Three new species of the genus Aphidius (Hymenoptera, Braconidae, Aphidiinae) from South Korea

Several species of the genus Aphidius are well known as commercial biocontrol agents of pest aphids, and more than 130 species of the genus have been recorded worldwide. To date, only 15 Aphidius species have been recorded in South Korea. Using the DNA barcode region (ca. 658 bp) of mitochondrial cytochrome c oxidase subunit I (COI), we amplified sequences of 15 Aphidius Korean species, aligned them in combination with 23 reference species retrieved from GenBank for comparison and identification, and then reconstructed a barcode phylogeny by the neighbour-joining method. As a result, three Aphidius species were found to be new to Science. Descriptions and illustrations of the three species new to Science – Aphidius longicarpus sp. nov., A. longistigmus sp. nov., and A. asiaticus sp. nov. – are provided, together with their phylogenetic position within the genus Aphidius. In addition, a redescription of A. areolatus, a parasitoid of maple aphids (Peryphillus spp.), is also given.


Introduction
The genus Aphidius consists of more than 130 species around the world and belongs to the subfamily Aphidiinae, which includes approximately 63 genera and 650 species (Yu et al. 2016). All Aphidius species are solitary koinobiont endoparasitoids of aphids, and many of them (e.g. A. colemani, A. gifuensis) are commercially used and produced as biocontrol agents against pest aphids worldwide (Hågvar and Hofsvang 1991;Blackman and Eastop 2000). As a species-rich genus with a huge diversity of host aphids, many new species have been discovered and described in recent studies (Tomanović et al. 2007; Davidian and Gavrilyuk 2010;Petrović et al. 2020).
Members of the genus Aphidius are medium-sized wasps, only 15 species of which have been recorded in South Korea (Starý and Choi 2000;Yu et al. 2016;Hwang et al. 2018;NIBR 2019;Kim et al. 2020). Since Aphidius species such as A. colemani and A. ervi are used as imported biocontrol agents against pest aphids worldwide, especially in agriculture and horticulture (Sequeira and Mackauer 1992;Henter and Via 1995;Fernandez and Nentwig 1997;Takada 1998), it is important to explore indigenous Aphidius species and to confirm their diversity in South Korea and surrounding regions.
In this study, we describe and diagnose three new Aphidius species and present their phylogenetic relationships with other congenerics. We also confirm the presence of A. areolatus, which was poorly known and often misidentified, in South Korea and redescribe it.

Materials and methods
Samples were borrowed from the Korean National Arboretum (Pocheon, South Korea). All of them were obtained with Malaise traps in South Korea. They are stored in 95% ethyl alcohol at -19 °C.
Specimen morphological identification was based on Shaw and Huddleston 1991;Wharton et al. 1997;Yu et al. 2016;and Rakhshani et al. 2019. We first performed morphological sorting of similar phenotypes and labeling of these samples using a dissecting microscope (OLYMPUS SZX16, Leica M205C, NIKON SMZ 1500), after which DNA extraction was performed. Total genetic DNA extraction was performed using a LaboPass Tissue Kit (COSMOgenetech, Korea) following the manufacturer's protocol. For DNA extraction, samples consisted of single or several individuals from the same colony.
The target site for molecular identification was the front partial region of mitochondrial COI, viz., a 658-bp fragment, amplified using primers LCO1490 (forward) 5'-GGTCAACAAATCATAAAGATATTGG-3' and HCO2198 (reverse) 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (Folmer et al. 1994) and Accu-Power PCR PreMix (Bioneer Corp., Daejeon, Korea). Polymerase chain reaction (PCR) amplification was conducted with 20 μl of a reaction mixture consisting of 3 μl of DNA extract, 2 μl of primer, and 15 μl of H 2 O. It was carried out as follows: denaturation for 5 min at 95 °C; 38 cycles of 20 s at 95 °C, 30 s at 45 °C, and 40 s at 72 °C; and final extension at 72 °C for 5 min. PCR products were tested by electrophoresis on agar gel and if a band existed, we commissioned Bionics (Korea) for analysis and purification.
Sequences were aligned using Clustal W default setting and their frame-shifts were checked to avoid pseudogenes. Alignments were translated to amino acids using MEGA, version 7.0. We calculated sequence divergences using the 'p-distance' model commonly employed to analyze COI barcoding data. A phylogeny tree was constructed using the neighbor-joining method with 1,000 bootstrapping replications and complete deletion in data gaps. After morphological and molecular identification, measurements of the new species were carried out. A LEICA DMC2900 digital camera and a LEICA M205 C microscope (Leica Geosystems AG) were used for photography and characterization, several pictures being taken for each height using multifocusing technology. LAS V4.11 (Leica Geosystems AG) and HeliconFocus 7 (Helicon Soft) software were used for stacking work. After stacking work, illustrations were created using Adobe Photoshop CS6. LAS V4.11 (Leica Geosystems AG) was used to ascertain the shape of specimens (Berkovitch et al. 2009;Arias-Penna et al. 2013).

Results
A total of 110 COI MOTUs (≥525) of 28 species, including the outgroup, were used to establish the phylogenetic tree. Fourteen species recorded in South Korea (but not A. pleotricophori because of a lack of sequence data) were used in phylogenetic analysis. Altogether, sequences of 28 species containing 72 reference sequences of 22 Aphidius species from GenBank (Suppl. material 1: Table S1), were used to explore the phylogenetic position of the three newly described species. Diaeretiella rapae was used as an outgroup.

Diagnosis.
In some morphological characters (shape of the first flagellomere and antennae, number of antennal segments, shape of the pterostigma, and number of maxillary and labial palps), Aphidius longicarpus sp. nov. is similar to A. funebris and A. balcanicus. However, it clearly differs from A. balcanicus in having a shorter R1 vein (the R1 vein is subequal to pterostigma length in Aphidius longicarpus sp. nov., while in A. balcanicus the ratio of pterostigma length to R1 vein length is 1.4-2.0). It differs from A. funebris in possessing a more elongate pterostigma (the pterostigma length/width ratio is 4.06 in Aphidius longicarpus sp. nov., vs. 3.0-3.5 in A. funebris) and a more elongate petiole (the petiole length/ width ratio at the spiracle level is about 3.14 in Aphidius longicarpus sp. nov., vs. 2.5-2.9 in A. funebris). The new species is distinguished from all other congeneric Aphidius species by a combination of the following characters: 16-segmented antennae, short first flagellomere (length/width ratio of about 2.6), and subequal length of the R1 vein and pterostigma.
Colour. Antenna black; scape yellowish-brown; pedicel brown to black from base to apex. Head black. Face brown, clypeus with mouthparts yellowish-brown. Dorsal side of mesoscutum and metasoma dark brown, except yellowish brown propodeum and petiole. Legs yellowish-brown with dark apices.
Etymology. The name of the new species refers to the very long fore wing R1 vein (=metacarpus).

Diagnosis.
On the basis of the number of maxillary (three) and labial (two) palpomeres, number of antennal segments, and possession of an elongate pterostigma, the new species is morphologically related to A. matricariae. However, it differs clearly from A. matricariae in having a very elongate fore wing pterostigma (fore wing pterostigma Description. Female. Length of body about 1.85 mm (Fig. 3A). Length of fore wing 1.45 mm (Fig. 3B).
Etymology. The name of the new species refers to the very long fore wing pterostigma.

Diagnosis.
On the basis of the number of antennal segments and wing venation pattern, the new species is morphologically related to A. matricariae and Aphidius longistigmus sp. nov.. However, it differs clearly from A. matricariae in having a more elongate fore wing pterostigma (the fore wing pterostigma length/width ratio is 4.62-4.79 in Aphidius asiaticus sp. nov., vs. 3.50-4.00 in A. matricariae) and a shorter flagellomere 1 (The F1 length/width ratio is 2.23-2.49 in Aphidius asiaticus sp. nov., vs. 2.50-3.00 in A. matricariae). Aphidius asiaticus sp. nov. differs from Aphidius longistigmus sp. nov. in having a less elongate pterostigma (the pterostigma length/width ratio is 4. 62-4.79 in A. asiaticus sp. nov., vs. 4.96-5.46 in Aphidius longistigmus sp. nov.). Additionally, A. asiaticus sp. nov. has four maxillary palpomeres (or three when the last one is very long and undivided), while A. longistigmus sp. nov. has three maxillary palpomeres.
Colour. Antennal scape, pedicel, and flagellomere 1 yellow, flagellomere 2 yellow at the base, remaining parts of antennae yellowish to light-brown. Head black. Face with clypeus dark-brown, mouth parts yellow. Dorsal side of mesoscutum and metasoma dark-brown except for the yellow to light-brown propodeum (propodeum sometimes dark-brown) with yellow petiole. Legs yellow with dark apices.
Metasoma. Petiole 2.17 times as long as wide at spiracles (Fig. 5F, G), with about 10 straight costulae in anterolateral area (Fig. 5G) Remarks. Morphologically, the examined specimens exhibited more variability than was known before (Starý and Schlinger 1967). The pterostigma is more triangular and the petiole more quadrate than in the original description. Chang and Youn (1983) reported A. areolatus from South Korea (one male specimen), but their record of this endemic Japanese species is doubtful. Due to misidentification (Chang and Youn 1983), Aphidius areolatus should be deleted from the previously known Korean parasitoid fauna (personal communication with Prof. Paik). It follows that A. areolatus is recorded from South Korea for the first time in the present study.

Discussion
It is well known that parasitoids, as a hyperdiverse and understudied group of organisms, generally are characterized by cryptic speciation (Derocles et al. 2012). Important biocontrol agents, aphidiine parasitoids in particular remain a largely unexplored group. In the last decades, through an integrative approach including DNA barcoding, there has been a rapid increase of knowledge about aphidiine diversity and cryptic species (Tomanović et al. 2018;Čkrkić et al. 2019;Kocić et al. 2020).
Here, we use DNA barcoding to describe three new Aphidius species from the Korean Peninsula, an area with a poorly known aphidiine fauna that includes only 58 known species (Starý and Choi 2000;Starý et al. 2001;Yu et al. 2016;Choi et al. 2017;Choi and Kim 2018;Hwang et al. 2018;Kim et al. 2020), which is not consistent with their economic and ecological importance. Aphidius longicarpus sp. nov. is a sister species to the A. absinthii -A. funebris -A. longipetiolus clade, with a long and narrow pterostigma as a clear apomorphic character, along with a short flagellomere 1 and long R1 vein as clear plesiomorphies. Aphidius longistigmus sp. nov. and Aphidius asiaticus sp. nov. are clustered with A. silvaticus, a member of the A. urticae group (Jamhour et al. 2016). Both species share a long and narrow pterostigma, along with three maxillary and two labial palpomeres in A. asiaticus sp. nov. as apomorphies and a short flagellomere 1 as a plesiomorphic character. In addition, we here redescribe A. areolatus, an interesting species related to Periphyllus aphids as hosts in the Far East (Starý and Schlinger 1967). This species is morphologically similar to the European and Central Asian A. setiger, replacing it in the forest type of habitats in the Far East, where it parasitizes Periphyllus aphids on Acer spp. Correspondingly, in our phylogeny, A. areolatus is clustered with A. setiger and A. cingulatus, both parasitoids of aphids on Acer spp. (Periphyllus spp. and Pterocomma spp., respectively).
We presume that the Korean parasitoid fauna is extremely rich due to habitat and plant diversity (NIE 2017a(NIE , 2017bNIBR 2019). This group is very important because most of its members are already being used or are tentatively applicable as biological control agents. Further research should therefore be conducted to explore this rich parasitoid biodiversity and detect tritrophic (host plant-aphidparasitoid) interactions.