Phylogenetic insights based on the first complete mitochondrial genome of Isomyia nebulosa (Diptera: Calliphoridae)

Abstract To investigate the phylogenetic position of Isomyia Walker, 1860, a genus that suffered from frequent revisions of the taxonomic status following the subfamily Rhiniinae (Diptera, Calliphoridae), we sequenced, assembled, annotated, and analyzed the first complete mitochondrial genome (mitogenome) of Isomyia nebulosa (Townsend, 1917) in this study. This mitogenome is 16,438 bp in length, with a standard set of 13 protein-coding genes (PCGs), 22 tRNAs, two rRNAs, and an A + T riched non-coding region without genetic rearrangement as most dipteran mitogenomes, but long intergenic nucleotides (IGNs) between trnQ and trnM are found. The phylogeny yielded by both Bayesian inference and maximum-likelihood analysis for all mitochondrial PCGs and rRNAs of 23 mitogenomes supports the monophyly of the family Calliphoridae and the subfamilies Calliphorinae, Chrysomyinae, and Luciliinae. In addition, I. nebulosa diverged anterior to the above-mentioned three calliphorid subfamilies with high genetic distances.


Introduction
Due to the metallic appearance and sarcosaprophagy, the members of the genus Isomyia Walker, 1860 are particularly similar to those common blow flies in the family Calliphoridae and are considered to have medical importance and forensic potential (Yan et al. 2021).The Isomyia has long been classified as the largest genus in the subfamily Rhiniinae of the Calliphoridae (Heo et al. 2013;Bunchu et al. 2014;Thomas-Cabianca et al. 2023).Benefiting from the rapid development of molecular biology and sequencing technologies, the phylogenetic framework within the superfamily Oestroidea has caused widespread controversy in recent years, leading to frequent revisions of the taxonomic status of the Rhiniinae (Marinho et al. 2012;Singh and Wells 2013;Yan et al. 2021).However, the Isomyia is currently inclined to be regarded as a genus of unassigned subfamily in the Calliphoridae in taxonomy (B� anki et al. 2023), on account of the lack of sampling of Isomyia species in related molecular phylogenetic studies.
In this study, we sampled Isomyia nebulosa (Townsend, 1917), a common Isomyia species distributed in South Asia (southwestern China, India, and Myanmar), obtained its complete mitochondrial genome (mitogenome) sequence (NCBI accession number OR497843), and performed a phylogenetic analysis for exploring its phylogenetic position and the calliphorid monophyly using available mitogenomes.

Materials and methods
We collected three female adults (Figure 1) around a snake carcass in Cangyuan, Lincang, Yunnan, China (23 � 54 0 28ʺN, 99 � 13 0 52ʺE, this location was open and no permission was required for insect collection) in May 2016.Jia Huang identified this species using systematic morphological identification keys of the Calliphoridae (Fan et al. 1997).I. nebulosa is characterized by the dark pattern on the distal part of wing (Figure 1) in the dotata species group Peris, 1952.All of these specimens were deposited at the Department of Entomology (Jia Huang, hj@scau.edu.cn),South China Agricultural University (SCAU), Guangzhou, China under the voucher number (H000041-43).The total DNA of one of the specimens was extracted from its thoracic muscles using the HiPure Tissue DNA Mini Kit (#D3121, Magen Biotech., Guangzhou, China) following the manufacturer's protocol.Beijing Geneplus Technology Co., Ltd.(China) performed DNA Nano Ball (DNB) library construction and quality control for the provided DNA sample.The main steps of the DNB library construction included denaturation, single-strand circularization, exonuclease digestion, and DNB amplification.
The mitogenome of I. nebulosa was assembled from the raw sequencing data (NCBI Short Read Archive, SRA accession number SRR25915118) using Geneious Prime v2023.0.4 and annotated following the study of Zhang et al. (2023) except for using the mitogenome of Polleniopsis mongolica S� eguy, 1928 (MT017721) (Shang et al. 2022) as a reference.We depicted the circular mitogenome using the Proksee server (Stothard et al. 2019).Its nucleotide composition was calculated in MEGA v7.0.26 (Kumar et al. 2016).
In this study, we employed the other 22 complete or almost complete mitogenomes for subsequent phylogenetic analyses (Table 1), including 18 Calliphoridae species, two Sarcophagidae species (as outgroup taxa in the superfamily Oestroidea), and two Muscidae species (as outgroup taxa in the Calyptratae).Although some genera (i.e. the Calliphora, Chrysomya, and Lucilia) have more available mitogenomes in NCBI, only two representatives of each of them were selected due to their mitogenomic monophyly has been wellsupported in previous studies (e.g.Shang et al. 2022) and reduced sampling in our analyses will not change the topology of the final trees.The nucleotide sequences of concatenated 13 protein-coding genes (PCGs) and two rRNAs of the 23 mitogenomes were aligned, partitioned, analyzed, and visualized following the methods in Zhang et al. (2023).Briefly, we used PartitionFinder v2.1 (Lanfear et al. 2017) for selecting best-fit partitioning schemes and models, and used MrBayes v3.2.7a (Ronquist et al. 2012) for Bayesian inference (BI) and IQ-TREE v2.2.2.7 (Minh et al. 2020) for maximum likelihood (ML) analysis.

Mitogenomic characteristics
The complete I. nebulosa mitogenome assembled in this study is 16,438 bp in length (Figure 2), with 1,173 to 40,076� coverage depth (supplementary material: Figure S1).This is currently the sixth-largest mitogenome among 65 known calliphorid mitogenomes in NCBI.It contains the typical set of 37 genes (13 PCGs, 22 tRNAs, and two rRNAs) and an A þ T riched non-coding region without genetic rearrangement.The lengths of the PCGs, tRNAs, and rRNAs range from 165 (ATP8) to 1,738 bp (ND5), 62 (trnR) to 77 bp (trnV), and 785 (srRNA) to 1,300 bp (lrRNA), respectively.Twelve PCGs share    and ND5).The intergenic nucleotides (IGNs) of I. nebulosa mitogenome spread over 18 pairs of adjacent genes and range in size from 1 to 71 bp with a total length of 263 bp.The longest IGNs have high A þ T content and are located between trnQ and trnM (Figure 2).Similarly, a total of seven overlapping regions ranging from 1 bp to 8 bp with a total length of 20 bp are identified in this mitogenome.The nucleotide composition of its majority strand is computed as A ¼ 40.82%,T ¼ 38.32%, G ¼ 8.46%, and C ¼ 12.40%, as a consequence, with a significant bias toward A and T (79.14%) especially in the non-coding region (87.67%).

Phylogenetic analyses
In this study, we performed both BI and maximum likelihood analysis for the 15-gene dataset of 23 mitogenomes (Table 1).

Discussion and conclusion
In this study, we provided and analyzed the first complete mitogenome of I. nebulosa as a representative for the Rhiniinae.This newly assembled mitogenome contains the typical mitogenomic gene set as most dipteran species (Zhang et al. 2015;Huang and Ma 2018)  study, along with more available mitogenomes of other related taxa, will contribute to a clearer understanding of the phylogenetic framework within the Oestroidea.

Figure 2 .
Figure 2. Graphical map of the complete mitochondrial genome (mitogenome) of I. nebulosa assembled in this study.Arrows indicate the directions of transcription.

Figure 3 .
Figure 3. Phylogenetic tree reconstructed based on all mitochondrial protein-coding genes (PCGs) and rRNAs of 23 mitogenomes by Bayesian inference (BI).numbers around nodes are posterior probabilities (PPs, left) of the BI and ultrafast bootstrap percentages (UBPs, right) inferred from the maximum likelihood (ML) analysis.Asterisks ( � ) indicate full support (PP ¼ 1.00, UBP ¼ 100).the bar indicates the estimated number of substitutions per site.

Table 1 .
Details of the mitogenomes used in the phylogenetic reconstructions.