The Evolution of Dark Matter in the Mitogenome of Seed Beetles
AbstractAnimal mitogenomes are generally thought of as being economic and optimized for rapid replication and transcription. We use long-read sequencing technology to assemble the remarkable mitogenomes of four species of seed beetles. These are the largest circular mitogenomes ever assembled in insects, ranging from 24,496 to 26,613 bp in total length, and are exceptional in that some 40% consists of non-coding DNA. The size expansion is due to two very long intergenic spacers (LIGSs), rich in tandem repeats. The two LIGSs are present in all species but vary greatly in length (114–10,408 bp), show very low sequence similarity, divergent tandem repeat motifs, a very high AT content and concerted length evolution. The LIGSs have been retained for at least some 45 my but must have undergone repeated reductions and expansions, despite strong purifying selection on protein coding mtDNA genes. The LIGSs are located in two intergenic sites where a few recent studies of insects have also reported shorter LIGSs (>200 bp). These sites may represent spaces that tolerate neutral repeat array expansions or, alternatively, the LIGSs may function to allow a more economic translational machinery. Mitochondrial respiration in adult seed beetles is based almost exclusively on fatty acids, which reduces the need for building complex I of the oxidative phosphorylation pathway (NADH dehydrogenase). One possibility is thus that the LIGSs may allow depressed transcription of NAD genes. RNA sequencing showed that LIGSs are partly transcribed and transcriptional profiling suggested that all seven mtDNA NAD genes indeed show low levels of transcription and co-regulation of transcription across sexes and tissues.
[…] We used a Pacific Biosciences RSII sequencer to sequence the mitogenomes of the three Callosobruchus species, employing the SMRT-analysis HGAP3 pipeline for assembly, and a Pacific Biosciences Sequel sequencer to sequence the mitogenome of A. obtectus, using the SMRT-analysis HGAP4 pipeline for assembly. The mitogenomes were annotated using DOGMA () and MITOS (). An Illumina HiSeq2500 platform (v4 sequencing chemistry) was used for the resequencing of the three C. maculatus populations which were assembled using MITObim V 1.8 () and MIRA V 4.0.2 (). The RNA sequencing involved standard RNA extraction and library preparation protocols followed by Illumina HiSeq2500 sequencing, described in detail in and ( online). […]