JHR 97: 399-439 (2024) ee, JOURNAL OF Areminrcrassen doi: 10.3897/jhr.97.121918 RESEARCH ARTICLE (=f) Hymenopter a > https://jhr.pensoft.net Thelmternaonl Society of Hymenopexriss, RESEARCH Cryptic or underworked? Taxonomic revision of the Antistrophus rufus species complex (Cynipoidea, Aulacideini) Louis FE. Nastasi', John E Tooker', Charles K. Davis', Cecil N. Smith', Timothy S. Frey’, M. J. Hatfield’, Tara M. Presnall, Heather M. Hines'?, Andrew R. Deans! | Department of Entomology, The Pennsylvania State University 501 Agricultural Science and Industries Building, University Park, PA, 16802, Pennsylvania, USA 2. Department of Plant Pathology, The Ohio State University, 1680 Madison Ave, Wooster, OH, 44691, USA 3 3079 Coldwater Creek Road, Cresco, IA, 52136, USA 4 Department of Mechanical Engineering, The Pennsylvania State University 137 Reber Building, University Park, PA, 16802, Pennsylvania, USA 5 Department of Biology, The Pennsylvania State University, 208 Mueller Labs, University Park, PA 16802, Pennsylvania, USA Corresponding author: Louis E. Nastasi (fnastasi@gmail.com) Academic editor: Miles Zhang | Received 1 March 2024 | Accepted 29 April 2024 | Published 21 May 2024 https://zoobank. org/9675 CE30-77A6-458A-A3EE-56E032633E9C Citation: Nastasi LE, Tooker JE Davis CK, Smith CN, Frey TS, Hatfield MJ, Presnall TM, Hines HM, Deans AR (2024) Cryptic or underworked? Taxonomic revision of the Antistrophus rufus species complex (Cynipoidea, Aulacideini). Journal of Hymenoptera Research 97: 399-439. https://doi.org/10.3897/jhr.97.121918 Abstract Cryptic species present challenges across many subdisciplines of biology. Not all “cryptic” species, how- ever, are truly cryptic; many are simply underexplored morphologically. We examined this idea for the An- tistrophus rufus species complex, which previously contained three species thought to be morphologically cryptic. To determine whether the A. rufus complex are truly cryptic species, we assessed species bounda- ries of members of the A. rufus species complex using morphological, ecological, and DNA barcode data, and tested whether a set of 50 morphological characters could adequately diagnose these species. We revealed that this complex includes five species, and that there are useful phenotypic diagnostic characters for all members of this species complex. This enabled redescription of four species and the description of Antistrophus laurenae Nastasi, sp. nov., which induces externally inconspicuous galls in stems of Si/phium integrifolium Michx., a host not associated with other members of the complex. We use these new diag- nostic characters to construct a key to the five species of the rufus complex. We conclude that the A. rufus complex was not a true case of cryptic species. Our Bayesian analysis of DNA barcode data suggests pos- sible cospeciation of members of the rufus complex and their Si/phium host plants, but further study is necessary to better understand the evolution of host use in the lineage. Copyright Louis F Nastasi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License (CC BY 4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 400 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) Keywords Cryptic species, gall wasp, morphology, Si/phium, superficial description impediment Introduction Cryptic species are those that cannot readily be distinguished on the basis of pheno- typic variation alone (Struck et al. 2018); they have created challenges in myriad areas of the life sciences, especially in agro-economic systems (Andrews et al. 2020; Hansen et al. 2021; MacLeod et al. 2021). One such system of emerging significance is that of the genus Si/phium L., a genus of composite herbs in the sunflower tribe (Asteraceae: Heliantheae) that is currently being investigated for use as biofuels, oilseeds, and addi- tional economic purposes (Kowalski et al. 2013; Jasinskas et al. 2014; Peni et al. 2020; Vilela et al. 2020; von Cossel et al. 2020; Titei et al. 2021). Despite recent interest in cultivating and domesticating various Si/phium species, as well as their prominence in threatened tallgrass prairie habitats, their associated arthropod fauna remains poorly studied (Henderson and Sauer 2010; Buffington et al. 2017). A major component of Silphium communities is herb gall wasps of the genus Antistrophus Walsh, 1869 (Fig. 1), which includes a known case of cryptic species (Tooker et al. 2004). Six described species of Antistrophus induce galls in the disc flowers and stems of four Silphium species (Nastasi and Deans 2021). Perhaps the most intriguing Si/phium gall wasps are three cryptic species composing the rufus species complex (A. jeanae Tooker and Hanks, A. meganae Tooker and Hanks, and A. rufus Gillette). These spe- cies are not only thought to be morphologically cryptic, but also induce galls that are inconspicuous and do not externally deform the host plant’s tissues (often referred to as “cryptic galls” in the literature; e.g., Ronquist and Liljeblad 2001); each species induces these galls within stems of their respective host species of the genus Si/phium (Tooker et al. 2004; Nastasi and Deans 2021). The complex was described by Tooker et al. (2004), who found each species to be distinct based on allozyme and ecological data, including use of host plant species (Tooker et al. 2004); A. jeanae is associated with S. perfoliatum L., A. meganae is associated with S. terebinthinaceum Jacq., and A. rufus is associated with S. laciniatum L. These species would be considered cryptic, or func- tionally so, based on the diagnostic morphological criteria which included averages of precise ratios of antennomere dimensions, lengths of dissected ovipositors, depths of galls within host stems, and masses of mature larvae (Tooker et al. 2004). These metrics are hard to replicate and do not fully diagnose the species, thus the original diagnoses are arguably insufficient to reliably arrive at a species identification without host plant data. While these are the only morphological diagnostic characters given by Tooker et al. (2004), the descriptions of these species, including Gillette’s 1891 description of A. rufus, are fairly limited in the number of morphological characters examined. Due to the brief original descriptions of species composing the A. rufus complex, it seems Cryptic or underworked? Antistrophus rufus complex 401 Figure |. Examples of Si/phium-galling Antistrophus wasps and their galls A adult female A. jeanae Took- er & Hanks, 2004 B galls and larvae of A. jeanae in stem pith of S. perfoliatum L. C adult female A. silphii Gillette, 1891 D galls of A. si/phii on terminal stem of S. integrifolium Michx E adult female A. daciniatus Gillette, 1891 F galls of A. laciniatus in disc flower of S. laciniatum L. adult gall wasps photographed by Antoine Guiguet. Galls photographed by Andrew R. Deans. 402 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) likely that this is a case of the so-called “superficial description impediment”, a taxo- nomic phenomenon in which species are not readily identifiable based on characteriza- tion in existing literature (Meier et al. 2021), rather than a true case of cryptic species. The potential for a superficial description impediment in the A. rufus complex is supported by the minimal history of taxonomic investigation of herb gall wasps in North America; among the early diverging lineages of gall wasps are several tribes of non-oak herbaceous gallers (Aylacini sensu lato), but these remain more poorly under- stood than the species-rich oak gallers (Cynipini). While the herb gall wasp fauna of the Western Palearctic has been comparatively well studied (e.g., Nieves-Aldrey 1994; Nieves-Aldrey et al. 2008; Nieves-Aldrey 2012; Nieves-Aldrey 2022), those in North America remain poorly investigated. Eighteen valid native species are known (Nastasi and Deans 2021; Nastasi et al. 2024); only 14 published papers explore genus- or species-level taxonomy of these taxa, and many of these papers treat only a single species (Table 1). Of these works, only two (Nieves-Aldrey 1994; Tooker et al. 2004) have been published in the last 100 years. When combined with the known diagnostic characters for the rufus complex, the paucity of taxonomic work on this group further increases the likelihood of a taxonomic impediment rather than a true case of the cryp- tic species phenomenon. In our analysis of the Antistrophus rufus complex, we included A. jeanae (gall inducer on S. perfoliatum), A. meganae (gall inducer on S. terebinthinaceum), and A. rufus (gall inducer on S. laciniatum) by sampling individuals reared from stems of these three Si/phium host species. In the process we reveal in the stems of S. inte- grifolium the presence of an additional species that we newly describe, Antistrophus laurenae Nastasi sp. nov.; S. integrifolium is a new host plant for the rufus complex. We include in this analysis A. minor Gillette, 1891; we treat this species in the ru- Jus complex based on its adherence to the diagnostic criteria for the rufus complex Table |. Taxonomic treatments of North American herb gall wasps, 1869—present. Names later trans- ferred to other genera marked with (*). Names later synonymized marked with (0). Species that are non- native to North America (or questionably so) are excluded. Author and year Description of taxonomic work Riley and Walsh 1869 Described Antistrophus pisum Ashmead 1887 Described Aylax harringtoni* Bassett 1890 Described Aylax tumidus* and Aylax podagrae* Gillette 1891 Described Aylax bicolor*) and five species of Antistrophus Brodie 1892 Described Aylax nabali* Ashmead 1896 Described Aylax ambrosiaecola*, A. cavicola*), A. mulgediicola*)), and A. sonchicola*) Ashmead 1897 Described the genus Aulacidea for some species placed in Aylax Beutenmiiller 1908 Described Aylax chrysothamni* Beutenmiiller 1910a Reviewed North American species of Aylax sensu lato Beutenmiiller 1910b Reviewed North American species of Aulacidea Kinsey 1920 Described Aulacidea abdita and A. annulata McCracken and Egbert 1922 Described Aylax microseris* Nieves-Aldrey 1994 Revised genera of “Aylacini” including those in North America Tooker et al. 2004 Described Antistrophus rufus species complex including A. jeanae and A. meganae Cryptic or underworked? Antistrophus rufus complex 403 we propose here along with that of Tooker et al. (2004). Antistrophus minor induces inconspicuous, externally imperceptible galls like those of A. rufus in the same host plant species (S. laciniatum). While some authors have discussed these species as po- tential synonyms (e.g., Beutenmiiller 1910a), no nomenclatural actions have been published. Antistrophus minor was excluded from Tooker et al.’s (2004) description of the A. rufus complex, but because its status as a valid species remained unclear, we opted to treat A. minor here. Methods Gall collection and rearing In late autumn and winter of 2020, 2021, and 2022, we collected entire, senesced stems of Silphium integrifolium, S. laciniatum, S. perfoliatum, and S. terebinthinaceum from sites in Illinois, Indiana, lowa, and Ohio (details below). We identified plants in the field based on external morphology, as the Si/phium species we studied are eas- ily diagnosable in our sampling region by characters of the leaves and stems that are observable even in senescent plants (e.g., Brock and Weakley 2020). At each site, we confirmed via stem dissection that externally inconspicuous stem galls were present in each host plant species. We then collected additional stems, cut them into evenly sized pieces, labeled them, and stored them in plastic zip bags, which we aerated by poking holes in them using insect pins. We returned stems to lab facilities at The Pennsylvania State University (University Park, Pennsylvania, USA), where we stored bagged stem samples in a barn (without climate controls) to expose occupants to natural environ- mental conditions. From April through September, we checked stems every two to three days for emerging insects, which we collected into vials containing ethanol and stored at -20 °C. For morphological study, we air-dried and mounted selected indi- viduals. Nastasi (2023) provides further recommendations for rearing cynipid galls on herbaceous plants. Museum collection material We examined specimens from the following collections: AMNH_ American Museum of Natural History, New York, NY, USA INHS _ Illinois Natural History Survey, University of Illinois, Champaign, IL, USA PSUC Frost Entomological Museum, The Pennsylvania State University, University Park, PA, USA USNM_ National Museum of Natural History, Washington, DC, USA WIRC Wisconsin Insect Research Collection, University of Wisconsin, Madison, WI, USA 404 Louis E Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) Using Darwin Core biodiversity data standards (Wieczorek et al. 2012), we digi- tized label data of all specimens that we examined. Digitized specimen data for all individuals we examined, including plant sample numbers and exact emergence dates for newly reared material, are available in Suppl. material 1: table 1. Morphological character selection, description, and examination To assess morphological boundaries of species belonging to the rufus complex, we selected 50 morphological characters (Suppl. material 1: table 2) from contemporary works on taxonomy of herb gall wasps (i.e., Nieves-Aldrey 1994; Tooker et al. 2004; Melika 2006; Ronquist et al. 2015; Azmaz and Katilmis 2020; Buffington et al. 2020; Nieves-Aldrey 2022; Tavakoli et al. 2022). We selected characters based on perceived potential for species-level diagnosis as well as ubiquity across the taxonomic treat- ments that we considered. We matched anatomical terms for adult wasps to concepts in the Hymenoptera Anatomy Ontology (Yoder et al. 2010) and provide a URI table (see Seltmann et al. 2012) outlining morphological terminology (Suppl. material 1: table 3). Our terminology relating to cuticular surface sculpture follows Harris (1979). We used the following additional abbreviation that is absent from the Hymenoptera Anatomy Ontology: - DLO (diameter of lateral ocellus) for the largest possible diameter of either lateral ocellus. We evaluated each character for five females and five males of each species (“pri- mary morphological exemplars;” Suppl. material 1: table 1); we putatively identified these individuals based on the associated host plant species and characters presented in the original descriptions and scored the primary type and some secondary types for each species. We selected specimens based on geographic origin and used mate- rial from various counties across four states to ensure that we accounted for different populations and intraspecies variability. In addition to “target” specimens that we used in the primary morphological analysis, we examined an additional 20 females and 20 males of each species (“secondary morphological exemplars;” Suppl. material 1: table 1) to confirm the characters given in descriptions and obtain replicate measurements of body length for each species. We provide raw morphological data (Suppl. material 1: table 4), and a summary of characters, states, and corresponding diagnostic utility (Suppl. material 1: table 5). We report morphological characters in taxon treatments as character-state pairs matching the findings of the morphological “test”. We performed morphological observations and measurements of mounted speci- mens with an Olympus SZX16 stereo microscope (Olympus Life Science, Tokyo, Ja- pan) fitted with an optical micrometer. We measured antennae at a resolution of 0.005 (1/200) millimeters using 10x magnification in combination with the 2x objective. Other measurements were taken at an appropriate magnification using the 1x objec- tive. We used a gooseneck illuminator fitted with mylar strips to diffuse light, which Cryptic or underworked? Antistrophus rufus complex 405 was especially helpful for discerning minute patterns involving sculpture and other aspects of the cuticular surface. Terms relating to gall phenotypes follow Deans et al. (2023). Imaging and drawings We took images of point-mounted adult wasps using a Macroscopic Solutions ‘micro- kit (Tolland, CT) imaging system. Additional images were captured from mounted wasps using Olympus SZX16 microscope (Olympus Life Science, Tokyo, Japan). We stacked images using Zerene Stacker LLC (Richland, WA), edited them using Adobe Photoshop (Adobe Inc.), and prepared plates using Adobe Illustrator (Adobe Inc.). Nomenclature of host plants For taxonomy of Si/phium host plants we follow Brock and Weakley (2020). While taxonomy of Si/phium is widely considered unsettled, the four species considered here (Silphium integrifolium, S. laciniatum, S. perfoliatum, and S. terebinthinaceum) are to- bust in the geographic area that we considered (M. Brock, in litt.). Distribution of gall wasps and host plants We generated maps showing confirmed and potential distribution of each gall wasp species using MapChart ( figures are licensed via CC BY-SA 4.0). We defined potential distribution of each gall wasp species as the known native range of its host plant; dis- tribution of Si/phium species follows Kartesz (2015). Molecular phylogenetics We sequenced and analyzed the cytochrome c oxidase subunit I (COI) gene to test the species concepts suggested by our morphological analysis and host plant data. We se- quenced the COI gene of three individuals of each species of the Antistrophus rufus complex. We also sequenced single specimens of three additional Antistrophus species: A. silphii Gillette, 1891 reared from a terminal stem gall on Si/phium integrifolium Michx. (Fig. 1D), A. /aciniatus Gillette, 1891 reared from a flower gall on Silphium la- ciniatum (Fig. 1F), and A. microseris (McCracken & Egbert, 1922) reared from a stem gall on Microseris douglasii (DC.) Sch.Bip. We also included a single sequence of Jsoco- lus leuzeae Nieves-Aldrey, 2003 from GenBank (DQ012643) to serve as outgroups to the A. rufus complex. We performed DNA extraction using an E.Z.N.A. Microelute Genomic DNA Kit (Omega Biotek Inc., Norcross, Georgia, USA) following kit protocols and elut- ing in 30 ul of buffer (15 pL eluted in two steps). We extracted DNA from wasps either entirely destructively, by grinding the entire body, or minimally destructively, by incubating the entire insect. We amplified the COI gene using primer pairs LEPR 406 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) and LEPF (Hebert et al. 2004) or LCO1490 and HCO2198 (Folmer et al. 1994), fol- lowing Hebert et al.’s PCR conditions for LEP primers and following those of Pang et al. (2020) for Folmer primers. Sequencing of PCR products was performed using The Huck Institute’s Genomics Core Facility (The Pennsylvania State University, Univer- sity Park, PA). We edited and aligned sequence data using Geneious (Biomatters Ltd., Auckland, New Zealand, http://www.geneious.com/) using the Geneious alignment function. Aligned sequence ends were trimmed to exclude primer sequences. We cal- culated genetic distances in Geneious using the Tamura-Nei distance model and 1,000 bootstrap replications. We determined the number of variable and parsimony informa- tive sites in our alignment using AMAS v1.0 (Borowiec, 2016). We estimated phylogenetic relationships using MrBayes v3.2.7a (Ronquist et al. 2012) using the following parameters: ngen=15000000; samplefreq=1000; nchains=4; nruns=3. We used two partitions for the first+second and third codon positions, re- spectively, and selected nucleotide substitution models for the two partitions using PartitionFinder 2 (Lanfear et al. 2017) using a greedy search (Lanfear et al. 2012) and specifying for models available in MrBayes; PartitionFinder 2 recommended GTR+G for the first+second positions and HKY+G for the third position. We specified /. leuze- ae as the outgroup and used a burnin value of 3750000 corresponding to 25% of samples. We visualized our consensus tree using Fig Tree v1.4.4 (https://tree.bio.ed.ac. uk/software/figtree/). All new sequences generated for this study were deposited in GenBank, accessions PP739172—PP739189. We provide collection data for sequenced specimens (Suppl. material 1: table 6) and DNA barcode divergence data (Suppl. material 1: table 7). We deposited DNA vouchers and additional material from gall samples and/or rearing events at PSUC. Identification of additional museum material After developing diagnostic characters for each studied species of Antistrophus, we identified additional material of the A. rufus species complex from collections we refer- enced, with the goal of checking previous determinations of identified specimens and identifying undetermined specimens for the first time. Results Morphological evaluation Of the 50 characters that we assessed, 13 had utility as diagnostic characters for at least one species of the rufus complex. Two of the diagnostic characters, both of which concerned dimensions of the first two flagellomeres, were used previously by Tooker et al. (2004) in their treatment of the complex; our observations of those characters were generally consistent with those of Tooker et al. (2004). Cryptic or underworked? Antistrophus rufus complex 407 While the specimens that we reared from S. laciniatum, S. perfoliatum, and S. terebinthinaceum all matched existing species concepts, the wasps associated with S. integrifolium appeared to compose an independent species, and our morphological observations suggest significant differences between these individuals and those of the described members of the rufus complex. Based on these differences, and the results of our molecular phylogenetic analysis, we describe below the species associated with S. integrifolium as Antistrophus laurenae Nastasi, sp. nov. Molecular phylogenetics Our trimmed alignment was 623 bp in length and contained 216 variable sites and 187 parsimony informative sites. Our Bayesian phylogenetic analysis of the 19 sequences (Fig. 2) provided clear evidence of distinct species matching those found by our mor- phological data, as each of the species had very short branch lengths within species (maximum COI divergence of 1.4% in A. meganae) and very long branch lengths be- tween them. COI species divergences between the closest species pair (A. meganae and A, minor) averaged 10.6% (Suppl. material 1: table 7), well beyond the often contested 2% ‘suggestion’ for distinguishing species. Each species was supported by a posterior winiydyig "yes wniydys 071 minor c Host: S. /aciniatum ~ meganae_CYNHG0119 0.74 ~~ rufus_CS102 x9|dwoo snjns snydoujshuy W = = =) meganae 8 ~ rufus_CS107 Tooker & Hanks, 2004 oe Host: S. terebinthinaceum a: s Mea a nt ewan atoyp MeSH AES] 2 0.64 rufus = ) Host: S. laciniatum ~ minor_CS124 ~~ Antistrophus_laciniatus_CYNHG0116 Antistrophus_silphii_CYNHG0181 ~ Antistrophus_microseris_CYNHG0138 Isocolus_leuzeae_DQ012643 0.05 Figure 2. Bayesian phylogenetic tree of DNA barcodes for the Antistrophus rufus complex. Support values shown only for nodes with posterior probability <1. Host plant clade associations are indicated by colored boxes (Silphium sect. Silphium in orange and S. sect. Composita in green) A adult female Antistro- phus jeanae Tooker & Hanks, 2004 B adult female Antistrophus rufus Gillette, 1891. 408 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) probability of 1, and A. daurenae and A. jeanae were strongly supported as sister species, but the remainder of the between-species relationships are less robust. Each recognized species has a distinct host plant except for A. rufus and A. minor, which are both associ- ated with Silphium laciniatum but were not supported as sister species. Our analysis also retrieved the Antistrophus rufus complex as monophyletic within the sampled spe- cies of Antistrophus with a posterior probability of 1. Taxonomy of the Antistrophus rufus species complex Antistrophus rufus species complex Diagnosis. Overall, the A. rufus complex is best diagnosed by the following combina- tion of characters: head and mesosoma mostly reddish-brown in color (Figs 3A—D); facial radiating striae complete (Fig. 3A); compound eye longer than malar space in anterior view (Fig. 3A); F2 much longer than F1 (e.g., Fig. 4B); mesopleuron sculpture reticulate with fine intermediate striae (Fig. 3B); notauli incomplete, indistinct in ante- rior third of mesoscutum (Fig. 3C); female fore wing without apparent marginal setae (Fig. 3D); galls inconspicuous, induced in stems of Si/phium species (e.g., Fig. 4F). Comparative diagnostic characters for the Antistrophus rufus species complex relative to other Antistrophus are presented in Fig. 3 and Table 2 below. Table 2. Diagnostic characters of the Antistrophus rufus complex. Morphological data for species outside the A. rufus complex are from ongoing revisionary work on Aulacideini (Nastasi et al., unpublished data). Biological data are from Nastasi and Deans (2021). * = Gall and host unconfirmed for A. bicolor Gillette, 1891; see Nastasi and Deans (2021). + = Coloration of fresh material unknown; based on description by Beutenmiiller (1908). Antistrophus Head and Facial Malar F2:Fl Mesopleuron Notauli Fore wing Gall species mesosoma radiating space:eye length sculpture marginal morphotype coloration striae setae (2) and host plant rufus complex Mostly Complete, Eyelonger F2 much Reticulate Incomplete, | Absent —Inconspicuous, (jeanae, reddish-brown reaching eye (Fig. 3A) longer with fine indistinct in (Fig. 3D) in stems of laurenae, (Fig. 3A) (e.g., intermediate anterior third Silphium meganae, minor, Fig. 4B) striae (Fig. 3C) (e.g., Fig. 4F) & rufus) (Fig. 3B) bicolor, Entirely black Complete Eye longer F2 Reticulate Complete, Absent Conspicuous, laciniatus, subequal with fine _—_ distinct across on stems or & silphii toFl — intermediate mesoscutum flowers of striae length Silphium* chrysothamni Black to Absent Eye longer F2 much Strongly Incomplete, Absent Conspicuous, reddish- longer striate with — distinct only apparently on brownt interspaces in posterior Chrysothamnus reticulate third microseris Entirely black Complete Eyelonger F2 much Reticulate Incomplete, Present © Conspicuous, longer with fine distinct only on stems of intermediate in posterior Microseris striae third pisum Mostly Incomplete, Malar = F2 much Entirely Incomplete, Absent Conspicuous, reddish-brown reaching space longer reticulate —_—_ distinct only on stems of halfway longer in posterior Lygodesmia to eye third Cryptic or underworked? Antistrophus rufus complex 409 Figure 3. Diagnostic characters of the Antistrophus rufus complex A head, anterior view, A. meganae female (PSUC_FEM_247320); frs = facial radiating striae B mesopleuron, lateral view, A. laurenae female (PSUC_FEM_248174) C mesoscutum, dorsal view, A. rufus female (PSUC_FEM_253129); not = no- tauli; arrow indicates end of distinct portion D fore wing, A. minor female (PSUC_FEM_253176). Remarks. The Antistrophus rufus complex includes all species of Antistrophus known to induce inconspicuous galls in stems of Si/phium species. While other spe- cies of Antistrophus induce galls on Silphium, they induce perceptible galls on the apical stems or in the flowerheads (Gillette 1891; Nastasi and Deans 2021) and are generally distinct morphologically (Table 2). Other species of Antistrophus induce galls on different host genera. A complete revision of Antistrophus and other North American Aulacideini is being undertaken by the authors (Nastasi et al., unpub- lished data). Antistrophus jeanae Tooker & Hanks, 2004 Fig. 4 Antistrophus jeanae Tooker & Hanks, 2004 in Tooker et al. 2004: 132. 9, J (type locality: Buckley Railroad Prairie, Iroquois Co., Illinois, USA). Material examined. Holotype (deposited at INHS). USA ¢ 9; Illinois, Iroquois Coun- ty, Buckley Railroad Prairie; 40°34.88'N, 88°02.70'W; J. Tooker leg.; reared from stem of Silphium perfoliatum in June 2000; INHS Insect Collection 25845. 410 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) Figure 4. Anztistrophus jeanae Tooker & Hanks, 2004 A female lateral habitus (PSUC_FEM_248169) B female proximal antennomeres (PSUC_FEM_248395) C female dorsal head (PSUC_FEM_247240) D female dorsal mesosoma (PSUC_FEM_247240) E female mesoscutum (PSUC_FEM_247240); not = notaulus F female dorsal scutellum (PSUC_FEM_247240); scf = mesoscutellar fovea G male lateral habitus (PSUC_FEM_248416) H gallss and larvae of A. jeanae in a stem of Silphium perfoliatum L. Cryptic or underworked? Antistrophus rufus complex 411 Paratypes (14 9 and 8 @). Deposited at INHS: USA * 5 2 and 2 3; same data as for holotype; INHS Insect Collection 52846-52852 ¢ 4 9 and 3 6; Illinois, Ford County, Paxton Railroad Prairie; 40°26.17'N, 88°06.36'W; J. Tooker leg.; reared from stems of Silphium perfoliatum, emerging in June 2000; INHS Insect Collection 52853-52859. Deposited at USNM: USA ¢ 2 9 and 1 C; same data as for holotype; USNMENT 01790168-01790170 © 3 Q and 2 @; Illinois, Ford County, Paxton Railroad Prairie; 40°26.17'N, 88°06.36'W; J. Tooker leg.; reared from stems of Si/phium perfoliatum, emerging in June 2000; USNMENT 00961127; 01790165-01790167; 01790171. Other material (10 2 and 17 4). Deposited at PSUC: USA * 3 9 and 1 <4; IL linois, Champaign County, Mahomet, Buffalo Trace Prairie; 40.208, -88.411; galled plant material collected 11 Nov 2020; JF Tooker and AR Deans leg.; reared from stems of Silphium perfoliatum, emerging in May or June 2021; PSUC_FEM_248395; 248398; 248413; 248423 © 4 9 and 3 CQ; Indiana, Parke County; 39.661, -87.371; galled plant material collected 12 Nov 2020; JF Tooker and AR Deans leg.; reared from stems of Silphium perfoliatum, emerging in May or June 2021; PSUC_FEM_248169-— 248170; 248396-248397; 248401—-248403 © 2 9 and 3 3; Iowa, Winneshiek Coun- ty, Plymouth Rock Prairie; 43.437, -92.005; galled plant material collected 19 Nov 2020; MJ Hatfield leg.; reared from stems of Silphium perfoliatum, emerging in May 2021; PSUC_FEM_ 248406; 248416; 248418; 248420; 248422. Deposited at WIRC: 4 3G; Wisconsin, Dane County, Anthony Branch FA.; 42.896072, -89.340018; galled plant material collected 4 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium perfoliatum, emerging in Apr 2012; WIRC 00171031—00171032; 00171035—00171036 © 2 4; Wisconsin, Dane County, Badger Pr. Park - North; galled plant material collected 3 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium perfoliatum, emerging in Apr 2012; WIRC 00171041; 00171043 ¢ 1 3; Wisconsin, Dane County, E-way at Mooreland; galled plant mate- rial collected 4 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium per- foliatum, emerging in Apr 2012; WIRC 00171047 © 1 9; Wisconsin, Iowa County, Noll Valley; galled plant material collected 4 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium perfoliatum, emerging in Apr 2012; WIRC 00170518 ¢ 3 3; Wisconsin, lowa County, Pr. Grove Rd at stream; galled plant material collected 5 Apr 2012; DNR Study SSGB leg.; reared from stems of Si/phium perfoliatum, emerging in Apr 2012; WIRC 00171027—-00171029. Diagnosis. A. jeanae is most similar to A. laurenae sp. nov. but is best distinguished by the dimensions of F2 in females, which in A. jeanae is 3.8x as long as wide (Fig. 4B) but 3.3x as long as wide in A. daurenae (Fig. 5B). The sculpture of the mesoscutel- lar disc (Fig. 4F) is also useful; the mesoscutellar disc is more or less rugose-reticulate throughout in A. jeanae but only has rugose-reticulate sculpture toward the outer mar- gins in A. laurenae. Description. Female (Fig. 4A)—Body length: 1.9-3.2 mm (X = 2.7 mm; n = 25; holotype = 2.6 mm). Color: Antenna color: red brown throughout, at most slightly darker distally than proximally. Head color: vertex and occiput dark red brown, mandi- 412 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) bles red brown basally to darker red brown apically, rest of head red brown throughout. Mesosoma color: pronotum and propodeum red brown laterally to dark red brown medially, mesoscutum dark red brown, scutellum dark red brown, and mesopleuron dark red brown dorsally and ventrally but red brown medially. Wing membrane color: hyaline throughout. Wing vein color: light brown. Leg color: red brown throughout, except for apical tarsomere which is conspicuously darker. Metasoma color: red brown to dark red brown. Antennae (Fig. 4B): Antennomere count: 13. Fl length: 2.4x as long as wide. F2 length: 3.8x as long as wide. F2:F1 length ratio: 1.5. Placodeal sen- silla on F2: absent; sensilla present only on F3 and following antennomeres. Head (Fig. 4C): Upper frons sculpture: reticulate. Gena posterolateral projection in anterior view: distinctly projecting past compound eyes. Facial radiating striae: distinct and complete, reaching compound eyes. Supraclypeal area sculpture: reticulate. Supracl- ypeal area projection: slightly projecting. OOL vs POL: OOL distinctly longer. OOL vs LOL: OOL greater than twice LOL. POL vs LOL: POL greater than twice LOL. LOL vs DLO: LOL longer. Vertex sculpture: reticulate throughout. Clypeus sculpture: reticulate. Mesosoma (Figs 4D-F): Pronotum pilosity: densely pilose along anterior margin and with only sparse setae elsewhere. Pronotum excluding pronotal plate sculp- ture: reticulate. Pronotal plate sculpture: reticulate. Mesopleuron excluding speculum sculpture: reticulate with fine intermediate striae. Speculum sculpture: reticulate. Mesopleuron pilosity: ventral margin and mesopleural triangle densely pilose and bare elsewhere. Mesoscutum pilosity: sparsely pilose. Mesoscutum sculpture: reticulate. Ap- parent length of anterior parallel lines: reaching one third across mesoscutum. Mor- phology of anterior parallel lines: narrow, distinct throughout perceptible length. Ap- parent length of parapsidal grooves: reaching halfway across mesoscutum. Morphology of parapsidal grooves: narrow, distinct throughout perceptible length. Morphology of median mesoscutal impression: apparent as a shallow impression extending across most of mesoscutum. Notauli completeness: incomplete, distinct posteriorly to indistinct in anterior third. Morphology of notauli: appearing as wide indentations with sloping edges throughout distinct portions. Metapleural sulcus: meeting posterior mesopleu- ron at about one third of its height. Lateral propodeal carinae: distinct and subparallel. Metapleuron sculpture: reticulate. Mesoscutellar foveae distinction: distinct, relatively deep anteriorly to shallower and inconspicuously delimited posteriorly. Mesoscutel- lar foveae sculpture: reticulate. Mesoscutellar disc sculpture: usually rugose-reticulate throughout; primarily reticulate, but usually with distinct rugose sculpture apparent throughout most of mesoscutellar disc. Mesoscutellar foveae length: reaching about one third across mesoscutellar disc. Mesoscutellar foveae shape: subquadrate, about as wide as long, separated by a narrow linear carina. Mesoscutellar disc shape: subcircular, about as wide as long. Wings: Marginal cell length: 3.2x as long as wide. Fore wing distal fringe of marginal setae: absent. Metasoma: Punctation of metasomal terga: T3 punctate only in posterior third and with T4 and following punctate throughout. Male (Fig. 4G)—Same as female except for the following: Body length: 1.8- 2.6 mm (x = 2.1 mm; n = 25). Antennae: Antennomere count: 14. F1 length: 2.4x as long as wide. F2 length: 2.9x as long as wide. F2:F1 length ratio: 1.4. Placodeal sensilla Cryptic or underworked? Antistrophus rufus complex 413 on F2: present. Wings: Fore wing distal fringe of marginal setae: present. Metasoma: Metasoma size: conspicuously smaller than in female. Biology. A. jeanae induces inconspicuous, externally imperceptible galls in stems of Silphium perfoliatum (Fig. 4H) (Tooker et al. 2004; Nastasi and Deans 2021). Distribution. Tooker et al. (2004) reported this species only from several prairie sites in Illinois (USA). Nastasi and Deans (2021) did not report additional localities. However, specimens we examined revealed records from three additional state records: Indiana, Iowa, and Wisconsin. A verifiable iNaturalist observation (https://www.inatu- ralist.org/observations/114414672) also records this species from Ohio; we have since examined adult gall wasps reared from the plant material from the same site, which confirmed their suspected identity (in litt.). Lastly, a specimen identified during this study (USNMENT 01822302; see complete specimen data in Suppl. material 1: table 1) confirms the occurrence of this species in Missouri (Columbia, Boone County) (see discussion). Known and potential distribution are summarized in Fig. 9. Antistrophus laurenae Nastasi, sp. nov. https://zoobank.org/3EA96644-BC93-4B03-908C-C62BEAABDCDF Fig. 5 Material examined. Holotype (deposited at PSUC). USA ¢ 9; Illinois, Ford and Iro- quois Counties, Paxton Railroad Prairie; 40.359, -88.106; galled plant material col- lected 11 November 2020; JF Tooker and AR Deans leg.; reared from stems of Si/- phium integrifolium, emerging in May or June 2021; PSUC_FEM_248174. Paratypes (24 2 and 25 4). Deposited at PSUC: USA * 2 3; same data as for holotype; PSUC_FEM_248338—248339 ¢ 1 @ and 4 4; Illinois, Iroquois County, Loda Cemetery Prairie; 40.528, -88.074; galled plant material collected 10 Novem- ber 2020; JF Tooker and AR Deans leg.; reared from stems of Silphium integrifolium, emerging in May or June 2021; PSUC_FEM_248173 5 9 and 4 CG: Illinois, McLean County, Weston Cemetery Prairie; 40.725, -88.606; galled plant material collected 11 November 2020; JF Tooker and AR Deans leg.; reared from stems of Silphium inte- grifolium, emerging in May or June 2021; PSUC_FEM_248178; 248325—248327; 248329-248330; 248333; 248336 © 2 9 and 2 G; Iowa, Story County, Grant, inter- section of I-35 and E 13" St. prairie restoration; galled plant material collected 28 No- vember 2020; MJ Hatfield leg.; reared from stems of Silphium integrifolium, emerging in June 2021; PSUC_FEM_248176; 248331; 248334; 248337. Deposited at USNM: USA ¢ 1 Q; Illinois, McLean County, Weston Cemetery Prairie; 40.725, -88.606; galled plant material collected 11 November 2020; JF Tooker and AR Deans leg.; reared from stems of Si/phium integrifolium, emerging in May or June 2021; PSUC_FEM_248332 © 1 9 and 2 @; same data as for holotype; PSUC_ FEM_ 248175; 248335; 248340. Deposited at WIRC: 2 9 and 1 ; Iowa, Delaware County, Dyersville West; 42.48564, -91.15425; galled plant material collected 2 May 2009; DNR Study SSGB 414 Louis E Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) Figure 5. Antistrophus laurenae Nastasi, sp. nov. A paratype female lateral habitus (PSUC_FEM_248173) B holotype female proximal antennomeres (PSUC_FEM_248174) C holotype female dorsal head (PSUC_FEM_248174) D holotype female dorsal mesosoma E holotype female dorsal mesoscutum; not = notaulus G holotype female dorsal scutellum; scf = mesoscutellar fovea. Paratype male lateral habi- tus (PSUC_FEM_248334) H galls and larva of A. laurenae in a stem of Silphium integrifolium Michx. Cryptic or underworked? Antistrophus rufus complex 415 leg.; reared from stems of Silphium integrifolium, emerging in May 2009; WIRC 00171299-00171301 ¢ 2 9 and 1 3; Iowa, Delaware County, Dyersville West Prai- rie; 42.47330, -91.138372; galled plant material collected 2 May 2009; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in May 2009; WIRC 00171267—00171268; 00171283 * 1 9 and 2 3; Iowa, Marshall County, Twinkle Hill Restoration; 42.064396, -92.854943; galled plant material collected 2 May 2009; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in May 2009; WIRC 00171284; 00171306-00171307 * 1 9; Wisconsin, Colum- bia County, Goose Pond Jill’s Pr.; galled plant material collected 3 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in Apr 2012; WIRC 00171314 © 1 3; Wisconsin, Dane County, Anthony Branch F.A.; 42.896072, -89.340018; galled plant material collected 4 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in Apr 2012; WIRC 00171322 ¢ 2 9 and 2 3; Wisconsin, Dane County, Badger Pr. Park - North; 42.896072, -89.340018; galled plant material collected 3 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in Apr 2012; WIRC 00170523; 00170837; 00171315; 00171331 © 1 9; Wisconsin, Dane County, Black Earth Prairie; 43.140146, -89.773011; galled plant material collected 5 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in Apr 2012; WIRC 00170838 ¢ 1 9 and 1 @; Wisconsin, Dane County, E-way at Mooreland; 43.030588, -89.349114; galled plant material collected 4 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in Apr 2012; WIRC 00170524; 00171329 1 9 and 1 3; Wisconsin, Dane County, Festge Roadside; 43.118267, -89.683665; galled plant material collected 18 Apr 2009; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in May 2009; WIRC 00171227; 00171289 ¢ 1 3; Wisconsin, Dane County, Garfoot Road; galled plant material collected 5 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in Apr 2012; WIRC 00171337 © 2 QO and 2 4; Wisconsin, Dane County, Gov Nel- son 1997; 43.137321, -89.437257; galled plant material collected 3 Apr 2012; DNR Study SSGB leg.; reared from stems of Silphium integrifolium, emerging in Apr 2012; WIRC 00170839; 00171303; 00171330; 00171341 ¢ 2 9 and 1 4; Wisconsin, Dane County, TNC Waubesa Wetland; galled plant material collected 22 Nov 2008; DNR Study SSGB leg.; reared from stems of Si/phium integrifolium, emerging in May 2009; WIRC 00171271; 00171274; 00171286 * 1 4; Wisconsin, Iowa County, TNC/ Lease; 42.97357, -89.88900; galled plant material collected 17 Apr 2009; DNR Study SSGB leg.; reared from stems of Si/phium integrifolium, emerging in May 2009; WIRC 00171287 * 1 3; Wisconsin, Iowa County, Underwood Prairie; galled plant material collected 9 May 2009; DNR Study SSGB leg.; reared from stems of Si/phium integri- folium, emerging in May 2009; WIRC 00171298. Diagnosis. See diagnosis to Antistrophus jeanae Tooker & Hanks, 2004. Description. Female (Fig. 5A)—Body length: 1.8—2.6 mm (x = 2.2 mm; n = 25; holotype = 2.6 mm). Color: Antenna color: red brown throughout, at most slightly darker distally than proximally. Head color: vertex and occiput dark red brown, mandi- 416 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) bles red brown basally to darker red brown apically, rest of head red brown throughout. Mesosoma color: pronotum and propodeum red brown laterally to dark red brown medially, mesoscutum dark red brown, scutellum dark red brown, and mesopleuron dark red brown dorsally and ventrally but red brown medially. Wing membrane color: hyaline throughout. Wing vein color: light brown. Leg color: red brown throughout, except for apical tarsomere which is conspicuously darker. Metasoma color: red brown to dark red brown. Antennae (Fig. 5B): Antennomere count: 13. F1 length: 2.2x as long as wide. F2 length: 3.3x as long as wide. F2:F1 length ratio: 1.4. Placodeal sensilla on F2: absent; sensilla present only on F3 and following antennomeres. Head (Fig. 5C): Upper frons sculpture: reticulate. Gena posterolateral projection in ante- rior view: distinctly projecting past compound eyes. Facial radiating striae: distinct and complete, reaching compound eyes. Supraclypeal area sculpture: reticulate. Su- praclypeal area projection: slightly projecting. OOL vs POL: OOL distinctly longer. OOL vs LOL: OOL greater than twice LOL. POL vs LOL: POL greater than twice LOL. LOL vs DLO: LOL longer. Vertex sculpture: reticulate throughout. Clypeus sculpture: reticulate. Mesosoma (Figs 5D-—F): Pronotum pilosity: densely pilose along anterior margin and with only sparse setae elsewhere. Pronotum excluding pronotal plate sculpture: reticulate. Pronotal plate sculpture: reticulate. Mesopleuron excluding speculum sculpture: reticulate with fine intermediate striae. Speculum sculpture: re- ticulate. Mesopleuron pilosity: ventral margin and mesopleural triangle densely pilose and bare elsewhere. Mesoscutum pilosity: sparsely pilose. Mesoscutum sculpture: retic- ulate. Apparent length of anterior parallel lines: reaching one third across mesoscutum. Morphology of anterior parallel lines: narrow, distinct throughout perceptible length. Apparent length of parapsidal grooves: reaching halfway across mesoscutum. Morphol- ogy of parapsidal grooves: narrow, distinct throughout perceptible length. Morphol- ogy of median mesoscutal impression: apparent as a shallow impression extending across most of mesoscutum. Notauli completeness: incomplete, distinct posteriorly to indistinct in anterior third. Morphology of notauli: appearing as wide indentations with sloping edges throughout distinct portions. Metapleural sulcus: meeting poste- rior mesopleuron at about one third of its height. Lateral propodeal carinae: distinct and subparallel. Metapleuron sculpture: reticulate. Mesoscutellar foveae distinction: distinct, relatively deep anteriorly to shallower and inconspicuously delimited poste- riorly. Mesoscutellar foveae sculpture: reticulate. Mesoscutellar disc sculpture: rugose- reticulate, primarily reticulate, but with distinct rugose-reticulate sculpture seemingly restricted to outer margins. Mesoscutellar foveae length: long, reaching about one third across mesoscutellar disc. Mesoscutellar foveae shape: subquadrate, about as wide as long, separated by a narrow linear carina. Mesoscutellar disc shape: subcircular, about as wide as long. Wings: Marginal cell length: 3.1x as long as wide. Fore wing distal fringe of marginal setae: absent. Metasoma: Punctation of metasomal terga: T3 punc- tate only in posterior third and with T4 and following punctate throughout. Male (Fig. 5G)—Same as female except for the following: Body length: 1.3- 2.1 mm (x = 1.8; n = 25). Antennae: Antennomere count: 14. F1 length: 2.3x as long as wide. F2 length: 3.2x as long as wide. F2:F1 length ratio: 1.5. Placodeal sensilla Cryptic or underworked? Antistrophus rufus complex 417 on F2: present. Wings: Fore wing distal fringe of marginal setae: present. Metasoma: Metasoma size: conspicuously smaller than in female. Etymology. Named for Lauren Ahlert, a biology teacher at Wayne Valley High School in Wayne, New Jersey, USA, who has served as a tremendous source of inspira- tion and passion for the author of this species. Biology. Antistrophus laurenae induces inconspicuous, externally imperceptible galls in stems of Silphium integrifolium (Fig. 5H). Distribution. Material examined in this study reveals that this species occurs in Illinois, lowa, and Wisconsin (USA; see Suppl. material 1: table 1). A verified iNatu- ralist record (https://www.inaturalist.org/observations/136446787) also suggests that this species occurs in Mississippi. Known and potential distribution are summarized in Fig. 9. Antistrophus meganae Tooker & Hanks, 2004 Fig. 6 Antistrophus meganae Tooker & Hanks, 2004 in Tooker et al. 2004: 132. 9, J (type locality: East St. Joseph Railroad Prairie, Iroquois Co., Illinois, USA). Material examined. Holotype (deposited at INHS). USA ¢ 9; Illinois, Champaign County, St. Joseph, roadside prairie; J. Tooker leg.; reared from stem of Si/phium ter- ebinthinaceum, emerging in June 2000; INHS Insect Collection 52830. Paratypes (14 9 and 8 4). Deposited at INHS: USA * 8 2 and 4 3; same data as for holotype; INHS Insect Collection 52831-52837 * 4 9 and 3 6; Illinois, Ford County, Paxton, Paxton Railroad Prairie; J. Tooker leg.; reared from stem of Silphium terebinthinaceum, emerging in June 2000; INHS Insect Collection 52838-52844. Deposited at USNM: USA ¢ 2 9 and 1 G; Illinois, Ford County, Paxton, Paxton Railroad Prairie; J. Tooker leg.; reared from stem of Silphium terebinthinaceum, emerg- ing in June 2000; USNMENT 01790188-01790190. Other material (10 2 and 17 3). Deposited at PSUC: USA * 1 Q; Illinois, Champaign County, St. Joseph, roadside prairie; JF Tooker and AR Deans leg.; 40.113, -88.064; galled plant material collected 10 Nov 2020; reared from stem of Si/phium terebinthinaceum, emerging in June 2021; PSUC_FEM_248429 ¢ 1 @ and 1 @; IL linois, McLean County, Chenoa, Weston Cemetery Prairie; 40.725, -88.606; galled plant material collected 11 Nov 2020; reared from stem of Silphium terebinthinaceum, emerging in June 2021; PSUC_FEM_248165-248166. Deposited at WIRC: USA ¢ 2 2; Wisconsin, Columbia County, Mass Rd.; galled plant material collected Fall 2008; reared from stem of Silphium terebinthinaceum, emerging in June 2009; WIRC 00171156; 00171166 * 2 9 and 2 4; Wisconsin, Dane County, Co A and Oak Ridge (Anthony Branch EA.); 42.892454, -89.320011; galled plant material collected 15 Apr 2009; reared from stem of Silphium terebinthinaceum, emerging in June 2009; WIRC 00170514; 00171140; 00171154—-00171155 « 1 3; 418 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) 1 = y : .: SUN \ = < Ww @ 5 ws ~S She : : Figure 6. Antistrophus meganae Tooker & Hanks, 2004 A female lateral habitus (PSUC_FEM_248165) B female proximal antennomeres (PSUC_FEM_247325) C female dorsal head (PSUC_FEM_247325) D female dorsal mesosoma (PSUC_FEM_247230) E female dorsal mesoscutum (PSUC_FEM_ 247230); not = notaulus F female dorsal scutellum (PSUC_FEM_247230); scf = mesoscutellar fovea G male lateral habitus (PSUC_FEM_248498) H gallss and larvae of A. meganae in a stem of Silphium terebinthinaceum Jacq. Cryptic or underworked? Antistrophus rufus complex 419 Wisconsin, Dane County, Kelly Road and RR, roadside; galled plant material collected Fall 2008; reared from stem of Silphium terebinthinaceum, emerging in July 2009; WIRC 00170513 © 1 3; Wisconsin, Dane County, Malone Road; galled plant mate- rial collected 17 Apr 2009; reared from stem of Silphium terebinthinaceum, emerging in June 2009; WIRC 00171112 © 1 4; Wisconsin, Dane County, Noll Valley; galled plant material collected 5 Apr 2012; reared from stem of Si/phium terebinthinaceum, emerging in June 2012; WIRC 00171114 1 &; Wisconsin, Dane County, Prairie Ridge City Park; galled plant material collected 3 Apr 2012; reared from stem of Si/- phium terebinthinaceum, emerging in May 2012; WIRC 00171124 © 1 9 and 1 3; Wisconsin, Dane County, Sugar Ridge Savanna; galled plant material collected 20 Mar 2012; reared from stem of Silphium terebinthinaceum, emerging in May 2012; WIRC 00171122; 00171134 © 1 3; Wisconsin, Dane County, TNC Waubesa Wet- land; galled plant material collected 22 Nov 2008; reared from stem of Silphium ter- ebinthinaceum, emerging in May 2009; WIRC 00170515 e 1 3; Wisconsin, Jefferson County, Bluejoint Prairie; 43.163398, -88.938815; galled plant material collected 16 Apr 2009; reared from stem of Silphium terebinthinaceum, emerging in July 2009; WIRC 00170517 * 1 3; Wisconsin, Jefferson County, Bluejoint Prairie; 43.163398, -88.938815; galled plant material collected 16 Apr 2009; reared from stem of Si/phium terebinthinaceum, emerging in May 2009; WIRC 00170512 * 1 9 and 2 &; Wiscon- sin, Jefferson County, Cold Spring Prairie; 42.872556, -88.770194; galled plant mate- rial collected 15 Apr 2009; reared from stem of Silphium terebinthinaceum, emerging in June 2009; WIRC 00171109; 00171137; 00171141 * 3 4; Wisconsin, Walworth County, Skoponong Prairie; 42.829155, -88.620586; galled plant material collected 4 Oct 2011; reared from stem of Silphium terebinthinaceum, emerging in May or June 2012; WIRC 00170749; 00171113; 00171120 © 1 2; Wisconsin, Walworth Coun- ty, Young Pr. East Annex; 42.839419, -88.63002; galled plant material collected 16 Apr 2009; reared from stem of Silphium terebinthinaceum, emerging in June 2009; WIRC 00170750 © 1 & and 2 3; Wisconsin, Winnebago County, Oshkosh-Larsen Trl mid (B); 44.13912, -88.624272; galled plant material collected 10 Oct 2011; reared from stem of Silphium terebinthinaceum, emerging in June 2012; WIRC 00171115- 00171116; 00171118. Diagnosis. A. meganae is the only species of the rufus complex in which the mes- oscutellar foveae (Fig. GF) are long and ovate, reaching nearly halfway across the mes- oscutellar disc. A. meganae also differs from most other members of the rufus complex by the POL (Fig. 6C), which is longer than the OOL in A. meganae and A. minor but shorter than the OOL in the other species. Description. Female (Fig. G6A)—Body length: 1.8-3.2 mm (X = 2.5 mm; n = 25; holotype = 2.3 mm). Color: Antenna color: red brown throughout, at most slight- ly darker distally than proximally. Head color: vertex and occiput dark red brown, mandibles red brown basally to darker red brown apically, rest of head red brown throughout. Mesosoma color: pronotum and propodeum red brown laterally to dark red brown medially, mesoscutum dark red brown with distinct posterolateral red brown spots, scutellum dark red brown, and mesopleuron dark red brown dorsally 420 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) and ventrally but red brown medially. Wing membrane color: hyaline throughout. Wing vein color: light brown. Leg color: red brown throughout, except for apical tar- somere which is conspicuously darker. Metasoma color: red brown to dark red brown. Antennae (Fig. 6B): Antennomere count: 13. Fl length: 2.6x as long as wide. F2 length: 3.6x as long as wide. F2:F1 length ratio: 1.4. Placodeal sensilla on F2: absent; sensilla present only on F3 and following antennomeres. Head (Fig. GC): Upper frons sculpture: reticulate. Gena posterolateral projection in anterior view: distinctly pro- jecting past compound eyes. Facial radiating striae: distinct and complete, reaching compound eyes. Supraclypeal area sculpture: reticulate. Supraclypeal area projection: slightly projecting. OOL vs POL: POL distinctly longer. OOL vs LOL: OOL less than twice LOL. POL vs LOL: POL twice LOL. LOL vs DLO: LOL longer. Ver- tex sculpture: reticulate throughout. Clypeus sculpture: reticulate. Mesosoma (Figs 6D-F): Pronotum pilosity: densely pilose along anterior margin and with only sparse setae elsewhere. Pronotum excluding pronotal plate sculpture: reticulate. Pronotal plate sculpture: reticulate. Mesopleuron excluding speculum sculpture: reticulate with fine intermediate striae. Speculum sculpture: reticulate. Mesopleuron pilosity: ventral margin and mesopleural triangle densely pilose and bare elsewhere. Mesoscutum pi- losity: sparsely pilose. Mesoscutum sculpture: reticulate. Apparent length of anterior parallel lines: reaching one third across mesoscutum. Morphology of anterior parallel lines: narrow, distinct throughout perceptible length. Apparent length of parapsidal grooves: reaching halfway across mesoscutum. Morphology of parapsidal grooves: narrow, distinct throughout perceptible length. Morphology of median mesoscutal impression: apparent as a shallow impression extending across most of mesoscutum. Notauli completeness: incomplete, distinct posteriorly to indistinct in anterior third. Morphology of notauli: appearing as wide indentations with sloping edges through- out distinct portions. Metapleural sulcus: meeting posterior mesopleuron at about one third of its height. Lateral propodeal carinae: distinct and subparallel. Metapleu- ron sculpture: reticulate. Mesoscutellar foveae distinction: distinct, relatively deep anteriorly to shallower and inconspicuously delimited posteriorly. Mesoscutellar fo- veae sculpture: reticulate. Mesoscutellar disc sculpture: rugose-reticulate, primarily reticulate, but with distinct rugose-reticulate sculpture seemingly restricted to outer margins. Mesoscutellar foveae length: long, reaching nearly halfway across mesoscu- tellar disc. Mesoscutellar foveae shape: ovate, slightly longer than wide, separated by a subtriangular carina slightly wider posteriorly than anteriorly. Mesoscutellar disc shape: subcircular, about as wide as long. Wings: Marginal cell length: 3.3x as long as wide. Fore wing distal fringe of marginal setae: absent. Metasoma: Punctation of metasomal terga: [3 punctate only in posterior third and with T4 and following punctate throughout. Male (Fig. GG)—Same as female except for the following: Body length: 1.3—2.5 mm (x = 2.1 mm; n = 25). Antennae: Antennomere count: 14. F1 length: 2.5x as long as wide. F2 length: 3.0x as long as wide. F2:F1 length ratio: 1.4 (as in female). Placo- deal sensilla on F2: present. Wings: Fore wing distal fringe of marginal setae: present. Metasoma: Metasoma size: conspicuously smaller than in female. Cryptic or underworked? Antistrophus rufus complex 421 Biology. Antistrophus meganae induces inconspicuous, externally imperceptible galls in stems of Silphium terebinthinaceum (Fig. 6H) (Tooker et al. 2004; Nastasi and Deans 2021). Distribution. Tooker et al. (2004) reported this species only from several prairie sites in Illinois (USA). Nastasi and Deans (2021) did not report additional localities; however, the specimens we examined revealed novel records from Wisconsin (Suppl. material 1: table 1). We also sequenced DNA barcodes for individuals from Ohio (Suppl. material 1: table 6), and a series of specimens identified using the key repre- sents a new state record from Michigan (see Discussion; Suppl. material 1: table 1). Known and potential distribution are summarized in Fig. 9. Antistrophus minor Gillette, 1891 Fig. 7, Antistrophus minor Gillette, 1891: 195. 9, J (type locality: unknown location in II- linois, USA). Aulax gillettei (Gillette, 1891): Kieffer 1902: 93. Material examined. Lectotype (deposited at INHS; designated by Frison [1927]). USA ¢ Q; Illinois; reared from stem of Silphium laciniatum; record number 5500; INHS Insect Collection 212949. Lectoallotype (deposited at INHS; designated by Frison [1927]). USA * 3; same data as lectotype; INHS Insect Collection 212950. Other material (24 2 and 24 3). Deposited at PSUC: USA * 3 Q; Illinois, Champaign County, Gifford, Shortline Railroad Prairie; 40.305, -87.999; JF Took- er and AR Deans leg.; galled plant material collected 10-11 Nov 2020; reared from stem of Silphium laciniatum, emerging in May or June 2021; PSUC_FEM_248257; 253063; 253067 * 2 Q; Illinois, Iroquois County, Loda, Loda Cemetery Prairie; 40.528, -88.074; JF Tooker and AR Deans leg.; galled plant material collected 10 Nov 2020; reared from stem of Silphium laciniatum, emerging in June 2021; PSUC_ FEM_248273; 253060 © 2 9 and 1 Q; Illinois, McLean County, Chenoa, Weston Cemetery Prairie; 40.725, -88.606; JF Tooker and AR Deans leg.; galled plant material collected 11 Nov 2020; reared from stem of Silphium laciniatum, emerging in May or June 2021; PSUC_FEM_248278; 248283; 248288; 248303 * 3 9 and 3 6; Illinois, McLean County, Chenoa, Weston Cemetery Prairie; 40.725, -88.606; LF Nastasi and AR Casadei leg.; galled plant material collected 16 Oct 2021; reared from stem of Si/- phium laciniatum, emerging in June 2022; PSUC_FEM_253166—253168; 253170; 253180; 253192 © 1 9 and 2 &; Iowa, Story County, Grant, Interstate 35 and E 13" St prairie restoration; MJ Hatfield leg.; galled plant material collected 30 Nov 2020; reared from stem of Silphium laciniatum, emerging in May or June 2021; PSUC_ FEM_248265; 248291; 248295 ¢ 1 4; Iowa, Winneshiek County, Decorah Commu- nity Prairie; MJ Hatfield leg.; galled plant material collected 18 Apr 2022; reared from 422 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) e 1mm Figure 7. Anztistrophus minor Gillette, 1891 A female lateral habitus (PSUC_FEM_248273) B female proximal antennomeres (PSUC_FEM_253176) C female dorsal head (PSUC_FEM_253091) D female dorsal mesosoma (PSUC_FEM_ 248257) E female dorsal mesoscutum (PSUC_FEM_ 248257); not = no- taulus F female dorsal scutellum (PSUC_FEM_248257); scf = mesoscutellar fovea G male lateral habitus (PSUC_FEM_253191) H gallss and larvae of A. minor or A. rufus in a stem of Silphium laciniatum L. Cryptic or underworked? Antistrophus rufus complex 423 stem of Silphium laciniatum, emerging in May or June 2022; PSUC_FEM_253229 ¢ 3 QO and 2 @; Iowa, Winneshiek County, Plymouth Rock Prairie; 43.438, -92.006; MJ Hatfield leg.; galled plant material collected 17 Apr 2016; reared from stem of Silphium laciniatum, emerging in May 2016; PSUC_FEM_253085; 253088; 253089; 253090; 253094. Deposited at USNM: USA ¢ 1 9; Illinois, McLean County, Chenoa, Weston Cemetery Prairie; 40.725, -88.606; JF Tooker and AR Deans leg.; galled plant mate- rial collected 11 Nov 2020; reared from stem of Silphium laciniatum, emerging in May or June 2021; PSUC_FEM_248286 ¢ 1 Q; Illinois, McLean County, Chenoa, Weston Cemetery Prairie; 40.725, -88.606; JF Tooker and AR Deans leg.; galled plant material collected 11 Nov 2020; reared from stem of Silphium laciniatum, emerging in May or June 2021; PSUC_FEM_248303 ¢ 3 ©; Illinois, McLean County, Chenoa, Weston Cemetery Prairie; 40.725, -88.606; LF Nastasi and AR Casadei leg.; galled plant ma- terial collected 16 Oct 2021; reared from stem of Silphium laciniatum, emerging in June 2022; PSUC_FEM_253190; 253191; 253194 © 1 Q; Illinois, McLean County, Chenoa, Weston Cemetery Prairie; 40.725, -88.606; LF Nastasi and AR Casadei leg.; galled plant material collected 16 Oct 2021; reared from stem of Silphium laciniatum, emerging in June 2022; PSUC_FEM_253165. Deposited at WIRC: USA * 1 9; Iowa, Blackhawk County, Hudson Railroad Prairie; 42.436349, -92.427034; DNR Study SSGB leg.; galled plant material col- lected 2 May 2009; reared from stem of Silphium laciniatum, emerging in June 2009; WIRC 00170819 © 1 Q; Iowa, Linn County, Walford 2; 41.883014, -91.825962; DNR Study SSGB leg.; galled plant material collected 3 May 2009; reared from stem of Silphium laciniatum, emerging in June 2009; WIRC 00170772 1 ; Wisconsin, Columbia County, Goose Pd. Sue Ames; DNR Study SSGB leg.; galled plant material collected Fall 2008; reared from stem of Si/phium laciniatum, emerging in July 2009; WIRC 00170816 * 1 9; Wisconsin, Columbia County, Hopkins Restoration; DNR Study SSGB leg.; galled plant material collected Fall 2008; reared from stem of Si/- phium laciniatum, emerging in July 2009; WIRC 00170661 ¢ 1 4; Wisconsin, Dane County, Badger Pr. Park North; DNR Study SSGB leg.; galled plant material col- lected 3 Apr 2012; reared from stem of Silphium laciniatum, emerging in May 2012; WIRC 00170757 * 1 4; Wisconsin, Dane County, E-way @ Mooreland; DNR Study SSGB leg.; galled plant material collected 4 Apr 2012; reared from stem of Silphium laciniatum, emerging in Apr 2012; WIRC 00170683 ¢ 2 9; Wisconsin, Dane County, Smith Drumlin; 42.98988, -89.06097; DNR Study SSGB leg.; galled plant material collected 15 Apr 2009; reared from stem of Silphium laciniatum, emerging in May 2009; WIRC 00170786; 00170788 * 2 3; Wisconsin, Dane County, Waubesa Wet- lands; WDNR Misc/SB Sauer leg.; galled plant material collected Apr 2007; reared from stem of Silphium laciniatum, emerging before 19 May 2007; WIRC 00170805; 00170807 * 2 3; Wisconsin, Grant County, Dewey Heights Prairie; 42.734647, -91.020306; DNR Study SSGB leg.; galled plant material collected 14 Oct 2011; reared from stem of Silphium laciniatum, emerging in May 2012; WIRC 00170664; 00171400 * 2 9; Wisconsin, Green County, Green Cemetery Prairie; DNR Study 424 Louis E Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) SSGB leg.; galled plant material collected 4 Apr 2012; reared from stem of Silphium laciniatum, emerging in May 2012; WIRC 00170663; 00170758 ¢ 1 4; Wisconsin, Green County, Muralt - South; 42.695505, -89.49367; DNR Study SSGB leg.; galled plant material collected 4 Apr 2012; reared from stem of Silphium laciniatum, emerg- ing in May 2012; WIRC 00170754 @ 1 &; Wisconsin, Iowa County, Hollandale Seed Orchard; DNR Study SSGB leg.; galled plant material collected 5 Apr 2012; reared from stem of Silphium laciniatum, emerging in May 2012; WIRC 00170756 * 1 <; Wisconsin, lowa County, TNC/Lease; 42.97357, -89.88900; DNR Study SSGB leg.; galled plant material collected 17 Apr 2009; reared from stem of Silphium laciniatum, emerging in May 2009; WIRC 00170678 ¢ 1 4; Wisconsin, Kenosha County, Bain Station; 42.556352, -87.893763; DNR Study SSGB leg.; galled plant material col- lected 30 Sep 2011; reared from stem of Silphium laciniatum, emerging in May 2012; WIRC 00170778 ¢ 1 9; Wisconsin, Waukesha County, Scuppernong Prairie State Natural Area; 42.89947, -88.50180; DNR Study SSGB leg.; galled plant material col- lected 15 Apr 2009; reared from stem of Silphium laciniatum, emerging in June 2009; WIRC 00170637. Diagnosis. A. minor is the only described species of Antistrophus in which the mes- oscutellar disc is strongly ovate and distinctly wider than long (Fig. 7F). Additionally, A. minor is easily separable from A. rufus (which also induces inconspicuous galls in stems of the S. laciniatum) and A. meganae by the shape and length of the mesoscutel- lar foveae; in A. minor, the mesoscutellar foveae are subquadrate and reach about one third across the mesoscutellar disc (Fig. 7F) but are shorter and subrectangular in A. ru- Jus (Fig. 8F) and longer and ovate in A. meganae (Fig. 6F). A. minor and A. meganae are also the only species of the rufus complex in which the POL is longer than the OOL. See additional comments in the diagnosis to A. rufus. Description. Female (Fig. 7A)—Body length: 1.4—2.4 mm (x = 2.0 mm; n = 25; lectotype = 2.3). Color: Antenna color: red brown throughout, at most slightly darker distally than proximally. Head color: vertex and occiput dark red brown, mandibles red brown basally to darker red brown apically, rest of head red brown throughout. Color: pronotum and propodeum mostly red brown, with some dark red brown col- oration medially, mesoscutum dark red brown with at least distinct red brown spots posterolaterally, scutellum red brown to dark red brown, and mesopleuron dark red brown to red brown. Wing membrane color: hyaline throughout. Wing vein color: light brown. Leg color: red brown throughout, except for apical tarsomere which is conspicuously darker. Metasoma color: red brown to dark red brown. Antennae (Fig. 7B): Antennomere count: 13. Fl length: 2.6x as long as wide. F2 length: 3.6x as long as wide. F2:F1 length ratio: 1.4. Placodeal sensilla on F2: absent; sensilla pre- sent only on F3 and following antennomeres. Head (Fig. 7C): Upper frons sculpture: reticulate. Gena posterolateral projection in anterior view: distinctly projecting past compound eyes. Facial radiating striae: distinct and complete, reaching compound eyes. Supraclypeal area sculpture: reticulate. Supraclypeal area projection: slightly pro- jecting. OOL vs POL: POL distinctly longer. OOL vs LOL: OOL twice LOL. POL vs LOL: POL greater than twice LOL. LOL vs DLO: LOL longer. Vertex sculpture: Cryptic or underworked? Antistrophus rufus complex 425 reticulate throughout. Clypeus sculpture: reticulate. Mesosoma (Figs 7D-F): Prono- tum pilosity: densely pilose along anterior margin and with only sparse setae elsewhere. Pronotum excluding pronotal plate sculpture: reticulate. Pronotal plate sculpture: re- ticulate. Mesopleuron excluding speculum sculpture: reticulate with fine intermedi- ate striae. Speculum sculpture: reticulate. Mesopleuron pilosity: ventral margin and mesopleural triangle densely pilose and bare elsewhere. Mesoscutum pilosity: sparsely pilose. Mesoscutum sculpture: reticulate. Apparent length of anterior parallel lines: reaching one third across mesoscutum. Morphology of anterior parallel lines: narrow, distinct throughout perceptible length. Apparent length of parapsidal grooves: reach- ing halfway across mesoscutum. Morphology of parapsidal grooves: narrow, distinct throughout perceptible length. Morphology of median mesoscutal impression: appar- ent as a shallow impression extending across most of mesoscutum. Notauli complete- ness: incomplete, distinct posteriorly to indistinct in anterior third. Morphology of no- tauli: appearing as wide indentations with sloping edges throughout distinct portions. Metapleural sulcus: meeting posterior mesopleuron at about one third of its height. Lateral propodeal carinae: distinct and subparallel. Metapleuron sculpture: reticu- late. Mesoscutellar foveae distinction: distinct, relatively deep anteriorly to shallower and inconspicuously delimited posteriorly. Mesoscutellar foveae sculpture: reticulate. Mesoscutellar disc sculpture: rugose-reticulate, primarily reticulate, but with distinct rugose-reticulate sculpture seemingly restricted to outer margins. Mesoscutellar foveae length: long, reaching about one third across mesoscutellar disc. Mesoscutellar foveae shape: subovate, slightly longer than wide, separated by a narrow linear carina. Mes- oscutellar disc shape: ovate, distinctly longer than wide. Wings: Marginal cell length: 3.1x as long as wide. Fore wing distal fringe of marginal setae: absent. Metasoma: Punctation of metasomal terga: T3 punctate only in posterior third and with T4 and following punctate throughout. Male (Fig. 7G)—Same as female except for the following: Body length: 1.1—-2.0 mm (x = 1.6; n = 25). Antennae: Antennomere count: 14. F1 length: 2.3x as long as wide. F2 length: 2.9x as long as wide. F2:F1 length ratio: 1.3. Placodeal sensilla on F2: pre- sent. Wings: Fore wing distal fringe of marginal setae: present. Metasoma: Metasoma size: conspicuously smaller than in female. Biology. Antistrophus minor induces inconspicuous, externally imperceptible galls in stems of Silphium laciniatum (Fig. 7H) (Tooker et al. 2004; Nastasi and Deans 2021). Weld (1926) reported A. minor from Silphium terebinthinaceum, but Weld’s specimens from this host plant truly represent Antistrophus meganae. Distribution. Antistrophus minor was described from adults reared alongside A. ru- Jus in Illinois. We examined specimens providing new state records from lowa and Wisconsin. Known and potential distribution are summarized in Fig. 9. It appears that A. minor is likely sympatric with A. rufus throughout the distribution of S. aciniatum, although further rearing of Si/phium species will be needed to better understand the role of geography in the distribution of Antistrophus, especially regarding these two species. Remarks. See remarks under Antistrophus rufus for notes on generic nomenclature and distinction of A. minor from A. rufus. 426 Louis F. Nastasi et al. / Journal of Hymenoptera Research 97: 399-439 (2024) Antistrophus rufus Gillette, 1891 Fig. 8 Antistrophus rufus Gillette, 1891: 195. 9, J (type locality: unknown location in II- linois, USA). Aulax rufa (Gillette, 1891): Kieffer 1902: 93. Material examined. Lectotype (deposited at INHS; designated by Frison [1927]). USA ¢ Q; Illinois; reared from stem of Silphium laciniatum; record number 5500; INHS Insect Collection 52812. Lectoallotype (deposited at INHS; designated by Frison [1927]). USA * 3; same data as lectotype; INHS Insect Collection 52813. Paralectotypes. Deposited at AMNH: USA ¢ 1 9; same data as lectotype; AMNHE_IZC 00393875. Deposited at INHS: USA * 3 & and 1 ; same data as lectotype; INHS Insect Collection 294742—294745. Deposited at USNM: USA ¢ 4 9 and 2 ; same data as lectotype; USNMENT 00961146; 01822098—01822102. Other material (16 2 and 21 3). Deposited at INHS: * 5 @ and 1