Separatatus megagnathus sp. nov. is recorded as new to science from South Korea. Due to this record, the genus Separatatus Chen & Wu, 1994 (Braconidae: Alysiinae) is recognized for the first time from South Korea. The genus and species are described and illustrated herein plus an identification key including the Korean new species is provided. In addition, the DNA barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) has been analyzed for the new species.

COI barcode, cyclostome, koinobiont, natural enemy, parasitoid wasp, systematics, taxonomy

The subfamily Alysiinae is a relatively large taxon among the family Braconidae, which contains over 2450 valid species and is subdivided into two tribes, Alysiini with 76 genera and Dacnusini with 31 genera (Yu et al. 2016; Peris-Felipo and Belokobylskij 2020). There are 180 species of 21 genera listed in the National Species List of South Korea, South Korea (NIBR 2020). It is known that Alysiini includes mostly koinobiont endoparasitoids of cyclorrhaphous dipteran larvae, which are recognized by using their mandible (usually with 3–4 teeth or lobes) to break open the puparium of the host (Wharton 1977). Some species of Alysiinae are utilized for biological control (Abd-Rabou 2006).

The genus Separatatus Chen & Wu, 1994 is a small group of Alysiinae, which includes five Oriental or East Palaearctic species (Yu et al. 2016; Zhu et al. 2017; Zhang et al. 2020). This genus is easily characterized by the rugose second metasomal tergite, robust mandible and metanotum slightly or not protruding dorsally (Zhang et al. 2020). Chen and Wu (1994) created the genus Separatatus and described Separatatus carinatus Chen & Wu, 1994 as new species from China. Bobekioides sinicus, which had been described by Zheng et al. (2013) from China, was transferred to Separatatus by Zhu et al. (2017). Zhu et al. (2017) also described a new species, S. parallelus, from China. Yao et al. (2018) transferred Phasmidiasta malaysiae Fischer, 2006 to Separatatus, and added a new species, S. xuexincheni, from Thailand. Recently, Zhang et al. (2020) described a new species, S. yinshani, from China.

In this study, Separatatus megagnathus sp. nov. is recorded as new to science from South Korea. Simultaneously, the genus Separatatus Chen & Wu, 1994 (Braconidae: Alysiinae) is recognized for the first time from South Korea. We descried the morphological characters and the barcoding sequences of the COI region of this new species. A description, diagnosis and photographs of the diagnostic characters are also provided.

The holotype was collected with a Malaise trap 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 Biological Science, Kunsan National University (KSNU) at Gunsan, South Korea. Zhu et al. (2017), Yao et al. (2018) and Zhang et al. (2020) were followed for the morphological identification. Morphological characters were observed with a Leica M205C stereo microscope. The Taxapad database (Yu et al. 2016) was used for checking valid species and references. We followed the terminology of Wharton (2002) and van Achterberg (1993). The holotype is deposited in the Insect Museum, Department of Biological Science, KSNU.

A LEICA DMC2900 digital camera and a LEICA M205 C microscope (Leica Geosystems AG, Wetzlar, Germany) were used for photography and several pictures being taken for each height using multifocusing technology. LAS V4.11 (Leica Geosystems AG, Wetzlar, Germany) and HeliconFocus 7 (Helicon Soft, Kharkiv, Ukraine) software were used for image stacking. After stacking, plates were created using Adobe Photoshop CS6.

Extraction of DNA was done in ASL, KSNU. Whole genomic DNA was extracted from the specimen by using a DNeasy Blood & Tissue kit (QIAGEN Inc., Dusseldorf, Germany) following the manufacturer’s protocol. In order to conserve the morphologically complete voucher specimen, DNA extraction was slightly modified from the ‘non-destructive method’ of Favret (2005) and the ‘freezing method’ of 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, following by three hours over incubation at 55 °C. In our 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 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 primerset of LCO-1490 (5’-GGTCAACAAATCATAAAGATATTGG-3’) and HCO-2198 (5’-TAAACTTCAGGGTGACCAAAAAATCA-3’) (Folmer et al. 1994) 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 AccuPowerH PCR PreMix (BIONEER, Corp., Daejeon) in 20 μl reaction mixtures containing 0.4 μM of each primer, 20 μM of the dNTPs, 20 μM of the MgCl₂, and 0.05 μg of the genomic DNA template. PCR amplification was performed using a GS1 thermo-cycler (Gene Technologies, Ltd., Essex, U.K) according to the following procedure: initial denaturation at 95 °C for 5 min, followed by 34 cycles at 94 °C for 35 s; an annealing temperature of 48 °C for 25 s; an extension at 72 °C for 45 s, and a final extension at 72 °C for 5 min. The PCR products were visualized 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, ABI, Waltham, MA, USA) at Macrogen Inc. (Seoul, South Korea).

A total of 620 bp of the COI fragments were sequenced from Separatatus megagnathus sp. nov. which was deposited in GenBank (accession number MZ717197). Unfortunately, it could not be used to measure genetic distance between related taxa because there is no sequence of a congeneric species in GenBank. However, the sequence will facilitate the recognition of the new species if other sequences of Separatatus are added in future.

 Separatatus Chen & Wu, 1994

Separatatus Chen & Wu, 1994: 132. Type species: Separatatus carinatus Chen & Wu, 1994.

Diagnosis

Antenna 1.0–1.3 times longer than body; first flagellomere slightly shorter than second (Fig. 1B), face with setae (Fig. 1E), eye slightly oval and glabrous; clypeus semicircular; labrum long and triangularly shape, mandible with 3–4 teeth or lobes (Fig. 1J), fourth ventral tooth (if present) small; maxillary palp with 6 segments; notauli present on anterior third of mesoscutum; medio-posterior depression distinct, round or longitudinal; scutellar sulcus distinct; fore wing (Fig. 1C) vein 2-SR slightly bent, vein 2-SR slightly shorter than vein 3-SR; vein 2-SR+M not sclerotized; hind wing vein 1-M longer than vein 1r-m; second tergite rugose, longer than first (Fig. 1H); ovipositor sheath slightly shorter than metasoma; tarsal claws slender.

Figure 1. 

Separatatus megagnathus sp. nov., ♀ A habitus, lateral view B antennae C wings D head, dorsal view E head, front view F mesosoma, dorsal view G mesosoma, lateral view H anterior half of metasoma, dorsal view I ovipositor sheath, lateral view J mandible, lateral view K mandible, antero-lateral view L mandible with teeth. Scale bars: 1 mm (A, C); 0.5 mm (B, I); 0.3 mm (D–H); 0.1 mm(J–L)

Biology

Unknown.

Distribution

Oriental and Palaearctic region.

This work was supported by a grant from the National Institute of Biological Resources (NIBR), funded by the Ministry of Environment (MOE) of the Republic of Korea (NIBR202102204). This work was also supported by the Korea National Arboretum (KNA1-1-20). This work was supported by Korea Environment Industry & Technology Institute (KEITI) through Exotic Invasive Species Management Program, funded by Korea Ministry of Environment (MOE)(2018002270005).