DNA barcoding of Aphelopus Dalman (Hymenoptera, Dryinidae) from China, with descriptions of four new species

Species of the genus Aphelopus Dalman (Hymenoptera, Dryinidae) are important natural enemies of leafhoppers. The genus is relatively diverse in China, with 35 recorded species. In order to further make use of these important parasitoids in biological control programs, species of Aphelopus collected across China are studied using an integrative approach (combined DNA barcoding and morphology). Of the 17 studied species, two are newly recorded from China: A. nivealis Mita & Olmi, 2014 and A. prolatus Mita & Olmi, 2014, and four are described as new to science: A. incognitus Chen, Olmi & Guglielmino sp. nov., A. maculiala Olmi, Chen & Ødegaard sp. nov., A. taianensis Olmi, Ødegaard & Chen sp. nov., and A. zaifui Olmi, Chen & Liu sp. nov. The total number of Aphelopus species known from China is raised from 35 to 39. Keys to the Oriental and Eastern Palaearctic species OLMI M. et al., Barcoding of Aphelopus from China 41 of Aphelopus are modified to include the new species. Application of DNA barcoding in the species delimitation of Dryinidae is discussed.


Introduction
Aphelopus Dalman, 1823 (Hymenoptera, Dryinidae) is a cosmopolitan genus of parasitoids attacking Typhlocybinae Kirschbaum, 1868 (Hemiptera, Cicadellidae). Many of their hosts are important insect pests (Guglielmino et al. 2013). The genus comprises 78 species (Guglielmino et al. 2017), present in all the zoogeographic regions except Antarctica. Xu et al. (2013) and Guglielmino et al. (2017) listed 30 species from the Oriental region, of which 29 are from China. Olmi & Xu (2015) added further six species from Eastern Palaearctic China, bringing the total number of species known from China to 35.
Further development of these important parasitoids in biological control programs against leafhoppers requires accurate identification of the species. However, the taxonomy of Dryinidae Haliday, 1833 is challenging because many species are morphologically similar and require a careful examination of detailed morphological characters, especially the male genitalia. Intraspecific morphological variation and extreme sexual dimorphism are the two main issues which hamper research on the systematics of Dryinidae (Olmi et al. 2021), including that of the genus Aphelopus. In addition, too few researchers rear dryinids and many species were described based on a single specimen or a small number of specimens, making it difficult to understand if a morphological or colour difference falls within the range of variability of a species or indicates a new taxon. The lack of rearing also means that it is difficult to associate conspecifics based on morphology, as males and females of the same species are usually completely different. DNA markers, such as the mitochondrial cytochrome c oxidase 1 (COI) gene, have become an important species identification tool for insects (Hebert et al. 2003a(Hebert et al. , 2003b. The importance of DNA sequences in the taxonomy and systematics of Dryinidae as well as the molecular identification of their host associations has been recognized by some researchers (see Mita & Matsumoto 2012;Mita et al. 2013;Tribull 2015;Chen et al. 2020;Olmi et al. 2021). By analyzing COI sequences, Mita & Matsumoto (2012) discovered the male of Gonatopus javanus (Perkins, 1912), a species previously known only from females. Similarly, based on COI sequences, Mita et al. (2013) found that Haplogonatopus oratorius (Westwood, 1833) and H. apicalis Perkins, 1905, whose females and males had previously been confused, were separate species. In addition, Chen et al. (2020) discovered the previously unknown host of Gonatopus viet Olmi, 1986, by a comparison of COI sequences of females and immature larvae extracted from host thylacia. More recently, Olmi et al. (2021) associated female, male, larva, and the host of Bocchus scobiolae Nagy, 1967, by COI sequences.
However, a DNA barcode database is still lacking for the Chinese fauna of Dryinidae. To fill this gap, one of the coauthors (HC) started a research campaign with the objectives to collect specimens of dryinids across China, identify the species by morphological characters and build a DNA database for these important parasitoids. This paper is the first result of the ongoing research campaign.

Descriptions
Species descriptions follow the terminology used by Olmi et al. (2019). The measurements reported are relative, except for the total body length (head to abdominal tip, without the antenna and the sting), expressed in millimeters. In the descriptions POL is the distance between inner edges of the lateral ocelli; OL is the distance between inner edges of a lateral ocellus and the median ocellus; OOL is the distance from the outer edge of a lateral ocellus to the eye; OPL is the distance from the posterior edge of a lateral ocellus to the occipital carina; TL is the distance from the posterior edge of an eye to the occipital carina. The term "metapectal-propodeal disc" is here used in the sense of Kawada et al. (2015). It corresponds to the term "dorsal surface of propodeum" sensu Olmi & Xu (2015) and Xu et al. (2013). The term "propodeal declivity" sensu Kawada et al. (2015), used here, corresponds to the term "posterior surface of propodeum", sensu Xu et al. (2013) and Olmi & Xu (2015).

Imaging
Multifocal images were made using a mirrorless Sony Alpha 6300 camera with cross table Proxxon KT 70 or a Leica M205C multifocal equipment and a Nikon SMZ25 microscope with a Nikon DS-Ri 2 digital camera system. Images were then post-processed with Adobe Photoshop CS6 Extended.

COI barcoding
Genomic DNA of the material from China was extracted from the entire specimen using a DNeasy Blood & Tissue Kit (QIAGEN, Inc.), following a nondestructive DNA extraction protocol as described in Taekul et al. (2014). Voucher specimens (Supp. File 1) are deposited in the Museum of Biology at Sun Yat-sen University, Guangzhou, China (SYSBM). The ‛barcode' region of the mitochondrial cytochrome oxidase subunit 1 (COI) was amplified using the LCO1490/HCO2198 primer pair (Folmer et al. 1994). Polymerase chain reactions (PCRs) were performed using Tks Gflex™ DNA Polymerase (Takara) and conducted in a T100™ Thermal Cycler (Bio-Rad). Thermocycling conditions were as follows: an initial denaturing step at 94°C for 1 min, followed by 5 cycles of 98°C for 10s, 45°C for 15s, 68°C for 30s; 35 cycles of 98°C for 10s, 52°C for 15s, 68°C for 30s and an additional extension at 68°C for 5 mins. Amplicons were directly sequenced in both directions with forward and reverse primers on an Applied Biosystem (ABI) 3730XL by TsingKe Biological Technology (Beijing, China). Chromatograms were assembled into contigs in Geneious ver. 11.0.3. The assembled sequences were translated to amino acids using the invertebrate mitochondrial code to check for stop codons and frame shifts, and were blasted against the GenBank database to check for contamination and pseudogenes (e.g., nuclear mitochondrial DNA, NUMT) as implemented in Geneious ver. 11.0.3. All sequences generated from this study are deposited in GenBank (accession numbers see Table 1), except the sequences of Aphelopus atratus (Dalman, 1823) and A. prolatus Mita & Olmi, 2014, respectively from Norway and Sweden, which are uploaded in the BOLD System (Ratnasingham & Hebert 2007): sequence ID for Aphelopus prolatus: NSMTP249-15.COI-5P; for Aphelopus atratus: NODRY067-14.COI-5P. COI sequences were aligned by codons using MUSCLE implemented in MEGA7 (Kumar et al. 2016). The aligned sequences were then analyzed using RAxML as implemented in Geneious ver. 11.0.3 to generate a maximum likelihood (ML) tree. The model used was GTRGAMMA+I. Automatic bootstopping criterion was selected as the appropriate number of bootstraps; 300 replicates were run. Bocchus thorpei Olmi, 2007 (Hymenoptera, Dryinidae), from New Zealand, was used as an out-group based on the phylogenetic topologies recovered by Tribull (2015) (COI sequence of B. thorpei was downloaded from GenBank, NZAC04036596).

Molecular analysis
The present study generated 23 COI sequences with an average of 658 bp. Including the sequences of A. atratus and A. prolatus, the 25 studied COI sequences were found to belong to 17 species, of which four are described as new below. Genetic distances of the sequences are in Supp. file 1. Intraspecific distances of the COI sequences generally are less than 3%, with the exception of A. prolatus, which has two haplogroups (from Sweden and China, respectively) and the distance between the two haplogroups is 5.6%. Interspecific distances range between 5.6% and 25.4%. Each species recovered on the tree is clearly separated from all neighboring species, as shown in Fig. 2 Perkins, 1912 Genus Aphelopus Dalman, 1823 See Xu et al. (2013) and Olmi & Xu (2015) for taxonomic details on the genus.  Chen, Olmi & Guglielmino sp. nov. urn:lsid:zoobank.org:act:A917F029-3AF2-451F-9B6B-CC215432441C Figs 3-4, 8A

Etymology
The species is named ‛incognitus' (Latin adjective meaning ‛unknown') because it was first recognized as a new species by COI sequences. Otherwise it would remain unnoticed, because morphologically it is extremely similar to closely related species such as A. maculiclypeus Olmi, 1999 andA. spadiceus Xu &He, 1997. Metapectal-propodeal complex with disc dull, reticulate rugose; propodeal declivity with two longitudinal keels, median area shiny, unsculptured and lateral areas rugose. Fore wing hyaline, without dark transverse bands. Basivolsella ( Fig. 8A) with two subdistal bristles and outer medial process. Tibial spurs 1/1/2.

Remarks
The female and male association of the new species is supported by the COI sequences, which are 99.2% identical between both sexes. This new species has been collected both in the Oriental (Yunnan) and Eastern Palaearctic (Beijing) region. In the Oriental region, following the above diagnosis, the female of A. incognitus Chen, Olmi & Guglielmino sp. nov. is close to that of A. ochreus Olmi, 1984. However, in the new species, the notauli reach about 0.6 × length of mesoscutum (Fig. 4D); OPL is longer than OOL (Fig. 4C); the frontal line is incomplete, absent in front of the anterior ocellus (in A. ochreus, the notauli are complete or reaching about 0.75-0.80 × length of mesoscutum; OPL is shorter than OOL; the frontal line is complete). Following the description of A. incognitus Chen, Olmi & Guglielmino sp. nov., the key to females of Oriental Aphelopus published by Xu et al. (2013) should be modified by replacing couplet 4 as follows.

Female
Unknown.

Remarks
According to Xu et al. (2013), the male of Aphelopus spadiceus usually has a hyaline fore wing, except a specimen from China (Yunnan, Tengchong, Jietou, 12.v.2009, Jie Zeng leg., 1 ♂, SCAU) showing a small infuscate patch beneath the pterostigma. The above specimen has been considered only a variety of A. spadiceus by Xu et al. (2013). However, a comparison between COI sequences of two males of A. spadiceus from China, 26.324161° N, 99.256617° E, one with a hyaline fore wing and the other with an infuscate patch (Fig. 5F), showed that the two specimens belong to two separate species. This result persuaded us to describe the specimen with an infuscate patch on the fore wing as a new species named A. maculiala Olmi, Chen & Ødegaard sp. nov. (although the genitalia of the new species is similar to that of A. spadiceus). The fore wing of the new species shows a small infuscate patch, so that it is more similar to A. xanthopus Xu, He & Olmi, 1999, than to A. spadiceus. However, in A. maculiala Olmi, Chen & Ødegaard sp. nov. the face is completely black (Fig. 5D), whereas in A. xanthopus it is whitish between the antennal toruli. Following the description of A. maculiala Olmi, Chen & Ødegaard sp. nov., the key to males of the Oriental Aphelopus published by Xu et al. (2013) should be modified by replacing couplet 21 as follows: 20. Fore wing with one infuscate patch beneath pterostigma (Fig. 5A)

Diagnosis
Male with head black, except mandible testaceous; mesosoma black; notauli incomplete, reaching approximately 0.5× length of mesoscutum; aedeagus distally not tridentate (Fig. 8C); basivolsella very narrow (Fig. 8C), without distal outer process, with two subdistal bristles and one lateral outer pointed apophysis; distivolsella not in the form of a long straight rod.

Etymology
The new species is named after Taian City, where it has been collected. lateral areas rugose. Fore wing hyaline, without dark transverse bands. Basivolsella (Fig. 8C) very narrow, without distal outer process, with one lateral outer pointed apophysis and two subdistal bristles situated on top of each other. Tibial spurs 1/1/2.

Female
Unknown.

Remarks
From the above diagnosis, A. taianensis Olmi, Ødegaard & Chen sp. nov. is close to A. nepalensis Olmi, 1984. However, in the new species, the basivolsella (Fig. 8C) is very narrow and with one lateral pointed apophysis (very wide and without lateral pointed apophysis in A. nepalensis (Olmi & Xu 2015: pl. 4k

Etymology
The species is named after the late Prof. Zaifu Xu (SCAU), a famous specialist of Chinese Dryinidae.

Female
Unknown.

Remarks
The distivolsella in the form of a long straight rod indicates that the new species is different from all known Aphelopus species, except A. serratus Richards, 1939, from the Palaearctic region. However, in A. zaifui Olmi, Chen & Liu sp. nov. the basivolsella has a widened apex (Fig. 8D), whereas in A. serratus it has a slender apex (Fig. 8F). Following the description of A. zaifui Olmi, Chen & Liu sp. nov., the key to males of Oriental Aphelopus published by Xu et al. (2013) should be modified by replacing couplet 1 as follows: 1. Distivolsella in the form of a long straight rod (Fig. 8D); basivolsella long and narrow, with distal apex very widened (Fig. 8D)

Remarks
Aphelopus nivealis Mita & Olmi, 2014 was originally described from the Eastern Palaearctic region (Honshu, Japan) (see Olmi & Xu 2015). In this species there are no notauli. One of the two specimens above (from Guangdong, 22.570411639° N, 113.99317344° E) matches completely with the description of A. nivealis, including the body colour and the absence of notauli ( Fig. 15A-B). On the contrary, the other specimen (from Guangdong, 23°3′9″ N, 113°23′23″ E) is whiter and seems to have shallow traces of notauli reaching about 0.5 × length of mesoscutum ( Fig. 15C-D). The genetic distance between the two specimens is only 0.6%, suggesting that the variations are just intraspecific. These records indicate that A. nivealis is present both in the Eastern Palaearctic and the Oriental regions. Olmi, 1984 Fig. 16 Aphelopus penanganus Olmi, 1984

Distribution
Widely spread across all of the Palaearctic region, from Europe to Russian Far East, South Korea and Japan (Olmi & Xu 2015;Kim & Lee 2016). Probably present in northeastern China, but not found so far.

Discussion
In the present study, we discovered that A. incognitus Chen, Olmi & Guglielmino sp. nov. shows an evident sexual dimorphism in head colour pattern (female with largely testaceous head, while male with head black), but the COI sequences generated from female and male specimens allowed us to confirm their association. In A. nivealis and A. zaifui Olmi, Chen & Liu sp. nov., notauli seem to be variable in appearance and length, but COI sequences indicated that these variations are just intraspecific. Considering that colour patterns and the length of the notauli have previously been used to separate most species of Aphelopus, the validity of these species should be tested by other methods such as rearing and DNA barcoding.
Our current study demonstrates that DNA barcoding is a powerful tool to enhance species delimitation and the female-male association of the same species. A more complete DNA barcode database of Dryinidae would also accelerate the discovery of host-Dryinidae associations using molecular methods (Chen et al. 2020;Olmi et al. 2021).