The monitoring, restoration, and conservation of coastal marine biodiversity are crucial ecological priorities to counteract the degradation of hard-bottom habitats. This thesis work is part of the REMEDY project, aimed at combating the desertification of rocky seabeds in the Conero Promontory caused by illegal fishing of the date mussel Lithophaga lithophaga, through ecological restoration interventions based on the transplantation of the habitat-forming sponge Chondrosia reniformis. The main objective was the development, optimization, and comparison of bioinformatic pipelines based on environmental DNA (eDNA) metabarcoding, to validate a non-invasive biomonitoring tool capable of tracking community structure and assessing restoration efficacy. The experimental design involved sampling the water column at a depth of 5 meters across three sites with different environmental matrices: Grotta Azzurra (Site A, degraded area subjected to experimental restoration), Passetto (Site C, natural rocky control), and Out (Site B, offshore sandy reference). Sampling was replicated across three months in 2025: July (T0, pre-restoration), September (T1, post-restoration), and October (T2, autumn phase). In the laboratory, eDNA was extracted using an optimized DNeasy Blood and Tissue kit and amplified targeting a 313 bp hypervariable region of the mitochondrial Cytochrome c Oxidase subunit I (COI) gene using Leray universal primers, followed by high-throughput sequencing on the Illumina MiSeq platform. Raw sequences were processed in QIIME2 using the DADA2 algorithm to generate Amplicon Sequence Variants (ASVs). The pipeline was optimized by implementing BlastN against the MIDORI database with a 94% identity threshold. To eliminate negative control contamination, a statistical algorithm named SD/Gaussian was developed and coded based on biological replicate variability. Methodological comparison demonstrated the superiority of this approach, which explained 91% of the total dataset variability compared to traditional filtering methods. Ecologically, taxonomic characterization successfully outlined metazoan and phytoplankton composition, highlighting a strong biological affinity between rocky sites compared to the sandy bottom. Alpha and Beta diversity analyses registered an initial positive restoration effect: Grotta Azzurra, initially degraded and species-poor in July, showed accelerated biological recolonization in September, nearly matching the ecological complexity of the natural control concurrently with the stabilization of the C. reniformis transplant. Global statis tical models identified seasonality as the main driver of species turnover, culminating in October in a community homogenization driven by autumn microalgal blooms. This study validates a high-performing bioinformatic pipeline, proving the effectiveness of eDNA as a sensitive tool supporting the management and conservation of vulnerable marine habitats.
Il monitoraggio e la conservazione della biodiversità marina costiera sono priorità ecologiche cruciali per contrastare la degradazione degli habitat a substrato duro. Il presente lavoro di Tesi si inserisce nel progetto REMEDY, volto a contrastare la desertificazione dei fondali rocciosi nel Promontorio del Conero causata dalla pesca illegale di Lithophaga lithophaga, mediante interventi di restauro ecologico basati sul trapianto della spugna habitat-former Chondrosia reniformis. L'obiettivo principale e stato lo sviluppo, l'ottimizzazione e il confronto di pipeline bioinformatiche basate sull'analisi del DNA ambientale (eDNA) mediante metabarcoding, per validare uno strumento di biomonitoraggio non invasivo capace di tracciare la struttura di comunita e valutare l'efficacia del restauro. Il disegno sperimentale ha previsto il campionamento della colonna d'acqua a 5 metri di profondita in tre siti con differenti matrici ambientali: Grotta Azzurra (Sito A, area degradata sottoposta a trapianto), Passetto (Sito C, controllo naturale) e Out (Sito B, riferimento sabbioso in mare aperto). I campionamenti sono stati replicati lungo tre mesi del 2025: luglio (T0, pre-restauro), settembre (T1, post-restauro) e ottobre (T2, fase autunnale). In laboratorio, l'eDNA e stato estratto mediante kit DNeasy Blood e Tissue ottimizzato e amplificato prendendo come target una porzione di 313 bp del gene mitocondriale Citocromo c Ossidasi subunità I (COI) tramite primer universali di Leray, con successivo sequenziamento massivo su piattaforma Illumina MiSeq. Le sequenze grezze sono state elaborate in QIIME2 con algoritmo DADA2 per la generazione di Amplicon Sequence Variants (ASVs). La pipeline e stata ottimizzata implementando BlastN sul database MIDORI con soglia di identita al 94%. Per eliminare le contaminazioni dei controlli negativi e stato sviluppato un algoritmo statistico denominato SD/Gaussiano, basato sulla variabilita delle repliche biologiche. Il confronto metodologico ha dimostrato la superiorita di questo approccio, capace di spiegare il 91% della variabilita totale del dataset rispetto ai metodi tradizionali. Dal punto di vista ecologico, la caratterizzazione tassonomica ha delineato con successo la composizione dei metazoi e del fitoplancton, evidenziando una forte affinita biologica tra i siti rocciosi rispetto al fondale sabbioso. Le analisi di Alpha e Beta Diversity hanno registrato un effetto positivo iniziale del restauro: la Grotta Azzurra, inizialmente degradata e povera di specie a luglio, ha mostrato una ricolonizzazione biologica accelerata a settembre, eguagliando la complessita ecologica del controllo naturale in concomitanza con la stabilizzazione del trapianto di C. reniformis. I modelli statistici globali hanno individuato nella stagionalita la forza principale nel determinare il turnover delle specie, culminando ad ottobre in una omogenizzazione guidata da fioriture microalgali autunnali. Lo studio convalida cosi una pipeline performante, dimostrando l'efficacia dell'eDNA come strumento sensibile a supporto della gestione e conservazione degli habitat marini vulnerabili.
Approcci bioinformatici in eDNA metabarcoding (COI) per il monitoraggio della biodiversità marina e la valutazione del restauro ecologico nel Parco del Conero (Marche).
TROPI, LINDA
2025/2026
Abstract
The monitoring, restoration, and conservation of coastal marine biodiversity are crucial ecological priorities to counteract the degradation of hard-bottom habitats. This thesis work is part of the REMEDY project, aimed at combating the desertification of rocky seabeds in the Conero Promontory caused by illegal fishing of the date mussel Lithophaga lithophaga, through ecological restoration interventions based on the transplantation of the habitat-forming sponge Chondrosia reniformis. The main objective was the development, optimization, and comparison of bioinformatic pipelines based on environmental DNA (eDNA) metabarcoding, to validate a non-invasive biomonitoring tool capable of tracking community structure and assessing restoration efficacy. The experimental design involved sampling the water column at a depth of 5 meters across three sites with different environmental matrices: Grotta Azzurra (Site A, degraded area subjected to experimental restoration), Passetto (Site C, natural rocky control), and Out (Site B, offshore sandy reference). Sampling was replicated across three months in 2025: July (T0, pre-restoration), September (T1, post-restoration), and October (T2, autumn phase). In the laboratory, eDNA was extracted using an optimized DNeasy Blood and Tissue kit and amplified targeting a 313 bp hypervariable region of the mitochondrial Cytochrome c Oxidase subunit I (COI) gene using Leray universal primers, followed by high-throughput sequencing on the Illumina MiSeq platform. Raw sequences were processed in QIIME2 using the DADA2 algorithm to generate Amplicon Sequence Variants (ASVs). The pipeline was optimized by implementing BlastN against the MIDORI database with a 94% identity threshold. To eliminate negative control contamination, a statistical algorithm named SD/Gaussian was developed and coded based on biological replicate variability. Methodological comparison demonstrated the superiority of this approach, which explained 91% of the total dataset variability compared to traditional filtering methods. Ecologically, taxonomic characterization successfully outlined metazoan and phytoplankton composition, highlighting a strong biological affinity between rocky sites compared to the sandy bottom. Alpha and Beta diversity analyses registered an initial positive restoration effect: Grotta Azzurra, initially degraded and species-poor in July, showed accelerated biological recolonization in September, nearly matching the ecological complexity of the natural control concurrently with the stabilization of the C. reniformis transplant. Global statis tical models identified seasonality as the main driver of species turnover, culminating in October in a community homogenization driven by autumn microalgal blooms. This study validates a high-performing bioinformatic pipeline, proving the effectiveness of eDNA as a sensitive tool supporting the management and conservation of vulnerable marine habitats.| File | Dimensione | Formato | |
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Descrizione: Tesi di Laurea Magistrale in Biologia Marina. Linda Tropi S1115989
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https://hdl.handle.net/20.500.12075/28160