Two new records of the genus Trioxys (Hymenoptera, Braconidae, Aphidiinae) parasitic on bamboo aphids from South Korea

Abstract Background The genus Trioxys Haliday, 1833 consists of more than 80 species worldwide with three species being recorded in South Korea. In this study, we report the first observation of the two additional species, T.liui Chou & Chou, 1993 from Takecallisarundinariae (Essig, 1917) on Phyllostachysbambusoides Siebold & Zucc., 1843 and T.remaudierei Starý & Rakhshani, 2017 from T.taiwana (Takahashi, 1926) on Sasaborealis (Hack.) Makino & Shibata, 1901. New information Trioxysliui and T.remaudierei are described and reported with phototographs of the diagnostic morphological characters and the mitochondrial cytochrome c oxidase subunit I (COI) data (barcode region) and Bayesian tree of the phylogenetic analysis amongst the closely-related taxa are provided.


Introduction
The genus Trioxys Haliday, 1833 (Hymenoptera, Braconidae, Aphidiinae) consists of more than 80 known species around the world (Rakhshani et al. 2012, Yu et al. 2016, Rakhshani et al. 2017, Čkrkić et al. 2021, Davidian 2021, Petrović et al. 2021), with three species being recorded in South Korea (National Institute of Biological Resources 2022).As a key character for classification to the two morphologically similar genera, Trioxys Haliday, 1833 and Binodoxys Mackauer, 1960, a pair of accessory prongs are evident on the abdominal sternite.The former has only a spiracular (primary) tubercle, while the latter has not only a spiracular tubercle, but also has a secondary tubercle (Mackauer 1961, Starý 1981).

Field and Taxonomic works
Samples were collected by searching for Takecallis mummies ( T. taiwana (Takahashi, 1926) and T. arundinariae (Essig, 1917)) on various bamboo species ( Phyllostachys bambusoides and Sasa borealis).Leaves containing mummified aphids were then collected and placed in a clean insect breeding dish (SPL Life Sciences, Korea).To ensure a sufficient number of samples, these dishes were kept in the laboratory at room temperature.The emergence of parasitoid wasps was monitored daily and they were collected using an insect aspirator.Subsequently, the collected wasps were preserved in 80% ethyl alcohol at -19℃.
After conducting both morphological and molecular identification, measurements of unrecorded species were carried out.A LEICA DMC2900 digital camera and a LEICA M205 C microscope (Leica Geosystems AG) were utilised for photography and characterisation.Multiple pictures were taken at various heights using multifocusing technology.LAS V4.11 (Leica Geosystems AG) and HeliconFocus 7 (Helicon Soft) software were used for the stacking process.After the stacking procedure, illustrations were generated using Adobe Photoshop CS6.LAS V.4.11 (Leica Geosystems AG) was utilised to determine the shape of the specimens (Berkovitch et al. 2009).

Molecular analysis
Total genomic DNA extraction was performed using a LaboPass Tissue Kit (COSMOgenetech, Korea) following the manufacturer's protocol.To preserve a morphologically complete specimen, the DNA extraction method was slightly modified from the "freezing method" used by Yaakop et al. (2013).In the original protocol, the sample was incubated for 30 minutes at 56°C with 200 μl of TL buffer + 20 μl of proteinase K.In the slightly modified DNA extraction methods, a 2 hour incubation period at the same temperature was used.Genomic DNA was extracted individually from each sample.
The target site for molecular identification was the front partial region of mitochondrial COI, a 658-bp fragment, amplified using primers, LCO1490 (forward) 5'-GGTCAACAAATCA TAAAGATATTGG-3' and HCO2198 (reverse) 5'-TAAACTTCAGGGTGACCAAAAAATCA-3' (Folmer et al. 1994), with AccuPower PCR PreMix (Bioneer Corp., Daejeon, Korea).Polymerase chain reaction (PCR) amplification was conducted with 20 ml of a reaction mixture consisting of 3 ml of DNA extract, 2 ml of primer and 15 ml of ddH O.It was carried out as follows: denaturation for 5 min at 95℃; 4 cycles of 20 s at 95℃, 30 s at 55℃ (decreasing incrementally by 2℃ per cycle) and 40 s at 72℃, 31 cycles of 20 s at 95℃, 30 s at 48℃ and 40 s at 72℃; and final extension at 72℃ for 5 min.PCR products were visualised by electrophoresis on agar gel and, if a band existed, we commissioned Macrogen (Daejeon, Korea) for purification and sequencing analysis.
Using MEGA version 7.0 (Kumar et al. 2016), sequences were aligned by ClustalW default settings and their frame-shifts checked to avoid pseudogenes.Alignments were translated to amino acids using MEGA version 7.0.We calculated sequence divergences using the 'pdistance' model commonly with 1,000 bootstrapping replications and complete deletion in data gaps.
A phylogenetic tree was constructed with the Bayesian method using BEAST2 (Bouckaert et al. 2014).To produce dated phylogenies, we used an optimised relaxed clock model ( Drummond et al. 2006) in BEAUti and other options were set at default.MCMC analysis was performed and checked using Tracer, DensiTree.After that, we constructed the consensus tree using TreeAnnotator with posterior probability limit 1.0 setting.Ratnasingham and Herbert (2007).The front partial region 658 bp of the COI fragment was sequenced from T. liui and T. remaudierei and deposited in GenBank.Altogether, 14 sequences of six species, containing the outgroup, were retrieved from GenBank and BOLD (http://www.boldsystems.org)and were used to compare them with T. liui and T. remaudierei (Table 1).
Metasoma.Petiole 1.5 times as long as wide at spiracles (Fig. 1I and J).Ovipositor sheath stout, concave on ventral margin.Ratio of ovipositor sheath width/length 2.0 at base (Fig. 1G).Anal prongs (= accessory prongs) almost straight, slightly curved upwards at apex.Dorsal side of prongs four setae, with one claw-like apical bristle and two setae at apex (Figure 2H).
Metasoma.Petiole 2.0 times as long as wide at spiracles (Fig. 2I and J).Ovipositor sheath elongate, gently concave on ventral margin.Ratio of ovipositor sheath width/ length 2.8 at base (Fig. 2G).Anal prongs (= accessory prongs) long and straight upwards, bifurcated on apical one-third to two-thirds (apical one-third are four, apcial half is two, apical two-third is one), each having one claw-like apical bristle and one seta at apex with two or three setae at dorsal side (Fig. 2H).
Colour.Antenna brown; scape, pedicel, F1 and F2 yellowish-brown, sometimes dorsal part of F2 brown; Head, face and clypeus with mouth-parts dark brown.Dorsal side of mesoscutum and metasoma dark brown, except light brown propodeum and yellowishbrown petiole.Legs yellowish-brown with dark apices.

Notes
In the original description, the antenna of this species was 13-segmented.However, it was a mistake in the email from the author.This species is really 11-segmented, but two segments are artificially repeated in the line drawing.

Analysis
A Bayesian tree was constructed with twelve sequences from seven species, including an outgroup.Three clades were identified: Clade A represented Trioxys sunnysidensis, Clade B included T. remaudierei, both identified as monophyletic and the remaining species formed Clade C (Fig. 3).Within Clade C, T. liui was observed as the sister group to T. pallidus, T. companatus and T. ulmi.In comparison to a previous study by Čkrkić et al. (2021), T. liui exhibited a consistent pattern, but in this study, it was positioned between T. ulmi and the T. pallidus-complanatus group.Trioxys remaudierei was situated between Clade A and Clade C (Fig. 3).
Schlinger and Hall 1961, Shaw and Huddleston 1991) and may have evolved into two strands to maximise it.Therefore, such prong morphology was considered a potential indicator of evolutionary ancestral traits even although it seemed to be not a clear correlation between taxa due to the limited number of samples in this study.
In case of T. liui, it is parasitic on Cranaphis formosanus (Takahashi) (Liu, 1975) 1978) and both species show a strong host specificity for Tacekallis species.Futhermore, since Takecallis species have been recorded only on bamboo species (Lee and Lee 2018), the distribution of T. liui and T. remaudierei parasitising on it seems to be limited to areas where bamboo is located.In the natural range of Tacekallis species, all known species of this genus are restricted to East Asia (China, India, Japan, Korea, Manchurian subregion, Taiwan), T. arundicolens (Clarke, 1903), T. arundinariae and T. taiwana, T. nigroantennatus Wieczorek, 2023 are now widely distributed and introduced to other countries, including Africa (Algeria), USA (California), England, Netherlands, Hungary, Madeira, Australia, New Zealand, Argentina, Spain and Poland (Blackman andEastop 2023, Wieczorek andSawka-Gądek 2023).Consequently, parasitoids of Takecallis species are likely to be invasive species outside East Asia, with invasive countries potentially hosting distributions of these species.