This data was collected to explore the founding strategies used by S. geminata to minimize inbreeding costs which cause some queens to produce sterile diploid males instead of workers.
I. Lab experiment on S. geminata colony founding
Methods: We collected newly mated queens of S. geminata after their nuptial flight, between March and April 2016 at Humpty Doo, Northern Territory, Australia (-12.5722°, 131.0842°). The 1187 queens collected in 2016 yielded 115±12 replicates per treatment. The queens were weighed and established in trials of 1, 2, 3 or 5 queens per colony within 15 to 40 hours after collection. Nests consisted of a 15-mL centrifuge tube, half filled with water retained by a cotton plug. Queens were retained by a second cotton plug inserted in the tube mouth. We inserted the nesting tubes into a polystyrene sheet to keep the nesting chambers in the dark. We allowed queens to initiate nests for 23 days, which was 1 day after the emergence of the first generation of workers in our pilot study. We recorded queen mortality and the cause of death three times per week. We considered dismembered queens to have been executed by other queens and dead intact queens to have died of natural causes. We also recorded the presence of distinctly large larvae (presumable diploid male) and their position within the nest. On the 24th day we terminated the colonies by freezing, weighed each queen and all brood, and counted the number of eggs, larvae, worker pupae, and adult workers of the colonies in which all the queens survived (n=380, 95±9 replicate per treatment. Unusually large larvae distinguishable from the second instar larval stage were counted and weighed separately from the rest of the brood.
Data description: The dataset consists of one excel document entitled “Colony founding lab experiment” and comprising six excel spreadsheets. The first spreadsheet (Main) contains data on the replicates for which queens survived until the end of the experiment. It details brood and workers stages counts as well as their weight, the mean queen weight before and after the experiment for each replicate, the presence or absence of large larvae with the brood and, large larvae weight (if large larvae were present by the end of the experiment). The second spreadsheet (Queen weight detail) gives the weight for each queen before and after the experiment. The third spreadsheet (Observation large larvae) contains data on whether large larvae disappeared from the nests during the experiment, the date of disappearance and, notes on the position of the large larvae during the experiment. The fourth spreadsheet (Large larvae disappearance) contains the dates at which large larvae were first seen in each nest and when the first disappearance was observed. The fifth spreadsheet (Queen death) contains data on replicates for which not all queens survived until the end of the experiment. It gives information on whether the queen was executed and whether the colony produced large larvae.
II. Microsatellite DNA data on S. geminata in Northern Australia
Methods: Adult males: We extracted DNA from 80 adult males collected from ten field colonies (8 males per colony) using the Zymo Research Tissue and Insect DNA MiniPrepTM kit following the supplier’s instructions. We used six microsatellite DNA markers to be able to determine whether males were haploid or diploid.Queens and spermatheca: We isolated sperm from 40 mated queens collected in 2015 and extracted the sperm DNA using the Qiagen DNeasy Blood and Tissue kit and the Zymo Research DNA Clean and ConcentratorTM following the supplier’s instructions. We also extracted DNA from the 40 queens that were dissected for sperm sampling, to enable distinction of multiple mating from potential maternal contamination. We used the Zymo Research Tissue and Insect DNA MiniPrepTM kit following the supplier’s instructions to extract DNA. We used three microsatellite DNA markers to determine whether queens were single of multiple mated and if our sperm DNA had been contaminated with maternal DNA during the sperm isolation procedure.Large larvae: We opportunistically selected 15 large larvae out of the 109 we observed during our colony founding experiment. We extracted their DNA using the Zymo Research Tissue and Insect DNA MiniPrepTM kit following the supplier’s instructions. We used three microsatellite DNA markers for ploidy determination.PCR conditions: All DNA fragments were amplified using PCR based on the methods of Tay & Crozier (2000), modified and optimized for S. geminata microsatellite markers. Each amplification (in a final 10µL reaction volume) required 1.5 µL of DNA (undiluted for queens, larvae and males, 1:10 diluted for sperm), 0.2mM dNTPs, 0.2µM of each forward primer and reverse primer, 0.5 unit of Phusion® Taq DNA polymerase (5u/µL), 1mM MgCl2, and 1x Phusion® Buffer. The PCR conditions were as follows: initial denaturation step at 95˚C for 2.5 minutes; 37 cycles comprising 30 seconds of DNA denaturing at 95˚C, specific primer annealing temperature (see Microsat DNA data excel file) for 30 seconds, 30 seconds DNA extension at 72˚C; and a final extension step of 5 minutes at 70˚C. Post PCR incubation was at 10˚C. For each individual sample, we multiplexed and submitted for genotyping 2.5 µL of each of the PCR amplicons for Ms19c367, Ms14c334 and Ms4Sol55 and, 2 µL for Ms16 C121 and 2.5 µL for Ms33 Sol11 and Ms41 i134. PCR for the queen, sperm and larvae were multiplexed and submitted to the Australian Cancer Research Foundation Biomolecular Resource Facility at the John Curtin School of Medical Research, Australian National University, for genotyping.
Literature cited: Tay WT, Crozier RH. 2000 Microsatellite analysis of gamergate relatedness of the queenless ponerine ant Rhytidoponera sp. 12. Insectes Soc. 47, 188–192. (doi:10.1007/PL00001700)
Data analysis: We used the proprietary software ‘Geneious®’ to visualize trace files, fit the internal ladder, and identify the microsatellite alleles. The highest peaks within the allele size range for S. geminata were determined.
Data description: The dataset consists of one excel document entitled “Microsat DNA data” and comprising seven excel spreadsheets. The first spreadsheet (Adult males) contains the microsatellite allele data of adult males collected in the field. The second spreadsheet (Queen) contains microsatellite allele data of queens collected in the field as well as the name of the corresponding sperm sample for each queen. The third spreadsheet (Sperm) contains microsatellite allele data of sperm dissected isolated from queens collected in the field as well as the name of the corresponding queen. The fourth spreadsheet (large larva) contains microsatellite allele data of large larvae collected from large larvae producing colonies in the colony founding experiment, the number of queens in the colony and the replicate number. The fifth spreadsheet (Male collection sites) contains the GPS coordinates of the adult male collection sites. The sixth spreadsheet (Other samples collection sites) contains the GPS coordinates of the queens and large larvae collection sites. The last spreadsheet (Annealing temperature) contains the annealing temperature for each microsatellite locus and their allele size range.