﻿A review of the genus Zygota (Hymenoptera, Diapriidae) in Germany with taxonomic notes on this genus and its distinction from Pantoclis

﻿Abstract This study provides a comprehensive overview of the genus Zygota Förster combining DNA barcoding and current morphology. Nineteen species of Zygota were found throughout Germany, including the newly described species Zygotawallisp. nov. First species records for Germany are: Zygotabalteata Macek, 1997; Z.comitans Macek, 1997; Z.spinosipes (Kieffer, 1908); Z.sordida Macek, 1997; Z.angularis Macek, 1997 and Z.vigil Nixon, 1957. We also clarify diagnoses for the two related genera, Pantoclis Förster and Zygota to designate the boundaries of the Zygota genus and propose new synonymies: Zygotacaligula Buhl, 1997 is a junior synonym of Z.congener (Zetterstedt, 1840); Z.reticulata Kozlov, 1978 is a junior synonym of Z.ruficornis (Curtis, 1831). Thirteen species of Zygota sensu Nixon (1957) are transferred to the genus Pantoclis with the following new combinations proposed: Zygotabrevinervis (Kieffer, 1908) (= Pantoclisbrevinervis (Kieffer, 1909), comb. nov.); Z.brevipennis (Kieffer, 1908) (= P.brevipennis (Kieffer, 1908), comb. nov.); Z.caecutiens (Kieffer, 1908) (= P.caecutiens (Kieffer, 1908), comb. nov.); Z.cursor (Kieffer, 1908) (= P.cursor (Kieffer, 1908), comb. nov.); Z.fossulata (Thomson, 1858) (=P.fossulata (Thomson, 1858), comb. nov.); Z.fuscata (Thomson, 1858) (= P.fuscata (Thomson, 1858), comb. nov.); Z.hemiptera (Thomson, 1858) (= P.hemiptera (Thomson, 1858), comb. nov.); Z.microtoma (Kieffer, 1909) (= P.microtoma (Kieffer, 1909), comb. nov.); Z.soluta (Kieffer, 1907) (= P.soluta (Kieffer, 1907), comb. nov.); Z.striata (Kieffer, 1909) (= P.striata (Kieffer, 1909), comb. nov.); Z.subaptera (Thomson, 1858) (= P.subaptera (Thomson, 1858), comb. nov.); Z.sulciventris (Kieffer, 1909) (= P.sulciventris (Kieffer, 1909), comb. nov.), and Z.unicolor (Kieffer, 1908) (= P.unicolor (Kieffer, 1908), comb. nov.).


Introduction
This article deals with the parasitoid wasps of the genus Zygota Förster (Diapriidae, Belytinae, Belytini), comprising mostly medium-sized (2.5-4.0 mm long) melanic and pubescent specimens with brightly colored appendages.ZooKeys 1207: 325-353 (2024), DOI: 10.3897/zookeys.1207.121725 Jeremy Hübner et al.: Zygota review The genus has 75 described species worldwide, of which most are described from the Palearctic and Nearctic (Johnson 1992;Buhl 1995Buhl , 1997Buhl , 1998;;Macek 1997).Although common, little is known about their biology and their hosts.In the past, morphology-based taxonomy of Zygota led to confusion and many reinterpretations of the genus.The generic diagnosis, key to the species of Central Europe, and diagnostic remarks based on available types were given by Macek (1997).According to the original description of the genus given by Förster (1856) Zygota can be easily distinguished from other Belytinae genera by the strengthened marginalis, open radial cell, and emarginated fore tibiae in males (Förster 1856).Förster's vague diagnosis was misinterpreted by the later authors Ashmead (1893Ashmead ( , 1902) ) and Kieffer (1909), which Macek (1997Macek ( , 2007) ) has pointed out in his revisionary works.He clarified the identity based on the designation of the neotype of Zygota abdominalis (Nees, 1834), and completed a revision of available types.However, the boundary between Zygota and its sister genus Pantoclis Förster is still unclear, as some species remained falsely placed inside Zygota.Nixon (1957) and later Kozlov (1978) placed all Belytini species with an open radial cell and unpunctured scutellum [except some few Belyta species (Macek 1995)] in the genus Zygota.The same genus concept was applied in Johnson's (1992) world catalog.Although the diagnosis of the genus Zygota was given by Macek (1997), the generic affiliation of many species was not discussed.For example, the taxonomy of the 14 species from 39 Palearctic species of Zygota listed by Johnson (1992) is still questionable.The genus Pantoclis has never been defined conclusively to exclude it from other Belytinae, because the diversity and lack of knowledge of Pantoclis species makes it extremely difficult to define.To understand the genus concept of Zygota, it must be distinguished from Pantoclis.We will, therefore, present a diagnosis for each.
Currently, there are 38 known species of Zygota in the Palearctic Region (Johnson 1992, Buhl 1995, 1997, Macek 1997).Full taxonomic treatments of the genera are given by Macek (1997) (only Zygota) and cataloged by Johnson (1992) (both, Zygota and Pantoclis).Macek (1997) has given a taxonomic interpretation only for 18 of these species.The present study thus aims to clarify the diagnosis of Zygota and the taxonomic position of the remaining 20 species, which are not discussed in Macek (1997).This revision is mostly based on material collected in Bavaria, Germany, in the framework of the German Barcode of Life (GBOL) III: Dark Taxa project (Hausmann et al. 2020).The most recent diversity evaluation that has been conducted for Germany was done over twenty years ago by Blank (2001).In his work, twenty Zygota taxa were recovered, of which two, Z. excisipes (Kieffer, 1916) and Z. norvegica (Kieffer, 1912), have been synonymized with Z. excisor (Zetterstedt, 1840) and Z. ruficornis (Curtis, 1831), respectively.For Zygota subclausa (Kieffer, 1907), Macek (1995Macek ( , 1997) ) proposed the new combination Belyta subclausa (Kieffer, 1907).In total, 19 species of Zygota were reliably identified for the German fauna.

Material and methods
Most of the examined material was collected within the GBOL III project as well as from earlier collecting events in Bavaria and Baden-Wuerttemberg (Germany) led by the Bavarian State Collection of Zoology in Munich (SNSB-ZSM).Further material originates from the collection of the National Museum in Prague (NMPC) and the Russian collections in St. Petersburg (ZISP).In addition, type material from the Zoological Museum in Copenhagen (ZMUC) and the Natural History Museum (NHM) in London was examined.All specimens were morphologically identified as far as possible, including the closely related genus Pantoclis.Afterwards, individuals were Sanger sequenced under the usage of a voucher recovery approach.The genetic information was obtained at the Canadian Centre for DNA Barcoding (CCDB) in Guelph by the application of a voucher recovery protocol (https://ccdb.ca/).All mitochondrial CO1 sequences were aligned in MEGA11 (Tamura et al. 2021), and the alignment was then used to construct maximum likelihood trees with the online program IQ TREE version 2.0 (Trifinopoulos et al. 2016) using the default settings (1000 bootstrap alignments, substitution model: TIM+F+I+G4, 1000 iterations).Editing was done using FIGTREE version 1.4.4 (Rambaut 2010) and INKSCAPE version 1.1.1(2021, available from: https:// inkscape.org/de).Clustering and BIN-distance-analyses were conducted to infer species barriers among the CO1 barcodes using MEGA11 as well as ASAP (Puillandre et al. 2021).Suppl.material 3 gives an overview of the genetically examined material and the clustering results.All molecular data and collection metadata are publicly available on the Barcode of Life Data System (BOLD) platform (http://www.barcodinglife.org,Ratnasingham and Hebert 2007) in the dataset [DS-ZYGPAN dx.doi.org/10.5883/DS-ZYGPAN].It is important to note that analysis was conducted on data that was downloaded from BOLD on 27 February 2024.Therefore, the results are based on the BIN-statuses of that time.
The morphological terminology and abbreviations follow those proposed by Yoder (2004) and as used in Hymenoptera Anatomy Ontology (Yoder et al. 2010); the measurements follow Yoder (2004) and Chemyreva (2015Chemyreva ( , 2018)).Terms of relative position follow Goulet and Huber (1993).The terms of sculpture description follow Eady (1968).The accurate taxonomic treatments of the genera and species Zygota and Pantoclis are given in Macek (1997) and Johnson (1992).Taxa that have received an updated taxonomic treatment, such as new species or synonyms, are newly diagnosed here.Sufficiently detailed diagnoses for all other species were given by Maсek (1997).The general distribution of species was obtained and updated from Blank (2001), Wall (1963), Buhl (1995Buhl ( , 1997)), Macek (1997), andChemyreva et al. (2023).New records are marked with an asterisk (*).The following abbreviations for locations in Germany are used: BW= Baden-Württemberg, BY= Bavaria.Museum acronyms: SNSB-ZSM -Bavarian State Collection of Zoology, Munich; ZISP -Zoological Institute of the Russian Academy of Sciences, St. Petersburg, Russia; ZMUC -Zoological Museum, University of Copenhagen.A series of images were taken using an Olympus OM-D camera mounted on a Leica M125 C binocular and stacked using HELICON FOCUS (Version 8).
Remarks.The female of Zygota congener is best recognized by the large propodeal spiracles (Fig. 6A) and the sharp median keel between the cerci on the apical tergite of the female (Fig. 6E).These two characters, together with other peculiarities of the morphology of Z. congener, correspond to the  characters of the holotype of Z. caligula Buhl.For this reason, Z. caligula is considered here to be a junior synonym of Z. congener.
Diagnosis.Both sexes: postmarginal vein distinctly shorter than radial cell length (Fig. 8F); occipital pit present; mesopleuron with only small bare area on it medially or entirely pubescent (Fig. 8D); axillar depression with scattered setae and only 2 verriculate tubercles; base of T2 with small lateral corners (Fig. 8A).Female: T2 finely granulate (Fig. 8A); Т8 without transverse or elongate carinae on it (Fig. 8B); S2 with a small pit in anteriorly half (as in Fig. 4C, green arrow).Male: A3 weakly emarginate (Fig. 8H); fore tibia broadened, with sharp projection and a row of strong setae on the top of it, bare at the apex on its anterior surface (Fig. 8G); S2 with a small area of micropuncture in anteriorly half (as in Fig. 4E, green arrow); digitus armed with 1 long curved spine; spine extending from digitus at significant angle and not pushed towards it (Fig. 8C).
This species is very similar to Z. pubescens except as follows: female antenna stout, with A6-A14 distinctly transverse (A6-A14 subquadrate in Z. pubescens); male genitalia armed with a spine, which extends from digitus at significant angle (this spine pushed towards digitus in Z. pubescence).Both species are very common in Germany.
Description.Female (holotype).Body length 3.2 mm, antenna length 2 mm, wing length 2.6 mm.Body mainly black with metasoma dark brown; antennae, palpi, mandibles, tegula, legs and venation brown (Fig. 11B).Head in dorsal view as long (measured with antennal shelf) as wide.Toruli separated from each other by narrow and shallow furrow and from front posteriorly with deep pubescent depression.Ocelli small, OOL twice as long as POL.Eye densely pubescent.Eye diameter 1.2 as long as malar space.Pleurostomal distance as long as malar space.Occipital carina narrow, almost smooth, without occipital pit (Fig. 11C).Head in lateral view as high as long, in frontal view subtriangular, with face smooth and shining.Antennal shelf rugose below toruli in frontal view.Subantennal furrows very short (Fig. 11A).Epistomal sulcus distinct, clypeus convex and smooth.Tentorial pits situated in small hollows.Mandibles not prominent.
Mesosoma convex, 1.2 times as wide as the head.Pronotal shoulders weakly convex, with transverse carina between them.Epomia with long lower branch and short lateral branch.Lateral part of pronotum strongly impressed, smooth and shining.Mesonotum convex, with percurrent notauli, converging posteriorly.Scutellum convex, smooth, with oval anterior scutellar pit.Axillar depressions smooth, densely pubescence, with a pair of vericulate tubercles.Mesopleuron smooth with deep mesopleural pit, with epicnemial and acetabular bridges (Fig. 11D).Metascutellum with strong median carina and lateral carinas.Metanotal trough smooth and bare.Propodeum slightly transverse, with round posterior rim.Median keel of propodeum simple.Both plicae parallel to each other, slightly projecting posteriorly.Lateral side of propodeum below plicae with lateral longitudinal carina, slightly projecting posteriorly.Fore tibia simple with homogeneous strengthened bristles on the inner side.
Wings.Marginal vein strongly developed, 3.9 times as long as wide (measured medially) and 1.45 times as long as distance from it to basal vein.Radial cell open, radialis long and nebulous (Fig. 11B).Postmarginal vein slightly shorter than stigmal vein; stigmal and postmarginal veins form 65° angle, stigmal vein 0.5 times as long as marginal vein.
Petiole cylindrical, entirely covered with semi-erect pubescence and elongate keels, ventrally with a row of verriculate tubercles.Base of T2 with slightly indicated lateral corners, short medial furrow and straight striation flanked at each side (Fig. 12B).S2 entirely pubescent, base of S2 with group of verriculate tubercles.Apical tergite (T8) with transverse sharp keel (Figs 11E,12A), smooth and bare anteriorly and smooth and setose posteriorly from the transverse keel.
Male.Head distinctly transverse, as wide as mesosoma.Antennae 14-segmented with A4-A14 cylindrical, A3 with keel and emargination extending to 0.35-0.40 of the segment length (Fig. 12C, E).Fore tibia modified, acutely angled on the inner side and covered at the top with several minute bristles (Fig. 3F).Excavation on the fore tibia bare and shining in frontal view.Postmarginal vein 0.5-1.5 times as long as marginal vein (Fig. 3D).Marginal vein 1.3 times as long as distance from it to basal vein or slightly shorter.Petiole 1.5-2.1 times as long as its median width.
Etymology.This newly described species is named after the diapriid taxonomist Ingmar Wall who made himself a name in the Diapriidae research for years.

Discussion
As a result of our study, new combinations were proposed for 13 of 20 species which have a yet questionable taxonomic position, and two names (Zygota caligula Buhl and Z. reticulata Kozlov) were considered synonyms.One species of the genus Zygota, Z. maura (Kieffer, 1910) remains unstudied and inexplicable.Based on the emarginated fore tibia in males, mentioned in the original description, this species should be without doubt classified in the genus Zygota (Kieffer 1910).However, the type specimen of this species has not been found, and the description is not detailed enough to allow further conclusions at the species level or potential synonymies.The types of the two species Z. strigata Kozlov, 1978 andZ. groenlandica Buhl, 1995 were examined, and both are valid taxa of Zygota.Zygota cilla Nixon, 1957 andZ. vigil Nixon, 1957 were not included in Macek ś (1997) revision because of the lack of relevant material.Nixon (1957) based both species on a single female (Z.cilla) and a single male specimen (Z.vigil), yet neither type has been found.The first discovery of a male Z. vigil since the description of the species is given here.A female of Z. cilla, which is unique in its morphology (Nixon 1957), was not found during this research.Thus, the taxonomic position of all Palearctic species (Johnson 1992, Buhl 1995, 1997, Macek 1997) listed in Zygota but not mentioned in Macek ś (1997) revision, are discussed in this article.Molecular-based analysis, which was conducted in the framework of this and previous works of GBOL III, has recovered rather poor results for the genus Zygota (and others of the Belytinae tribes Cinetini and Belytini; ~68% sequencing success rate) when compared to other diapriid taxa (~90%).Therefore, we recommend future studies invest their efforts into the development of a specific primer set to improve sequencing success.Nevertheless, we significantly improved the amount of genetic information that is available online.Prior to this study, BOLD listed a total of 391 public records that were assigned to 26 BINs globally.Our dataset DS-ZYGPAN presents 178 Zygota records and 19 BINs from Germany alone (see also Suppl.material 3).
In this study, some Zygota morphospecies were assigned to more than one BIN.This can happen for a variety of reasons: incomplete lineage sorting, heteroplasmy, NUMTs, hybridisation, recent speciation, cryptic species, phylogeographic effects, introgression or endosymbionts or their combinations can influence the outcome of genetically sorting of different OTUs (Raupach et al. 2016).Another factor that plays a key role in the construction of a BIN is the DNA barcoding gap difference between the highest intra-and smallest interspecific variation of a certain taxon.A typical threshold in the genetic distance between two species ranges from 10-15%, but this can vary immensely (Meier et al. 2006, Hebert et al. 2016, Raupach et al. 2016).In our case, 10-15% was indeed a fitting value to delimit species with CO1.A MEGA mean group distance analysis (Suppl.material 3) confirmed our morphological findings, namely, that specimens assigned to the same morphological species all displayed smaller genetic distances between one another than between other morpho-species: Z. comitans (mean group distance within all sequences of the BIN: 7%), Z. spinosa (5.4%), Z. parallela (5.8%), Z. spinosipes (6.3%), Z. ruficornis (three BINs; 5.3%, 3.5%, 4.3%) and Z. walli sp.nov.(2.6%).The corresponding specimens of each BIN cluster together in the taxonomic MLtree (see Suppl.material 1).An ASAP analysis of the genetic material confirmed the BIN clusters for the genus Zygota.The highly variable genus Pantoclis, on the other hand, displayed less resemblance when comparing the BINs with ASAP clusters.All of those questionable records were only represented by one or two sequences in our dataset which might explain their uncertain placement.
A subset of the available CO1 sequence data of species of the tribe Belytini was used to construct a phylogenetic ML-tree (Fig. 13).Here, the genera Zygota and Pantoclis were displayed as well-supported sister groups within the Belytini.Fig. 14 shows a more detailed tree with records from all Pantoclis BINs we investigated.The data show that some species with an open radial cell are grouped and demonstrate close genetic relationships with species that clearly belong to Pantoclis and have a closed radial cell.These findings suggest that the character state of the radial cell reduction cannot be used as an appropriate feature for genus designation.Nixon (1957) also noticed these differences between Zygota species and the group of Pantoclis species with an open radial cell.He proposed to aggregate them into the Z. fuscata -species group "... because of the form of the radial cell and better development of the radialis, this group is transitional between Pantoclis and Zygota and has perhaps more relationships to the former genus [Pantoclis] than to Zygota s. str." (Nixon 1957).Nixon placed six species (Z.fuscata, Z. microtoma, Z. striata, Z. brevinervis, Z. soluta, Z. fossulata) in the Z. fuscata -species group which have been transferred to Pantoclis here.
In addition, the species transferred to the genus Pantoclis in this research are not similar to Zygota species in other key characteristics.Unlike Zygota species, males of Pantoclis never display a modified fore tibia and most of them have slender genitalia with lanceolate apex of aedeagus and a diminished digitus.On the contrary, some Zygota males have the digitus with a single strong curved spine, while similar structures are not known for the Pantoclis species.All females of Zygota show a very short ovipositor, while many Pantoclis females (with closed or  open radial cell) show a long ovipositor (Fig. 1A).Thus, combining this morphological information with our understanding of the genus Pantoclis (see the diagnosis of the genus proposed above), and taking data on the venation variability based on the molecular data into consideration, we propose in this study, new combinations for 13 species previously listed in the genus Zygota (Suppl.material 2).
Because a detailed revision of Pantoclis is still lacking, it is important to note that the diagnosis presented here is preliminary.The high amount of variation in the morphology and the large species richness of the genus suggest that Pantoclis is paraphyletic.On the other hand, as a consequence of the taxonomic changes proposed here, the monophyly of the Zygota is now less controversial based on species morphology.

Figure 1 .
Figure 1.Morphological characters to identify the closely related genera Zygota and Pantoclis A, E female B, C, D males A, B P. barycera C Z. walli sp.nov.D, E Z. abdominalis.Scale bars: 1 mm (A); 0.5 mm (B-F).

Figure 6 .
Figure 6.Zygota congener, male (B-D, F) and female (A, E) A mesosoma and petiole in dorsal view B A1-A5 in ventral view C fore tibia D head and mesosoma in lateral view E apex of metasoma in dorsal view (Z.caligula Buhl, holotype) F antennae in ventral view.Scale bars: 0.5 mm (D); 1 mm (F).

Figure 7 .
Figure 7. Holotype of the Zygota caligula Buhl A face B body in dorsal view C body in lateral view D metasoma, ventral view E type material labels F fore wing venation.Scale bar: 0.5 mm.

Figure 8 .
Figure 8. Zygota ruficornis male (C, G, H) and female (Z.reticulata Kozlov, holotype) (A, B, D, E, F) A metasoma, dorsal view B apex of metasoma, dorsal view C genitalia, lateral view D head and mesosoma, lateral view E antennae, dorsal view F fore wing G fore tibia H antenna, proximal part I label of the holotype.Scale bar: 0.5 mm.

Figure 9 .
Figure 9. Zygota vigil Nixon, male A whole insect in lateral view B male genitalia C fore wing venation.Scale bar: 1 mm.

Figure 10 .
Figure 10.Zygota vigil Nixon, details of morphology, male A, B head and mesosoma in dorsal (A) and lateral (B) views C, E metasoma, in ventral (C) and dorsal (E) views D fore tibia F, G antennae in dorsal view.Scale bars: 0.5 mm (B); 1 mm (F).

Figure 11 .
Figure 11.Zygota walli sp.nov.female holotype (ZSMHYM42437-A07) A face B whole body in dorsal view C head, dorsal view D head and mesosoma in lateral view E apex of metasoma, dorso-lateral view F head and mesosoma in lateral view.Scale bar: 1 mm.

Figure 12 .
Figure 12.Details of Zygota walli sp.nov.morphology, female (A, B, D) and male (C, D) A apex of metasoma B metasoma in dorsal view C A1-A4 in dorsal view D antenna in lateral view E antenna in dorsal view.Scale bar: 0.5 mm.

Figure 13 .
Figure 13.Phylogenetic ML consensus tree of barcoded Belytini specimens with bootstrap/jackknife values and Cinetus cameroni as an outgroup.

Figure 14 .
Figure 14.Phylogenetic ML tree of barcoded Pantoclis material and the polyphyletic appearance of their wing venation.Green represents the taxa with a closed radial cell while species with an open cell are color-coded red.Each node's support is displayed by the bootstrap and the jackknife values.Aclista was used as an outgroup.