Illumina Short-Read Sequencing of the Mitogenomes of Novel Scarites subterraneus Isolates Allows for Taxonomic Refinement of the Genus Scarites Fabricius 1775, within the Carabidae Family

SIMPLE SUMMARY: Ground beetles (Carabidae) have important ecological functions and serve as food, biological pest control, and models in biological research. Although there are over 40,000 ground beetle species worldwide, only a small fraction of those has genomic information currently available, which limits their classification and understanding of diversity. Since next-generation genome sequencing has become more mainstream, we used Illumina short-read sequencing to obtain complete mitogenomes from two Scarites beetles that we collected ourselves in Nebraska and Arkansas. Scarites are large ground beetles that resemble tropical beetles with a big head and large mandibles, and their role as predator and prey helps maintain sustainability in local ecosystems. This straightforward sequencing and analysis was found to be accurate and sufficient to help classify these isolates to the subspecies level. This is the first report of mitogenomes for Scarites subterraneus and only the second in that genus. This method is easily applicable to more beetle species and can be used to increase our understanding of beetles worldwide. ABSTRACT: We sequenced the complete mitogenomes, 18S and 28S rRNA of two new Scarites isolates, collected in Eastern Nebraska and Northern Arkansas (US). Based on molecular sequence data comparison and morphological characteristics, the new isolates were identified as a subspecies of Scarites subterraneus Fabricius 1775, for which we propose the subspecies names ‘nebraskensis’ and ‘arkansensis’. The new 18S and 28S rRNA sequences were found to be 99% and 98% identical to Scarites subterraneus. There are no other Scarites 18S or 28S rRNA sequences in the Genbank database, however, phylogenetic analysis of the Cox1 genes showed S. vicinus Chaudoir, 1843, and S. aterrimus Morawitz, 1863, as the closest relatives. This is the first report of a mitogenome for S. subterraneus, and only the second mitogenome for that genus. The nucleotide sequence identity between the mitogenomes of the two isolates is 98.8%, while the earlier sequenced S. buparius Forster 1771 mitogenome is more distantly related, with only 90% (to ssp. nebraskensis) and 89% (to ssp. arkansensis) overall nucleotide sequence identity. These new mitogenomes, and their phylogenetic analysis, firmly establish the position of Scarites on the Carabidae family tree and further refine the genus. In addition to the molecular data provided for the Scarites species, this approach also allowed us to identify bacterial and viral signatures for Providencia, Myroides, Spiroplasma, and a giant Nucleocytoviricota virus, associated with the Scarites species. We hereby present a simple and efficient protocol for identification and phylogenetic analysis of Scarites, that is applicable to other Coleoptera, based on total DNA extraction and Illumina short-read Next-Gen sequencing.