A new species of the genus Hylcalosia Fischer (Hymenoptera: Braconidae: Alysiinae) from South Korea, with a key to the Korean species

Abstract The species of the genus Hylcalosia Fischer, 1967 (Braconidae: Alysiinae) from South Korea are revised. One species, Hylcalosiabicolorsp. nov., is new to science. They are described and illustrated herein and an identification key to the Korean species is added. In addition, the DNA barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) has been analysed for the new species and H.sutchanica is used for genetic comparison.


Introduction
The subfamily Alysiinae, which is one of the large taxa in the family Braconidae, occurs worldwide and contains over 2,440 valid species (Yu et al. 2016). In Korea, 180 species in 21 genera are listed in the National Species List of South Korea (NIBR 2019). This group can be discriminated from other subfamilies by having non-overlapping mandibles and is subdivided into two tribes, Alysiini and Dacnusini, which are distinguished from each other by the presence or absence of fore wing vein r-m, respectively (Shaw and Huddleston 1991). Alysiinae are known as koinobiont endoparasitoids of dipteran larvae, characteristically using their mandible (with three or four teeth, rarely more or less) to break open the puparium of the host.
Hylcalosia Fischer, 1967 is a small genus of Alysiinae, which includes 18 species (Yu et al. 2016, Yao et al. 2020). This genus is easily diagnosed by the rugose or granulated second and third metasomal tergites combined by the acutely protruding clypeus and enlarged upper valve of the ovipositor (van Achterberg 1983). Fischer (1967) re-described the type species from Myanmar Holcalysia ruficeps Cameron, 1910. Van Achterberg (1983 revised the genus Hylcalosia and described two new species: H. maetoi and H. hemiflava from Japan and Indonesia, respectively. Belokobylskij (1992) added two new species from Russia: H. hymaenei and H. sutchanica. Papp (1994) Yao et al. (2020).
In this study, we present new morphological characters and the barcoding sequences of the COI region of H. bicolor sp. nov. and one previously-recorded species, H. sutchanica. Descriptions, diagnoses, an identification key and photographs of the diagnostic characters are provided.

Materials and methods
Samples used in this study were collected with Malaise traps in South Korea at the DMZ Botanical Garden, Mandae-ri, Haean-myeon, Yanggu-gun, Gangwon-do. Sorting and preparation were done at the Animal Systematics Lab. (ASL), Department of Biology, Kunsan National University (KSNU) at Gunsan. For morphological identification, Zhu et al. (2017Zhu et al. ( , 2018 and Yao et al. (2020) were used. Morphological characters were observed with a Leica M205C stereomicroscope. The Taxapad database (Yu et al. 2016) was used for references. We followed the terminology of Wharton (2002) (1993). The type specimens are deposited in Korea National Arboretum (KNA). A LEICA DMC2900 digital camera and a LEICA M205C stereomicroscope (Leica Geosystems AG) were used for photography and several pictures were taken for each height using multi-focusing technology. LAS V4.11 (Leica Geosystems AG, Wetzlar, Germany) and HeliconFocus 7 (Helicon Soft, Kharkiv, Ukraine) 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., Dusseldorf, Germany) following the manufacturer's protocol. In order to conserve morphologically-complete voucher specimens, the DNA extraction method was used slightly modified from the '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 μl of buffer ATL + 20 μl of proteinase, followed by three hours incubation at 55°C. In slightly modified DNA extraction methods, samples were soaked with 180 μl of buffer ATL + 20 μl of proteinase K without destroying the sample, followed by 20 minutes incubation at 55°C and then kept in a freezer at -21°C overnight. After that, the general protocol was used for the remaining steps. The primer-set of LCO-1490 (5'-GGTCAACAAATCATAAAGA-TATTGG-3') and HCO-2198 (5'-TAAACTTCAGGGTGACCAAAAAATCA-3') was used to amplify approximately 658 bp as the partial front region of the COI. The polymerase chain reaction (PCR) products were amplified by using AccuPow-erH PCR PreMix (BIONEER, Corp., Daejeon, Korea) in 20 μl reaction mixtures containing 0.4 μM of each primer, 20 μM of dNTPs, 20 μM of MgCl 2 and 0.05 μg of the genomic DNA template. PCR amplification was performed using a GS1 thermo-cycler (Gene Technologies, Ltd., Essex, UK) according to the following 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 visualised by electrophoresis on a 1.5% agarose gel. A single band was observed, purified using a QIAquick PCR purification kit (QIAGEN, Inc., Milan, Italy) and then sequenced directly using an automated sequencer (ABI Prism 3730 XL DNA Analyzer) at Macrogen Inc. (Seoul, South Korea).
Sequence alignments were performed in MEGA version 7 (Kumar et al. 2016) with the ClustalW tool. To estimate the pairwise genetic distances, the P-distance model was conducted using MEGA version 7.

Results
A total of 563 bp of the COI barcode region were sequenced from H. bicolor sp. nov. and H. sutchanica which were deposited in GenBank (accession numbers MZ717196, MZ717194). Pairwise distances were estimated by using the P-distance model with the option for pairwise deletion. As a result, H. bicolor sp. nov. showed a fairly large genetic difference of 6% from H. sutchanica.
Description. ♀. Length of body in lateral view 2.6 mm, length of antenna 4.7 mm and length of fore wing 2.9 mm.
Colour. Body (Fig. 1A) mainly reddish-brown; head black; antenna brown basally; mandible reddish-brown. Head. Head (Fig. 1D) width 1.6 times median length in dorsal view. Antenna (Fig. 1B) 1.8 times longer than body in female, 43-segmented. First flagellomere 0.7 times longer than second. Eye slightly oval, 1.1 times as long as wide in lateral view. Width of face (Fig. 1E) 2.1 times its height from ventral rim of antennal sockets to upper margin of clypeus; face with long setae. Eye in dorsal view 1.7 times as long as temple. Ocello-ocular line (OOL) 4.5 times longer than diameter of anterior ocellus; OOL:antero-posterior ocellar line (AOL):postero-ocellar line (POL) = 18:6:7. Stemmaticum concave. Vertex smooth and shiny with groove. Mandible with four teeth or lobes (Fig. 1J); dorsal tooth large and lobe-shaped; ventral tooth lobe-shaped, middle of tooth curved. Medial length of mandible 1.7 times longer than maximum width. Labrum small, 1.4 times longer than wide. Maxillary palp 0.5 times longer than mesosoma.

Material
Head. Head (Fig. 2D) width 1.6 times median length in dorsal view. Antenna (Fig.  2B) 1.6 times longer than body, 40 or 42 segmented. First flagellomere 0.7 times as long as second, second flagellomere 1.1 times longer than third. Eye slightly oval, 1.1 times as long as wide in lateral view. Width of face (Fig. 2E) 2.0 times its height from ventral rim of antennal sockets to upper margin of clypeus; face with long setae. Eye in dorsal view 2.4 times as long as temple. Ocello-ocular line (OOL) 4.0 times longer than diameter of anterior ocellus; OOL:antero-posterior ocellar line (AOL):posteroocellar line (POL) = 12:5:5. Vertex smooth and shiny with groove. Mandible (Fig. 2J) with four teeth and setae; dorsal tooth large and lobe-shaped and distinctly surpassing apex of first tooth, ventral tooth lobe-shaped, middle of tooth curved; second tooth narrow and sharp with dark brown tip and separated from first tooth by incision in lateral view. Medial length of mandible 1.7 times longer than maximum width. Labrum small, 1.3 times longer than wide. Maxillary palp 0.7 times as long as mesosoma. Mesosoma. Mesosoma (Fig. 2G) 2.1 times longer than wide in dorsal view; notauli moderately crenulated, but situated far from comparatively small medio-posterior depression (Fig. 2F); scutellar sulcus with four carinae; laterally mesopleuron and metapleuron with long setae, metapleuron distinctly rugose. Anterior half of propodeum smooth, posterior of median carina reticulate-rugose (Fig. 2H), lateral view of propodeum not curved dorsally; precoxal sulcus ( Fig. 2F) shallow and with 14 crenulae. Fore wing (Fig. 2C) 2.4 times longer than wide; pterostigma long and thick, 3.4 times longer than wide; vein r of fore wing 2.9 times longer than wide; vein 2-SR slightly bent; vein 2-SR+M and r-m not sclerotised; veins 2-SR: r: 3-SR = 11:2:7; first subdiscal cell of fore wing 1.5 times longer than wide. Hind wing veins M+CU:1-M:1r-m = 11:7:5.
Leg. Hind coxa smooth and 1.2 times longer than hind trochanter; hind femur 0.9 times as long as hind tibia and 8.5 times longer than wide; hind tibia 0.9 times longer than hind tarsus, tarsal claws slender (Fig. 2L).