JHR 92: 173-184 (2022) or JOURNAL OF > 4?eertevewed open-access Jourel doi: 10.3897/jhr.92.86954 RESEARCH ARTICLE () Hymenopter a . https://jhr.pensoft.net The Iarational Sociery of Hymenoperiss, RESEARCH Additions to the genus Cratospila Foerster (Hymenoptera, Braconidae, Alysiinae) from South Korea Ju-Hyeong Sohn", Cornelis van Achterberg?*’, Yeonghyeok Yu', Hyojoong Kim! | Animal Systematics Lab., Department of Biological Science, Kunsan National University, Gunsan, 54150, Republic of Korea 2. State Key Laboratory of Rice Biology and Ministry of Agriculture, Hangzhou, 310058, China 3 Key Lab of Agricultural Entomology, Institute of Insect Science, Zhejiang University, Hangzhou, 310058, China Corresponding author: Hyojoong Kim (hkim@kunsan.ac.kr) Academic editor: Jose Fernandez-Triana | Received 25 May 2022 | Accepted 30 June 2022 | Published 31 August 2022 Attps://zoobank.org/B301EDB5-B06D-4219-8D0A-6308B1B8ACBD Citation: Sohn J-H, van Achterberg C, Yu Y, Kim H (2022) Additions to the genus Cratospila Foerster (Hymenoptera, Braconidae, Alysiinae) from South Korea. Journal of Hymenoptera Research 92: 173-184. https://doi.org/10.3897/ jhr.92.86954 Abstract Two new species of the genus Cvatospila Foerster, 1863 (Braconidae: Alysiinae), Cratospila albosignata sp. nov. and C. longivena sp. nov., are described and illustrated. In addition, the DNA barcode region of the mitochondrial cytochrome c oxidase subunit I (COL) of both species has been sequenced with three previously described species (C. albifera, C. luteocephala and C. syntoma). Alysia ponerola Papp, 2009 which was recorded from North Korea is transfered in Cratospila (C. ponerola (Papp, 2009) comb. nov.). All species validly recorded from Korea are included in a revised key. Keywords Alysiini, COI, Hymenoptera, new combination, new record, new species, taxonomy Introduction The subfamily Alysiinae is a large taxon of the family Braconidae, which includes two tribes, Alysiini and Dacnusini with 76 genera and 31 genera, respectively (Yu et al. 2016). Alysiinae contains over 2,440 valid species and the subfamily occurs worldwide * These authors contributed equally to this paper. Copyright Ju-Hyeong Sohn 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. 174 Ju-Hyeong Sohn et al. / Journal of Hymenoptera Research 92: 173-184 (2022) (Yu et al. 2016), of which 180 species in 21 genera are listed in the National Species List of South Korea (NIBR 2021). Although, Alysiinae are very similar to members of the subfamily Opiinae both genetically and morphologically, the former can be distin- guished from the latter easily by having the exodont (= non-overlapping) mandibles. Alysiinae is known as a group of koinobiont endoparasitoids of cyclorraphous dipter- ous larvae. They use the outward-curved teeth of the mandibles to break open the host puparium (Docavo et al. 2002). Some species are commercially utilized for biological control (Ozawa et al. 2001; Chabert et al. 2012). The genus Cratospila Foerster, 1863, is a small and rather isolated taxon in the subfamily Alysiinae, including 18 species worldwide (Yu et al. 2016; Sohn et al. 2021). This genus is easy to be diagnosed by having the first flagellomere at least 1.5 times longer than the second flagellomere and often with white antennal segments apically. So far, four species have been known in the Oriental region but two species of them are doubtful. Bhat (1980) reported C. curvabilis from India and C. bhutanensis from Bhutan; considering the original description it is questionable if the first species be- longs to Cratospila. Tobias (1990) described C. alboapicalis from Vietnam. Additional- ly, Wharton (2002) described six species (C. confusa, C. difficis, C. dracula, C. elongata, C. masneri, C. neocirce and C. storeyi) from Australia. Cratospila circe (Haliday, 1838) was reported from North Korea by Papp (1994) and also from Malaysia by Yaakop and Aman (2012), but both records are likely to concern one of the very similar local spe- cies of the genus (Sohn et al. 2021). So far, C. circe has been found only in the Western Palaearctic region. Checking figures of the holotype of Alysia ponerola Papp, 2009 (Papp 2009; NIBR 2012) made us aware that likely this species was classified into the wrong genus and should be transferred to Cratospila (C. ponerola (Papp, 2009) comb. nov.). Fortunately, it is different from the very similar and recently described C. albifera Sohn & van Achterberg, 2021 from S. Korea as indicated in the added key. In this study, we present new morphological characters and the barcoding se- quences of the CO/ region of both new species together with three previously de- scribed species (C. albifera, C. luteocephala and C. syntoma). Descriptions, diagnoses, species identification key, and photographs of the diagnostic characters of the new species are provided. Materials and methods Samples used in this study were collected with Malaise traps in South Korea at the Ne- bang-ri, Sudong-myeon, Nanyangju-si, Gyeonggi-do and Unilam Banilam, Jucheon- myeon, Jinan-gun, Jeollabuk-do. Sorting and preparation were done at the Animal Systematics Lab. (ASL), Department of Biology, Kunsan National University (KSNU) at Gunsan. For morphological identification, Wharton et al. (1997) and Zhu et al. (2017) were used. Morphological characters were observed with a Leica M205C ste- reo microscope. The Taxapad database (Yu et al. 2016) was used for references. We Review of the genus Cratospila (Hymenoptera, Braconidae, Alysiinae) from S. Korea 175 followed the terminology of Wharton (2002) and van Achterberg (1993). The type specimens are deposited KNA (Korea National Arboretum). A Leica DMC2900 digital camera and a Leica M205 C microscope (Leica Geosys- tems AG) were used for photography and several pictures being taken for each height using multi-focusing technology. LAS V4.11 (Leica Geosystems AG) and Helicon- Focus 7 (Helicon Soft) software were used for stacking work. After stacking work, illustrations were created using Adobe Photoshop CS6. Extraction of DNA was done in ASL, KSNU. Whole genomic DNA was extracted from the specimens by using a DNeasy Blood & Tissue kit (QIAGEN Inc., Dussel- dorf, Germany) following the manufacturer’s protocol. In order to conserve morpho- logically complete voucher specimens, DNA extraction method was used slightly modi- fied from ‘non-destructive method’ by Favret (2005) and ‘freezing method’ by Yaakop et al. (2009). In the original protocol, the sample was crushed or wounded, and then soaked with 180 ul of buffer ATL + 20 pl of proteinase, following by three hours over incubation at 55 °C. In the slightly modified DNA extraction methods, samples were soaked with 180 ul of buffer ATL + 20 ul of proteinase K without destroying the sam- ple, followed by 10 minutes incubation at 55 °C and then kept in a freezer at -22 °C overnight. After that the general protocol was used for the remaining steps. The primer set of LCO-1490 (5°--GGTCAACAAATCATAAAGATATTGG-3’) and HCO-2198 (5’°-TAAACTTCAGGGTGACCAAAAAATCA-3’) was used to amplify approximately 658 bp as the partial front region of the COM. The polymerase chain reaction (PCR) prod- ucts were amplified by using AccuPowerH PCR PreMix (BIONEER, Corp., Daejeon) in 20 ul reaction mixtures containing 0.4 uM of each primer, 20 uM of the dNTPs, 20 uM of the MgCL,, and 0.05 ug of the genomic DNA template. PCR amplification was per- formed using a GS1 thermo-cycler (Gene Technologies, Ltd., U.K) according to the fol- lowing procedure: initial denaturation at 95 °C for 5 min, followed by 34 cycles at 94 °C for 35 sec; an annealing temperature of 48 °C for 25 sec; an extension at 72 °C for 45 sec, and a final extension at 72 °C for 5 min. The PCR products were visualized by electro- phoresis on a 1.5% agarose gel. A single band was observed, purified using a QlAquick PCR purification kit (QIAGEN, Inc.), and then sequenced directly using an automated sequencer (ABI Prism 3730 XL DNA Analyzer) at Macrogen Inc. (Seoul, South Korea). Sequence alignment was performed in MEGA version 7 (Kumar et al. 2016) with ClustalW method. To estimate the pairwise genetic distances, the P-distance model was conducted using MEGA version 7. Results Total of 630 bp of the COI locus were sequenced for Cratospila albosignata sp. nov. (GenBank accession no. ON504323), Cratospila longivena sp. nov. (GenBank accession no. ON504322), C. albifera Sohn & van Achterberg, 2021 (GenBank accession no. MW376064), C. luteocephala Sohn & van Achterberg, 2021 (GenBank accession no. 176 Ju-Hyeong Sohn et al. / Journal of Hymenoptera Research 92: 173-184 (2022) MW376065) and C. syntoma Sohn & van Achterberg, 2021 (GenBank accession no. MW3760066). Pairwise genetic distances were caculated by using ‘P-distance’ model with option for pairwise deletion; C. albosignata differed by 7% from C. longivena, by 7% from C. albifera, by 9% from C. luteocephala and by 6% from C. syntoma (Table 1). Taxonomy Cratospila Foerster, 1863 Figs 1, 2 Cratospila Foerster, 1863: 265; Shenefelt. 1974: 985; Wharton 1980: 84; Tobias 1990; Belokobylskij 1998: 287; Yao et al. 2016: 1; Zhu et al. 2017: 60. Type species (by monotypy): Alysia circe Haliday, 1838. Hedylus Marshall, 1891: 14-15 (not Foerster, 1868); Papp 2009: 29-30 (as synonym of Cratospila because of synonymising both type species). Type species (by mono- typy): Hedylus habilis Marshall, 1894 (examined; = Alysia circe Haliday, 1838). Diagnosis. First flagellomere 1.5—2.1 times longer than second (Figs 1B, 2B), most species with 8—13 white segments in apical part of antenna (unknown of C. longivena, but has reddish brown head, morphologically related to C. albifera and has accord- ing to the COI analysis a derived position compared to other species), face with setae (Figs 1E, 2E), eye slightly oval, clypeus protruding anteriorly (Figs 1E, 2E), clypeus large, triangularly shaped and ventrally truncate, mandible with three teeth, second tooth narrow and sharp, maxillary palp with six segments, as long as mesosoma; no- tauli at least present anteriorly, scutellar sulcus distinct, precoxal sulcus medially deep- ly impressed and coarsely crenulate, more or less reduced anteriorly and posteriorly (Figs 1G, 2G); fore wing (Figs 1C, 2C) vein 2-SR slightly bent, vein 3-SR shorter than vein 2-SR; veins 2-SR+M and r-m not sclerotized, hind wing vein 1-M shorter than vein 1r-m; first tergite longer than second (Figs 1H, 2H). Biology. Rather small genus, of which the biology is unknown. Distribution. Cosmopolitan, except Neotropical region. Key to species of Cratospila Foerster from Korea 1 Mesoscutum medio-posteriorly and scutellum reddish brown; notauli on middle of mesoscutum comparatively coarsely crenulate; pterostigma rather slender and narrowly yellow basally; vein 1-SR+M of fore wing slightly sinu- ate; mesosoma 1.5—1.6 times longer than high in lateral view and anterior half of propodeum less sloping; propodeum less extensively rugose medially; antennal sockets comparatively close to level of inner side of eyes; [head in dorsal -viewruzellowésly brow | iecnew ves nee onertece vor oo sacey Sage ong onvcenttne vontncicemevtecusens 2 = Mesoscutum medio-posteriorly and scutellum black; head in dorsal view more transverse and at least posteriorly darkened; notauli on middle of Review of the genus Cratospila (Hymenoptera, Braconidae, Alysiinae) from S. Korea 177 mesoscutum narrowly crenulate; pterostigma rather robust and brown basal- ly; vein 1-SR+M of fore wing nearly straight; mesosoma 1.4—1.5 times longer than high in lateral view and anterior half of propodeum largely sloping; pro- podeum more extensively rugose medially; antennal sockets more removed FrOMIR CVE ROL MINS SIME VCSr. 9: acta rca daetctypuseseteneeeadee somave costs weiss ee + Minimum width of face 0.9 times its height (measured from lower rim of antennal socket to upper medio-dorsal margin of clypeus); vein r of fore wing approx. 3 times longer than wide; first subdiscal cell of fore wing approx. 7.5 times longer than wide; [colour of apical antennal segments unknown]........ L en Ateee mau cael eet See ce C. luteocephala Sohn & van Achterberg, 2021 Minimum width of face 1.2 times its height; vein r of fore wing 4—5 times longer than wide; first subdiscal cell of fore wing 4—5 times longer than wide; [antenna ot 9 with*lO—1l white segments] oie, Se ee ee ee 3 First tergite about twice as long as wide apically; eye in dorsal view approx. 2.4 times longer than temple and head in dorsal view more transverse (Fig. 2 in Papp 2009); apical antennal segment dark brown; vein r of fore wing leSS