Laura Carugati

Metagenetic tools for the census of marine meiofaunal biodiversity: An overview

Marine organisms belonging to meiofauna (size range: 20-500 μm) are amongst the most abundant and highly diversified metazoans on Earth including 22 over 35 known animal Phyla and accounting for more than 2/3 of the abundance of metazoan organisms. In any marine system, meiofauna play a key role in the functioning of the food webs and sustain important ecological processes. Estimates of meiofaunal biodiversity have been so far almost exclusively based on morphological analyses, but the very small size of these organisms and, in some cases, the insufficient morphological distinctive features limit considerably the census of the biodiversity of this component. Molecular approaches recently applied also to small invertebrates (including meiofauna) can offer a new momentum for the census of meiofaunal biodiversity. Here, we provide an overview on the application of metagenetic approaches based on the use of next generation sequencing platforms to study meiofaunal biodiversity, with a special focus on marine nematodes. Our overview shows that, although such approaches can represent a useful tool for the census of meiofaunal biodiversity, there are still different shortcomings and pitfalls that prevent their extensive use without the support of the classical taxonomic identification. Future investigations are needed to address these problems and to provide a good match between the contrasting findings emerging from classical taxonomic and molecular/bioinformatic tools.

Implementing and Innovating Marine Monitoring Approaches for Assessing Marine Environmental Status

Marine environmental monitoring has tended to focus on site-specific methods of investigation. These traditional methods have low spatial and temporal resolution and are relatively labor intensive per unit area/time that they cover. To implement the Marine Strategy Framework Directive (MSFD), European Member States are required to improve marine monitoring and design monitoring networks. This can be achieved by developing and testing innovative and cost-effective monitoring systems, as well as indicators of environmental status. Here, we present several recently developed methodologies and technologies to improve marine biodiversity indicators and monitoring methods. The innovative tools are discussed concerning the technologies presently utilized as well as the advantages and disadvantages of their use in routine monitoring. In particular, the present analysis focuses on: (i) molecular approaches, including microarray, Real Time quantitative PCR (qPCR), and metagenetic (metabarcoding) tools; (ii) optical (remote) sensing and acoustic methods; and (iii) in situ monitoring instruments. We also discuss their applications in marine monitoring within the MSFD through the analysis of case studies in order to evaluate their potential utilization in future routine marine monitoring. We show that these recently-developed technologies can present clear advantages in accuracy, efficiency and cost.

Unveiling the biodiversity of deep-sea nematodes through metabarcoding: are we ready to bypass the classical taxonomy?

Nematodes inhabiting benthic deep-sea ecosystems account for >90% of the total metazoan abundances and they have been hypothesised to be hyper-diverse, but their biodiversity is still largely unknown. Metabarcoding could facilitate the census of biodiversity, especially for those tiny metazoans for which morphological identification is difficult. We compared, for the first time, different DNA extraction procedures based on the use of two commercial kits and a previously published laboratory protocol and tested their suitability for sequencing analyses of 18S rDNA of marine nematodes. We also investigated the reliability of Roche 454 sequencing analyses for assessing the biodiversity of deep-sea nematode assemblages previously morphologically identified. Finally, intra-genomic variation in 18S rRNA gene repeats was investigated by Illumina MiSeq in different deep-sea nematode morphospecies to assess the influence of polymorphisms on nematode biodiversity estimates. Our results indicate that the two commercial kits should be preferred for the molecular analysis of biodiversity of deep-sea nematodes since they consistently provide amplifiable DNA suitable for sequencing. We report that the morphological identification of deep-sea nematodes matches the results obtained by metabarcoding analysis only at the order-family level and that a large portion of Operational Clustered Taxonomic Units (OCTUs) was not assigned. We also show that independently from the cut-off criteria and bioinformatic pipelines used, the number of OCTUs largely exceeds the number of individuals and that 18S rRNA gene of different morpho-species of nematodes displayed intra-genomic polymorphisms. Our results indicate that metabarcoding is an important tool to explore the diversity of deep-sea nematodes, but still fails in identifying most of the species due to limited number of sequences deposited in the public databases, and in providing quantitative data on the species encountered. These aspects should be carefully taken into account before using metabarcoding in quantitative ecological research and monitoring programmes of marine biodiversity.

Indicator-Based Assessment of Marine Biological Diversity–Lessons from 10 Case Studies across the European Seas

The Marine Strategy Framework Directive requires the environmental status of European marine waters to be assessed using biodiversity as one out of 11 descriptors, but the complexity of marine biodiversity and its large span across latitudinal and salinity gradients have been a challenge to the scientific community aiming to produce approaches for integrating information from a broad range of indicators. The Nested Environmental status Assessment Tool (NEAT), developed for the integrated assessment of the status of marine waters, was applied to ten marine ecosystems to test its applicability and compare biodiversity assessments across the four European regional seas. We evaluate the assessment results as well as the assessment designs of the ten cases, and how the assessment design, particularly the choices made regarding the area and indicator selection, affected the results. The results show that only 2 out of the 10 case study areas show more than 50 % probability of being in good status in respect of biodiversity. No strong pattern among the ecosystem components across the case study areas could be detected, but marine mammals, birds, and benthic vegetation indicators tended to indicate poor status while zooplankton indicators indicated good status when included into the assessment. The analysis shows that the assessment design, including the selection of indicators, their target values, geographical resolution and habitats to be assessed, has potentially a high impact on the result, and the assessment structure needs to be understood in order to make an informed assessment. Moreover, recommendations are provided for the best practice of using NEAT for marine status assessments.

A comparison of the degree of implementation of marine biodiversity indicators by European countries in relation to the Marine Strategy Framework Directive (MSFD)

The degree of development and operability of the indicators for the Marine Strategy Framework Directive (MSFD) using Descriptor 1 (D1) Biological Diversity was assessed. To this end, an overview of the relevance and degree of operability of the underlying parameters across 20 European countries was compiled by analysing national directives, legislation, regulations, and publicly available reports. Marked differences were found between countries in the degree of ecological relevance as well as in the degree of implementation and operability of the parameters chosen to indicate biological diversity. The best scoring EU countries were France, Germany, Greece and Spain, while the worst scoring countries were Italy and Slovenia. No country achieved maximum scores for the implementation of MSFD D1. The non-EU countries Norway and Turkey score as highly as the top-scoring EU countries. On the positive side, the chosen parameters for D1 indicators were generally identified as being an ecologically relevant reflection of Biological Diversity. On the negative side however, less than half of the chosen parameters are currently operational. It appears that at a pan-European level, no consistent and harmonized approach currently exists for the description and assessment of marine biological diversity. The implementation of the MSFD Descriptor 1 for Europe as a whole can therefore at best be marked as moderately successful.

Impact of breakwater relocation on benthic biodiversity associated with seagrass meadows of northern Adriatic Sea

Breakwaters are man-made constructions utilized for preventing coastal erosion primarily from wave action. At the same time, defence structures, modifying currents and circulation can affect water quality and benthic assemblages. Assessing and minimizing the impact of these structures is a priority in human-modified coastal ecosystems, such as the central northern Adriatic where breakwaters extend for hundreds of kilometres. We investigated the effects of breakwater relocation on benthic features and meiofaunal diversity. To do this, we conducted a before–after comparison of (2011–2017) the relocation, which occurred in 2015. The analysis was conducted comparing a sheltered site, characterized by the presence of seagrass meadows and a wave-exposed site. Sediment features and meiofaunal variables were altered by the relocation, especially in the areas colonized by seagrass meadows. Results presented here pointed out to an enrichment in organic matter, the loss of two sensitive taxa (Cumacea and Ostracoda) and a shift in the assemblage structure with the increase of the relevance of Copepoda and Polychaeta. These results indicate that the careful management of breakwater is crucial for planning adequate conservation practices and protecting seagrass habitats and their biodiversity.

Impact of mangrove forests degradation on biodiversity and ecosystem functioning

Mangroves are amongst the most productive marine ecosystems on Earth, providing a unique habitat opportunity for many species and key goods and services for human beings. Mangrove habitats are regressing at an alarming rate, due to direct anthropogenic impacts and global change. Here, in order to assess the effects of mangrove habitat degradation on benthic biodiversity and ecosystem functioning, we investigated meiofaunal biodiversity (as proxy of benthic biodiversity), benthic biomass and prokaryotic heterotrophic production (as proxies of ecosystem functioning) and trophic state in a disturbed and an undisturbed mangrove forests. We report here that disturbed mangrove area showed a loss of 20% of benthic biodiversity, with the local extinction of four Phyla (Cladocera, Kynorincha, Priapulida, Tanaidacea), a loss of 80% of microbial-mediated decomposition rates, of the benthic biomass and of the trophic resources. The results of this study strengthen the need to preserve mangrove forests and to restore those degraded to guarantee the provision of goods and services needed to support the biodiversity and functioning of wide portions of tropical ecosystems.

Limited impact of beach nourishment on macrofaunal recruitment/settlement in a site of community interest in coastal area of the Adriatic Sea (Mediterranean Sea)

Beach nourishment is a widely utilized solution to counteract the erosion of shorelines, and there is an active discussion on its possible consequences on coastal marine assemblages. We investigated the impact caused by a small-scale beach nourishment carried out in the Western Adriatic Sea on macrofaunal recruitment and post-settlement events. Artificial substrates were deployed in proximity of nourished and non-manipulated beaches and turbidity and sedimentation rates were measured. Our results indicate that sedimentation rates in the impacted site showed a different temporal change compared to the control sites, suggesting potential modifications due to the beach nourishment. The impact site was characterized by subtle changes in terms of polychaete abundance and community structure when compared to controls, possibly due to beach nourishment, although the role of other factors cannot be ruled out. We conclude that small-scale beach nourishments appear to be an eco-sustainable approach to contrast coastal erosion.

A comparative analysis of metabarcoding and morphology-based identification of benthic communities across different regional seas

Abstract In a world of declining biodiversity, monitoring is becoming crucial. Molecular methods, such as metabarcoding, have the potential to rapidly expand our knowledge of biodiversity, supporting assessment, management, and conservation. In the marine environment, where hard substrata are more difficult to access than soft bottoms for quantitative ecological studies, Artificial Substrate Units (ASUs) allow for standardized sampling. We deployed ASUs within five regional seas (Baltic Sea, Northeast Atlantic Ocean, Mediterranean Sea, Black Sea, and Red Sea) for 12–26 months to measure the diversity and community composition of macroinvertebrates. We identified invertebrates using a traditional approach based on morphological characters, and by metabarcoding of the mitochondrial cytochrome oxidase I (COI) gene. We compared community composition and diversity metrics obtained using the two methods. Diversity was significantly correlated between data types. Metabarcoding of ASUs allowed for robust comparisons of community composition and diversity, but not all groups were successfully sequenced. All locations were significantly different in taxonomic composition as measured with both kinds of data. We recovered previously known regional biogeographical patterns in both datasets (e.g., low species diversity in the Black and Baltic Seas, affinity between the Bay of Biscay and the Mediterranean). We conclude that the two approaches provide complementary information and that metabarcoding shows great promise for marine monitoring. However, until its pitfalls are addressed, the use of metabarcoding in monitoring of rocky benthic assemblages should be used in addition to classical approaches rather than instead of them.