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

4. McFrederick QS, Rehan SM (2016) Characterization of pollen and bacterial community composition in brood provisions of a small carpenter bee. Molecular Ecology, 25, 2302–2311. https://doi.org/10.1111/ mec.13608 PMID: 26945527 5. Potter C, de Vere N, Jones LE, Ford CR, Hegarty MJ, Hodder KH, et al. (2019) Pollen metabarcoding reveals broad and species-specific resource use by urban bees. PeerJ, 7, e5999. https://doi.org/10. 7717/peerj.5999 PMID: 30809427 6. Suchan T, Talavera G, Sa´ ez L, Ronikier M, Vila R (2019) Pollen metabarcoding as a tool for tracking long-distance insect migrations. Molecular Ecology Resources, 19, 149–162. https://doi.org/10.1111/ 1755-0998.12948 PMID: 30267472 7. Bell K, de Vere N, Keller A, et al. (2016) Pollen DNA barcoding: Current applications and future pros- pects. Genome, 59, 629–640. https://doi.org/10.1139/gen-2015-0200 PMID: 27322652 8. Rahl M (2008) Microscopic identification and purity determination of pollen grains. In: Allergy Methods and Protocols (eds Jones MG, Lympany P), Humana Press, Totowa, NJ. 9. Kraaijeveld K, de Weger LA, Ventayol Garcı´a M, et al. (2015) Efficient and sensitive identification and quantification of airborne pollen using next-generation DNA sequencing. Molecular Ecology Resources, 15, 8–16. https://doi.org/10.1111/1755-0998.12288 PMID: 24893805 10. Richardson RT, Lin C-H, Quijia JO, et al. (2015a) Rank-based characterization of pollen assemblages collected by honey bees using a multi-locus metabarcoding approach. Applications in Plant Sciences, 3, 1500043.

11. Richardson RT, Lin C-H, Sponsler DB, et al. (2015b) Application of ITS2 metabarcoding to determine the provenance of pollen collected by honey bees in an agroecosystem. Applications in Plant Sciences, 3, 1400066. 12. Pauw A, Stanway R (2015) Unrivalled specialization in a pollination network from South Africa reveals that specialization increases with latitude only in the Southern Hemisphere (Burns KC, Ed,). Journal of Biogeography, 42, 652–661. 13. Johnson SD (1997) Pollination ecotypes of Satyrium hallackii (Orchidaceae) in South Africa. Botanical Journal of the Linnean Society, 123, 225–235. 14. Pauw A (2006) Floral syndromes accurately predict pollination by a specialized oil-collecting bee (Redi- viva peringueyi, Melittidae) in a guild of South African orchids (Coryciinae). American Journal of Botany, 93, 917–926. https://doi.org/10.3732/ajb.93.6.917 PMID: 21642155 15. Welsford MR, Johnson SD (2012) Solitary and social bees as pollinators of Wahlenbergia (Campanula- ceae): single-visit effectiveness, overnight sheltering and responses to flower colour. Arthropod-plant interactions 6, 1–14. 16. Stanley DA, Msweli S, Johnson SD (2020). Native honeybees as flower visitors and pollinators in wild plant communities in a biodiversity hotspot. Ecosphere (in press). 17. Van Wyk AE, Smith GF (2001) Regions of Floristic Endemism in Southern Africa: A Review with Emphasis on Succulents (du Plessis E, Steffens F, Eds,). Umdaus Press, Hatfield. 18. Hamer M (2013) A national strategy for zoological taxonomy (2013–2020). South African National Bio-

diversity Institute, Pretoria. 19. Da Silva JM, Willows-Munro S (2016) A review of over a decade of DNA barcoding in South Africa: a faunal perspective. African Zoology, 51. 20. Eardley CD (1989) Diversity and endemism of southern African bees. Plant Protection News, 18, 1–2. 21. Eardley CD, Gikungu M, Schwarz MP (2009) Bee conservation in Sub-Saharan Africa and Madagas- car: diversity, status and threats. Apidologie, 40, 355–366. 22. Kuhlmann M (2009) Patterns of diversity, endemism and distribution of bees (Insecta: Hymenoptera: Anthophila) in southern Africa. South African Journal of Botany, 75, 726–738. 23. Packer L, Zayed A, Grixti JC, et al. (2005) Conservation genetics of potentially endangered mutualisms: Reduced levels of genetic variation in specialist versus generalist bees. Conservation Biology, 19, 195–202. 24. Zayed A (2009) Bee genetics and conservation. Apidologie, 40, 237–262. 25. Pennisi E (2000) Taxonomic Revival. Science, 289, 2306–2308. https://doi.org/10.1126/science.289. 5488.2306 PMID: 11041799 26. Gous A, Swanevelder ZH, Eardley C, Willows-Munro S (2019) Plant-pollinator interactions over time: Pollen metabarcoding from bees in a historic collection. Evolutionary Applications. https://doi.org/10. 1111/eva.12707 PMID: 30697333 27. White TJ, Bruns T, Lee S, Taylor J (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: PCR Protocols: A Guide to Methods and Applications, p. Academic Press Inc., New York. PLOS ONE Floral hosts of Megachilidae revealed by metabarcoding

PLOS ONE | https://doi.org/10.1371/journal.pone.0244973 January 21, 2021 15 / 16