Integrated taxonomy unveils three new species of Foenobethylus (Hymenoptera, Bethylidae) from China

Species of the genus Foenobethylus Kieffer, 1913 are parasitoids wasps rarely collected and are only found in the Oriental region. In this study, based on both morphological and molecular evidence, we describe three new species from China: F. robusta Li & Chen, sp. nov., F. xinglongsensis Wang & Chen, sp. nov., and F. yunkaishanensis Chen & Luo, sp. nov. An updated key to species of the genus is provided. Additionally, the phylogenetic relationships between Foenobethylus and other three morphologically similar genera are discussed based on the analyses of COI and 28S genes.


Introduction
Foenobethylus Kieffer is a rare genus of Pristocerinae in the flat wasp family Bethylidae, with only 11 described species Oriental (Várkonyi and Polaszek 2007;Liu et al. 2011;Savergnini and Azevedo 2013;Chen and Azevedo 2020). Of the 11 described species, 10 species were described based on only males. Until recently, the first female of the genus was discovered by Chen and Azevedo (2020). The new finding suggested that Foenobethylus might be a synonym under Parascleroderma. Although the morphological differences between Foenobethylus and Parascleroderma were intensely discussed (Azevedo and Lanes 2007;Várkonyi and Polaszek 2007;Chen and Azevedo 2020), Chen and Azevedo (2020) concluded that the precise taxonomic delimitation of both genera only could be solved under phylogentic analyses. Interestingly, before the discovery of the female of Foenobethylus, considering the extreme sexual dimorphism in Pristocerinae, Várkonyi and Polaszek (2007) also suspected that the females of Foenobethylus might be already known to science under a different generic name and the exact phylogenetic status of Foenobethylus could be resolved by preferably molecular evidence.
Recently, we have accumulated some fresh specimens of several Foenobethylus species collected by Malaise traps in South China. In this study, we aims to identify this new material to species using an integrated taxonomic approach that combines both morphology and molecular data and to conduct a preliminary phylogenetic analysis between Foenobethylus and morphologically similar genera based on DNA sequences.

Collection and identification
This work is based on specimens of Foenobethylus collected by Malaise traps (MT) set up across southern China. Specimens were identified using the keys of Chen and Azevedo (2020). All studied specimens are deposited in the Insect Collection of South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China (SCBG, curator: Huayan Chen). The morphological terms generally follow Lanes et al. (2020) and Brito et al. (2021), and the sculptural and texture of integument nomenclature follows Harris (1979).
Abbreviations and morphological terms used in text are: WH width of the head; LH length of the head; WF width of the frons; HE height of the eye; OOL ocello-ocular line; WOT width of the ocelar triangle.
The genitalia and subgenitial plate of a male paratype were removed and cleared using 10% potassium hydroxide solution, and mounted in glycerol on slides, when examined and photographed. Images and measurements were made using Nikon SMZ25 microscope with a Nikon DS-Ri 2 digital camera system. Images were postprocessed with Abobe Photoshop 2022.

DNA extraction, amplification, and sequencing
In total, 7 specimens of 4 morphospecies were used for DNA acquisition (see Table 1). Collecting information of the studied specimens are avaiable in the material examined section by the associated codes. Genomic DNA was extracted from entire specimens using a DNeasy Blood & Tissue Kit (QIAGEN, Inc.), following a nondestructive DNA extraction protocol as described in Taekul et al. (2014). Following DNA extraction, the "barcode" region of the mitochondrial cytochrome oxidase subunit 1 (COI) and nuclear 28S rRNA D1-2 (28S) were amplified using the LCO1490/HCO2198 (Folmer et al. 1994) and D2-3551F/D2-4057R (Gillespie et al. 2005) primer pairs, respectively. Polymerase chain reactions (PCRs) were performed using Tks Gflex DNA Polymerase (Takara), and conducted in a T100 Thermal Cycler (Bio-Rad). Thermocycling conditions were: an initial denaturing step at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 50 °C for 30 s, 72 °C for 30s and an additional extension at 72 °C for 5 min. Amplicons were directly sequenced in both directions with forward and reverse primers on an Applied Biosystems (ABI) 3730XL by Guangzhou Tianyi Huiyuan Gene Technology Co., Ltd. (Guangzhou, China). Chromatograms were assembled with Geneious 11.0.3. All sequences generated from this study are deposited in GenBank (accession numbers see Table 1). All residual DNAs are archived (-30 °C) in the molecular laboratory of SCBG, Guangzhou, China, and are available for further study upon request.

Molecular species delimitation and phylogenetic analysis
All sequences were blasted in BOLD (Barcode of Life Database, http://www. barcodinglife.org/index.php/IDS_OpenIdEngine, only for COI) and GenBank. Sequences were aligned using MAFFT v7.470 by the G-INS-I strategy for 28S and G-INS-I strategy for COI (Katoh and Standley 2013). Genetic Kimura-2 parameter (K2P) distances of COI sequences within and between species were calculated in MEGA 7 with pairwise deletion for gaps (Kumar et al. 2016 (Waterston, 1923) and Sierola gracilis Fullaway, 1920 (Hymenoptera, Bethylidae) selected as outgroups as used by Alencar et al. (2018). The concatenated sequences of 28S and COI were then analyzed using RAxML as implemented in Geneious 11.0.3 under the GTRGAMMA evolutionary model to generate a maximum likelihood (ML) tree.

Results
This study generated seven sequences of COI and six sequences of 28S for seven specimens. These seven voucher specimens were subjected to further morphological examination and four species were recognized, of which three are described as new. The COI sequences do not show a high match with sequences in both BOLD and GenBank databases. The closes match is an undetermined species of Parascleroderma, with 86.4% identical base pairs. Genetic distances of COI sequences among Foenobethylus species and representative species of four other morphologically similar or purported phylogenetically close genera and outgroups are in Table 2. Intraspecific distances of the COI sequences of Foenobethylus are identical. Interspecific distances among Foenobethylus speces range between 10.3% and 13.6%. Intergeneric distances between Foenobethylus and the four potential close genera and the outgroups range between 14.3% and 28.6%, with Parascleroderma is the most close genus, which shows 14.8-   18.6% divergence. The morphology-based delimitations of species are congruent with the molecular species identification based on COI sequences. Phylogenetic relationships between Foenobethylus and the studied genera are shown in Fig. 1. Foenobethylus was recovered as a monophyletic clade, with Parascleroderma as a sister group. Metafemur with only one acute spine in ventral midline, 0.26× as long as metafemoral width, touching apical margin of metatrochanter, and with one small, broad and dentate protuberance ventrally, 0.04× metafemoral width, located on second quarter of metafemur (Fig. 3G). Posterior hypopygeal margin strongly incurved, lateral lobe without conical protuberance (Fig. 3E). Basivolsella large, 0.6× as long as gonostipe, basal half about as wide as apical half, basal margin incurved ( Fig. 3C, D). Description. Male holotype. Body length 4.52 mm. Forewing length 3.10 mm. Colors. Head, mesosoma, metasoma, antenna and apex of mandible dark castaneous; palpi, base of mandible, all tibiae and tarsi castaneous; wings subhyaline, fore wing somewhat infuscate medially.
Variation. Mesosoma more polished, expecially pronotum and propodeum. Etymology. The specific epithet derived from the Latin word for robust, and refers to the robust body of this species.
Variation. Body size maller and color lighter. Etymology. The specific epithet refers to the locality (Mt. Yunkaishan) where the type specimens were collected.
Remarks. This new species is very similar to F. bidentatus Várkonyi & Polaszek [Brunei, Thailand], but can be separated from the latter by the following characters: distance between posterior margin of compound eye and occipital carina longer length of compound eye in dorsal view (as long as in F. bidentatus); seventh sternum with distal margin strongly emarginated (narrowly emarginated in F. bidentatus); posterior margin of hypopygium strongly incurved (broadly and almost evenly emarginate in F. bidentatus).

Discussion
Based on a preliminary phylogenetic analysis using morphological data, Várkonyi and Polaszek (2007) first assigned Foenobethylus to the subfamily Pristocerinae. However, the phylogenetic position of Foenobethylus within Pristocerinae was still unclear in their study. According to Azevedo and Lanes (2007), Afgoiogfa, Foenobethylus and Parascleroderma are closely related genera. However, currently no molecular sequences of Afgoiogfa are available for phylogenetic analysis. From the results of Alencar et al. (2018), the clade of Foenobethylus + Parascleroderma + Cleistepyris was recovered as the sister-group of Apenesia. In the light of the first discovery of Foenobethylus female, Chen and Azevedo (2020) suggested that Foenobethylus might be a synonym under Parascleroderma due the extreme similarity they share in females. Foenobethylus also shares some similar characters with Apenesia and Dissomphalus (Chen and Azevedo 2020), although Dissomphalus was never found close to Foenobethylus in any phylogenetic analyses (Alencar et al. 2018). The molecular data seem to support the relatedness between Foenobethylus and Parascleroderma, as suggested by their similar morphology. In the present study, all of the five studied Foenobethylus species form a monophyletic clade, with Parascleroderma as a sister group (Fig. 1). This result is congruent with that of Alencar et al. (2018), in which Parascleroderma is sister to Foenobethylus. The intergeneric distances between Foenobethylus and Parascleroderma ranged between 14.8-18.6%, generally higher than interspecific distances within Foenobethylus. This high genetic distance suggests that Foenobethylus is likely a distinct genus, as similar intergeneric distances have been found in Epyrinae of Bethylidae (Colombo et al. 2020). However, given that the two Parascleroderma species together with all the Foenobethylus also form a monophyletic group in this study, the possibility that these two genera are synonymous cannot be ruled out. The precise taxonomic delimitation of both genera could be resolved by accumulating molecular data of more Parascleroderma taxa for phylogenetic analyses. The relatively high interspecific genetic distances (10.3%-13.6%) indicate that the use of DNA barcoding for delimitating morphologically similar species of Foenobethylus may be promising. For example, F. robusta is very similar to F. syndesis, but the genetic distance between them is up to 11.3% and they are well supported as different species. Future comprehensive taxon sampling and molecular analyses should be able to test the power of DNA barcoding in delimitating morphologically similar species.
With the three newly described species, the total number of Foenobethylus is raised from 11 to 14 (Table 3). Species of Foenobethylus mainly occur in tropical forests (Savergnini and Azevedo 2013). All of the three new species described in this study were also collected in tropical or subtropical forests, indicating that species diversity of Foenobethylus from tropical and subtropical forests of Southeast Asia is still undersampled and intensive study is required. The wingless feature of the females allows these parasitoids to adapt to the restricted environments such as tunnels under the tree bark, where the females look for preys (Chen and Azevedo 2020). However, the host of Foenobethylus speices is still unknown. As other members of Pristocerinae, species of Foenobethylus are likely ectoparasitoids of bettle larvae in concealed habitat (Terayama 2006). Searching for wood boring bettle larvae in tropical and subtropical forests should be a promsing direction in finding the hosts of these parasitoids. Table 3. List of the world species of Foenobethylus (updated from Chen and Azevedo 2020).