DOI: 10.5061/dryad.wh70rxws9
title: Environmental adaptation in an alpine ground beetle

description: We test for evidence of adaptive evolution in the alpine ground
 beetle Nebria vandykei. First, we explore the genetic pathways
 induced during environmental stress responses through RNA sequencing,
 showing that cold, heat and desiccation stress activate a largely
 non-overlapping set of molecular pathways. Using additional transcriptome
 sequencing, we estimate the evolutionary relationship of N. vandykei to
 related species in the subgenus Catonebria and several outgroups.
 Phylogenetic analyses suggest a history of admixture or very rapid
 diversification underlies the evolution of N. vandykei. Finally, using
 tests for positive selection polarized by high and low elevation
 relatives, we demonstrate selection acting on stress response pathways, as
 well as pathways known to function in tolerance to cold and hypoxic
 environments. 
description: We sampled four closely related
 <em>Nebria</em> (<em>Catonebria</em>) taxa from
 western North America: <em>N. meanyi, N. piperi, N.
 riversi,</em> and <em>N. vandykei</em>. The species
 <em>N. meanyi</em> and <em>N. piperi</em> are
 found in small mid-elevation streams and large low-elevation rivers,
 respectively, while the other two taxa are alpine specialists that are
 found inhabiting talus, riparian, and seep habitats on or near
 high-elevation snowfields. We sampled 20 <em>N. vandykei
 </em>(grouped by physiological assay: control, cold stress, heat
 stress, and desiccation stress) and a single adult specimen from each of
 the other species, while three <em>N. riversi</em> were
 available from a previous project (NCBI accessions: SRR12967585,
 SRR12967587 and SRR12967588). We additionally sampled
 <em>N</em>. (<em>Nivalonebria</em>)
 <em>paradisi</em> and <em>N</em>.
 (<em>Neaptenonebria</em>) <em>spatulata</em>
 <em>sierrae</em>, the closest two outgroup lineages to
 <em>Nebria</em> (<em>Catonebria</em>), as well as
 outgroups that span the tribe Nebriini by including
 <em>Leistus</em> (<em>Leistus</em>)
 <em>frater</em>, <em>Nippononebria</em>
 (<em>Vancouveria</em>) <em>altisierrae</em>, and
 <em>Nebria</em> (<em>Nakanebria</em>)
 <em>shiretokoana</em>. Total RNA was extracted using Trizol
 regeant from each beetle. Sequencing libraries were
 prepared for short-read RNA sequencing. Most samples were prepared using
 the TruSeq RNA library Prep kit (Illumina Corp.), as 2 x 50 base pair (bp)
 libraries and sequenced on an Illumina HiSeq2500. Another set of samples
 (<em>N. vandykei</em> desiccation stress, <em>N.
 piperi</em>, and <em>N. riversi</em>) were prepared
 using TruSeq Stranded Total RNA Library Prep kit (Illumina Corp.), as 2 x
 150 bp libraries and sequenced on the Illumina Novoseq 6000. 
 We generated <em>de novo</em> transcriptome
 assemblies for each species using the program Trinity v2.10.0 (Haas et
 al., 2013), based on default settings. When more than one individual was
 available, all individuals were combined to generate the most complete
 transcriptome. 
description: Use text editor to open all files. Excel can be used to open the
 annotation files (.xlsx). R scripts (.R) can be opened in R.