DNA Barcoding Unveils New Species of the Sexually Dimorphic Genus Anteon Jurine (Hymenoptera, Dryinidae) from China

Simple Summary Anteon Jurine, 1807 is a cosmopolitan genus of dryinid parasitoids that attack leafhoppers. Sexual dimorphism is extreme in Anteon species, rendering the morphological taxonomy of these parasitoids difficult. This paper employs an integrated taxonomic approach that combines morphology with DNA barcoding to investigate the species delimitation of Anteon from China. Of the twenty-nine morphospecies examined, twenty-four species were identified as previously known species and five were described here as new species. The sexual association of six species was confirmed by DNA barcoding, indicating that such tools are powerful to tackle the taxonomic challenges in parasitoids with sexual dimorphism. Abstract Species of Anteon Jurine, 1807 are a large group of parasitoids attacking leafhoppers, which are important insect pests. Despite their great potential in pest biological control, the taxonomy and biology of these parasitoids are far from clear. Sexual dimorphism is extreme in Anteon species and has hampered the taxonomy of these parasitoids, resulting in many species described based on a single sex. In this paper, we employed an integrated taxonomic approach for delimitating species, combining morphological examinations with DNA barcoding, to investigate Anteon species from China. In total, 53 COI sequences representing 29 species of Anteon were obtained and analyzed. On the basis of both morphology and DNA barcoding, five new species of Anteon were discovered and described: A. clariclypeum sp. nov., A. maguanense sp. nov., A. parafidum sp. nov., A. shaanxianum sp. nov., and A. shandonganum sp. nov. The neotype of A. claricolle Kieffer is designated. The sexual association of six species was confirmed by DNA barcoding, which led to the synonymy of Anteon liui Xu, Olmi & He 2010, new syn., under Anteon meifenganum Olmi, 1991. Keys to species of Anteon from the Oriental and Eastern Palaearctic are updated to contain the five new species. Our study demonstrates that DNA barcoding is a potent tool for tackling the taxonomic challenges in parasitoids with extreme sexual dimorphism.


Introduction
The genus Anteon Jurine, 1807 (Hymenoptera, Dryinidae) is a large group of parasitoids attacking leafhoppers of the families Cicadellidae and Eurymelidae (Hemiptera, Cicadomorpha) [1].Since many leafhoppers are important insect pests, these parasitoids are economically important in the biological control of leafhoppers [2].The genus comprises 465 species distributed worldwide, except Antarctica [3].In total, 125 species have been reported from China [2], with 116 species belonging to the Oriental region [4,5] and 9 species belonging to the Eastern Palaearctic region [6].
As has been pointed out by many studies, the taxonomy of Dryinidae is challenging due to the extreme sexual dimorphism in these parasitoids, resulting in many species described based on a single sex because the sexual association of the same species is difficult [7][8][9][10].This situation is especially true in Anteon.However, this kind of taxonomic obstacle could be overcome by employing DNA barcoding methods such as the application of the mitochondrial cytochrome c oxidase 1 (COI) gene [9,10].In fact, DNA sequences have been used in the taxonomy, phylogenetics and the confirmation of host associations in Dryinidae and have become an important tool [8,9,[11][12][13].
As the second paper of an ongoing research campaign started by the first author (HC), with the objective to collect specimens of dryinids across China and identify the species using an integrated taxonomic approach that combines morphology and DNA barcoding, this study presents the results for the genus Anteon.

Insect Specimens and Identification
This study is based upon specimens collected by Malaise traps set up in many provinces of China (See Figure 1 in Olmi et al. [10]).Monthly collected specimens from Malaise traps were kept in 100% ethanol.Dryinidae specimens were sorted out and used for DNA sequencing and morphological examinations.The species of Anteon were identified by morphology with the keys provided by Xu et al. [2] and Olmi & Xu [6].

Repositories
The specimens examined in this study are deposited in the following collections, with the abbreviations used in the text: NTNU, Norwegian University of Science and Technology, Department of Natural History, NO-7491 Trondheim, Norway; SCAU, South China Agricultural University, Guangzhou, China; SCBG, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China; ZJUC, Zhejiang University, Hangzhou, Zhejiang, China.Regarding the Scandinavian specimens, one mid leg of each individual was torn off and submitted to the University of Guelph in Canada for COI barcoding and the generated sequences were uploaded to the BOLD system [14].All the types of Oriental and Palaearctic species of Anteon were examined.

Morphology
The terminology follows Olmi et al. [3].Abbreviations and morphological terms used in the text are as follows: OL: the distance between inner edges of a lateral ocellus and the median ocellus; OOL: the distance from the outer edge of a lateral ocellus to the eye; OPL: the distance from the posterior edge of a lateral ocellus to the occipital carina; POL: the distance between inner edges of the lateral ocelli; and TL: the distance from the posterior edge of an eye to the occipital carina.Body length was measured from the head to abdominal tip and expressed in millimeters; other measurements are reported in relative values.The terms "metapectal-propodeal disc" and "dorsal surface of the propodeum" are here used in the sense of Kawada et al. [15] and respectively correspond to the "dorsal surface of propodeum" and "posterior surface of propodeum" used by Olmi and Xu [6] and Xu et al. [2].

Imaging
Multifocal images of the new species and representatives of the known species (Figures S1-S25) were made using a mirrorless camera Sony Alpha 6300 (Sony, Tokyo, Japan) with cross table Proxxon KT 70 (Proxxon, Wecker, Luxemburg) or Leica M205C multifocal equipment (Leica, Wetzlar, Germany) and a Nikon SMZ25 microscope mounted with a Nikon DS-Ri 2 digital camera system (Melville, NY, USA).Image plates were made with Adobe Photoshop CS6 Extended.

. Sequence Analysis and Molecular Species Delimitation
Genomic DNA of the Chinese specimens was nondestructively extracted from the entire wasp using the TIANamp Micro DNA Kit (Tiangen Biotech, Beijing, China) following the methods used by Taekul et al. [16].Voucher specimens (Table S1) are deposited in the insect collection of South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China (SCBG).The COI gene was amplified using the LCO1490/HCO2198 primer pair [17].PCRs, sequencing and sequence analysis were conducted as in the Olmi et al. study [10].All the newly generated sequences were deposited into GenBank (Table 1), and sequences of six Anteon species were downloaded from the BOLD system: A. claricolle Kieffer, 1906 (HYMNI1041), Anteon collare (Dalman, 1818) (HYMNI945), Anteon ephippiger (Dalman, 1818) (HYMNI1151), Anteon exiguum (Haupt, 1941) (HYMNI1251), Anteon fulviventre (Haliday, 1828) (HYMNI390), Anteon gaullei Kieffer, 1905 (HYMNI430).Sequences were aligned by codons using MUSCLE implemented in Geneious 11.0.3.The K2P distances within and between species were calculated in MEGA 11 [18].A maximum likelihood (ML) tree was generated based on the aligned sequences using the RAxML plugin in Geneious 11.0.3.A sequence of Aphelopus niger Xu & He, 1999 (Hymenoptera, Dryinidae) (MZ151323) was downloaded from GenBank and used as an outgroup, according to the phylogenetic topologies present in Tribull [12].To explore the molecular species delimitation among the studied Anteon species, the distance-based barcode gap approach using the Automatic Barcode Gap Discovery (ABGD, Puillandre et al. [19]) and the updated Poisson tree processes model (bPTP, Zhang et al. [20]) were tested.The ABGD method sorts the sequences to hypothetical species by partitioning and comparing the difference between sequences to identify a "barcode gap (a given threshold distance)" (Puillandre et al. [19]).The ABGD analysis was performed on the web interface (https://bioinfo.mnhn.fr/abi/public/abgd/)(accessed on 15 November 2023) using the default priors, Pmin = 0.001, Pmax = 0.1, Steps 10, and with a barcode relative gap width = 1.00,Nb bins (for distance distribution = 20).The bPTP method tests species boundaries on non-ultrametric phylogenetic trees by detecting significant differences in the numbers of substitutions between species and within species (Zhang et al. [20]).For the bPTP analysis, the ML tree generated above was used to perform the analysis on the bPTP web server (https://species.h-its.org/ptp/)(15 November 2023) with default parameters.

Morphological Identification
In this study, we identified 28 morphospecies based on 53 specimens collected from China and Thailand.Twenty-three species were identified as previously known species and five are described below as new species (Table 1).With the help of DNA barcoding, the sexual association of six species was confirmed, leading to the synonymy of Anteon liui Xu, Olmi & He 2010 to Anteon meifenganum Olmi, 1991.

Molecular Analysis
The 53 newly generated COI sequences in the present study ranged from 576 bp to 674 bp.When analyzed in the BOLD system and GenBank, the sequence of A. exiguum (Haupt, 1941) received a close match (around 99%) with multiple sequences labeled as A. exiguum, while the sequences of Anteon claricolle Kieffer, 1906 received close matches with A. albidicolle Kieffer, 1905, which was a synonym of A. ephippiger (Dalman, 1818).Other species received no similar sequences in BOLD or GenBank.The intraspecific pairwise distances ranged from 0 to 3% (Table S2).The interspecific pairwise distances ranged from 3.1% to 20.7% (Table S3).The ABGD method produced 31 groups at a priori genetic distance thresholds of 0.002-0.036.Anteon achterbergi Olmi, 1991 was grouped together with all three specimens of A. claricolle, but genetic distances between these two species ranged from 3.1% to 4.6%.Based on the ML tree, the bPTP method delimited 33 putative species.The two specimens of Anteon nanlingense Xu, Olmi & He, 2011, collected from south China and Thailand, respectively, were assigned as putative species.For all the other species, both ABGD and bPTP returned congruent results with the morphological identification, as shown in Figure 1.
tance thresholds of 0.002-0.036.Anteon achterbergi Olmi, 1991 was grouped together with all three specimens of A. claricolle, but genetic distances between these two species ranged from 3.1% to 4.6%.Based on the ML tree, the bPTP method delimited 33 putative species.The two specimens of Anteon nanlingense Xu, Olmi & He, 2011, collected from south China and Thailand, respectively, were assigned as putative species.For all the other species, both ABGD and bPTP returned congruent results with the morphological identification, as shown in Figure 1.

Remarks.
A. blanduscutum was known only by females [2,22], the male of this species is here confirmed by DNA barcoding.Therefore, we present the following description and diagnosis of the male.
Description.Male.Body length 4.1 mm; fully winged (Figure 2A,B).Head black with mandible testaceous; antenna brown, but proximal half of scape testaceous; mesosoma black; metasoma brown; legs testaceous, except the basal extremity of metacoxa is darkened.Antenna filiform; antennomeres in the following proportions: 16:8:14:13:13:13:12:12:11:13.Head (Figure 2E) shiny, punctate, areas between punctures smooth; frontal line absent; frons with two short longitudinal keels near orbits directed towards antennal toruli; OL = 4; OOL = 9; OPL = 8; POL = 8; TL = 8; greatest breadth of lateral ocellus shorter than OL (3:4); occipital carina complete.Mesoscutum and mesoscutellum shiny, punctate, areas between punctures smooth.Notauli incomplete, present at the anterior half of mesoscutum.Metanotum shiny, largely smooth.Metapectal-propodeal complex with strong transverse keel between disc and propodeal declivity; disc reticulate rugose; propodeal declivity rugose, with two complete longitudinal keels.Forewing hyaline; distal part (Rs) of stigmal vein (2r-rs&Rs) more than 0.5, as long as the proximal (2r-rs) part (8:13).Paramere (Figure 2F) about as long as the aedeagus, without a distal inner pointed process.Tibial spurs 1/1/2.Diagnosis.The diagnosis of the male of A. blanduscutum is as follows: head black with mandible testaceous; head punctate, areas between punctures smooth; mesoscutum punctate, areas between punctures smooth; notauli present at the anterior half of the mesoscutum; propodeal declivity rugose, with two complete longitudinal keels; distal part of the stigmal vein (2r-rs&Rs) more than 0.5 as long as the proximal part; paramere (Figure 2F) about as long as the aedeagus, without a distal inner pointed process; inner side of the paramere not sculptured by papillae (Figure 2F).Because of the above diagnosis, the male of A. blanduscutum can be inserted in the key to the males of the Oriental Anteon published by Xu et al. [2] by modifying couplet 57 as follows: Diagnosis.The diagnosis of the male of A. blanduscutum is as follows: head black with mandible testaceous; head punctate, areas between punctures smooth; mesoscutum punctate, areas between punctures smooth; notauli present at the anterior half of the mesoscutum; propodeal declivity rugose, with two complete longitudinal keels; distal part of the stigmal vein (2r-rs&Rs) more than 0.5 as long as the proximal part; paramere (Figure 2F) about as long as the aedeagus, without a distal inner pointed process; inner side of the paramere not sculptured by papillae (Figure 2F).Because of the above diagnosis, the male of A. blanduscutum can be inserted in the key to the males of the Oriental Anteon published by Xu et al. [2] by modifying couplet 57 as follows:

Male. Unknown.
Diagnosis.Female with head (Figure 3E) punctate and unsculptured among the punctures; clypeus testaceous; frons without lateral keels along orbits; OL shorter than POL, OPL more than twice as long as the greatest breadth of lateral ocellus; mesosoma totally black; propodeal declivity with two longitudinal keels and median area almost completely unsculptured; protarsomere 4 shorter than 1; basal region of the protarsomere 5 (Figure 3F) about as long as the distal region.A. clariclypeum sp.nov. is similar to A. kresli Olmi, 2008.However, in A. clariclypeum, the clypeus is testaceous; the frons has no lateral keels along

Male. Unknown.
Diagnosis.Female with head (Figure 3E) punctate and unsculptured among the punctures; clypeus testaceous; frons without lateral keels along orbits; OL shorter than POL, OPL more than twice as long as the greatest breadth of lateral ocellus; mesosoma totally black; propodeal declivity with two longitudinal keels and median area almost completely unsculptured; protarsomere 4 shorter than 1; basal region of the protarsomere 5 (Figure 3F) about as long as the distal region.A. clariclypeum sp.nov. is similar to A. kresli Olmi, 2008.However, in A. clariclypeum, the clypeus is testaceous; the frons has no lateral keels along the orbits directed towards the antennal toruli; protarsomere 4 is shorter than 1; the basal region of the protarsomere 5 (Figure 3F) is about as long as the distal region (in A. kresli, the clypeus is black; the frons has two lateral keels along the orbits directed towards antennal toruli; the protarsomere 4 is longer than 1; the basal region of the protarsomere 5 is much shorter than the distal region (figure 32A in Xu et al. 2013) [2]).Following the description of A, clariclypeum, the key to the females of the Oriental species of Anteon published by Xu et al. (2013) [2] should be modified by replacing couplet 67 as follows: 67. Propodeal declivity with median region shiny, punctate and areas between punctures smooth. ..67 ′ -Propodeal declivity with median region dull and rugose. ..68 67 ′ .Clypeus black; frons with two lateral keels along orbits directed towards antennal toruli; protarsomere 4 longer than 1; basal region of protarsomere 5 much shorter than the distal region (figure 32A in Xu et al. [2]) . ..A. kresli Olmi -Clypeus testaceous; frons without two lateral keels along orbits directed towards antennal toruli (Figure 3E); protarsomere 4 shorter than 1; basal region of protarsomere 5 about as long as the distal region (Figure 3F) .[6,27].The main reason for this omission is that the type was considered lost (see also Richards [26]) and the original description is unreliable.Kieffer examined six females of this species, all deposited in Marshall's collection [25].In the labels, the localities of capture were indicated by the following abbreviations: Bugsby, Bfm., B.T. and N. (all localities apparently situated in England).However, these specimens are not deposited in the main European collections, including the Museum of Natural History of Paris (France) and the Natural History Museum of London (UK).Richards [26] wrote that the types were lost and considered A. claricolle as a junior synonym of A. ephippiger (Dalman, 1818) var.collaris (Dalman, 1818).In recent years, one of the authors (Frode Ødegaard) collected some female specimens with a pale yellow prothorax, whose morphology and color corresponded to the description of A. claricolle.COI sequences of these specimens supported that they were different from specimens of A. ephippiger var.collaris (i.e., females of A. ephippiger with head and mesosoma black, except that the prothorax is ferruginous or reddish).In addition, Frode Ødegaard also collected males of Anteon with COI sequences identical to those of the females of A. claricolle and different from the COI sequences of males of A. ephippiger var.collaris.Therefore, we decided to resurrect A. claricolle by designating a female neotype (see the description below).

Male. Unknown.
Diagnosis.Female with face partly reticulate rugose and partly punctate, areas between punctures smooth; head vertex without oblique keels connecting lateral ocelli to occipital carina; posterior surface of the pronotum about as long as the anterior surface, about 0.5 as long as the mesoscutum; forewing hyaline without dark transverse bands; propodeal declivity with two longitudinal keels and median area as rugose as lateral areas; protarsomere 4 longer than segment 1; protarsomere 5 with the basal part much shorter than the distal part; protarsomere 5 is straight before the distal apex (Figure 6F). A. maguanense sp.nov. is similar to A. semirugosum Xu, Olmi, Guglielmino & Chen, 2012.However, in the new species, the head vertex has no oblique keels connecting lateral ocelli to occipital carina and the protarsomere 5 is straight before the distal apex (Figure 6F) (in A. semirugosum, head vertex with two oblique keels connecting lateral ocelli to occipital carina and protarsomere 5 curved before the distal apex (Plate 44A in Xu et al. [2]).Following the description of A. maguanense, the key to the females of the Oriental species of Anteon published by Xu et al. [2] should be modified by replacing couplet 98 as follows: 98. Face completely punctate, areas between punctures smooth; posterior surface of pronotum about as long as mesoscutum.Remarks.Anteon meifenganum Olmi, 1991 and A. liui Xu, Olmi & He 2010 were described, respectively, by males [28] and females [6].The COI sequences of the new material of these two species generated in this study are identical, indicating that they are just the opposite sex of the same species.Therefore, A. liui is here treated as a junior synonym of A. meifenganum.1 male, same locality as holotype, but 28.V-28.VI.2019,SCAU 3044066 (SCBG); 1 female, Yunnan, Xishuangbanna, Menghai, Bulangshan Village, 1683 m, Area A2, grass, 21 • 44.981 ′ N, 100 • 26.907 ′ E, 20.IV-28.V.2019, MT, Li Ma leg., 3044033 (SCBG).

Diagnosis.
Male with head black and punctate, areas between punctures smooth; clypeus ferruginous; frontal line incomplete, present in front of the anterior ocellus and absent in the anterior half of frons; notauli present at the anterior 0.7 of mesoscutum; propodeal declivity with two complete longitudinal keels and median area as rugose as lateral areas; distal part of the stigmal vein (2r-rs&Rs) less than 0.5 as long as the proximal part; paramere with pointed distal inner process (Figure 12F), provided with inner distal margin slightly excavated; dorsal process of paramere shorter than volsella (Figure 12F); legs yellow.-Notauli present at about the anterior 0.7 of the mesoscutum (Figure 12D); frontal line incomplete, present in front of the anterior ocellus and absent in the anterior half of frons; clypeus ferruginous.

Female. Unknown.
Diagnosis.Male with head black and punctate, areas between punctures smooth; clypeus ferruginous; frontal line incomplete, present in front of the anterior ocellus and absent in the anterior half of frons; notauli present at the anterior 0.7 of mesoscutum; propodeal declivity with two complete longitudinal keels and median area as rugose as lateral areas; distal part of the stigmal vein (2r-rs&Rs) less than 0.5 as long as the proximal part; paramere with pointed distal inner process (Figure 12F), provided with inner distal  Distribution.China (Shandong).
Etymology.The species is named after Shandong Province, the locality of the holotype.

Male. Unknown.
Diagnosis.Female with head testaceous, punctate and areas between punctures smooth; frontal line almost completely absent, only very shortly present in front of anterior ocellus; ocellar triangle not delimited by keels; mesosoma mostly testaceous; posterior surface of the pronotum about as broad as long; mesoscutum slightly longer than the posterior region of pronotum; notauli present at about the anterior 0.6 of the mesoscutum; propodeal declivity reticulate rugose, with two longitudinal keels; forewing hyaline; protarsomere 4 slightly longer than 1; protarsomere 5 with the basal part much shorter than the distal part (Figure 13F).
Anteon shandonganum sp.nov. is similar to A. quatei Olmi, 1991.However, in A. shandonganum, the frontal line is almost completely absent, the mesoscutum is slightly longer than the posterior region of the pronotum, the notauli (Figure 13D) reach about 0.6 × length of the mesoscutum (in A. quatei, the frontal line is complete; mesoscutum about twice as long as the posterior region of pronotum; notauli reaching about 0.9 × length of the mesoscutum).Following the description of A. shandonganum, the key to the females of the Oriental Anteon published by Xu et al. [2] should be modified by replacing couplet 63 as follows: 63. Head black with mandible, clypeus and anterior region of face between antennal toruli brown-testaceous; notauli reaching about 0.25 × length of mesoscutum. . .

Discussion
The pronounced sexual dimorphism in Dryinidae makes it difficult to associate the opposite sexes of the same species.In the past taxonomic practice of the sexually dimorphic genus Anteon, many species were described based on a single sex.Such a practice might result in that one species would have been described twice, leading to the exaggeration of a higher diversity.On the other hand, while some species were described by both sexes, the female and male were often associated tentatively, i.e., by the comparison of some morphological characters, or because they were collected in the same locality.However, such tentative associations might result in two species that have been lumped into one species, underestimating the real diversity of these parasitoids.DNA barcode-based methods have become powerful in delimitating species boundaries and the confirmation of female-male associations for parasitoid groups with extreme sexual dimorphism [10,30,31].Our present study on Anteon further demonstrates that DNA barcoding is useful to enhance species delimitation and the sexual association of the same species in Dryinidae.The interspecific distances are larger than intraspecific distance for the COI sequences, with the intraspecific distances generally lower than 3%, while the interspecific distances are higher than 3.1% (Tables S2 and S3).The results produced by the two molecular species delimitation methods, i.e., ABGD and bPTP, are mostly congruent with the morphological identification.In addition, the sexual association of six species was confirmed by DNA sequences and lead us to synonymize A. liui with A. meifenganum.
Given the great potential of Anteon in the biological control of leafhoppers, the confirmation of the host association is also important.As indicated by a few researchers, DNA barcoding can be useful for clarifying host-parasitoid relationships in Dryinidae [9,13], which requires a more complete DNA barcode database as a reference.This study represents another effort towards the achievement of this goal.

Figure 1 .
Figure 1.RAxML tree and result of the species delimitation of Anteon based on COI barcodes.Bootstraps values of 50 and above are indicated.The scale bar represents 0.08 substitutions per site.

Figure 1 .
Figure 1.RAxML tree and result of the species delimitation of Anteon based on COI barcodes.Bootstraps values of 50 and above are indicated.The scale bar represents 0.08 substitutions per site.

Author Contributions:Funding:
Conceptualization, H.C. and M.O.; methodology, all authors; software, H.C.; investigation, H.C., M.O., F.Ø., L.C. and J.L.; data curation, H.C. and M.O.; writing-original draft preparation, all authors; writing-review and editing, all authors; supervision, H.C. and J.L.; funding acquisition, J.L.All authors have read and agreed to the published version of the manuscript.This research was funded by the National Natural Science Foundation of China (No. 31472027) to Jing-Xian.Data Availability Statement: All data are available in this paper.

Table 1 .
List of sequenced species and accession numbers.