Boris Yagound, Kathleen A. Dogantzis, Amro Zayed, Julianne Lim, Paul Broekhuyse, Emily J. Remnant, Madeleine Beekman, Michael H. Allsopp, Sarah E. Aamidor, Orly Dim, Gabriele Buchmann, Benjamin P. Oldroyd

 Experimental Model and Subject Details

We used two subspecies of the honeybee Apis mellifera. Arrhenotokous A. m. scutellata (Scutellata) bees were obtained from Douglas, Northern Cape (29°02′S, 23°45’E), well north of the hybrid zone. Thelytokous A. m. capensis (Capensis) bees were obtained from the Stellenbosch area, Western Cape (33°56’S, 18°52′E), well south of the hybrid zone.

For transects we collected single workers foraging on flowers in locations removed from known apiaries, or single bees from domestic but sedentary colonies throughout South Africa, Botswana and Eswatini over two years. We collected workers north of the hybrid zone in Aliwal North (30°42′S, 26°43’E), Ballito/Dolphin Coast (29°32′S, 31°13’E), Bergville KZN (28°43’S, 29°21E), Burgersdorp (30°59’S, 26°20’E), Champagne Castle (29°05’S, 29°20’E), Douglas (29°07’S, 23°45’E), Gege (26°58’S, 30°60’E), Gharagab camp (25°02′S, 20°04’E), Golden Gate Highlands Ntl Park (28°30’S, 28°36’E), Hilton (29°33’S, 30°17’E), Kokstad (30°30’S, 29°24’E), Mankayane (26°40’S, 31°03’E), Manzini (26°30’S, 31°22′E), Meerkat Manor (26°58’S, 21°49’E), Port Shepstone (30°43’S, 30°26’E), Shakaskraal (29°27’S, 31°13’E), Steynsburg (31°18’S, 25°49’E), Tswalu Nature Reserve (27°14’S, 22°24’E), Umdloti (29°40’S, 31°07’E), Vanstadensrus (29°59’S, 27°00’E), and Winterton (28°49’S, 29°31’E). We collected workers south of the hybrid zone in Aberdeen (32°48’S, 24°04’E), Addo Nlt Park Colchester Gate (32°24’S, 20°10’E), Buffeljagsrivier (34°12′S, 21°16’E), Caledon (34°23’S, 19°43’E), Cape Point (34°00’S, 18°63’E), Cederberg Wilderness Area (32°33’S, 19°12′E), Cradock (32°18’S, 25°65’E), Graaff-Reinet (32°25’S, 24°55’E), Grootvadersbosch (33°59’S, 20°49’E), Harkerville (34°03’S, 23°23’E), Idutywa (32°10’S, 28°31’E), Karoo Ntl Park (32°36’S, 22°54’E), Mossel Bay (32°24’S, 20°10’E), Mount Frere (30°89’S, 28°98’E), Mountain Zebra Ntl Park (32°14’S, 25°51’E), Mountain Zebra Ntl Park Craddock (33°48’S, 25°75’E), Port Elizabeth (33°96’S, 25°60’E), PPRI Stellenbosch (33°93’S, 18°87’E), Riviersonderend (34°36’S, 18°50’E), Robben Island (33°81’S, 18°37’E), Tankwa Ntl Park (32°14’S, 20°05’E), West Coast Ntl Park (33°17’S, 18°15’E), and Wilderness Ntl Park (33°99’S, 22°61’E). We collected drones north of the hybrid zone in Kwara (19°11’S, 23°27’E), Johannesburg (26°20’S, 28°05’E), Louis Trichardt (23°05’S, 29°90’E), and Molopo (25°72′S, 25°04’E). We collected drones south of the hybrid zone in Helderberg (34°05’S, 18°93’E), Kogelberg (34°29’S, 18°92′E), and Somerset West (34°08’S, 18°84’E).

 Method Details

 Crosses

We used an arrhenotokous Scutellata queen as the mother of the drones used in our crosses. To obtain Capensis queens that were assuredly homozygous th/th we dequeened three Capensis colonies from the Stellenbosch area, and removed any developing queen cells one week later. The three colonies produced virgin queens from the progeny of the thelytokous workers that developed within the colonies. We instrumentally inseminated three of the Capensis queens, each with the semen of a different Scutellata male using standard methods [

Propagation and instrumental insemination.