Molecular procedures
To extract DNA from the gut of each spider, the opisthosoma (abdomen)
was first cleaned using 70% ethanol and water then removed using a
sterile scalpel blade and placed in Qiagen cell lysis solution. The
opisthosoma was then ground using two 3 mm steel beads on a Genogrinder
(Spex SamplePrep, Matuchen, NJ, USA) for 2 min at 1,200 hz. DNA from
ground samples was extracted using the Qiagen Puregene kit (Qiagen,
Hilden, Germany) according to the manufacturer’s protocol. DNA was then
amplified using primer sets for 28s, 18s, and 16s (Primers listed in S1
of Supplemental Information). 28s has been used for fungal
identification (Xu 2016; Zhao et al. 2011) and was therefore
appropriate for detecting both arthropod and fungi. Amplification was
performed using the Qiagen multiplex PCR kit (Qiagen, Hilden, Germany)
in 10 ul reactions with 1ul of template DNA, and 1ul 10uM primer
dilutions for 35 PCR cycles at 46°C annealing temperature. A second
round of PCR was performed to add 8bp indexing tails on 5‘ end and
Illumina (Illumina San Diego, CA, USA) TruSeq adapters. PCR products
were visualized using a 1.5% agarose gel. Products were pooled in
approximately equal amounts based on band strength. 1X AmpureBeads were
used to clean the pooled products. The cleaned library was then
sequenced on an Illumina Miseq using V3 chemistry. Negative controls
were included on the sequencing run.