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. 

We sampled four closely related Nebria (Catonebria) taxa from western North America: N. meanyi, N. piperi, N. riversi, and N. vandykei. The species N. meanyi and N. piperi 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 N. vandykei (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 N. riversi were available from a previous project (NCBI accessions: SRR12967585, SRR12967587 and SRR12967588). We additionally sampled N. (Nivalonebria) paradisi and N. (Neaptenonebria) spatulata sierrae, the closest two outgroup lineages to Nebria (Catonebria), as well as outgroups that span the tribe Nebriini by including Leistus (Leistus) frater, Nippononebria (Vancouveria) altisierrae, and Nebria (Nakanebria) shiretokoana. 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 (N. vandykei desiccation stress, N. piperi, and N. riversi) 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 de novo 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. 

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