Chinese Species of the Genus Pseudanaphes Noyes & Valentine (Hymenoptera: Mymaridae) with Description of a New Species

Simple Summary Parasitoids of the family Mymaridae (Hymenoptera: Chalcidoidea) are difficult to collect and study, in part because of their small body size of 0.2–1.5 mm. Currently, more than 1500 species have been described and recorded. However, molecular data are rarely generated and used in relation to these small parasitoids. The genus Pseudanaphes is one of the smaller genera, with only five known species, for which molecular data were not available until this study. In this study, a new species of Pseudanaphes, P. yadongicus Aishan & Cao sp. nov., is separated from the known species P. zhaoi Lin by combining morphological and molecular data, both collected in Tibet, China. The DNA barcode and illustrations of P. zhaoi are also provided simultaneously. The DNA barcodes, morphological diagnosis, and a key to the world species of this genus will facilitate studies on this genus. Abstract The fairyfly Mymaridae (Hymenoptera: Chalcidoidea) are widely distributed worldwide, but species of this family have rarely been collected and recorded from the Qinghai–Tibet Plateau. In this study, mymarids collected in Tibet, China, are identified based on morphology and molecular data. Two species of the genus Pseudanaphes Noyes & Valentine are treated and illustrated here, including a known species, P. zhaoi Lin, and a new species, P. yadongicus Aishan & Cao sp. nov. In addition, a key to the world species of Pseudanaphes (females) and DNA barcodes for P. yadongicus and P. zhaoi are provided.


Introduction
The genus Pseudanaphes Noyes & Valentine is one of several smaller genera in the family Mymaridae (Hymenoptera: Chalcidoidea), species of which are not commonly or numerously collected. This genus is characterized by relatively hairy fore wings and long marginal veins and was established by Noyes and Valentine (1989), who recorded an Australian species, Pseudanaphes hirtus Noyes & Valentine, from Titirangi, New Zealand [1]. After that, another species, P. zhaoi Lin, was discovered in the Palaearctic region (Shaanxi, China) [2]. Two Australian species, P. lincolni (Girault) and P. particoxae (Girault), were originally described briefly without illustrations based on a female specimen under the generic name Polynemoidea Girault [3,4]. New (1974) provided measurements and illustrations for these two species by examining the type specimens, and Lin (2007) transferred these two species to Pseudanaphes [5,6]. Later, Rehmat and Anis (2011) described another new species, P. sikkimianus, from India, based on only a single female specimen [7]. As a result, five 2 of 9 species were known in the genus Pseudanaphes prior to this study. Nevertheless, the host information of Pseudanaphes is currently unknown, with only the type species of this genus, P. hirtus, being possibly associated with the eggs of weevils (Coleoptera: Curculionidae) [8].
Currently, the systematic relationship of Pseudanaphes with other mymarids remains unclear due to the lack of comprehensive molecular evidence and insufficient collections of this genus and possibly related groups. Usually, Pseudanaphes is placed in the Anaphes group, along with Anaphes Haliday, Dorya Noyes & Valentine, and Erythmelus Enock [6]. Among these genera, the species of Pseudanaphes are most similar to some species of Anaphes Haliday and can be distinguished from the latter by the rounded apex of the fore wing with a longer marginal vein and the absence of a longitudinal medina groove on the propodeum.
Species of the genus Pseudanaphes are rarely reported, either because they are not frequently discovered and collected in the field or because their identification is difficult. This genus also remains poorly studied in China. In this study, a new species from Tibet, China, is described, and diagnosis and illustrations are provided for a known Chinese species, P. zhaoi Lin. In addition, DNA barcodes of the new species and P. zhaoi are provided here, which are also reported for the first time for this genus.

Specimens Collection
All examined specimens of the two Chinese Pseudanaphes species were collected using Malaise traps and preserved in 99% ethanol at -20 • C until use. The holotype and one paratype of the newly described species were deposited in the Institute of Zoology, Chinese Academy of Sciences, Beijing, China (IZCAS), and the remaining paratypes and specimens of P. zhaoi were deposited in the Insect Collection of the College of Life Science and Technology, Urumqi, Xinjiang, China (ICXU).

Taxonomic Studies
Specimens were dissected and mounted on slides by using Canada balsam. All measurements were taken from slide-mounted specimens at 65×, 100×, 250×, or 400× magnification with a Leica compound microscope fitted with an eyepiece reticle. Body length, in micrometers (µm), was measured from the transverse trabecula to the metasomal apex, excluding the exserted part of the ovipositor. The remaining measurements are given either in micrometers or as ratios. Photographs were taken from slide-mounted specimens using a Nikon Ni-E system, and images were processed by Adobe Photoshop.

DNA Extraction, Amplification, and Sequencing
Genomic DNA was extracted from 16 specimens (Table 1) using the whole body of the specimens and following the protocol of the DNeasy Blood & Tissue Kit (Qiagen, Germany). After DNA extraction, the voucher specimens were dissected and mounted on slides for further morphological examination. The voucher specimens were deposited in IZCAS.

Sequence Analysis
All sequences were verified by the NCBI Nucleotide Blast tool. Sequences from both directions were assembled and edited in Sequencher version 4.5 (Gene Codes Corporation, Ann Arbor, MI, USA) and aligned in BioEdit version 7.0.9.0 [10] to create the COI matrix used for distance calculations. The COI matrix was translated into amino acids in MEGA7.0 [11] to check for stop codons. A neighbor-joining (NJ) tree based on the Kimura two-parameter (K2P) distances was constructed using MEGA7.0 with 1000 bootstrap replicates to generate support values for the nodes. The COI sequences generated in this study were deposited in GenBank under accession numbers ON834699-ON834714.

Results
The genus Pseudanaphes, with one described species and one new species from China, was treated in this study. The DNA barcodes (COI) of seven females of P. yadongicus and nine females of P. zhaoi were successfully obtained. After alignment and trimming, the COI matrix without insertions or deletions, with a length of 401 base pairs, was used to obtain a NJ tree ( Figure 1) by K2P distances. Based on 401 bp COI sequences, these two species were separated with high bootstrap support of 100%. The interspecific divergence (K2P distance) between these two species varied from 11.7% to 12.5%. Pseudanaphes yadongicus displayed no intraspecific variation, and P. zhaoi showed the maximum intraspecific distance at 0.8%. Diagnosis. The genus Pseudanaphes Noyes & Valentine can be characterized by the following characters: female antenna with six-segmented funicle, three-segmented clava; clava large and slightly shorter than the combined length of six funiculars, and with one row of campaniform sensilla; fore wing apex evenly rounded, with venation extending at least 0.4× wing length and with disc cilia dense and with a slight brownish tinge and curved, infuscate band below proximal half of marginal vein; four-segmented tarsi. In males, antenna with 11 flagellomeres; body color and size similar to females. Diagnosis. The genus Pseudanaphes Noyes & Valentine can be characterized by the following characters: female antenna with six-segmented funicle, three-segmented clava; clava large and slightly shorter than the combined length of six funiculars, and with one row of campaniform sensilla; fore wing apex evenly rounded, with venation extending at least 0.4× wing length and with disc cilia dense and with a slight brownish tinge and curved, infuscate band below proximal half of marginal vein; four-segmented tarsi. In males, antenna with 11 flagellomeres; body color and size similar to females.

Pseudanaphes Noyes & Valentine
The Anaphes group consists of four genera, Anaphes, Erythmelus, Dorya, and Pseudanaphes. The genus Pseudanaphes can be distinguished from the other three genera by the combination of the following characters: large and three-segmented antenna clava; fore wing apex evenly rounded, with a curved, dark mark below parastigma and a longer venation that extends at least 0.4× wing length. These characters are also useful in distinguishing species of Pseudanapahes from the most similar genus, Anaphes Haliday. Compared to the females, the species of Anaphes have a one-or two-segmented clava and an asymmetrical fore wing, often with an oblique row of microtrichia extending from just below the apex of the stigmal vein towards the wing apex. Lin (2007) stated that, for Pseudanapahes, there is no longitudinal median groove on the propodeum [6]. In contrast, a median longitudinal groove was observed on the propodeum (Figures 2c and 3c) for the Pseudanapahes specimens examined in this study. Along with this character, the clava with one row of campaniform sensilla (Figure 3b) was suggested to be diagnostic for the genus Pseudanapahes. Diagnosis. Antenna with the first claval segment the longest, apical segment the shortest, the first and second segments of clava with two longitudinal sensilla on each, and the third segment of clava with three longitudinal sensilla. Fore wing disc densely The Anaphes group consists of four genera, Anaphes, Erythmelus, Dorya, and Pseudanaphes. The genus Pseudanaphes can be distinguished from the other three genera by the combination of the following characters: large and three-segmented antenna clava; fore wing apex evenly rounded, with a curved, dark mark below parastigma and a longer venation that extends at least 0.4× wing length. These characters are also useful in distinguishing species of Pseudanapahes from the most similar genus, Anaphes Haliday. Compared to the females, the species of Anaphes have a one-or two-segmented clava and an asymmetrical fore wing, often with an oblique row of microtrichia extending from just below the apex of the stigmal vein towards the wing apex. Lin (2007) stated that, for Pseudanapahes, there is no longitudinal median groove on the propodeum [6]. In contrast, a median longitudinal groove was observed on the propodeum (Figures 2c and 3c) for the Pseudanapahes specimens examined in this study. Along with this character, the clava with one row of campaniform sensilla (Figure 3b) was suggested to be diagnostic for the genus Pseudanapahes.

Pseudanaphes yadongicus Aishan & Cao, sp. nov.
Diagnosis. Antenna with the first claval segment the longest, apical segment the shortest, the first and second segments of clava with two longitudinal sensilla on each, and the third segment of clava with three longitudinal sensilla. Fore wing disc densely setose with setae extending to base of submarginal vein. Ovipositor long and exserted beyond apex of metasoma; ovipositor length equal to length of metasoma.
Description. Female (holotype and paratypes). Body length 1450-1470 µm. Body color brown. Antenna brown, except scape, and pedicel yellowish brown. Fore wing hyaline, slightly infuscate below submarginal vein, and with a usual, curved, infuscate band below proximal half of marginal vein. Legs yellow brown, except tarsi yellow.
Head (Figure 2a Biology. Unknown. Comments. The specimens studied here differ from the holotype in the following characters: F6 with one longitudinal sensillum; fore wing 3.25-3.58× as long as wide, with longest marginal seta 0.42-0.52× greatest width of the fore wing; ovipositor 1.39-1.42× as long as the mesotibia. In addition, for one specimen of the material examined above, the apical two claval segments, F2 and F3, are coalesced (Figure 4a,b). These characteristics fall within the range of intraspecific variations.  Biology. Unknown.

Distribution. China (Tibet).
Comments. Females of P. yadongicus can be recognized by the combination of characters described in the diagnosis. Accordingly, the females of this species resemble those of P. lincolni (Girault). In females, P. yadongicus differs from the latter species in antenna color, relative length of F1 and the pedicel, relative length of the three claval segments, and relative length of the extended ovipositor. Females of P. yadongicus have brown antenna with yellowish brown scape and pedicel, F1 and pedicel of subequal length, the longest F1 of the funicle, and the longest first segment of the clava (Figure 2b); they have an ovipositor notably exserted beyond the apex of the metasoma (Figure 2g), whereas, in comparison to P. yadongicus, females of P. lincolni have black antennae, F1 longer than pedicel, F2 and pedicel of subequal length, the longest F2 of the funicle, and the ovipositor not notably exserted beyond the apex of the metasoma as in the former species. Distribution. China (Tibet, Shaanxi). Biology. Unknown. Comments. The specimens studied here differ from the holotype in the following characters: F6 with one longitudinal sensillum; fore wing 3.25-3.58× as long as wide, with longest marginal seta 0.42-0.52× greatest width of the fore wing; ovipositor 1.39-1.42× as long as the mesotibia. In addition, for one specimen of the material examined above, the apical two claval segments, F2 and F3, are coalesced (Figure 4a,b). These characteristics fall within the range of intraspecific variations.

Discussion
Almost all species of Mymaridae are important parasitoids of the eggs of a wide variety of insect pests [12]. Currently, more than 1500 species in 115 genera of Mymaridae are known worldwide [2,9]. Although a total of 31,839 COI sequences of Mymaridae have been recorded in GenBank (National Center for Biotechnology Information, NCBI), most of them were sequenced from unnamed species with a high repeat rate of 80%, and 673 of them were sequenced from only thirteen named species of Anaphes Haliday, Anagrus Haliday, Cosmocomoidea Howard, and Ooctonus Haliday [13][14][15][16][17][18][19][20]. In this study, COI primers for the genus Pseudanaphes and COI sequences of Pseudanaphes were developed and generated for the first time, which provide a reference for studies on the family Mymaridae. The results of molecular analysis support the species delimitation of these two Pseudanaphes species, P. yadongicus and P. zhaoi. In addition, it is suggested that the antennal clava with one row of campaniform sensilla could be used as a diagnosis for the genus Pseudanaphes. This will facilitate the identification and discovery of Pseudanaphes species or even other mymarids.