JHR 96: 1061-1072 (2023) or JOURNAL OF 4 Pereewed openscess ural doi: 10.3897/jhr.96.1 15001 RESEARCH ARTICLE ) Hymenopter a The International Society of Hymenopterists RESEARCH https://jhr.pensoft.net Limited phylogeographic structure in a flightless, Appalachian chalcidoid wasp, Dipara trilineata (Yoshimoto) (Hymenoptera, Diparidae), with reassessment of the male of the species Michael S. Caterino', Nathan C. Arey? | Department of Plant and Environmental Sciences, Clemson University, Clemson, SC 29634, USA 2. Depart- ment of Entomology and Plant Pathology, University of Tennessee, Knoxville, TN 37996, USA Corresponding author: Michael S. Caterino (mcateri@clemson.edu) Academic editor: Ankita Gupta | Received 1 November 2023 | Accepted7 December 2023 | Published 19 December 2023 Attps://zoobank.org/792C4DBB-2F49-4724-8928-201 BBE945797 Citation: Caterino MS, Arey NC (2023) Limited phylogeographic structure in a flightless, Appalachian chalcidoid wasp, Dipara trilineata (Yoshimoto) (Hymenoptera, Diparidae), with reassessment of the male of the species. Journal of Hymenoptera Research 96: 1061-1072. https://doi.org/10.3897/jhr.96.115001 Abstract Dipara trilineata (Diparidae) is a widespread eastern North American parasitoid with apterous females and winged males. Despite its seemingly limited dispersal capabilities, phylogeographic analysis over southern Appalachia reveals little structure, with only limited population level isolation. DNA barcoding surveys also definitively associate the male of the species, which had previously been misattributed, and a description of the correctly associated male is provided. Keywords aptery, megabarcoding, parasitoid, phylogeography Introduction Dipara Walker, 1833 is a member of the Diparidae, a globally distributed family of about 130 species of Chalcidoidea (Desjardins 2007), recently elevated to family status from a subfamily of Pteromalidae (Burks et al. 2022). Among North American Chalcidoidea, Dipara are unusual with females that are flightless and ant-like in morphology. ‘There is little literature on Dipara biology. For several years, Diparinae were thought solely to Copyright M. S. Caterino & N. C.Arey. 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. 1062 M.S. Caterino & N. C. Arey / Journal of Hymenoptera Research 96: 1061-1072 (2023) parasitize soil dwelling Curculionidae (Coleoptera) based on the first documented host record in 1988 (Boucek 1988). However, several species have been successfully reared from mantid egg cases and tsetse fly puparia (Desjardins 2007). The host range of Di- paridae needs further research to gain a better picture of parasitoid-host relationships. There are presently three native species of the genus Dipara known from North Amer- ica (Desjardins 2007), D. canadensis Hedqvist, 1969, D. nigriceps (Ashmead, 1904), and D. trilineata (Yoshimoto, 1977). The European D. petiolata Walker, 1833, has also apparently been introduced to the region (Garrido Torres and Nieves-Aldrey 1999; Wisniowski and Jirak-Leszczyniska 2021), though we aren't aware of specific records. Dipara trilineata is the most common species of Dipara in the eastern United States. Described from Kentucky, there are also published records from Missouri, North Caro- lina, Florida, Arkansas, Texas, Oklahoma, Tennessee, and the District of Columbia (sev- eral of these under the now synonymous Trimicrops bilineatus Yoshimoto 1977 (Yoshi- moto 1977; Boucek 1993; Desjardins 2007), and online photographic records from Louisiana and Quebec (based on identifiable photographic vouchers on BugGuide.net). This is a remarkably broad range for a species whose females are wingless and flightless. Finding the species to be abundant in leaf litter samples from the higher elevations of the southern Appalachian mountains, which function as a series of sky islands for many inhabitants (Browne and Ferree 2007; Hedin et al. 2015; Caterino and Recuero 2023), it seemed likely that D. trilineata would exhibit considerable genetic structure, and po- tentially cryptic species over its range. Using mitochondrial, barcode-region sequences from numerous southern Appalachian populations, we examine this hypothesis here. We also address a mistaken attribution of males to this species. We have associ- ated three male specimens from multiple populations unambiguously with females of Dipara trilineata through DNA barcodes, and find them to differ significantly from males originally described by Yoshimoto (1977). We rectify this error, and provide a new description of male morphology. Methods New data for this paper include 69 Dipara trilineata COI sequences, generated as part of an ‘all-arthropods’ metabarcoding study on the fauna of leaf litter in the high Appa- lachians, plus a small selection of other Chalcidoidea outgroups for rooting. Specimens of D. trilineata were identified using keys in Yoshimoto (1977). Descriptions of all de- scribed Diparidae with flightless females occurring in North America were carefully compared to our specimens. Significant character conflicts are found for all but D. tri- lineata (and its well-justified synonym D. bilineatus (Yoshimoto)), and the type and oth- er known localities for these names correspond closely to the species as we treat it here. In preliminary analyses we included selected “Diparidae’ specimens from the Barcoding of Life Database (BoLD). However, finding that none of these affected the monophyly or polarity of the D. trilineata topology, we conducted most analyses without these. Sequenced specimens came from our own recent collections (sampling map shown in Fig. 1), where we sifted leaf litter at sites ranging in elevation from 1300-2000 m Phylogeography of Dipara trilineata 1063 (~4500-6600 ft). The highest elevation sites (> 1500 m) were dominated by a canopy of red spruce (Picea rubens) and Fraser fir (Abies fraseri), with a litter layer composed mainly of their shed foliage. Lower localities were associated with more typical south- eastern deciduous forest, with litters of oak, maple, birch, and Rhododendron. Litter samples were Berlese extracted until dry, and all specimens were collected into and preserved in 100% ethanol until extraction. Prior to extraction, each specimen was imaged (images available at https://www. flickr.com/search/?user_id=183480085%40N02 &desc&text=Dipara&view_all=1). Abdomens were subsequently punctured for digestion, and moved to a 96-well plate. Tissues were digested with lysis buffer and proteinase K (Omega BioTek, Norcross, GA), the liquid fraction then removed to a new plate and extracted using Omega BioTek’s MagBind HDQ Blood and Tissue kit, eluting with 150 uL elution buffer. Voucher specimens were retained, labelled, assigned unique identifiers, and deposited in the Clemson University Arthropod Collection. The data set includes sequences produced by Illumina and Nanopore methods. In both cases, mini- (421 bp) barcodes were amplified from the mitochondrial COI gene using the primers BF2-BR2 (GCHCCHGAYATRGCHTTYCC & TCDGGRI- GNCCRAARAAYCA; Elbrecht and Leese 2017), corresponding to the downstream two-thirds of the standard barcoding region. Each PCR reaction was tagged with a unique combination of 9 bp indexes (Meier et al. 2016). All PCRs were conducted in 12.5 ul volumes (5.6 pL water, 1.25 pL Taq buffer, 1.25 pL dNTP mix [2.5 mM each], 0.4 pL MgCl [50 mM], 1.5 pL each primer, 0.05 wL Platinum Taq polymerase, 1 pL DNA template, with a 95 °C initial denaturation for 5 minutes, followed by 35 cycles of 94 °C (30 sec), 50 °C (30 sec), 72 °C (30 sec), and a 5 minute 72 °C final extension on an Eppendorf Gradient Mastercycler. PCR products were combined and purified using Omega Bio-Tek’s Mag-Bind To- tal Pure NGS Kit, in a ratio of 0.7:1 (enriching for fragments >300 bp). Illumina adapters and sequencing primers were ligated to PCR products using New England BioLab’s Blunt/TA Ligase Master Mix. Resulting libraries were purified using Mag- Bind Total Pure NGS, quantified using a Qubit fluorometer, and sequenced on an II- lumina MiSeg using a v.3 2 x 300 paired-end kit. For Nanopore MinION sequencing, libraries were prepared using the ligation sequencing kit LSK-112 (Oxford Nanopore Technologies, Oxford, UK), and loaded onto a v10.4 flowcell. Illumina reads were processed with bbtools software package (https://jgi.doe.gov/ data-and-tools/bbtools/; v38.87; Bushnell et al. 2017), trimming adapters, removing PhiX control reads, merging paired-end reads, filtering reads for the correct size, re- moving reads with quality score < 30, clustering sequences by similarity allowing 5 mismatches (~1%), and generating a final matrix in FASTA format. Nanopore reads were basecalled using the ‘super-accurate’ algorithm of Guppy (v6.1.2) running on Clemson’s Palmetto cluster, then demultiplexed using ONTbarcoder v0.1.9 (Srivath- san et al. 2021), with minimum coverage set at 5. FASTA files from all sequencing runs were combined and aligned with the online version of Mafft v7 (Katoh et al. 2017) using the auto strategy. All barcode sequences have been deposited in GenBank, with accession #s listed in Suppl. material 1. 1064 M.S. Caterino & N.C. Arey / Journal of Hymenoptera Research 96: 1061-1072 (2023) Figure |. Map of all localities represented by COI sequences in the present study. Colors refer to those in trees in Figs 2, 3. We produced a phylogeny using W-IQ-Tree (Nguyen et al. 2015; Trifinopoulos et al. 2016) under maximum likelihood criteria, applying a GTR+gamma model, with empirical base frequencies. Branch support was estimated using 10000 replicates of ul- trafast bootstrapping (Minh et al. 2013). To assess relationships among haplotypes un- der a population genetic framework, a TCS haplotype network (Clement et al. 2000) was constructed using Popart (Leigh and Bryant 2015). Results Phylogenetic analyses that included a broader selection of Diparidae (not shown) from BOLD invariably resolved southern Appalachian D. trilineata as monophyletic, with no other available sequences very closely related. Sequences unidentified beyond ‘Di- paridae’ from Thailand and Western Australia appeared more closely related to D. tri- lineata than did sequences of the Palaeartic Dipara petiolata or what appears (from a voucher photo in the BOLD database) to represent D. canadensis (from Virginia, USA). Within D. trilineata, 69 individuals resolved into 35 distinct haplotypes. Divergences among them were remarkably low, with most less than 2% (uncorrected). The largest diver- gences were between a single individual from Brasstown Bald, Georgia (BBld.A.048) and most other sequences, at 4-6%. Comparisons to a couple other more divergent and well supported lineages (those from the Black Mts. in North Carolina and those from White- top Mt. in southwestern Virginia) were intermediate, ranging from 2—3.6%. Phylogenetic resolution was low and mostly weakly resolved (see Fig. 2). The deeply divergent individual from Brasstown Bald in northeastern Georgia was resolved as the sister to all other popula- tions, although it differs in no obvious morphological characteristics. Among the latter, a single individual from a lower elevation locality in south-central North Carolina (Green Phylogeography of Dipara trilineata 1065 misc. Chalcidoidea outgroups BBid A 048 92 NC GreenRiver WR A 055 RHB A 038 RHB A 038d RHB A 038¢ 96 BgBid B 368 BgBid A 053 RHB A 039 - MALE RHB A 038 GRB A 044c GRB A 044f GRB A 044d | _ GRBA 044e GRB A 044 RHB A 040 GRB A 0445 BigButt152 CK B 476 BigButt150 9: BrK B 345 WrKk A 028 BT A 027b BT B 337 BT A 027d BT A 027e BT B 339 - MALE 29] MM A 044b 96 BT A 027c 99— MH A 020 049 MH A 020b MH A 020d CK A 056 CB 072 CB B 368 JonesGap CB B 368 Sass B 464 RC 030 Sass A 086 CD A 056 91] CD A 056b CD A 056c RB A 029c RB A 029d RB A 029e RB B 329 . MLc 053 97, CD B 329 [1 CDB 333 - MALE Le _ss« BCat A 083 NC WayahBaild BBK A 047 BBK B 348 CrB 044 93 MHy A 037 MHy B 355 97, MK A054 gof | MKA054c | MK A 054b L___. MK B 400 98, GrM B 335 DBS B 349 NC LinvilleGorge WTA 032c 95] WTB 318 WT A 032b 96] wT A032d WT A 032 Figure 2. Maximum likelihood phylogeny of Dipara trilineata individuals, with locality abbreviations as in Suppl. material 1, and colors of OTUs keyed to localities shown in Fig. 1. 1066 M.S. Caterino & N. C. Arey / Journal of Hymenoptera Research 96: 1061-1072 (2023) River) was sister to the remainder. More northern populations (Roan Highlands, Big Bald, Black Mountains, Grandfather Mt., and Whitetop) were broadly paraphyletic with respect to populations southwest of the Asheville Depression. Populations in the latter region were resolved into a few moderately to weakly supported lineages (mostly from single localities: Mt. Kephart, Mt. Hardy), but relationships among most are unresolved. The haplotype network (Fig. 3) uncovers little population level structuring. Although only a couple haplotypes are shared across populations (Black Mts. and Roan Highlands by one haplotype, Nantahala Mts. and Great Balsam Mts. by another), few populations form tight clusters, and haplotypes from some widely separated localities (e.g., Celo Knob in the northern Black Mts. and Rabun Cliffs in north Georgia) are quite closely related (differing in that case by only two mutations). Three male specimens (fully winged, with long filiform antennae), representing three different populations, were resolved as identical to one or more females from their respective populations, and can be considered definitively associated. These specimens conflict in several characters with the descriptions and figures presented in Yoshimoto (1977), then described as the males of the now synonymous Trimicrops bilineatus. The clearest point of contrast is in the antennal flagellum, shown in fig. 25 of Yoshimoto (1977: p.1053) as moderately elongate, with evenly cylindrical flagellomeres with surfac- es covered with short setae, described as “filiform, densely pubescent with a single short annellus”. In the specimens we attribute to D. trilineata (Fig. 4C—F) the antennae are WT_A_032 CD_A_056c¢ WT_A_032d BBld_A_048 cD NC_LinvilleGorgg C) BgBld_A_053 ©) BgBld_B_368 MLc_053 WrK_A_028 BCat_A_0831 MHy BigButt1 50 NC_GreenRiver MK_A_054b BBK_B_348 JonesGap MK_B_400 CrB_044 MH_A_020d MH Black Mts. Figure 3. Haplotype network from TCS analysis, with sizes of circles proportional to number of indi- viduals with that haplotype, and colors of circles keyed to localities as shown in Fig. 1. Phylogeography of Dipara trilineata 1067 much more slender, every flagellomere tapered basally and distally, and verticillate, with few very long setae borne in whorls (Fig. 4E). Yoshimoto also figures the wings in his Fig- ure 8 (1977: p. 1050), showing the hind wing to be broadly rounded apically, while our D. trilineata has much narrower, apically subacute hind wings (Fig. 4F). Those are the only characters illustrated by Yoshimoto, but it is apparent that the described “allotype” male from the type locality represents a distinct species. The male he attributed to Dipara pedunculata (with antennae figured in Yoshimoto’s Fig. 27; 1977: p. 1053), now con- sidered a synonym of D. canadensis, matches our D. trilineata males much better than it does D. canadensis (the male antenna of which is shown in his fig. 26: (1977: p. 1053). Heydon and Bouéek (1992), when synonymizing D. pedunculata with D. canadensis, previously noted some inconsistencies between Yoshimoto’s (1977) description and fe- male holotype. We suggest that the male presumed to represent Yoshimoto's D. peduncu- lata was a misidentified D. trilineata. Dipara pedunculata was described from Kentucky, well within the range of D. trilineata, so the two valid species must be sympatric there, and the original series of D. pedunculata a mix of D. canadensis and D. trilineata. Comparing our confirmed males of D. trilineata directly to Yoshimoto’s (1977) description of D. pedunculata, we note several other points of difference, and provide a brief re-description here (with slightly updated terminology). Male (Fig. 4C-F): Head, mesosoma, and metasoma fuscous; legs (except meso- coxa), petiole, and bases of antennae yellowish, the antennae gradually darker from 3“ flagellar segment distad, mesocoxa also darker toward base; head almost hemispherical, very shallowly depressed above toruli, smooth and shining above, finely transversely re- ticulate below toruli, with scattered setae throughout; eyes prominent, eye height slightly more than half lateral head height, coarsely faceted; ocellar triangle wide, individual ocelli oval; clypeus outlined by disconnected series of punctures, convex, apical mar- gin evenly rounded; mandibles tridentate; antennae inserted in front of middle of eye, slightly above middle of frons, toruli approximately equally separated from each other as from inner edge of eye; scape cylindrical, slightly curved, almost as long as pedicel and flagellomeres 2 and 3 combined; pedicel short, expanded to slightly wider than scape at apex, flagellomeres narrow basally and apically (‘pedunculate’), but bulbous in basal half, tapered apically, with few (~6) long setae (about 1.5 times as long as flagellomere) inserted in an uneven series around bulbous base; entire antenna nearly as long as rest of body; neck transversely reticulate, bounded posteriorly by evenly curved, weakly im- pressed collar; notauli subcrenulately impressed, curving to meet along finely and deeply impressed mesoscutum-scutellar suture, the mesoscutum polygonally microsculptured between; frenal groove of scutellum only weakly indicated, but frenum smoother than polygonally microsculptured scutellum; propodeum with coarsely raised reticulate mi- crosculpture; anterior insertion of petiole slightly narrower than posterior insertion, petiole about 3 times as long as maximum width, with weak longitudinal carinae; 1“ gastral segment nearly half entire gastral length, 2"“-5" gastral segments subequal in length; forewing widening only slightly beneath costal cell, widening more abruptly be- yond, anterior margin bent slightly forward at this point; submarginal vein bearing two conspicuous dorsal setae; marginal vein more densely setose, the setae directed distad at about 45°, their maximum length about 4 maximum wing width; postmarginal vein 1068 M.S. Caterino & N. C. Arey / Journal of Hymenoptera Research 96: 1061-1072 (2023) wee SQW Figure 4. Female (A, B) and male (C-F) Dipara trilineata (Yoshimoto). weak, fading evenly beyond short stigmal vein, stigma slightly expanded, uncus poorly developed; wing with sublinear series of short setae in basal cell, bare briefly within spec- ular area, densely and evenly setose beyond; setae of apical and posteroapical margins of wing long, nearly half maximum wing width; hind wing about three-fourths length of forewing, posterior margin rounded, widened slightly beyond midpoint, narrowed to subacute apex, setae along posterior margin longer than width of hindwing membrane. Material examined (males): North Carolina, Yancey County, Mt. Mitchell State Park, Big Tom near summit (35.7799, -82.2596), 7-Sep-2021 (CUAC000135520); North Carolina, Swain County, Great Smoky Mountains National Park, Clingmans Dome (35.5589, -83.4983), 14-Sep-2021 (CUAC000157203); North Carolina, Mitch- ell County, Roan High Bluff (36.0931, -82.1453), 15-Aug-2018 (CUAC000002974). Other taxonomic remarks: No recent authors have addressed the mismatch in gen- der of Dipara trilineatus (sic). Walker’s (1833) genus name would be feminine, appearing to be based on a Greek adverb used as a singular noun (S. Chatzimanolis, pers. comm.), Phylogeography of Dipara trilineata 1069 and virtually all usage from Walker’s onward has used feminine species names. It is unfor- tunate that when synonymizing Trimicrops Keiffer with Dipara Walker, Desjardins (2007) did not properly amend ‘trilineatus’ to the singular feminine ending, but we do that here. Discussion Dipara trilineata is a remarkably widespread species for one having such seemingly limited dispersal capabilities. Our collections, along with reliable records from other sources reveal the species to cover much of the eastern US, extending from central Tex- as into southeastern Canada. As to state records, the species was previously unreported for Mississippi, Indiana, Georgia, South Carolina, Virginia, and West Virginia (Fig. 5). Even more surprising is the relatively limited degree of population structuring, at least over the range we sampled. Some geographic clustering is evident, and a number of populations exhibit haplotype monophyly, but the overall patterns exhibit only loose cor- respondence with geography. One potential confounding factor is the relatively high hap- lotype diversity, as would be expected for a species with large population sizes. This could slow coalescence and limit phylogenetic resolution even if populations are largely isolated. But based on available data, there are no indications that D. trilineata represents a cryptic species complex, despite its flightlessness. If additional individuals from the more divergent lineages (BBld.A.048 or NC_GreenRiver) showed comparable genetic difference, more systematic morphological comparisons may reveal subtle differences not yet apparent. y’ P g p y. yet app Z WY Dipara published * Dipara sequenced fi xe Dipara not sequenced * Dipara - online records —_ 0 140280420 km Figure 5. Total known distribution of Dipara trilineata, based on a combination of published, online, and newly contributed records. 1070 M.S. Caterino & N. C. Arey / Journal of Hymenoptera Research 96: 1061-1072 (2023) The lack of phylogeographic structuring may provide some indirect hints as to host breadth. Even though individual Dipara females may not themselves be capable of long-distance dispersal, it is worth suggesting the potential for dispersal in the larval stage by a more mobile, flying or ballooning host, which would serve to reduce effec- tive isolation (as has been shown for Dryinidae parasitoids of leafhoppers; Mita et al. 2012). Host records for Dipara to date include only non-mobile stages, eggs, larvae, and pupae (Desjardins 2007). But these already cover a considerable range, and more mobile hosts should not be ruled out. As to potential host identities for Dipara trilineata, its general abundance over a wide range argues against any close host specificity. There are few other arthropod spe- cies in eastern US leaf litter that have so wide a distribution, occurring in such a wide range of microhabitats, although perhaps some of the spider species do (Recuero et al. 2023). Previous suggestions of weevil associations would not seem likely, at least not as a primary host, as weevils are poorly represented in our highest elevation samples. There are intriguing possibilities to better understand host/parasitoid relationships through metabarcoding approaches, such as detecting the DNA of a parasitoid as co- amplifying with that of its host (Miller et al. 2021), and the Dipara system would be a promising one to explore such potential. Acknowledgments This study was funded by the U.S. National Science Foundation (Award DEB-1916263 to MSC) and the Clemson University Experiment Station (SC-1700596 to MSC). We also acknowledge the support of the John and Suzanne Morse Endowment for Arthropod Biodiversity. For permissions and assistance with field work we are grateful to the North Carolina State Parks, Great Smoky Mountains National Park, Blue Ridge Parkway National Park, Monica Martin, Frank Etzler, Ernesto Recuero, Curt Harden, Patricia Wooden, Adam Haberski, Roy Kucuk, Laura Vasquez-Vélez, Laary Cushman, Paul Marek, Michael Ferro, and Will Kuhn. Mary Atieh, Caroline Dukes, Caroline Mc- Cluskey, Grace Holliday, Grace Arnold, Hannah Skinner, Alejandra Carranza, and An- thony Villanueva provided valuable assistance in the lab. We thank Fernando Farache and Mircea-Dan Mitroiu for comments that improved the manuscript, and Stylianos Chatzimanolis for providing insight into the nomenclature. This paper represents Tech- nical Contribution No. 7248 of the Clemson University Experiment Station. References Boucek Z (1988) Australasian Chalcidoidea (Hymenoptera): A biosystematic revision of genera of fourteen families, with a reclassification of species. 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Arey Data type: xlsx Explanation note: Fields include source of record, project morphospecies code (search- able on Flickr), sex, Caterino lab DNA extraction number, GenBank accession number (where sequenced successfully), verbal locality description, decimal lati- tude/longitude, date collected, and unique CUAC voucher code. Copyright notice: This dataset is made available under the Open Database License (http://opendatacommons.org/licenses/odbl/1.0/). The Open Database License (ODDbL) is a license agreement intended to allow users to freely share, modify, and use this Dataset while maintaining this same freedom for others, provided that the original source and author(s) are credited. Link: https://doi.org/10.3897/jhr.96.115001.suppl1