DOI: 10.5061/dryad.1vhhmgqzd
title: Data from: Complex evolutionary processes maintain an ancient chromosomal inversion

description: Genome re-arrangements such as chromosomal inversions are often involved
 in adaptation. As such, they experience natural selection, which can erode
 genetic variation. Thus, whether and how inversions can remain polymorphic
 for extended periods of time remains debated. Here we combine genomics,
 experiments, and evolutionary modeling to elucidate the processes
 maintaining an inversion polymorphism associated with the use of a
 challenging host plant (Redwood trees) in Timema stick insects. We show
 that the inversion is maintained by a combination of processes, finding
 roles for life-history trade-offs, heterozygote advantage, local
 adaptation to different hosts, and gene flow. We use models to show how
 such multi-layered regimes of balancing selection and gene flow provide
 resilience to help buffer populations against the loss of genetic
 variation, maintaining the potential for future evolution. We further show
 that the inversion polymorphism has persisted for millions of years and is
 not a result of recent introgression. We thus find that rather than being
 a nuisance, the complex interplay of evolutionary processes provides a
 mechanism for the long-term maintenance of genetic variation.
description: We conducted a laboratory experiment to test for a potential
 effect of the Perform locus on performance (here growth and survival) in
 <em>T. knulli</em> reared on <em>Ceanothus</em> or
 Redwood. For this experiment, each of the 138 <em>T.
 knulli</em> collected were placed individually in 500 mm plastic
 containers, with air holes for breathing punched into the lid containers
 using a needle. Each stick insect was then fed fresh plant material from
 either<em> Ceanothus </em>or Redwood every second day (when
 survival was recorded, see below). Host-plant treatment was determined
 randomly and was independent of the host from which the stick insect was
 collected. We then measured weight and survival at 15 and 21 d as metrics
 of performance, and survival (dead or alive) was monitored every second
 day for the course of the 21-d experiment.  After the
 performance experiment, we isolated DNA from each of 138 <em>T.
 knulli</em>. Frozen legs from each individual were ground into
 powder form using a Qiagen TissueLyser (Qiagen Inc., Valencia, CA).
 Genomic DNA was then extracted using Qiagen DNeasy Blood and Tissue kits,
 using a protocol with slightly altered incubation temperatures and times.
 We used a reduced- representation technique (i.e.,
 genotyping-by-sequencing or GBS) to construct DNA sequencing libraries.
 Genomic DNA from each individual was digested with two restriction
 endonucleases, MseI (four base recognition site) and EcoRI (six base
 recognition site). Illumina adaptors with unique 8 to 10 bp DNA barcodes
 for each individual were ligated to EcoRI cut sites, and a base Illumina
 adaptor was ligated to MseI cut sites. Barcoded fragment libraries were
 then PCR amplified using Illumina primers and a high-fidelity proofreading
 polymerase (Iproof, BioRad, Hercules, CA). PCR products were pooled into a
 single library which was then quality screened using an Agilent
 BioAnalzyer automated electrophoresis device. To reduce the portion of the
 genome targeted for sequencing, the reduced-representation library was
 then size-selected for DNA fragments 350 to 450 bp in length using a
 Pippin Prep quantitative electrophoresis unit (Sage Science, Beverly, MA)
 at the University of Texas Genome Sequencing and Analysis Facility
 (UTGSAF). The size-selected library was then sequenced using S2 chemistry
 and a single lane on an Illumina NovaSeq 4000 at UTGSAF.
 We aligned the newly acquired <em>T. knulli</em> GBS
 reads to our new <em>T. knulli</em> reference genome for SNP
 calling. We generated the <em>T. knulli</em> reference genome
 using a combination of PacBio and Illumina reads from Chicago and Hi-C
 genomic libraries. DNA extraction, library preparation, DNA sequencing,
 and de novo genome assembly were performed by Dovetail Genomics (now
 Cantata Bio). Specifically, 105.4 Gbp of PacBio data (∼75.2× coverage)
 were generated over two SMRT cells and used to build an initial assembly
 with Falcon assembler (with default options). This initial assembly was
 further improved and scaffolded using the HiRise assembler (with default
 options). To do so, 58.8 Gbp of Chicago DNA sequence data and 72.9 Gbp of
 Hi-C DNA sequence data were generated on a HiSeqX machine (150 bp
 paired-end reads). A total of three female stick insects were used for the
 assembly, and the individuals were chosen based on a preliminary analysis
 (i.e., PCA on genotypes obtained by GBS) that suggested they were
 homozygous for the Perform PRW allele. The final assembly created using
 Dovetail’s HiRise Assembly pipeline comprised 1,322,373,696 base pairs
 (bps) with an N50 of 83,614,905 bps. Based on BUSCO version 4.0.5 with the
 eukaryota_odb10 database (70 species, 255 BUSCOs), the assembly included
 216 complete BUSCOs (212 single copy and four duplicated; 84.7%), 15
 fragmented BUSCOs (5.9%) and 24 missing BUSCOs (9.4%). We then used the
 BRAKER2 pipeline to annotate this genome.
description: This data set contains only ASCII text files.