Research Article |
Corresponding author: Dong Zhang ( zhangdong_bjfu@bjfu.edu.cn ) Academic editor: Brian Wiegmann
© 2023 He-Nan Li, Wen-Ya Pei, Ming-Fu Wang, Bang-Qing Chen, Hong-Lin Peng, Rong-Jun Cao, Ming-Teng Zhao, Jun Yang, Xiao-Chen Zhang, Dong Zhang.
This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Citation:
Li H-N, Pei W-Y, Wang M-F, Chen B-Q, Peng H-L, Cao R-J, Zhao M-T, Yang J, Zhang X-C, Zhang D (2023) Mitochondrial genomes provide new insights into the phylogeny and evolution of Anthomyiidae (Insecta: Diptera). Arthropod Systematics & Phylogeny 81: 1051-1062. https://doi.org/10.3897/asp.81.e106356
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Anthomyiidae is a cosmopolitan and diverse family of Calyptratae, and is routinely considered to play key roles in both ecology and agriculture. The higher-level phylogenetic classification of Anthomyiidae has been highly controversial, necessitating further molecular data for precise reconstruction of phylogenetic relationships. In this study, we successfully acquired and annotated 18 new mitogenomes of anthomyiids. Moreover, the mitogenomes of the following genera Eustalomyia, Hyporites, Leucophora, Emmesomyia and Eutrichota are reported for the first time. The 18 mitogenomes are compared with confamilial species to assess genetic variation and to better understand evolutionary relationships within the family Anthomyiidae. In comparisons among 13 mitochondrial protein coding genes (PCG), the calculation of evolutionary rate exhibited nad1 as the fastest evolving gene in Anthomyiidae. Among the anthomyiids investigated, cox2 and nad4 had the lowest genetic distance across the 13 PCGs, suggesting a high degree of conservation for these two genes. Herein, we conducted phylogenetic analyses of the newly sequenced mitogenomes along with 11 known anthomyiids to investigate the interrelationships of Anthomyiidae. Our results indicate that Anthomyiidae is a monophyletic lineage and sister group to Scathophagidae, confirming prior findings based on morphological and molecular analyses. We recovered two subfamilies as monophyletic (Myopininae, Pegomyinae) while Anthomyiinae was polyphyletic. The great species diversity of anthomyiid flies limits the availability of mitogenomes for accurately resolving the phylogeny of Anthomyiidae. Nonetheless, our study provides novel insight into the molecular taxonomy, evolution, and phylogeny of the family Anthomyiidae.
Calyptratae, evolutionary rate, mitogenome, molecular analysis, Muscoidea, phylogenetics
Anthomyiidae (Diptera: Calyptratae, Anthomyiidae) are the second-most speciose family in a grade of flies called the the Muscoidea (
Taxonomy of Anthomyiidae is challenging due to a reliance on male genitalia for most morphological diagnoses. A systematic classification for Anthomyiidae is currently deficient and no comprehensive experiments have been conducted using rigorous cladistic argumentation to systematize this family (
Notwithstanding the economic and ecological significance, only few molecular studies have treated the Anthomyiidae (
Mitochondrial genomes have been shown to supply an increase in molecular information content as compared to individual genes, making them conducive to investigations of phylogeny and evolution across a broad diversity of insects (
To expand the available coverage of anthomyiid mitogenomes for comparison and analysis across various taxonomic levels, we sequenced multiple newly sampled anthomyiid mitogenomes to compare these with publicly available sequences. We used a method of next-generation sequencing of multispecies pooled genomic DNA to acquire mitogenomes for 18 anthomyiids, belonging to three subfamilies: Anthomyiinae (eleven species), Pegomyinae (six species) and Myopininae (one species). Additionally, we constructed phylogenetic relationships using maximum likelihood (ML) and Bayesian inference (BI) methods, to investigate higher-level phylogeny within Anthomyiidae. This approach provides novel insights into the phylogenetics and classification of Anthomyiidae and can be used to support their morphological identification.
All anthomyiids were captured by malaise traps in the Baihua Mountain (39°50′11.04″N, 115°34′41.52″E) and Dalaoling National Natural Reserve (31°4′35.6″N and 110°56′11.6″E), from 2017 to 2019 in China. All experimental materials were preserved in absolute ethanol and cryopreserved at –20°C until further processing in the
Museum of Beijing Forestry University (
Taxonomic information and GenBank accession numbers of mitochondrial genomes used in the study. *Species documented in this study.
Family | Subfamily | Species | Accession No. |
Anthomyiidae | Anthomyiinae | Anthomyia confusanea | OP616801* |
Anthomyia illocata | MW296030 | ||
Anthomyia oculifera | OP616786* | ||
Anthomyia pluvialis | OP616785* | ||
Anthomyia procellaris | MT584110 | ||
Botanophila fugax | MT410801 | ||
Botanophila sp. | OP616795* | ||
Delia antiqua | NC028226 | ||
Delia longitheca | OP616787* | ||
Delia platura | MT483617 | ||
Delia takizawai | OP616791* | ||
Eustalomyia hilaris | OP616792* | ||
Eustalomyia vittipes | OP616796* | ||
Fucellia costalis | MH823369 | ||
Hydrophoria lancifer | OP616790* | ||
Hydrophoria linogrisea | MT483657 | ||
Hylemya vagans | MT410822 | ||
Hylemyza partita | MT584149 | ||
Hyporites sp. | OP616793* | ||
Leucophora shanxiensis | OP616797* | ||
Myopininae | Pegoplata annulata | OP616788* | |
Pegoplata infirma | MT410786 | ||
Pegomyinae | Emmesomyia oriens | OP616789* | |
Eutrichota similis | OP616798* | ||
Pegomya bicolor | MT410802 | ||
Pegomya exilis | OP616794* | ||
Pegomya flaviprecoxa | OP616799* | ||
Pegomya quadrivittata | OP616784* | ||
Pegomya sp. | OP616800* | ||
Outgroups | |||
Calliphoridae | Luciliinae | Lucilia sericata | AJ422212 |
Drosophilidae | Drosophilinae | Drosophila mercatorum | MK575470 |
Fanniidae | Fannia scalaris | MT017706 | |
Muscidae | Muscinae | Musca domestica | NC024855 |
Sarcophagidae | Sarcophaginae | Sarcophaga crassipalpis | NC026667 |
Scathophagidae | Scathophaginae | Scathophaga inquinata | MT483619 |
Scathophaga stercoraria | KM200724 | ||
Tachinidae | Phasiinae | Subclytia rotundiventris | MN199029 |
We used the DNeasy Blood and Tissue kit (Qiagen, Hilden, Germany) according to manufacturer’s protocol for DNA extracted from individual adult flies. Qubit 3.0 was used to quantify the concentration of the DNA samples. To enhance sequencing efficiency and minimize resource waste, hybrid libraries were adopted (
Sequence comparisons were carried out in PhyloSuite software (
The 29 complete mitogenomes from three subfamilies of Anthomyiidae were chosen to construct the phylogenetic tree, including 18 new mitogenomes documented in this study. Eight outgroups were selected to represent seven outgroup families of Diptera, with the placed between Drosophilidae (Drosophila mercatorum) and all other sampled flies. Phylogenetic relationships were inferred from analyses of a dataset of the 13 mitochondrial PCGs. To construct this dataset, each PCG of 37 mitogenomes was individually aligned using MAFFT (
Our newly sequenced mitogenomes of Anthomyiidae show some variation in genome size, ranging from 15,635 bp to 21,098 bp in length. They are compact circular, double-stranded molecules, and are composed of the core 37 genes and a control region. The majority strand (J-strand) encoded 23 genes (9 PCGs, and 14 tRNAs), while the remaining genes were transcribed on the minority strand (N-strand) (4 PCGs, 8 tRNAs, and 2 rRNAs). All newly sequenced Anthomyiidae mitogenomes were conserved in gene order and orientation, consistent with previously published Muscoidea mitogenomes (
Relative synonymous codon usage (RSCU) values of all three subfamilies are illustrated in Figure
The AT content of PCGs from all anthomyiids varies between 76.7% (Anthomyiinae) and 77.7% (Pegomyinae) with a mean value of 77%, whereas the basic composition of all PCGs is homogeneous. Third codon positions possess a substantially higher AT content than first and second codon positions, according to analyses of the average base composition at each codon position (Table
Nucleotide composition of mitochondrial genomes of anthomyiid flies at subfamily level.
Regions | Feature | Anthomyiinae | Myopininae | Pegomyinae |
Whole genome | A+T(%) | 78.6 | 78.4 | 80.5 |
AT-Skew | 0.016 | 0.007 | 0.021 | |
GC-Skew | –0.166 | –0.154 | –0.137 | |
PCGs | A+T(%) | 76.7 | 77.2 | 77.7 |
AT-Skew | –0.152 | –0.157 | –0.154 | |
GC-Skew | 0.028 | 0.028 | 0.038 | |
1st codon | A+T(%) | 70.9 | 71.1 | 71.5 |
AT-Skew | –0.125 | –0.137 | –0.134 | |
GC-Skew | 0.187 | 0.193 | 0.205 | |
2nd codon | A+T(%) | 71.0 | 71.0 | 71.4 |
AT-Skew | –0.305 | –0.303 | –0.306 | |
GC-Skew | –0.135 | –0.141 | –0.134 | |
3rd codon | A+T(%) | 88.3 | 89.5 | 90.2 |
AT-Skew | –0.050 | –0.057 | –0.049 | |
GC-Skew | 0.037 | 0.039 | 0.054 |
Additionally, saturation plots reveal that only the third codon locations in all of the PCGs exhibit notable heterogeneity, implying that levels of heterogeneity in the PCG123 are exceedingly low (Fig.
To explore sequence evolution among the 13 PCGs sampled in anthomyiids, the values of Ka, Ks, and Ka/Ks (ω) for each PCG were computed, respectively (Fig.
Nucleotide diversity among the 13 PCGs is shown in Figure
Overall, we find that the cox1 gene evolves at a considerably higher rate and under comparatively relaxed purifying selection among anthomyiids, manifesting that nad1 gene could be a suitable candidate marker for clarifying the phylogenetic relationships among taxa with morphological traits that are difficult to interpret.
Phylogenetic analyses were carried out on mitogenomes from 29 anthomyiids and eight outgroups, including our 18 newly sequenced Anthomyiidae mitogenomes. Both ML and BI methods were conducted on 13 PCGs and produced completely resolved trees with identical topologies and with most branches receiving strong support. The muscoids were confirmed as a non-monophyletic group or grade, with Scathophagidae plus Anthomyiidae placed as sister to the clade Oestroidea ((Sarcophagidae + Calliphoridae) + Tachinidae), congruent with previous studies (
Anthomyiidae and Scathophagidae are clearly very closely related, with various forms of evidence supporting either a sister-group relationship or placement of the latter family within a more broadly defined Anthomyiidae. They are morphologically similar, with both families possessing a long anal vein, usually reaching wing edge at least as a fold (
In this study, we provide a systematic analysis of 18 mitogenomes representing three subfamilies of Anthomyiidae. This is the first report of mitogenomes from the three genus Eustalomyia, Hyporites and Leucophora of the subfamily Anthomyiinae, and two genus Emmesomyia and Eutrichota of the subfamily Pegomyinae. Our study reveals conserved traits among anthomyiid mitogenomes, including strongly biased A + T richness, a more rapidly evolving nad1 gene and a positive GC skew among the 13 PCGs. Both ML and BI phylogenetic trees using the 13 PCGs yield an identical topology, with most divergences possessing strong bootstrap and posterior probability support. These results provide fundamental information on mitogenome organization and reinforce an increased understanding of phylogenetic relationships within the family Anthomyiidae.
Conceptualization, D.Z.; Methodology, H.L., W.P. and M.W.; Software, H.L. and W.P.; Validation, H.L.; Formal Analysis, H.L. and M.W.; Investigation, B.C., H.P., R.C., M.Z., J.Y. and X.Z.; Resources, B.C., H.P., R.C., M.Z., J.Y. and X.Z.; Data Curation, H.L., W.P. and M.W.; Writing—Original Draft Preparation, H.L. and W.P.; Writing—Review and Editing, H.L.; Visualization, W.P. and M.W.; Supervision, D.Z.; Project Administration, D.Z.; Funding Acquisition, D.Z. All authors have read and agreed to the published version of the manuscript.
The authors declare no conflict of interest.
Prof. Brian Wiegmann and two anonymous reviewers are acknowledged for their contribution in improving this manuscript. This research was funded by the National Natural Science Foundation of China (No. 32170450, 31872964), the Beijing Forestry University Outstanding Young Talent Cultivation Project (No. 2019JQ0318) and the Beijing Forestry University Outstanding Postgraduate Mentoring Team Award.