Pollen metabarcoding of bees from a historic collection reveales provenance and possible microbial connections

Pollination is a key component in both agricultural food production and ecosystem maintenance. Natural history collections containing pollinators can provide us with unique access to pollen samples collected at different spatial and temporal scales. Identification of the plant origins of pollen trapped on the bodies of pollinators in these collections can provide insight into historic plant communities and pollinators' preferred floral taxa. In this study, pollen has been sampled from Megachile venusta Smith bees taken from the National Collection of Insects, South Africa, spanning 93 years. Two barcode regions were sequenced on an Illumina MiSeq, namely the internal transcribed spacer 1 (ITS1) and the internal transcribed spacer 2 (ITS2). Reference sequence databases were generated by mining data for Viridiplantae from GenBank for both barcodes, and comparing the ITS2 database with a previously published ITS2 database for plants. Amplification universality of the barcode primers enabled the investigation of both pollen and microbial microbiomes from pollen samples collected. Plant classification was more diverse with ITS2 than with ITS1 barcode data. Limited local plant sequence representation in reference databases resulted in higher-level taxon classifications being more confidently interpreted. Sequences that were not of plant origin were mostly assigned to fungi, especially Malassezia. This study successfully used pollen from bee specimens collected from as early as 1914 to obtain both the pollen and microbial metabarcodes, thereby allowing the identification of the pollen's plant origin and microbiological compositions of the samples.