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

strategies. To address these predictions, the pollen loads from megachilid specimens were metabarcoded targeting the ITS2 region of the pollen nuclear genome to reveal plant prove- nance and plant family affinities for each species. Materials and methods Specimen selection Bee specimens sampled for pollen were already housed in the National Collection of Insects of the Agricultural Research Council, at the Biosystematics unit in Pretoria, South Africa. No specimens were collected for this study, which relied on the use of historical specimens. Taxo- nomic experts previously identified all specimens housed in the collection and the collection contains many type specimens. Six bee species from the genus Megachile were selected for analysis (Table 1). Two species, Megachile karooensis Brauns, 1912 and Megachile murina Fri- ese, 1913 are endemic to the Succulent Karoo biome. The semiarid Succulent Karoo biome is distributed in the south-western portion of southern Africa and forms part of the Greater Cape Floristic Region. This biome is renowned as a globally important centre of endemism and biodiversity. The region is also well-known for high bee diversity and endemism [17, 20]. Megachile felina Gerstaecker, 1857 and Megachile maxillosa Gue´rin-Me´neville, 1845 are restricted to the Savanna biome. This biome is the largest biome in South Africa. It is a grass- land ecosystem distributed primarily in the eastern summer rainfall region of South Africa. Two widely distributed species, Megachile niveofasciata Friese, 1904 and Megachile venusta

Smith, 1853 were selected as examples of generalist species. All specimens with some pollen visible on their bodies when viewed under a dissecting microscope were selected regardless of when they were collected. One of the two widespread species selected, Megachile venusta, was used in a previous pollen metabarcoding study [26], where it was shown that that DNA could be successfully sequenced from a wide temporal range of samples, including pollen sampled from specimens which were collected in 1914. The data produced in this study are also used here. Information on all bee specimens, including accession numbers, original sampling dates of bees, sampling regions and GPS coordinates (where available) is provided as supplementary information (S1–S5 Tables, and S1 Table from [26]). A stereo dissection microscope (SteREO Discovery.V8 microscope, Carl Zeiss Microscopy GmbH, Jena, Germany) was used to view each specimen to confirm the presence of pollen, as well as to scrape pollen off abdominal scopae. Pollen samples were transferred to sterile 1.5 ml Eppendorf tubes and pollen crushed with the micropipette tip while still under magnification. The micropipette tip for each sample was left inside the tube to include any pollen that inad- vertently entered the micropipette tip during scraping. Table 1. Megachile (Megachilidae) selected for study from different biomes in South Africa. The number of specimens in the National Collection of Insects that con- tained pollen for sampling is also indicated, as well as the age range of specimens based on collection dates.

Species Biome Number of specimens Bee specimen collection years Megachile karooensis Succulent Karoo 20 1982–1990 Megachile murina Succulent Karoo 27 1982–1990 Megachile felina Savanna 17 1966–1990 Megachile maxillosa Savanna 32 1914–2003 Megachile niveofasciata Widespread 10 1984–2000 Megachile venusta Widespread 21 1914–2007 https://doi.org/10.1371/journal.pone.0244973.t001 PLOS ONE Floral hosts of Megachilidae revealed by metabarcoding PLOS ONE | https://doi.org/10.1371/journal.pone.0244973 January 21, 2021 3 / 16