Complete mitochondrial genome and phylogenetic position of the gall aphid Chaetogeoica ulmidrupa (Hemiptera: Aphididae)

Abstract

We sequenced two complete mitochondrial genomes of Chaetogeoica ulmidrupa, a gall-forming aphid on Pistacia chinensis. Each genome consists of 13 protein-coding genes, 22 tRNA genes, two rRNA genes and a control region, with a strong A+T bias (∼85%). All protein-coding genes initiate with ATN codon and terminate with TAA, except for ND4 and COX1 ending with a single T. Phylogenetic analysis strongly supported that C. ulmidrupa was sister to C. yunlongensis and together they formed a clade with Slavum lentiscoides, all three species of which feed on the primary host plant P. chinensis to form galls.

Introduction

The gall aphid Chaetogeoica ulmidrupa Zhang (Hemiptera: Aphididae), a Fordini species specifically feeding on Pistacia chinensis Bunge (1835), was originally described as new from the aphid specimens stored at the Museum of the Institute of Zoology, Chinese Academy of Sciences (Zhang and Qiao 1998). This species later was cited in the review article on the diversity and host specificity of gall aphids in China (Chen and Qiao 2012). However, no further updates were published on this species to date. Seven species were identified in the genus Chaetogeoica (Aphididae: Fordini) (Favret 2025), and they all choose the Pistacia species as the primary host plant. In recent years, mitogenome has been employed as a powerful tool to analysis the molecular phylogenetics and population genetics (Kim et al. 2024; Wang et al. 2024). To date, only one mitochondrial genome in this genus, leaving the phylogenetic position and genetic distinctness of other members, like C. ulmidrupa, unresolved (Ren et al. 2017). The mitochondrial genes of C. yunlongensis along with those of other Aphididae taxa were used to reconstruct the phylogeny (Liang et al. 2024). In addition, one chromosome-level genome for C. ovagalla was assembled with a size of 289 Mb, containing 14,492 genes (Xu et al. 2024). As gall aphids, they are highly limited in molecular analyses, yet crucial to the study of the genetics, evolution and potential implications for the coevolutionary model with its host plant.

In this study, we sequenced two complete mitochondrial genomes of C. ulmidrupa and analyzed its phylogenetic position. This study provides valuable genetic data and resources information for further research on aphid phylogeny.

Materials and methods

We collected two individuals of Chaetogeocia ulmidrupa with the vouchers Ren_A1094 (Shiyan, Hubei, China; 32.63 N, 110.79E; July 2014) and Ren_A4512 (Baoji, Shaanxi, China; 34.36 N, 107.23E; June 2016). The aphid sample (sample Ren_A4512 as representative) is shown in Figure 1. All samples are stored at School of Life Science, Shanxi University (contact Zhumei Ren at zmren@sxu.edu.cn). The mitochondrial genome sequences of other aphid species were downloaded from GenBank.

Figure 1.

Reference image of Chaetogeoica ulmidrupa with sample ren_4512 as a representative (the picture taken by Yukang Liang in 2016).

DNA extraction was performed using the Tissue Genomic DNA Extraction Kit from TIANGEN Biotech (Beijing) Co, Ltd. The genomic DNA was sent to Shanghai Majorbio Bio-Pharm Technology Co., Ltd. for library construction and sequenced using the shotgun genome skimming method on an Illumina HiSeq 4000 platform (Zimmer and Wen 2012). The raw data, consisting of 2 × 150 bp paired-end reads and respectively yielding 7.0 and 4.6 GB of data, were filtered using Trimmomatic 3.0 to obtain clean reads (Bolger et al. 2014). De novo assembly was conducted using SPAdes software (Bankevich et al. 2012). The scaffold data were imported into Geneious v10.2.4 for further analysis (Kearse et al. 2012). Using Chaetogeocia yunlongensis (Accession No. MF043988) as reference sequences, the two complete mitochondrial genomes of C. ulmidrupa were obtained through sequence comparison and alignment. Finally, the physical map of the complete mitochondrial genomes were generated using the Proksee online platform (Grant et al. 2023). The read coverage plots for the complete mitochondrial genomes of C. ulmidrupa were presented in Figure S1.

Phylogenetic analysis

To infer the phylogenetic position of C. ulmidrupa, we used a total of 92 mitochondrial genomes from the family Aphididae species with the two species Adelges laricis and A. tsugae from the family Adelgidae as outgroups. We separately aligned 13 protein-coding genes (PCGs) and two rRNAs of all the aphid sequences. Then, the concatenated alignment matrix was used to construct the molecular phylogenetic tree using the maximum likelihood (ML) method in IQ-TREE v2.1.4 (Nguyen et al. 2015) with the automated substitution model selected by ModelFinder and branch supports quantified exclusively through 1000 UFBoot2 replicates (Hoang et al. 2018) and 1000 SH-aLRT replicates. The tree was then visualized and formatted using the program FigTree v1.4.4 (Rambaut 2018).

Results

These complete mitochondrial genomes of Chaetogeocia ulmidrupa were 15,549 and 15,682 bp (Accession number PQ613859 and PV031690) in length (Figure 2), respectively, including 13 PCGs, 22 tRNA genes, two rRNA genes and one control region. The nucleotide composition for the sample Ren_A4512 is 46.3% A, 5.4% G, 9.8% C, and 38.4% T, with an A + T bias of 84.7%. Similarly, the composition for the Ren_A1094 is 46.4% A, 5.4% G, 9.7% C, and 38.4% T, with an A + T bias of 84.8%. The cumulative length of the 13 PCGs is 10,922 bp, with each gene length ranging from 150 bp to 1641 bp. The cumulative length of the tRNA genes is 1469 bp for Ren_A4512 and 1463 bp for Ren_A1094. All the tRNAs have a cloverleaf structure, except for tRNA-Ser losing TψC arm. The two rRNA genes, i.e. rrnL and rrnS, are respectively 1266 bp and 777 bp in length. The control region is located between rrnS and tRNA-Ile, is 681 bp for Ren_A4512, with the nucleotide composition of 45.8% A, 42.6% T, 6.9% C, 4.7% G, and a GC content of 11.6%. For Ren_A1094, the control region is 723 bp, with the nucleotide composition of 46.1% A, 43.0% T, 6.5% C, 4.4% G, and a GC content of 10.9%. All the PCGs use ATN as start codon, among which the four genes, i.e. COX3, ND4L, ND6, CYTB, start with ATG, five genes, i.e. ND3, ATP8, COX1, COX2, ND2, start with ATA, and the other four genes, i.e. ND4, ND1, ND5, ATP6, start with ATT. All the PCGs terminate with TAA except for COX1 and ND4 with a single T.

Figure 2.

Graphical map of the complete mitochondrial genomes of Chaetogeoica ulmidrupa.

The ML phylogenetic tree (Figure 3) supported the monophyly of Hormaphidinae and Aphidinae, while Eriosomatinae and Calaphidinae showed paraphyly. The subfamily Hormaphidinae is located at the base of the tree, and the tribe Fordini (subfamily Eriosomatinae), to which the species Chaetogeoica ulmidrupa belongs, is sister to the other 13 subfamilies in the family Aphididae. Additionally, our current species C. ulmidrupa is sister to C. yunlongensis, and clusters with Slavum lentiscoides Mordvilko. These three species all feed on the primary host plant Pistacia chinensis and form galls. They are most closely related to the gall aphids that live on the genus Rhus, which include six genera and ten species, marked as yellow in Figure 3.

Figure 3.

Maximum likelihood phylogenetic tree of aphididae based on 13 PCGs and two rRNA genes. Nodes are labeled with ultrafast bootstrap support values >75%, and ‘★’ represents nodes with 100% BS. The following sequences were used: Hormaphis betulae NC_029495 (Li et al. 2017), Hamamelistes spinosus MT010853 (Lu et al. 2020), Schizoneuraphis gallarum MT822308 (Zhang et al. 2021), Neothoracaphis yanonis KP722574 (unpublished), Pseudoregma bambucicola MN820984 (Nong et al. 2020), (Ceratovacuna keduensis OL069341, Pseudoregma panicola OL069342) (Zhang et al. 2022), Ceratovacuna lanigera KP722586 (unpublished), (Pterocomma pilosum KC840676, Cavariella salicicola KC332935) (Wang et al. 2013), (Acyrthosiphon pisum FJ411411, Acyrthosiphon caraganae MW724715, Macrosiphum albifrons MW659868, Macrosiphum rosae MW724716) (unpublished), Sitobion avenae KJ742384 (Zhang et al. 2016), (Uroleucon erigeronensis MZ695840, Uroleucon sonchi MT533446) (unpublished), Indomegoura indica MK258689 (Hong et al. 2019), (Neotoxoptera formosana MW534268, Myzus persicae KU236024) (unpublished), Diuraphis noxia KF636758 (Zhang et al. 2014), Brevicoryne brassicae MT900510 (Li et al. 2021), Lipaphis pseudobrassicae NC_072150 (unpublished), Rhopalosiphum padi KT447631 (Song et al. 2016), Rhopalosiphum rufiabdominalisMN876840 (unpublished), Schizaphis graminum NC_006158 (Thao et al. 2004), (Rhopalosiphum maidis NC_081990, Rhopalosiphum nymphaeae MZ420705) (unpublished), (Hyalopterus pruni OK641614, Hyalopterus arundiniformis OK274075, Hyalopterus amygdali OK641613) (Zhang et al. 2024), Aphis gossypii NC_024581 (Zhang et al. 2016), Aphis glycines MK111111 (Song et al. 2019), (Aphis craccivora NC_031387, Aphis solanella OM894973, Aphis fabae mordvilkoi MG897128) (unpublished), Aphis spiraecola NC_053819 (Du et al. 2019), Aphis aurantii NC_052865 (Pu et al. 2020), Aphis coreopsidis OM894972 (unpublished), Aphis citricidus MK540501 (Wei et al. 2019), Melanaphis sacchari MW811104 (unpublished), Nippolachnus piri OL069343 (Zhang et al. 2022), Tuberolachnus salignus OK642815 (unpublished), Stomaphis sinisalicis MW006542 (Zhang et al. 2021), (Cinara tujafilina KP722583, Kurisakia onigurumii KP722578, Phloeomyzus passerinii signoret KP722571, Aiceona himalaica KP722590, Laingia psammae KX822747, Chaetosiphella stipae KX822748, Periphyllus diacerivorus MZ665537, Periphyllus koelreuteriae KP722572, Chaitophorus saliniger KP722584, Mindarus keteleerifoliae KP722576, Appendiseta robiniae MH643884, Takecallis arundinariae KP722568) (unpublished), Therioaphis tenera MH643885 (Voronova et al. 2019), Therioaphis trifolii MK766411 (Liu et al. 2019), Saltusaphis sp. KP722569, Thripsaphis sp. KP722567, Macropodaphis sp. KP722577, Phyllaphis fagi KP722570, (unpublished), Greenidea ficicola MN704283 (Liu et al. 2019), Greenidea psidii MH844624 (Chen et al. 2019), Greenidea kuwanai KP722580 (unpublished), Eutrichosiphum pasaniae MT883997 (Li et al. 2020), Mollitrichosiphum tenuicorpus MW123009 (Li et al. 2021), Schoutedenia ralumensis MT381994 (Chen et al. 2020), Cervaphis quercus KF254841 (Wang et al. 2014), Eriosoma lanigerum KP722582 (unpublished), Paracolopha morrisoni MN167467 (Lee et al. 2019), (Eucallipterus tiliae KP722581, Anoecia fulviabdominalis KP722588, Slavum lentiscoides OK323378, Chaetogeoica yunlongensis MF043988, Kaburagia rhusicola ovogallis MF043986, Kaburagia rhusicola rhusicola MF043987, Kaburagia rhusicola ensigallis MF043984, Kaburagia rhusicola ovatirhusicola MF043985, Meitanaphis flavogallis MF043982, Meitanaphis elongallis MF043989) (unpublished), Meitanaphis microgallis MK948431 (Liang et al. 2019), (Floraphis meitanensis MF043980, Floraphis meitanensis MF043990, Schlechtendalia peitan MF043979) (unpublished), Schlechtendalia chinensis KX852297 (Ren et al. 2016), Melaphis rhois KY624581 (Ren and Wen 2017), Nurudea shiraii MF043978 (Ren et al. 2017), Nurudea yanoniella MK435595 (Ren et al. 2019), (Nurudea yanoniella MF043983, Adelges tsugae MT263947, Adelges laricis KP722589) (unpublished).

Discussion and conclusion

In this study, we successfully sequenced and annotated two complete mitochondrial genomes of Chaetogeoica ulmidrupa. The gene order and composition are identical to the mitogenome of the same genus species C. yunlongensis, which showed the mitochondrial genomic conservation in the same genus.

Our phylogenetic analysis strongly supported the sister relationship between two C. ulmidrupa samples, which subsequently clustered with C. yunlongensis (BS value of 100%).

The clade consisting of Chaetogeoica + Slavum lentiscoides was sister to another one composed of ten species, which use Rhus species as their host plants. While, the two genus Pistacia and Rhus are sister relationship (Yi et al. 2008). Thus, our current results showed a relative phylogenetic relationship between the group of aphids and their host plants, and further to show the effects of the herbivores to insect evolution, i.e. related insect lineages tend to feed on related plants (Lopez-Vaamonde et al. 2003; Sword et al. 2005). Notably, the control region exhibits a low GC content, which may be associated with its high variability and play a role as a transcription initiation site (Dial et al. 2023).

Our study is the first to publish the mitochondrial genome of C. ulmidrupa, and the second one in the genus Chaetogeoica. Adding more mitochondrial genomes of representative species of the genus Chaetogeoica will help clarify the internal evolutionary relationships within the genus. These findings provide molecular data for studying the evolution of Aphididae, and also shed light on the coevolution of aphids and their host plants.

Funding Statement

This work was supported by the Joint Funds of the National Natural Science Foundation of China (U24A20358), Central Guidance Fund for Local Science and Technology Development Project (YDZJSX2024D012), National Natural Science Foundation of China (31870366).

Ethical approval

The insect specimen was not collected from a natural reserve, so the collection did not require any specific permissions or licenses. The field studies did not involve endangered or protected species. The insect species sequenced is a common Aphididae species in China and is not included in the ‘List of Protected Animals in China’.

Disclosure statement

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

Data availability statement

The genome sequence data that support the findings of this study are available in GenBank of National Center for Biotechnology Information (NCBI, https://www.ncbi.nlm.nih.gov/) under the accession numbers PQ613859 and PV031690. The associated BioProject, SRA, and BioSample numbers are as follows: PRJNA1193212 and PRJNA1208921; SRR31578817 and SRR32023171; SAMN45133405 and SAMN46192941, respectively.