1. Multi-marker metabarcoding approach to study mesozooplankton at basin scaleSergio Stefanni, David Stanković, Diego Borme, Alessandra De Olazabal, Tea Juretić, Alberto Pallavicini, Valentina Tirelli, 2018, izvirni znanstveni članek Povzetek: Zooplankton plays a pivotal role in marine ecosystems and the characterisation of its biodiversity still represents a challenge for marine ecologists. In this study, mesozooplankton composition from 46 samples collected in summer along the western Adriatic Sea, was retrieved by DNA metabarcoding analysis. For the first time, the highly variable fragments of the mtDNA COI and the V9 region of 18S rRNA genes were used in a combined matrix to compile an inventory of mesozooplankton at basin scale. The number of sequences retrieved after quality filtering were 824,148 and 223,273 for COI and 18S (V9), respectively. The taxonomical assignment against reference sequences, using 95% (for COI) and 97% (for 18S) similarity thresholds, recovered 234 taxa. NMDS plots and cluster analysis divided coastal from offshore samples and the most representative species of these clusters were distributed according to the dominant surface current pattern of the Adriatic for the summer period. For selected sampling sites, mesozooplankton species were also identified under a stereo microscope providing insights on the strength and weakness of the two approaches. In addition, DNA metabarcoding was shown to be helpful for the monitoring of non-indigenous marine metazoans and spawning areas of commercial fish species. We defined pros and cons of applying this approach at basin scale and the benefits of combining the datasets from two genetic markers.
Ključne besede: zooplankton, genetics, biodiversity
Objavljeno v DiRROS: 24.07.2024; Ogledov: 66; Prenosov: 23
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2. ITS2 metabarcoding analysis complements lichen mycobiome diversity dataElisa Banchi, David Stanković, Fernando Fernández-Mendoza, Fabrizia Gionechetti, Alberto Pallavicini, Lucia Muggia, 2018, izvirni znanstveni članek Povzetek: Lichen thalli harbor complex fungal communities (mycobiomes) of species with divergent trophic and ecological strategies. The complexity and diversity of lichen mycobiomes are still largely unknown, despite surveys combining culture-based methods and high-throughput sequencing (HTS). The results of such surveys are strongly influenced by the barcode locus chosen, its sensitivity in discriminating taxa, and the depth to which public sequence repositories cover the phylogenetic spectrum of fungi. Here, we use HTS of the internal transcribed spacer 2 (ITS2) to assess the taxonomic composition and diversity of a well-characterized, alpine rock lichen community that includes thalli symptomatically infected by lichenicolous fungi as well as asymptomatic thalli. Taxa belonging to the order Chaetothyriales are the major components of the observed lichen mycobiomes. We predict sequences representative of lichenicolous fungi characterized morphologically and assess their asymptomatic presence in lichen thalli. We demonstrated the limitations of metabarcoding in fungi and show how the estimation of species diversity widely differs when ITS1 or ITS2 are used as barcode, and particularly biases the detection of Basidiomycota. The complementary analysis of both ITS1 and ITS2 loci is therefore required to reliably estimate the diversity of lichen mycobiomes.
Ključne besede: ascomycetes, basidiomycetes, endophytes, fungal isolates, ion torrent, ITS1
Objavljeno v DiRROS: 24.07.2024; Ogledov: 52; Prenosov: 29
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3. DNA metabarcoding uncovers fungal diversity of mixed airborne samples in ItalyElisa Banchi, Claudio Gennaro Ametrano, David Stanković, Pierluigi Verardo, Olga Moretti, Francesca Gabrielli, Stefania Lazzarin, Maria Francesca Borney, Francesca Tassan, Mauro Tretiach, Alberto Pallavicini, Lucia Muggia, 2018, izvirni znanstveni članek Povzetek: Fungal spores and mycelium fragments are particles which become and remain airborne and have been subjects of aerobiological studies. The presence and the abundance of taxa in aerobiological samples can be very variable and impaired by changeable climatic conditions. Because many fungi produce mycotoxins and both their mycelium fragments and spores are potential allergens, monitoring the presence of these taxa is of key importance. So far data on exposure and sensitization to fungal allergens are mainly based on the assessment of few, easily identifiable taxa and focused only on certain environments. The microscopic method used to analyze aerobiological samples and the inconspicuous fungal characters do not allow a in depth taxonomical identification. Here, we present a first assessment of fungal diversity from airborne samples using a DNA metabarcoding analysis. The nuclear ITS2 region was selected as barcode to catch fungal diversity in mixed airborne samples gathered during two weeks in four sites of North-Eastern and Central Italy. We assessed the taxonomic composition and diversity within and among the sampled sites and compared the molecular data with those obtained by traditional microscopy. The molecular analyses provide a tenfold more comprehensive determination of the taxa than the traditional morphological inspections. Our results prove that the metabarcoding analysis is a promising approach to increases quality and sensitivity of the aerobiological monitoring. The laboratory and bioinformatic workflow implemented here is now suitable for routine, high-throughput, regional analyses of airborne fungi.
Ključne besede: fungi, fungal spores, taxonomy, sequence databases
Objavljeno v DiRROS: 24.07.2024; Ogledov: 62; Prenosov: 36
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4. PLANiTS : a curated sequence reference dataset for plant ITS DNA metabarcodingElisa Banchi, Claudio Gennaro Ametrano, Samuele Greco, David Stanković, Lucia Muggia, Alberto Pallavicini, 2020, izvirni znanstveni članek Povzetek: DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called ‘better clustering for QIIME’ (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100 224 sequences), PLANiTS2 (96 771 sequences) and PLANiTS (97 550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering.
Ključne besede: DNA metabarcoding, reference datasets
Objavljeno v DiRROS: 19.07.2024; Ogledov: 79; Prenosov: 54
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5. Detection of the endangered stone crayfish Austropotamobius torrentium (Schrank, 1803) and its congeneric A. pallipes in its last italian biotope by eDNA analysisChiara Manfrin, Massimo Zanetti, David Stanković, Umberto Fattori, Victoria Bertucci-Maresca, Piero G. Giulianini, Alberto Pallavicini, 2022, izvirni znanstveni članek Povzetek: The stone crayfish, Austropotamobius torrentium, is a European freshwater crayfish. Although this species is relatively widespread throughout the continent, it is undergoing significant declines throughout its range. However, as the decline rates have not been quantified in detail, this species is classified as data deficient by the IUCN Red List of Threatened Species. The present study describes the development and validation of two species-specific assays based on hydrolysis probe chemistry for the detection of A. torrentium and A. pallipes environmental DNA (eDNA) in water samples collected in the Julian Alps of Italy (Friuli Venezia Giulia). The eDNA-based method was applied to 14 sites within the Danubian Slizza basin, known to be inhabited by A. torrentium, but with insufficient information on their distribution. In addition, one station in the Tagliamento River basin was sampled to test the performance of the A. pallipes probe. The presence of A. torrentium is confirmed at 6 out of 15 sites. At four of these sites, A. torrentium is detected for the first time. In contrast, the presence of A. torrentium was not detected at two sites already known to harbour the species. Finally, the presence of A. pallipes was confirmed in the station belonging to the Tagliamento basin. The methodology described, which allows the distinction between the two species, paves the way for the parallel detection of the stone crayfish and the white-clawed crayfish (A. pallipes) through eDNA analysis.
Objavljeno v DiRROS: 16.07.2024; Ogledov: 80; Prenosov: 31
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6. Contribution to the knowledge of Gongolaria barbata (Sargassaceae, Fucales) from the Mediterranean : insights into infraspecific diversityAnnalisa Falace, Chiara Manfrin, Giovanni Furnari, Sara D’Ambros Burchio, Alberto Pallavicini, Emmanuelle Patricia Descourvières, Sara Kaleb, Ana Lokovšek, Daniele Grech, Giuseppina Alongi, 2024, izvirni znanstveni članek Povzetek: Gongolaria barbata (Sargassaceae, Fucales) is a widespread species for which several infraspecific taxa have been described, indicating its polymorphism. This study contributes to the understanding of the molecular, nomenclatural, morphological and ecological aspects of G. barbata in the Mediterranean and sheds light on the infraspecific diversity and its implications for the taxonomy of this species. Molecular analyses were performed using sequencing of the mitochondrial cox1 gene on both haptophytic and pleustophytic forms from different sites in the Adriatic and Tyrrhenian Seas. Vegetative and reproductive morphology was studied on thalli samples from the Adriatic. Our results showed that there are different morphotypes within G. barbata populations related to specific environmental conditions, suggesting infraspecific variation. In contrast, molecular analyses showed no differences between samples, regardless of whether individuals are growing “attached” to a substrate or “unattached”. We also discussed the taxonomic status and nomenclatural issues related to certain infraspecific taxa previously proposed for G. barbata. In particular, the confusion surrounding Cystoseira aurantia is clarified.
Ključne besede: ecotypes, taxonomy, Northern Adriatic, Cystoseira s.l.
Objavljeno v DiRROS: 16.05.2024; Ogledov: 208; Prenosov: 107
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