Seed Bank Conservation and Incipient Seed Development in Orchids Colonizing Mining Wastes: Results of a Field Pilot Experiment.

As they represent actual or potential risks to human and environmental safety and health, abandoned mines are a major global problem. The heavy metal-polluted tailings dump of Barraxiutta (Domusnovas, southwestern Sardinia, Italy) is home to a metallicolous population of Epipactis tremolsii (Orchidaceae). A reclamation of the abandoned mine area seems to be approaching, and such an intervention may pose a serious risk for the maintenance of the unique orchid population colonizing the mine wastes. In the present work, the seed packet technique was implemented for the first time to observe orchid seed development in mine wastes. This approach allowed us to explore different seed-based conservation options for the metallicolous orchid population and to gain a deeper grasp of population dynamics and ecology. Four different sowing treatments were set up in the tailing dump and in a near unpolluted site (control site). The field phase of the experiment lasted for 10 months, a period in which the experimental seed bank preservation and incipient seed development were observed and statistically approached. Our findings observed no significant seed loss happening during the experiment, demonstrating the suitability of the seed packet technique to also explore seed bank conservation and development in extreme environmental conditions (i.e., polluted mine wastes). This field method will be a useful tool to further explore the more effective translocation and quasi in situ conservation alternatives for the E. tremolsii metallicolous population. Incipient and site-specific seed development (non-mycorrhizal stage) was observed during the experiment. A plant-soil fungus interaction at the seed level was also observed, the nature of which remains to be ascertained in further studies providing a longer duration for the field phases.

Rapid In Situ Detection of THC and CBD in Cannabis sativa L. by 1064 nm Raman Spectroscopy.

The need to find a rapid and worthwhile technique for the in situ detection of the content of delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD) in Cannabis sativa L. is an ever-increasing problem in the forensic field. Among all the techniques for the detection of cannabinoids, Raman spectroscopy can be identified as the most cost-effective, fast, noninvasive, and nondestructive. In this study, 42 different samples were analyzed using Raman spectroscopy with 1064 nm excitation wavelength. The use of an IR wavelength laser showed the possibility to clearly identify THC and CBD in fresh samples, without any further processing, knocking out the contribution of the fluorescence generated by visible and near-IR sources. The results allow assigning all the Raman features in THC- and CBD-rich natural samples. The multivariate analysis underlines the high reproducibility of the spectra and the possibility to distinguish immediately the Raman spectra of the two cannabinoid species. Furthermore, the ratio between the Raman bands at 1295/1440 and 1623/1663 cm-1 is identified as an immediate test parameter to evaluate the THC content in the samples.

Chemical Composition of Essential Oil from Four Sympatric Orchids in NW-Italy.

Orchidaceae is a flowering plant family worldwide distributed known for producing volatile organic compounds (VOCs) which can act as olfactory signals for pollinators. Despite the importance of VOCs in the different reproductive strategies, in the literature there are only a few publications on the characterization of orchids' volatile profiles. In this study, the essential oils from fresh inflorescences of sympatric orchids Anacamptis morio, Himantoglossum robertianum, Ophrys sphegodes and Orchis purpurea, naturally growing in Piedmont (Italy) were isolated by steam distillation and characterized by GC/FID and GC/MS. A number of compounds were identified, with a peculiar distribution in the species: alcohols (range 16.93-50.60%), from which p-cresol (range 12.75-38.10%) was the most representative compound; saturated hydrocarbons (range 5.81-59.29%), represented by pentacosane (range 2.22-40.17%) and tricosane (range 0.78-27.48%); long-chain monounsaturated hydrocarbons (range 0.29-5.20%) represented by 9-pentacosene, 11-tricosene, and 1-heneicosene. The structure of positional isomers in linear alkenes was elucidated by derivatization with dimethyl disulfide and MS fragmentation patterns. Coumarin (68.84%) was the dominant compound in O. purpurea and was detected in lower concentrations (range 0.21-0.26%) in the other taxa. These volatile compounds may represent a particular feature of these plant species and play an essential role in pollinator interaction.

Do floral and ecogeographic isolation allow the co-occurrence of two ecotypes of Anacamptis papilionacea (Orchidaceae)?

Ecotypes are relatively frequent in flowering plants and considered central in ecological speciation as local adaptation can promote the insurgence of reproductive isolation. Without geographic isolation, gene flow usually homogenizes the allopatrically generated phenotypic and ecological divergences, unless other forms of reproductive isolation keep them separated. Here, we investigated two orchid ecotypes with marked phenotypic floral divergence that coexist in contact zones. We found that the two ecotypes show different ecological habitat preferences with one being more climatically restricted than the other. The ecotypes remain clearly morphologically differentiated both in allopatry and in sympatry and differed in diverse floral traits. Despite only slightly different flowering times, the two ecotypes achieved floral isolation thanks to different pollination strategies. We found that both ecotypes attract a wide range of insects, but the ratio of male/female attracted by the two ecotypes was significantly different, with one ecotype mainly attracts male pollinators, while the other mainly attracts female pollinators. As a potential consequence, the two ecotypes show different pollen transfer efficiency. Experimental plots with pollen staining showed a higher proportion of intra- than interecotype movements confirming floral isolation between ecotypes in sympatry while crossing experiments excluded evident postmating barriers. Even if not completely halting the interecotypes pollen flow in sympatry, such incipient switch in pollination strategy between ecotypes may represent a first step on the path toward evolution of sexual mimicry in Orchidinae.

The Impact of Fruit Etiolation on Quality of Seeds in Tobacco.

Seed's maturity and integrity are essential requirements for germination, and they rely on nutrients availability and a correct phytohormones' balance. These aspects are prerequisites for prompt germination at the end of the dormancy period and strictly depend on chloroplast metabolism and photosynthesis. In the present work, capsules of Nicotiana tabacum were grown in dark during the whole post-anthesis period. Among others, photosynthetic rates, dormancy, and phytohormones levels in seeds were found to be significantly different with respect to controls. In particular, etiolated capsules had expectedly reduced photosynthetic rates and, when compared to controls, their seeds had an increased mass and volume, an alteration in hormones level, and a consequently reduced dormancy. The present findings show how, during fruit development, the presence of light and the related fruit's photosynthetic activity play an indirect but essential role for reaching seeds maturity and dormancy. Results highlight how unripe fruits are versatile organs that, depending on the environmental conditions, may facultatively behave as sink or source/sink with associated variation in seed's reserves and phytohormone levels.

Epipactis tremolsii Seed Diversity in Two Close but Extremely Different Populations: Just a Case of Intraspecific Variability?

Analysis of the seed morphology is a widely used approach in ecological and taxonomic studies. In this context, intraspecific variability with respect to seed morphology (size, weight, and density) was assessed in two close Epipactis tremolsii Pau. populations sharing the same ecological conditions, except for the soil pollution distinguishing one of them. Larger and heavier seeds were found in plants growing on the heavy metal polluted site, while no differences in seed density were detected between seeds produced by plants growing on the contaminated and the control site. Moreover, seed coats and embryos varying together in their dimensions were described in the control population, while coats varying in their size independently from embryos were described in plants growing on the polluted site. Seeds from the two studied populations significantly differed in several parameters suggesting that intraspecific seed variability occurred in the case study.

A Synopsis of Sardinian Studies: Why Is it Important to Work on Island Orchids?

Biological and ecological investigations of islands are crucial to explain ecosystem functioning. Many studies on island biodiversity are carried out on oceanic islands. In contrast, information on continental islands, such as those in the Mediterranean Sea, is very often fragmented in space and time. Here, a synopsis of the Orchidaceae of Sardinia is presented based on literature surveys and recent botanical field studies. Our final list comprises of 64 species and 14 genera: thirteen species and subspecies were recognized as endemic and four new species were recorded for the flora of the island: Anacamptis palustris (Jacq.) R.M. Bateman, Pridgeon & M.W. Chase; Himantoglossum hircinum (L.) Spreng; Orchis italica Poir.; and Platanthera kuenkelei subsp. kuenkelei var. sardoa R.Lorenz, Akhalk., H.Baumann, Cortis, Cogoni & Scrugli. This orchid richness reflects the geological history of the island that was linked to the mainland several times, facing long periods of isolation. We also discuss a critical point-of-view of the biodiversity shortfalls still problematic for insular orchids. Indeed, within the Mediterranean Basin, the greatest amount of endemism occurs mainly on large islands, and, despite a long history of botanical exploration in European countries, many of them are scarcely investigated. This annotated synopsis shows the potential of continental islands to understand trends in ecology and evolution. Further studies are required to complete our knowledge of the orchid diversity on continental islands in order to propose scientific-based conservation programs to preserve these unique taxa.

Heavy metal tolerance of orchid populations growing on abandoned mine tailings: A case study in Sardinia Island (Italy).

Understanding how environmental pollutants influence plant occurrence, growth, and development is key for effective management plans and potential bioremediation. Rare plants, such as orchids, may occur in modified habitats and on soils containing heavy metals, yet their ecological and physiological responses to heavy metals is poorly understood. We investigated the influence of heavy metal pollution on orchid growth rates and interactions with soil fungal mutualists by comparing a large population of the orchid Epipactis helleborine (L.) Crantz subsp. tremolsii (Pau) E. Klein that grows on mine tailings in south-west Sardinia (Italy) with a population that grows on non-contaminated soils in central Sardinia. Soils of the contaminated site had high levels of heavy metals and low organic matter and nutritive elements content. We performed a morphological analysis on twenty individuals that have been subjected to measurement of bioaccumulation and translocation of heavy metals. Fungi associated with the roots of plants from the contaminated and uncontaminated site were grown and identified by DNA barcoding approach. Plants from the contaminated site were smaller than the ones growing in the uncontaminated site and were found to be able to tolerate heavy metals from the soil and to accumulate and translocate them into their organs. Fungi belonging to the genus Ilyonectria (Ascomycota) were found both in contaminated and uncontaminated sites, while an unidentified fungus was isolated from roots in the contaminated site only. These results are discussed in terms of orchids' tolerance to heavy metals and its physiological and ecological mechanisms. The role of contaminated habitats in harbouring orchids and peculiar taxa is also discussed.

Prenylated phloroglucinols from Hypericum scruglii, an endemic species of Sardinia (Italy), as new dual HIV-1 inhibitors effective on HIV-1 replication.

In a search for new potential multitarget anti-HIV compounds from natural products, we have identified in Hypericum scruglii, an endemic and exclusive species of Sardinia (Italy), a potent plant lead. The phytochemical study of the hydroalcoholic extract obtained from its leaves led to the isolation of its most abundant secondary metabolites, belonging to different chemical classes. In particular, three phloroglucinols derivatives were identified, confirming their significance as chemotaxonomic markers of the Hypericum genus. Among them, the 3-(13-hydroxygeranyl)-1-(2'-methylbutanoyl)phloroglucinol was reported here for the first time. All six isolated compounds have been evaluated firstly for the inhibition of both Human Immunodeficiency Virus type 1 (HIV-1) Reverse Transcriptase (RT)-associated DNA Polymerase (RDDP) and Ribonuclease H (RNase H) activities, for the inhibition of HIV-1 integrase (IN) in biochemical assays, and also for their effect on viral replication. Among the isolated metabolites, three phloroglucinol derivatives and quercitrin were effective on both RT-associated RDDP and RNase H activities in biochemical assays. The same active compounds affected also HIV-1 IN strand transfer function, suggesting the involvement of the RNase H active site. Furthermore, phloroglucinols compounds, included the newly identified compound, were able to inhibit the HIV-1 replication in cell based assays.

Chemical and biomolecular analyses to discriminate three taxa of Pistacia genus from Sardinia Island (Italy) and their antifungal activity.

This work reports the results and the comparison concerning the chemical and biomolecular analyses and the antifungal activity of three wild Pistacia species (Anacardiaceae) from Sardinia. Volatile oils from leaves and twigs of Pistacia x saportae, Pistacia lentiscus and Pistacia terebinthus were characterised using GC-FID and GC-MS techniques and tested against some fungal strains. Two DNA nuclear regions (ITS and 5S-rRNA-NTS) were amplified through PCR technique and sequenced. The three **Pistacia have similar chemical profile, although there are some important quantitative differences. The analysis of ITS and 5S-rRNA-NTS regions, reveals a species-specific nucleotide variation among the three **taxa. This method could emerge as a powerful tool for the species identification, especially because the discrimination of these three **taxa appears difficult for non-expert botanists. Concerning the antifungal activity, P. lentiscus and P. x saportae show the highest activity against Cryptococcus neoformans, with a MIC value of 0.32 µL/mL.

Chemical, molecular, and proteomic analyses of moss bag biomonitoring in a petrochemical area of Sardinia (Italy).

In this study, Hypnum cupressiforme moss bags were used to examine the atmospheric deposition of trace elements in the oil refinery region of Sardinia (Italy) compared with surrounding natural zones. The concentrations of 13 elements [arsenic (As), calcium (Ca), cadmium (Cd), chromium (Cr), copper (Cu), iron (Fe), potassium (K), magnesium (Mg), sodium (Na), nickel (Ni), lead (Pb), vanadium (V), and zinc (Zn)] were determined using inductively coupled plasma optical emission spectrometry. A significant accumulation of pollutants was detected using active biomonitoring with moss bags compared with a control site. The most relevant contaminants for all of the tested sites were Cr, Cu, Ni, and Zn. Moreover, the accumulation of Cr and Zn in the refinery industrial areas, IA1 and IA2, was more than five times greater than that detected at the control site. Levels of Cd, Mg, and Pb were also higher at all of the monitored sites compared with the control site. Both genomic and proteomic methods were used to study the response of H. cupressiforme to air pollution. No DNA damage or mutations were detected using the amplified fragment length polymorphisms (AFLP) method. At the protein level, 15 gel spots exhibited differential expression profiles between the moss samples collected at the IA1 site and the control site. Furthermore, among the 14 spots that showed a decrease in protein expression, nine were associated with ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) and proteins of the light-harvesting complexes of photosystem (PS) II, three were associated with protein synthesis, and three were stress-related proteins. Thus, some of these proteins may represent good moss biosensors which could be used as pre-alert markers of environmental pollution.

Ménage à trois-two endemic species of deceptive orchids and one pollinator species.

In the sexually deceptive orchid genus Ophrys, reproductive isolation is based on the specific attraction of males of a single pollinator species by mimicking the female species-specific sex pheromone. Changes in the odor composition can lead to hybridization and speciation by the attraction of a new pollinator that acts as an isolation barrier toward other sympatrically occurring Ophrys species. On Sardinia, we investigated the evolutionary origin of two sympatrically occurring endemic species, Ophrys chestermanii and O. normanii, which are both pollinated by males of the cuckoo bumblebee Bombus vestalis. Chemical and electrophysiological analyses of floral scent and genetic analyses with amplified fragment length polymorphisms and plastid-markers clearly showed that O. normanii is neither a hybrid nor a hybrid species. The two species evolved from different ancestors, viz. O. normanii from O. tenthredinifera and O. chestermanii from O. annae, and converged to the same pollinator attracted by the same bouquet of polar compounds. In spite of sympatry, pollinator sharing and overlapping blooming periods, no evidence has been obtained for gene flow between O. chestermanii and O. normanii indicating an unusual case among sexually deceptive orchids in which postmating rather than premating reproductive isolation mechanisms strongly prevent interspecific gene flow.