The use of environmental DNA (eDNA) over the past decade for monitoring aquatic biodiversity has surged due to advancements in molecular methods enabling the extraction of information from tiny traces of DNA shed by aquatic organisms into their environment. eDNA is proving to be increasingly time and cost effective for monitoring, with as little as a few hundred milliliters of filtered water being sufficient to identify everything from microscopic species of plankton up to larger organisms such as fish. eDNA is particularly useful for elusive and taxonomically complex species not easily identified through morphology alone.

By contrast, traditional methods applied to aquatic biodiversity studies require substantial volumes of water (e.g., at least 30 liters when sampling zooplankton in a lake) to be collected and filtered with time consuming microscopic analysis requiring taxonomic expertise to follow. Even then, elusive species may be missed and taxonomically challenging samples such as damaged individuals, developmental stages, and cryptic and non-indigenous species, may remain unidentified to the species level.

Figure 1: Non-indigenous species (NIS) found/recorded in Thessaloniki Bay; (a) the calanoid copepod Pseudodiaptomus marinus and (b) the cyclopoid copepod Oithona davisae; bar scale 50 μm

In the ZooPlankton Lab within the Laboratory of Ichthyology of the School of Biology in the Aristotle University of Thessaloniki, we have been monitoring the zooplankton community in Thessaloniki Bay (40°37′52.7″N; 22°56′06.1″E) since 2018. This bay is next to the port of Thessaloniki, one of the main Mediterranean ports, receiving a significant number of commercial maritime vessels that discharge their ballast tanks into the bay, thereby increasing the risk of biological invasions. We use traditional methods for biodiversity monitoring that involve deploying a plankton net to a depth of 3 m, which is then vertically hauled to filter and retain zooplankton within the water column. This sampling is typically only performed during daytime (in the morning) but we also carried out some nighttime samplings. Back in the laboratory we microscopically identify the animals (as small as 50 μm), which in many cases we need to dissect, to look at the discriminating morphological characteristic that could be a spine, a seta, cilia, appendages, etc. Through this process we have recorded so far two non-indigenous species which are also first records in the Hellenic seas. These are two tiny microscopic copepods Oithona davisae (size of about 0.5 mm) (Anadoli & Michaloudi, 2019) and Pseudodiaptomus marinus (size of about 1 mm) (Kourkoutmani & Michaloudi, 2022). The latter could have easily gone undetected because it is a species that stays in the bottom during daytime only moving up in the water column during nighttime, which is when we collected it.

Figure 2: Sampling procedure when collecting eDNA with the vampire sampler in the field

eDNA would likely have detected P. marinus even during daytime sampling, highlighting the value of this monitoring tool for revealing non-indigenous invasive species. Early detection of invasives is of great importance, since in many cases these species compete with native species and cause economic and environmental harm. eDNA monitoring can be built into early detection programs, to provide data underpinning effective control measures, to limit the spread and minimize the impacts of invasive species. The BGE (Biodiversity Genomics Europe) project has a task focusing on the sampling of ports and harbors aimed at building capacity in the early detection of marine invasives using eDNA. Sampling by both scientists and citizen scientists has been carried out across Europe following standardized protocols – the simplicity of the sample collection makes it possible for non-specialist citizens and even children to participate. Our team participated in this pan-European project and collected eDNA samples in the port of Thessaloniki, and we are going to sample other ports and harbors with the help of citizens in the future.

The analysis of the eDNA samples collected during the BGE project may reveal further invasive species beyond the two we already encountered through our traditional monitoring. More generally, it will also help to identify the full spectrum of species that constitute the indigenous zooplankton community in the sampled ports. This will include at-risk species for which distributional data are much needed to support conservation measures. Using eDNA to increase the scale and speed of research into biodiversity will help us to tackle the urgent problem of massive biodiversity loss and contribute to our still limited knowledge about the numerous species that are waiting to be described and discovered. Here the results from eDNA analysis can feed back to inform the work of ecologists and taxonomists using traditional approaches, whose knowledge and judgment remain invaluable to the conservation of marine biodiversity.

Figure 3: Location of the sampling sites in Thessaloniki Bay (northern part of Thermaikos Gulf, Aegean Sea)

References

Anadoli O & Michaloudi E (2019). Oithona davisae Ferrari & Orsi, 1984 (Copepoda: Cyclopoida: Oithonidae): Α newly recorded species in the North Aegean Sea. In: Dragicevic et al. New Mediterranean Biodiversity Records (December 2019). Mediterranean Marine Science 20 (3): 645-656. https://doi.org/10.12681/mms.20913

Kourkoutmani P & Michaloudi E (2022). First record of the calanoid copepod Pseudodiaptomus marinus Sato, 1913 in the North Aegean Sea. Thessaloniki Bay, Greece. BioInvasions Records 11: 738-746. https://doi.org/10.3391/bir.2022.11.3.15

Guest blog by Jessica Frigerio, Andrea Galimberti & Massimo Labra

Mediterranean biodiversity is under threat due to phenomena of global and local concern such as climate change, sea-land use alteration and the introduction and outbreaks of invasive species. Immediate actions informed by reliable monitoring tools and strategies are required to conserve and restore Mediterranean biodiversity. This requirement complies with the EU 2030 Biodiversity Strategy, which intends to address the main drivers of biodiversity loss and set legally binding targets.

Identifying even described species is often difficult, and knowledge of their distributions, variation, properties, interdependencies, and conservation status remains patchy and incomplete. While museum collections and morphology-based studies have been invaluable for biodiversity studies to date, these need refinement and expansion to address our patchy knowledge on a wider, territory-based scale to mitigate the loss of habitats and species diversity. Large-scale DNA barcoding approaches have transformed the speed at which the inventory of life will be completed and have provided the foundations of a global, standardized bio-surveillance system for biodiversity. This includes several complete DNA barcoding studies published by Italian researchers on groups including the dragonflies and damselflies, butterflies, and bats [1-4]. In addition, many other taxa have been partially studied from a ‘molecular’ point of view, but a considerably portion of Italy’s biodiversity remains understudied.

Italian biodiversity is remarkably rich and varied due to its geographical location and the diversity of climates and habitats, ranging from the forests and mountains of the Alps and the Apennines to the Mediterranean coast. Italy is considered one of the most biodiverse countries in Europe, hosting a wide range of animal and plant species. The Italian flora comprises over 7,000 species of vascular plants, many of which are endemic to the Italian territory. The Italian fauna is among the richest in Europe, representing more than a third of all European fauna with 57,000 species, 10% of which are endemic species (especially invertebrates). Italy hosts 340 species protected under the Habitats Directive – approximately 25% of the total of the Habitats Directive’s species – and 297 protected birds, roughly 65% of the species protected under the Birds Directive.

To preserve this incredible richness, the Italian National Biodiversity Future Center NBFC (https://www.nbfc.it/en) has been funded under the National Recovery and Resilience Plan (PNRR) as the first national research center dedicated to the conservation, restoration and management of Italian and Mediterranean biodiversity. It is composed of about 2,000 researchers belonging to 48 partner organizations (universities and research institutes) working on biodiversity topics. One of the goals of the NBFC is to develop a molecular repository to address species identification and monitoring by leveraging expertise in DNA barcoding, metabarcoding and whole genome sequencing of emblematic species. The project aims to collect and analyse samples of the majority of Italian species including endemic, vulnerable, rare and/or those relevant to ecosystem functioning.

The DNA barcode sequences will be generated from herbaria and museum collections, supplemented with fresh samples. In the case of herbarium and museum samples, a digitalized reference image database will also be assembled. Additionally, a national effort will be conducted to aggregate Italian DNA barcode sequences already deposited in international databases.

The NBFC Molecular Platform will be integrated with tools for genomic analysis and for processing DNA barcoding and metabarcoding data, with the option to utilize validated restricted databases (e.g. for pollinators, urban tree species, etc.). These genetic reference databases will support researchers who are interested in studying Italian biodiversity at the local or national scale to address theoretical or applicative purposes (e.g. conservation, evolution, early-warning detection of rare or problematic species). Moreover, the molecular platform will provide a connection to large international initiatives in molecular characterization of biodiversity, such as the International Barcode of Life (iBOL) and BOLD Systems. Finally, it will support public and private institutions and industrial stakeholders for biodiversity monitoring, molecular traceability, bioprospecting, and industrial biotechnology.

If you would like to contact the NBFC, please get in touch at molecular_biodiversity@nbfc.it

References:

1. Galimberti, A., Assandri, G., Maggioni, D., Ramazzotti, F., Baroni, D., Bazzi, G., … & Casiraghi, M. (2021). Italian odonates in the Pandora’s box: A comprehensive DNA barcoding inventory shows taxonomic warnings at the Holarctic scale. Molecular Ecology Resources, 21(1), 183-200.
2. Galimberti, A., Martinoli, A., Russo, D., Mucedda, M., & Casiraghi, M. (2010). Molecular identification of Italian Mouse-eared bats (genus Myotis).
3. Dapporto, L., Cini, A., Vodă, R., Dincă, V., Wiemers, M., Menchetti, M., … & Vila, R. (2019). Integrating three comprehensive data sets shows that mitochondrial DNA variation is linked to species traits and paleogeographic events in European butterflies. Molecular Ecology Resources, 19(6), 1623-1636.
4. Dapporto, L., Menchetti, M., Vodă, R., Corbella, C., Cuvelier, S., Djemadi, I., … & Vila, R. (2022). The atlas of mitochondrial genetic diversity for Western Palaearctic butterflies. Global Ecology and Biogeography, 31(11), 2184-2190.