Research Article
Research Article
Description and mitochondrial genome sequencing of a new species of inquiline gall wasp, Synergus nanlingensis (Hymenoptera, Cynipidae, Synergini), from China
expand article infoYu-Bo Duan, Yan-Jie Wang, Dao-Hong Zhu, Yang Zeng, Xiu-Dan Wang
‡ Central South University of Forestry and Technology, Changsha, China
Open Access


A new species of inquiline gall wasp, Synergus nanlingensis Wang & Zeng, sp. nov., which was reared from galls on Castanopsis eyrei Tutch (Fagaceae) collected in Guangdong Province, China, is described and illustrated herein along with its mitochondrial genome. The mitogenome of S. nanlingensis is 16,604 base pairs in length and comprises 37 genes, which is typical of mitogenomes. One large control region was detected in the S. nanlingensis mitogenome, which differed from that reported for other Cynipidae species. Similar to other Cynipidae species, S. nanlingensis has the same four common gene rearrangement events; however, it shows some differences, as follows: trnS1 is downstream of Cytb; trnS2 is upstream of nad1; and trnC is downstream of rrnS. Phylogenetic analysis using COI, CytB, and 28S-D2 sequences confirmed that S. nanlingensis is a distinct species belonging to the genus Synergus Hartig.


Castanopsis, gall wasp, mitogenome, morphology, phylogenetic analysis


Cynipids or gall wasps (Hymenoptera: Cynipoidea, Cynipidae) are the second largest radiation of gall-inducing insects, with about 1400 described species (Ronquist et al. 2015; Lobato-Vila et al. 2022a). They are widely distributed worldwide, mainly throughout the Holarctic region (Nearctic and Palearctic), and most species are gall inducers on different host plants (Melika and Abrahamson 2002; Ronquist et al. 2015; Lobato-Vila et al. 2022b). Gall induction starts after the oviposition where this interaction between the female wasp and the plant tissue triggers gall formation, and larval activity (chewing, feeding) promotes gall growth and subsequent transformations of the gall structure (Csóka et al. 2005). This can affect the growth of the host plant, even causing host death (Duffet 1968). The gall protects the larvae from not only predatory insects, but also insecticides, posing difficulties for their chemical control (Moriya et al. 2003; Chiara et al. 2018). Some cynipoids can significantly impact the forestry industry. For example, Dryocosmus kuriphilus Yasumatsu is a worldwide invasive pest that causes serious damage to chestnut trees (Zhu et al. 2007; Yang et al. 2021), while damage by Diplolepis abei Pujade-Villar & Wang causes significant economic losses to the rose horticulture industry in Northwest China (Guo et al. 2013; Lobato-Vila et al. 2020).

By contrast, nearly 240 species of cynipids (Lobato-Vila et al. 2022b), termed inquilines, are unable to trigger gall growth; instead, they develop inside galls induced by other gall wasps, forming an advantageous relationship that benefits only the inquilines and that can even cause the death of the gall inducer (Duffet 1968; Pénzes et al. 2009; Bozsó et al. 2015). Inquilines are distributed into four tribes: Synergini sensu stricto, Ceroptresini, Diastrophini, and Rhoophilini (Ronquist et al. 2015; Lobato-Vila et al. 2022a). Synergus Hartig is the most speciose genus in Synergini, with about 130 species known worldwide (Schwéger et al. 2015; Pujade-Villar et al. 2017; Lobato-Vila et al. 2020). Most Synergus species are associated with galls induced by gall wasps of the tribe Cynipini on Fagaceae (principally Quercus spp.). In Europe, the 22 species of Fagaceae (Schwarz 1993; Tutin 1993; Tutin and Akeroyd 1993) are known to host at least 30 species of Synergus (Melika 2006; Pénzes et al. 2012). By comparison, there are 294 species of Fagaceae in seven genera in mainland China, including 163 endemic and at least three introduced (Huang et al. 1999). However, only 17 Synergus species are known from mainland China (Lobato-Vila et al. 2022a), thus, it is thought that the species diversity of Synergus in mainland China is likely to be higher than current estimates (Abe 2007; Liu et al. 2012).

The mitochondrial genome of most insects is a double-stranded circular structure DNA molecule comprising 13 protein-coding genes, 22 transfer RNAs (tRNAs), two ribosomal RNA (rRNA) genes, and a major noncoding sequence called ‘Control Region’ (CR) (Cameron 2014). Given the maternal mode of inheritance and conserved gene components, the insect mitochondrial genome is a molecular marker widely used in phylogenetic construction (Cameron 2014). However, gene rearrangements have been found frequently in Hymenoptera (Wei et al. 2014; Chen et al. 2018), not only for tRNA genes but also for protein-coding genes (PCGs) (Simon et al. 2006; Tang et al. 2019). Gene rearrangements, including transpositions, inversions, and inverse transpositions in the mitogenome, are common in certain insect groups, can be an informative feature for phylogenetic reconstruction (Cameron 2014; Feng et al. 2020). For example, Tang et al. (2019) analyzed 83 full or partial mitochondrial genomes to resolve relationships among all major clades of Hymenoptera with high support, confirming the phylogenetic position of Cynipoidea in Proctotrupomorpha as previously hypothesized by Heraty et al. (2011). Despite these advances, complete or nearly complete mitogenome sequences remain scarce for Cynipidae, with just seven species documented, including only two from Synergini (Tang et al. 2019; Xue et al. 2020; Pang et al. 2022; Shu et al. 2022; Zhong and Zhu 2022; Mozhaitseva et al. 2023; Su et al. 2023).

In this study, we describe a new species of the genus Synergus from China. The completed mitogenome of this new species was sequenced and annotated, and mitogenome structure and gene rearrangements in this lineage were analyzed. Additionally, phylogenetic analyses were conducted using COI, Cytb, and 28S-D2 sequences to delineate the evolutionary relationships between this new species and existing species from the Palearctic region within Synergus.

Materials and methods

Specimen collection

A total of 142 galls were collected in September 2023, from branches of Castanopsis eyrei Tutch on the summit of Xiaohuang Mountain, Guangdong Province, China. The galls were kept in insect mesh bags with moistened cotton and placed in meshed rearing cages. These cages were placed in the laboratory environment under room temperature conditions. To maintain humidity, the cages were misted with water every 1.5 days, and the humidifying cotton was frequently replaced until the emergence of insects. Adult wasps were directly preserved in 100% ethanol within two days after emergence and frozen at –80 °C for morphological and molecular studies.

Morphological observations

Specimens for conventional morphological examination were air-dried at room temperature and mounted to pinned triangular card paper. They were then photographed with a Leica M205C microscope system equipped with Leica DMC6200 digital camera (Leica Inc.,Wetzlar, Germany) attached to a computer. The illustration was made using the Procreate application on an iPad Air 3, utilizing an Apple Pencil and based on a magnified photograph of the tarsal claw.

The terminology used to describe the morphology of specimens follows that used in other studies on gall wasps (Harris 1979; Ronquist and Nordlander 1989; Ronquist 1995; Melika 2006) as follows: abbreviations: F1–F13 = 1st and subsequent flagellomeres; post-ocellar distance (POL) = distance between the posterior ocelli; ocellar-ocular distance (OOL) = distance from the outer margin of a posterior ocellus to the inner margin of the compound eye; lateral-frontal ocelli distance (LOL) = distance between the lateral and frontal ocellus. The width of the radial cell of the forewing was measured from the margin of the wing to the Rs vein.

Type specimens are housed in the Insect Collection of the Central South University of Forestry and Technology (CSUFT), Changsha city, Hunan province, China.

DNA extraction and sequencing

Before DNA extraction, specimens were washed in sterile water to avoid surface contamination. Total DNA was then extracted using SDS/proteinase K digestion and phenol-chloroform extraction. The extracted DNA pellets were air-dried, resuspended in 20 µL sterile water, and stored at 4 °C for PCR and sequencing. Insect universal primers designed by Folmer et al. (1994), Simon et al. (1994), Schwéger et al. (2015), and Tavakoli et al. (2019) (Suppl. material 1) were used to amplify partial fragments of the mitochondrial rrnL, COI, Cytb, and 28s-D2 genes. The PCR products were purified and sequenced using the Sanger method by Wuhan Icongene Co, Ltd (Wuhan, China). GDcox1F, GDcox1R, GDrrnLF, GDrrnLR, and GDcytbR were designed to amplify the remaining genome by long PCR (Suppl. material 2). The reaction mixture comprised: 0.4 µL APEX (AG, Dalian, China), 10 µL buffer mixture, 0.4 µL of each primer, and 0.5 µL of DNA; water was added to each reaction to a final volume of 20 µL. Amplification was conducted using a C1000 Touch Thermal Cycler (Bio-Rad, Hercules, CA, USA). The cycling conditions were as follows: 98 °C for 1 min, 34 cycles of 98 °C for 10 s, 55 °C for 30 s, and 68 °C for 10 min. Two amplification strategies were used to obtain the complete mitogenome sequence. First, PCR amplification was performed using four specific long PCR primer combinations: GDrrnLF/GDcox1F, GDrrnLR/GDcox1R, GDrrnLF/GDcox1R, and GDrrnLR/GDcox1F. However, only GDrnnLF/GDcox1F resulted in a desired outcome. A clear single band was obtained using the primer combination GDcytbR/GDcox1R. These PCR products were then purified and sequenced.

The primer walking method was used to determine the sequence for each long PCR product using an ABI 3730XL DNA sequencer (Applied Biosystems, Foster City, CA, USA) by Wuhan Icongene Co, Ltd. Long PCR fragments were sequenced directly with the PCR primers and internal primers (Suppl. material 3). Sequences were assembled using SeqMan Pro 7.1.0 (Burland, 2000), then checked and corrected manually. The same site with different nucleotides was used to check the original sequencing peak map or to resequence the products to determine the nucleotides of the site.

Genome annotation and analyses

The initial mitogenome annotations were conducted using MITOS on Galaxy ( PCGs were identified by ORFFinder in NCBI ( rRNA genes were confirmed by sequence comparison with published mitochondrial rRNA sequences from Synergus sp. (Tang et al. 2019), and Dryocosmus liui Pang, Su & Zhu (Hymenoptera: Cynipidae) (Su et al. 2023). Control regions (CRs) were confirmed by the boundaries of trnS2 and trnC. Codon usage and relative synonymous codon usage (RSCU) of 13 PCGs in the specimens were calculated using PhyloSuite v1.2.2. The RSCU figure was drawn using the ggplot2 package (Hadley 2009), a plugin of Rscript 3.4.4 (Zhong and Zhu 2022). The nucleotide composition and AT/GC skew were calculated using PhyloSuite.

Analyses of phylogenetic relationship and pairwise genetic distance

To assess the taxonomic position of Synergus nanlingensis within the genus Synergus, we incorporated S. nanlingensis into the clade of Synergus species from the Palearctic region as recovered by Lobato-Vila et al. (2022a). This clade was strongly supported as monophyletic. New species specificity and whether the morphological similarities reflected the phylogenetic relationship based on molecular data were determined using the method of Lobato-Vila et al. (2021). Specifically, the COI, Cytb, and 28S-D2 sequences of 31 Palearctic Synergus species and two additional species of other cynipid genera were used as outgroups (Suppl. material 4). Sequences were aligned using MAFFT (Katoh et al. 2002) and those from each gene (660 bp of COI, 450 bp of Cytb, and 574 bp of 28S-D2) were concatenated in a single matrix (1684 bp) using PhyloSuite.

This concatenated matrix of molecular data sets was analyzed based on the model-based phylogenetic approaches Bayesian Inference (BI) and Maximum Likelihood (ML). To determine the best partitions and models, the data sets were also analyzed using ModelFinder (Kalyaanamoorthy et al. 2017). For BI analysis, four simultaneous Markov chains were run for 10 million generations, with tree sampling occurring every 1,000 generations, and a burn-in of 25% of the trees in MrBayes 3.2.7 (Huelsenbeck and Ronquist 2001). For ML analyses, a total of 10,000 bootstrap replicates were obtained with the auto model applied to all partitions in IQ-tree2.2.2.7 (Nguyen et al. 2015). The final tree was rooted using the outgroup.


Morphology-based taxonomy

Synergus nanlingensis Wang & Zeng, 2023, sp. nov.

Figs 1, 2


Female, China, Guangdong Province, Shaoguan City, 24-09-2022, reared from galls collected in 1-9-2022, leg. Y. Zeng, L. Liu and Y. Duan. Paratypes: three females and 13 males, same as holotype, housed in CSUFT (the holotype and two male paratypes were dried and mounted, while the other paratypes were deposited in 99% ethanol in a freezer at –80 °C).


Synergus nanlingensis Wang & Zeng, sp. nov., most closely resembles Synergus hupingshanensis (Liu, Yang & Zhu) is part of a group characterized by a completely opened radial cell, tarsal claws with a basal lobe and lateral pronotal carina present. However, it can be differentiated from S. hupingshanensis by the following morphological features: (1) The first flagellomere (F1) of S. nanlingensis is nearly equal in length to the second flagellomere (F2), whereas in S. hupingshanensis, F1 1.3× as long as F2; (2) the head of S. nanlingensis reddish brown with the frons and the center of the occiput being black, whereas head of S. hupingshanensis entirely orange without such black markings; and (3) scutellar foveae in S. nanlingensis are smooth and shiny at the bottom, whereas in S. hupingshanensis are roughly sculptured.


Female; body length: 2.6–3.2 mm (N = 10).

Color (Figs 1a, 2c): head reddish brown, except frons, mandible teeth, and center of occiput black; antennae reddish brown. Mesosoma, legs, and metasoma black, with tarsus and distal part of body reddish brown. Wings hyaline with distinct brown veins.

Figure 1. 

Synergus nanlingensis Wang & Zeng, 2023, sp. nov. a general habitus (♀) b general habitus (♂) c antenna (♀) d antenna (♂) e head in anterior view (♀) f head in anterior view (♂) g head in dorsal view (♀) h head in dorsal view (♂).

Figure 2. 

Synergus nanlingensis Wang & Zeng, 2023, sp. nov. a head in lateral view (♀) b fore wing (♀) c head in posterior view (♀) d mesosoma, lateral view (♀) e mesosoma, dorsal view (♀) f propodeum, dorsal view (♀) g metasoma, lateral view (♀) h metasoma, lateral view (♂) i tarsal claw.

Head (Figs 1e, g, 2a, c): transverse ellipse in front view (the widest of head near middle), 1.2× as wide as high, slightly broader than mesosoma in the anterior view, 1.2× wider than long as seen from above; frons slightly elevated from lateral view; lateral frontal carinae inconspicuous or absent, with rugose sculpture between the compound eye and frons; frons surface densely punctate with deep punctures and sparse setae (Fig. 1g). Eyes 1.6× as high as wide; height of eye 1.5× as high as length of malar space (Fig. 1e). Lower face densely setose, radiating from the clypeus toward basal margin of compound eye and antennal toruli. Gena broadened behind eyes, with punctures and white sparse setae. Middle of clypeus slightly impressed; anterior tentorial pit large and distinct; epistomal sulcus and clypeopleurostomal line indistinct; malar sulcus absent. Transfacial distance longer than the height of the compound eye; diameter of torulus shorter than the diameter of toruli and about half the distance between the inner margin of the eye and torulus (Fig. 1e). POL: OOL: LOL=2.2:1.8:1; LOL approximately as long as the diameter of the lateral ocellus. Ocelli ovate, all three similar in size (Fig. 1e). Occiput smooth; postgena with setae.

Antenna (Fig. 1c): 12 flagellomeres, pedicel 1.8× as long as broad, F1 longer than F2. F1–F12:14:13:13:13:11:10:9:8:8:7:7:10. Placoid sensillae distinct on F5–F12.

Mesosoma (Fig. 2d–f): 1.3× as long as high on the lateral view (Fig. 2d), with dense pubescence. Length of the middle part of pronotum is one-third that of the outer lateral margin; pronotum punctate, laterally areolate-rugulose, lateral carina distinct. Mesoscutum 1.4× as wide as long (measuring along the anterior edge of tegulae), surface areolate-rugose, center with a transverse rugae, covered with densely yellow setae. Notauli percurrent and distinct, somewhat convergent posteriorly; anterior parallel line, parapsidal line, and median mesoscutal line indistinct, barely traceable (Fig. 2e). Scutellar foveae elongate ovate, bottom smooth and shiny, deeply impressed, with short sparse white setae, separated by distinct central carina. Mesopleuron hairless, finely striated ventrally and carinate-rugose dorsally. Metapleural sulcus reaches posterior margin of mesopectus in the most upper 1/4 of its height (Fig. 2d). Propodeum smooth coriaceous, with short sparse white setae. Lateral propodeal carinae slightly impressed basally and slightly convergent distally (Fig. 2f).

Legs : Tarsal claws with a small basal lobe (Fig. 2i).

Forewing (Fig. 2b): hyaline and densely setose, approximately as long as body length. All veins well pigmented. Radial cell open, about 2.9× as long as broad; R1 does not reach wing margin; Rs curved toward to posterior distally.

Metasoma (Fig. 2g): slightly shorter than the head and mesosoma combined, and 1.2× as long as high; petiole sulcate; syntergite almost completely covering remaining tergites, surface smooth and mainly glabrous, with few white setae anterolaterally, and a posterodorsal area without setae and micropunctures. Subsequent tergites and hypopygium micropunctate; prominent part of ventral spine of hypopygium small, with few lateral setae.

Male (Figs 1b, d, f, g, 2h): similar to the female, but body length 1.9–2.2 mm (N = 6); head, legs, and distal part of abdomen yellowish brown; frons, mandible teeth, mesosoma, basal part of abdomen, and hind coax black.

Antenna : 13 flagellomeres, pedicel 1.4 times as long as broad. F1–F13: 16:13:14:14:14:13:13:13:12:11:10: 11. Metasoma elongated, shorter than the head and mesosoma combined.


Specimens of S. nanlingensis were collected from galls found on branches of Castanopsis eyrei on the summit of Xiaohuang Mountain 1,600 m above sea level. Galls are nearly spherical in shape, range in diameter from 15 to 35 mm, and are hard and strongly lignified (Fig. 3). Galls appear in July and inquilines emerged from late September to October. The gall inducer of the gall which yielded S. nanlingensis is unconfirmed.

Figure 3. 

Gall of Synergus nanlingensis Wang & Zeng, 2023, sp. nov. on Castanopsis eyrei Tutch.


Shaoguan City, Guangdong Province, China.


The specific epithet refers to the type locality.

Genome organization and base composition

The total length of the complete mitogenome of S. nanlingensis is 16,604 bp (GenBank accession OR978581). The mitochondrial genome contains the typical gene repertoire of 13 PCGs, two rRNA genes, and 22 tRNA genes (Fig. 4). There are eight overlapping regions, ranging in size from 1 to 7 bp. The mitogenome contains 20 intergenic spacers, with lengths ranging from 1 to 336 bp. The longest gene spacer is between Cox2 and trnD (Table 1). The nucleotide content of the S. nanlingensis mitogenome is as follows: 44.2% A, 5.6% G, 42% T, and 8.2% C; the total A + T percentage is 86.2%,.AT skew is 0.026 and GC skew is –0.191, which is consistent with that in other Hymenoptera (Wei et al. 2010; Chen et al. 2018).

Table 1.

Annotation of the Synergus nanlingensis Wang & Zeng, 2023, sp. nov. mitochondrial genome.

Gene Positions Size Strand Nucleotides Intergenic Anti or Start codon Stop codon A+T(%)
trnS2 1–68 68 -2 TGA 89.7
nad1 67–1005 939 73 ATT TAG 85
trnL1 1079–1144 66 1 TAG 92.4
trnI 1146–1215 70 5 GAT 85.7
trnL2 1221–1291 71 -1 TAA 90.1
trnW 1291–1358 68 3 TCA 91.2
trnM 1362–1427 66 + -5 CAT 89.4
trnQ 1423–1491 69 2 TTG 87
nad2 1494–2501 1008 24 ATT TAA 91.7
trnY 2526–2592 67 4 GTA 86.6
trnV 2597–2664 68 + 12 TAC 94.1
cox1 2677–4212 1536 + 117 ATT TAA 77.4
cox2 4330–5016 687 + 336 ATA TAA 83.9
trnK 5353–5424 72 + 6 TTT 87.5
trnD 5431–5503 72 + 0 GTC 94.5
atp8 5504–5665 161 + -6 ATT TAA 87.7
atp6 5659–6333 675 + 1 ATG TAA 83.7
cox3 6335–7122 788 + 3 ATG TA 80.5
trnG 7126–7198 73 + 0 TCC 94.5
nad3 7199–7534 336 + 31 ATT TAA 87.8
trnA 7566–7636 71 + -3 TGC 87.3
trnR 7634–7703 70 + 0 TCG 88.6
trnN 7704–7771 68 + 1 GTT 83.8
trnF 7773–7836 64 + -2 GAA 92.2
trnE 7835–7901 67 0 TTC 97
nad5 7902–9572 1671 0 ATT TAA 87.4
trnH 9573–9646 74 7 GTG 89.2
nad4 9654–10967 1314 -7 ATG TAA 85.5
nad4L 10961–11236 276 12 ATT TAA 90.6
trnT 11249–11312 64 + -1 TGT 92.2
trnP 11312–11378 67 81 TGG 88.1
nad6 11460–11969 510 + 3 ATT TAA 91.8
cytb 11973–13109 1137 + 0 ATG TAA 80.9
trnS1 13110–13171 62 + 76 TCT 87.1
rrnL 13248–14628 1381 0 88.8
rrnS 14629–15461 833 0 90.1
trnC 15462–15530 69 + 0 GCA 91.3
CR 15531–16604 1066 84.6
Figure 4. 

Mitochondrial genome of Synergus nanlingensis Wang & Zeng, 2023, sp. nov. sequenced in this study. Genes outside the circle are encoded by the majority strand, and genes inside are encoded by the minority strand. The tRNA genes are indicated by their one-letter corresponding amino acids. The GC content is plotted using a black sliding window. Abbreviations: atp6 and atp8, ATP-synthase subunits 6 and 8; cob, cytochrome b; cox1–3, cytochrome oxidase subunits 1–3; nad1–6 and nad4L, NADH dehydrogenase subunits 1–6 and 4 L; rrnL and rrnS, large and small rRNA subunits.

Protein-coding genes and codon usage

The total length of the 13 PCGs of S. nanlingensis is 11,037 bp. Five PCGs (nad1, nad2, nad4L, nad4, and nad5) are encoded by the minority strand (N-strand), and the other eight genes are encoded by the majority strand (J-strand) (Table 1). The overall A + T content in PCGs is 84.7%, ranging from 77.4% (cox1) to 91.8% (nad6). The AT skew of the PCGs is –0.102, and the GC skew is 0.03. A very high A + T content (94.9%) is found at the third codon of PCGs.

In S. nanlingensis, eight genes (cox1, nad1, nad2, nad3, nad4L, nad5, nad6, atp8) are initiated with ATT, four genes (atp6, nad4, cob, and cox3) with ATG, and Cox2 initiated with ATA. All PCGs use ATN as the starting codon, similar to that reported for other Hymenoptera (Tang et al. 2019). Most PCGs from S. nanlingensis terminate with stop codons TAA, whereas Cox3 ends with TA and nad1 with TAG.

The relative synonymous codon usage in all 13 PCGs is shown in Fig. 5. As reported for previously studied Cynipidae species, the most common amino acids are leucine (Leu2) and serine (Ser2). The least common codons are CUG-Leu1 and CUC-Leu1.

Figure 5. 

Relative synonymous codon usage (RSCU) of Synergus nanlingensis Wang & Zeng, 2023, sp. nov. mitochondrial genome. Codon families are labeled on the x-axis. Values on the top of the bars indicate the percentage of each amino acid used for the construction of 13 protein-coding genes (PCGs).

tRNA and rRNA

In total, 22 tRNA genes were identified in the mitogenome of S. nanlingensis, ranging in size from 62 bp to 74 bp and accounting for 1,507 bp in total concatenated length (Table 1). Of the tRNA genes, 12 are located on the H-strand whereas ten tRNA genes are located on the L-strand. Of these tRNA genes, 21 can be folded into a conventional cloverleaf secondary structure, whereas trnS1 lack the dihydrouridine arm (D-arm). This feature has also been reported for Andricus mairei (Kieffer) (Zhong and Zhu 2022). The lack of the D-arm in trnS1 is a common feature of most metazoans (Kahnt et al. 2015; Du et al. 2017). In the mitochondrial tRNA secondary structures of S. nanlingensis, seven mismatched base pairs were detected: five G‐U pairs, one G‐A pair, and one A‐A pair. As reported for other Cynipoidea species (Mao et al. 2015; Tang et al. 2019; Xue et al. 2020; Su et al. 2023), rrnL and rrnS are next to each other and both are located in the L-strand in S. nanlingensis, with lengths of 1,381 bp and 833 bp, respectively.

Noncoding sequences (CR)

A large CR was detected in S. nanlingensis mitogenome, located between trnC and trnS2. The CR is 1073 bp in length and its AT content was 84.6%. It has three 166-bp non-tandem repeat units, one 36-bp A + T-rich region (AT% = 94.3%) and one 32-bp A + T-rich region (AT% = 90.6%) (Fig. 6).

Figure 6. 

Structures of control regions in the mitogenome of Synergus nanlingensis Wang & Zeng, 2023, sp. nov. Abbreviation: NTR, nontandem repeat. Yellow shows A + T-rich regions.

Gene arrangements

Compared with the ancestral mitogenome arrangement, rearrangements of S. nanlingensis mitogenome involve tRNA genes, rRNA genes, and PCGs. Su et al. (2023) compared the reported mitochondrial gene rearrangements of gall wasps and found four rearrangement events: trnE and trnF had inverted and swapped positions; rrnL and rrnS moved into the cobnad1 junction; a novel tRNA gene cluster trnL1trnItrnL2trnWtrnMtrnQ was formed between nad1 and nad2; and trnV was inverted and moved to the nad2cox1 junction. These four rearrangements are also found in S. nanlingensis. However, unlike gall wasps with two CRs (Xue et al. 2020; Pang et al. 2022; Zhong and Zhu 2022; Su et al. 2023), mitochondrial genes of S. nanlingensis have the following differences: trnS1 is downstream of Cytb; trnS2 is upstream of nad1; and trnC is downstream of rrnS (Fig. 7).

Figure 7. 

Mitochondrial genome organization and gene rearrangement in Synergus nanlingensis Wang & Zeng, 2023, sp. nov. compared with the ancestral type of the insect mitochondrial genome. All abbreviations are the same as in Table 1 in the main text. Arrow pointing to the right represents the J-strand and arrow pointing to the left represents the N-strand. Genes are drawn in their original order; intergenic distances are not included, and sizes of genes are not to scale. Yellow boxes indicate genes with different positions from two control regions reported in Cynipidae. and white boxes indicate genes that are different in terms of both position and strand associations from the putative ancestral pattern. Gray boxes show conserved gene blocks.

DNA taxonomy and phylogenetic relationship

The genetic distance between Synergus nanlingensis and other Synergus species is long (Suppl. material 5). The topology of our phylogenetic tree mostly coincides with that recovered by Lobato-Vila et al. (2022a) (Fig. 8) and supports S. nanlingensis as a distinct species, clustering it with other Palearctic Synergus. Different analytical approaches (BI and ML) did not affect the topology but did affect the level of node support (Suppl. material 7). Synergus nanlingensis is recovered as a sister species of Synergus itoensis Abe, Ide & Wachi, although this relationship is not highly supported.

Figure 8. 

Bayesian analysis of the MAFFT alignment data set inferred from the COI + Cytb + 28S-D2 data sets. Posterior probabilities are shown at each node.


The discovery of Synergus nanlingensis, a new species found in China, marks a significant contribution to the biodiversity of the family Cynipidae, especially among inquilines. Currently, little is known about the gall wasp species associated with Castanopsis, with only two Synergus species, S. hupingshanensis and S. kawakamii (Tang & Melika), reported so far (Schwéger et al. 2015; Lobato-vila et al. 2022a). Given the rich diversity of species and endemics within Castanopsis and Fagaceae in China (Xu et al. 2022), further research in this region is expected to uncover more new species associated with these hosts.

Phylogenetic tree analysis robustly confirms the status of S. nanlingensis as a member of the genus Synergus. Although an open radial cell in the forewing is not a typical characteristic of Synergus, the species was confirmed as a member based on the presence of the female antenna with 12 flagellomeres, the complete notaulus, and presence of an incomplete lateral frontal carina (Schweger et al. 2015); placing it within a group characterized by a fully open radial cell, basally lobed claws, and the presence of a lateral thoracic carina (Lobato-Vila et al. 2020). Interestingly, no Cynipini were reared from the same galls as S. nanlingensis, despite three years of collections made in different months. This phenomenon was also observed in the breeding records of S. hupingshanensis, where no expected gall-inducers were reared from two years of field collections (Liu et al. 2012). The reasons for the lack of expected gall inducers for these two species are still unknown and warrant further experimental investigation to determine whether or not they are inducers or inquilines as its currently known sister species S. itoensis is one of the few known rare cases of Synergus that have secondarily reverted back to gall induction (Abe and Wachi 2011). Gobbo et al. (2020) have compared the genome of S. itoensis with that of three other related Synergus inquilines, and found that there were distinct genetic differences between gall inducers and inquilines. Therefore, further study on the mitogenome or genome comparison between Chinese Synergus and known gall inducers and inquilines will provide molecular evidence for speculating whether they have gall inducing ability.

This study presents the first complete mitochondrial genome reported for a species of Synergus. In the mitochondrial genome of S. nanlingensis, general characteristics and typical rearrangement events of Cynipidae species were observed (Zhong and Zhu 2022; Su et al. 2023), but some differences were also noted. For instance, a long intergenic spacer of 366 bp between Cox2 and trnD was observed in S. nanlingensis. Such long intergenic spacers have also been found in the mitochondrial genomes of other Hymenoptera insects, possibly as a result of gene rearrangement (Chen et al. 2018; Zhong and Zhu 2022). While TAA is commonly used as a stop codon in arthropod mitogenomic PCGs, variations such as TA, a single T, and more uniquely, TAG, have been observed (Yamauchi et al. 2002). In S. nanlingensis, Cox3 ends with TA and nad1 with TAG, aligning with stop codon usage in two previously known Synergini mitogenomes (Tang et al. 2019; Shu et al. 2022). Prior research identified two control regions (CR1 and CR2) in Cynipoidea, with CR2 being a partially inverted repeat of CR1 (Mao et al. 2015; Zhong and Zhu 2022; Su et al. 2023). This led to speculation that inverted, duplicated CRs might be characteristic of the Cynipoidea mitochondrial genome. However, only one control region was found in the S. nanlingensis mitogenome, consistent with the two known mitogenomes of Synergini. Remarkably, this study provides a complete sequencing of the control region, which were never described before. Whether these features and gene rearrangement serve as distinguishing characteristics within Synergini requires further data support.


This study is supported by the National Key Research and Development Program of China (2018YFE0127100). We thank International Science Editing ( for editing this manuscript. We sincerely thank Zong-jun Liu, Yong Xie, Jia-dong Zhang, Zhi-ping Zhou and other staff of the Nanling National Nature Reserve for their selfless help in collecting specimens.


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Supplementary materials

Supplementary material 1 

List of universal insect mitochondrial short fragments of the cox1, cob, rrnL and D2 genes primers used for long PCR primer developments

Yu-Bo Duan, Yan-Jie Wang, Dao-Hong Zhu, Yang Zeng, Xiu-Dan Wang

Data type: docx

This dataset is made available under the Open Database License ( 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.
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Supplementary material 2 

List of PCR primers used in this study

Yu-Bo Duan, Yan-Jie Wang, Dao-Hong Zhu, Yang Zeng, Xiu-Dan Wang

Data type: docx

This dataset is made available under the Open Database License ( 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.
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Supplementary material 3 

List of PCR primers and sequencing primers used in this study

Yu-Bo Duan, Yan-Jie Wang, Dao-Hong Zhu, Yang Zeng, Xiu-Dan Wang

Data type: docx

This dataset is made available under the Open Database License ( 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.
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Supplementary material 4 

Summary of taxonomic groups used in Fig. 8

Yu-Bo Duan, Yan-Jie Wang, Dao-Hong Zhu, Yang Zeng, Xiu-Dan Wang

Data type: docx

This dataset is made available under the Open Database License ( 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.
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Supplementary material 5 

Pair-wise COI sequence distances in Synergus

Yu-Bo Duan, Yan-Jie Wang, Dao-Hong Zhu, Yang Zeng, Xiu-Dan Wang

Data type: docx

This dataset is made available under the Open Database License ( 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.
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Supplementary material 6 

Predicted folding pattern for tRNAs of Synergus nanlingensis mitochondrial genome

Yu-Bo Duan, Yan-Jie Wang, Dao-Hong Zhu, Yang Zeng, Xiu-Dan Wang

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This dataset is made available under the Open Database License ( 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.
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Supplementary material 7 

Maximum Likelihood tree were inferred from the datasets COI + Cytb + 28S-D2 using IQ-tree

Yu-Bo Duan, Yan-Jie Wang, Dao-Hong Zhu, Yang Zeng, Xiu-Dan Wang

Data type: doc

This dataset is made available under the Open Database License ( 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.
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