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A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens.

ABSTRACT: Herbarium specimens represent important records of morphological and genetic diversity of plants that inform questions relevant to global change, including species distributions, phenology and functional traits. It is increasingly appreciated that plant microbiomes can influence these aspects of plant biology, but little is known regarding the historic distribution of microbes associated with plants collected in the pre-molecular age. If microbiomes can be observed reliably in herbarium specimens, researchers will gain a new lens with which to examine microbial ecology, evolution, species interactions. Here, we describe a method for accessing historical plant microbiomes from preserved herbarium specimens, providing a proof of concept using two plant taxa from the imperiled boreal biome (Andromeda polifolia and Ledum palustre subsp. groenlandicum, Ericaceae). We focus on fungal endophytes, which occur within symptomless plant tissues such as leaves. Through a three-part approach (i.e. culturing, cloning and next-generation amplicon sequencing via the Illumina MiSeq platform, with extensive controls), we examined endophyte communities in dried, pressed leaves that had been processed as regular herbarium specimens and stored at room temperature in a herbarium for four years. We retrieved only one endophyte in culture, but cloning and especially the MiSeq analysis revealed a rich community of foliar endophytes. The phylogenetic distribution and diversity of endophyte assemblages, especially among the Ascomycota, resemble endophyte communities from fresh plants collected in the boreal biome. We could distinguish communities of endophytes in each plant species and differentiate likely endophytes from fungi that could be surface contaminants. Taxa found by cloning were observed in the larger MiSeq dataset, but species richness was greater when subsets of the same tissues were evaluated with the MiSeq approach. Our findings provide a proof of concept for capturing endophyte DNA from herbarium specimens, supporting the importance of herbarium records as roadmaps for understanding the dynamics of plant-associated microbial biodiversity in the Anthropocene.This article is part of the theme issue 'Biological collections for understanding biodiversity in the Anthropocene'.

SUBMITTER: Daru BH

PROVIDER: S-EPMC6282087 | biostudies-literature | 2018 Nov

REPOSITORIES: biostudies-literature

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A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens.

Daru Barnabas H BH Bowman Elizabeth A EA Pfister Donald H DH Arnold A Elizabeth AE

Philosophical transactions of the Royal Society of London. Series B, Biological sciences 20181119 1763

Herbarium specimens represent important records of morphological and genetic diversity of plants that inform questions relevant to global change, including species distributions, phenology and functional traits. It is increasingly appreciated that plant microbiomes can influence these aspects of plant biology, but little is known regarding the historic distribution of microbes associated with plants collected in the pre-molecular age. If microbiomes can be observed reliably in herbarium specimen ...[more]

PMID: 30455213

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Project description:BackgroundA catalogue of types from the Herbarium of the University of Granada has not previously been compiled. As a result, a search of these collections in order to compile digital images for preservation and publication yielded a large number of formerly unrecognized types.New informationThis dataset contains the specimen records from the catalogue of the nomenclature types of fungi and lichens in the Herbarium of the University of Granada, Spain. These herbarium specimens are included in the GDA and GDAC collections, acronyms from Index Herbariorum (Thiers 2014). At this time, the type collection of fungi and lichens contains 88 type specimens of 49 nominal taxa, most from Agaricales and the genus Cortinarius, described from the western Mediterranean, mainly Spain, by the following authors: V.Antonin, J.Ballarà, A.Bidaud, G.F.Bills, M.Bon, C.Cano, M.Casares, G.Chevassut, M.Contu, F.Esteve-Raventós, R.Galán, L.Guzmán-Dávalos, R.Henry, E.Horak, R.Mahiques, G.Malençon, P.Moënne-Loccoz, G.Moreno, A.Ortega, F.Palazón, V.N.Suárez.-Santiago, A.Vêzda, J.Vila, and M.Villareal. For each specimen, the locality indication, species name, observation date, collector, type status, related information, associated sequences, other catalogue numbers related to each type, and image URL are recorded. The dataset is associated with an image collection named "Colección de imágenes de los tipos nomenclaturales de hongos, líquenes, musgos y algas incluidos en el Herbario de la Universidad de Granada (GDA y GDAC)" (Vizoso and Quesada 2013) which is housed and accessible at the Global Biodiversity Information Facility in Spain (GBIF.ES) Hosting and Publishing Service "Biodiversity Image Portal of Spanish collections" and is also available at the Herbarium of University of Granada institutional web (Vizoso 2014a, Vizoso 2014b). That image collection contains 113 images, of which 56 correspond to the nomenclature types of 49 taxa (47 fungi, 2 lichens), the rest of the images in this collection correspond to documents and specimens or microscopy photographs which are included in the herbarium specimens of fungi. These complement and document the process of the typification.

Project description:The world's herbaria collectively house millions of diverse plant specimens, including endangered or extinct species and type specimens. Unlocking genetic data from the typically highly degraded DNA obtained from herbarium specimens was difficult until the arrival of high-throughput sequencing approaches, which can be applied to low quantities of severely fragmented DNA. Target enrichment involves using short molecular probes that hybridise and capture genomic regions of interest for high-throughput sequencing. In this study on herbariomics, we used this targeted sequencing approach and the Angiosperms353 universal probe set to recover up to 351 nuclear genes from 435 herbarium specimens that are up to 204 years old and span the breadth of angiosperm diversity. We show that on average 207 genes were successfully retrieved from herbarium specimens, although the mean number of genes retrieved and target enrichment efficiency is significantly higher for silica gel-dried specimens. Forty-seven target nuclear genes were recovered from a herbarium specimen of the critically endangered St Helena boxwood, Mellissia begoniifolia, collected in 1815. Herbarium specimens yield significantly less high-molecular-weight DNA than silica gel-dried specimens, and genomic DNA quality declines with sample age, which is negatively correlated with target enrichment efficiency. Climate, taxon-specific traits, and collection strategies additionally impact target sequence recovery. We also detected taxonomic bias in targeted sequencing outcomes for the 10 most numerous angiosperm families that were investigated in depth. We recommend that (1) for species distributed in wet tropical climates, silica gel-dried specimens should be used preferentially; (2) for species distributed in seasonally dry tropical climates, herbarium and silica gel-dried specimens yield similar results, and either collection can be used; (3) taxon-specific traits should be explored and established for effective optimisation of taxon-specific studies using herbarium specimens; (4) all herbarium sheets should, in future, be annotated with details of the preservation method used; (5) long-term storage of herbarium specimens should be in stable, low-humidity, and low-temperature environments; and (6) targeted sequencing with universal probes, such as Angiosperms353, should be investigated closely as a new approach for DNA barcoding that will ensure better exploitation of herbarium specimens than traditional Sanger sequencing approaches.

Project description:BACKGROUND:The present study involves diversity and bioactivity of the endophytic fungal community from Catharanthus roseus inhabiting the coastal region. This study has been conducted hypothesizing that the microbial communities in the coastal regions would tolerate a range of abiotic stress such as salinity, humidity, temperature and soil composition, and it may produce new metabolites, which may possess bioactive property. Therefore in the current study, the cytotoxicity and free radical scavenging potential of the fungal organic extracts have been investigated. Moreover, the apoptotic and the antioxidant potential of the fungus that exhibited the best activity in preliminary screening has also been demonstrated. RESULTS:Twenty endophytic fungal isolates were obtained from different parts of the plant, and identified using internal transcribed spacer region analysis. Based on the colonization frequency, the dominant genera were found to be Colletotrichum, Alternaria and Chaetomium with colonization frequency % of 8.66, 7.00 and 6.33, respectively. It was observed that the species diversity and richness was the highest in bark followed by leaf and stem regions of the plant. On screening the fungal ethyl acetate extracts for cytotoxicity against the HeLa cells, the Chaetomium nigricolor extract exhibited potent cytotoxic activity of 92.20% at 100??g?mL-?1 concentration. Comparison between the different organic extracts (ethyl acetate, chloroform, dichloromethane and hexane) of Chaetomium nigricolor mycelial and culture filtrate, it was observed that the mycelial as well the culture filtrate ethyl acetate extracts and the culture filtrate hexane extract showed significant cytotoxic potential against the HeLa and MCF-7 cells, respectively. The apoptotic- and mitochondrial membrane depolarisation-induction potential of the Chaetomium nigricolor ethyl acetate extract has also been demonstrated in this study. Further the screening of antioxidant potential of the ethyl acetate fungal extracts using DPPH scavenging assay showed that Chaetomium nigricolor extract exhibited potential activity with a significant EC50 value of 22??g?mL-?1. The ethyl acetate extract of Chaetomium nigricolor also exhibited superoxide radical scavenging potential. CONCLUSION:These results indicated that diverse endophytic fungal population inhabits Catharanthus roseus. One of the fungal isolate Chaetomium nigricolor exhibited significant cytotoxic, apoptotic and antioxidant potential.

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A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens.

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A novel proof of concept for capturing the diversity of endophytic fungi preserved in herbarium specimens.

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