Floral hosts of leaf-cutter bees (Megachilidae) in a biodiversity hotspot revealed by pollen DNA metabarcoding of historic specimens

16 plant species. The numbers of plant families and species visited by each bee species in each area studied can be seen in Fig 2. The mean number of angiosperm plant families detected in pollen loads varied significantly among bee species (χ2 = 130.51, P <0.0001, Fig 3a). Similarly, the mean number of angio- sperm species detected in pollen loads varied significantly among the different bee species (χ2 = 115.49, P <0.0001, Fig 3b). The highest diversity of pollen OTUs and families was recorded for the widespread species M. niveofasciata, but sample-based rarefaction of species OTU data indicated that the other widespread species M. venusta has higher expected species richness when compared to the other bee species (Fig 4). There was no significant effect of time since bee collection for either angiosperm plant families (χ2 = 0.56, P = 0.454) or the number of OTUs (χ2 = 2.28, P = 0.131). We recorded a small, but significant difference in pollen composition (plant species recorded) between the three groups of bees as shown by NMDS (Fig 5) and ANOSIM analysis (R = 0.24, P < 0.0001). Uncorrected significance values between the different groups com- pared in ANOSIM (S6 Table) indicated that M. venusta and M. niveofasciata were the only two species which showed significantly different overall pollen composition. The plant families identified in each bee group were compared to the angiosperm plant family list created from the POSA database to confirm their occurrence in South Africa. All but three angiosperm families (87.5%) in all the regions studied were found to occur in the

specific region detected. Magnoliaceae was not found in any of the regions searched. Magno- liaceae species are widely cultivated which could explain their presence in the samples. Simi- larly, Annonaceae and Caprifoliaceae are not native to the succulent Karoo, but are widely Fig 1. Visual representation of the proportion of bee specimen samples in which each plant family was identified in each biome group. Families from the phylum Chlorophyta were removed prior to plotting the graph. The family Pteridaceae was identified in nearly all samples in all three bee groups (Savanna, Succulent Karoo and widespread), with clear differences in the prevalence for plants belonging to Asteraceae, Fabaceae and Magnoliaceae by the widespread bee group. The absence of a group marker in a column indicates that the particular plant family was not detected in pollen loads from any bee specimens in that biome group. https://doi.org/10.1371/journal.pone.0244973.g001 PLOS ONE Floral hosts of Megachilidae revealed by metabarcoding PLOS ONE | https://doi.org/10.1371/journal.pone.0244973 January 21, 2021 7 / 16