DNA extraction, amplification and sequencing
Representative DNA barcode sequences for the cytochrome c oxidase
subunit I (COI) (~658 bp) of species captured as adults
were available from a previous study (Crespo et al. 2018) (see Table S1
of Supporting information and Data Accessibility).
For each plot, we homogenised all the collected juveniles with the help
of liquid nitrogen. We obtained two extraction replicates from each
homogenised plot sample, and we extracted a fraction of 0.3 g from each
replicate using a PowerSoil DNA Isolation Kit (QIAGEN, Valencia, CA,
USA). We added one negative (distilled water) and one positive control,
a specimen of the cobweb spider Simitidion simile (C. L. Koch,
1836), in the extraction protocol. These controls were included in the
batch, processed and sequenced along with the rest of the samples. We
cleaned and sterilised all the equipment with diluted sodium
hypochlorite between successive sample extractions. We amplified the COI
“Leray fragment” of 313 bp using the degenerate primer set Leray-XT
(Wangensteen, Palacín, Guardiola, Turon, 2018). This set includesthe
reverse primer jgHCO2198 5′-TAIACYTCIGGRTGICCRAARAAYCA-3′ (Geller,
Meyer, Parker, & Hawk, 2013) and the forward primer mlCOIintF-XT
5′-GGWACWRGWTGRACWITITAYCCYCC-3′, modified from the mlCOIintF primer
(Leray et al., 2013). Each primer pair included twin 8-bp sample tags
(the same tag in the forward and reverse primers) and a lead of 2–4
random Ns in the 5’ end for increasing sequence variability of the
library. The PCR mix included 10 μl AmpliTaq Gold 360 Master mix
(Applied Biosystems, Foster City, CA, USA), with 1 µl of each 5 µM
forward and reverse primers, 0,16 µl of bovine serum albumin, 2 μl of
DNA template and DNase-Free water to adjust the volume up to 20 uL per
sample. The PCR profile included 10 min at 95 °C, 35 cycles of 94 °C 1
min, 45 °C 1 min and 72 °C 1 min, and 5 min at 72 °C. We used two PCR
replicates of each extraction replicate in the study, giving a total of
4 replicates per plot (except for plots PA1 and PS1, for which we
obtained 3 replicates as they were the first to be processed and served
as a test). We evaluated the quality of amplifications by
electrophoresis in 1% agarose in Tris-borate-EDTA buffer and stained
with GelRed® Nucleic Acid Gel Stain (Biotium, Hayward, California, USA).
We pooled all PCR products by equal volume (including two PCR-negative
controls and one PCR-positive control) and purified them using a
MinElute PCR Purification Kit (Qiagen, Valencia, CA, USA). Three µg of
the purified pool (determined by Qubit fluorometric quantitation dsDNA
BR Assay Kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA)
were used to build a library using the NextFlex PCR-free DNA-seq kit
(Perkin-Elmer, Waltham, Massachusetts, USA). The multiplexed library was
sequenced in an Illumina MiSeq with a V3 2x250 bp paired-end partial run
at the University of Salford, UK.