Scuola di Dottorato di Ricerca in SCIENZE Curriculum BIOLOGIA ed ECOLOGIA MARINA XIV° ciclo n.s. Molecular analysis of marine benthic biodiversity: methodological implementations and perspectives Tesi di Dottorato di: Tutor: Laura Carugati Prof. Roberto Danovaro Co-Tutor: Prof. Antonio Dell’Anno A.A. 2016/2017 Index RIASSUNTO ............................................................................................................................................ 3 ABSTRACT .............................................................................................................................................. 4 CHAPTER 1. INTRODUCTION ............................................................................................................. 5 1.1 A comparison of the degree of implementation of marine biodiversity indicators by European countries in relation to the MSFD ....................................................................................................... 13 1.2 Indicator-based assessment of marine biological diversity – lessons from 10 case studies across the European Seas ............................................................................................................................... 39 CHAPTER 2. METHODOLOGICAL IMPLEMENTATION ............................................................... 92 2.1 Implementing and innovating marine monitoring approaches for the assessment of the environmental status ............................................................................................................................ 92 2.2 Metagenetic tools for the census of marine meiofaunal biodiversity: An overview ................... 149 CHAPTER 3. IMPLEMENTATION OF METAGENETIC ANALYSIS OF MEIOFAUNAL BIODIVERSITY ................................................................................................................................... 180 3.1 Sensitivity of metagenetic analysis of meiofaunal biodiversity .................................................. 180 3.2 Unveiling the Biodiversity of Deep-Sea Nematodes through Metabarcoding: Are We Ready to Bypass the Classical Taxonomy? ...................................................................................................... 201 CHAPTER 4. CASE STUDY AND APPLICATION TO MEIOBENTHIC BIODIVERSITY .......... 234 4.1 Meiofaunal biodiversity in the Ross Sea continental shelf, Antarctica ....................................... 234 CHAPTER 5. CONCLUSIONS ........................................................................................................... 259 5.1 Perspectives in the use of molecular tools for the analysis of marine benthic biodiversity ........ 259 Annex 1- Publications on ISI journal .................................................................................................... 263 2 RIASSUNTO La biodiversità marina regola il funzionamento ecosistemico, responsabile della produzione di beni e servizi importanti per la biosfera e il benessere umano. La stima della biodiversità è uno degli obiettivi fondamentali della scienza. I cambiamenti globali e le attività antropiche stanno avendo un impatto crescente sugli ecosistemi. Le valutazioni d'impatto e i programmi di monitoraggio si basano su specie di grandi dimensioni, trascurando gli organismi di piccola taglia come la meiofauna (20-500 µm), a causa delle difficoltà connesse con la tradizionale identificazione morfologica. L’obiettivo di questo lavoro è quello di implementare i metodi per lo studio della meiofauna, combinando l’approccio morfologico e molecolare. Al fine di rispondere alle esigenze della Direttiva Quadro sulla Strategia Marina sono stati testati, in diversi ecosistemi marini, uno strumento per la valutazione dello stato ambientale e varie metodologie innovative di campionamento. Gli strumenti molecolari, come il metabarcoding, insieme ai nuovi sistemi di campionamento, mostrano un grande potenziale per lo studio della biodiversità marina. Il protocollo molecolare qui messo a punto, permette di analizzare la diversità di organismi di piccola taglia in modo rapido ed economicamente efficiente, migliorando la capacità di studiare la biodiversità della meiofauna anche in ecosistemi marini profondi. Nonostante i numerosi vantaggi, l'applicazione del metabarcoding al monitoraggio richiede il superamento di alcuni limiti. L'incompletezza delle banche dati e la presenza di copie multiple di geni in individui della stessa specie ostacolano l'interpretazione dei dati e non consentono di effettuare stime quantitative di biodiversità. Per garantire che le analisi di metabarcoding possano essere ulteriormente convalidate, risulta cruciale conservare la competenza nell’identificazione morfologica. L’approccio tradizionale e quello molecolare forniscono informazioni diverse, quindi dovrebbero essere usati entrambi per ottenere stime accurate di biodiversità. 3 ABSTRACT Marine biodiversity regulates ecosystem functions that are responsible for the production of goods and services for the entire biosphere and human wellbeing. Censusing species inhabit oceans is among the most fundamental questions in science. Global changes and human activities are having an increasing impact on ocean biodiversity and ecosystem functioning. Impact assessments and monitoring programmes are almost based on large and conspicuous species. Small and cryptic organisms including eukaryotic meiobenthic fauna (20-500 µm) remain overlooked due to the difficulties associated with the traditional, morphology-based identification. We here aim to implement methods for the study of meiofaunal biodiversity, combining classical morphological and molecular approaches. In order to answer to the needs of the Marine Strategy Framework Directive, an environmental status assessment tool and a variety of innovative sampling methodologies have been tested in different ecosystems. Along with new sampling approaches, molecular tools, as metabarcoding, have a great potential in innovating the analysis of marine biodiversity. Molecular protocol set up in this work allows to analyse the diversity of tiny organisms in a rapid and cost-effective way, thus increasing our ability to investigate meiofaunal biodiversity, also in the deep sea. Beyond its many advantages, the routine application of metabarcoding for monitoring requires overcoming some limitations. The incompleteness of public databases and the presence of multi-copy genes within meiofaunal species hamper the interpretation of metabarcoding data and do not allow to infer biodiversity quantitative estimates. It is thus critically important to maintain expertise in morphological identification to ensure that metabarcoding analyses can be further validated. Morphological- and molecular-based approaches provide different information, thus they should be combined to obtain better evaluations of the actual marine biodiversity. 4 CHAPTER 1. INTRODUCTION Almost 20 years ago, Gray stated: “Complete loss of habitat is the most serious threat to marine biodiversity” (Gray, 1997). Although habitat loss would have dramatic effects on marine biodiversity, nowadays, it is recognized that pressures and threats due to anthropogenic activities and climate changes can erode biodiversity before habitat is completely lost. These stressors can act in synergy and cause changes in biodiversity that are more pervasive than those caused by single disturbances (Sala and Knowlton, 2006). Human activities such as overfishing, agricultural expansion, dumping, mining, waste, pollution, species introductions can reduce marine biodiversity, by i) accelerating food webs (increasing the turnover of communities via fishing down food webs and enhancing microbial activity), ii) causing pollution-mediated mass mortalities of marine organisms (e.g., dead zones), and iii) facilitating the dominance of invasive species (Halpern et al., 2008). While anthropogenic activities are considered the main direct drivers of biodiversity loss during recent years (Hoffmann et al., 2010), climate change is likely to become the main cause of extinction over the coming century (Burrows et al., 2011; Doney et al., 2012). In addition, human-forced climate changes, such as those provoked by anthropogenic greenhouse gas emissions, have already caused increased temperatures, sea level rise, altered rainfall patterns and the frequency and severity of extreme events (IPCC, 2014). Climate changes induced by human activities may also have an indirect impact on marine ecosystems, altering how and where people interact with environment (Watson, 2014). The increasing attitude to build seawalls to protect against sea-level rise, has led to habitat loss (Dugan et al., 2008; Grantham et al., 2011) and changes in fish distribution (Pinsky and Fogarty, 2012). The impacts of relocation of urban areas and agricultural land due to future sea level rise may be more severe than the direct influence of sea-level rise (Wetzel et al., 2012). Widespread impacts of human activities and climate changes on marine biodiversity can threaten ecosystem functions and thus ecosystem services, reduce ecosystem stability and resilience and hinder the recovery of biodiversity (Lotze et al., 2006; Sala and Knowlton, 2006; Worm et al., 2006; Danovaro et al., 2008; Halpern et al., 2008; Butchart et al., 2010). These effects have been reported for different geographic areas (Duffy, 2003; Springer et al., 2003; Sala and Knowlton, 2006). With the increasing awareness about the benefits that healthy marine ecosystems provide to people (MEA,