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.