Type it master mix kit

We determined individual bee genotypes at five microsatellite loci according to the protocol described by Evans et al. (2013 ) using the primers detailed in Supplementary Table S7. These microsatellite loci have been used in other honeybee population genetic studies (Muñoz et al., 2009 , 2012 ). We ran the microsatellites as two separate multiplexes, with primers A113, AP043, and AP055 in multiplex 1 and primers A007 and B124 in multiplex 2, using the Qiagen Type‐It master mix kit (Qiagen, Valencia, CA) in a final reaction volume of 10 μl. PCR amplification involved denaturation at 94°C for 5 min, followed by 30 cycles of 95°C for 30 s, 57°C for 30 s, and 72°C for 30 s, with a final elongation step at 72°C for 30 min. The size of the microsatellites was determined by capillary electrophoresis on an ABI 3730 DNA Analyser (Applied Biosystems, CA, USA) at the University of Manchester Sequencing Facility, using the GeneScan™ LIZ500 (Thermo Fisher Scientific, USA) size standard. The output peaks were scored using Genemapper v5.0 software (Applied Biosystems) and sorted into allele bins using the MsatAllele package v1.05 (Alberto, 2009 (link)) in R (R Core Team, 2017 ). A subset of samples were repeated to ensure consistency of allele calling. The function hw.test in the package pegas (Paradis, 2010 (link)) was used to test whether the alleles were in Hardy–Weinberg equilibrium.

We conducted paternity analysis, whenever two eggs in a brood hatched on the same day in Experiment 2, making it impossible to determine identity of the foster chick. A small blood sample (5–10 µL) was taken from both chicks and parents via brachial venepuncture using a 26-gauge needle and stored in 70% ethanol. Total genomic DNA was extracted using an adapted phenol-chloroform protocol. Each sample was then genotyped at 8 polymorphic microsatellite loci^{55 (link),56 (link)}: Indigo41, Tgu01, Tgu03, Tgu04, Tgu05, Tgu08, Tgu09 and Tgu12 using a Qiagen Type-it Mastermix kit. The following PCR-profile was used: 95 °C for 5 min, 8 cycles of 95 °C for 30 seconds, 60 °C for 90 seconds (minus 1 °C per cycle) and 72 °C for 60 seconds, followed by 30 cycles of 95 °C for 30 seconds, 56 °C for 90 seconds and 72 °C for 60 seconds. Final extension was performed at 70 °C for 15 minutes. Fragment sizes were scored using the automated software Genemarker v1.7 (Softgenetics). All of the scores were checked manually and adjusted wherever necessary. Paternities were assigned manually to one of the two potential parent pairs by comparing the fragment sizes. Paternities were assigned unambiguously in all cases (n = 7 foster chicks; 4 in the experiment with the maternal scent and 3 in the experiment with the paternal scent).