Introduction
Beetles (Coleoptera) are the most diverse animal group on the planet, with more than 386,000 species described worldwide (Bouchard et al. 2017; Forbes et al. 2018). The extremely high diversity and abundance of beetles plays a critical role in most terrestrial ecosystems (Brooks et al. 2012; Erwin 1997; Schowalter 2013; Slade et al. 2019), with further significance to human society as they serve as food, pests, pets, biological control agents, and models in biological and engineering research (Adamski et al. 2019). Despite their importance, relatively few genomic studies have been conducted on beetles to date (McKenna 2018).
Coleoptera are divided into four suborders including Polyphaga, Adephaga, Myxophaga, and Archostemata, based on both morphological and molecular evidence (Beutel & Haas 2000; Zhang et al. 2018). Among the four suborders, Polyphaga is the most diverse group, comprising approximately 90% of coleopteran species, while most of the remaining 10% comprise Adephaga (Grimaldi & Engel 2005). Carabidae is the largest family of Adephaga, consisting of around 40,000 species, and is known for its broad use of terrestrial habitats, ecosystem services, and environmental sensitivity as bioindicators (Brooks et al.2012; Lorenz 2020). Except for a few species that are considered agricultural pests (e.g. Zabrus tenebrioides ), most carabid species are predators or scavengers that serve as beneficial biological control agents and play key roles in local food webs (Georgescu et al. 2017; Pizzolotto et al. 2018). Carabid beetles are found in a wide range of habitats, spanning all terrestrial biomes, from tropical and desert habitats, to arctic and alpine regions. As a result, they are frequently used in studies of community ecology, conservation, and biogeography (Kotze et al. 2011). Recent phylogenomic work has provided an understanding of the evolutionary relationships of Carabidae to other Coleoptera lineages and some patterns of gene family diversification (Gustafson et al. 2019; McKenna et al.2019; Misof et al. 2014; Seppey et al. 2019a), but has not provided a high-quality genome assembly for comparative analysis.
Carabid beetles are one of the few insect lineages that thrive in cold habitats (Mani 1968). A strong positive relationship exists between minimal environmental temperature and minimal temperature tolerance in insects (Kimura 2004), because insects, as ectotherms, do not regulate body heat through metabolism, but must instead physiologically adjust to the surrounding environment (Addo‐Bediako et al. 2002). Cold environments consequently pose a significant challenge to insects, with few species surviving these conditions. Less than 2% of North American insect species are found in the arctic biome (Danks et al. 2017), and insect species richness declines dramatically in alpine and sub-nival habitats (Nagy & Grabherr 2009). Carabid beetles, however, are notable as arctic and alpine insects, comprising ~40-50% of beetle species richness in the arctic (Chernov et al. 2000; Danks 1981), and setting the highest altitudinal distribution record of Coleoptera, with three taxa (Amara altiphila , Bembidion maddisoni , and Trechus astrophilus ) reaching 5,600 m above sea level (a.s.l.) in the Himalayas (Schmidt 2009; Schmidt et al. 2017). The carabid beetlePterostichus brevicornis additionally sets a record among beetles in demonstrating extreme cold tolerance, where the LD50of overwintering adults occurs at -87°C (Baust 1972). The carabid genusNebria (> 500 species) is a notable example of a cold-specialized beetle lineage, which exhibits its greatest diversity and abundance at high latitude and high elevation (Lorenz 2020). For example, 12 species of Nebria are found around Mount Rainier, Washington, with four species inhabiting the alpine and sub-nival zone (> 2000 m a.s.l., Kavanaugh 1985; Slatyer & Schoville 2016). Comparative genomic analysis of other insects has shown that adaptation to cold environments might involve constraints in genome architecture instead of gene content (Kelley et al. 2014; Kimet al. 2017), although other studies have mostly found evidence of accelerated protein evolution (Cicconardi et al. 2020; Parkeret al. 2018). With the dual purpose of generating genomic resources to investigate Carabid beetle genome evolution and cold specialization, we assemble the genome of Nebria riversiVan Dyke (Carabidae: Nebriinae), an alpine ground beetle endemic to the Sierra Nevada in California (2900–3910 m a.s.l.). Considering its highly divergent taxonomic position from other Coleoptera genomes, and as the only cold-specialized beetle genome sequenced to date, we investigate genome architecture, gene family diversity, and differential gene expression across life-stages and sexes. Finally, as the microbial associates of high elevation insects have rarely been examined (Receveuret al. 2020), we scan genomic evidence for symbiotic microbes inN. riversi .