Description of one new species of Agriotypus Curtis, 1832 (Hymenoptera, Ichneumonidae, Agriotypinae) from South Korea

Two species of the genus Agriotypus Curtis, 1832, A. jilinensis Chao, 1981 and A. wangpiensis sp. nov. , are added to the South Korean fauna. In total, four species, A. gracilis , A. jilinensis , A. silvestris , and A. wangpien-sis sp. nov. , have now been found in South Korea. The female of A. jilinensis is described for the first time and the species is redescribed based on the first fully eclosed specimens. Photographs of collecting sites, the larva and the adult of A . wangpiensis sp. nov. , and a key to the South Korean species of Agriotypus are provided.


Introduction
The subfamily Agriotypinae (Hymenoptera: Ichneumonidae) is a small group of aquatic parasitoid wasps which are known to parasitize trichopteran pupae and prepupae in fast-running water but, some species (A.armatus) can attack hosts in lakes and slow-moving rivers (Townes 1969;Bennett 2001).The subfamily is monotypic with Agriotypus Curtis as the only recognized genus.Until recently, the genus was comprised of 16 species from the Palaearctic and Oriental Regions (Bennett 2001) however, five species were just reported from China (Tang et al. 2022), thus, a total of 21 species of Agriotypus are currently known.In South Korea, two species, A. gracilis Waterston and A. silvestris Konishi and Aoyagi, were previously recorded and A. armatus Curtis was excluded from the South Korean fauna (Kim et al. 2018).
This genus can be easily identified by the following characteristics: a strongly distinct spine on the scutellum; all metasomal sternites fully sclerotized; fused 2 nd and 3 rd metasomal tergites and sternites.Because of the latter two characteristics, Mason (1971) proposed Agriotypus should be given family status in the superfamily Proctotrupoidea.However, all other taxonomists preferred to recognize Agriotypus within Ichneumonidae (Short 1952;Townes 1969;Sharkey and Wahl 1992;Bennett 2001).Also, Chao (1992) described the genus Atopotypus Chao as a new monotypic Agriotypinae genus, but Bennett (2001) synonymized Atopotypus with Agriotypus forming Agriotypus succinctus (Chao), based on the results of a cladistic analysis of morphological characters of adults and larvae.Bennett (2001) also defined two species groups within Agriotypus, the Palaearctic armatus species group and the Oriental himalensis species group (based on the absence or presence, respectively of longitudinal carinae at the base of the second metasomal tergite).Prior to this study, the armatus group included A. armatus Curtis, A. changbaishanus Chao, A. gracilis Waterston, A. jilinensis Chao, A. silvestris Konishi & Aoyagi and A. succinctus (Chao).
In this study, a new species, A. wangpiensis sp.nov., and the female of A. jilinensis are described, including the larva of A. wangpiensis sp.nov.We also provide DNA barcodes and an illustrated identification key to the South Korean species.

Materials and methods
Type specimens of the new species are preserved in the Insect Inquiry Education Institute in Daegu National University of Education (DNUE-IIEI, Daegu, Korea), Nakdonggang National Institute of Biological Resources (NNIBR, Sangju, Korea), and Ehime University of Matsuyama (EUM, Japan).Other examined material is deposited in the Canadian National Collection of Insects, Arachnids and Nematodes (CNC, Ottawa, Canada).Specimens used in this study were collected by rearing from trichopteran pupae and prepupae (Figs 2, 3) and Malaise trapping.Most of the images of specimens of the new species were taken using an AxioCam MRc5 camera attached to a stereo microscope (Zeiss SteREO Discovery.V20; Carl Zeiss, Göttingen, Germany), processed using AxioVision SE64 software (Carl Zeiss), and optimized with a Delta imaging system (i-solution, IMT i-Solution Inc.Vancouver, Canada).Some images of the new species were taken using a Leica MC190 HD Camera attached to a Leica M125 Microscope (Leica Microsystems, Germany) and processed using LEICA LAS X software (Leica).The epomia is not visible in Figs 5E, 8D, but we verified that it extends dorsal to pronotal furrow.This character is very hard to photograph because of two factors: first, the region is highly setose which obscures the epomia and second the area is often in shadow because of the head and mesoscutum.The morphological terminology follows Broad et al (2018), except for the terms for the male genitalia that follow Konishi (2005).
The examined Agriotypus wangpiensis sp.nov.specimens for DNA barcoding are deposited in DNUE-IIEI.Genomic DNA from two adult females and five adult males, one pupa, and one larva were extracted using a DNeasy Blood & Tissue Kit (Qiagen, Valencia, California).Each PCR was performed in a 30µl volume consisting of 15µl of premix (Solg TM 2× Taq PCR Pre-Mix: 0.5× Band Doctor TM with dye, Taq DNA polymerase (5U/µl), 10× Taq Reaction Buffer (25 mM MgCl 2 included), 10 mM each dNTP Mix), 2µl of DNA template, 1µl of each primer as 10 pmol, and 11µl of DNase free water.An approximately 650-700 bp piece of the mitochondrial cytochrome oxidase I(COI) gene was amplified using the primers LCO1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and HCO2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') (Folmer et al. 1994).Polymerase chain reactions were run with an initial predenaturation step at 94 °C for 5 min, followed by 35 cycles of denaturation for 1 min at 94 °C, 1 min of annealing at 50 °C, and 1 min of extension at 72 °C; the last cycle was followed by a final 5 min extension step.The amplified fragments were cleaned and sequenced by BIONEER (Daejeon, South Korea), after which the sequences were edited and aligned.Sequences have been deposited in GenBank under accession numbers OQ981233.1-OQ981241.1.Agriotypus silvestris and A. gracilis were barcoded with the following modifications to the above protocol.The amplification components were 2µl 2.5 mM dNTPs.0.2 µl TaKaRa Ex Taq® Hot Start, 2.5 µl 10× Ex Taq buffer, 2 µl 25 mM MgCl2, 1µl of 10 µM primers LCO1490 and HCO2198, 1-4 µlgenomic DNA and water to 25 µl.The amplification protocol was as follows: 95 °C for 1 minute, 35 cycles of 95 °C for 15 s, 49 °C for 15 s, and 72 °C for 45 s, and 72 °C for 4 minutes.The amplified product was viewed on a 2% agarose gel with GelRed, then cleaned with ExoSAP-IT (PE Applied Biosystems, Foster City, CA, USA).Cycle sequencing was completed with the BigDye Terminator v3.1 kit (PE Applied Biosystems, Foster City, CA, USA) in 10 µl reactions.Sequencing was completed at the Agriculture & Agri-Food Canada Ottawa Research and Development Centre Core Sequencing Facility (Ottawa, ON, Canada) on a 3500 xl DNA Genetic Analyzer (PE Applied Biosystems, Foster City, CA, USA).All sequences obtained were assembled and manually edited using Bioedit 7.2.5 (Hall 1999) and finally aligned using ClustalW implemented in MEGA 11 (Tamura et al. 2021).COI sequences were aligned using MEGA 5.2 (Tamura et al. 2011) and Clustal W. A dataset consisting of 25 COI sequences was finally trimmed to 537 bp in length.
The final tree was visualized using FigTree v1.4.4 (http://tree.bio.ed.ac.uk/software/figtree).The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree.The evolutionary distances were computed using the Kimura 2-parameter method.The evolutionary distance is omitted in the Fig. 10  Synonymized by Bennett, 2001.Face with median vertical ridge.Fore wing with abscissa of vein M between 2rs-m and 2m-cu 0.3-0.9times as long as 2rs-m.Digitus elongated, but shorter than lamina volsellaris (Fig. 7C Head.Face width (minimum length of inner orbits) 1.1 times as wide as its median height (length between antennal socket and clypeal margin), with a median vertical ridge (see Fig. 4G in Bennett 2001), with dense punctures and hairs except area between lateral ocellus and eye impunctate (Fig. 1B).Antenna with 20-22 flagellomeres, 0.5 times as long as fore wing.Temple behind eyes slightly roundly narrowed in dorsal view.Antennal scrobe deep.POL: OOL = 1:1-1:2.Face with median longitudinal ridge, coarse rugosity and dense pubescence.Area between antennal sockets with a glabrous short longitudinal tubercle.Clypeus in anterior view with apical margin truncate, not pointed; anterior edge in profile moderately convex; summit of convexity in lateral view angulate; shape of angular summit of clypeus in anterior view rounded medially (Fig. 1B).Distance between anterior tentorial pits 1.2-1.3times as long as length between a tentorial pit and eye.Occipital carina weak, but complete.Malar space 1.4 times as long as basal width of mandible; mandible twisted, lower tooth longer than upper one.

Key to the species of South Korean
Mesosoma.Pronotum with long and strong epomia extending dorsal of furrow and almost straight.Mesoscutum shiny, anterior 1/2-2/3 of median lobe moderately punctate and setose, the rest of medial lobe and all lateral lobes completely impunctate and lacking setae; notaulus distinct, meeting at around middle (Fig. 1D).Scutellum in dorsal view, long triangle-shaped, 1.6 times as long as its basal width including spine, 0.9-1.1 times as long as propodeum; scutellum with lateral carinae at base, convex medially, taper ing toward apex, spine-like apical half, smooth (Fig. 1E); spine 0.5-0.6 times as long as propodeum (distance as long as lateral longitudinal carinae from base to apex); in lateral view roundly curved medially, weakly up-curved (Fig. 8D).Mesopleuron finely punctate and pubescent, with longitudinal groove complete, without longitudinal carina on anterior edge at mid-height; epicnemial carina curving sharply anteriorly to meet anterior edge of mesopleuron and with a vertical extension dorsally; sternaulus present as a weak and wide groove.Metapleuron finely punctate and pubescent Propodeum with laterome dian longitudinal carinae almost parallel; lateral longitudinal carinae straight, complete and parallel to lateromedian longitudinal carinae (Fig. 1F, G).
Colour.Generally reddish brown.Head, pronotum, mesoscutum and metasoma reddish brown.Scutellum and metasomal tergite I dark reddish brown.All legs, mesopleuron and propodeum almost black.Fore wing with fuscous longitudinal bands and with wide hyaline spots occupying middle of basal cell, discosubmarginal cell and distal half of marginal and basal of fourth submarginal cells (Fig. 6C); hind wing hyaline (Fig. 1I).
Male.Agrees with the above-mentioned description of the female, except for the following character states: Body length 5.5-7.4mm (holotype: 7 mm); fore wing length 4.7-6.0mm; antenna with 29-34 flagellomeres (holotype: 32 flagellomeres); mesoscutum entirely punctate with setae; spine of scutellum laterally compressed and slightly up-curved or straight (Fig. 8B); abscissa of vein M between 2rs-m and 2cu-m 0.3-0.9times as long as 2rs-m (Fig. 6D); metasomal tergite I 5.4 times as long as apical width, lateromedian carina incomplete posterior to 0.2 length of segment; body, antenna and legs black; wings hyaline except apical marginal area of fore wing weakly tinged with brown.Male genitalia and hypopygium shown in Fig. 7A, C, with digitus relatively weakly elongated but shorter than half of paramere, somewhat broadened toward apex (Fig. 7C).Apex of paramere almost truncate with corners rounded.Penis valve slightly curved ventrally; basal apodeme of aedeagus striate dorsally.
Variation.The scutellar spine of the male holotype is straight and relatively stout apically compared to some South Korean A. jilinensis specimens that are more upcurved and tapered (although some are straight and non-tapered as in Fig. 8B).
Region.Eastern Palaearctic.Remarks.This species has been known previously from only two pharate males (holotype and paratype).Thus, this is the first description of the female and of the wings of the male.(Korean name: Am-bul-eun-bae-mul-beol).Diagnosis.Females of Agriotypus wangpiensis sp.nov.can be distinguished from all other species for which the female is known by the combination of the following characters: 1) metasomal tergite II lacking dorsal and dorsolateral carinae anteriorly (Fig. 5F) (present in all species of the A. himalensis species group); 2) clypeus in lateral view with summit of convexity angulate (Fig. 5B) (rounded in A. armatus and A. silvestris); 3) clypeus in anterior view with summit of convexity rounded medially (Fig. 5C) (acutely pointed medially in A. gracilis); 4) pronotum with a long and strong epomia  wing with complete longitudinal, hyaline bands proximal and distal to pterostigma in A. jilinensis as in Fig. 6C).Note that the female of A. succinctus (A.armatus group) is not known.In addition, females of several species of the A. himalensis species group are unknown, but it is likely they could be distinguished from the new species by character 1 (above).Males of Agriotypus wangpiensis sp.nov.can be distinguished from all other species for which the male is known by the combination of characters 1-4 in the female diagnosis as well as the face lacking a medial, vertical ridge (ridge present in A. jilinensis).All males of the A. armatus group are known.

Agriotypus wangpiensis
Description.Female (Adult).Body length 5.0-6.8mm; fore wing length 4.0-4.9mm.Head.Head width 1.0 times as wide as its median height (Fig. 5C).Face width (minimum length of inner orbit) 1.57 times as wide as its median height (length between antennal socket and clypeal margin), convex medially, without a median vertical ridge, with coarse rugosity and dense pubescence (Fig. 5C).Antenna with 20 flagellomeres, 0.6 times as long as fore wing.Temple behind eyes roundly narrowed in dorsal view.Transverse diameter of eye 0.9 times as wide as temple in dorsal view.Frons finely densely punctulate-reticulate.Area between antennal sockets lacking a glabrous short longitudinal tubercle.Antennal scrobe deep.POL: OOL = 1: 2. Clypeus in anterior view with apical margin round; anterior edge in profile moderately convex; summit of convexity in lateral view angulate; shape of angular summit of clypeus in anterior view rounded medially (Fig. 5C).Distance between anterior tentorial pits shorter than length between a tentorial pit and eye; malar space 1.3 times as long as basal width of mandible; mandible twisted, lower tooth longer than upper one.Occipital carina strong.
Mesosoma.Pronotum with long and strong epomia, upper area of pronotum with more than 10 fine carinae dorso-posteriorly.Mesoscutum shiny, anterior half of median lobe and outer side of lateral lobes punctate and setose; notauli distinct, meeting in posterior 0.3 (Fig. 5D, E).Scutellum in dorsal view triangular, 1.8 times as long as its anterior width including spine, 1.25 times as long as length of propodeum; scutellum with lateral carina at base, and spine smooth in lateral view, distinctly upcurved (Figs 5E, 8D).Mesopleuron and metapleuron densely punctate, reticulate and pubescent, with longitudinal groove deep and wide, without longitudinal carina on anterior edge at mid-height; epicnemial carina curving sharply anteriorly to meet anterior edge of mesopleuron and with a vertical extension dorsally.Sternaulus present.Dorsal area of mesopleuron with transverse carinae.Propodeum finely coriaceous-punctate; lateromedian longitudinal and lateral longitudinal carinae weakly convergent posteriorly (Fig. 5F).Wings.Fore wing with 1cu-a distad of M&Rs; abscissa of vein M be tween 2rs-m and 2cu-m 2.5-2.7 times as long as 2rs-m.Hind wing with 8 distal hamuli; vein 1/Cu & 1cu-a intercepted by 2/Cu in lower 0.25-0.35(Fig. 6G).
Metasoma.Metasomal tergite I 3.8 times as long as its apical width, 1.7 times as long as length of propodeum; metasomal tergite I finely punct ulate-reticulate, spiracles situated in anterior 0.45, lateromedian and dorsolateral carinae complete and strong, reaching to apex.Metasomal tergite II lacks dorsal and dorsolateral carinae.Ovipositor sheath 0.7 times as long as length of hind basitarsus.
Colour.Almost black.Apical spine of scutellum, posterior metasomal tergites slightly reddish brown.Fore wing generally infuscate, with small hyaline spots occupying middle of basal cell, discosubmarginal cell and marginal cell: hyaline spot of basal cell at basal 0.6 on the basal cell of fore wing, hyaline spots of discosubmarginal cell narrow, distal half of marginal cell with hyaline spot, fourth submarginal cell without hyaline spot (Fig. 6G).
Distribution.South Korea.
Etymology.The species is named after 'Wangpicheon', from the location where the type specimens was collected.(Korean name: Wang-pi-mul-beol).
Remarks.This new species is similar to A. silvestris Konishi & Aoyagi, 1994, but the female can be distinguished from the latter by the profile of the clypeus (convex with angulation but, roundly convex without angulation in A. silvestris); the ratio of the lengths of the scutellar spine and the propodeum (scutellar spine length 1.25 times as long as propodeum but only 0.75 times as long as in A. silvestris); the colour of the female fore wing (basal areas of fourth submarginal cell and third discal cells of fore wing almost completely dark without hyaline spot (arrows in Fig. 6G) compared to A. silvestris that has the basal 1/4 of these cells hyaline (arrows in Fig. 6B)); length of metasomal tergite I of female (3.8 times as long as length of posterior width but 4.6 times as long as posterior width in A. silvestris); hypopygium (evenly round median dorsal margin but, emarginate in median dorsal margin in A. silvestris).

Discussion
The first study on South Korean Agriotypinae by Kim (1963) reported Agriotypus armatus from South Korea.However, Konishi and Aoyagi (1994) and Bennett (2001) mentioned that A. armatus is very similar to A. gracilis but clearly distinguished by the clypeus, mesonotum, metasomal tergite I, host records, and geographical distribution.Kim et al. (2018) excluded the record of A. armatus from the South Korean insect fauna based on the literature review and specimen examination.Therefore four species, A. gracilis, A. jilinensis, A. silvestris, and A. wangpiensis sp. nov., are now known to be found in South Korea.All of these species belong to the A. armatus species group of Bennett (2001) on the basis of the lack of the longitudinal carina at the anterior of metasomal tergite II.Among them, A. wangpiensis sp.nov. is a parasitoid of Goera japonica Banks, 1906 in Wangpicheon in the northeast of Gyeongsangbuk-do Province.The bootstrap support for nodes is displayed if higher than 75%.
In the DNA barcode analysis, Agriotypus gracilis is highly divergent from the other species (8.5-10% different DNA barcode) and Agriotypus wangpiensis sp.nov. is most closely related to A. silvestris (Fig. 9).The species status of A. wangpiensis sp.nov. is confirmed by both the unique combination of morphological characters (see the species diagnosis) and the molecular analysis that found that the DNA barcode region of the new species is 3.4-3.6%different from A. silvestris (Fig. 9).In a ML analysis (Fig. 10), the four species in the genus Agriotypus [Agriotypus wangpiensis sp.nov., A. silvestris, A. gracilis and A. armatus] clustered as a monophyletic group with strong support (100%).Also, the clustering of all specimens of A. wangpiensis sp.nov.together (monophyly of the new species) is supported by the 99% bootstrap score, while the sister relationship of the new species and A. silvestris is supported with 98% bootstrap score.

Figure 1 .
Figure 1. Adult of A. jilinensis (female) (non-type) A habitus in dorsal view B head in frontal view C metasomal tergite I in dorsal view D head and mesoscutum in dorsal view E head and mesosoma in lateral view F propodeum and metasomal tergite I in dorsal view G propodeum in dorsal view H hind tarsomeres and tarsal claw I wings J metasomal tergites in lateral view.Scale bars: 1 mm (A); 0.5 mm (D, E, F, I, J); 0.2 mm (B, C, G, H).

Figure 2 .
Figure 2. Habitat and collecting sites of A. wangpiensis sp.nov.A distant view of Wangpicheon stream B close range view of Wangpicheon stream C parasitized hosts D parasitized trichopteran pupae.

Figure 3 .
Figure 3. Reared A. wangpiensis sp.nov.A-B breaking case of trichopteran species C exiting trichopteran case D male adult following exit from trichopteran case.

Figure 7 .
Figure 7. Male genitalia and hypopygium of Agriotypus jilinensis (A, C non-type) and A. wangpiensis sp.nov.(B, D paratype) A hypopygium, ventral view of A. jilinensis B hypopygium, ventral view of A. wangpiensis sp.nov.C genitalia of A. jilinensis D genitalia of A. wangpiensis sp.nov.Scale bar: 0.3 mm.

Figure 10 .
Figure 10.Maximum Likelihood tree (ML) of the successfully DNA barcoded specimens of Agriotypus.The bootstrap support for nodes is displayed if higher than 75%.