Description of two new species of Conostigmus Dahlbom, 1858 (Hymenoptera, Megaspilidae) from Yintiaoling Nature Reserve, Chongqing, China

379 Description of two new species of Conostigmus Dahlbom, 1858 (Hymenoptera, Megaspilidae) from Yintiaoling Nature Reserve, Chongqing, China Yuanhan Yang1, Fang Li2, Yixin Huang1, Xu Wang2, Zhisheng Zhang3, Huayan Chen4 1 Collaborative Innovation Center of Recovery and Reconstruction of Degraded Ecosystem in Wanjiang Basin Co-founded by Anhui Province and Ministry of Education; School of Ecology and Environment, Anhui Normal University, Wuhu, Anhui 241000, China 2 Anhui Provincial Key Laboratory of the Conservation and Exploitation of Biological Resources, Key Laboratory of Biotic Environment and Ecological Safety in Anhui Province, College of Life Sciences, Anhui Normal University, Wuhu, Anhui 241000, China 3 Key Laboratory of Eco-environments in Three Gorges Reservoir Region (Ministry of Education), School of Life Sciences, Southwest University, Chongqing 400715, China 4 Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China Corresponding authors: Xu Wang ([email protected]); Zhisheng Zhang ([email protected]); Huayan Chen (huay[email protected]) Copyright: © Yuanhan Yang et al. This is an open access article distributed under terms of the Creative Commons Attribution License (Attribution 4.0 International – CC BY 4.0). Research Article Abstract Two new species of Conostigmus Dahlbom, 1858, C. rotundus Yang & Wang, sp. nov. and C. clayulatus Yang & Wang, sp. nov. are described from the Yintiaoling Nature Reserve, Chongqing, China. The 28S rDNA sequences of the new species were generated to associate the female and male of the same species. Key words: 28S rDNA, Ceraphronoidea, identification key, morphology, parasitoid wasps, taxonomy Introduction Megaspilidae belongs to Ceraphronoidea of Hymenoptera. It is widely distributed throughout the world except for the polar regions (Johnson and Musetti 2004; Mikó et al. 2011; Bijoy et al. 2014; Pezzini and Köhler 2020). More than 450 species of 12 genera are known, and recent studies indicate that a very high number of undescribed species exist in Megaspilidae (Iemma et al. 2016; Salden and Peters 2023). Conostigmus was established by Dahlbom (1858) and proposed as a subgenus of Megaspilus. Kieffer (1907) erected Conostigmus as a genus from Megaspilus. Conostigmus is the most species-rich and widely distributed group in the family Megaspilidae, with about 180 species recorded worldwide (Johnson and Musetti 2004; Trietsch et al. 2020; Cui et al. 2023; Qian et al. 2024; Wang et al. 2024). Considering that sexual dimorphism is common in Conostigmus, it is difficult to match male and female individuals of the same species by morphological identification alone. Molecular identification is necessary to associate both sexes of the same species. In the present paper, we describe two new species of Conostigmus, C. rotundus Yang & Wang, sp. nov., and C. clayulatus Yang & Wang, sp. nov. from Academic editor: Jonah M. Ulmer Received: 3 April 2025 Accepted: 22 September 2025 Published: 15 October 2025 ZooBank: https://zoobank. org/8A3626C2-9F67-4A4B-B37AFEB586E4703D Citation: Yang Y, Li F, Huang Y, Wang X, Zhang Z, Chen H (2025) Description of two new species of Conostigmus Dahlbom, 1858 (Hymenoptera, Megaspilidae) from Yintiaoling Nature Reserve, Chongqing, China. ZooKeys 1255: 379–394. https://doi. org/10.3897/zookeys.1255.154710 ZooKeys 1255: 379–394 (2025) DOI: 10.3897/zookeys.1255.154710 380 ZooKeys 1255: 379–394 (2025), DOI: 10.3897/zookeys.1255.154710 Yuanhan Yang et al.: Two new species from Yintiaoling, China Chongqing, China, bringing the Chinese species number of Conostigmus up to ten (Dessart 1997; Cui et al. 2023; Qian et al. 2024; Wang et al. 2024). In addition, we provide 28S rDNA sequences of the two new species, which contribute to the molecular systematics of Conostigmus. Material and methods The specimens in this study were collected from sweep-net and yellow pan traps. Specimens were deposited in the Insect Collection of Anhui Normal University (AHNU), Wuhu, China. All voucher specimens were then observed and dried in the air. The specimens were photographed using a Leica M205A stereo microscope and a Leica DFC-500 digital camera. Image stacking was performed with Leica Microsystems CMS GmbH. To facilitate the preparation of male genitalia for morphological study, apical metasomal segments were dissected from specimens and subjected to a graded chemical processing protocol: first incubated in 35% H2O2 for 24 h, then transferred to 5% acetic acid for 24 h of further treatment, followed by a 1-h rinse in distilled water. After post-processing, the segments were transferred to a glycerin microdroplet on a concavity slide, and subsequent microdissection was performed in glycerin using #5 fine forceps and #2 insect pins as dissecting instruments. Measurements are given in microns. Morphological terminology follows the Hymenoptera Anatomy Ontology (Yoder et al. 2010) (Table 1). According to previous studies, sexual dimorphism is prevalent in Conostigmus (Trietsch et al. 2020; Wang et al. 2024). In order to match females and males of the same species, we sequenced the 28S rDNA of each species. A total of eight male and female sequences were generated for five species, including the two new species (two of which were published only for males). DNA was extracted from each putative species using the TIANamp Genomic DNA Kit (TIANGEN, Changping District, Beijing, cat. no. DP3400), following the protocol used by Taekul et al. (2014). Primers used for 28S amplification were D2-3549 (5’-AGTCGTGTTGCTTGATAGTGCAG-3’) and D2-4068 (5’-TTGGTCCGTGTTTCAAGACGGG-3’) (Zhang et al. 2014). The polymerase chain reactions (PCRs) were performed in a 25 μL reaction volume in a T100 Thermal Circulator (Bio-Rad). The thermal cycle conditions were: an initial denaturation step of 94 °C for 1 min, followed by 35 cycles of Table 1. Abbreviations and morphological terms used in text. F1, F2, ..., F9 Flagellum 1, 2, ..., F9. LOL Lateral ocellar length, shortest distance between inner margins of median and lateral ocelli. OOL Ocular ocellar length, minimum distance between a posterior ocellus to the eye margin. POL Posterior ocellar length, shortest distance between inner margins of posterior ocelli. HH Head height, lateral. EHf Eye height, anterior view. HL Head length. HW Head width. IOS Interorbital space. AseW Anterior mesoscutal width. PscW Posterior mesoscutal width. 381 ZooKeys 1255: 379–394 (2025), DOI: 10.3897/zookeys.1255.154710 Yuanhan Yang et al.: Two new species from Yintiaoling, China 94 °C for 1 min, 50 °C for 30 s, 72 °C for 30 s, with an additional extension at 72 °C for 5 min. Amplicons were directly sequenced in both directions with forward and reverse primers by GENERAL BIOL (Anhui, China). Chromatograms were assembled with Sequencing Analysis 6 (Thermo Fisher Scientific, Gloucester, UK). Sequences generated from this study were deposited in GenBank (accession numbers are listed in Table 2). Conostigmus sequences from the previous studies were downloaded from GenBank (Table 2). The genetic distances were calculated under the Kimura 2-parameter (K2P) model in MEGA X (Kumar et al. 2018). The sequences were aligned using the MUSCLE (Multiple Sequence Comparison by Log-Expectation) model of MEGA X (Pais et al. 2014). The alignment was used to reconstruct a maximum-likelihood (ML) tree by using the Auto-detect model of IQ-TREE (Minh et al. 2020). The specific parameters were set to Ultrafast for Bootstrap analysis with 1000 bootstrap alignments and 1000 maximum iterations. The minimum correlation coefficient was set to 0.99, and the SH-aLRT branch test replicates were set to 1000. The ingroup consists of five Conostigmus and five Dendrocerus species. Sequences of two Ceraphron species (accession Nos. MH733890 and GQ374733) were downloaded from GenBank and used as outgroups. Results Taxonomy Conostigmus Dahlbom, 1858 Conostigmus Dahlbom, 1858: 291. Dichogmus Thomson, 1858: 287. Eumegaspilus Ashmead, 1888: 48. Eumegalospilus: Schulz, 1906: 152. Conostigmoides Dodd, 1914: 88. Table 2. Detailed information of sequenced samples and accession numbers. Species Sex GenBank accession No. Reference D. carpenteri male MZ340592 Wang et al. (2021) female MZ340590 Wang et al. (2021) D. laticeps male MZ340593 Wang et al. (2021) female MZ340624 Wang et al. (2021) D. bellus male MZ344975 Wang et al. (2021) female MZ344976 Wang et al. (2021) D. anisodontus male MZ344977 Wang et al. (2021) female MZ344978 Wang et al. (2021) D. lui male OR120392 Li et al. (2023) female OR120391 Li et al. (2023) C. xui male SAMN44283636 this study C. nankunensis male SAMN44283637 this study C. ampullaceus male SAMN44283638 this study female SAMN44283639 this study C. rotundus sp. nov. male SAMN44283640 this study female SAMN44283641 this study C. clayulatus sp. nov. male SAMN44283642 this study female PQ686133 this study 382 ZooKeys 1255: 379–394 (2025), DOI: 10.3897/zookeys.1255.154710 Yuanhan Yang et al.: Two new species from Yintiaoling, China Ecnomothorax Dessart & Masner, 1965: 276. Dolichoceraphron Hellén, 1966: 15. Szelenyides Dessart, 1974: 43. Key to the species of Conostigmus from China (male) 1 Mesosoma distinctly elongate, length more than 2.0 × maximum width .... ....................................................................... C. ampullaceus Dessert, 1997 – Mesosoma moderately narrow, length 2.0 × or less than maximum width . ........................................................................................................................2 2 Facial pit absent ............................................................................................3 – Facial pit present ...........................................................................................6 3 Anteromedian projection of metanoto-propodeo-metapecto-mesopectal complex present ............................................................................................ 4 – Anteromedian projection of metanoto-propodeo-metapecto-mesopectal complex absent .............................................................................................5 4 Sternaulus extending to 2/3 length of mesopleuron .................................... ..................................................................... C. asperatus Wang & Zhu, 2025 – Sternaulus extending to 4/5 length of mesopleuron; facial sulcus present .............................................................. C. clayulatus Yang & Wang, sp. nov. 5 Harpe shorter than gonostipes ...........C. nankunensis Qian & Wang, 2024 – Harpe longer than gonostipes ................... C. abdominalis Boheman, 1832 6 Flagellomere 1 (F1) longer than scape .......... C. longus Wang & Zhu, 2025 – Flagellomere 1 (F1) shorter than or equal in length to scape ....................7 7 Body length (excluding antennae) ≥ 2.0 mm ...............................................8 – Body length (excluding antennae) < 2.0 mm .............................................10 8 Basal gastral carinae length ≥ 1/3 of syntergum length ............................. 9 – Basal gastral carinae length < 1/3 of syntergum length; harpe longer than gonostipes .............................................................. C. xui Cui & Wang, 2023 9 F1 subequal to scape; head and mesosoma reddish brown ........................ ...................................................................C. electrinus Wang & Chen, 2024 – F1 shorter than scape; head and mesosoma black ...................................... ................................................................................ C. villosus Dessert, 1997 10 Preoccipital furrow present and extends into the ocellar triangle ............... ........................................................................ C. acutus Wang & Chen, 2024 – Preoccipital furrow present but does not extend into the ocellar triangle .. ......................................................................................................................11 11 Scutellum as long as wide; dorsal margin of S9 straight ............................. ................................................................ C. rotundus Yang & Wang, sp. nov. – Scutellum longer than wide; dorsal margin of S9 protruded ........................ ............................................................ C. quadripetalus Wang & Chen, 2024 Conostigmus rotundus Yang & Wang, sp. nov. https://zoobank.org/C2EF15F6-E88E-4800-A927-D6CD28221BBD Diagnosis. The new species can be distinguished from other Conostigmus species by the following characters: facial sulcus absent; sternaulus present, elongate and reaching 4/5 of mesopleuron length; scutellum as long as wide; 383 ZooKeys 1255: 379–394 (2025), DOI: 10.3897/zookeys.1255.154710 Yuanhan Yang et al.: Two new species from Yintiaoling, China harpe simple, not bilobed, distal margin of harpe straight, and pointed laterally; and S9 bowl shaped, concave tip. Material examined. Holotype. ♂, China • Chongqing Municipality, Wuxi County, Yintiaoling Nature Reserve, Mt. Guanshan, 31°31'5.65"N, 109°42'59.07"E, alt. 1978 m, 22 July 2024, F. Li, W. J. Zhao, Y. C. Li leg. (CQWX-2402-0701-29). Paratypes. 1♂1♀, China • Chongqing Municipality, Wuxi County, Yintiaoling Nature Reserve, Mt. Guanshan, 31°31'5.65"N, 109°42'59.07"E, alt. 1978 m, 22 July 2024, F. Li, W. J. Zhao, Y. C. Li leg. (CQWX-2402-0701-28/32). Description. Male: Body length 1.4–1.6 mm (N = 2). Coloration (Fig. 1). Cranium, mesosoma and metasoma black. Mandibles brown palps yellow. Scape and pedicel brown, F1 to F9 black. Coxa of fore, mid and hind legs black to brown; rest of legs brownish yellow. Syntergum black; posterior metasomal segments black (Fig. 1G). Pterostigma, costal vein, Figure 1. Conostigmus rotundus Yang & Wang, sp. nov., male, holotype. A. Dorsal habitus; B. Head and mesosoma, dorsal view; C. Lateral habitus; D. Head and mesosoma, lateral view; E. Head, anterior view; F. Wings; G. Metasoma, dorsal view. 384 ZooKeys 1255: 379–394 (2025), DOI: 10.3897/zookeys.1255.154710 Yuanhan Yang et al.: Two new species from Yintiaoling, China and radial vein light brown (Fig. 1F). Body pubescence pale yellowish brown; marginal fringes of wings light brown. Head (Fig. 1B, D, E). About the width of mesosoma (about 1.02 × as wide as mesosoma). HH: EHf = 1.7–1.9. HH: HL = 1.6–1.7. HW: IOS = 1.4–1.6. HW: HH = 1.1. OOL longer than POL and ocellar triangle with narrow base. OOL: LOL = 2.9–3.0. POL: OOL = 0.6–0.7. Head oval with pubescence. Preoccipital lunula present. Preoccipital furrow present, ending inside ocellar triangle, but posterior to anterior ocellus. Median process on intertorular carina present, process not extending across intertorular area towards dorsal margin of clypeus. Facial sulcus absent. Facial pit present. Intertorular carina present. Antennae (Fig. 1C). Scape 1.1 × as long as the combined length of pedicel and F1. Scape length vs. pedicel length: 3.5–3.6. Scape length vs. F1 length: 1.5. F1 length vs. pedicel length: 2.5–2.6. F1 length vs. F2 length: 1.1. Flagellum cylindrical. Setae on flagellomeres shorter than the width of flagellomeres. Mesosoma (Fig. 1B, D). Pronotum shorter than the mesoscutum along the midline. AscW/PscW = 1.1. Mesosoma 1.3 × as long as wide (Length/ width/height = 526/396/402 μm), densely pubescent. Mesoscutum 2.0 × as wide as long (Width/length = 397/195 μm). Transscutal articulation present. Median mesoscutal sulcus present. Notaulus present, foveate and extends the length of the mesoscutum (percurrent), not adjacent to the median mesoscutal sulcus posteriorly. Scutoscutellar sulcus present and foveolate. Scutoscutellar sulcus adjacent and contiguous to transscutal articulation. Axillular carinae present. Scutellum as long as wide. Sternaulus present, elongate and reaching 4/5 of mesopleuron length. Anterior mesopleural sulcus present. Mesopleural sulcus shape: straight. Mesopleural pit present. Lateral propodeal carina forming an inverted “Y” shape. Wing (Fig. 1F). Fore wing total length 1.2–1.3 mm, translucent with nearly triangular pterostigma. Pterostigma length vs. width: 3.1. Radius (242 μm), slightly curved medially, 1.2 × as long as the length of the pterostigma. Fore wing with translucent orange-brown irregular stripes and dense light brown pubescence; wing marginal hairs longer than those on the inner wing surface; hind wing venation reduced, translucent. Metasoma (Fig. 1G). Metasoma 1.9 × as long as wide (Length/width/height = 768/413/322 μm). Metasoma shape: ovoid. Syntergum with five distinct gastral carinae, reaching 1/3 of syntergum length; syntergal translucent patch transverse. Rest of tergites smooth, with sparse hairs on both sides. Male genitalia (Fig. 2). Harpe slightly longer than gonostipes, with numerous slender apical setae; harpe orientation: medial; harpe shape: simple and not bilobed, distal margin straight dorsally and ventrally, and pointed; lateral setae of harpe present, but sparse. Parossiculus not fused medially. Gonostipes longer than wide, not fused with parossiculus. Penisvalva curved ventrally. Gonossiculus and gonossiculus spine present; apical parossiculal setae present. The middle part of male S9 slightly concave. The spiculum of S9 short, reaching 1/3 of S9. Female (Fig. 3). Same as the males, except for the following characters: body length = 1.9 mm; antennal pedicel long, slightly longer than any individual flagellomere from F1 to F8 of the same individual; legs tawny, coxa blackish-brown; wider mesosoma (Length/width = 554/451 μm). 385 ZooKeys 1255: 379–394 (2025), DOI: 10.3897/zookeys.1255.154710 Yuanhan Yang et al.: Two new species from Yintiaoling, China Remarks. This new species is similar to C. acutus Wang & Cui, 2024 in body length, preoccipital lunula, preoccipital furrow and lateral propodeal carina but can be distinguished by the almost circular scutellum (subcircular, subequal in length and width, more distinct than that in C. acutus); and the harpe straight apically and distal margin pointed in lateral view (harpe outward apically and distal margin beveled in lateral view). And this new species is similar to C. quadripetalus Wang & Chen, 2024 in the preoccipital lunula, preoccipital furrow, syntergum and harpe shape, but can be distinguished by body length (a longer body length than C. quadripetalus); S9 shape (male S9 shape of C. quadripetalus convex in distal margin but concave in C. rotundus); and HH: HL (a higher head height than C. quadripetalus). Distribution. China (Chongqing). Etymology. Consistent with the genus name, the species name is a Latin masculine adjective meaning “rounded”, referring to the nearly round scutellum of this species. Figure 2. Conostigmus rotundus Yang & Wang, sp. nov., male, holotype, genitalia. A. Dorsal view; B. Ventral view; C. Lateral view D S9. 386 ZooKeys 1255: 379–394 (2025), DOI: 10.3897/zookeys.1255.154710 Yuanhan Yang et al.: Two new species from Yintiaoling, China Conostigmus clayulatus Yang & Wang, sp. nov. https://zoobank.org/11E831F1-6770-49DE-AEC3-144A21A7BB05 Diagnosis. The new species can be distinguished from other Conostigmus species by the following characters: facial sulcus present and extends from the median ocellus to the intertorular carina; sternaulus present, elongate and reaching 3/4 of mesopleuron length; harpe with numerous long and slender apical setae and sparse lateral setae; and S9 cup-shaped with a straight tip bearing six setal patches at the end. Material examined. Holotype. ♂, China • Chongqing Municipality, Wuxi County, Yintiaoling Nature Reserve, Cotton Hill, 31°30'38.35"N, 109°42'4.54"E, alt. 2188 m, 22 July 2024, F. Li, W. J. Zhao, Y. C. Li leg. (CQWX-2402-0707-18). Paratypes. 1♂, China • Chongqing Municipality, Figure 3. Conostigmus rotundus Yang & Wang, sp. nov., female, holotype. A. Dorsal habitus; B. Head and mesosoma, dorsal view; C. Lateral habitus; D. Head and mesosoma, lateral view; E. Head, anterior view; F. Wings; G. Metasoma, dorsal view. 387 ZooKeys 1255: 379–394 (2025), DOI: 10.3897/zookeys.1255.154710 Yuanhan Yang et al.: Two new species from Yintiaoling, China Wuxi County, Yintiaoling Nature Reserve, Mt. Guanshan, 31°32'12.43"N, 109°41'58.38"E, alt. 2155 m, 21 July 2024, F. Li, W. J. Zhao, Y. C. Li leg. (CQWX-2402-0702-13). 1♀, China • Chongqing Municipality, Wuxi County, Yintiaoling Nature Reserve, Cotton Hill, 31°30'38.35"N, 109°42'4.54"E, alt. 2188 m, 22 July 2024, F. Li, W. J. Zhao, Y. C. Li leg. (CQWX-2402-0707-20). Description. Male: Body length 2.1–2.2 mm (N = 2). Coloration (Fig. 4). Cranium, mesosoma and metasoma black. Mandibles brown and palps yellowish brown. Scape, pedicel and F1 brown, F2 to F9 black. Legs and joint brownish yellow. Syntergum black (Fig. 4G); metasoma gaster black. Pterostigma, costal vein, and radial vein brown (Fig. 4F). Body pubescence pale yellowish brown; marginal fringes of wings light brown. Head (Fig. 4B, D, E). About the width of mesosoma. (about 0.96 × as wide as mesosoma). HH: EHf = 1.5–1.7. HH: HL = 0.7–1.1. HW: IOS = 1.4. HW: Figure 4. Conostigmus clayulatus Yang & Wang, sp. nov. male, holotype. A. Dorsal habitus; B. Head and mesosoma, dorsal view; C. Lateral habitus; D. Head and mesosoma, lateral view; E. Head, anterior view; F. Wings; G. 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