Towards reviving post-Olympic Athens as a cultural destination

This paper examines the effects of global change on the status and qualities of the Greek national capital, Athens, focusing on how they affect the development of cultural tourism in the city. Although Athens constituted one of the most significant destinations for Greek tourism in the past, in recent years it started to weaken. Athens is characterised by a series of problems, among them are the degradation of its environment and quality of life and traffic congestion. However, in terms of tourism development, the Olympic Games helped in re-imaging the city and upgrading its infrastructure. This study based on semi-structured interviews with top officials reveals how global change has affected Athens’ socio-cultural/economic status, identity and image. Despite the tourism policy/planning responses to global changes, Athens’ tourism continues to decline leaving unexplored potential such as its rich cultural heritage, new multicultural identity and the New Acropolis Museum. The paper suggests that cultural elements of capital cities must be multidimensional including a variety of attractions and amenities. The use of cultural heritage assets needs to be in line with global developments in order for cities to effectively leverage heritage for cultural tourism

Droplet-based microfluidic screening and sorting of microalgal populations for strain engineering applications

The application of microfluidic technologies to microalgal research is particularly appealing since these approaches allow the precise control of the extracellular environment and offer a high-throughput approach to studying dynamic cellular processes. To expand the portfolio of applications, here we present a droplet-based microfluidic method for analysis and screening of Phaeodactylum tricornutum and Nannochloropsis gaditana, which can be integrated into a genetic transformation workflow. Following encapsulation of single cells in picolitre-sized droplets, fluorescence signals arising from each cell can be used to assess its phenotypic state. In this work, the chlorophyll fluorescence intensity of each cell was quantified and used to identify populations of P. tricornutum cells grown in different light conditions. Further, individual P. tricornutum or N. gaditana cells engineered to express green fluorescent protein were distinguished and sorted from wild-type cells. This has been exploited as a rapid screen for transformed cells within a population, bypassing a major bottleneck in algal transformation workflows and offering an alternative strategy for the identification of genetically modified strains

Air Biodiversity: a high throughput plant species identification on environmental DNA

The aerobiological spectrum is characterized by different types of biological particles (pollens, spores, bacteria, etc.), with a variability linked to site and environmental factors. Data on airborne pollen reflect differences in the species composition of the local flora and may capture the spreading of alien species. The air biomonitoring may also detect the flowering season of anemophilous taxa as well as the reproductive response of plants to environmental changes at a temporal and spatial scale. Aim of this research is to characterize the air biodiversity of different ecosystems through a DNA-based metabarcode analysis applied on complex air samples. The metabarcoding of environmental DNA will allow the taxonomic identification based on specific genetic markers, leading to an estimation of the biodiversity. Since loss in biodiversity can endanger ecosystem health, natural ecosystems of different vegetation zones will be selected on the basis of their putative degradation and invasion by alien species, such as Ambrosia (ragweed), to highlight differences in species composition and richnes

Airborne pollen patterns along an altitudinal gradient of the Italian Alps: combination of classical pollen identification methods and next generation sequencing on environmental DNA

With the combination of classical pollen identification methods and next generation sequencing on environmental DNA, airborne pollen patterns were investigated in six habitats along an altitudinal gradient of Pale di San Martino-Paneveggio National Park (Italian Alps). Pollen was identified from environmental samples that were collected with gravimetric Tauber traps. Results of both methods were evaluated to investigate how similarly they can capture pollen spatio-temporal patterns and how they can be used for different applications, such as for diversity assessment and monitoring studies. For the taxonomic identification of pollen DNA, a reference database of DNA sequences was first constructed. ΤrnL sequences were downloaded from Genbank for most of the anemophilous taxa present in the study area (Trentino, northern Italy) and they were processed and stored in a local database. For plant species without available sequences, plant material was collected, the trnL gene was targeted and sequenced and the new sequences were integrated in the final database, in total 1188 sequences corresponding to 403 species of 198 genera and 46 families; from these 44 were new sequences, corresponding to 26 species. Preliminary experiments for metabarcoding analysis οf airborne pollen were designed in order to develop appropriate lab procedures. The experimental design included sample collection from volumetric and gravimetric traps at the Aerobiological Monitoring Centre of Fondazione Edmund Mach, which were utilized both for microscope and molecular analysis. Experiments were performed for the steps of sample processing to retrieve pollen pellets and of DNA extraction of optimal yield. The protocols were applied across a complexity of samples from single-species pollen to environmental multiple- species samples. It was found that a number of factors like the mechanic disruption of the pollen cell walls or the extraction kit used influence significantly the DNA yield. On the basis of the results taken, the optimal combination was selected (Nucleomag kit and disruption with steel beads). Also, it was found that the DNA yield decreases after chemical treatment of the sample, which suggests that better washing of the pollen pellet should be applied instead. A small fragment (about 150 base pairs) of the chloroplast trnL gene was amplified and sequenced (with cloning and classic Sanger sequencing) using universal primers for plants. For the taxonomic identification we used a custom trnL database as reference. The results showed that 75% and 37% of the taxa identified by microscope were revealed by metabarcoding for the volumetric and the gravimetric samplings, respectively. The whole procedure allowed the identification of pollen from environmental samples. The taxonomic information showed higher resolution via metabarcoding, while the amount and quality of information could increase with the application of next generation sequencing. Having defined the optimal methodology, we could proceed to the analysis of the samples from the park (54 in total). Sequencing was performed this time with the Illumina next generation sequencing platform. The sequencing data resulted in a total of 11,137,178 sequences, clustered in 140 Operational Taxonomic Units which were assigned taxonomically to 32 families, 55 genera (or group of genera). According to the plant growth form, 37 taxa represent woody and 25 herbaceous plants. Thirteen of these species (21%) are not present in the plant checklist of the park; Cedrus and Cupressus sempervirens pollen, in particular, had a considerable contribution of >1%. There are 13 main pollen taxa contributing at least 0.5% to the total number of the sequence reads. These are Pinus (36.8%), Larix decidua (14.5%), Cedrus (12.4%), Picea (11.6%), Abies (5.4%), Corylus/Ostrya/ Carpinus (5%), Alnus viridis (2.9%), Urtica dioica (2.8%), Juniperus communis (0.7%), Taxus baccata (0.6%), Chenopodium album (0.6%), Festuca/Trisetum/Lolium (0.5%) and Cupressus sempervirens (0.5%). When we used concurrently metabarcoding and classical microscopic analysis of the pollen trapped, almost all non-rare families were commonly identified by the two methods, but the molecular method could discern more genera. Nevertheless, Cyperaceae and Polygonaceae, although with considerable abundance in the microscopic dataset, did not feature in the metabarcoding results. Compared to the total pollen recorded, Poaceae Betulaceae, Corylaceae and Oleaceae were found to contribute less with the metabarcoding method than with the microscopic one, and Pinaceae more. For the main pollen season, Pinus is the most abundantly represented taxon in the pollen spectrum after both methods and similarly at high concentrations in the aerobiological data (Lanzoni sampler). Nevertheless, its contribution to the park’s vegetation is lower than that of the dominant Picea (85%), the pollen of which consists only 12% of the annual sum. Regarding the biodiversity assessment, metabarcoding could discern the sampling periods. It detected March-July 2015 as the period with the highest number of taxa (alpha diversity), and revealed significant changes in diversity (beta diversity) among sampling periods. These results matched the features of the pollen season, as defined by aerobiological studies running in parallel. Spatial patterns could not be clearly defined; nevertheless, results of metabarcoding were in accordance to the ones obtained with the microscopic method. Optimized molecular protocols can increase our potential for time-efficient analysis of pollen datasets. Providing high resolution taxonomic results, the molecular method that we applied can be used for biodiversity assessments and floral surveys or for monitoring vegetation changes, particularly those expressed in species composition rather than in species abundance. On the basis of our results and previous reports, we can argue that the metabarcoding and the microscopic methods have each their weak and strong points and they should be applied in a complementary way, at least until the quantitative and qualitative issues associated with metabarcoding are adequately addressed

Plant biodiversity estimation through pollen spectra in Natura 2000 habitats: preliminary methodological steps for implementation of eDNA metabarcoding

Airborne pollens reflect differences in the species composition of the local flora and may capture the spreading of alien species. The air biomonitoring may detect the flowering season of anemophilous taxa as well as the reproductive response of plants to environmental changes at a temporal and spatial scale. Our study aims at characterizing the air biodiversity of different habitats - as coded by Natura 2000 and Corine Land Cover - through a DNA metabarcoding approach on environmental samples collected at ground level. Metabarcoding is promising for identifying airborne pollen from environmental samples, with potential advantages over microscopic methods. There is still no consensus in sample preparation and DNA extraction, especially for gravimetric pollen samplers. As a first step, we established protocols to process environmental samples for extracting DNA to be analyzed by metabarcoding, and to verify the efficacy of this approach for the taxonomic assignment of airborne pollens, collected by both gravimetric (Tauber trap) and volumetric samplers (Burkard spore trap). Protocols were tested across an increasing complexity of samples, from pure pollen to environmental samples, considering the variability in pollen grain structure, the mixture of different pollen species, the influence of adopted sampling medium. A short fragment (about 150 base pair of trnL P6 loop) of chloroplast DNA was amplified by universal primers for plants. Amplicons were Sanger-sequenced and taxonomic assignment was accomplished through comparison to a custom-made reference database - 46 families representing all the anemophilous plants occurring in the study area (Trentino, Italy, Eastern Italian Alps). By comparison to the classical morphological pollen analysis, it emerged that DNA metabarcoding is valid across a complexity of samples, from pure pollen of single species to environmental samples, provided that sample preparation, DNA extraction and amplification protocols are specifically optimized

Plant biodiversity and airborne pollen: next-generation sequencing of environmental DNA

Airborne pollens reflect differences in the species composition of the local flora and may capture the spreading of alien species. The air biomonitoring may also detect the flowering season of anemophilous taxa as well as the reproductive response of plants to environmental changes at a temporal and spatial scale. Aim of this research is to characterize the air biodiversity of different habitats - as coded by Natura 2000 and Corine Land Cover - through a DNA metabarcoding approach on environmental samples collected at ground level. The metabarcoding of environmental DNA will allow the taxonomic identification based on specific genetic markers, leading to an estimation of the biodiversity. The information gathered, complemented with results from traditional approaches, will eventually lead to a characterization of functional and compositional biodiversity in the study area, Eastern Italian Alps. This area can be considered as a case study for Alpine regions where land is exploited for different purposes: residential, cultural (including leisure, recreation and amenity) and natural environment quality (conservation and management

DNA metabarcoding of airborne pollen: new protocols for improved taxonomic identification of environmental samples

Metabarcoding is a promising DNA-based method for identifying airborne pollen from environmental samples with advantages over microscopic methods. Sample preparation and DNA extraction are of fundamental importance for obtaining an optimal DNA yield. Currently, there is no standard procedure for these steps, especially for gravimetric pollen samplers. Therefore, the aim of this study was to develop protocols for processing environmental samples for pollen DNA extraction and for metabarcoding analysis and to assess the efficacy of these protocols for the taxonomic assignment of airborne pollen collected by gravimetric (Tauber trap) and volumetric (Hirst-type trap) samplers. Protocols were tested across an increasing complexity of samples, from pure single-species pollen to environmental multi-species samples. A short fragment (about 150 base pairs) of the chloroplast trnL gene was amplified using universal primers for plants. After PCR amplification, amplicons were Sanger-sequenced and taxonomic assignment was accomplished by comparison with a custom-made reference database including chloroplast DNA sequences from most of the anemophilous taxa occurring in the study area (Trentino, northern Italy), representing 46 plant families. Using the classical morphological pollen analysis as a benchmark, we show that DNA metabarcoding is efficient and applicable even in complex samples, provided that protocols for sample preparation, DNA extraction, and metabarcoding analysis are carefully optimize