Lichens – as self-sustaining, mutualistic and symbiotic systems – house a multiplicity of microorganisms (prokaryotes, microalgae and microfungi), whose biodiversity and roles in the symbioses are still largely unknown. Until recently, only a few studies focused on the overall diversity of the lichen-associated fungi which constitute the lichen mycobiome. In this research, I characterized the diversity of the lichen mycobiome in two cosmopolitan, epilithic lichens, i.e. Rhizoplaca melanophthalma and Tephromela atra, that were chosen as study organisms. Their mycobiomes were investigated for the first time at a global scale, as lichen samples were collected across their entire geographic range and both a culture-dependent and environmental DNA (eDNA) metabarcoding approaches were performed. I aimed at i) characterizing if any stable ‘core mycobiome’ can be detected for each lichen species and if a shared mycobiome exists; ii) understanding the role of the environmental conditions in determining the lichen mycobiome composition. By culture-dependent approach, I isolated and identified, using an integrated approach based on morphological and phylogenetic data, 76 basidiomycete yeasts (belonging to the five classes Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes) and 131 ascomycetes fungi (belonging to the three classes Eurotiomycetes, Dothideomycetes and Sordariomycetes). Furthermore, within the ascomycetes fungi I identified two new lineages in Chaetothyriales. Most of the isolated fungi, either the ascomycetes and basidiomycetes, have been previously found in lichens, or resulted to be taxa closely related to species which usually occupy other ecological niches (i.e. plants, rocks and soil) and which adopt different lifestyles (i.e. plant pathogens, opportunists and saprotrophs). By DNA metabarcoding, I first tried to design and test a set of blocking primers, which were specifically designed to reduce the amplification of R. melanophthalma and T. atra mycobionts and to enhance the amplification of all the other fungi present within the lichen thalli. However, these primers did not prevent the amplification of the mycobionts and I carried out the metabarcoding approach without using them. The comparison of the fungal communities highlighted low diversity within single thalli with the presence of few ascomycetes consistently present in both lichen species, mostly belonging to Chaetothyriales, Capnodiales and Helotiales. Basidiomycetes account only for 7-14% of the sequences, mostly from Tremellales. The environmental conditions, especially when harsh (e.g. extreme elevation) and the geographic isolation possibly played the main role in shaping the mycobiome composition, however also the lichen host seemed to influence the mycobiome community, though in a lesser extent. A stable species-specific core mycobiome was hardly identifiable both in R. melanophthalma and T. atra as most of the mycobiome taxa were present with low frequency of occurrence and no taxon was ubiquitously present in neither lichen. I also compared the fungal diversity detected by DNA metabarcoding with the one obtained by the culture-dependent approach, to understand which fraction of the mycobiome was only detected either by culture isolation or by eDNA analyses. Even if DNA metabarcoding described most of the diversity of the lichen mycobiome, and many of the metabarcoded taxa were also isolated in culture, surprisingly there were some fungal species which grew in culture but were not detected by the metabarcoding analysis. In conclusion, both the culture-dependent and the DNA metabarcoding approaches well complemented each other and allowed to obtain a fairly complete estimation of the global lichen mycobiome diversity in R. melanophthalma and T. atra.

I licheni – essendo sistemi autosufficienti, mutualistici e simbiotici – ospitano una molteplicità di microrganismi (procarioti, microalghe e microfunghi), la cui biodiversità e ruolo nella simbiosi è ancora ampiamente sconosciuta. Fino ad oggi, pochi studi si sono concentrati sulla diversità complessiva dei funghi associati ai licheni che costituisce il micobioma lichenico. In questa ricerca, ho caratterizzato la diversità del micobioma di due licheni epilitici e cosmopoliti, i.e. Rhizoplaca melanophthalma e Tephromela atra, che sono stati scelti come organismi studio. Avendo campioni di licheni provenienti da tutto il loro areale geografico è stato possibile per la prima volta analizzare il micobioma su scala globale. Per fare questo è stato eseguito sia l’approccio di coltura-dipendente che quello di metabarcoding del DNA ambientale (eDNA). L’obiettivi erano i) caratterizzare possibili comunità fungine che si mantengono stabili per una data specie di lichene e verificare se una parte di questa comunità fosse condivisa tra i due organismi studio; ii) capire se le condizioni ambientali influenzano la composizione e specificità del micobioma dei licheni. Utilizzando l’approccio di coltura-dipendente, ho isolato e identificato sulla base di dati morfologici e genetici, 76 lieviti basidiomiceti (appartenenti alle classi Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes) e 131 funghi ascomiceti (appartenenti alle classi Eurotiomycetes, Dothideomycetes e Sordariomycetes). Inoltre, per i funghi ascomiceti ho descritto due nuove specie nell’ordine Chaetothyriales. La maggior parte dei funghi isolati, sia ascomiceti che basidiomiceti, erano già stati precedentemente descritti in licheni, o risultavano filogeneticamente vicini a specie che solitamente occupano altre nicchie ecologiche (i.e. piante, rocce e suolo) e che hanno evoluto stili di vita diversi (patogeni di piante, opportunisti o saprotrofi). Tramite il metabarcoding del DNA, prima di tutto ho disegnato e testato un set di cosiddetti ‘blocking primers’ i quali riducono specificatamente l’amplificazione dei micobionti di R. melanophthalma e T. atra e di conseguenza permettono l’identificazione di tutti gli altri funghi presenti nei licheni. Tuttavia, questo esperimento non ha funzionato e ho eseguito l’approccio di metabarcoding senza utilizzarli. Il confronto della comunità fungine ha mostrato una bassa diversità per i singoli talli con la comparsa di pochi ascomiceti costantemente presenti in entrambe le due specie di licheni, la maggior parte appartenenti all’ordine Chaetothyriales, Capnodiales and Helotiales. I basidiomiceti rappresentano solo il 7-14% delle sequenze, e la maggioranza appartiene all’ordine Tremellales. Le condizioni ambientali, soprattutto le più rigide (e.g. elevate altitudini) e isolamento geografico sono i principali fattori che influiscono sulla composizione del micobioma dei licheni, mentre il micobionte sembra avere un ruolo minore. In R. melanophthalma e T. atra non è stata identificata una comunità fungina stabile e specifica poiché soli pochi taxa sono presenti nei licheni con bassa frequenza e nessuno di questi è presente in entrambe le specie studio. Infine, ho confrontato la diversità fungina ottenuta applicando l’approccio di metabarcoding con quella ottenuta con metodo di coltura-dipendente, per capire quale frazione del micobioma è identificabile utilizzando diversi approcci. Anche se il DNA metabarcoding permette di catturare una maggiore diversità, è interessante notare come alcune specie di funghi che sono cresciuti in coltura non è stata identificata dalle analisi di metabarcoding. Per concludere, l’approccio di coltura-dipendente e il DNA metabarcoding integrandosi l’uno con l’altro hanno permesso di ottenere una visione completa della diversità del micobioma in R. melanophthalma e T. atra.

Stabilità e variabilità del micobioma lichenico / Cometto, Agnese. - (2023 Mar 03).

Stabilità e variabilità del micobioma lichenico

COMETTO, AGNESE
2023-03-03

Abstract

Lichens – as self-sustaining, mutualistic and symbiotic systems – house a multiplicity of microorganisms (prokaryotes, microalgae and microfungi), whose biodiversity and roles in the symbioses are still largely unknown. Until recently, only a few studies focused on the overall diversity of the lichen-associated fungi which constitute the lichen mycobiome. In this research, I characterized the diversity of the lichen mycobiome in two cosmopolitan, epilithic lichens, i.e. Rhizoplaca melanophthalma and Tephromela atra, that were chosen as study organisms. Their mycobiomes were investigated for the first time at a global scale, as lichen samples were collected across their entire geographic range and both a culture-dependent and environmental DNA (eDNA) metabarcoding approaches were performed. I aimed at i) characterizing if any stable ‘core mycobiome’ can be detected for each lichen species and if a shared mycobiome exists; ii) understanding the role of the environmental conditions in determining the lichen mycobiome composition. By culture-dependent approach, I isolated and identified, using an integrated approach based on morphological and phylogenetic data, 76 basidiomycete yeasts (belonging to the five classes Agaricostilbomycetes, Cystobasidiomycetes, Microbotryomycetes, Tremellomycetes and Ustilaginomycetes) and 131 ascomycetes fungi (belonging to the three classes Eurotiomycetes, Dothideomycetes and Sordariomycetes). Furthermore, within the ascomycetes fungi I identified two new lineages in Chaetothyriales. Most of the isolated fungi, either the ascomycetes and basidiomycetes, have been previously found in lichens, or resulted to be taxa closely related to species which usually occupy other ecological niches (i.e. plants, rocks and soil) and which adopt different lifestyles (i.e. plant pathogens, opportunists and saprotrophs). By DNA metabarcoding, I first tried to design and test a set of blocking primers, which were specifically designed to reduce the amplification of R. melanophthalma and T. atra mycobionts and to enhance the amplification of all the other fungi present within the lichen thalli. However, these primers did not prevent the amplification of the mycobionts and I carried out the metabarcoding approach without using them. The comparison of the fungal communities highlighted low diversity within single thalli with the presence of few ascomycetes consistently present in both lichen species, mostly belonging to Chaetothyriales, Capnodiales and Helotiales. Basidiomycetes account only for 7-14% of the sequences, mostly from Tremellales. The environmental conditions, especially when harsh (e.g. extreme elevation) and the geographic isolation possibly played the main role in shaping the mycobiome composition, however also the lichen host seemed to influence the mycobiome community, though in a lesser extent. A stable species-specific core mycobiome was hardly identifiable both in R. melanophthalma and T. atra as most of the mycobiome taxa were present with low frequency of occurrence and no taxon was ubiquitously present in neither lichen. I also compared the fungal diversity detected by DNA metabarcoding with the one obtained by the culture-dependent approach, to understand which fraction of the mycobiome was only detected either by culture isolation or by eDNA analyses. Even if DNA metabarcoding described most of the diversity of the lichen mycobiome, and many of the metabarcoded taxa were also isolated in culture, surprisingly there were some fungal species which grew in culture but were not detected by the metabarcoding analysis. In conclusion, both the culture-dependent and the DNA metabarcoding approaches well complemented each other and allowed to obtain a fairly complete estimation of the global lichen mycobiome diversity in R. melanophthalma and T. atra.
3-mar-2023
MUGGIA, LUCIA
PALLAVICINI, Alberto
35
2021/2022
Settore BIO/01 - Botanica Generale
Università degli Studi di Trieste
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Descrizione: Stability and variation of lichen mycobiome
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/11368/3041119
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