Over the past decade the mitochondrial (mt) genome has become the most widely used genomic resource available for systematic entomology. While the availability of other types of ‘–omics’ data – in particular transcriptomes – is increasing rapidly, mt genomes are still vastly cheaper to sequence and are far less demanding of high quality templates. Furthermore, almost all other ‘–omics’ approaches also sequence the mt genome, and so it can form a bridge between legacy and contemporary datasets. Mitochondrial genomes have now been sequenced for all insect orders, and in many instances representatives of each major lineage within orders (suborders, series or superfamilies depending on the group). They have also been applied to systematic questions at all taxonomic scales from resolving interordinal relationships (e.g. Cameron et al., 2009; Wan et al., 2012; Wang et al., 2012), through many intraordinal (e.g. Dowton et al., 2009; Timmermans et al., 2010; Zhao et al. 2013a) and family-level studies (e.g. Nelson et al., 2012; Zhao et al., 2013b) to population/biogeographic studies (e.g. Ma et al., 2012). Methodological issues around the use of mt genomes in insect phylogenetic analyses and the empirical results found to date have recently been reviewed by Cameron (2014), yet the technical aspects of sequencing and annotating mt genomes were not covered. Most papers which generate new mt genome report their methods in a simplified form which can be difficult to replicate without specific knowledge of the field. Published studies utilize a sufficiently wide range of approaches, usually without justification for the one chosen, that confusion about commonly used jargon such as ‘long PCR’ and ‘primer walking’ could be a serious barrier to entry. Furthermore, sequenced mt genomes have been annotated (gene locations defined) to wildly varying standards and improving data quality through consistent annotation procedures will benefit all downstream users of these datasets. The aims of this review are therefore to: 1. Describe in detail the various sequencing methods used on insect mt genomes; 2. Explore the strengths/weakness of different approaches; 3. Outline the procedures and software used for insect mt genome annotation, and; 4. Highlight quality control steps used for new annotations, and to improve the re-annotation of previously sequenced mt genomes used in systematic or comparative research.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/syen.12071

How to sequence and annotate insect mitochondrial genomes for systematic and comparative genomics research

Lepidoptera encompasses more than 160,000 described species that have been classified into 45–48 superfamilies. The previously determined Lepidoptera mitochondrial genomes (mitogenomes) are limited to six superfamilies of the lineage Ditrysia. Compared with the ancestral insect gene order, these mitogenomes all contain a tRNA rearrangement. To gain new insights into Lepidoptera mitogenome evolution, we sequenced the mitogenomes of two ghost moths that belong to the non-ditrysian lineage Hepialoidea and conducted a comparative mitogenomic analysis across Lepidoptera. The mitogenomes of Thitarodes renzhiensis and T. yunnanensis are 16,173 bp and 15,816 bp long with an A + T content of 81.28 % and 82.34 %, respectively. Both mitogenomes include 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and the A + T-rich region. Different tandem repeats in the A + T-rich region mainly account for the size difference between the two mitogenomes. All the protein-coding genes start with typical mitochondrial initiation codons, except for cox1 (CGA) and nad1 (TTG) in both mitogenomes. The anticodon of trnS(AGN) in T. renzhiensis and T. yunnanensis is UCU instead of the mostly used GCU in other sequenced Lepidoptera mitogenomes. The 1,584-bp sequence from rrnS to nad2 was also determined for an unspecified ghost moth (Thitarodes sp.), which has no repetitive sequence in the A + T-rich region. All three Thitarodes species possess the ancestral gene order with trnI-trnQ-trnM located between the A + T-rich region and nad2, which is different from the gene order trnM-trnI-trnQ in all previously sequenced Lepidoptera species. The formerly identified conserved elements of Lepidoptera mitogenomes (i.e. the motif ‘ATAGA’ and poly-T stretch in the A + T-rich region and the long intergenic spacer upstream of nad2) are absent in the Thitarodes mitogenomes. The mitogenomes of T. renzhiensis and T. yunnanensis exhibit unusual features compared with the previously determined Lepidoptera mitogenomes. Their ancestral gene order indicates that the tRNA rearrangement event(s) likely occurred after Hepialoidea diverged from other lepidopteran lineages. Characterization of the two ghost moth mitogenomes has enriched our knowledge of Lepidoptera mitogenomes and contributed to our understanding of the mechanisms underlying mitogenome evolution, especially gene rearrangements.

/pdf/the-complete-mitochondrial-genomes-of-two-ghost-moths-2j78rgnd1p.pdf

The complete mitochondrial genomes of two ghost moths, Thitarodes renzhiensis and Thitarodes yunnanensis: the ancestral gene arrangement in Lepidoptera.

The complete mitochondrial genome (mitogenome) of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae) was determined. The genome is a circular molecule 15 481 bp long. It presents a typical gene organization and order for completely sequenced lepidopteran mitogenomes, but differs from the insect ancestral type for the placement of tRNAMet. The nucleotide composition of the genome is also highly A + T biased, accounting for 80.38%, with a slightly positive AT skewness (0.010), indicating the occurrence of more As than Ts, as found in the Noctuoidea species. All protein-coding genes (PCGs) are initiated by ATN codons, except for COI, which is tentatively designated by the CGA codon as observed in other lepidopterans. Four of 13 PCGs harbor the incomplete termination codon, T or TA. All tRNAs have a typical clover-leaf structure of mitochondrial tRNAs, except for tRNASer(AGN), the DHU arm of which could not form a stable stem-loop structure. The intergenic spacer sequence between tRNASer(AGN) and ND1 also contains the ATACTAA motif, which is conserved across the Lepidoptera order. The H. cunea A+T-rich region of 357 bp is comprised of non-repetitive sequences, but harbors several features common to the Lepidoptera insects, including the motif ATAGA followed by an 18 bp poly-T stretch, a microsatellite-like (AT)8 element preceded by the ATTTA motif, an 11 bp poly-A present immediately upstream tRNAMet. The phylogenetic analyses support the view that the H. cunea is closerly related to the Lymantria dispar than Ochrogaster lunifer, and support the hypothesis that Noctuoidea (H. cunea, L. dispar, and O. lunifer) and Geometroidea (Phthonandria atrilineata) are monophyletic. However, in the phylogenetic trees based on mitogenome sequences among the lepidopteran superfamilies, Papillonoidea (Artogeia melete, Acraea issoria, and Coreana raphaelis) joined basally within the monophyly of Lepidoptera, which is different to the traditional classification.

/pdf/the-complete-mitochondrial-genome-of-the-fall-webworm-1a0r5d2fqt.pdf

The complete mitochondrial genome of the fall webworm, Hyphantria cunea (Lepidoptera: Arctiidae)

Reconstructing intraordinal relationships in Lepidoptera using mitochondrial genome data with the description of two newly sequenced lycaenids, Spindasis takanonis and Protantigius superans (Lepidoptera: Lycaenidae).

A comparative analysis of complete mitochondrial genomes among Hexapoda.

In this paper, the complete mitochondrial genome of Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini) is reported; a circular molecule of 15,245 bp in size. For A. issoria, genes are arranged in the same order and orientation as the complete sequenced mitochondrial genomes of the other lepidopteran species, except for the presence of an extra copy of tRNAIle(AUR)b in the control region. All protein-coding genes of A. issoria mitogenome start with a typical ATN codon and terminate in the common stop codon TAA, except that COI gene uses TTG as its initial codon and terminates in a single T residue. All tRNA genes possess the typical clover leaf secondary structure except for tRNASer(AGN), which has a simple loop with the absence of the DHU stem. The sequence, organization and other features including nucleotide composition and codon usage of this mitochondrial genome were also reported and compared with those of other sequenced lepidopterans mitochondrial genomes. There are some short microsatellite-like repeat regions (e.g., (TA)9, polyA and polyT) scattered in the control region, however, the conspicuous macro-repeats units commonly found in other insect species are absent.

The complete mitochondrial genome of the yellow coaster, Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini): sequence, gene organization and a unique tRNA translocation event

The Apollo butterfly, Parnassius apollo is a representative species of the butterfly subfamily Parnassiinae. This charming species is one of the most endangered butterfly species in the world. In this study, we sequenced its complete mitochondrial genome (mitogenome), with the aim of accumulating genetic information for further studies of population genetics and mitogenome evolution in the Papilionidae. The 15,404-bp long mitogenome harbors a typical set of 37 genes and is the largest butterfly mitogenome determined, except for Papilio maraho (16,094 bp). Like many other sequenced lepidopteran species, one tRNA Trp -like and one tRNA Leu (UUR)-like sequences were detected in the AT-rich region. A total of 164 bp of non-coding sequences are dispersed in 14 regions throughout the genome. The longest intergenic spacer (68 bp) is located between tRNA Ser (AGN) and tRNA Glu , and is the largest spacer at this location among Papilionidae species. This spacer may have resulted from an 8-fold repetition of a TTTCTTCT motif or a 4-fold repetition of a CTTTATTT motif.

The complete mitochondrial genome of the endangered Apollo butterfly, Parnassius apollo (Lepidoptera: Papilionidae) and its comparison to other Papilionidae species

The mitochondrial genome (mitogenome) of Eurytoma sp. (Hymenoptera: Eurytomidae: Eurytominae) was determined in this study. The sequenced region is 14,946 bp with an A + T content of 82.65%, including 13 protein-coding genes, 2 ribosomal RNAs, 18 transfer RNAs, and a partial sequence of AT-rich region. All PCGs begin with typical ATN codons and use standard canonical TAA as their termination codons, except for atp6 gene, which stops with an incomplete codon T. A comparison of all the sequenced hymenopteran mitogenomes revealed that Eurytoma sp. has dramatic gene rearrangements, some of which is peculiar to Chalcidoidea. What’s more, Maximum likelihood (ML) analysis highly supported the monophyly of both Chalcidoidea and Apocrita.

Sequencing and characterization of mitochondrial genome of Eurytoma sp. (Hymenoptera: Eurytomidae).

The mitochondrial genome (mitogenome) of Megachile sculpturalis (Hymenoptera: Megachilidae) has been reported in this study. This is the first sequenced mitogenome from the family Megachilidae. The sequenced region of this mitochondrial genome is determined to be 16 581 bp in length, including 13 protein-coding genes, 2 ribosomal RNAs, 22 transfer RNAs, and a partial sequence of the AT-rich region, and the AT-rich region contains several characteristic repeated sequences. Additionally, we conducted a phylogenetic analysis using all the mitochondrial genomes of Apoidea that are available in Genbank. Bayesian method supported the monophyly of both Apidae and Colletidae. And Megachilidae was inferred as the sister group of Apidae.

Dunyuan Huang

Papers

The complete mitochondrial genome of the yellow coaster, Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini): sequence, gene organization and a unique tRNA translocation event

The complete mitochondrial genome of the yellow coaster, Acraea issoria (Lepidoptera: Nymphalidae: Heliconiinae: Acraeini): sequence, gene organization and a unique tRNA translocation event

The complete mitochondrial genome of the endangered Apollo butterfly, Parnassius apollo (Lepidoptera: Papilionidae) and its comparison to other Papilionidae species

Sequencing and characterization of mitochondrial genome of Eurytoma sp. (Hymenoptera: Eurytomidae).

Sequencing and characterization of the Megachile sculpturalis (Hymenoptera: Megachilidae) mitochondrial genome