Toxares koreanus sp. nov. – a new Toxares species from South Korea (Hymenoptera, Braconidae, Aphidiinae)

The genus Toxares Haliday, 1840 is a small taxon of Aphidiinae, consisting four valid species in the world. One Toxares species is recorded as new to science from South Korea, in this study. Descriptions and illustrations of the new species, T. koreanus sp. nov. , are provided, together with their mitochondrial cytochrome c oxidase subunit I ( COI ) and D2 region of the nuclear gene for 28S rRNA ( 28S ) sequences. The phylogenetic tree reconstructed using a combination of COI and 28S revealed the phylogenetic position of the genus Toxares within Aphidiinae.

Based on the forewing venation being related to braconid ancestors, the genus Toxares is classified within the Ephedrini tribe (Mackauer 1961), which has sometimes been considered basal within Aphidiinae (Belshaw and Quicke 1997;Sanchis et al. 2000;Derocles et al. 2012). Some other studies showed that the tribe Praini is basal (Smith et al. 1999). However, there is very little evidence about molecular data of Toxares species, and its phylogenetic position is still unknown. Derocles et al. (2012) determined that the phylogenetic position of Toxares deltiger is between Ephedrini and Praini based on sequences of the Cytochrome c Oxidase subunit I (COI) gene. Ye et al. (2017) analysed molecular markers for identification of primary parasitoids of cereal aphids. Within their analysis, T. deltiger clustered as a sister group of the Trioxini tribe based on COI sequences and as a sister group of Aphidiini based on 16S ribosomal RNA (Ye et al. 2017).
The aim of this study is to present additional knowledge about the diversity of Toxares species. After initial research of Korean aphid parasitoid fauna, we recognized a new Toxares species which is herein described and diagnostified using morphological and molecular characters. We also analysed phylogenetic relationships among genera Toxares, Ephedrus Haliday, 1833 and Praon Haliday, 1833 and discussed the phylogenetic position of the genus Toxares within Aphidiinae.

Specimen collection and morphological analysis
Specimens were collected by Malaise trap in a deciduous forest habitat (mostly Quercus spp.) in Mt. Beophwa which is about 450 m.a.s.l. Rosa multiflora, Cirsium japonicum, and Urtica thunbergiana were the dominant plant species. Two specimens were slidemounted with Hoyer medium and one preserved in 70% ethanol. External structure was studied and measurements taken with a LEICA DM LS phase-contrast microscope. Morphological terminology used in this paper regarding diagnostic characters is based on that of Sharkey and Wharton (1997).

Molecular analysis
DNA extraction was performed using a LaboPass Tissue Kit (COSMOgenetech, Korea) following the manufacturer's protocol. In order to conserve morphologically complete voucher specimens, the DNA extraction method was slightly modified from the 'nondestructive method' by Favret (2005) and 'freezing method' by Yaakop et al. (2009). In the original protocol, the sample was crushed and then soaked in 180 μl of TL buffer + 20 μl of proteinase-K, followed by three hours of incubation at 55 °C. In the slightly modified DNA extraction methods, samples with all specimens were soaked in 180 μl of TL buffer + 20 μl of proteinase-K without destroying the sample, followed by 10 minutes incubation at 55 °C and kept in a freezer at -22 °C overnight. After that the general protocol was used for the remaining steps. The target site for molecular identification was the front partial region of mitochondrial COI, amplified using the primers LCO1490 (forward) 5'-GGTCAACAAATCATAAAGATAT-TGG-3' and HCO2198 (reverse) 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (Folmer et al. 1994). The molecular marker used for comparing with other Toxares species and species of Ephedrus and Praon was the D2 region of the nuclear gene for 28S rRNA (28SrDNA), amplified using primers 28SD2f (forward) 5'-AGAGAGAGTTCAAGAGTA-CGTG-3' (Belshaw and Quicke 1997) and 28SD2r (reverse) 5'-TTGGTCCGTGTTTCAAGACG-GG-3' (Campbell et al. 1993). We used heterogenous F/R primers as referred to by Tomanović et al. (2018).
Polymerase chain reaction (PCR) amplification of COI and 28S was conducted by using AccuPower PCR PreMix (Bioneer Corp., Daejeon, Korea) in 20 μl of a reaction mixture consisting of 3 μl of DNA extract, 2 μl of primer, and 15 μl of H 2 O. Thermal profile for COI was 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. Thermal profile for 28S was as follows: denaturation for 3 min at 95 °C; 32 cycles of 30 s at 95 °C, 30 s at 48 °C, and 30 s at 72 °C; and final extension at 72 °C for 10 min. The PCR products were tested by electrophoresis on agar gel and if a band existed, we commissioned Bionocs (Korea) for sequencing and purification.
Average genetic distances were calculated using MEGA X and Kimura's two-parameter method of base substitution (K2P, Kimura 1980) (Table 1).
MEGA X was used to construct phylogenetic trees based on each gene used in the study, as well as a combined tree employing concatenated sequences of both genes.
Phylogenetic relationships were reconstructed using Maximum Likelihood (ML) and Maximum Parsimony (MP) methods.
Body length: about 1.70-2.20 mm. Coloration. General body color light brown to yellow. Scape, pedicel and F 1 -F 3 yellow, F 4 basally light brown, remaining antennal parts brown. Mouthparts yellow. Head brown. Mesoscutum light brown to brown. Propodeum light brown. Legs yellow with brown apices. Petiole yellow to light brown, other metasomal terga light brown. Ovipositor sheath yellow.
Male (Fig. 2): Antenna 19-segmented with shorter flagellomeres (Fig. 2A). F 1 about 2.60 times as long as wide and longer than F 2 (Fig. 2B). Number of longitudinal placodes on F 1 and F 2 , 3 and 5, respectively. Maxillary palps with four palpomeres, labial palps with three palpomeres. Pterostigma shorter than in female and about 4.7 times as long as wide. Mesosoma with small mid pit . Petiole shorter than in female and about 2.55 times longer than width at spiracles level. Male genitalia (Fig. 2C). Body generaly darker than in female. Scapus and pedicel light brown. F 1 yellow, remaining antennal parts brown. Legs yellow to light brown with dark apices. Petiole and first half of metasomal terga light brown, remaining part of metasoma brown. Legs and mouthparts light brown.
Molecular analysis. Obtained phylogenetic trees reconstructed based on COI, 28S and the combination of both genes showed identical topology, and the tree based on the combination of both genes is shown on Fig. 3. Toxares koreanus sp. nov. groups with the only other Toxares species used in the analysis, while this clade is sister to the clade of Praon species. Ephedrus species basally form a separate clade on the tree.

Discussion
The genus Toxares is considered as one of the most basal within the subfamily Aphidiinae, classified within the tribe Ephedrini (Mackauer 1961), and sharing a braconid ancestral wing venation pattern with species of the genus Ephedrus. Except for the forewing venation pattern as a clear plesiomorphy, the newly described species, along with other congeners (e.g. T. deltiger), shares additional plesiomorphic character states, such as a large number of placodes on F 1 and F 2 , areolated propodeum, and 4-maxillary and 3-labial palpomeres. On the other hand, the elongated flagellomeres and petiole represent apomorphic characters (Tomanović et al. 2006). Toxares koreanus sp. nov. also possesses a small mid pit on the mesoscutum. This is a unique character present only in some Ephedrus species from the subgenus Fovephedrus (Chen 1986;Kocić et al. 2019), as well as in all known Toxares species. Toxares koreanus sp. nov. along with other congeners (e.g. T. deltiger) possesses a divided scutellar sulcus (Fig. 1D), a character state present in the subgenus Breviephedrus (e.g. E. brevis) (Kocić et al. 2019), which supports the phylogenetic position of the genus Toxares within the tribe Ephedrini. Toxares koreanus sp. nov. is the fifth known member of the genus Toxares and fourth species described from Asia. Based on the currently available data about the distribution of described species, we can assume that the origin of this genus should be Far Eastern Asia. Considering the habitat and plant diversity in Far Eastern Asia, we can expect to discover additional species of the genus Toxares. Molecular analysis using COI and 28S supports the description of the new species. Toxares koreanus sp. nov. is clearly separated from T. deltiger by both genes (Fig. 3, Table 1), in addition to morphological differences.
Molecular markers employed in this study show some incongruence with morphological characters. While Toxares is morphologically most similar to Ephedrus, molecular data suggests the genus is closer to Praon (Fig. 3, Tables 1, 2). Calculated genetic distances between all three genera are very high, based on both genes used in the analysis (Table 2). Although those between Toxares and Ephedrus are slightly higher than those between Toxares and Praon, it is still advisable to interpret these results carefully, and use an integrative approach including biological and ecological traits when making conclusions about the relatedness of groups. The discrepancy between morphological and molecular data is a fairly common occurence in Aphidiinae research and numerous studies have shown that molecular and morphological analyses often give somewhat conflicting results (Tomanović et al. 2013(Tomanović et al. , 2018Petrović et al. 2015;Jamhour et al. 2016;Čkrkić et al. 2020). One possible solution to this ongoing dilemma could be the use of more molecular markers or increasing the number of molecular operational taxonomic units, in an effort to uncover the mechanisms underlying the differences in multi-locus determined morphological traits (Zimmerman et al. 2000;Mezey et al. 2005;Čkrkić et al. 2020) and more emphasis on functions and adaptiation of morphological characters.
Although the genus Toxares, as a member of Ephedrini tribe, is already considered as basal within Aphidiinae (Mackauer 1961), our molecular data do not confirm it. We believe that discoveries of more species of this poorly known genus, along with appropriate molecular studies (which will include "ancient" genera Pseudephedrus and Choreopraon) should allow us to determine the exact phylogenetic position of Toxares.