Three new species and two new records of the genus Phaenocarpa Foerster (Hymenoptera, Braconidae, Alysiinae) from South Korea

The species of the genus Phaenocarpa Foerster, 1863 (Braconidae: Alysiinae) from South Korea are revised, and the genus is recorded for the first time from South Korea. Three species, Phaenocarpa artotemporalis sp. nov., P. brachyura sp. nov. and P. lobata sp. nov., are new to Science, and two species, P. masha Belokobylskij, 1998 and P. fidelis Fischer, 1970, are newly recognized in South Korea. They are described and illustrated herein with a provision of the identification key to the Korean species. In addition, the DNA barcode region of the mitochondrial cytochrome c oxidase subunit I (COI) has been analyzed for the six species including P. ruficeps for genetic comparison.


Introduction
The subfamily Alysiinae is a relatively large taxon among the family Braconidae, and is subdivided into two tribes; the tribes Alysiini with 76 genera and Dacnusini with 31 genera (Yu et al. 2016). The two tribes are distinguished by the presence (or absence) of fore wing vein r-m as a main key character (Shaw and Huddleston 1991). Alysiinae occurs worldwide and contains over 2,440 valid species (Yu et al. 2016), of which 180 species in 21 genera are listed in the National Species List of South Korea (NIBR 2019). This group is known as koinobiont endoparasitoids of dipterous larvae, using their mandible (with three or four teeth, rarely more or less) to break open the puparium of the host. Some of them, such as Dacnusa sibirica in Dacnusini, are commercially utilized in biological control for Liriomyza trifolii (Abd-Rabou 2006).

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, Wharton et al. (1997) and Zhu et al. (2017) were used. Morphological characters were observed with a Leica M205C stereo microscope. The Taxapad database (Yu et al. 2016) was used for references. We 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 Geosystems 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., Dusseldorf, Germany) following the manufacturer's protocol. In order to conserve morphologically complete voucher specimens, DNA extraction method was used slightly modified 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 μl of buffer ATL + 20 μl of proteinase, following by three hours over incubation at 55 °C. In the 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 primer set of LCO-1490 (5'-GGT-CAACAAATCATAAAGATATTGG-3') and HCO-2198 (5'-TAAACTTCAGGGT-GACCAAAAAATCA-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 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 MgCl 2 , and 0.05 μg of the genomic DNA template. PCR amplification was performed using a GS1 thermo-cycler (Gene Technologies, Ltd., U.K) 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 visualized by electrophoresis on a 1.5% agarose gel. A single band was observed, purified using a QIAquick PCR purification kit (QIA-GEN, 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 tool. To estimate the pairwise genetic distances, the P-distance model was conducted using MEGA version 7.
Description. Holotype, ♀: length of body in lateral view 2.0 mm, length of antenna 3.2 mm, and length of fore wing 2.6 mm.
Leg. Hind coxa compressed, grooved and 1.4 times longer than hind trochanter; hind femur 0.8 times longer than hind tibia; hind tibia 1.2 times longer than hind tarsus.
Male. Unknown. Distribution. South Korea. Etymology. From "artus/arctus" (Latin for narrow) and "tempus" (Latin for area behind eyes) because of the enlarged temples. Comparative diagnosis. Runs to the East Palaearctic P. basarukini Belokobylskij, 1998 and to the West Palaearctic P. curticauda van Achterberg, 1998 because of the short ovipositor sheath (approx. 0.7 times as long as hind tibia). The new species differs from P. basarukini by the slightly longer ovipositor sheath (0.5-0.6 times as long as hind tibia in P. basarukini), first mandibular tooth distinctly lobe-shaped and middle tooth slenderer, pterostigma and hind femur wider. Differs from P. curticauda by the wider pterostigma, longer 1r-m of hind wing and 1-CU1 of fore wing, second tooth less widened dorsally, complete notauli, first tergite 1.4 times longer than its apical width and areola of propodeum wider.

Phaenocarpa brachyura
Colour. Head black, around eye brown; antenna yellowish brown; mandible brown and apically dark brown. First tergite dark brown and mesonotum entirely black.
Head. Head (Fig. 2D) width 1.6 times median length in dorsal view. Antenna (Fig. 2B) twice as long as body, 41 segmented. Fourth segment 1.4 times longer than third segment and 1.2 times longer than fifth segment. Eye slightly oval, 1.2 times as long as wide in lateral view. Width of face (Fig. 2E) 1.6 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 3.0 times longer than diameter of anterior ocellus; OOL:AOL:POL = 21:5:7. Stemmaticum concave. Mandible with three teeth (2L), subparallel-sided, first mandibular tooth distinctly lobe-shaped and separated from second tooth by incision. Labrum 2.9 times longer than wide. Maxillary palp 0.4 times longer than mesosoma.
Leg. Hind coxa compressed, grooved and 1.3 times longer than hind trochanter; hind femur 0.7 times longer than hind tibia; hind tibia 0.8 times longer than hind tarsus.
Male. Unknown. Distribution. South Korea. Etymology. From "brachys" (Greek for short) and "oura" (Greek for tail) because of the comparatively short ovipositor sheath. Comparative diagnosis. Differs from other species treated in this paper by the long pterostigma of the fore wing (approx. 5.5 times as long as wide; 2.9-4.4 times in other species). The orange antenna and the yellow and robust third and fourth antennal segments distinguish it from other Phaenocarpa species. Closely related to P. masha Belokobylskij, 1998, for differences see key above.

Phaenocarpa lobata
Description. Holotype, ♀; length of body in lateral view 3.3 mm (Fig. 3A), length of antenna 6.1 mm and length of fore wing 3.6 mm.
Colour. Head (Fig. 3D) black; antenna orange (third and fourth antennal segments yellow); mandible orange brown. First tergite dark brown and mesonotum entirely reddish brown, area around of medio-posterior depression orangish brown.
Head. Width 1.9 times median length in dorsal view. Antenna (Fig. 3B) twice as long as body, 39 segmented. Fourth segment 1.5 times longer than third segment and 1.3 times longer than fifth; third and fourth segments robust. Eye slightly oval, 1.3 times as long as wide in lateral view. Width of face (Fig. 3E) 1.6 times its height from ventral rim of antennal sockets to upper margin of clypeus. Eye in dorsal view 2.6 times as long as temple. Ocello-ocular line 4.1 times longer than diameter of anterior ocellus; OOL:AOL:POL = 25:6:9. Stemmaticum concave. Vertex smooth, glossy. Mandible with three teeth and first tooth separated from second tooth by incision (Fig. 3L); middle tooth of mandible widened dorsally, with black tip. Labrum 2.0 times longer than wide. Maxillary palp 0.8 times longer than mesosoma.
Mesosoma. Mesosoma (Fig. 3G) 1.8 times longer than wide in dorsal view. Notauli comparatively coarsely crenulate near middle of mesoscutum and reaching Figure 3. Phaenocarpa lobata sp. nov., ♀ A body B antennae C wings D head, dorsal E head, anterior F mesosoma, lateral G mesosoma, dorsal H propodeum and metasoma, dorsal I ovipositor and its sheaths, lateral J tarsus, lateral K tarsus, dorsal L mandible, lateral. medio-posterior depression; medio-posterior depression nearly reaching transscutal articulation; scutellar sulcus with two carinae; metanotum obtuse dorsally in lateral view; small bump on hind coxa adjacent to metapleuron; in lateral view metapleuron with distinct setae. Anterior half of propodeum less sloping; propodeum bent in lateral view; precoxal sulcus ( Fig. 3F) deep and distinct, with about eight crenulae. Fore wing (Fig. 3C) 2.4 times as long as wide; pterostigma long and narrow, 5.5 times longer than wide; vein r of fore wing 1.5 times longer than wide; vein 2-SR slightly bent; vein r-m not sclerotized; vein 2-SR:vein r:vein 3-SR = 11:2:16; first subdiscal cell of fore wing ca 0.9 times longer than wide.
Leg. Hind coxa compressed, grooved and 1.2 times longer than hind trochanter; hind femur 0.6 times longer than hind tibia; hind tibia 1.2 times longer than hind tarsus; tarsal claws robust.
Male. Unknown. Distribution. South Korea. Etymology. From "lobus" (Latin for lobe), because of the lobe-shaped third mandibular tooth. Description. ♀; length of body in lateral view 2.9 mm (Fig. 4A), length of antenna 4.7 mm, length of fore wing 3.1 mm.
Head. Width 1.9 times median length in dorsal view. Antenna (Fig. 4B) 1.6 times longer than body, 28 segmented. Fourth segment 1.6 times longer than third segment, 1.1 times longer than fifth. Eye slightly oval, 1.2 times longer than wide in lateral view. Width of face (Fig. 4E) 1.3 times its height from ventral rim of antennal sockets to upper margin of clypeus. Face with dense setae. Eye in dorsal view 2.6 times longer than temple. Ocello-ocular line 2.6 times longer than diameter of anterior ocellus; OOL:AOL:POL = 17:5:7. Stemmaticum concave. Mandible robust with three teeth (Fig. 4L) and first tooth gradually connected to second tooth, forming a straight or arcuate connection. Labrum 1.8 times longer than wide. Maxillary palp 0.7 times longer than mesosoma.
Leg. Hind coxa compressed, grooved and 1.1 times longer than hind trochanter; hind femur 0.7 times longer than hind tibia; hind tibia 0.9 times longer than hind tarsus.
Male. Unknown. Distribution. Eastern Palaearctic, Western Palaearctic, Europe; South Korea (new record). Description. ♀; length of body in lateral view 2.8 mm (Fig. 5A), length of antenna 4.7 mm and length of fore wing 3.3 mm.
Head. Width 1.7 times median length in dorsal view. Antenna (Fig. 5B) 1.6 times longer than body, 27-segmented. Fourth segment 1.6 times longer than third segment and 1.2 times longer than fifth, third and fourth segments dark brown and slender. Eye slightly oval, 1.2 times as long as wide in lateral view. Width of face (Fig.  5E) 1.5 times its height from ventral rim of antennal sockets to upper margin of clypeus. Face with dense setae. Eye in dorsal view 3.0 times as long as temple. Ocelloocular line 4.1 times longer than diameter of anterior ocellus; OOL:AOL:POL = 21:5:7. Stemmaticum concave. Mandible with three teeth (Fig. 5L) and first tooth separated from second tooth by incision; third tooth bent outside, middle tooth not widened dorsally. Labrum 2.4 times longer than wide. Maxillary palp 0.9 times longer than mesosoma.
Leg. Hind coxa compressed, grooved and 1.4 times longer than hind trochanter; hind femur 0.6 times longer than hind tibia; hind tibia 1.2 times longer than hind tarsus; tarsal claws slender.