Two new species of Ooencyrtus (Hymenoptera, Encyrtidae), egg parasitoids of the bagrada bug Bagrada hilaris (Hemiptera, Pentatomidae), with taxonomic notes on Ooencyrtus telenomicida

In support of a biological control program in California, USA, against the bagrada bug, Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae), an invasive pest of Asian origin, colonies of two species of Ooencyrtus Ashmead (Hymenoptera, Encyrtidae) are maintained using B. hilaris eggs as host. One of them, Ooencyrtus mirus Triapitsyn & Power, sp. nov., is of Pakistani origin. It displays natural preference for bagrada bug eggs and is being evaluated in quarantine as a candidate for classical biological control. The other, Ooencyrtus lucidus Triapitsyn & Ganjisaffar, sp. nov., appears to be native to California, and we believe it switched to B. hilaris from native pentatomid hosts. Both new species are described and illustrated, as is the Old World species Ooencyrtus telenomicida (Vassiliev), for which a neotype is designated. The presented morphometric evidence as well as mitochondrial and nuclear ribosomal DNA sequence data separate Ooencyrtus mirus from O. telenomicida. A lectotype is designated for Ooencyrtus californicus Girault from California, which is morphologically similar to O. lucidus.


Introduction
The painted bug, also known as bagrada bug, Bagrada hilaris (Burmeister) (Hemiptera, Pentatomidae) (Fig. 1A), is native from western Asia to southern Africa (Howard 1907;Mahmood et al. 2015). It first was discovered in the United States in 2008 in Los Angeles County, California (Arakelian 2008). Since then, its range in the United States has expanded: it has been reported from other regions of California , Arizona (Palumbo and Natwick 2010;Palumbo et al. 2016), New Mexico (Bundy et al. 2012), Texas (Vitanza 2012), Nevada , Utah , and Hawaii (Matsunaga 2014). It also has been identified in Mexico (Sánchez-Peña 2014;Lomeli-Flores et al. 2019). Bagrada hilaris is a major pest of cole crops (Palumbo et al. 2016;Bundy et al. 2018), and there is no established biological control program for it. In 2014, in response to the rapid spread of B. hilaris, a search was conducted for egg parasitoids within its native range in Pakistan. Three species of parasitoid wasps, Trissolcus hyalinipennis Rajmohana & Narendran (Hymenoptera, Scelionidae, Telenominae) (Rajmohana 2006;Ganjisaffar et al. 2018), Gryon gonikopalense Sharma (Scelionidae, Scelioninae) (Sharma 1982;Martel et al. 2019), and Ooencyrtus sp. (Hymenoptera, Encyrtidae), were collected from B. hilaris eggs (Mahmood et al. 2015). Live specimens of the recovered parasitoids were shipped to the United States Department of Agriculture, Agricultural Research Service Quarantine Facility at the National Biological Control Laboratory in Stoneville, Mississippi (Mahmood et al. 2015) to be evaluated as classical biological control candidate agents for B. hilaris. In 2016, Ooencyrtus sp. was transported under permit to the University of California Riverside (UCR) Quarantine Facility, and we have been studying its biology, host specificity, and risk assessment. In concert with these biological studies, we addressed the taxonomic identification of this insect and describe it as a new species herein.
In addition to the classical biological control efforts with the exotic Ooencyrtus sp., surveys for resident egg parasitoids of B. hilaris have been conducted throughout California since 2017 using sentinel egg cards. In this project, we collected a variety of parasitoid species from the B. hilaris eggs. A species of Ooencyrtus Ashmead was recovered from sentinel cards in Riverside, California. Because the taxonomy of the Nearctic species of Ooencyrtus is in flux and there are no keys to the 11 described species, the first author physically compared specimens collected in our study to the types and specimens of all described species from North America. In addition, he tried to identify our specimens with keys from other regions (i.e. Neotropical, Oriental, and Palearctic). These efforts were unsuccessful, suggesting that our insect was an undescribed native species. Therefore, we also describe this parasitoid as a new taxon.
During our investigations, we attempted to compare our insects to Ooencyrtus telenomicida (Vassiliev) and to other egg parasitoids in the O. telenomicida species complex. However, we were unable to locate the type series of Encyrtus telenomicida Vassiliev (now Ooencyrtus telenomicida). Therefore, we collected Ooencyrtus sp. from eggs of Eurygaster Laporte, the host genus of Encyrtus telenomicida, in the same general habitat as indicated in the original description and matched the morphology of the new collections both with that description and with non-type specimens reared before 1950 from the original host in both Russia and Ukraine, not too far from its type locality. Based on the morphological congruence, we designated a neotype for Encyrtus telenomicida and supplement this designation with DNA sequence data necessary for the differentiation of Ooencyrtus telenomicida from morphologically similar species in the complex. Throughout the paper we are using the term 'O. telenomicida species complex' for those species that are genetically and morphologically close to O. telenomicida. The group was defined morphologically by Hayat et al. (2014) and later expanded by Samra et al. (2018) using molecular and morphological data.

Sources of specimens Ooencyrtus species of Pakistan origin
Compared to other pentatomids, B. hilaris has an unusual ovipositional behavior of laying eggs singly or in small clusters on live plant material, in detritus and in soil (Taylor et al. 2014). Knowing this behavior, researchers working in the Toba Tek Singh District of the Punjab Plain in Pakistan noticed B. hilaris adults congregating on dry debris of Brassica juncea (L.) Czernajew and Brassica napus L. The plant debris was collected and shaken onto a plastic sheet, and the resulting leaves, stems and soil were transported to the laboratory and examined for B. hilaris eggs (Mahmood et al. 2015). Eggs were collected and placed in glass vials to wait for parasitoid emergence. Mahmood et al. (2015) recovered a uniparental strain of Ooencyrtus sp. from the B. hilaris eggs on B. napus but not from the eggs on B. juncea. The emerging parasitoids were reared and shipped to Dr. Walker Jones at the USDA-ARS Quarantine Facility, National Biological Control Laboratory in Stoneville, Mississippi, USA (Mahmood et al. 2015). From there a colony was sent to the UCR Quarantine Facility, where it has been reared continuously on B. hilaris eggs since January 2016. The colony is maintained at 26 °C, 14:10 L:D and ~50% RH.

Ooencyrtus species native to California
Sampling surveys for B. hilaris parasitoids were initiated in October 2017 and are still in progress. For the present study, B. hilaris adults were collected from a greenhouse colony where they were raised on seedlings of broccoli (Brassica oleracea L. var. italica), canola (Brassica napus L.), mustard greens (Brassica juncea L.), and sweet alyssum (Lobularia maritima (L.) Desvaux). Thirty adult mating pairs were placed in round plastic containers (15 cm diameter × 6 cm depth) (Durphy Packaging Co., Warminster, Pennsylvania, USA) with 2 screen openings on opposite sides for air circulation (Fig. 1B). Paper towels were placed in the bottom of the containers as an ovipositional substrate. The insects were provided organic broccoli florets and moved to new containers daily. Eggs were collected from the plastic containers and paper towels and glued to sentinel cards as described by Ganjisaffar et al. (2018). The cards were deployed in a squash field infested with shortpod mustard weeds, Hirschfeldia incana (L.) at the UCR Agricultural Operations on October 26, 2018. One of the cards with 15 glued B. hilaris eggs had 12 eggs parasitized, and from these 11 adult parasitoids emerged between November 13 and 15, 2018. These adults were placed in a vial, provided with honey, and given access for 24 hours to 50 B. hilaris eggs that had been glued (Elmer's) to a 1.5 × 4 cm white card. Following the 24 hour access period, the card with parasitized eggs was transferred to a new vial for rearing the parasitoids. The original egg parasitoids then were collected and placed in vials containing 95% ethanol and stored in a freezer at -20 °C until they were used for morphological studies or DNA extraction. Primary molecular voucher specimens were slide-mounted in Canada balsam.

Ooencyrtus telenomicida (Vassiliev) from Europe
Specimens of O. telenomicida, reared in Russia and Ukraine from eggs of Eurygaster integriceps Puton (Hemiptera, Scutelleridae) in 1948 and 1950, were borrowed from the Zoological Institute, Russian Academy of Sciences, Saint Petersburg, Russia. Unfortunately, PCRs failed on all the specimens that were extracted. Therefore, new specimens of O. telenomicida were reared in Ipatele, Iaşi County, Romania, from eggs of Eurygaster sp. found on wheat. We were able to extract DNA from these specimens, and the primary molecular vouchers were individually slide-mounted in Canada balsam or chemically dried and point mounted.

Taxonomic studies
For the taxonomic descriptions of the new species, the morphological terms of Gibson (1997) were used, with a few modifications. All wing measurements of length or length:width are given in micrometres (µm). Abbreviations used in the descriptions are: F = funicle segment of the female antenna or flagellomere of the male antenna; mps = multiporous plate sensillum or sensilla on the antennal flagellar segments (= longitudinal sensillum or sensilla, or sensory ridge(s)).
Specimens for morphometric studies were dried from ethanol using a critical point drier, then point-mounted and labeled. Selected specimens then were dissected and slide-mounted in Canada balsam. Slide mounts were examined under a Zeiss Axioskop 2 plus compound microscope (Carl Zeiss Microscopy, LLC, Thornwood, New York, USA) and photographed using the Auto-Montage system (Syncroscopy, Princeton, New Jersey, USA). Photographs were retouched where necessary using Adobe Photoshop (Adobe Systems, Inc., San Jose, California, USA). In addition, the body length of 24 male and 24 female O. mirus wasps were measured from the anterior end of the head to the posterior end of the gaster, not including the ovipositor or aedeagus, with a Leica Wild M10 stereoscope using a Bausch & Lomb 0.1 mm and 0.01 mm micrometer.
For the morphometric analysis, we measured characters in adult females and males to determine the following ratios: 1) ovipositor length to mesotibia length; 2) fore wing length to maximum width; 3) scape length to width (excluding the radicle); 4) clava length to width; 5) F1 length to pedicel length; and 6) F2 length to F1 length. For all parameters, the ranges, means, and standard deviations were determined.
For O. mirus and O. telenomicida we also used multivariate ratio analysis (MRA) (Baur and Leuenberger 2011) because these two species differ mostly in color and very little in body ratios. For this analysis, 15 measurements per specimen were used and 9 female specimens for each species were included in the analysis. Following the approach of Baur et al. (2014) the MRA also served to place the neotype of O. telenomicida in the morphospace of specimens from Russia and Ukraine. The complete set of measurements is available as Suppl. material 1 data from the publisher's website.
Specimens examined are deposited in the collections with the following acronyms:  (Truett et al. 2000) as described in Andreason et al. (2019aAndreason et al. ( , 2019b to preserve specimen integrity for subsequent slide mounting and morphological study. DNA from the neotype female specimen of O. telenomicida (UCRC ENT 311776) and a non-type female from the same collection event (UCRC ENT 311775) was extracted using the non-destructive method described in Triapitsyn et al. (2019). Three more females and one male from the same collection event (AICF vouchers OoIs0101, OoIs0102, OoIs0201, OoIs0202) were extracted with another non-destructive method as detailed in Cruaud et al. (2019); several other specimens are kept at AICF in 96% ethanol at -20 °C to preserve DNA integrity for future investigations. DNA extracts were stored at -20 °C until PCR amplification was performed. Paired with morphological descriptions, confirmation of novel species was based on analysis of a fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene and the nuclear internal transcribed spacer 2 (ITS2) region. PCRs were performed using the reagents at concentrations described in Andreason et al. (2019b) with the primers of Samra et al. (2018), synthesized by Integrated DNA Technologies, Inc. (Coralville, IA, USA), with modified thermal cycling conditions. For COI amplification, thermal cycling was performed at 95 °C for 3 m, 40 cycles of 95 °C for 30 s, 30 °C for 1 m, and 72 °C for 1 m 15 s, with a final extension at 72 °C for 5 m. For ITS2 reactions, thermal cycling was performed similarly but at a 54 °C annealing temperature. Confirmation of amplification by gel electrophoresis, product cleaning, and sequencing were all performed according to Andreason et al. (2019b).

AICF
For O. telenomicida sequencing of the ITS2 with the primers in Samra et al. (2018) failed, thus we used the primers described in Yara (2006) to obtain a sequence from specimen OoIs0102. For the same species we obtained standard DNA barcodes for specimens OoIs0101, OoIs0102 and OoIs0201 with the primers LCO1490 and HCO2198 (Folmer et al. 1994). Molecular protocols described in Fusu and Ribes (2017) were used, except for the annealing temperature for ITS2 that was set at 45 °C. All sequences were deposited in GenBank (accession numbers MN933499-MN933500; MN935769-MN935775; MN947512-MN947518; MN945949-MN945951 [CO1]; MN946502 [ITS2]).

Genetic analysis
Interspecific variation among O. lucidus, O. mirus, O. telenomicida, and other populations and species of Ooencyrtus was estimated by calculating uncorrected pairwise distances (p-distances) of the COI fragment and ITS2 region and by phylogenetic analysis. MEGA X (Kumar et al. 2018) was used to trim and analyze sequence files obtained in this study, to perform multiple sequence alignments using ClustalW (Thompson et al. 2003), and to construct a phylogenetic tree. For comparisons of the COI and ITS2 regions, Ooencyrtus sequences deposited in GenBank by Samra et al. (2018) were aligned with O. lucidus, O. mirus, and O. telenomicida from Romania, and p-distances were calculated using the p-distance model with pairwise deletion of gaps. When comparing COI fragments, all available sequences were analyzed to account for intraspecific differences within species; for ITS2, one sequence was selected from Gen-Bank because the ITS2 region generally has very little, if any, intraspecific variation in Hymenopterans (Campbell et al. 1994;Stouthamer et al. 1999). A phylogeny of the studied species based on concatenated and unpartitioned COI and ITS2 sequences was inferred using the Maximum Likelihood method based on the Tamura-Nei model with 1000 bootstrap replications (Tamura and Nei 1993). The tree was drawn to scale with the number of substitutions per site estimating the branch lengths. Ooencyrtus kuvanae (Howard) was included as an outgroup because it is not part of the O. telenomicida complex and for which reliable sequences are available (Samra et al. 2018).  Diagnosis. There are no comprehensive keys for Ooencyrtus in North America and only 3 described species have been identified from California (Zuparko 2015(Zuparko , 2018. Therefore, to confirm that O. lucidus was not already collected in North America, the first author visited USNM in February 2019 and compared O. lucidus specimens with all the available types of Ooencyrtus species; no match was found. Morphologically, O. lucidus is most similar to the Nearctic species O. californicus, to which its female specimens key in both Noyes (2010) (to the Neotropical species) and Zuparko (2018) (to species in California). However, females of O. lucidus differ from O. californicus in having the scape at most 7.5× as long as wide (average of 6.6× as long as wide, Table 1) and the F1 is about 1.5× as long as wide (Fig. 2C). For O. californicus the scape (Fig. 5C) is about 8.8× as long as wide (as measured from the slide-mounted syntypes, with no significant difference between the four scapes measured; however, these measurements could very well be inaccurate because of the way the specimens were crushed, and the antennae were slide-mounted), and the F1 is a little more than 2.0× as long as wide. In addition, the "base of abdomen encircled by a narrow golden band" described by Girault (1917: 22)  In Noyes (1985), O. lucidus keys to the New World species O. johnsoni (Howard), whose entire gaster is shining black, perhaps with a slight greenish tinge. The entire type series of the latter taxon, 2 females and 1 male syntypes, were examined by the first author at USNM; the females are on points, with some parts of them mounted on a slide, and the male is on a slide. It also does not fit any of the described Old World species keyed in the publications mentioned below in the diagnosis of O. mirus, and is presumed to be native to the USA.
Color. Body ( Fig. 2A) mostly shining black with some metallic reflections, particularly on mesosoma, except base of gaster always with a distinct yellow, dorsal spot medially (on gastral tergites 1-3) and often with either yellow or light brown areas laterally and ventrally (always separated from medial yellow spot by a brown area); antenna brown; legs mostly yellow to light brown except coxae brown to dark brown basally and protibia and tarsi brownish.
Sculpture. Head with faint, inconspicuous sculpturing; mesoscutum reticulate, with sculpture cells mostly wider than long; axilla and anterior 1/3 or so of scutellum with a rather weak cell-like sculpture, remainder of body smooth.
Pubescence. Frontovertex, pronotum, mesoscutum, axilla, and scutellum with short, dark setae except scutellum with a few pairs of long, dark setae in posterior half.
Head ( Fig. 3A) about 1.2× as wide as high. Minimum width of frontovertex about 0.3× head width. Toruli just below level of lower eye margin. Ocelli in an obtuse triangle. Maxillary palpus 4-segmented, labial palpus 3-segmented. Mandible with 2 teeth and a broad truncation.
Legs. Mesotibial spur about as long as mesobasitarsus. Gaster (Fig. 3C) longer than mesosoma. Ovipositor occupying 0.6-0.7 length of gaster, a little exserted beyond its apex, and 1.0-1.2× (about 1.1× in the holotype) as long as mesotibia. Male (paratypes). Body length of dry-mounted, critical point-dried paratypes 595-795 µm, and of slide-mounted paratype 940 µm. Head and mesosoma shining black with metallic reflections (Fig. 4B), gaster dark brown; legs mostly yellow or light brown except coxae brown to dark brown and tarsi brownish. Head with toruli slightly above lower eye margin. Antenna (Fig. 4C) with scape minus short radicle 3.7-4.0× as long as wide (Table 2); funicle segments all longer than wide and more or less subequal in length (proximal segments a little shorter), F1-F3 apparently without mps, F4-F6 with at least 2 mps each; clava entire, 3.1-3.2× as long as wide, with several mps; flagellar segments all with numerous long setae. Fore wing (Fig. 4D) 5B) on this slide (because 4 scapes are present), only parts of 4 antennae and a slightly damaged fore wing (Fig. 5D) remain; the lectotype is constituted by the remains of one of them, circled in India ink, with the most intact antenna (Fig. 5C); remains of the other specimen are those of the paralectotype, and the single fore wing (Fig. 5D) can belong to either of them. The species was poorly described (Girault 1917: 22 [as Oenocyrtus californicus, sic]) from the unspecified number of "Types" under this catalog number in USNM; the type series was reared in Sacramento, California, USA from bug eggs on Pinus sabiniana (Douglas) D. Don (Pinaceae). The whereabouts of the other specimens of the type  series, if they ever existed, are unknown; however, it is quite likely that these two females were the only original "types". Thus, all other identifications of this species could be regarded to be tentative at best: for instance, specimens belonging to   Diagnosis. This new species is close to a small group of species of Ooencyrtus which are similar to O. telenomicida (Vassiliev), as defined by Hayat et al. (2014), although its female legs are entirely yellow. Ooencyrtus mirus keys to O. telenomicida in Ferrière and Voegelé (1961), Trjapitzin (1989), Huang and Noyes (1994), Zhang et al. (2005), Hayat and Mehrnejad (2016), and Samra et al. (2018). Morphologically, females of O. mirus differ from those of O. telenomicida mainly in having at least the proximal half of the gaster yellow, with only the apex (from the cercal plates) being brown to dark brown (Figs 6, 7E). In O. telenomicida, the yellow or light brown is present as a narrow, transverse basal band (Figs 10A, C, 12A, B, 13C), and this band is practically never extending to the cercal plates. Otherwise, females of these two species are quite similar although there are some differences in the lengths of their funicular segments (Table 3). In the multivariate ratio analysis O. mirus is well separated from O. telenomicida using the shape PCA (Fig. 16B). However, the scatterplot of isosize against the first shape PC (Fig. 16A) shows that O. mirus is also slightly smaller than O. telenomicida. This plot thus shows a certain amount of allometric variation and part of the separation is probably based on size rather than shape, and this might be a case of allometric scaling rather than true separation. The next two analyses indicated the same aspect. The PCA ratio spectrum for PC1 (Fig. 16C) identified as most relevant the ratio between propodeum length and scape width (variables lying at the opposite ends of the spectrum are the most relevant), while at the same time this is also the most allometric ratio as shown by the allometry ratio spectrum (Fig. 16D).
The LDA ratio extractor, which is a tool for identifying the best ratios for separating two groups, found that the best ratio to separate the two species is scape width / F5 length, the ratios being almost non-overlapping (Table 8).
Because the commonly used morphometric parameters and ratios of O. telenomicida and O. mirus are so similar, the importance of their clear separation based on the presented genetic data can not be overestimated. Description. Female (holotype and paratypes). Body length of dry-mounted, critical point-dried paratypes 595-1025 µm.
Color. Head and mesosoma (Fig. 6) mostly black with some metallic reflections, particularly on mesosoma, except mesopleuron with a strong violet luster; most of gaster yellow except brown to dark brown apically (from cercal plates); antenna brown; legs yellow.
Sculpture. Head with faint cell-like sculpture; mesoscutum reticulate, more so anteriorly; axilla reticulate; scutellum more strongly reticulate than mesoscutum or axilla (except sometimes almost smooth at apex), remainder of body more or less smooth.
Antenna (Fig. 7B) with radicle about 2.8× as long as wide, rest of scape slender, a little wider in the middle and narrowing towards apex, 5.6-6.9× (6.3× in the holotype) as long as wide; pedicel about 2.0× as long as wide, longer than any funicular segment (F1 0.5-0.6× length of pedicel, Table 3); funicle segments all longer than wide, F1 usually about as long as F2 and slightly shorter than following funicular segments (F2 0.9-1.1× length of F1, Table 3), F3-F6 subequal in length although F3 usually slightly shorter than following funicular segments (Table 3), F1-F2 without mps, F3-F4 each with 1 mps, F5-F6 each with 2 mps; clava 3-segmented, 3.0-3.7× (3.1× in the holotype) as long as wide and almost as long as combined length of F4-F6, each claval segment with several mps. Mesosoma (Fig. 7D, E). Mesoscutum about 2.8× as wide as long; scutellum wider than long and a little shorter than mesoscutum, placoid sensilla close to each other and closer to posterior margin of scutellum. Propodeum smooth and very narrow medially, less than 0.1× as long as scutellum.
Measurements ( Male (paratypes). Body length of dry-mounted, critical point-dried paratypes 660-890 µm, and of slide-mounted paratypes 950-960 µm. Head and mesosoma black with metallic reflections (Fig. 8B), gaster mostly dark brown to black except yellow to light brown or brown basally; antenna brown except scape light brown ventrally and often dark brown dorsally; legs yellow. Antenna (Fig. 9A) with scape minus short radicle 3.4-3.8× as long as wide (Table 4); funicle segments all longer than wide, more or less subequal in length and each with several mps; clava entire, 3.6-3.8× as long as wide, with several mps; flagellar segments all with numerous long setae. Fore wing (Fig. 9B) 2.2-2.4× as long as wide; hind wing 4.6-4.8× as long as wide. Genitalia (Fig. 9C) length 171-191 µm.
Variation (female and male body length, non-type specimens from the colony in UCR quarantine laboratory). The female body lengths, male body lengths, and paired differences, analyzed by the Shapiro-Wilks normality test in R (R Core Team 2018), all had normal distributions. The mean lengths were 849 µm for the females and 795 µm for the males, with a mean difference of 54 µm. A paired t-test in R showed that the males were significantly shorter in length than the females (P<0.001).
Etymology. The name is an adjective meaning "remarkable" or "amazing." The name is given to this species because the authors find its biology to be quite remarkable.
Distribution. Oriental region: Pakistan. The population in the quarantine laboratory in UC Riverside that served for the description of this species originated from the Toba Tek Singh District, Punjab, Pakistan.
Hosts. Pentatomidae: Bagrada hilaris (Burmeister). We conducted host studies on O. mirus and found it to reproduce on the eggs of eight other species in Pentatomidae, one species in Rhopalidae, and one species in Coreidae (Hemiptera), as well as on one species in Noctuidae (Lepidoptera). Of all the potential host species we evaluated, only one, in Pyralidae (Lepidoptera), was not utilized as a host, likely because its eggs were too small. These findings show O. mirus to be a generalist parasitoid, although it prefers and reproduces more successfully on B. hilaris than on the other hosts evaluated.
Biology. Ooencyrtus mirus, a uniparental species, typically produces about 99% females. However, the percentage of males can be increased by providing new eggs to the same female wasps daily for more than two weeks. This depletes the supply of Wolbachia bacteria in the ovaries (Lindsey and Stouthamer 2017), and the eggs, all unfertilized, then produce males instead of females.
Comments. This species was initially identified from digital images of both dryand slide-mounted specimens as Ooencyrtus telenomicida sensu lato (J. S. Noyes and E. Guerrieri, personal communications). This determination was ambiguous, however, since O. telenomicida was not clearly defined prior to this communication, despite the availability of its numerous diagnoses and redescriptions (e.g., Ferrière and Voegelé 1961;Huang and Noyes 1994;Hayat and Mehrnejad 2016). Thus, until a neotype of O. telenomicida was properly designated, and respective DNA sequences were ob-  ( Description of the neotype female. Color. Body (Fig. 10A) mostly very dark brown with some metallic reflections (mainly dark bluish and some greenish) on fron- Figure 10. Ooencyrtus telenomicida female (from Romania) A habitus in lateral view (neotype, prior to DNA extraction) B habitus in dorsal view (non-type) C mesosoma and metasoma (neotype). tovertex, mesoscutum, and scutellum except tegula brown and base of gaster with a narrow, light brown band on the first gastral tergite; antenna brown except radicle dark brown; legs mostly yellow except meso-and metacoxa brown basally and tarsi partially light brown.
Antenna (Fig. 11B) with radicle 2.5× as long as wide, rest of scape slender, slightly wider in the middle and narrowing towards apex, 6.3× as long as wide; pedicel 2.0× as long as wide, longer than any funicular segment (F1 0.6× length of pedicel); funicle segments all longer than wide, F1 as long as F2 and slightly shorter than following funicular segments, F3, F4 and F6 about equal in length, and F5 the longest funicular segment, F1-F2 without mps, F3-F4 each with 2 mps, F5-F6 each with 3 mps; clava 3-segmented, 3.0× as long as wide and almost as long as combined length of F4-F6, each claval segment with several mps. Mesosoma (Fig. 10C). Mesoscutum about 2.3× as wide as long; scutellum (Fig. 11D) slightly wider than long and a little longer than mesoscutum, placoid sensilla close to each other and closer to posterior margin of scutellum. Propodeum (Fig. 11D) smooth and very narrow medially, less than 0.1× as long as scutellum.
Wings not abbreviated, fore wing extending well beyond apex of gaster. Fore wing (Fig. 11E) 2.4× as long as wide, its disc hyaline; costal cell about 11× as long as wide; marginal vein punctiform; postmarginal vein a little shorter than stigmal vein; linea calva almost closed posteriorly by a row of short, inconspicuous setae; filum spinosum with 3 setae on one wing and 5 on the other; longest marginal seta 0.09× maximum wing width. Hind wing 5.4× as long as wide, disc hyaline.
Measurements ( Taxonomic notes. Female. Variation (non-type specimens from Romania, Russia, and Ukraine). Body length of dry-mounted, air-dried specimens 860-925 µm. Body (Figs 10B, 12A, B, 14B) mostly very dark brown with some bluish and greenish metallic reflections on mesoscutum, except tegula and mesopleuron brown and base of gaster usually with a complete, narrow, yellowish or light brown band (dorsally almost Figure 11. Ooencyrtus telenomicida female (neotype) A slide B antenna C head in frontal view D axillae, scutellum and propodeum E fore wing F mesotibia and mesotarsus. always at most on the first and second gastral tergites, usually only on the first) and often brown (but never yellow) between the yellow basal band and cercal plates dorsally, but occasionally base of gaster entirely dark (Fig. 10B) or, very rarely (observed only in one specimen from Ukraine) the yellow band extends almost to cercal plates (in the absence of molecular data for this historical specimen, it cannot be excluded that it might belong to another species); antenna brown except apex of pedicel a little lighter (light brown); legs mostly yellow except meso-and metacoxa often brown basally, tarsi partially light brown. Minimum width of frontovertex 0.25-0.28× head width (Figs 13A, 14A). Antenna (Fig. 12C) with scape minus radicle 6.0-8.75× as long as wide; F1 the shortest funicular segment, 0.5-0.65× length of pedicel; F2 1.0-1.1× length of F1 (Tables 5, 7), F1-F2 without mps, F3-F6 each with at least 2 mps; clava 2.6-4.1× as long as wide. Fore wing (Fig. 13D) 2.2-2.7× as long as wide; filum spinosum with 3-5 setae. Hind wing 4.2-4.4× as long as wide, its disc hyaline. Ovipositor occupying 0.7-0.9 length of gaster (Fig. 13C), at most barely exerted beyond its apex, and 0.9-1.0× as long as mesotibia.
Male (non-type specimens from Russia). Body length of dry-mounted, air-dried specimens 600-900 µm. Body (Fig. 15A) black with metallic reflections, particularly on mesosoma; antenna brown except scape light brown ventrally and dark brown dorsally; legs yellow except most of coxae and metafemur brown. Antenna (Fig. 15B) with scape minus short radicle 3.6-3.7× as long as wide; funicle segments all longer than Table 5. Morphometric ratios and measurements (µm) of morphological characters of female Ooencyrtus telenomicida from Russia and the Ukraine. All measurements are from slide-mounted specimens.   Samra et al. (2018), but many of them will need to be verified using molecular methods.
Biology. Ooencyrtus telenomicida is a facultative hyperparasitoid of Eurygaster integriceps, being either a primary egg parasitoid (more so earlier in the season when unparasitized eggs of the host are readily available and prevalent) or a secondary parasitoid via the telenomine primary egg parasitoids, particularly later in the season when many of the host eggs are parasitized (Romanova 1953).
Comments. According to V. A. Trjapitzin (personal communication), the entire type series of O. telenomicida, if such ever existed, has never been located and is certainly lost. The dire necessity of a proper recognition of this nominal species, which has been impossible with any confidence from some other members of the O. telenomicida species complex (e.g., according to Huang and Noyes (1994), from O. gonoceri Viggiani and O. acastus Trjapitzin), leaves no choice but to designate a neotype for O. telenomicida, complemented with the much needed DNA sequence data from it. That is done herein from the specimen reared from an egg of a species of Eurygaster Laporte,    which is the genus from which the originally described O. telenomicida emerged. Furthermore, the insects were collected in northeastern Romania which is relatively close to the original collection site in Kharkov oblast' of Ukraine. Importantly, the collections were made in the same general habitat (sylvo-steppe biome) as the originally described species. Morphologically, female specimens from Romania (Figs 10, 11; Ta-ble 7) are identical to those from Russia and Ukraine reared from eggs of Eurygaster integriceps in the late 1940s and early 1950s (Figs 12, 13A, C, D; Table 5). The neotype and especially a second specimen from the same collecting event ('topotype') grouped together with the specimens from Russia and Ukraine (Fig. 16A, B) in the shape PCA of the multivariate ratio analysis. Based on this information, a genetic library of other members of the complex can be constructed, and their identity determined.  (Table 9). In contrast, the neotype and one non-type O. telenomicida specimens from Romania had 3.1% pairwise sequence differentiation (Table 9), indicating high intraspecific variation (these two specimens were obtained from two distinct egg masses found in close proximity). The standard barcode region, obtained from other three specimens but from the same two egg masses, confirms this genetic differentiation (4.4% p-distance). This genetic divergence is at a level that has been demonstrated for other Ooencyrtus species, e.g. O. pistaciae clades a and b at 4.4% (Samra et al. 2018). ITS2 sequences of O. lucidus and O. mirus also had no intraspecific differences among specimens. The ITS2 sequence of only one O. telenomicida specimen from Romania was obtained. Intraspecific variation could not be determined, although high variation in this region is not expected within the species.

Molecular analyses
COI alignment and p-distance calculations among the Ooencyrtus species revealed at least 5.9% and 7.3% genetic divergence in O. mirus and O. lucidus, respectively, Table 8. First five best ratios found by the LDA ratio extractor for separating O. mirus sp. nov. and O. telenomicida. Standard distance indicates how well one ratio discriminates compared to another; δ indicates how well shape discriminates compared to size (values close to 0 indicate no influence of size and those close to 1 indicate separation based mainly on size). Ranges were calculated on all available measurements, not only on those from the complete dataset used in the analysis. The ratio marked with * has very little overlap.  Samra et al. (2018). This is supported by 6.7-7.4% and 6.0-7.0% genetic divergence, respectively. Ooencyrtus lucidus had high genetic separation from all compared species. Analysis of the ITS2 region further demonstrated genetic separation of these species. Intraspecific variation in this region is absent to extremely low, while interspecific variation is expected to be high. Our analysis was based on a partial fragment of the ITS2 region because the full sequence was not obtained for every specimen; however, the region analyzed was flanked by regions of congruence (16 bases at the 5' end and 22 bases at the 3' end; average 385 bp region analyzed for each species). The lowest p-distance between O. mirus and all compared species in this region was 0.069 (6.9% pairwise distance), which was demonstrated with O. pistaciae (Table 9). Sequence divergence for O. lucidus was extremely high with the lowest p-distance at 0.202.
Phylogenetic analysis of Ooencyrtus species, inferred using concatenated COI and ITS2 genetic regions, supported O. mirus as a sister taxon to O. telenomicida from Romania (Fig. 17). These two species formed a larger clade with the sister taxa O. pistaciae and East Mediterranean O. telenomicida, separated from O. zoeae and O. mevalbelus. Basal to this clade were O. pityocampae and O. lucidus; O. kuvanae was used to root the tree. Using concatenated COI and ITS2 sequences resulted in a phylogenetic tree with a topology that combined the two separate COI and ITS2 trees of Samra et al. (2018). East Mediterranean Ooencyrtus telenomicida and O. pistaciae branched in a clade separate from O. zoeae and O. mevalbelus as seen in both the COI and ITS2 phylogenetic Figure 16. Multivariate ratio analysis for Ooencyrtus mirus sp. nov. and O. telenomicida A scatterplot of isosize against first shape PC B shape PCA, scatterplot of first against second shape PC C PCA ratio spectrum for PC1, bars represent 68% confidence intervals D allometry ratio spectrum; bars represent 68% confidence intervals.
trees, and O. pityocampae branched basally as in the ITS2 tree. As suspected, inferring the phylogenetic placement of O. mirus and the Romanian O. telenomicida (including the neotype) with these species resulted in a clade for the O. telenomicida species complex. Almost certain of New World origin and completely different from the O. telenomicida species complex morphologically, O. lucidus, with its high genetic divergence in both COI and ITS2 regions, branched separately. It is basal to all other species in the concatenated phylogenetic tree (as well as in our earlier analyses on single-gene COI and ITS2 trees; data not shown).

Conclusion
Members of the speciose genus Ooencyrtus, in which more than 300 currently valid species are known, are notoriously difficult to identify morphologically. That is particularly true for the taxa within the O. telenomicida species complex in the Old World. In the New World, identification keys exist only for some Neotropical species (Noyes 1985(Noyes , 2010 but not for those in the Nearctic region. Moreover, many undescribed species have been recognized (e.g., Zuparko 2015Zuparko , 2018, and misidentifications of Ooencyrtus species are quite common. Whereas molecular methods with both mitochondrial and nuclear gene regions often are necessary for providing reliable identifica-tion or separation of morphologically similar species, using these methods for positive identifications is useful only if those taxa were correctly identified based on morphological studies of the type specimens and reared material from known hosts. Here we provide both morphological and genetic evidence that has helped to untangle the true identity of the common Old World parasitoid, O. telenomicida, and also of two primary egg parasitoids of the bagrada bug, one from California and the other from Pakistan. Traditionally, the standard DNA barcode region of the COI gene described in Folmer et al. (1994) is analyzed to estimate intraspecific and interspecific difference when comparing metazoan invertebrate species. However, in order to compare our new species with recently described Ooencyrtus species, we sequenced the COI region analyzed by Samra et al. (2018). This region is 946-bp in length, whereas the Folmer region is 648-bp long, and these regions share approximately 400-bp overlap allowing comparison of a portion of the two regions. However, we also obtained the standard barcode sequence for three specimens of O. telenomicida in order to maximize compatibility with standard DNA barcodes libraries. We found well-supported genetic separation of the two new species described herein from all compared Ooencyrtus species in both the full Samra et al. (2018) proposed COI region and the overlapping Folmer region. ITS2 sequences reinforced O. lucidus and O. mirus as distinct species with high levels of genetic differentiation. Our COI and ITS2 sequence analyses supported and confirmed the morphological differences and morphometric separation observed for these new species. Interestingly, our analysis also demonstrated significant genetic divergence of the neotype of O. telenomicida from the likely misidentified O. telenomicida specimens previously sequenced. This observation emphasizes the fact that additional work remains to sort out and properly describe and re-describe the species of the O. telenomicida species complex, including proposing possible synonymies. That difficult and laborious task, however, is well beyond the scope of this study.