Capturing the diversity of endophytic fungi preserved in herbarium specimens

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 five 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 data set, 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.