Next Generation Sequencing of Ancient Fungal Specimens: The Case of the Saccardo Mycological Herbarium

Despite their essential role in the environment, the number of known fungal species is still low compared to the recent estimates of the fungal biodiversity, principally because of their often cryptic or ambiguous morphological traits. Recent studies have reported that the number of fungal DNA sequences deposited in public DNA databases and representing correctly a species appears dramatically low (approx. 120,000) when compared with the estimated total number of species (approx. from 2.2 to 3.8 million). Thus, the linkage of curated DNA sequence data to expertly identified voucher specimens is of fundamental importance to fill the present gap between the different sizes of described and sequenced fungal diversity. To this purpose, the mycological herbarium collections are considered an important source for fungal DNA-barcoding, and collection-based sequencing is a relevant priority for the coming decades. Unfortunately, ancient herbarium samples have both time and conservation related DNA damages, besides exogenous DNA contamination, that make nucleic acid extraction and amplification challenging. Here, we present the results of DNA extraction, ITS2 amplification and Illumina MiSeq sequencing of 36 specimens from the Saccardo Mycological Herbarium that were collected in the late XIX century and assigned to the genus Peziza. High-throughput sequencing was chosen as an alternative to the conventional Sanger- and cloning-based methods to overcome the high fragmentation of the ancient DNA and the massive occurrence of non-target DNA from fungal contaminants. Our approach has permitted to assign ITS2 sequences to 23 out of the 36 specimens studied in this work, thus providing a univocal DNA sequence for those one century old samples. Furthermore, the ITS2 sequence analysis has permitted a taxonomic study of the samples that has resulted in a revaluation of 5 samples at the species level and 18 samples at genus or higher level. Our results highlight the possibility to apply the technique presented in this work also to the old and more precious type specimens in order to relate a DNA sequence to the species distinctive sample, coupling the traditional morphological description of the species with a DNA sequence

A next generation sequencing approach for the study of ancient fungal specimens belonging to the Pier Andrea Saccardo collection preserved at the University of Padua

The mycological collections represent a huge source of molecular information that may be exploited to obtain important DNA data. Indeed, it has been demonstrated that DNA barcoding projects of fungarium material have the potential to enlarge the coverage of species-level DNA sequence information deposited in public databases. However, these collections are an underused resource for building up voucher-based reference datasets, due to the difficulty to obtain DNA sequences from herbarium material. The over one century old Saccardo mycological collection preserved in the herbarium of the Botanical Garden of Padua contains about 70,000 specimens including more than 4,000 type specimens. The types in this collection have been borrowed by mycologists from all over the world for morphological revisions and consequent taxonomic reclassifications, but they have never been involved in sequencing projects so far. Accordingly, the aim of this research was to apply a DNA barcoding approach to obtain internal transcribed spacer (ITS) sequences, the consensus barcode for fungal species identification, from specimens preserved in this collection. This DNA region has been identified as a suitable marker for molecular studies that involve ancient mycological material, because only short DNA regions can be obtained from ancient and degraded DNA. In addition, in case of initial PCR failure of the ITS region, it is possible to increase the amplification/sequencing success by analyzing separately the two non-coding regions ITS1 and ITS2 that form the entire ITS. In this thesis work, an Illumina MiSeq sequencing method was applied to recover ITS1/ITS2 sequences overcoming the problems of the high level of DNA degradation of the Saccardo fungarium samples and the presence of contaminations by exogenous fungal DNA. The method required the setup of the steps involved in the samples preparation for the sequencing and in the bioinformatic data analysis, and then its efficacy was first tested to obtain ITS2 sequences from 36 non-type Peziza specimens. Despite the presence of both external fungal contamination and cross-contamination between fungarium specimens, this high-throughput sequencing method has permitted to recover ITS2 sequences from 23 out of the 36 specimens studied and also a taxonomic re-evaluation of some samples at the species level and others at genus or higher taxonomic level. Then, this next-generation sequencing (NGS) approach was used to retrieve ITS1/ITS2 sequences from type specimens belonging to the genus Nectria and from Nectria-like types classified in the collection as members of other genera. Several of these types were morphologically revised in the past by expert mycologists and placed in synonymy with other species or reclassified as members of new genera. The ITS1/ITS2 sequences were obtained for 25 different types (30 in total considering multiple specimens) out of 76 specimens involved in the study. The combined morphological and molecular data analysis suggests that there is a need to reclassify some Nectria/Nectria-like types previously reclassified only on a morphological basis and some types never considered for taxonomic revisions. In fact, for 11 types the original species name has been confirmed, for four and five types new nomenclature combinations and synonymies have been proposed respectively, while for other five types the taxonomic assignment has been possible only at genus level. Since type specimens constitute an integral part of fungal classification and nomenclature and given the outstanding and worldwide importance of the Saccardo collection, these findings provide material for a taxonomic revision of invaluable types. Moreover, the method proposed in this research not only has provided an additional scientific value to the Saccardo collection, but it can be applied to obtain important voucher-sequences from problematic herbarium material, thus expanding the databases with well-annotated ITS barcode sequences.Le collezioni micologiche rappresentano un’enorme risorsa di informazioni molecolari che può essere sfruttata per ottenere importanti sequenze di DNA. Infatti, è stato dimostrato che i progetti di DNA barcoding che coinvolgono materiale fungino proveniente da collezioni micologiche possono aumentare la quantità di informazioni molecolari relative alle diverse specie fungine nei database pubblici. Tuttavia, queste raccolte sono una risorsa poco sfruttata per la creazione di datasets di riferimento basati su campioni accuratamente identificati, a causa della difficoltà nell’ottenere sequenze di DNA dal materiale conservato all’interno degli erbari. La raccolta micologica di Pier Andrea Saccardo, di oltre un secolo, conservata nell'erbario dell'Orto Botanico di Padova, contiene circa 70.000 campioni fungini di cui oltre 4.000 sono esemplari tipo. I tipi di questa collezione sono stati spesso richiesti da micologi di tutto il mondo per revisioni morfologiche e conseguenti riclassificazioni tassonomiche, ma non erano mai stati coinvolti in progetti di sequenziamento finora. Di conseguenza, lo scopo di questa ricerca è stata quella di applicare un approccio di DNA barcoding per ottenere sequenze della regione ITS (internal transcribed spacer), il barcode utilizzato per l'identificazione delle specie fungine, dai campioni conservati in questa collezione. Questa regione è considerata un marcatore adatto per studi molecolari che coinvolgono materiale micologico antico, poiché solo corte regioni di DNA possono essere ottenute da DNA antico e degradato. Inoltre, in caso di fallimento nell’amplificazione della regione ITS, è possibile aumentare il successo dell'amplificazione/sequenziamento analizzando separatamente le due regioni non codificanti ITS1 e ITS2 che formano l'intera regione ITS. In questo lavoro di tesi, è stato applicato un metodo di sequenziamento Illumina MiSeq per ottenere sequenze ITS1/ITS2 superando i problemi relativi all'alto livello di degradazione del DNA dei campioni della collezione micologica e la presenza di contaminazioni da DNA fungino esogeno. Il metodo ha richiesto l'ottimizzazione delle diverse fasi coinvolte nella preparazione dei campioni per il sequenziamento e nell'analisi bioinformatica dei dati, e la sua efficacia è stata prima testata per ottenere sequenze ITS2 da 36 esemplari fungini non-tipo appartenenti al genere Peziza. Nonostante la presenza sia di contaminazioni fungine esterne che di contaminazione incrociata tra i campioni della collezione, questo metodo di sequenziamento ha permesso di recuperare sequenze della regione ITS2 da 23 dei 36 campioni studiati e anche una rivalutazione tassonomica di alcuni campioni a livello di specie e altri a livello di genere o livello tassonomico superiore. Successivamente, questo approccio di sequenziamento di nuova generazione è stato utilizzato per ottenre sequenze ITS1/ITS2 da campioni tipo appartenenti al genere Nectria e da tipi Nectria-simili classificati nella collezione come membri di altri generi. Molti di questi tipi furono morfologicamente revisionati in passato da esperti micologi e posti in sinonimia con altre specie o riclassificati come membri di nuovi generi. Le sequenze ITS1/ITS2 sono state ottenute per 25 diversi tipi (30 in totale considerando campioni multipli) su 76 campioni coinvolti nello studio. L'analisi combinata dei dati morfologici e molecolari suggerisce la necessità di riclassificare alcuni tipi di Nectria/Nectria-simili precedentemente riclassificati solo su base morfologica e alcuni tipi mai considerati per una revisione tassonomica. Infatti, per 11 tipi è stato confermato il nome originale della specie, per quattro e cinque tipi sono state proposte rispettivamente nuove combinazioni di nomenclatura e sinonimie, mentre per altri cinque l'assegnazione tassonomica è stata possibile solo a livello di genere. Poiché i campioni tipo costituiscono una parte integrante della classificazione e della nomenclatura dei funghi e data l'importanza eccezionale e mondiale della collezione Saccardo, questi risultati forniscono materiale per una revisione tassonomica di inestimabili campioni tipo. Inoltre, il metodo proposto in questa ricerca non solo ha fornito un valore scientifico aggiuntivo alla collezione Saccardo, ma può essere applicato per ottenere importanti sequenze da materiale d’erbario, espandendo così i database con sequenze ITS ben annotate

Taxonomic Re-Examination of Nine Rosellinia Types (Ascomycota, Xylariales) Stored in the Saccardo Mycological Collection

In a recent monograph on the genus Rosellinia, type specimens worldwide were revised and re-classified using a morphological approach. Among them, some came from Pier Andrea Saccardo’s fungarium stored in the Herbarium of the Padova Botanical Garden. In this work, we taxonomically re-examine via a morphological and molecular approach nine different Roselliniasensu Saccardo types. ITS1 and/or ITS2 sequences were successfully obtained applying Illumina MiSeq technology and phylogenetic analyses were carried out in order to elucidate their current taxonomic position. Only the ITS1 sequence was recovered for Rosellinia areolata, while for R. geophila, only the ITS2 sequence was recovered. We proposed here new combinations for Rosellinia chordicola, R. geophila and R. horridula, while for R. ambigua, R. areolata, R. australis, R. romana and R. somala, we did not suggest taxonomic changes compared to the current ones. The name Rosellinia subsimilis Sacc. is invalid, as it is a later homonym of R. subsimilis P. Karst. & Starbäck. Therefore, we introduced Coniochaeta dakotensis as a nomen novum for R. subsimilis Sacc. This is the first time that these types have been subjected to a molecular study. Our results demonstrate that old types are an important source of DNA sequence data for taxonomic re-examinations

CoCoNet: Towards coast to coast networks of marine protected areas (From the shore to the high and deep sea), coupled with sea-based wind energy potential

This volume contains the main results of the EC FP7 "The Ocean of Tomorrow" Project CoCoNet, divided in two sections: 1) a set of guidelines to design networks of Marine Protected Areas in the Mediterranean and the Black Seas; 2) a smart wind chart that will allow evaluating the possibility of installing Offshore Wind Farms in both seas. The concept of Cells of Ecosystem Functioning, based on connectivity, is introduced to define natural units of management and conservation. The definition of Good Environmental Status, as defined in the Marine Strategy Framework Directive, is fully embraced to set the objectives of the project, by adopting a holistic approach that integrates a full set of disciplines, ranging from physics to bio-ecology, economics, engineering and many sub-disciplines. The CoCoNet Consortium involved scientist sfrom 22 states, based in Africa, Asia, and Europe, contributing to build a coherent scientific community