Characterization of the complete mitochondrial genome and phylogenetic analysis of Coelophora saucia (Mulsant, 1850) (Coleoptera: Coccinellidae)

Abstract

Coelophora saucia is an important natural enemy insect in agricultural production. We determined the complete mitochondrial genome of the C. saucia by high-throughput sequencing, and the mitogenome was 18,068 bp in length, with a GC content of 20.9%, encodes 2 ribosomal RNA genes, 22 transporter RNA genes, 13 protein-coding genes and with a non-coding control region. A phylogenetic tree was constructed using the maximum likelihood (ML) method, and the results indicated that the C. saucia was most closely related to Propylea japonica and Propylea quattuordecimpunctata.

Introduction

Coelophora saucia (Mulsant, 1850) belongs to the family Coccinellidae in Coleoptera. Its body is nearly round and black, with a white circular spot on each side of the pronotum and an orange-red elongated patch in the center of the forewings (Doeurk et al. 2024). C. saucia, mainly preying on aphids, whiteflies and other pests, is an important predatory natural enemy insect in agriculture, widely distributed in China (Dai 1990a). It is highly predatory, with a maximum daily predation of 256 individuals of aphids (Chen et al. 2024a), and plays an important role in agro-ecological regulation. C. saucia has 5–6 generations per year in the Jianghuai Region, overwintering and over-summering as an adult, with overlapping generations often occurring due to the long egg-laying period (Dai 1990b). Despite extensive studies on its life history, reproductive characteristics, and predation potential (Chen et al. 2024b), there remains a need for further characterization of its mitochondrial genome and phylogenetic relationships within the broader context of Coccinellidae.

In this study, we determined the complete mitochondrial genome of C. saucia and clarified its phylogenetic status by constructing a phylogenetic tree using the maximum likelihood method. The results will not only contribute to our understanding of the genetic diversity and evolutionary relationships within the Coccinellidae family, but also provide valuable information for pest management and agroecological conservation.

Materials

The C. saucia specimens used in this study were collected on September 10, 2024 by Jing-Qiang Zhou at the Broccoli Selenium-enriched Ecological Planting Base, Longgui Township, Shaoguan City, Guangdong Province, China (24°44′N 113°25′E). The specimen (Figure 1) and its genomic DNA are stored at the Insect Collections of Hunan Agricultural University, Changsha, China. The material is cataloged under the voucher code HAUHL143796, with contact information provided to Guo-Hua Huang at ghhuang@hunau.edu.cn for further inquiries or assistance.

Figure 1.

The adult of C. saucia. (A) the dorsal view; (B) the ventral view. Photographed by Jing-Qiang zhou.

Methods

DNA was extracted from muscle tissues of C. saucia adult using the SteadyPure Universal Genomic DNA Extraction Kit (Changsha, China). Subsequent to extraction, clustering of the index-coded samples was performed on a cBot Cluster Generation System using NovaSeq 6000 S4 Reagent Kit (Illumina), and 2 × 150 bp short reads were generated through paired-end sequencing via the Illumina NovaSeq 6000 platform. Raw reads underwent filtering using FASTQ (Chen et al. 2018). The complete mitochondrial genome of C. saucia was assembled utilizing NOVOPlasty (Dierckxsens et al. 2017) and GetOrganelle v1.7.6 (Jin et al. 2020), with adjustments made post-assembly. The average sequencing depth of the genome was 4782x, with a minimum depth of 2324x and a maximum depth of 10525x (Figure S1). Annotation of the mitochondrial genome was performed through MITOS (Bernt et al. 2013), employing Geneious (version 5.4.2) (Kearse et al. 2012), Final visualization was achieved using CGview, generating a circular map accessible via [https://proksee.ca/].

For sequence analysis, mitochondrial genomes from 22 published species within the family Coccinellidae and two outgroup species from the family Endomychidae were retrieved from the NCBI database (Table S1). A total of 13 protein-coding genes (PCGs) were manually extracted and concatenated using Geneious Prime (v2024.0.5). Multiple sequence alignment was performed using MAFFT v1.5.0, with model selection performed through ModelFinder v2.2.0 (Kalyaanamoorthy et al. 2017). The optimal molecular evolution model (GTR + F + I + R4) was chosen for the analysis. A phylogenetic tree was constructed employing IQ-TREE v2.4.0 (Minh et al. 2020), and bootstrap values were calculated with 1000 iterations. The tree topology was visualized using FigTree v1.4.4 and iTOL (https://itol.embl.de/) (Ivica and Peer 2024). Species divergence times were estimated using MCMCTree (Yang and Rannala 2006). Calibration points were selected based on the fossil record of Coccinellidae.

Results

Structural organization of the Coelophora saucia mitogenome

The complete mitochondrial genome of C. saucia (GenBank accession: PQ668616) comprises a circular DNA molecule spanning 18,068 bp. This sequence includes 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a single non-coding control region (D-loop) (Table S2). Genomic organization was asymmetric with respect to gene distribution across strands: nine PCGs and fourteen tRNAs were encoded on the heavy strand, while the remaining genes were localized on the light strand (Figure 2).

Figure 2.

Mitogenome pattern map of coelophora saucia. Genes located within the gray circle are coded into the light strand (L-strand), while those outside the black circle are encoded in the heavy strand (H-strand). The Middle black circle denotes the GC content, while the innermost circle signifies the GC skew.

Nucleotide composition and codon usage

The overall nucleotide composition exhibited a bias toward purines (A = 41.6%, T = 37.5%; C = 11.9%, G = 9.0%), resulting in a GC content of 20.9%. Among the PCGs, twelve utilize ATN (N: A/T/C/G) as initiation codons, while COX1 uniquely employs AAG. Termination codon analysis revealed TAA/TAG as stop codons for eight PCGs, whereas five PCGs displaying incomplete termination via single T residues.

Non-coding and structural features

The 22 tRNA genes exhibited variable lengths, ranging from 58 bp (trnR) to 69 bp (trnK). Ribosomal RNA components included a 799 bp 12S rRNA and a 1325 bp 16S rRNA. Intergenic spacers spanned approximately 1–1290 bp between 12 adjacent genes, with one pair of genes exhibiting overlapping regions (1–38 bp), totaling 60 bp of sequence overlap. A non-coding control region (D-loop) measuring 2186 bp was located between the rRNAs and tRNAs.

Phylogenetic relationships and divergence

The molecular phylogenetic tree in Figure 3 was constructed using concatenated sequences of 13 PCGs from C. saucia. The results reveal that C. saucia is more closely evolutionarily related to the Propylea spp. forming a distinct clade with strong phylogenetic support (genetic distance of 0.066, bootstrap value = 100%). These findings are consistent with previous study by Zhou et al. (2019), which further validate the close evolutionary relationships between C. saucia and Propylea spp. This topology highlights the close phylogenetic affinity between the three species, as evidenced by Bayesian divergence time estimates that indicate a shared most recent common ancestor with the Propylea lineage (posterior probability = 1.0).

Figure 3.

Maximum likelihood (ML) tree of 23 species within the tribe coccinellidae based on 13 PCGs of the mitogenome with 2 endomychidae species as outgroups. The numerical values positioned above the branches denote genetic distances, whereas the values displayed below correspond to bootstrap support percentages.

The high level of genetic similarity between C. saucia and P. species (average nucleotide identity: 98.2% across PCGs) further corroborates their sister-group relationship. This clade is distinct from other members of the Coccinellidae tribe, with branch lengths suggesting a divergence event approximately 18.01 million years ago, this divergence time estimation is calibrated based fossil records within Coccinellidae, including Coccinella spp. (14.12 Ma) Propylea japonica (7.15 Ma), Vibidia duodecimoguttata (19.20 Ma), and Illeis koebelei (1.63 Ma) (Huang et al. 2025). These findings resolve uncertainties in the systematic position of C. saucia and extend our understanding of its evolutionary relationships within the broader context of Coccinellidae.

Discussion and conclusion

The present study reports the first complete mitochondrial genome sequence of C. saucia. Unlike previous studies by Zhou et al. (2019), which identified several mitochondrial genes, this study refined the sequence to include a complete mtDNA genome, thereby providing a more accurate representation of the species’ genetic makeup.

The Maximum Likelihood phylogenetic tree robustly placed C. saucia as an outgroup to the genus Propylea. This clear topological structure indicates that C. saucia is phylogenetically distinct from the core Propylea species, adds nuance to the previous classification proposed by Zhu et al. (2022). Based on comparisons of gene content, AT content, and gene order with other members of the tribe (Coccinellidae), the newly sequenced C. saucia mitochondrial genome shows remarkable similarity to P. quattuordecimpunctata (MF992931) and P. japonica (KM244660). Additionally, it shares several conserved features with other members of the tribe, such as the majority of mitochondrial protein coding genes starting in tRNA regions (Ge et al. 2021). These findings highlight the need for taxonomic review of the tribe, contributing to a better understanding of its evolutionary history. To conclusively resolve the taxonomic status of C. saucia, future studies should incorporate a broader sampling of related genera and employ phylogenomic approaches.

Funding Statement

This study was also supported by the China Agriculture Research System [CARS-23-C08].

Ethics statement

The focal species used in this study is not under protection of CITES or wildlife laws in China, its status is not assessed by IUCN. The collecting and handling of the specimen was conducted in accordance with the ‘Regulation on Experimental Animals of Hunan Province’.

Author contribution statement

CRediT: Jing-Qiang Zhou: Conceptualization, Data curation, Formal analysis, Investigation, Writing – original draft, Writing – review & editing; Yi Li: Conceptualization, Data curation, Formal analysis, Writing – original draft, Writing – review & editing; Xue-Fei Cai: Conceptualization, Writing – original draft, Writing – review & editing; Feng Li: Conceptualization, Data curation, Formal analysis, Supervision, Writing – review & editing; Guo-Hua Huang: Conceptualization, Data curation, Formal analysis, Investigation, Resources, Writing – original draft, Writing – review & editing.

Disclosure statement

The authors declare that they have no known competing financial interests. The second author, Yi Li, and the fourth author, Feng Li, are affiliated with the Amway (China) Botanical R&D Center. The third author, Cai XueFei, is affiliated with Amway (Shanghai) Innovation & Science Co., Ltd. All authors declare that they have no financial, commercial, legal, or non-financial conflicts of interest pertaining to the research content of this article. We hereby confirm that no potential conflicts of interest exist among the authors that could influence the research outcomes presented in this article.

Data availability statement

The genome sequence data that support the findings of this study are openly available in GenBank of NCBI under the accession number PQ668616. The associated BioProject, SRA, and BioSample numbers are PRJNA1187021, SRR31442579, and SAMN44780754, respectively.