for all analytes with limit of quantitation values in the range of 1-15 µg/mL, and demonstrated adequate intra- and inter-precision (< 15 % RSD) and accuracy (< 7 % RE). The method was applied to the analysis of various commercial saffron samples and of indigenous Crocus taxa and allowed for the first time the absolute quantitation of several Crocus components.

Phytochemical composition of "mountain tea" from Sideritis clandestina subsp. clandestina and evaluation of its behavioral and oxidant/antioxidant effects on adult mice.

PURPOSE: The goals of this study were to monitor the effect of drinking of herbal tea from Sideritis clandestina subsp. clandestina for 6 weeks on behavioral and oxidant/antioxidant parameters of adult male mice and also to evaluate its phytochemical composition. METHODS: The phytochemical profile of the Sideritis tea was determined by liquid chromatography-UV diode array coupled to ion-trap mass spectrometry with electrospray ionization interface. The effects of two doses of the herbal infusion (2 and 4% w/v, daily) intake on anxiety-like state in mice were studied by the assessment of their thigmotactic behavior. The oxidant/antioxidant status of brain (-Ce), liver and heart of adult male Balb-c mice following the consumption of Sideritis tea was also evaluated via the measurement of malondialdehyde (MDA) and reduced glutathione (GSH) levels using fluorometric assays. Our study was further extended to determine the antioxidant effects of the herbal tea on specific brain regions (cerebral cortex, cerebellum and midbrain). RESULTS: The identified compounds were classified into several natural product classes: quinic acid derivatives, iridoids, phenylethanol glycosides and flavonoids. Our results showed that only the 4% Sideritis tea exhibited anxiolytic-like properties as evidenced by statistically significant (p < 0.05) decrease in the thigmotaxis time and increase in the number of entries to the central zone in comparison with the control group. Consumption of both tea doses (2 and 4% w/v) elevated GSH (12 and 28%, respectively, p < 0.05) and decreased MDA (16 and 29%, p < 0.05) levels in brain (-Ce), while liver and heart remained unaffected. In regard to the effect of herbal tea drinking (2 and 4% w/v) on specific brain regions, it caused a significant increase in GSH of cerebellum (13 and 36%, respectively, p < 0.05) and midbrain (17 and 36%, p < 0.05). Similarly, MDA levels were decreased in cerebellum (45 and 79%, respectively, p < 0.05) and midbrain (50 and 63%, respectively, p < 0.05), whereas cerebral cortex remained unaffected. CONCLUSIONS: Mountain tea drinking prevents anxiety-related behaviors and confers antioxidant protection to rodent's tissues in a region-specific, dose-dependent manner, and its phytochemical constituents are shown for the first time.

Differential antioxidant effects of consuming tea from Sideritis clandestina subsp. peloponnesiaca on cerebral regions of adult mice.

Oxidative stress is involved in the pathophysiology of neurodegenerative diseases and aging. Many species of the genus Sideritis (mountain tea) are widely consumed in the Mediterranean region as herbal tea. This study evaluated the effect of supplementation of mice with herbal tea from Sideritis clandestina subsp. peloponnesiaca on the antioxidant status of different brain regions. To select the most bioactive herbal tea, the polyphenolic content (Folin-Ciocalteu method) and the antioxidant properties (ferric reducing antioxidant power [FRAP] and 2,2-diphenyl-1-picrylhydrazyl assays) of several taxa and different populations of the S. clandestina infusions were measured in vitro. Male adult mice had ad libitum access to water (control) or the herbal tea (4% w/v) for 6 weeks. At the end of the treatment period we assessed the total antioxidant power (FRAP assay) and the levels of malondialdehyde (indicator of lipid peroxidation) and reduced glutathione in the cerebral cortex, cerebellum, and midbrain. These biochemical measures have also been determined in liver samples used as a comparative reference peripheral tissue. Consumption of 4% herbal tea increased the total antioxidant power of the midbrain by 72% (P<.05); a significant (P<.05) decrease in malondialdehyde levels and increase in reduced glutathione content of the cerebellum (78% and 27%, respectively) and midbrain (59% and 32%, respectively) were also observed. These findings indicate that mountain tea consumption enhances the antioxidant defense of the adult rodent brain in a region-specific manner.

Inhibition of breast cancer cell proliferation by style constituents of different Crocus species.

Among the different species of Crocus, only C. sativus has been extensively studied for the composition and the biological properties of its styles, since these constitute the well-known spice saffron, which is widely used in the Mediterranean, Indian and Chinese diet. With high performance liquid chromatography (HPLC) and UV/vis spectroscopy, the presence of hydrophilic carotenoids in the styles of three other Crocus taxa, endemic in Greece, C. boryi ssp. tournefortii, C. boryi ssp. boryi and C. niveus, is reported for the first time. Incubation of MCF-7 and MDA-MB-231 breast cancer cells for 48 h with different concentrations of all four Crocus style extracts showed a dose-dependent inhibitory effect on cell proliferation measured by the MTT assay. The antiproliferative effect was not related to the presence of estrogen receptors. Studies on the effect of trans-crocin-4 (the main carotenoid constituent of C. sativus styles, digentibiosylester of crocetin), crocetin and safranal showed that the antiproliferative effect is attributed to the constituent crocins irrespective of the degree of glycosylation. These results show that the styles of the various Crocus taxa merit further investigation of their composition and mechanisms of action of their carotenoid constituents in order to establish if they could be used as chemopreventive or anticancer agents.