Microbial diversity identification
Bacterial and fungal communities were identified for each colony by sequencing cuticular washes of three female alates, three male alates, and three workers per colony (N = 54). Individuals were collected using sterile tools and washed in 300μL of 0.1% Tween 80 solution. After 15 minutes of gentle rotation, the solution was removed for DNA extraction using a Phenol/Chloroform protocol. For the bacterial community, the v4 hypervariable region of 16S was amplified using the bacterial primers 515f and 806r (Kozich et al. 2013). For the fungal community, ITS was amplified using the primers CS1-ITS3 and CS2-ITS4 with Fluidigm CS1 and CS2 universal oligomers added to their 5’ end (White et al.1990). PCR protocols are provided in Supplementary Information S1 (Aguero et al. 2021a). Pooled amplicons were loaded onto an Illumina MiSeq Standard v2 flow cell and sequenced in a 2x250bp paired end format using a MiSeq.v2.500 cycles reagent cartridge. Base calling was performed by Illumina Real Time Analysis v1.18.54 and output was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v2.19.1. All analyses were performed using QIIME 2 (Bolyenet al. 2019). Paired-end reads were filtered for quality control and combined using the DADA2 pipeline (Callahan et al.2016). 16S and ITS sequences were joined at 250bp and identified as amplicon sequence variants. Samples with low coverage (<10,000 reads) were removed from further analyses; all samples were conserved for bacterial analyses, but 13 samples were discarded from fungal analyses. To estimate microbial difference within and between colonies, weighted and unweighted UniFrac distances between each individual were visualized using a principal coordinates analysis (PCoA) (Hamadyet al. 2010). Unweighted distances only consider the presence or absence of observed microbes, while weighted values also account for their abundance. Euclidean distances between pairs of individuals on the two PCs of the PCoA were used to build pairwise distance matrices and to compare differentiation among individuals within and between colonies using a Mann–Whitney U-test.