Application of noninvasive sampling technique in mitochondrial genome intraspecific phylogeny of the endangered butterfly, Teinopalpus aureus (Lepidoptera: Papilionidae).

The butterfly genus of Teinopalpus, endemic to Asia, embodies a distinct species of mountain-dwelling butterflies with specific habitat requirements. These species are rare in the wild and hold high conservation and research value. Similar to other protected species, the genetic analysis of the rare Teinopalpus aureus poses challenges due to the complexity of sampling. In this study, we successfully extracted DNA and amplified mitochondrial genomic DNA from various noninvasive sources such as larval feces, larval exuviae, larval head capsules, pupal exuviaes, and filamentous gland secretions, all integral parts of butterfly metamorphosis. This was conducted as part of a research initiative focused on the artificial conservation of T. aureus population in Jinggang Shan Nature Reserve. Our findings illustrated the successful extraction of DNA from multiple noninvasive sources, achieved through modified DNA extraction methodologies. Although the DNA concentration obtained from noninvasive samples was lower than that from muscle tissues of newly dead larvae during rearing, all samples met the requirements for PCR amplification and sequencing, yielding complete circular sequences. These sequences are pivotal for both interspecific and intraspecific genetic relationship analysis. Our methods can be extended to other insects, especially scarce species.

Phylogenetic relationships of Scelimeninae genera (Orthoptera: Tetrigoidea) based on COI, 16S rRNA and 18S rRNA gene sequences.

Scelimeninae is an important subfamily of Tetrigoidea; however, the phylogenetic relationships within Scelimeninae are poorly understood, and its generic classification has remained unstable. In this study, the COI, 16S rRNA and 18S rRNA genes from 24 species in 9 genera within Scelimeninae were amplified and sequenced, the base composition and inter-species genetic distance of the combined sequence of COI, 16S rRNA and 18S rRNA genes were analyzed, and the molecular phylogenetic relationships were reconstructed using Maximum Likelihood (ML) and Bayesian inference (BI) methods. The results of sequence analysis showed that the total length of the combined COI, 16S rRNA and 18S rRNA gene sequence was 3507 bp, including 2345 conservative sites, 1144 variable sites and 901 parsimony-informative sites. The average A+T content was 63.5% and 78.1% in the COI, 16S rRNA sequences, respectively, indicating A+T bias. The average genetic distance between all species was 0.134, and the average genetic distance in the inner group (Scelimeninae) was 0.126. A phylogenetic tree based on the combined sequences of the COI, 16S rRNA and 18S rRNA genes showed that the phylogenetic relationships among 9 Scelimeninae genera were as follows: Criotettix + (((Zhengitettix + Hebarditettix) + (Falconius + (Scelimena + Paragavialidium))) + ((Eucriotettix + Thoradonta) + Loxilobus)). The molecular phylogenetic results generally support the morphological taxonomy; at the genus level, Criotettix, Scelimena, Paragavialidium, Thoradonta and Eucriotettix are monophyletic groups, Scelimena and Paragavialidium form sister groups, and Thoradonta and Eucriotettix also form sister groups, but the relationship between Hebarditettix and Zhengitettix needs further study. At the species level, synonyms may exist between Thoradonta spiculoba and Thoradonta transpicula and Thoradonta nodulosa and Thoradonta obtusilobata, but more studies are required to confirm this inference.

Complete mitochondrial genomes of Teinopalpus imperialis (Lepidoptera: Papilionidae) and phylogenetic relationships analyses.

In this study, we sequenced the complete mitochondrial genome of Teinopalpus imperialis, which is a national butterfly of India, and a grade-II protected species in China. The complete mtDNA from T. imperialis was 15 299 base pairs in length and contained 13 protein-coding genes (PCGs), 22 tRNA genes, two rRNA genes, and 401 bp non-coding region. The T. imperialis genes were highly similar to those of sequenced mitogenomes of other lepidopteran species in the order and orientation. Twelve PCGs (ND2, ATP8, ND3, COII, ATP6, COIII, ND4, ND4L, CytB, ND1, ND5, and ND6) start with a typical ATN codon, only the COI gene starts with CGA codon. Eight PCGs (ND2, COI, ATP8, ATP6, COIII, ND5, ND6, and Cyt B) terminate in the common stop codon TAA, three PCGs (ND4L, ND3, and ND1) terminate in the stop codon TAG, and two PCGs (COII and ND4) terminate in a single T residue. The phylogenetic relationships were reconstructed with the concatenated sequences of the 13 PCGs of the mitochondrial genome, and phylogenetic results showed that Danaidae, Satyridae, Libytheidae, Nymphalidae, Acraeidae, Pieridae, Hesperiidae, Riodinidae, and Lycaenidae are monophyletic clades.

Complete mitochondrial genome of the Teinopalpus aureus guangxiensis (Lepidoptera: Papilionidae) and related phylogenetic analyses.

In this study, we sequenced the complete mitochondrial genome of Teinopalpus aureus guangxiensis (Lepidoptera: Papilionidae), which is considered as an endemic species in China. It is listed as a vulnerable species by International Union for Conservation of Nature and Natural Resources Red List and also a first class endangered species in China. The complete mtDNA from T. aureus guangxiensis was 15,235 base pairs in length and contained 13 protein-coding genes (PCGs), 2 rRNA genes, 22 tRNA genes, and a control region. The T. aureus guangxiensis genes were in the same order and orientation as the completely sequenced mitogenomes of other lepidopteran species. All PCGs of T. aureus guangxiensis mitogenome start with a typical ATN codon and terminate in the common stop codon TAA, except that ND1 gene uses TTA, ND3 gene uses ATT, and ND4 and ND4L gene use TAA. The phylogenetic relationships were reconstructed with the concatenated sequences of the 13 PCGs of the mitochondrial genome, and phylogenetic results confirmed that Nymphalidae, Lycaenidae, Papilionidae, Pieridae are monophyletic clades.