A morphological, biological and molecular approach reveals four cryptic species of Trissolcus Ashmead (Hymenoptera, Scelionidae), egg parasitoids of Pentatomidae (Hemiptera)

Accurate identification of parasitoids is crucial for biological control of the invasive brown marmorated stink bug, Halyomrpha halys (Stål). A recent work by Talamas et al. (2017) revised the Palearctic fauna of Trissolcus Ashmead, egg-parasitoids of stink bugs, and treated numerous species as junior synonyms of T. semistriatus (Nees von Esenbeck). In the present paper, we provide a detailed taxonomic history and treatment of T. semistriatus and the species treated as its synonyms by Talamas et al. (2017) based on examination of primary types, molecular analyses and mating experiments. Trissolcus semistriatus, T. belenus (Walker), T. colemani (Crawford), and T. manteroi (Kieffer) are here recognized as valid and a key to species is provided. The identification tools provided here will facilitate the use of Trissolcus wasps as biological control agents and as the subject of ecological studies.


Introduction
Taxonomy of the genus Trissolcus Ashmead has received renewed attention in recent years (Talamas et al. 2015, largely because accurate identification of these wasps is needed to use them as biological control agents against the invasive brown marmorated stink bug (Halyomorpha halys (Stål)) in Europe and North America. Morphological similarity, sharing of hosts by various species of Trissolcus, and the historical complications presented in Talamas et al. (2017) and Buffington et al. (2018) are some of the challenges faced by taxonomists working with this group.
In support of studies on the egg-parasitoid complex of European Pentatomoidea, a survey of egg masses was conducted and previously collected specimens were also examined. Using the key to species provided by Talamas et al. (2017), Trissolcus specimens that emerged from Aelia rostrata Boheman, Arma custos (F.), Carpocoris spp., Eurygaster maura (L.), Graphosoma lineatum (L.), Palomena prasina (L.) collected between 1996 and 2017 in Piedmont (NW Italy) were identified as T. semistriatus. However, some consistent morphological differences were detected among the specimens, which instigated closer examination using multiple methods. The focus of this paper is the morphological and molecular analysis of species synonymized under T. semistriatus by Talamas et al. (2017), and the integration of mating tests, when possible, to confirm species delimitation.

Species described by Walker
Telenomus belenus was described by Walker (1836), then transferred by Kieffer (1912) to Aphanurus Kieffer, then transferred to Microphanurus Kieffer (Kieffer 1926). Walker (1838) described Telenomus arminon but did not provide distinctive characters by which it could be identified or separated from Telenomus belenus. Kieffer (1912) transferred Te. arminon to Allophanurus Kieffer and provided a redescription. Kieffer did not mention if his treatment was based on type material, and we consider it unlikely that it was. Lectotypes for Te. belenus and Te. arminon were designated by Fergusson (1984Fergusson ( , 1983, respectively, from material housed in the National Museum of Ireland, Dublin. Despite their antiquity, and thus priority, these species received no further taxonomic treatment.

Trissolcus semistriatus vs. T. grandis
In taxonomic literature, the distinction between T. semistriatus and T. grandis has long been questioned. Mayr (1879) and Nixon (1939) ascertained T. semistriatus to be a highly variable species. Masner (1959) wrote "On base of the check of type of Asolcus grandis (Thomson), the latter species was synonymized with semistriatus". However, the meaning of this sentence is unclear because we have not found in the literature previous synonymy of T. grandis under T. semistriatus, and it is not clear that Masner sought to synonymize them for the first time. In this paper, Masner addressed characters considered to distinguish T. grandis and T. semistriatus (rugosity of the frons, leg color, longitudinal sculpture on the posterior mesoscutum, body length) based on the comparison of ~500 reared specimens and stated that these characters were variable within T. semistriatus. Viktorov (1967) considered T. grandis to be conspecific with T. semistriatus, but he did not formally treat it as a junior synonym. Subsequent authors considered T. semistriatus and T. grandis as different species, but without clearly defining the boundaries between them. Delucchi (1961) provided the first reliable character to distinguish T. semistriatus from T. grandis: the external surface of the hind femur is almost totally covered by setation in T. grandis (Figure 1), and he coupled this character with the color of the tibiae: reddish yellow in T. semistriatus, dark or black in T. grandis. Most authors continued to distinguish T. semistriatus and T. grandis by tibial color and ignored setation of the hind femora. This color-based distinction was employed in numerous previous and following papers (Delucchi 1961;Javahery 1968;Kozlov 1968Kozlov , 1978Safavi 1968;Voegelé 1969;Fabritius 1972;Kozlov and Kononova 1983), and no substantial change was indicated in keys to species by Kononova ( , 2014Kononova ( , 2015. Talamas et al. (2017) did not use tibial setation to differentiate between these species, but listed a new character, the form of the mesoscutal humeral sulcus, and mentioned setation of the first laterotergite, which was first presented as a character for species of Trissolcus by Johnson (1987). Although Talamas et al. (2017) treated these characters as variable within T. semistriatus, analysis of these characters in light of molecular and mating experiments has allowed us to use them for species delimitation.
In a study on larval stages, Voegelé (1964) provided information about pigmentation of the membrane secreted by the larvae of different Trissolcus species reared in eggs of Eurygaster austriaca (L.). He distinguished T. semistriatus from T. grandis by the width of the pigmented band close to the margin of host egg operculum (see fig. 4 in Voegelé 1964). In his key to species, Safavi (1968) coupled color of the hind tibia (instead of mid tibia), and width of the pigmented band in larval membrane shown by Voegelé (1964), also adding different length ratios of the first two flagellomeres in males.
Trissolcus artus was distinguished by Kozlov and Kononova (1983) and  from T. grandis (black tibiae) by its reddish-yellow tibiae, and from T. semistriatus by having a more elongate clava and infuscation in the fore wing. This last feature is used in the key by Kononova (2014Kononova ( , 2015 to distinguish T. artus from both T. grandis and T. semistriatus.

Trissolcus manteroi
Trissolcus manteroi was described by Kieffer (1909) as having the postmarginal vein (pm) slightly longer than the stigmal vein (st). In Kozlov and Kononova (1983), Koçak and Kilinçer (2003) and Kononova (2014Kononova ( , 2015, T. manteroi was distinguished by its postmarginal vein 1.3× as long as the stigmal vein, compared to 1.8× in Trissolcus rufiventris (Mayr), and 2× in T. grandis (=T. belenus) and T. semistriatus. Kononova  Delucchi (1961) where differences in the bare area of the external side of hind femora of Asolcus semistriatus (Fig III, I) and A. grandis (Fig. III, H) are shown. (2014,2015) also distinguished T. manteroi by the sculpture of T2, in which short longitudinal rugae are arranged medially and do not extend to the posterior half of the tergite, contrasting with longitudinal rugae throughout the anterior two thirds of T2 in T. belenus and T. semistriatus. Crawford (1912) described Telenomus colemani from specimens that emerged from an egg mass of Dolycoris indicus Stål, collected in India. Masner and Muesebeck (1968) transferred this species into Trissolcus and no other information was recorded until its treatment as a junior synonym of T. semistriatus in Talamas et al. (2017).

Trissolcus pseudoturesis and T. djadetshko
The original description of Microphanurus (=Trissolcus) pseudoturesis Rjachovskij (Rjachovskij 1959) Viktorov (1964) distinguished Asolcus (=Trissolcus) djadetshko and A. rufiventris by the lack of longitudinal striae on the posterior margin of the mesoscutum in contrast to their presence in A. pseudoturesis and A. semistriatus. Viktorov (1967) then modified his concept, considering the color of the hind tibia as a valid character to distinguish T. djadetshko from T. semistriatus and the color of femora to distinguish T. djadetshko from T. pseudoturesis. The keys to species by Kozlov (1968) and Fabritius (1972) distinguished T. djadetshko from T. grandis, T. pseudoturesis and T. semistriatus by the absence of longitudinal striation on the posterior mesoscutum and an absence of transverse striation on the frons, and T. pseudoturesis from T. grandis and T. semistriatus by color of the femora. Kozlov and Kononova (1983) separated T. djadetshko from both T. grandis and T. semistriatus by the absence of longitudinal striation on the posterior mesoscutum. Safavi (1968) and Voegelé (1969) separated T. djadetshko and T. pseudoturesis by their "ochraceous" femora from T. semistriatus and T. grandis (black femora), and separated T. djadetshko from T. pseudoturesis by longitudinal striae on the posterior margin of mesoscutum (vs. striate throughout) and the presence of parapsidal furrows. Koçak and Kilinçer (2003) distinguished T. djadetshko by its femora being reddish-yellow in contrast with dark brown or black femora in T. semistriatus and T. grandis, and separated T. djadetshko from T. pseudoturesis by sculpture on mesoscutum as in Voegelé (1969). Petrov (2013) again distinguished T. djadetshko on the basis of the mesoscutum without longitudinal wrinkles, contrasting with the clear longitudinal wrinkles of T. grandis, T. pseudoturesis and T. semistriatus, and he separated T. pseudoturesis from T. grandis and T. semistriatus by the color of femora. Kononova (2014Kononova ( , 2015 differentiated T. djadetshko by its yellow legs and mesoscutum without longitudinal rugae posteriorly from T. semistriatus and T. grandis having all femora black and mesoscutum with longitudinal rugae posteriorly, and T. pseudoturesis from T. grandis and T. semistriatus as in Kozlov (1968). Trissolcus djadetshko and T. pseudoturesis were treated as junior synonyms of T. semistriatus in Talamas et al. (2017). Javahery (1968) described and keyed T. waloffae (Javahery) using leg color (predominantly brownish to reddish-yellow) and weakly indicated parapsidal furrows to separate it from T. grandis, T. semistriatus, T. nixomartini and T. silwoodensis, which he considered to have black femora in both sexes and be without parapsidal furrows. Characters provided to distinguish each of the last four species from each other were black vs. brown front tibiae, presence of infuscation of wings, color of wing venation, ratio between first flagellar segment and pedicel of male, sculpture of the head, distance between lateral ocelli and compound eye, and 'weakly concave' vs. 'somewhat concave' head. Trissolcus silwoodensis and T. nixomartini were previously treated as synonyms of T. grandis by .

Trissolcus crypticus
During a program for classical biological control of Nezara viridula L. in Australia, several 'strains' of different geographical populations of Trissolcus basalis (Wollaston) were introduced, starting in the 1930s (Clarke 1990). Of the strains introduced in subsequent years to the interior of Australia, one population imported from Pakistan (1961) was not able to efficiently control N. viridula (Clarke 1990). Clarke (1993) demonstrated that this 'strain' was indeed a different species, which he described as Trissolcus crypticus Clarke. Comparing T. crypticus with T. basalis, he considered the complete netrion sulcus (figure 1 in Clarke 1993) as the main diagnostic character for T. crypticus. Clarke analyzed specimens of Trissolcus rungsi (Voegelé) labelled by Voegelé and deposited in NHMUK and concluded that they were not the same species as T. crypticus, but did not present characters to support his hypothesis (Clarke 1993).

Primary types
Due to the challenge of historic confusion regarding species close to T. semistriatus, we treat only species for which the primary types were directly examined, or the diagnostic characters are clearly visible in photographs.

Geographical distribution and host association
The identification tools of previous literature are not reliable for identifying the species that we treat here. Hence, the geographical distribution and host associations presented in Material Examined sections derive only from specimens examined as part of this study.

Cybertaxonomy
Specimens used in this study were assigned collecting unit identifiers (CUIDs) and their associated collection and host association data were deposited in Hymenoptera Online (hol.osu.edu). In addition to the abbreviated Material examined sections, a DarwinCore archive is provided for each species (Suppl. material: S2-S5). These files contain the totality of specimens for which data is deposited in Hymenoptera Online, including specimens for which updated identification has not yet occurred, which can be assessed by the dates of determination. Taxonomic synopses, descriptions, and material examined sections were generated in the online, matrix-based program vSysLab (vsyslab.osu.edu) with a matrix based on that of Talamas et al. (2017).

Photography
A Leitz Großfeld-Stereomikroskop TS with magnification up to 160×, a Stereomicroscope Wild M3B with oculars 15×, and a spot light Leica CLS 150× were used for biometric diagnosis. A semi-transparent light shield was used to reduce glare and to diffuse the light. The lectotypes of T. belenus and T. arminon were photographed with a Macroscopic Solutions Macropod MicroKit with individual slices rendered in Helicon Focus 6. All other images were produced using a Leitz Dialux 20 EB compound microscope with a Leica DFC 290 Camera with LED spot light or dome light based on different points of view after techniques summarized in Buffington et al. (2005), Kerr et al. (2008) and Buffington and Gates (2008). LEICA APPLICATION SUITE V 3.7.0 software was used to manage image acquisition and ZERENE STACKER was used for merging of the image series into a single in-focus image.

Morphology
Terminology for surface sculpture follows the glossary by Harris (1979), Mikó et al. (2007), Yoder et al. (2010) and Talamas et al. (2017). Measurements of the head, mesosoma, metasoma, total body, and wing venation follow Masner (1980) and Tortorici et al. (2016). In the wing ratio expressed as st:pm:mg, the stigmal vein is treated as the benchmark unit (=1). Morphological terms largely follow Mikó et al. (2007) and were matched to concepts in the Hymenoptera Anatomy Ontology (Yoder et al. 2010) using the text analyzer function and a table of these terms and URI links is provided in Suppl. material: S1. Additional abbreviations and terminology used in this paper: HL: head length; HW: head width; HH: head height, from vertex to distal end of clypeus; FCI: frontal cephalic index (HW/HH); LCI: lateral cephalic index (HH/HL); OOL:POL:LOL: ocular distance ratio, OOL as the benchmark unit (=1); IOS: interorbital space ; claval formula: the sequence of sensilla, from the apical antennomere (A11) to the last functional clavomere (Bin 1981), i.e. the last antennomere bearing one or two multiporous gustatory sensilla, as defined by Isidoro et al. (1996); compound eye height and width: measured when eye longitudinal axis is parallel to the focal plane.

Insect collecting and rearing
A host colony of E. maura used for rearing Trissolcus was established from adults collected on wheat in Piedmont (NW Italy) and maintained in cages under laboratory conditions (climatized chambers at 24 ± 1 °C, 65 ± 5% RH, L:D = 16:8). All eggs laid in the cages were collected and frozen at -20 °C. Because of the short egg-laying period of E. maura, freezing the eggs allowed the eggs to be used for a much longer time.
To obtain Trissolcus specimens, egg masses of E. maura and P. prasina were collected in the field in Piedmont (NW Italy) in the spring and summer of 2017. The fieldcollected egg masses were reared and checked daily. Trissolcus specimens that emerged from field-collected egg masses were allowed to mate. Some females were isolated in small plastic boxes (64.5 × 40.9 × 16 mm), fed with water and honey, and provided with E. maura frozen egg masses to produce progeny for use in subsequent tests.
For interbreeding experiments, specimens were isolated immediately following emergence to prevent mating, and females and males were maintained singly in plastic boxes as described above. When the parasitoids reach the early pupal stage inside the eggs, their red eyes are clearly visible through the transparent operculum of the host egg. Following observation of this feature (Figure 2), the eggs were checked at a frequency of 4-5 times per day to ensure that they were isolated prior to mating.
Some of the progeny from isolated, mated females were selected for preservation, identification and molecular analysis. The remaining progeny were used in breeding experiments.

Molecular analyses
Molecular analyses were performed to confirm morphological identification and characterize the species. Genomic DNA was extracted from the metasoma of specimens from rearing experiments and pinned collection specimens according to Kaartinen et al. (2010), but doubling the proteinase K dose (5 μl of 20 mg ml −1 proteinase K). The barcode region of the cytochrome oxidase I (COI) gene was amplified using universal PCR primers for insects LCO1490 (5'-GGT CAA CAA ATC ATA AAG ATA TTG G-3') and HCO2198 (5'-TAA ACT TCA GGG TGA CCA AAA AAT CA-3') (Folmer et al. 1994). The PCR was performed in a 50 μl reaction volume: 2 μl of DNA, 37.9 μl molecular grade water, 5 μl 10× Qiagen PCR buffer, 3 μl dNTPs (25 mM each), 1.5 μl MgCl2, 0.2 μl of each primer (0.3 μM each), 0.2 μl Taq DNA Polymerase (Qiagen, Hilden, Germany). Thermocycling conditions were optimized to shorten reaction times and included initial denaturation at 94 °C for 300 s, followed by 35 cycles of 94 °C for 30 s, annealing at 52 °C for 45 s and extension at 72 °C for 60 s; then further 600 s at 72 °C for final extension. PCR products were purified using a commercially available kit (QIAquick PCR Purification Kit, Qiagen GmbH, Hilden, Germany) following the manufacturer's instructions, and sequenced by a commercial service (Genechron S.r.l., Rome, Italy). The sequences were compared with the GenBank database and each other using the Basic Local Alignment Search Tool (http://www.ncbi.nlm. nih.gov/BLASTn). All sequences were aligned using ClustalW with default settings as implemented in Mega X. The pairwise nucleotide sequence distances among and within taxa were estimated using the Kimura 2-parameter model (K2P) of substitution (Kimura 1980) using Mega X (Kumar et al. 2018). The sequences generated from this study are deposited in the GenBank database. All residual DNA is archived at DISAFA.

Mating tests and reproductive isolation between T. belenus and T. semistriatus
For mating experiments, 1-2-day old virgin females and males were used. Four combinations for mating tests were done: . The total number of interbreeding tests was 24: four replicates for each intraspecific mating combination and eight replicates for each interspecific mating combination. Each pair of wasps was observed at the stereomicroscope until the end of copulation or for 10 minutes if copulation did not occur. The pair then remained together in isolation for 24 hours. After the mating test, an egg mass of E. maura was provided to each female wasp for 24 hours of exposure. The egg masses were then moved to other plastic boxes until offspring emergence. Each mating test was considered successful when emerged offspring included females, because in all known Trissolcus species, only mated females can produce female offspring. We compared the percentage of mating success among the four combinations and the significance of the results was assessed with a chi-square test.

Morphological analysis
The easiest task regarded the distinction of T. manteroi from T. semistriatus, T. belenus and T. colemani. Trissolcus manteroi clearly has a shorter postmarginal vein, only slightly longer than the stigmal vein; A7 has only one papillary sensillum instead of two in the other three species; and T. manteroi has no episternal foveae. The holotype of T. manteroi is thus morphologically very close to T. rufiventris, from which it can be differentiated by the length of the postmarginal vein.
The distinction of T. belenus and T. colemani from T. semistriatus is more nuanced and required an integrative approach to determine which morphological characters were congruent with the biological and molecular data. The results of this in-depth analysis demonstrate that some of the characters that Talamas et al. (2017) treated as intraspecifically variable have diagnostic power.
The presence or absence of setation on the external face of the hind femur, described in the key and figure III (I) (H) in Delucchi (1961), is a reliable character to distinguish T. grandis from T. semistriatus. However, in the lectotype of T. grandis and neotype of T. semistriatus this character is opposite to what was stated by Delucchi (1961). Furthermore, the holotype of T. colemani has the external surface of hind femur setose, as in the lectotype of T. grandis. The association proposed in Delucchi (1961): 'external face of hind femora uncovered by hair' -'reddish yellow tibiae' is the typical combination for T. colemani, while Delucchi (1961) proposed it for T. semistriatus, and 'external face of hind femora covered by hair' -'dark or black tibiae' is the typical combination for T. semistriatus. We conclude that this interpretation is contrary to what is found in type material.

Synonymy in T. belenus
In the analysis of original descriptions and images of lectotype of T. arminon and T. grandis, no remarkable characters were recognized to distinguish them from T. belenus, which we therefore consider it to be their senior synonym. In the analysis of type material of T. silwoodensis and T. nixomartini, previously considered junior synonym of T. grandis (Kozlov & Lê, 1977), we confirmed the findings of previous authors, and thus treat these species as junior synonyms of T. belenus. Mayr (1879) considered Telenomus ovulorum Thomson to be a junior synonym of Telenomus semistriatus Nees von Esenbeck, but through analysis of the photographs of type material of T. ovulorum Thomson, we recognized the character states of T. belenus, and therefore treat T. ovulorum as a junior synonym of T. belenus.

Synonymy in T. colemani
One paratype of T. djadetshko and three syntypes of T. pseudoturesis were analyzed via photographs and compared with the original description and photographs of the holo-type of T. colemani. The character states of the two first species matched perfectly with those of the latter, leading us to treat T. colemani as the senior synonym of T. djadetshko and T. pseudoturesis. We conclude that the characters of T. crypticus match those in the holotype of T. colemani based on examination of T. crypticus paratypes collected in Pakistan and its original description (see figs 1, 3, 5 in Clarke 1993). We thus treat T. crypticus as a junior synonym of T. colemani. Clarke (1993) also reported that "Examination of material of T. rungsi labelled by Voegelé (deposited in NHMUK) shows that this species is not the same of T. crypticus" but he did not provide any distinguishing characters between the two species. Contrary to what was reported by Clarke (1993), in our analysis of the material deposited at NHMUK, 37 specimens labelled as "Asolcus rungsi Voegelé" were identified as T. colemani and four specimens labelled as "rungsi 1965 Voegele" were identified as T. basalis, while other 25 with the same last cumulative label were identified as T. colemani. This confirms our interpretation of the description and analysis of figures regarding A. rungsi and demonstrates confusion of species in the Moroccan rearing efforts at École Nationale d'Agriculture in Meknès.
The original description of Asolcus rungsi mentioned the presence of short traces of notauli ( fig. 1, c. in Voegelé 1965); these traces are visible in all specimens T. colemani ( Figure 23). However, because the location of the holotype of A. rungsi is not known, we were unable to examine it and at this time do not treat this species name as a synonym. The morphological analyses of the holotype and paratypes of T. waloffae showed the conspecificity of this species with T. colemani.

Molecular analysis
Barcode sequences were obtained from 17 Trissolcus specimens (Table 1). The Blast search showed that the sequences of T. semistriatus from Italy and from Iran had a 98% sequence identity with the GenBank sequence from Trissolcus nigripedius (accession no. AB971830). The sequences from the two specimens of T. colemani showed a 98% identity with a GenBank sequence with a Platygastridae sp. (accession no. KY839581), while the sequences from the specimens of T. manteroi, T. belenus and T. rufiventris showed a lower similarity with GenBank sequences. The final alignment consisted of 548 characters. Pairwise distance values within and among analyzed species are shown in Table 2. The genetic distances between the specimens identified as of the same species (which averaged between 0.000 ± 0.000 and 0.005 ± 0.002), were much lower than the mean pairwise distances observed between the specimens identified as of different species (from 0.105 ± 0.001 to 0.149 ± 0.000).

Mating tests
Specimen pairs tested for intraspecific combination mated within ten minutes; pairs tested for interspecific combination did not mate within the 10-minute observation period.

Key to Trissolcus of the Palearctic region (females)
Modified couplets for the Key to Trissolcus of the Palearctic region (females) in Talamas et al. (2017) 29 Ventral mesopleuron distinctly bulging; mesocoxa oriented parallel to long axis of body; dorsal frons with sculpture effaced, sometimes entirely smooth and shining; A7 with two papillary (basiconic)

Discussion
More than 180 years have passed between the original descriptions of T. semistriatus and T. belenus and the development of identification tools that can reliably distinguish them. This can be viewed as a glacial rate of progress, but also as an indication that modern methods can resolve long-standing taxonomic challenges. The taxonomy of Trissolcus illustrates that the examination of primary types and detailed comparison of specimens across a broad geographical range is necessary to advance the field, and that further refinement may be required even when these practices are implemented. Talamas et al. (2017) significantly advanced the taxonomy of Palearctic Trissolcus but additional analysis was needed to distill diagnostic characters from those that were treated as intraspecifically variable. Specifically, setation on the first laterotergite, the form of the mesoscutal humeral sulcus, and the length of the anteroventral extension of the metapleuron were treated as variable within T. semistriatus. Although the utility of these characters for separating T. belenus and T. colemani was not recognized, Talamas et al. (2017) did bring attention to them, as they had not yet been used in the taxonomy of Palearctic Trissolcus. Setation of the hind femora, not mentioned by Talamas et al. (2017), represents a case in which a diagnostic character was previously recognized, but incorrectly associated with a taxon name (Delucchi 1961), and is now treated as useful for identifying T. belenus. Trissolcus manteroi is a different matter, in which reexamination of the type specimen was needed for its diagnostic characters (wing venation, claval formula, absence of episternal foveae) to be correctly characterized. These features place T. manteroi closer to T. rufiventris than to T. semistriatus, T. belenus or T. colemani.
The trail of photographic evidence provided by Talamas et al. (2017) enabled junior synonyms of T. semistriatus to be rapidly redistributed among T. belenus and T. colemani once the characters that delimit these species were identified, as well as the resurrection of T. manteroi. Given that producing a natural classification is an iterative process, explicit presentation of data that underlies taxonomic decisions accelerates further refinement. This is perhaps the only means by which the various quagmires of inadequate species descriptions in Platygastroidea can be transformed into a useful classification.
The need for reliable identification can be clearly seen in examples where quality taxonomy was absent. In the early part of the 20 th century, Trissolcus specimens identified as T. semistriatus or T. grandis were reared and released in Russia and Iran as classical biological contral agents against Eurygaster (Alexandrov 1947;Saakov 1903;Vaezi 1950;Vassiliev 1913;Zomorrodi 1959). Some of these authors did not indicate how they identified the species, and in any case, the characters that reliably separate these species were not established. It is only by retroactively identifying voucher specimens, if they exist, that the results of these efforts can be interpreted in a meaningful way. The presence of H. halys in Europe has created a similar situation, with the same species involved in studies of its biological control. The refined species concepts presented here are thus of immediate relevance, given that T. belenus was recorded from frozen sentinel eggs of H. halys in Europe, and was previously identified as T. semistriatus.
Finally, it should be noted that independent testing of species concepts, ideally using multiple methods, is the best means by which they can be verified or improved. This study employed such an approach, using morphology, mating studies and molecular analysis to resolve four species from the concept of T. semistriatus provided in Talamas et al. (2017). In a manner conforming with this perspective, our results have been confirmed by a concomitant study by Talamas et al. (2019), in which a phylogeny of Trissolcus based on five molecular markers retrieved T. belenus, T. colemani and T. semistriatus as distinct entities.
transliteration from Cyrillic to Latin alphabet of labels; David Notton (BMNH), who hosted a visit of Virgilio Caleca; Dr. Paolo Visconti, who hosted a visit of Elijah Talamas to NMID which enabled the lectotypes of T. belenus and T. arminon to be studied and photographed and Dr. Norman Johnson (The Ohio State University), for maintaining Hymenoptera Online and vSysLab and assisting with data processing. Elijah Talamas was supported in part by a cooperative agreement with Kim Hoelmer (USDA/BIIRU) and by the Florida Department of Agriculture and Consumer Services-Division of Plant Industry. This research was funded by Fondazione Cassa di Risparmio di Cuneo (project HALY-END) and Regione Piemonte (project BIOHALY).
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