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Research Article
The first two complete mitochondrial genomes for the genus Anagyrus (Hymenoptera, Encyrtidae) and their phylogenetic implications
expand article infoCheng-Hui Zhang‡, Hai-Yang Wang‡, Yan Wang‡, Zhi-Hao Chi‡, Yue-Shuo Liu‡, Guo-Hao Zu‡
‡ Tianjin Agricultural University, Tianjin, China

Corresponding author: Guo-Hao Zu ( zuguohao@tjau.edu.cn )

Academic editor: Zachary Lahey
© 2024 Cheng-Hui Zhang, Hai-Yang Wang, Yan Wang, Zhi-Hao Chi, Yue-Shuo Liu, Guo-Hao Zu.
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: Zhang C-H, Wang H-Y, Wang Y, Chi Z-H, Liu Y-S, Zu G-H (2024) The first two complete mitochondrial genomes for the genus Anagyrus (Hymenoptera, Encyrtidae) and their phylogenetic implications. ZooKeys 1206: 81-98. https://doi.org/10.3897/zookeys.1206.121923
ZooBank: urn:lsid:zoobank.org:pub:C6737E11-5992-4B82-B0C1-48C6C32DF550
Open Access

Abstract

Anagyrus, a genus of Encyrtidae (Hymenoptera, Chalcidoidea), represents a successful group of parasitoid insects that attack various mealybug pests of agricultural and forestry plants. Until now, only 20 complete mitochondrial genomes have been sequenced, including those in this study. To enrich the diversity of mitochondrial genomes in Encyrtidae and to gain insights into their phylogenetic relationships, the mitochondrial genomes of two species of Anagyrus were sequenced, and the mitochondrial genomes of these species were compared and analyzed. Encyrtid mitochondrial genomes exhibit similarities in nucleotide composition, gene organization, and control region patterns. Comparative analysis of protein-coding genes revealed varying molecular evolutionary rates among different genes, with six genes (ATP8, ND2, ND4L, ND6, ND4 and ND5) showing higher rates than others. A phylogenetic analysis based on mitochondrial genome sequences supports the monophyly of Encyrtidae; however, the two subfamilies, Encyrtinae and Tetracneminae, are non-monophyletic. This study provides valuable insights into the phylogenetic relationships within the Encyrtidae and underscores the utility of mitochondrial genomes in the systematics of this family.

Key words

Encyrtid, genome structure, mitogenome, protein-coding genes, phylogenetic analyses, Tetracneminae

Introduction

Encyrtidae is a large hymenopteran family in the superfamily Chalcidoidea, comprising 518 known genera, of which 495 are recognized as valid (totaling more than 4830 species), along with 23 fossil genera (26 species) worldwide (Simutnik et al. 2022; Simutnik et al. 2023; Simutnik and Perkovsky 2023; Wang et al. 2023). The genus Anagyrus Howard, 1986 is one of the largest genera in Encyrtidae, comprising 289 valid species (Noyes 2019). This genus was established by Howard and Ashmead (1896) based on the type species, Anagyrus greeni Howard, 1896. Diagnostics for the genus include a broadened, flattened scape (normally 2–3× as long as broad), funicle segments longer than broad, occipital margin normally quite sharp but often rounded, postmarginal vein normally not longer than the stigma vein, and ovipositor at least half the length of the mid tibia (Noyes 1980; Noyes and Hayat 1994). Anagyrus species are primary parasitoids of Pseudococcidae; for example, Anagyrus galinae has been utilized in classical biocontrol and integrated pest management of Trionymus copiosus (Japoshvili and Hansen 2015; Noyes 2019).

Insect mitochondrial genomes are usually small, circular molecules containing 37 genes: 13 protein-coding genes (PCGs), two ribosomal RNA genes (rRNAs), and 22 transfer RNA genes (tRNAs), as well as a large non-coding element known as the A+T-rich or control region (CR), which regulates transcription and replication (Wolstenholme 1992a, 1992b; Boore 1999; Cameron 2014). Due to their distinct characteristics, including gene-content conservation, maternal inheritance, and rapid evolutionary rate, mitogenome sequences serve as valuable molecular markers for various evolutionary studies (Boore 1999; Krzywinski et al. 2006). Although the mitochondrial genome of Chalcidoidea exhibits structural resemblance to other insects, significant rearrangements characterize it, along with a relatively high A+T content in its sequence composition, deviating from the presumed ancestral pattern (Brown et al. 1979; Cameron and Whiting 2008).

The exploration of hymenopteran mitochondrial genomes commenced with the sequencing of CYTB and ATP8 genes of Apis mellifera, and it was not until 1993 that the first complete mitochondrial genome was deciphered (Crozier and Crozier 1992, 1993). The first comprehensive phylogenetic analysis of Chalcidoidea based on molecular data was conducted using 18S and 28S rDNA (Munro et al. 2011). Subsequently, Heraty et al. (2013) conducted an in-depth exploration of the phylogenetic relationships within Chalcidoidea based on both morphological and molecular data. Zhang et al. (2020a) further reconstructed the phylogenetic relationships within Chalcidoidea using transcriptome data, providing valuable insights for achieving more accurate phylogenetic relationships. Recently, Cruaud et al. (2024) conducted a comprehensive phylogenetic study using data from PCGs and ultra-conserved elements (UCEs), while Zhu et al. (2023) conducted a comprehensive phylogenetic study using 139 mitochondrial genomes from the main clades of Chalcidoidea. These studies have significantly advanced our understanding of the phylogenetic relationships within Chalcidoidea. However, to obtain a more accurate reconstruction of evolutionary relationships, it is necessary to expand the sampling range to include more understudied species. This approach will help construct a more comprehensive and precise phylogenetic tree, revealing deeper levels of phylogenetic relationships. Additionally, integrating different types of data, such as rDNA genes, mitochondrial genomes, and UCEs, is crucial. By comprehensively utilizing morphological, biological, and molecular data and conducting multidimensional phylogenetic analyses, we can improve the accuracy of classification and phylogenetic research. Such integrative approaches will provide a more robust framework for understanding the evolutionary relationships within Hymenoptera.

At present, there are only morphology-based classification systems for Encyrtidae (Noyes and Hayat 1984, 1994; Trjapitzin 1989), lacking auxiliary verification from molecular data, particularly from the mitochondrial genome (mitogenome). Consequently, the monophyly and phylogenetic relationships of Encyrtidae have been controversial for a long time. Problems that are difficult to distinguish in taxonomy indicate the requirement for using various molecular data to understand the systematic position and the monophyly of Encyrtidae in Chalcidoidea. Mitogenome data seem sufficient to solve these problems (Wei et al. 2010; Li et al. 2016; Liu et al. 2023). There are currently only 1291 complete mitochondrial genomes of Hymenoptera on GenBank, and the number of encyrtid genomes is small (Sayers et al. 2024). This limited data negatively impacts our ability to resolve potential systematic ambiguity within Encyrtidae.

In this study, we conducted the sequencing and annotation of the mitogenomes of Anagyrus galinae (accession number: OR652687) and Anagyrus jenniferae (accession number: OR790122), analyzing their respective characteristics. In addition, we reconstructed the molecular phylogenetic relationships of these two new mitochondrial genomes and other species of Encyrtidae. The molecular data presented in this study will contribute to a better understanding of the characteristics of the Encyrtidae mitogenome. Further, a phylogenetic analysis was performed, including 19 uploaded mitogenomes together with our newly acquired data, which represented Encyrtidae. The goal of our study was to place two new species of Anagyrus within the context of the known mitogenome diversity of Encyrtidae by performing mitogenomic and phylogenetic analyses.

Materials and methods

Sample collection, DNA extraction and sequencing

The specimens, A. galinae and A. jenniferae, were collected from Tianjin Agricultural University (39°5′21″N, 117°5′38″E), Xiqing District, Tianjin City, China, in September 2022. Freshly collected specimens were promptly immersed in 100% ethanol for initial preservation and subsequently stored at −40 °C in the Insect Herbarium of Tianjin Agricultural University. Following morphological identification, total DNA from each specimen was extracted from the body, excluding the abdomen, using the DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purity and concentration of the extracted total DNA were assessed through 1% agarose gel electrophoresis and optical density value detection. The total DNA of two encyrtids underwent sequencing using the Illumina NovaSeq 6000 platform with a 350 bp insert size and a paired-end 150 bp sequencing strategy. Sequencing was conducted by Novogene Co., Ltd. (Beijing, China).

Mitogenome assembly, annotation and analysis

After initial data acquisition, with adapter sequences removed, additional filtering was carried out using fastp 0.23.4 (Chen et al. 2018) to filter low-quality reads (quality value <30), ensuring that each sample retained clean data of no less than 4 Gb. The software MitoZ v. 3.6 (Meng et al. 2019) and GetOrganelle v. 1.7.7.0 (Jin et al. 2020) were used for the de novo assembly of mitogenomes. Homologous sequences of other Encyrtidae species from GenBank were used for comparison, and the mitogenomes were annotated using the Mitos WebServer (Donath et al. 2019). The secondary structures of tRNAs were predicted using Mitos WebServer and further visualized using VARNA v. 3.9 (Darty et al. 2009). The structures of the mitochondrial genome were mapped using the online tool CGview Server. The nucleotide composition and relative synonymous codon usage (RSCU) of protein-coding genes were calculated and analyzed by MEGA v. 11.0.13 (Tamura et al. 2021). The skew analysis of nucleotide composition was calculated using the formulas: AT-skew = (A−T)/(A+T) and GC-skew = (G−C)/(G+C), where A, T, G and C were the base contents of the same chain (Perna and Kocher 1995; Hassanin et al. 2005). The nonsynonymous mutation rate (Ka) and synonymous mutation rate (Ks) of protein coding genes were calculated using DnaSP 6.12.03 (Rozas et al. 2017). Tandem repeats in the CR were identified by Tandem Repeats Finder (Benson 1999).

Molecular phylogenetic analyses

A total of 21 mitogenomes from two families of Chalcidoidea, including 20 Encyrtidae species and a Aphelinidae species as outgroup, were used for the phylogenetic analysis (Table 1). The phylogenetic trees were reconstructed using both maximum-likelihood (ML) and Bayesian-inference (BI) methods. For this, each PCG was individually aligned using the MAFFT 7 online service with the L-INS-i strategy, followed by optimization using MACSE (Ranwez et al. 2018; Katoh et al. 2019). The individual PCG alignments were trimmed using GBlocks and concatenated into a PCG dataset using PhyloSuite v. 1.2.3 (Talavera and Castresana 2007; Zhang et al. 2020b). The best nucleotide substitution model was obtained using ModelFinder v. 2.2.0 with Bayesian Information Criterion (BIC) (Kalyaanamoorthy et al. 2017). BI analysis was performed using MrBayes v. 3.2.7a with four chains (Ronquist et al. 2012). Two independent runs of 2,000,000 generations were carried out with sampling every 1,000 generations. The first 25% of trees were discarded as burn-in. After the average standard deviation of split frequencies fell below 0.01 and the potential scale reduction factor (PSRF) approached 1.0, stationarity was assumed. ML analysis was performed using IQ-TREE v. 2.2.0 (Nguyen et al. 2015) under the standard bootstrap approximation approach with 1,000 replicates.

Table 1.
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GenBank accession numbers of species used in phylogenetic reconstruction and their original publications.

Superfamily Family Species Accession Number References
Chalcidoidea Aphelinidae Encarsia formosa MG813797 Zhu et al. 2018
Encyrtidae Aenasius arizonensis NC_045852 Ma et al. 2019
Anagyrus galinae OR652687 This study
Anagyrus jenniferae OR790122 This study
Blastothrix speciosa NC_082111 Unpublished
Cheiloneurus chinensis NC_084192 Unpublished
Cheiloneurus elegans NC_071192 Unpublished
Diaphorencyrtus aligarhensis NC_046058 Du et al. 2019
Encyrtus aurantii OR120384 Unpublished
Encyrtus eulecaniumiae NC_051459 Rudoy et al. 2022
Encyrtus infelix NC_041176 Xiong et al. 2019
Encyrtus rhodococcusiae NC_051460 Rudoy et al. 2022
Encyrtus sasakii NC_051458 Rudoy et al. 2022
Exoristobia philippinensis NC_084171 Unpublished
Lamennaisia ambigua NC_082113 Unpublished
Lamennaisia nobilis NC_061411 Unpublished
Leptomastidea bifasciata OR790123 Unpublished
Metaphycus eriococci NC_056349 Zhou et al. 2021
Ooencyrtus plautus NC_068223 Xing et al. 2022
Psyllaephagus sp. OP787025 Unpublished
Tassonia gloriae NC_082112 Unpublished

Results

Mitogenome organization and nucleotide composition

The assembled mitochondrial genome of A. galinae was a 15,364 bp, and the A. jenniferae mitochondrial genome was 15,396 bp, which both had the same gene organization, including 13 PCGs, 22 tRNAs, two rRNAs and a control region located between trnM and trnI (Fig. 1). For the mitogenomes of two species, the majority strand (J-strand) encodes 10 PCGs (ND3, CO3, ATP6, ATP8, CO2, CO1, ND5, ND4, ND4L, ND1), 15 tRNAs (trnI, trnY, trnS1, trnC, trnR, trnG, trnD, trnL2, trnF, trnH, trnP, trnL1, trnA, trnV, trnM) and 2 rRNAs (lrRNA, srRNA), while the remaining three PCGs (ND2, ND6, CYTB) and seven tRNAs (trnW, trnN, trnK, trnE, trnT, trnS2, trnQ) are located on the minority strand (Table 2). Two mitogenomes both obtained 13 overlapping nucleotides, and up to 53 bp ranging from 1 to 16 bp. The longest overlap was located between CO1 and trnE in A. jenniferae. There were 17 and 16 intergenic spacers each from A. galinae and A. jenniferae, totaling 171 bp and 115 bp, ranging 1 to 77 bp and 1 to 27 bp, respectively.

Table 2.
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Gene organization of the mitochondrial genomes of Anagyrus galinae and Anagyrus jenniferae.

Gene Direction Anticodon Anagyrus galinae Anagyrus jenniferae
Position Length Start codon Stop codon Intergenic Nucleotides Position Length Start codon Stop codon Intergenic Nucleotides
trnI − GAU 1–70 70 1–67 67
ND2 + 98–1087 990 ATT TAA 27 74–1081 1008 ATT TAA 6
trnW + UCA 1087–1149 63 −1 1080–1146 67 -2
trnY − GUA 1155–1221 67 5 1148–1212 65 1
trnS1 − UCU 1222–1280 59 0 1216–1275 60 3
trnC − GCA 1283–1348 66 2 1293–1361 69 17
trnN + GUU 1369–1434 66 20 1368–1431 64 6
trnR − UCG 1433–1497 65 −2 1439–1504 66 7
ND3 − 1498–1842 345 ATT TAA 0 1505–1858 354 ATA TAA 0
trnG − UCC 1843–1906 64 0 1856–1919 64 -3
CO3 − 1911–2714 804 ATG TAA 4 1925–2710 786 ATG TAA 5
ATP6 − 2715–3387 673 ATG T 0 2710–3383 674 ATG TA -1
ATP8 − 3381–3542 162 ATT TAA −7 3377–3538 162 ATC TAA -7
trnD − GUC 3543–3608 66 0 3539–3602 64 0
trnK + UUU 3612–3683 72 3 3606–3676 71 3
CO2 − 3688–4365 678 ATT TAG 4 3678–4355 678 ATT TAA 1
trnL2 − UAA 4369–4434 66 3 4365–4428 64 9
CO1 − 4440–5987 1548 ATT TAA 5 4431–5969 1539 ATG TAA 2
trnE + UUC 5972–6036 65 −16 5972–6034 63 2
trnF − GAA 6036–6102 67 −1 6034–6099 66 -1
ND5 − 6102–7769 1668 ATA TAA −1 6099–7763 1665 ATT TAG -1
trnH − GUG 7767–7833 67 −3 7764–7829 66 0
ND4 − 7844–9169 1326 ATG TAG 10 7829–9156 1328 ATG TA -1
ND4L − 9163–9450 288 ATT TAA −7 9150–9437 288 ATT TAA -7
trnT + UGU 9453–9518 66 2 9440–9505 66 2
trnP − UGG 9520–9582 63 1 9506–9574 69 0
ND6 + 9584–10151 568 ATG T 1 9575–10143 569 ATG TA 0
CYTB + 10152–11300 1149 ATG TAA 0 10143–11285 1143 ATG TAA -1
trnS2 + UGA 11300–11365 66 −1 11290–11354 65 4
ND1 − 11356–12291 936 ATT TAG −10 11345–12283 939 ATA TAG -10
trnL1 − UAG 12292–12358 67 0 12284–12348 65 0
lrRNA − 12364–13674 1311 5 12353–13654 1302 4
trnA − UGC 13682–13744 63 7 13651–13719 69 -4
trnQ + UUG 13761–13831 71 16 13797–13864 68 77
srRNA − 13831–14602 772 −1 13891–14646 756 26
trnV − UAC 14602–14669 68 −1 14646–14710 65 -1
trnM − CAU 14668–14735 68 −2 14709–14770 62 -2
CR 14736–15364 629 0 14771–15396 626 0
Figure 1. 

Circular map of the mitochondrial genome of Anagyrus galinae and Anagyrus jenniferae.

The nucleotide composition of the mitogenome from A. galinae was biased toward A and T, with 83.12% of A+T content (A = 45.12%, T = 38.00%, C = 10.82%, G = 6.05%), A+T content was 82.94%, 87.20% in PCGs and rRNAs, respectively. The nucleotide composition of the mitogenome from A. jenniferae was biased toward A and T, with 82.64% of A+T content (A = 46.41%, T = 36.23%, C = 11.33%, G = 6.02%), A+T content was 82.32%, 85.20% in PCGs and rRNAs, respectively. The values of AT-skew and GC-skew were often used to indicate the nucleotide composition of the mitochondrial genome. In this study, the nucleotide features of two new mitogenomes were investigated by calculating the percentages of AT-skew and GC-skew (Table 3). The skew analysis showing the mitogenome of A. galinae had a positive AT-skew (0.086) and a negative GC-skew (−0.283), and the mitogenome of A. jenniferae had a positive AT-skew (0.123) and a negative GC-skew (−0.306).

Table 3.
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Nucleotide features of the mitochondrial genome across Anagyrus galinae and Anagyrus jenniferae.

Feature Length (bp) T% C% A% G% A+T% AT-Skew GC-Skew
Whole genome 15364/15396 38.00/36.23 10.82/11.33 45.12/46.41 6.05/6.02 83.12/82.64 0.086/0.123 −0.283/−0.306
ATP6 673/674 46.66/47.63 7.43/8.01 34.92/34.27 11.00/10.09 81.58/81.90 −0.144/−0.163 0.194/0.115
ATP8 162/162 48.77/48.77 4.32/4.94 43.83/36.42 3.09/9.88 92.59/85.19 −0.053/−0.145 −0.167/0.333
CO1 1524/1539 45.41/46.39 10.37/10.98 29.86/27.23 14.37/15.40 75.26/73.62 −0.207/−0.260 0.162/0.167
CO2 678/678 45.58/45.72 8.55/8.41 33.04/33.19 12.83/12.68 78.61/78.91 −0.159/−0.159 0.200/0.203
CO3 804/786 46.64/49.75 7.84/8.52 32.21/29.90 13.31/11.83 78.86/79.64 −0.183/−0.249 0.259/0.163
CYTB 1149/1143 43.69/41.91 14.36/14.7 32.64/34.82 9.31/8.57 76.33/76.73 −0.145/−0.092 −0.213/−0.263
ND1 936/939 46.47/48.35 7.05/6.71 32.37/31.31 14.1/13.63 78.85/79.66 −0.179/−0.214 0.333/0.340
ND2 990/1008 50.10/47.52 9.19/9.62 37.58/39.19 14.10/13.63 87.68/86.71 −0.143/−0.096 −0.492/−0.448
ND3 345/351 51.01/52.99 5.22/5.41 33.91/31.34 9.86/10.26 84.93/84.33 −0.201/−0.257 0.308/0.309
ND4 1326/1328 50.08/52.41 4.98/5.20 34.01/30.20 10.94/12.20 84.09/82.61 −0.191/−0.269 0.374/0.403
ND4L 288/288 53.13/53.82 2.78/2.08 34.03/36.46 10.07/7.64 87.15/90.28 −0.219/−0.192 0.568/0.571
ND5 1665/1665 50.81/51.11 5.77/5.77 33.09/32.61 10.33/10.51 83.90/83.72 −0.211/−0.221 0.284/0.292
ND6 568/569 46.13/45.34 8.45/10.54 42.25/41.48 3.17/2.64 88.38/86.82 −0.044/−0.045 −0.455/−0.600
srRNA 772/756 44.30/44.84 4.15/4.10 43.52/40.08 8.03/10.98 87.82/84.92 −0.009/−0.056 0.319/0.456
lrRNA 1311/1302 44.55/46.08 4.27/4.15 42.03/39.40 9.15/10.37 86.58/85.48 −0.029/−0.078 0.364/0.429
CR 629/626 42.61/40.57 7.15/8.47 46.26/48.72 3.98/2.24 88.87/89.29 0.041/0.091 −0.285/−0.582

Protein-coding genes and codon usage

By comparing the known mitochondrial genome structure of Encyrtidae, we found that the sequence of 13 PCGs was consistent, except for ND3 rearranged in Diaphorencyrtus aligarhensis and Leptomastidea bifasciata. The sequence of PCGs in these mitochondrial genomes were the same (Fig. 2). Additionally, this arrangement is consistent with the mitochondrial gene order in other Encyrtidae, which is also consistent with inferred ancestry.

Figure 2. 

Gene order of mitochondrial genomes of different Encyrtidae species.

The total lengths of 13 PCGs are 11,108 bp in A. galinae, 11,130 bp in A. jenniferae. In these mitochondrial genomes, the length of each PCG ranges from 162 bp (ATP8) to 1665 bp (ND5). Two mitogenomes of Anagyrus exhibited similar start and stop codons. All the initiation codons of PCGs were ATN (ATA, ATG and ATT). Three kinds of stop codons existed on the new mitogenomic sequences: TAA, TAG and truncated termination codons (TA existed on ATP6, ND4, ND6 in A. jenniferae, T existed on ATP6, ND6 in A. galinae), TAA were the most frequently used. Truncated termination codons are commonly used in metazoan mitogenomes, which could be completed by post-transcriptional poly-adenylation (Ojala et al. 1981).

The codon UUA (Leu2) was the most commonly used in both mitogenomes. Mitochondrial protein coding genes have obvious bias towards A and T, and for mitochondrial protein-coding gene of A. galinae the three most frequently used codons were UUA (Leu2) 469 times, AUU (Ile) 440 times and UUU (Phe) 432 times. For A. jenniferae, the three most used codons were UUA (Leu2) 463 times, UUU (Phe) 431 times and AUU (Ile) 393 times. Mitochondrial protein-coding genes in Encyrtidae prefer A and U in the third codon, which is like some hymenopteran insects (Fan et al. 2017; Peng et al. 2017). The RSCU values of A. galinae and A. jenniferae are shown in Fig. 3.

Figure 3. 

Relative synonymous codon usage in mitochondrial genomes of Anagyrus galinae and Anagyrus jenniferae.

In this study, based on 20 mitochondrial genomes of Encyrtidae, DnaSP was used to calculate the non-synonymous substitution rate, synonymous substitution, and Ka/Ks ratio of 13 PCGs in the mitochondrial genome and then to compare the evolution rate between genes (Fig. 4). The results showed that among the 13 protein-coding genes in the mitochondrial genome of encyrtids, CYTB had the highest Ks, whereas ATP8 had the highest Ka and Ka/Ks value, and ATP8 had the largest variation and COI had the slowest evolution rate. The evolution rate of 13 genes was in the order of ATP8 > ND2 > ND4L > ND6 > ND4 > ND5 > ND3 > ATP6 > ND1 > CO3 > CO2 > CYTB > CO1.

Figure 4. 

Evolutionary rates of protein-coding genes in the mitochondrial genomes of Encyrtidae.

Ka/Ks values of 12 PCGs (all PCGs except ATP8) were far lower than 1.0, indicating that they were subject to purifying selection, a phenomenon first discovered in Chalcidoidea. In addition, the Ka/Ks value of ATP8 is higher than 1.0, higher value of ATP8 was also found in other species (Ma et al. 2019; Jia et al. 2020; Xu et al. 2021). The reason for this phenomenon may be that the evolution speed of a gene is related to its function (Wang et al. 2011).

Transfer RNA genes, ribosomal RNA genes, and control region

The mitochondrial genomes of the two species both included 22 tRNA genes, and the total lengths of the tRNAs of A. galinae and A. jenniferae are 1455 bp and 1445 bp, respectively. The length of tRNA genes in two Anagyrus species ranged from 59 to 72 bp. The secondary structures of the 22 tRNAs of the two species are shown in Suppl. materials 1, 2. The 22 tRNA genes in the mitochondrial genome are identical with the anticodon of tRNA corresponding to the mitochondrial genome of other Hymenoptera, except that trnL and trnS have two tRNA structures, and the others only have one corresponding tRNA structure. Most tRNAs could be folded into a typical clover-leaf structure, except for trnS1 which lost a dihydrouridine (DHU) arm and became a simple loop. A lack of the DHU arm in trnS1 was found in the mitochondrial genomes of most insects (Dowton et al. 2002). Changes in the length of the DHU and TΨC arms led to differences in the size of the tRNA sequence (Shao et al. 2001). In addition, the anticodon of trnS1 became UCU instead of the more common GCU. In addition to typical Waston-Crick pairings (A-U and G-C), G-U pairings also exist, which are called atypical pairings or wobble base pairs. A total of 30 mismatched base pairs were found in the arm structures of the tRNAs.

Hymenopteran mitochondria have a high rearrangement rate, which mainly occurs in A+T-rich regions, ND2, ND2-CO2, CO2-ATP8, and ND3-ND5 regions (Wei 2009). The gene arrangement of the suborder Symphyta was conserved and less rearranged than that of suborder Apocrita. However, there are a large number of rearrangements in the Apocrita, including displacement, inversion in situ, and ectopic inversion (Song 2015; Zhao et al. 2021). The rearrangement of mitochondrial genomes in Encyrtidae species was compared (Fig. 2), and the rearrangement was mainly found in tRNA genes. The rearrangement of tRNA occurred at many sites, and the pattern was complicated. Except that trnD-trnK (trnK-trnD in Ooencyrtus plautus), trnL2, trnE-trnF (trnF-trnE in Aenasius arizonensis and Diaphorencyrtus aligarhensis), trnH, trnT-trnP (trnP-trnT in Aenasius arizonensis and Lamennaisia ambigua), trnS2 and trnL1 are stable between ATP8 to lrRNA, there was no exclusion, and the other tRNA genes had been rearranged.

As for the rRNAs of two Anagyrus species, both lrRNA and srRNA genes are encoded on the N-strand and have a heavy AT nucleotide bias. The lengths of lrRNA and srRNA in A. galinae are 1311 bp and 772 bp, with the different A+T contents of 86.58% and 87.82%, and in A. jenniferae are 1302 bp and 756 bp, with the different A+T contents of 85.48% and 84.92%.

In the mitogenome, the largest non-coding region is normally the A+T-rich region, also known as the control region, which regulates the replication and transcription of mitochondrial DNA (Boore 1999; Cameron 2014). In the mitogenomes of the two Anagyrus species sequenced in this study, the CR is located between trnM and trnI (Fig. 5). The length of the CR is 629 bp in A. galinae and 626 bp in A. jenniferae. The A+T content is 88.87% and 89.29% in the CR of A. galinae and A. jenniferae. Analysis of AT-skew and CG-skew indicates that both Anagyrus species exhibit A and C usage bias. Three structural elements were found in each CR of two Anagyrus species: (1) a leading sequence adjacent to trnM; (2) four tendem repeats (TPs); (3) the remaining area of the control region.

Figure 5. 

Control region structure of two Anagyrus species. TR, tandem repeat.

Phylogenetic relationships

The phylogenetic analysis of the concatenated dataset was conducted using BI and ML, which were shown in Fig. 6. With Encarsia formosa as an outgroup, the phylogenetic trees of Encyrtidae were constructed based on 13 protein-coding gene sequences of the 21 mitochondrial genomes, including NCBI data and the two newly sequenced Anagyrus genomes reported in this study.

Figure 6. 

Phylogenetic tree of Encyrtidae based on nucleotide sequence of PCGs. Numbers at the nodes are Bayesian posterior probabilities (left) and ML bootstrap values (right). Each color block represents the corresponding family and tribe.

The result of maximum-likelihood and Bayesian analysis both indicate that the taxonomic relationship of each genus of Encyrtidae is (Metaphycus + Aenasius) + (((Anagyrus + Leptomastidea) + Encyrtus) + ((Blastothrix + Psyllaephagus) + (((Cheiloneurus + Tassonia) + Diaphorencyrtus) + (Ooencyrtus + (Exoristobia + Lamennaisia))))).

Overall, the phylogenetic trees reconstructed by both methods indicate that species belonging to the same tribe are clustered into one or adjacent clades, while species belonging to the same genus are clustered into the same clade, consistent with the morphological classification system. At the subfamily level, according to the morphological classification system, Encyrtidae is divided into two subfamilies: Tetracneminae and Encyrtinae. Aenasius, Anagyrus, and Leptomastidea all belong to Tetracneminae, while the remaining genera belong to Encyrtinae. However, in the phylogenetic trees reconstructed in this study, the results of both methods show that, except for Encyrtus and Metaphycus, Encyrtidae is divided into two main parts, which essentially conforms to the morphological classification system. Metaphycus and Aenasius form a monophyletic clade as sister groups, which is consistent with the previous phylogenetic results (Zhao et al. 2021; Xing et al. 2022).

While the Anagyrus species were not clustered on one branch with Aenasius arizonensis but clustered with Encyrtus, this may be due to different dietary habits. The five genera Metaphycus, Aenasius, Anagyrus, Leptomastidea, and Encyrtus exclusively parasitize scale insects within Hemiptera. In contrast, other species of Encyrtinae have a broader host range, including species from Lepidoptera, Diptera, Coleoptera, Hymenoptera, and more families within Hemiptera (Noyes 2019). Specifically, Anagyrus jenniferae parasitizes Phenacoccus indicus, Anagyrus galinae parasitizes Trionymus copiosus, and Leptomastidea bifasciata parasitizes Phenacoccus aceris and Planococcus vovae (Noyes and Hayat 1994; Japoshvili and Hansen 2015; Trjapitzin 1989; Zhang and Xu 2009). These Anagyrini species, which exclusively parasitize the Pseudococcidae, form a distinct clade in both phylogenetic trees. The hosts of Encyrtus sasakii include Takahashia japonica and Eulecanium kuwanai; Encyrtus eulecaniumiae parasitizes Eulecanium kuwanai and Eulecanium giganteum; Encyrtus rhodococcusiae targets Rhodococcus sariuoni; and Encyrtus infelix parasitizes Ceroplastes destructor, Saissetia coffeae, and Saissetia oleae (Trjapitzin 1989; Öncüer 1991; Noyes and Hayat 1994; Zhang and Huang 2001; Gupta and Poorani 2009; Wang et al. 2016), which were exclusively parasitize the Coccidae. Additionally, Encyrtus aurantii can parasitize members of the Coccidae (Saissetia coffeae), Eriococcidae (Eriococcus buxi), and Pseudococcidae (Planococcus citri) (Hayat et al. 2003). Consequently, in the phylogenetic trees, the clustering of Anagyrini and Encyrtini species together in the phylogenetic analysis might be attributed to the close genetic relationship between Coccidae and Pseudococcidae (Cook et al. 2002). This phenomenon also indicates the need for further mitochondrial genome sequencing of Encyrtidae species to obtain a more accurate classification status.

Discussion

In this study, we determined two newly sequenced mitogenomes, which are from A. galinae and A. jenniferae, then found them consistent with previously reported mitogenomes of Encyrtidae. Two new mitogenomes exhibited quite similar features in the genome size, base content, AT nucleotide bias, AT-skew, GC-skew, codon usage of protein genes, secondary structure of tRNAs and gene rearrangement. The BI and ML phylogenetic analysis among the major lineages based on the concatenated datasets yielded well-resolved topologies with moderate to high support for most branches. These results provide a relatively holistic framework and valuable data toward the future resolution of phylogenetic relationships in Encyrtidae. This study provided insights into the phylogenetic relationships of certain taxa within Encyrtidae, the limited sample size and scarcity of molecular evidence remain challenges. Therefore, future studies should aim to augment the number of sampled species and expand the dataset of mitochondrial genomes, utilizing a broader range of data for robust phylogenetic analysis and a comprehensive assessment of the taxonomic status within Encyrtidae.

Acknowledgments

We extend our heartfelt gratitude to Tao Wang from the College of Life Sciences, Nankai University, China, for providing valuable assistance in software analysis. Special thanks also go to Miss Zi-Yan Wang from the University of Sheffield, UK, as well as Mr. Shuai Zhang and Mr. Mark Sharples from the University of Manchester, UK, for their dedicated efforts in reviewing and revising the article. Their contributions have significantly enriched the quality and clarity of our work.

Additional information

Conflict of interest

The authors have declared that no competing interests exist.

Ethical statement

No ethical statement was reported.

Funding

No funding was received for conducting this study.

Author contributions

Conceptualization: GHZ, CHZ. Data curation: YW, CHZ. Formal analysis: CHZ. Investigation: CHZ, HYW. Methodology: CHZ, YW. Project administration: YW, CHZ, HYW. Resources: GHZ. Software: CHZ, YSL, ZHC. Supervision: GHZ, CHZ. Validation: CHZ, HYW. Visualization: CHZ. Writing – original draft: YW, CHZ, HYW. Writing – review and editing: CHZ, HYW.

Author ORCIDs

Cheng-Hui Zhang https://orcid.org/0000-0001-8234-0903

Hai-Yang Wang https://orcid.org/0009-0007-5665-2111

Yan Wang https://orcid.org/0000-0002-5001-975X

Zhi-Hao Chi https://orcid.org/0009-0006-0447-6948

Guo-Hao Zu https://orcid.org/0000-0002-9892-2171

Data availability

Data presented in this study are openly available in the NCBI repository with accession numbers: OR652687, OR790122.

References

  • Brown WM, George Jr M, Wilson AC (1979) Rapid evolution of animal mitochondrial DNA. Proceedings of the National Academy of Sciences of the United States of America 76(4): 1967–1971. https://doi.org/10.1073/pnas.76.4.1967
  • Cameron SL, Whiting MF (2008) The complete mitochondrial genome of the tobacco hornworm, Manduca sexta, (Insecta: Lepidoptera: Sphingidae), and an examination of mitochondrial gene variability within butterflies and moths. Gene 408(1–2): 112–123. https://doi.org/10.1016/j.gene.2007.10.023
  • Cook LG, Gullan PJ, Trueman HE (2002) A preliminary phylogeny of the scale insects (Hemiptera: Sternorrhyncha: Coccoidea) based on nuclear small-subunit ribosomal DNA. Molecular Phylogenetics and Evolution 25(1): 43–52. https://doi.org/10.1016/S1055-7903(02)00248-8
  • Cruaud A, Rasplus JY, Zhang JX, Burks R, Delvare G, Fusu L, Gumovsky A, Huber JT, Janšta P, Mitroiu MD, Noyes JS, van Noort S, Baker A, Böhmová J, Baur H, Blaimer BB, Brady SG, Bubeníková K, Chartois M, Copeland RS, Dale-Skey Papilloud N, Dal Molin A, Dominguez C, Gebiola M, Guerrieri E, Kresslein RL, Krogmann L, Lemmon E, Murray EA, Nidelet S, Nieves-Aldrey JL, Perry RK, Peters RS, Polaszek A, Sauné L, Torréns J, Triapitsyn S, Tselikh EV, Yoder M, Lemmon AR, Woolley JB, Heraty JM (2024) The Chalcidoidea bush of life: Evolutionary history of a massive radiation of minute wasps. Cladistics 40(1): 34–63. https://doi.org/10.1111/cla.12561
  • Donath A, Jühling F, Al-Arab M, Bernhart SH, Reinhardt F, Stadler PF, Middendorf M, Bernt M (2019) Improved annotation of protein-coding genes boundaries in metazoan mitochondrial genomes. Nucleic Acids Research 47(20): 10543–10552. https://doi.org/10.1093/nar/gkz833
  • Dowton M, Castro LR, Austin AD (2002) Mitochondrial gene rearrangements as phylogenetic characters in the invertebrates: The examination of genome ‘morphology’. Invertebrate Systematics 16(3): 345–356. https://doi.org/10.1071/IS02003
  • Du YM, Song X, Liu XJ, Zhong BL (2019) Mitochondrial genome of Diaphorencyrtus aligarhensis (Hymenoptera: Chalcidoidea: Encyrtidae) and phylogenetic analysis. Mitochondrial DNA. Part B, Resources 4(2): 3190–3191. https://doi.org/10.1080/23802359.2019.1667913
  • Fan XL, Gong YJ, Chen PY, Tan QQ, Tan JL, Wei SJ (2017) Next-generation sequencing of the mitochondrial genome of Dolichovespula panda (Hymenoptera: Vespidae) with a phylogenetic analysis of Vespidae. Journal of Asia-Pacific Entomology 20(3): 971–976. https://doi.org/10.1016/j.aspen.2017.07.009
  • Gupta A, Poorani J (2009) Taxonomic studies on a collection of Chalcidoidea (Hymenoptera) from India with new distribution records. Journal of Threatened Taxa 1(5): 300–304. https://doi.org/10.11609/JoTT.o1861.300-4
  • Hassanin A, Léger N, Deutsch J (2005) Evidence for multiple reversals of asymmetric mutational constraints during the evolution of the mitochondrial genome of Metazoa, and consequences for phylogenetic inferences. Systematic Biology 54(2): 277–298. https://doi.org/10.1080/10635150590947843
  • Hayat M, Narendran TC, Remadevi OK, Manikandan S (2003) Parasitoids (Hymenoptera: Chalcidoidea; Ceraphronoidea) reared mainly from Coccoidea (Homoptera) attacking Sandalwood, Santalum album L. Oriental Insects 37(1): 309–334. https://doi.org/10.1080/00305316.2003.10417352
  • Heraty JM, Burks RA, Cruaud A, Gibson GAP, Liljeblad J, Munro J, Rasplus JY, Delvare G, Janšta P, Gumovsky A, Huber J, Woolley JB, Krogmann L, Heydon S, Polaszek A, Schmidt S, Darling DC, Gates MW, Mottern J, Murray E, Dal Molin A, Triapitsyn S, Baur H, Pinto JD, van Noort S, George J, Yoder M (2013) A phylogenetic analysis of the megadiverse Chalcidoidea (Hymenoptera). Cladistics 29: 466–542. https://doi.org/10.1111/cla.12006
  • Japoshvili O, Hansen LO (2015) New records of Encyrtidae (Hymenoptera, Chalcidoidea) from Norway VI. Norwegian Journal of Entomology 62(2): 174–179.
  • Jia CH, Zhang XM, Xu SY, Yang TY, Yanagimoto T, Gao TX (2020) Comparative analysis of the complete mitochondrial genomes of three rockfishes (Scorpaeniformes, Sebastiscus) and insights into the phylogenetic relationships of Sebastidae. Bioscience Reports 40(12): BSR20203379. https://doi.org/10.1042/BSR20203379
  • Jin JJ, Yu WB, Yang JB, Song Y, dePamphilis CW, Yi TS, Li DZ (2020) GetOrganelle: A fast and versatile toolkit for accurate de novo assembly of organelle genomes. Genome Biology 21(1): 241. https://doi.org/10.1186/s13059-020-02154-5
  • Kalyaanamoorthy S, Minh BQ, Wong TKF, von Haeseler A, Jermiin LS (2017) ModelFinder: Fast model selection for accurate phylogenetic estimates. Nature Methods 14(6): 587–589. https://doi.org/10.1038/nmeth.4285
  • Katoh K, Rozewicki J, Yamada KD (2019) MAFFT online service: Multiple sequence alignment, interactive sequence choice and visualization. Briefings in Bioinformatics 20(44): 1160–1166. https://doi.org/10.1093/bib/bbx108
  • Krzywinski J, Grushko OG, Besansky NJ (2006) Analysis of the complete mitochondrial DNA from Anopheles funestus: An improved dipteran mitochondrial genome annotation and a temporal dimension of mosquito evolution. Molecular Phylogenetics and Evolution 39(2): 417–423. https://doi.org/10.1016/j.ympev.2006.01.006
  • Li Q, Wei SJ, Tang P, Wu Q, Shi M, Sharkey MJ, Chen XX (2016) Multiple Lines of Evidence from Mitochondrial Genomes Resolve Phylogenetic Relationships of Parasitic Wasps in Braconidae. Genome Biology and Evolution 8(9): 2651–2662. https://doi.org/10.1093/gbe/evw184
  • Liu MD, Luo YF, Jallow BJJ, Meng FM (2023) Characterization of Complete Mitochondrial Genome and Phylogenetic Analysis of a Nocturnal Wasps – Provespa barthelemyi (Hymenoptera: Vespidae). Current Issues in Molecular Biology 45(12): 9368–9377. https://doi.org/10.3390/cimb45120587
  • Ma Y, Zheng BY, Zhu JC, Tang P, Chen XX (2019) The mitochondrial genome of Aenasius arizonensis (Hymenoptera: Encyrtidae) with novel gene order. Mitochondrial DNA. Part B, Resources 4(1): 2023–2024. https://doi.org/10.1080/23802359.2019.1617052
  • Meng GL, Li YY, Yang CT, Liu SL (2019) MitoZ: A toolkit for animal mitochondrial genome assembly, annotation and visualization. Nucleic Acids Research 47(11): e63. https://doi.org/10.1093/nar/gkz173
  • Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ (2015) IQ-TREE: A fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Molecular Biology and Evolution 32(1): 268–274. https://doi.org/10.1093/molbev/msu300
  • Noyes JS (1980) A review of the genera of Neotropical Encyrtidae (Hymenoptera: Chalcidoidea). Bulletin of the British Museum of Natural History 41: 107–253.
  • Noyes JS, Hayat M (1984) A review of the genera of Indo-Pacific Encyrtidae (Hymenoptera: Chalcidoidea). Bulletin of the British Museum of Natural History 48: 131–395.
  • Noyes JS, Hayat M (1994) Oriental mealybug parasitoids of the Anagyrini (Hymenoptera: Encyrtidae). CAB International, Wallingford, 554 pp.
  • Ojala D, Montoya J, Attardi G (1981) tRNA punctuation model of RNA processing in human mitochondria. Nature 290(5806): 470–474. https://doi.org/10.1038/290470a0
  • Öncüer C (1991) A catalogue of the parasites and predators of insect pests of Turkey. Ege University Agricultural Faculty Press No. 505, Izmir, 204 pp.
  • Peng Y, Chen B, Li TJ (2017) Sequencing and analysis of the complete mitochondrial genome of Parapolybia crocea (Hymenoptera: Vespidae). Acta Entomologica Sinica 60(4): 464–474. https://doi.org/10.16380/j.kcxb.2017.04.011
  • Perna NT, Kocher TD (1995) Patterns of nucleotide composition at fourfold degenerate sites of animal mitochondrial genomes. Journal of Molecular Evolution 41(3): 353–358. https://doi.org/10.1007/BF01215182
  • Ranwez V, Douzery EJP, Cambon C, Chantret N, Delsuc F (2018) MACSE v2: Toolkit for the Alignment of Coding Sequences Accounting for Frameshifts and Stop Codons. Molecular Biology and Evolution 35(10): 2582–2584. https://doi.org/10.1093/molbev/msy159
  • Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, Larget B, Liu L, Suchard MA, Huelsenbeck JP (2012) MrBayes 3.2: Efficient Bayesian phylogenetic inference and model choice across a large model space. Systematic Biology 61(3): 539–542. https://doi.org/10.1093/sysbio/sys029
  • Rozas J, Ferrer-Mata A, Sánchez-DelBarrio JC, Guirao-Rico S, Librado P, Ramos-Onsins SE, Sánchez-Gracia A (2017) DnaSP 6: DNA sequence polymorphism analysis of large data sets. Molecular Biology and Evolution 34(12): 3299–3302. https://doi.org/10.1093/molbev/msx248
  • Rudoy A, Zhu CD, Ferrari RR, Zhang YZ (2022) Integrative taxonomy based on morphometric and molecular data supports recognition of the three cryptic species within the Encyrtus sasakii complex (Hymenoptera, Encyrtidae). Journal of Hymenoptera Research 90: 129–152. https://doi.org/10.3897/jhr.90.75807
  • Sayers EW, Cavanaugh M, Clark K, Pruitt KD, Sherry ST, Yankie L, Karsch-Mizrachi I (2024) GenBank 2024 Update. Nucleic Acids Research 52(D1): D134–D137. https://doi.org/10.1093/nar/gkad903
  • Simutnik SA, Perkovsky EE (2023) Description of a new genus and species of Encyrtidae (Hymenoptera: Chalcidoidea) from Danish amber, based on a male specimen featuring an antenna with a distinct anellus. Zootaxa 5369(3): 437–445. https://doi.org/10.11646/zootaxa.5369.3.7
  • Simutnik SA, Perkovsky EE, Vasilenko DV (2022) Electronoyesella antiqua Simutnik, gen. et sp. nov. (Chalcidoidea, Encyrtidae) from Rovno amber. Journal of Hymenoptera Research 94: 105–120. https://doi.org/10.3897/jhr.94.94773
  • Simutnik SA, Perkovsky EE, Pankowski MV (2023) A new genus and species of Encyrtidae (Hymenoptera: Chalcidoidea) with a four-segmented funicle from late Eocene Baltic amber. Zootaxa 5389(1): 119–127. https://doi.org/10.11646/zootaxa.5389.1.6
  • Song SN (2015) Comparative mitogenomics and phylogeny of the Microsatrinae (Hymenoptera: Braconidae). PhD Thesis, Zhejiang University, Hangzhou, China.
  • Talavera G, Castresana J (2007) Improvement of Phylogenies after Removing Divergent and Ambiguously Aligned Blocks from Protein Sequence Alignments. Systematic Biology 56(4): 564–577. https://doi.org/10.1080/10635150701472164
  • Trjapitzin VA (1989) Parasitic Hymenoptera of the Fam. Encyrtidae of Palaearctics. Nauka, Leningrad, 489 pp.
  • Wang DP, Fei L, Wang L, Huang S, Yu J (2011) Nonsynonymous substitution rate (Ka) is a relatively consistent parameter for defining fast-evolving and slow-evolving protein-coding genes. Biology Direct 6(1): 13. https://doi.org/10.1186/1745-6150-6-13
  • Wang Y, Zhou QS, Qiao HJ, Zhang AB, Yu F, Wang XB, Zhu CD, Zhang YZ (2016) Formal nomenclature and description of cryptic species of the Encyrtus sasakii complex (Hymenoptera: Encyrtidae). Scientific Reports 6(1): 34372. https://doi.org/10.1038/srep34372
  • Wang HY, Zhang CH, Xue H, Xi CX, Yang ZN, Zu GH (2023) Encyrtidae (Hymenoptera: Chalcidoidea) from China, with the description of two new species. Zootaxa 5361(2): 237–251. https://doi.org/10.11646/zootaxa.5361.2.5
  • Wei SJ (2009) Characterization and evolution of hymenopteran mitochondrial genomes and their phylogenetic utility. PhD Thesis, Zhejiang University, Hangzhou, China.
  • Wei SJ, Shi M, Sharkey MJ, van Achterberg C, Chen XX (2010) Comparative mitogenomics of Braconidae (Insecta: Hymenoptera) and the phylogenetic utility of mitochondrial genomes with special reference to Holometabolous insects. BMC Genomics 11(1): 371. https://doi.org/10.1186/1471-2164-11-371
  • Xing ZP, Liang X, Wang X, Hu HY, Huang YX (2022) Novel gene rearrangement pattern in mitochondrial genome of Ooencyrtus plautus Huang & Noyes, 1994: New gene order in Encyrtidae (Hymenoptera, Chalcidoidea). ZooKeys 1124: 1–21. https://doi.org/10.3897/zookeys.1124.83811
  • Xu W, Lin SP, Liu HY (2021) Mitochondrial genomes of five Hyphessobrycon tetras and their phylogenetic implications. Ecology and Evolution 11(18): 12754. https://doi.org/10.1002/ece3.8019
  • Zhang YZ, Xu ZH (2009) A review of Chinese species of Leptomastidea Mercet (Hymenoptera: Encyrtidae). Acta Entomologica Sinica 52(4): 420–423.
  • Zhang JX, Lindsey ARI, Peters RS, Heraty JM, Hopper KR, Werren JH, Martinson EO, Woolley JB, Yoder MJ, Krogmann L (2020a) Conflicting signal in transcriptomic markers leads to a poorly resolved backbone phylogeny of chalcidoid wasps. Systematic Entomology 45(4): 783–802. https://doi.org/10.1111/syen.12427
  • Zhang D, Gao FL, Jakovlić I, Zou H, Zhang J, Li WX, Wang GT (2020b) PhyloSuite: An integrated and scalable desktop platform for streamlined molecular sequence data management and evolutionary phylogenetics studies. Molecular Ecology Resources 20(1): 348–355. https://doi.org/10.1111/1755-0998.13096
  • Zhao HF, Chen Y, Wang ZT, Chen HF, Qin YG (2021) Two complete mitogenomes of Chalcididae (Hymenoptera: Chalcidoidea): genome description and phylogenetic implications. Insects 12(12): 1049. https://doi.org/10.3390/insects12121049
  • Zhou QS, Xiong M, Luo AR, Zhang YZ, Zhu CD (2021) The complete mitochondrial genome of Metaphycus eriococci (Timberlake) (Hymenoptera: Encyrtidae). Mitochondrial DNA. Part B, Resources 6(2): 550–552. https://doi.org/10.1080/23802359.2021.1872450
  • Zhu JC, Tang P, Zhou BY, Wu Q, Wei SJ, Chen XX (2018) The first two mitochondrial genomes of the family Aphelinidae with novel gene orders and phylogenetic implications. International Journal of Biological Macromolecules 118(A): 386–396. https://doi.org/10.1016/j.ijbiomac.2018.06.087
  • Zhu JC, Xiao H, Tang P, Li XF, Li XK, Zhu CD, Wu Q, Xiao JH, van Achterberg C, Huang DW, Chen XX (2023) Evolutionary timescale of chalcidoid wasps inferred from over one hundred mitochondrial genomes. Zoological Research 44(3): 467–482. https://doi.org/10.24272/j.issn.2095-8137.2022.379

Supplementary materials

Supplementary material 1 

Secondary structures of 22 tRNA genes of Anagyrus galinae

Cheng-Hui Zhang, Hai-Yang Wang, Yan Wang, Zhi-Hao Chi, Yue-Shuo Liu, Guo-Hao Zu

Data type: jpg

Explanation note: Blue gene names indicate that in the major strand, and red names indicate that in the minor strand.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (25.89 MB)
Supplementary material 2 

Secondary structures of 22 tRNA genes of Anagyrus jenniferae

Cheng-Hui Zhang, Hai-Yang Wang, Yan Wang, Zhi-Hao Chi, Yue-Shuo Liu, Guo-Hao Zu

Data type: jpg

Explanation note: Blue gene names indicate that in the major strand, and red names indicate that in the minor strand.

This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited.
Download file (25.69 MB)
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