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

S5 Table. Collection information from the national insect collection, ARC, South Africa, of Megachile niveofasciata bee specimens from which pollen was collected for the wide- spread group in this study. Available information regarding the specimen collection, such as date, province, GPS coordinates and collection locality are given for each sample. (DOCX) S6 Table. Uncorrected significance (p) values between the different groups compared in ANOSIM. Groups were set as bee species. The only two bee species differing significantly from others were the two widespread species, M. venusta and M. niveofasciata. (DOCX) S7 Table. A list of plant species and families on which visits from the six studied bee spe- cies have been recorded. References from which data were sourced are given, as well as any additional information available. (DOCX) S1 Fig. Rarefaction curves for each bee species to determine whether sequence saturation has been reached with the amount of reads sequenced per pollen sample. The lines for all samples collected from specimens from all six of the bee species flattened when 1,000 sequence reads were reached, indicating that all possible plant families represented in the pollen samples have been identified. a) and b) represents bee species occurring in the Savanna biome, c) and d) represents bee species occurring exclusively in the Succulent Karoo biome, and e) and f) represents bee species occurring all over South Africa (cosmopolitan group). (DOCX) Acknowledgments Charles Hefer and Genevieve Thompson for their assistance with bioinformatics.

Author Contributions Conceptualization: Annemarie Gous, Connal D. Eardley, Dirk Z. H. Swanevelder, Sandi Wil- lows-Munro. Data curation: Annemarie Gous. Formal analysis: Annemarie Gous, Steven D. Johnson, Sandi Willows-Munro. Supervision: Connal D. Eardley, Dirk Z. H. Swanevelder, Sandi Willows-Munro. Writing – original draft: Annemarie Gous, Sandi Willows-Munro. Writing – review & editing: Connal D. Eardley, Steven D. Johnson, Dirk Z. H. Swanevelder. References 1. Bruni I, Galimberti A, Caridi L, et al. (2015) A DNA barcoding approach to identify plant species in multi- flower honey. Food Chemistry, 170, 308–315. https://doi.org/10.1016/j.foodchem.2014.08.060 PMID: 25306350 2. Keller A, Danner N, Grimmer G, et al. (2015) Evaluating multiplexed next-generation sequencing as a method in palynology for mixed pollen samples. Plant Biology, 17, 558–566. https://doi.org/10.1111/ plb.12251 PMID: 25270225 3. Sickel W, Ankenbrand MJ, Grimmer G, et al. (2015) Increased efficiency in identifying mixed pollen samples by meta-barcoding with a dual-indexing approach. BMC Ecology, 15. https://doi.org/10.1186/ s12898-015-0051-y PMID: 26194794 PLOS ONE Floral hosts of Megachilidae revealed by metabarcoding PLOS ONE | https://doi.org/10.1371/journal.pone.0244973 January 21, 2021 14 / 16