The complete mitochondrial genome and phylogenetic analysis of Illiberis pruni Dyar, 1905 (Lepidoptera: Zygaenidae)

Abstract Illiberis pruni is a leaf-eating pest that infests pear trees across all pear-producing regions of China. The present study, aimed to sequence the I. pruni mitochondrial genome (GenBank accession no. MZ726799) using the Illumina NovaSe Sequencing System to understand the population genetics, evolution, and taxonomy of I. pruni and other related species. The circular I. pruni mitochondrial genome was found to be 15,252 bps in length and comprised 38 sequence elements including 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a putative control region (CR). Phylogenetic analysis revealed that I. pruni and Illiberis ulmivora are closely related, thereby indicating that their mitochondrial genes may share common ancestry.

Illiberis pruni; mitochondrial genome; sequence; phylogenetic analysis Illiberis pruni classified under Zygaenidae, Lepidoptera, and commonly known as dumpling insect, is the primary leaf-eating pest of pear trees. The worm is endemic across all pearproducing regions of China, and mainly infests pear, peach, apple, and begonia trees among other fruit trees (Yan et al. 2020). The overwintering larvae feed on newly emerged flower and leaf buds (Li 2013). Consequently, the damaged flower buds mostly lose the ability to bloom, and retain brown wounds and holes. In summer, the newly hatched larvae feed on mesophyll. I. pruni infestations occur twice a year, the first round being more severe than the second. The former often results in drastic defoliation and fruit loss, thereby seriously impacting yield and tree potential. Scientists have proposed a number of preventive and control measures to combat damage caused by I. pruni infestations. For instance, natural enemies have been introduced in an attempt to suppress the population (Song 2014). Further, old bark is brushed, burned, or buried deep in autumn and winter to reduce resources available to overwintering insects (Wang 2017). The genetic structure and phylogenetic status of I. pruni have not been reported yet. This study, for the first time, reports the complete mitochondrial genome sequence of I. pruni and compares it with that of other insects to aid comprehension of the population genetics, evolution, and taxonomy of I. pruni and its related species.
The complete I. pruni (GenBank accession number: MZ726799) mitochondrial genome was of typical structure and 15,252 bps in length. In the context of nucleotide composition, it comprised 39.94% adenosine (A), 42.32% thymine (T), 7.41% guanine (G), and 10.33% cytosine (C). The A:T ratio of 82.26% was found to be significantly higher than that of the G:C ratio of 17.74%. The AT-skews and GC-skews calculated for the major strands of the mitogenome were approximately À0.029 and À0.165, respectively. There were 38 sequence elements in total that comprised 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a putative control region (CR). The 13 PCGs of I. pruni possessed the ATN (Met) start codon that is typical of invertebrate mitochondrial PCGs, except for cox1 that utilized a CGA initiation codon. Three of these genes (nad1, nad2, and atp8) used ATT, another four genes (cox2, nad5, nad6, and cob) used ATA, and the remaining five genes (atp6, cox3, nad3, nad4, and nad4l) had ATG. Of the 13 PCGs, 10 terminated with the typical TAN stop codon with nine genes (nad1, nad2, atp6, atp8, cox3, nad3, cob, nad4l, and nad6) using a TAA stop codon, while a single gene (nad4) used the TAT stop codon. Three genes (cox1, cox2, and nad5) ended with the incomplete codon T, which was completed by the addition 30 bp poly-A tail to the mRNA. All 22 tRNA genes that were scattered within the coding region, ranged from 63 to 74 bps in length, and could be folded into the typical clover secondary structure. The two rRNA genes of LrRNA and SrRNA genes were 1342 and 774 bps in length, respectively.
MEGA7 software was used to construct a phylogenetic tree and to analyze phylogenetic relationships between I. pruni and other insect species (Kumar et al. 2016). In addition, it was constructed using the maximum likelihood method, which was based on the 1,000 bootstraps, Jones-Taylor-Thornton model, uniform rates, partial deletion, nearest neighbor interchange heuristic method, and default automatic NJ/BioN. Figure 1 depicts the phylogenetic relationship between I. pruni and 19 other species. These included 4 members of Zygaenidae including I. ulmivora, Eterusia aedea, Amesia sanguiflua, and Histia rhodope; 3 species of Parnassiidae including Parnassius nomion, and Parnassius bremeri; 2 species of Gelechiidae including Phthorimaea operculella and Pectinophora gossypiella; 2 species of Nymphalidae including Limenitis elwesi and Junonia almana; 6 species of others Lepidoptera including Cnaphalocrocis exigua (Pyralidae), Ampelophaga rubiginosa (Sphingidae), Omiodes indicata (Crambidae), Thyatira batis (Thyatiridae), Grapholita delineana (Tortricidae), and Euschemon rafflesia (Euschemonidae). Two species of Diptera as outgroups, including Haematopota vexativa (Tabanidae) and Pseudolimnophila brunneinota (Limoniidae). All species were found to be clearly distinct from each other, with high bootstrap values, and the outgroups were grouped into small independent clades. We found that I. pruni and I. ulmivora formed a monophyletic clade with a bootstrapping rate of 100%, indicating that they are closely related, and that their mitochondrial genes may share a common ancestry. Illiberis pruni and H. rhodope belong to the same family, but the genomic sequence of H. rhodope has low similarity with that of I. pruni; thus, it is slightly distantly related to I. pruni. The results of this study will increase our understanding of, and drive further research in, insect population genetics.

Acknowledgment
No funding was received.

Ethical approval
The study not involving humans or animals. This study don't need ethical approval or permissions to collect the sample, which is a pest and we need proper control.

Author contributions
Yuantao Zhou, Yanbin Nan, Jingwen Peng and Kexing Cheng were involved in the conception and design, analysis and interpretation of the data; Jiahui Guo, Zhiao Pei and mingyan Ma collectted insects; Yanbin Nan draftted the paper, Yuantao Zhou revised it critically for intellectual content.Yuantao Zhou and Yanbin Nan final approval of the version to be published. All authors agree to be accountable for all aspects of the work.

Disclosure statement
No potential conflict of interest was reported by the author(s).

Funding
The author(s) reported there is no funding associated with the work featured in this article.