JHR 97: 105-126 (2024) gee JOURNAL OF *reermieretovensccese our doi: 10.3897/jhr.97.1 19433 RESEARCH ARTICLE () Hymenopter a 9 https://jhr.pensoft.net The Imarasional Society of Hymenopeeriss, RESEARCH Description and mitochondrial genome sequencing of a new species of inquiline gall wasp, Synergus nanlingensis (Hymenoptera, Cynipidae, Synergini), from China Yu-Bo Duan", Yan-Jie Wang!'’, Dao-Hong Zhu', Yang Zeng', Xiu-Dan Wang! | Laboratory of Insect Behavior and Evolutionary Ecology, College of Life Science and Technology, Central South University of Forestry and Technology, Changsha, Hunan, China Corresponding authors: Yang Zeng (zengyangsile@163.com); Xiu-Dan Wang (t20192464@csuft.edu.cn) Academic editor: Miles Zhang | Received 24 January 2024 | Accepted 26 February 2024 | Published 11 March 2024 https://z00bank. org/ DEOBABB9-F338-468A-8 148-90BD 1562E3A6 Citation: Duan Y-B, Wang Y-J, Zhu D-H, Zeng Y, Wang X-D (2024) Description and mitochondrial genome sequencing of a new species of inquiline gall wasp, Synergus nanlingensis (Hymenoptera, Cynipidae, Synergini), from China. Journal of Hymenoptera Research 97: 105-126. https://doi.org/10.3897/jhr.97.119433 Abstract 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: t7nS1 is downstream of Cytb; trnS2 is upstream of nadJ; and trnC is downstream of rrnS. Phylogenetic analysis using COJ, CytB, and 28S-D2 sequences confirmed that S. nanlingensis is a distinct species belonging to the genus Synergus Hartig. Keywords Castanopsis, gall wasp, mitogenome, morphology, phylogenetic analysis * These authors contributed equally. Copyright Yu-Bo Duan et al. 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. 106 Yu-Bo Duan et al. / Journal of Hymenoptera Research 97: 105-126 (2024) Introduction Cynipids or gall wasps (Hymenoptera: Cynipoidea, Cynipidae) are the second larg- est 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 interac- tion 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 (Csdka 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 in- quilines and that can even cause the death of the gall inducer (Duffet 1968; Pénzes et al. 2009; Bozso 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 (Schweger 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 Re- gion (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 Description and mitogenome of a new inquiline gall wasp 107 al. 2019). Gene rearrangements, including transpositions, inversions, and inverse trans- positions in the mitogenome, are common in certain insect groups, can be an informa- tive 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 hypoth- esized by Heraty et al. (2011). Despite these advances, complete or nearly complete mi- togenome 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 mi- togenome structure and gene rearrangements in this lineage were analyzed. Addition- ally, phylogenetic analyses were conducted using CO/, 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. [hese cages were placed in the laboratory environment under room tem- perature 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 tem- perature and mounted to pinned triangular card paper. They were then photographed with a Leica M205C microscope system equipped with Leica DMC6200 digital cam- era (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 = 1* and subsequent flagellom- eres; post-ocellar distance (POL) = distance between the posterior ocelli; ocellar-ocular 108 Yu-Bo Duan et al. / Journal of Hymenoptera Research 97: 105-126 (2024) 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 meas- ured from the margin of the wing to the Rs vein. Type specimens are housed in the Insect Collection of the Central South Univer- sity 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 con- tamination. Total DNA was then extracted using SDS/proteinase K digestion and phenol-chloroform extraction. The extracted DNA pellets were air-dried, resuspended in 20 ul 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, COL, Cytb, and 28s-D2 genes. The PCR products were puri- fied and sequenced using the Sanger method by Wuhan Icongene Co, Ltd (Wuhan, China). GDcox1K GDcox1R, GDrrnLF, GDrrnLR, and GDcytbR were designed to amplify the remaining genome by long PCR (Suppl. material 2). The reaction mixture comprised: 0.4 wL APEX (AG, Dalian, China), 10 uL buffer mixture, 0.4 pL of each primer, and 0.5 pL of DNA; water was added to each reaction to a final volume of 20 wL. 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/GDcox]1F 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 assem- bled 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 (https://usegalaxy.org/root?tool_id=toolshed.g2.bx.psu.edu%2Frepos%2Fiuc%2Fmit 0s2%2Fmitos2%2F2.1.3%20galaxy0). PCGs were identified by ORFFinder in NCBI (www.ncbi.nlm.nih.gov). rRNA genes were confirmed by sequence comparison with Description and mitogenome of a new inquiline gall wasp 109 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 calcu- lated using PhyloSuite v1.2.2. The RSCU figure was drawn using the ggplot2 pack- age (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 mono- phyletic. New species specificity and whether the morphological similarities reflect- ed the phylogenetic relationship based on molecular data were determined using the method of Lobato-Vila et al. (2021). Specifically, the COL, 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 COJ, 450 bp of Cyd, 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 mod- el-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 ob- tained 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. Results Morphology-based taxonomy Synergus nanlingensis Wang & Zeng, 2023, sp. nov. https://zoobank.org/982D4466-0B8A-4F8B-BD6B-93887 1C8417B Figs 1, 2 Holotype. Female, Cutna, 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 fe- males 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). 110 Yu-Bo Duan et al. / Journal of Hymenoptera Research 97: 105-126 (2024) 0.5mm wu $10 Figure |. Synergus nanlingensis Wang & Zeng, 2023, sp. nov. a general habitus (2) b general habitus (3) e antenna (Q) d antenna (<) e head in anterior view (2) f head in anterior view (@) g head in dorsal view (2) h head in dorsal view (<4). Description and mitogenome of a new inquiline gall wasp 111 Diagnosis. Synergus nanlingensis Wang & Zeng, sp. nov., most closely resembles Synergus hupingshanensis (Liu, Yang & Zhu) is part of a group characterized by a com- pletely 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 1.3x 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. Description. Female; body length: 2.6—3.2 mm (NV = 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. Head (Figs le, g, 2a, c): transverse ellipse in front view (the widest of head near mid- dle), 1.2x as wide as high, slightly broader than mesosoma in the anterior view, 1.2x 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 1.6x as high as wide; height of eye 1.5x as high as length of malar space (Fig. le). 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. le). 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. le). Occiput smooth; postgena with setae. Antenna (Fig. 1c): 12 flagellomeres, pedicel 1.8x 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): 1.3x 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 1.4x as wide as long (measuring along the anterior edge of tegulae), sur- face 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 poste- rior 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). He Yu-Bo Duan et al. / Journal of Hymenoptera Research 97: 105-126 (2024) imm )O.8 mi imm Figure 2. Synergus nanlingensis Wang & Zeng, 2023, sp. nov. a head in lateral view (Q) b fore wing (2) c head in posterior view (2) d mesosoma, lateral view (2) € mesosoma, dorsal view (2) f propodeum, dorsal view (2) g metasoma, lateral view (2) h metasoma, lateral view (@) i tarsal claw. Description and mitogenome of a new inquiline gall wasp 113 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 2.9x 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 1.2x as long as high; petiole sulcate; syntergite almost completely covering remaining tergites, surface smooth and mainly glabrous, with few white setae anterolaterally, and a postero- dorsal area without setae and micropunctures. Subsequent tergites and hypopygium mi- cropunctate; 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 (NV = 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 meso- soma combined. Biology. 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 Septem- ber to October. The gall inducer of the gall which yielded S. nanlingensis is unconfirmed. Distribution. Shaoguan City, Guangdong Province, China. Etymology. 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 overlap- ping 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). 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% (nado). 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. 114 Yu-Bo Duan et al. / Journal of Hymenoptera Research 97: 105-126 (2024) Figure 3. Gall of Synergus nanlingensis Wang & Zeng, 2023, sp. nov. on Castanopsis eyrei Tutch. jtrnM (cat) trnL1(tag) tmS2(tga) eae control region nad1 tmQ(ttg) | trni(gat) trnC(gca) hast trnl2(taa) Hi : a‘ Lu : Mi Ya Ary (gta) é ee ae col——= pen alt nad6 = \ tmP(tgg)— ~~ f Bre trnT(tgt) nad4l NQhtak ttt) atp8 trnD(gtc) atp6 i, . : = \ * ie tmH(gt ‘ , a | \\\\\ nad3, g) tmG(tcc) tmE(tty) trnA(tgc) tmN(gtt) trnF(gaa) 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 dehy- drogenase subunits 1—6 and 4 L; rrnL and rrnS, large and small rRNA subunits. Description and mitogenome of a new inquiline gall wasp 115 Table |. Annotation of the Synergus nanlingensis Wang & Zeng, 2023, sp. nov. mitochondrial genome. Gene Positions Size Strand Nucleotides Anti or Start codon Stop A+T(%) Intergenic codon trnS2 1-68 68 - -2 TGA 89.7 nad1 67-1005 939 — Ne ATT TAG 85 trnLl 1079-1144 66 - 1 TAG 92.4 trn] 1146-1215 70 — 5 GAT 85.7 trnL2 1221-1291 71 - -l TAA 90.1 tnW 1291-1358 68 — ) TCA 91.2, trnM 1362-1427 66 + -5 CAT 89.4 trnQ 1423-1491 69 2 2 ime 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 cox 2677-4212 1536 + abies ATT TAA 77.4 cox2 4330-5016 687 + 336 ATA TAA 83.9 trnk 5353-5424 72 + 6 TIT 87.5 trnD 5431-5503 72 + 0 GTC 94.5 atp8 5504-5665 161 + -6 ATT TAA 87 atp6 5659-6333 675 + 1 ATG TAA 83:7 cox3 6335-7122 788 + 3 ATG TA 80.5 trnG 7126-7198 rs, + 0 TCG 94.5 nad3 7199-7534 336 + 31 ATT TAA 87.8 trnA 7566-7636 ya + -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 HEARS 97 nad5 7902-9572 1671 = 0 ATT TAA 87.4 trnH 9573-9646 74 - as GTG 89.2 nad4 9654-10967 1314 - -7 ATG TAA 85.5 nad4L 10961-11236 276 - 12 ATT TAA 90.6 tnT 11249-11312 64 + ~] TGT 922 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 i 76 TCT 87.1 rrnoL 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 Notes: + indicates the gene is coded on majority strand while — indicates the gene is coded on minority strand. In S. nanlingensis, eight genes (coxl, nad1, nad2, nad3, nad4L, nad5, nado, 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 nadI1 with TAG. The relative synonymous codon usage in all 13 PCGs is shown in Fig. 5. As report- ed for previously studied Cynipidae species, the most common amino acids are leucine (Leu2) and serine (Ser2). The least common codons are CUG-Leul and CUC-Leul. 116 Yu-Bo Duan et al. / Journal of Hymenoptera Research 97: 105-126 (2024) 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 con- ventional cloverleaf secondary structure, whereas trmS1 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), rrmZ 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). RSCU 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). trnS 2 Figure 6. Structures of control regions in the mitogenome of Synergus nanlingensis Wang & Zeng, 2023, trnc sp. nov. Abbreviation: NTR, nontandem repeat. Yellow shows A + T-rich regions. Description and mitogenome of a new inquiline gall wasp 117 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; rrnZ and rrnS moved into the cob—nad1 junction; a novel tRNA gene cluster trnL1—trnl-trnL2-trnW-trnM-—trnQ was formed between nadI and nad2; and trnV was inverted and moved to the nad2-cox1 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 Cyt; trnS2 is upstream of nad1; and trnC is downstream of rrnS (Figs