SRX12601975 Paramycetophylax_bruchi_1366 SRP341325 bp0 To generate the UCE dataet we performed the following steps: DNA extraction, DNA shearing, library preparation, sample pooling, UCE enrichment, and sequencing. Ant DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA. U.S.A.). DNA extractions were performed non-destructively by opening small holes on the right side of the specimen, especially on the pronotum and propodeum, with a sterilized entomological pin to facilitate the lysis process. Cell lysis was performed overnight with 20 L of Proteinase-K in a dry bath shaker at 56 C and at 500 rpm. We followed the recommendations of the manufacturer for the extraction process except that we eluted the cleaned, extracted DNA from the spin-collection columns with two (rather than one) washes, each consisting of 65 L of nuclease-free water, differing from the manufacturers recommendation of 200 L of AE buffer. 50 ng of DNA template was sheared to an average fragment length of 300600 bp using a Qsonica Q800R2 Sonicator for 60 s. Libraries were prepared in 1.5 mL tubes on a rare magnet stand using the Kapa Hyper Prep Library Kit with the iTru Adapter protocol. We implemented all magnetic bead clean-up steps (Fisher et al. 2011) as described in Faircloth et al. (2015) and used dual-indexing TruSeq adapters (Faircloth and Glenn 2012, Glenn et al. 2019) for ligation. The ligation step was followed by PCR-amplification of 15 L of the library product using 25 L of KAPA HiFi ReadyMix (Kapa Biosystems, Wilmington, MA, U.S.A.), 2.5 L of each of Illumina TruSeq (i5 and i7) primers, and 5 L nuclease-free ddH2O. The following thermal cycler program was executed: 98 C for 45 s; 13 cycles of 98 C for 15 s, 60 C for 30 s, 72 C for 60 s; and final extension at 72 C for 5 m. Following PCR, we purified DNA products using 1.2x Kapa Pure beads and rehydrated the purified product in 22 L of Elution Buffer (pH= 8). Individual libraries were quantified using 2 L of library product in a Qubit 3.0 Fluorometer using the Broad Range Kit (Thermo Fisher Scientific, Inc.). Post-PCR libraries were pooled at equimolar concentrations into one pool containing the ant libraries. Pool concentration was adjusted to 71.5 ng/L by drying the sample in a vacuum centrifuge for 4560 min or until all liquid was evaporated at 60 C, and then by re-suspending the pool in nuclease-free water at the estimated value. The pool was enriched by using the ant-customized bait set (ant-specific-hym-v2) targeting 2524 conserved loci in Hymenoptera (Branstetter et al. 2017b) at an incubation temperature of 65 C for 24 h in a thermal cycler. Enrichment, bead-cleaning, and PCR reaction procedures partially followed the Arbor Biosciences v4.0.1 To obtain reliable estimates of DNA concentration for the enriched pool, we performed a quantitative qPCR on an ViiA7 Real-Time PCR System (Thermo Fisher Scientific, Inc.) using the KAPA Library Quantification Kit (Kapa Biosystems, Inc) with the KAPA SYBR FAST qPCR Master Mix, universal Illumina primers, and dilutions of 1:1,000,000 and 1:2,000,000. The final enriched pool of 100 L was submitted to the Laboratories for Analytical Biology (LAB) of the Smithsonian Institution National Museum of Natural History for quality control and sequencing on an Illumina MiSeq (employing the V3 600 MiSeq Kit). SRS10556371 Paramycetophylax_bruchi_1366 Paramycetophylax_bruchi_1366 WGS GENOMIC Hybrid Selection Illumina MiSeq SRX12601976 Paramycetophylax_bruchi_2350 SRP341325 bp0 To generate the UCE dataet we performed the following steps: DNA extraction, DNA shearing, library preparation, sample pooling, UCE enrichment, and sequencing. Ant DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA. U.S.A.). DNA extractions were performed non-destructively by opening small holes on the right side of the specimen, especially on the pronotum and propodeum, with a sterilized entomological pin to facilitate the lysis process. Cell lysis was performed overnight with 20 L of Proteinase-K in a dry bath shaker at 56 C and at 500 rpm. We followed the recommendations of the manufacturer for the extraction process except that we eluted the cleaned, extracted DNA from the spin-collection columns with two (rather than one) washes, each consisting of 65 L of nuclease-free water, differing from the manufacturers recommendation of 200 L of AE buffer. 50 ng of DNA template was sheared to an average fragment length of 300600 bp using a Qsonica Q800R2 Sonicator for 60 s. Libraries were prepared in 1.5 mL tubes on a rare magnet stand using the Kapa Hyper Prep Library Kit with the iTru Adapter protocol. We implemented all magnetic bead clean-up steps (Fisher et al. 2011) as described in Faircloth et al. (2015) and used dual-indexing TruSeq adapters (Faircloth and Glenn 2012, Glenn et al. 2019) for ligation. The ligation step was followed by PCR-amplification of 15 L of the library product using 25 L of KAPA HiFi ReadyMix (Kapa Biosystems, Wilmington, MA, U.S.A.), 2.5 L of each of Illumina TruSeq (i5 and i7) primers, and 5 L nuclease-free ddH2O. The following thermal cycler program was executed: 98 C for 45 s; 13 cycles of 98 C for 15 s, 60 C for 30 s, 72 C for 60 s; and final extension at 72 C for 5 m. Following PCR, we purified DNA products using 1.2x Kapa Pure beads and rehydrated the purified product in 22 L of Elution Buffer (pH= 8). Individual libraries were quantified using 2 L of library product in a Qubit 3.0 Fluorometer using the Broad Range Kit (Thermo Fisher Scientific, Inc.). Post-PCR libraries were pooled at equimolar concentrations into one pool containing the ant libraries. Pool concentration was adjusted to 71.5 ng/L by drying the sample in a vacuum centrifuge for 4560 min or until all liquid was evaporated at 60 C, and then by re-suspending the pool in nuclease-free water at the estimated value. The pool was enriched by using the ant-customized bait set (ant-specific-hym-v2) targeting 2524 conserved loci in Hymenoptera (Branstetter et al. 2017b) at an incubation temperature of 65 C for 24 h in a thermal cycler. Enrichment, bead-cleaning, and PCR reaction procedures partially followed the Arbor Biosciences v4.0.1 To obtain reliable estimates of DNA concentration for the enriched pool, we performed a quantitative qPCR on an ViiA7 Real-Time PCR System (Thermo Fisher Scientific, Inc.) using the KAPA Library Quantification Kit (Kapa Biosystems, Inc) with the KAPA SYBR FAST qPCR Master Mix, universal Illumina primers, and dilutions of 1:1,000,000 and 1:2,000,000. The final enriched pool of 100 L was submitted to the Laboratories for Analytical Biology (LAB) of the Smithsonian Institution National Museum of Natural History for quality control and sequencing on an Illumina MiSeq (employing the V3 600 MiSeq Kit). SRS10556373 Paramycetophylax_bruchi_2350 Paramycetophylax_bruchi_2350 WGS GENOMIC Hybrid Selection Illumina MiSeq SRX12601977 Paramycetophylax_bruchi_2380 SRP341325 bp0 To generate the UCE dataet we performed the following steps: DNA extraction, DNA shearing, library preparation, sample pooling, UCE enrichment, and sequencing. Ant DNA was extracted using the Qiagen DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA. U.S.A.). DNA extractions were performed non-destructively by opening small holes on the right side of the specimen, especially on the pronotum and propodeum, with a sterilized entomological pin to facilitate the lysis process. Cell lysis was performed overnight with 20 L of Proteinase-K in a dry bath shaker at 56 C and at 500 rpm. We followed the recommendations of the manufacturer for the extraction process except that we eluted the cleaned, extracted DNA from the spin-collection columns with two (rather than one) washes, each consisting of 65 L of nuclease-free water, differing from the manufacturers recommendation of 200 L of AE buffer. 50 ng of DNA template was sheared to an average fragment length of 300600 bp using a Qsonica Q800R2 Sonicator for 60 s. Libraries were prepared in 1.5 mL tubes on a rare magnet stand using the Kapa Hyper Prep Library Kit with the iTru Adapter protocol. We implemented all magnetic bead clean-up steps (Fisher et al. 2011) as described in Faircloth et al. (2015) and used dual-indexing TruSeq adapters (Faircloth and Glenn 2012, Glenn et al. 2019) for ligation. The ligation step was followed by PCR-amplification of 15 L of the library product using 25 L of KAPA HiFi ReadyMix (Kapa Biosystems, Wilmington, MA, U.S.A.), 2.5 L of each of Illumina TruSeq (i5 and i7) primers, and 5 L nuclease-free ddH2O. The following thermal cycler program was executed: 98 C for 45 s; 13 cycles of 98 C for 15 s, 60 C for 30 s, 72 C for 60 s; and final extension at 72 C for 5 m. Following PCR, we purified DNA products using 1.2x Kapa Pure beads and rehydrated the purified product in 22 L of Elution Buffer (pH= 8). Individual libraries were quantified using 2 L of library product in a Qubit 3.0 Fluorometer using the Broad Range Kit (Thermo Fisher Scientific, Inc.). Post-PCR libraries were pooled at equimolar concentrations into one pool containing the ant libraries. Pool concentration was adjusted to 71.5 ng/L by drying the sample in a vacuum centrifuge for 4560 min or until all liquid was evaporated at 60 C, and then by re-suspending the pool in nuclease-free water at the estimated value. The pool was enriched by using the ant-customized bait set (ant-specific-hym-v2) targeting 2524 conserved loci in Hymenoptera (Branstetter et al. 2017b) at an incubation temperature of 65 C for 24 h in a thermal cycler. Enrichment, bead-cleaning, and PCR reaction procedures partially followed the Arbor Biosciences v4.0.1 To obtain reliable estimates of DNA concentration for the enriched pool, we performed a quantitative qPCR on an ViiA7 Real-Time PCR System (Thermo Fisher Scientific, Inc.) using the KAPA Library Quantification Kit (Kapa Biosystems, Inc) with the KAPA SYBR FAST qPCR Master Mix, universal Illumina primers, and dilutions of 1:1,000,000 and 1:2,000,000. The final enriched pool of 100 L was submitted to the Laboratories for Analytical Biology (LAB) of the Smithsonian Institution National Museum of Natural History for quality control and sequencing on an Illumina MiSeq (employing the V3 600 MiSeq Kit). SRS10556372 Paramycetophylax_bruchi_2380 Paramycetophylax_bruchi_2380 WGS GENOMIC Hybrid Selection Illumina MiSeq