Environmental DNA detects biodiversity and ecological features of phytoplankton communities in Mediterranean transitional waters.

Climate changes and anthropogenic pressures are causing a biodiversity decline in terms of species number and genetic diversity, reducing the adaptability and evolvability of natural communities. Transitional water ecosystems are more sensitive to habitat reduction and degradation and, thus, are more exposed to biodiversity declines requiring biodiversity monitoring programs for their conservation. Environmental DNA (eDNA) metabarcoding represents a high-throughput tool for biodiversity assessment that is facilitating data collection for biodiversity monitoring. In this study, we applied, for the first time, eDNA metabarcoding in a Mediterranean coastal lagoon to assess the ecological features of eukaryotic phytoplankton communities. We sampled water in seven different lagoon sites and amplified the extracted DNA with primers targeting the variable region 4 (V4) of the 18S rRNA gene marker. The results demonstrated the validity of eDNA studies to provide insights into lagoon phytoplankton composition, establish the structure and spatial variation of phytoplankton communities, and evaluate its correlation to abiotic factors. Finally, the genetic distances analysis suggests that the different spatial distribution of OTUs, at least for the Tetraselmis genus, reflects the genetic background.

Phytoplankton Size Structure and Diversity in the Transitional System of the Aquatina Lagoon (Southern Adriatic Sea, Mediterranean).

The Aquatina Lagoon (Southern Adriatic Sea, Mediterranean Ecoregion) is a transitional water ecosystem with great ecological and socio-economic interest. Anthropogenic activities around the lagoon (e.g., agriculture and tourism) and hydrology can affect the environmental quality and biodiversity of the lagoon. Herein, the dynamics and diversity of phytoplankton communities were studied before and after the opening of a new canal connecting the lagoon with the sea, by using different approaches based on an evaluation of the size and structure of the phytoplankton as well as the taxonomic analyses. The lagoon depicted time-related fluctuations in chemical-physical parameters. The phytoplankton trend was characterized by an increase in abundance and biomass in summer, when pico-sized autotrophs dominated. Generally, nano-sized phytoflagellates dominated the community, while micro-sized dinoflagellates and diatoms were less abundant. An increase in the phytoplankton taxa number was observed throughout the years. All the analyzed parameters were generally relatively homogeneous before the opening of the channel, while some quantitative differences among stations were observed in the second sampling period. Considering the statistical evidence, both environmental and biological parameters were affected by the "dilution" effect exerted by marine water inputs. This research supports the evidence that phytoplankton is a good indicator of the environmental status, and the obtained results contribute to the implementation of management strategies for the conservation of transitional water ecosystems.

DNA Barcode Gap Analysis for Multiple Marker Genes for Phytoplankton Species Biodiversity in Mediterranean Aquatic Ecosystems.

The implementation of DNA metabarcoding and environmental DNA (eDNA) to the biodiversity assessment and biomonitoring of aquatic ecosystems has great potential worldwide. However, DNA metabarcoding and eDNA are highly reliant on the coverage of the DNA barcode reference libraries that are currently hindered by the substantial lack of reference sequences. The main objective of this study was to analyze the current coverage of DNA barcode reference libraries for phytoplankton species of the aquatic Mediterranean ecoregion in the southeast of Italy (Apulia Region) in order to assess the applicability of DNA metabarcoding and eDNA in this area. To do so, we investigated three main DNA barcode reference libraries, BOLD Systems, GenBank and SILVA, for the availability of DNA barcodes of the examined phytoplankton species. The gap analysis was conducted for three molecular gene markers, 18S, 16S and COI. The results showed a considerable lack of barcodes for all three markers. However, among the three markers, 18S had a greater coverage in the reference libraries. For the 18S gene marker, the barcode coverage gap across the three types of ecosystems examined was 32.21-39.68%, 60.12-65.19% for the 16S marker gene, and 72.44-80.61 for the COI marker gene. Afterwards, the interspecific genetic distance examined on the most represented molecular marker, 18S, was able to distinguish 80% of the species mined for lakes and 70% for both marine and transitional waters. Conclusively, this work highlights the importance of filling the gaps in the reference libraries, and constitutes the basis towards the advancement of DNA metabarcoding and eDNA application for biodiversity assessment and biomonitoring.

Testing the Influence of Incomplete DNA Barcode Libraries on Ecological Status Assessment of Mediterranean Transitional Waters.

The ecological assessment of European aquatic ecosystems is regulated under the framework directives on strategy for water and marine environments. Benthic macroinvertebrates are the most used biological quality element for ecological assessment of rivers, coastal-marines, and transitional waters. The morphological identification of benthic macroinvertebrates is the current tool for their assessment. Recently, DNA-based tools have been proposed as effective alternatives. The main current limits of DNA-based applications include the incompleteness of species recorded in the DNA barcode reference libraries and the primers bias. Here, we analysed the influence of the incompleteness of DNA barcode databases on species diversity indices, ecological indicators, and ecological assessment in transitional waters of the southeast Mediterranean, taking into account the availability of commonly sequenced and deposited genomic regions for listed species. The ecological quality status assigned through the potential application of both approaches to the analysed transitional water ecosystems was different in 27% of sites. We also analysed the inter-specific genetic distances to evaluate the potential application of the DNA metabarcoding method. Overall, this work highlights the importance to expand the barcode databases and to analyse, at the regional level, the gaps in the DNA barcodes.

Relationships between size and abundance in beach plastics: A power-law approach.

Sandy beaches are one of the most relevant coastal environments in terms of socio-ecological and economical value. So, the presence and accumulation of plastic litter determines a degradation of these values, and calls for management actions for cleaning are required. In this research, we investigated the features of plastic litter distribution on a Mediterranean beach in relation to size and abundance of the sampled items. Simple allometric models were applied with the aim to provide a parsimonious tool for estimating the amount and sizes of the beach plastic litter. The results show effective relations between size and abundance of plastic items according to the power-law distribution. This relationship could support decision-makers to estimate the total amount of beach plastics through the application of a simple model instead of more complex models requiring the estimation of many parameters and the availability of large datasets.

Coupling beach ecology and macroplastics litter studies: Current trends and the way ahead.

As sites of floating marine material deposition, sandy beaches accumulate marine litter. While research and assessment on beach litter is increasing and involves various actors (scientists, society and NGOs), there is the need to assess current and future dominant trends, directions and priorities in that research. As such, a textural co-occurrence analysis was applied to published scientific literature. Words were considered both singly and as part of compound terms related to concepts relevant to sandy beach ecology: morphodynamic state; Littoral Active Zone; indicator fauna. Litter as a compound term was also included. The main co-occurrences were found within compounds, with scarce interaction of "morphodynamic state" with the others, indicating the need for further integration of beach ecology paradigms into beached plastics studies. Three approaches are proposed to overcome the research limits highlighted: the unequivocation of terms, the consideration of adequate scales, and the attention to dynamics rather than just patterns.

Building a transnational biodiversity geo-database of the protected areas in the Adriatic-Ionian Macro-Region: approaches and results from the IMPRECO Project.

BACKGROUND: The main objective of the project Common strategies and best practices to IMprove the transnational PRotection of ECOsystem integrity and services - IMPRECO is to enhance the safeguarding of ecosystems and ecosystem services. Additionally, the aim of this project is to tackle their environmental vulnerability by strengthening the potential of the Protected Areas in biodiversity, ecosystems and ecosystem services conservation. This is expected to be addressed by maintaining it through their transnational networking located in the European Adriatic-Ionian Macro-Region. NEW INFORMATION: The aim of this research is: 1) to characterise the habitats and ecosystems involved in the coastal-marine protected areas considered; 2) to set a biodiversity baseline; 3) to understand what current ecosystems' conditions are; 4) to build up a transnational biomonitoring programme of target species and habitats and 5) to assess their response to pilot actions. To do so, a transnational inventory of species, habitats, ecosystems and ecosystem services was established, starting with the seven coastal-marine protected areas involved in the project. Data collection was carried out using different sources of information: scientific literature, officially available data from NATURA 2000 Standard Data Forms, checklists from local biomonitoring programmes, personal observations and citizen science, historical maps and data from new in-field analyses. Data were filled in the transnational biodiversity geo-databases according to the NATURA 2000 standards about habitat features, species protection level and species features. The presence of alien species (non-indigenous species, NIS) was also acknowledged and references about data collection were provided in the databases according to the Darwin Core standards.

A new extralimital sighting of Monachusmonachus (Hermann, 1779) in the Aquatina di Frigole NATURA 2000 site (IT9150003) beach (Salento peninsula, Apulia Region, Italy) after two decades: strategies for conservation are needed.

The Mediterranean monk seal Monachusmonachus (Hermann, 1779) is the most endangered pinniped in the world. In addition, its presence has not been documented for about two decades in the Apulian Region and about 10 years along the Italian coastline. In this work, we aim to describe an exceptional and well-documented observation of a subadult specimen of Monachusmonachus occurring during the last days of January 2020 in the Salento peninsula (Apulia Region, Italy) for the first time after two decades of local extinction in the south-western Adriatic Sea.

The future of biotic indices in the ecogenomic era: Integrating (e)DNA metabarcoding in biological assessment of aquatic ecosystems.

The bioassessment of aquatic ecosystems is currently based on various biotic indices that use the occurrence and/or abundance of selected taxonomic groups to define ecological status. These conventional indices have some limitations, often related to difficulties in morphological identification of bioindicator taxa. Recent development of DNA barcoding and metabarcoding could potentially alleviate some of these limitations, by using DNA sequences instead of morphology to identify organisms and to characterize a given ecosystem. In this paper, we review the structure of conventional biotic indices, and we present the results of pilot metabarcoding studies using environmental DNA to infer biotic indices. We discuss the main advantages and pitfalls of metabarcoding approaches to assess parameters such as richness, abundance, taxonomic composition and species ecological values, to be used for calculation of biotic indices. We present some future developments to fully exploit the potential of metabarcoding data and improve the accuracy and precision of their analysis. We also propose some recommendations for the future integration of DNA metabarcoding to routine biomonitoring programs.

Simultaneous determination of polyphenolic compounds in red and white grapes grown in Sardinia by high performance liquid chromatography-electron spray ionisation-mass spectrometry.

A new method for the identification and the quantification of nonanthocyanin phenolic compounds from six Vitis Vinifera grape varieties native to Sardinia (three native: Vermentino, Malvasia and Cannonau and three non-native types: Chardonnay, Sauvignon and Cabernet Sauvignon; Argiolas vineyard) was developed. This rapid and selective method employs LC/ESI-MS in negative mode. Different solvents extraction and different sorbents for purification were compared to the direct analysis of the initial extracts without further sample preparation. A total of 54 phenolic compounds were identified either in the freeze-dried skins or seeds, including nonflavonoids (hydroxybenzoic and hydroxycinnamic acids and their derivatives, stilbenes) and flavonoids (flavanols, flavonols, dihydroxyflavonols).

Spatial analysis of plant detritus processing in a Mediterranean river type: the case of the River Tirso Basin, Sardinia, Italy.

The river continuum concept represents the most general framework addressing the spatial variation of both structure and function in river ecosystems. In the Mediterranean ecoregion, summer drought events and dams constitute the main sources of local disturbance to the structure and functioning of river ecosystems occurring in the river basin. In this study, we analysed patterns of spatial variation of detritus processing in a 7th order river of the Mediterranean ecoregion (River Tirso, Sardinia-Italy) and in three 4th order sub-basins which were exposed to different summer drought pressures. The study was carried out on Phragmites australis and Alnus glutinosa leaf detritus at 31 field sites in seasonal field experiment. Detritus processing rates were higher for Alnus glutinosa than for Phragmites australis plant detritus. Processing rates of Alnus glutinosa leaves varied among seasons and study sites from 0.006 d(-1) to 0.189 d(-1) and those of Phragmites australis leaves ranged from 0.0008 d(-1) to 0.102 d(-1), with the lowest values occurring at sites exposed to summer drought. Seasons and sites accounted for a significant proportion of such variability. Alder detritus decay rates generally decreased with increasing stream order, while reed detritus decay rates generally increased on the same spatial gradient. Summer drought events affected these spatial patterns of variation by influencing significantly the decay rates of both plant detritus. The comparisons among and within sub-basins showed strong negative influence of summer drought on detritus processing rates. Similarly, in the entire River Tirso basin decay rates were always lower at disturbed than at undisturbed sites for each stream order; decay rates of reed detritus remained lower at those sites even after the end of the disturbance events, while alder decay rates recovered rapidly from the summer drought perturbations. The different recovery of the processing rates of the two leaves could also explain the different patterns of spatial variation observed between the two leaves.