title: Hypoponera pruinosa group UCEs
design: We extracted DNA from individual specimens per sample using a modified procedure adapted from Patzold et al. (2020), involving an overnight proteinase K digest in lysis buffer followed by oligonucleotide cleanup using the Monarch PCR & DNA Clean-up Kit. Each extract was quantified for DNA with a QubitTM flurometer (dsDNA HS Assay Kit, Life Technologies Inc.) before proceeding to the next stage. Extracts assessed to be of mostly high molecular weight and containing >100 ng mass were first sheared to the same target size range via enzymatic fragmentation or sonication. Sheared DNA products of both HM and HP taxa were channelled as input for a genomic DNA (gDNA) library preparation protocol (Kapa Hyper Prep Library Kit, Kapa Biosystems, Boston, MA). This included gDNA purification with a modified bead-based method (Fisher et al. 2011, Rohland and Reich 2012), followed by preparation into Illumina-compatible libraries with customized dual-indexing adapters (Glenn et al. 2019). Hybrid capture pools were prepared by combining 4-10 libraries per reaction, pooled roughly based on the input mass of gDNA to library preparation. Each pool was enriched following either the myBaits v5.03 protocol with overnight hybridization and washes at 62C (HP taxa), or a modified version of the myBaits v4.01 protocol with overnight hybridization and washes at 65C (HM taxa). The second day of the latter procedure is based on the original UCE enrichment protocol of Blumenstiel et al. (2010), instead of myBaits v4.01. Both protocols are in-solution and use a set of ant-specific custom-designed probes, either: 1) myBaits UCE Hymenoptera 2.5Kv2P (Daicel Arbor Biosciences) (HM taxa), or 2) myBaits Expert UCE Hymenoptera 2.5Kv2 (Daicel Arbor Biosciences)(HP taxa). Following enrichment, each capture pool was quantified using qPCR; pools were then further combined into final sequencing pools at approximately equimolar ratios.