Description of a new species of Euderus Haliday from the southeastern United States (Hymenoptera, Chalcidoidea, Eulophidae): the crypt-keeper wasp

Abstract A new species of the genus Euderus Haliday, Euderus set sp. n., is described and illustrated from the southeastern United States, where it parasitizes the crypt gall wasp, Bassettia pallida Ashmead, 1896, on live oaks in the genus Quercus (subsection Virentes). This is the 1st species of the genus reported from the southeastern United States to parasitize cynipid gall wasps and the 3rd species of the genus reported to attack cynipids in North America. Modified sections of the identification keys to subgenera and species of Euderus (Yoshimoto, 1971) are included to integrate the new species.


Introduction
The genus Euderus Haliday, 1844 is a group of parasitic wasps in the family Eulophidae (Hymenoptera) with approximately 77 described species (Yoshimoto 1971, Noyes 2016. The genus has a cosmopolitan distribution, where it is found in North and South America, Europe, Asia, Australia, and many isolated island archipelagos, including the Hawaiian islands, Micronesia, the Canary Islands, and the Seychelles archipelago (Ahmad 1976, Askew et al. 2001, Báez and Askew 1999, Gates et al. 2002, Gibson et al. 2006, Goolsby et al. 2001, Gunasena and Harris 1998, Herting 1973, Thompson 1955, Yoshimoto 1971. Yoshimoto (1971) produced the last revision of the genus Euderus for North America, north of Mexico. The revisions built off of Nearctic catalogues by Peck (1951Peck ( , 1963 and Burks (1967), with new additions based on material deposited in the Canadian National Collection, material loaned from the Natural History Museum, London, Philadelphia Academy of Sciences, and the Museum of Comparative Zoology, Harvard University. Based on this revision, there are 22 species reported from North America north of Mexico, with 12 residing in the coastal southeastern U.S. (Florida, Alabama, Mississippi, Louisiana, and Texas;Yoshimoto 1971, Noyes 2016. According to Yoshimoto (1971), the biological records of Euderus from the U.S. indicate that most species are host specific on pupae of leaf tying, leaf mining, twig and fruit boring Lepidoptera (Tortricidae or Gelechiidae) and stem boring and other herbivorous Coleoptera (Buprestidae, Cerambycidae, and Curculionidae). Rarely, Euderus has been reported to attack gall-making Hymenoptera (Cynipidae) or exhibit hyperparasitism on other Hymenopteran parasitoids (Ichneumonidae). Detailed host records from field observation across the genus are summarized in Table 1 of Yoshimoto (1971).
Here, we report the first species of this genus from the southeastern United States to attack cynipid gall wasps, where it is associated with the crypt gall wasp Bassettia pallida Ashmead, 1896 (Hymenoptera; Cynipidae) on live oaks (Quercus; subsection Virentes), including Quercus virginiana and Q. geminata. We modify the key published by Yoshimoto (1971) to include the new species and add a correction to the key to subgenera.

Field collections and lab husbandry
For the type locality, branches of the sand live oak, Quercus geminata, infested with the asexual generation of the crypt gall wasp, Bassettia pallida, were collected July 15, 2014, August 1, 2015, and October 1, 2015). Additional populations were collected across the U.S. Gulf coast on Q. geminata and Q. virginiana in 2014, 2015, and 2016 (see Table 1). Branches were placed in clear plastic cups, covered with a coffee filter and rubber band, and maintained outside in natural temperature and humidity conditions in a constantly shaded walkway at Rice University in Houston, Texas (Lat/Long: 29.717030, -95.401279). Emergence of all in-  Gibson et al. (1997), Yoder et al. (2010), and Hymenoptera Anatomy Consortium (2016). The identification key is modified from the key to subgenera and species of the genus Euderus in Yoshimoto (1971).

Complementing morphological taxonomy with molecular barcodes
When samples were of sufficient quality for genetic work, we complemented morphological taxonomy with molecular barcodes (e.g., Smith et al. 2008, Forbes and Funk 2013, Forbes et al. 2016. Genomic DNA from two individuals from the Inlet Beach, FL population were extracted using DNeasy Blood and Tissue kits (Qiagen Inc., Valencia, CA). We used a pair of degenerate primers to amplify a segment of the mitochondrial cytochrome oxidase (mtCOI) gene using standard PCR protocols (Smith et al. 2008). Primers used were COI pF2: 5' -ACC WGT AAT RAT AGG DGG DTT TGG DAA -3' and COI 2437d: 5' -GCT ART CAT CTA AAW AYT TTA ATW CCW G -3', developed by Simon et al. (1994) and modified by Kaartinen et al. (2010). We treated amplified fragments with Exonuclease I (New England Biolabs, Ipswich, MA) and Shrimp Alkaline Phosphatase (Fermentas Life Sciences, Glen Burnie, MD) and sequenced in both forward and reverse directions on an ABI 3730 DNA analyzer using BigDye 3.1 sequencing chemistry (ThermoFisher Inc., Waltham, MA). We edited raw sequences and assembled forward and reverse reads using Geneious v.6.1.8 (Kearse et al. 2012). The final sequences were 703bp and 745bp in length, a function of amplification and sequencing success. We ran each sequence through the "identification request" module on the Barcode of Life Database (BOLD; Ratnasingham and Hebert 2007) to identify the highest percentage matches from previously identified taxa. All sequences were deposited in GenBank (accession numbers provided below).

Results
Details on the on the type material and type localityare provided, with a thorough description with images, a differential diagnosis of the new species, and a modification to the identification key published by Yoshimoto (1971) that distinguishes this new species from closely related species. We also provide a correction to the key to subgenera published in Yoshimoto (1971). In addition, we provide a brief description of etymology and information on the distribution, natural history, biology, and results of the mtDNA DNA barcoding analysis. In Supplemental File 1, we provide further details of the morphological, geographic, and ecological distinguishing features of this new Euderus species, Euderus set, which differentiate it from (1) ecologically similar Euderus species attacking cynpid gall wasps, (2) geographically overlapping Euderus species in the coastal southeastern United States, (3) geographically proximate Euderus speices from the Caribbean, and (4) morphologically similar species within the Euderus genus. We also provide the DNA sequence data to complement the morphological taxonomy in Suppl. material 1. Head, scrobal depression, pedicel, flagellum, mesoscutum, scutellum, coxae and metasoma metallic, olive green to turquoise to iridescent blue (colors depends on lighting and age of specimen); antennal scape white to yellow; femora and tibiae concolorous with mesoscutum but color lightens apically; tarsi white, except terminal segment dark brown (Fig. 1A, B).

Taxonomy
Head. Head in fresh specimens as wide as mesosoma; in dorsal view 2.9 times as broad as long; eyes prominent and bare; vertex, frons, and clypeus reticulate; vertex and upper frons distributed with white bristles; scrobal depression extends from slightly below anterior ocellus to level of lower eye margin, smooth above torulus and striolate below; toruli located in lower third of scrobal depression; clypeus short, subquadrate, only slightly longer than wide; malus sulcus inconspicuous and 0.44 times eye length; mandibles with three teeth. Antennal scape 3.5 times as long as broad and 0.6 times eye length; Flagellum with nine segments, with anellus two-segmented, funicle four-segmented, and clava three-segmented. Funicular segments each with 2 rows of thick, mostly non-overlapping bristles (Fig. 2B). Relative length of scape, pedicel, anelli, funicle 1, 2, 3, and 4, and clava 1, 2, and 3 as 38, 12, 2, 24, 22, 22, 20, 16, 12, 8, respectively; two anelli with the same length but relative breadth of first anellus to second anellus as 6, 8.
Mesosoma. Pronotum, mesoscutum and scutellum reticulate; pronotum short in dorsal view, with six brown bristles at margin with mesoscutum; mesoscutum sparsely setose and setae inconspicuous; scutellum with many short setae and two pairs of strong setae in posterior third. Mesoscutum 0.95 as long as broad, mid-lobe convex, notauli complete and deep; axillae slightly advanced, their anterior tip extending to the approximate midpoint of the mesoscutum. Scutellum moderately convex and length 0.80 times length of mesoscutum. Propodeum 0.24 times length of scutellum and with strong median carina (Fig. 1B); callus with 6-7 bristles ( Fig. 2A).
Wings. Forewing broad, extending past apex of gaster, marginal ciliae short; basal cell bare; submarginal vein with 6 dorsal bristles; postmarginal vein 1.3x length of stigmal vein; 3 admarginal hairs on left wing, four admarginal hairs on right wing; stigmal vein short and with large stigmus with 6 hairs on surface; 5 strongly-defined hair lines reaching dorsal and apical margin of forewing, with 3-4 additional less strongly defined hair lines that may or may not reach wing margin; 1 hair at median of radial cell ( Fig. 2A). Hindwing 0.80 times length of forewing; hindwing moderately and evenly setose; hind marginal ciliae long. Metasoma. Metasoma with petiole hidden in dorsal view; gaster elongate, 2.6 times as long as broad, and as long or longer than head and mesosoma combined; scattered white setae at posterior margins of each tergite becoming more dense towards the apex of the gaster; each cercus with two long bristles. Ovipositor extends slightly beyond apex of gaster (Fig. 1A).
MALE. Length 1.2 -1.6 mm. Antennae inserted in middle of face (Fig. 2C). Funicular segments each with 3 loosely ordered rows of fine bristles. Gaster ovate; shorter than head and thorax combined. Other characters similar to female.
Etymology. Named after the ancient Egyptian god Set, whose mythological stories mirror the natural biology of E. set. Set was the god of evil and chaos (Pinch 2004) and was reported to have control over evil animals like hyenas and serpents, just as E. set manipulates the behavior of its host, which is a parasite of trees (see Biology section below). The god Set is also reported to have trapped his brother Osiris in a crypt to kill him, later retrieving the body and chopping it up into small piece, which also fits with E. set, the crypt-keeper wasp, which kills its host in a crypt, and devours the host from the inside out, leaving major sections of exoskeleton (i.e., body parts) chopped up and distributed in the crypt (Weinersmith et al., in revision).

Diagnosis.
Two changes are required for the new species, E. set, to be included in the North American key to species of the genus Euderus by Yoshimoto (1971). First, a change is required to the key to subgenera of Euderus, where characters referring to the male flagellum should be removed. Yoshimoto (1971) used whorls of long hairs on the male flagellum as a diagnostic trait to discriminate between subgenera Neoeuderus and Euderus, but at that time only a single male specimen of subgenus Neoeuderus was available for study, and this individual was from the species E. viridilineatus for which no females had been found. As the male E. set. do not have antennae with pronounced whorls of long hairs, this is not an appropriate diagnostic trait for the subgenus.
Biology. The genus Euderus Haliday is a small group of chalcidoid wasps belonging to the family Eulophidae (Burks 2003) where the majority are reported to be primary parasitoids of arthropods at all stages of development (Burks 1979, Burks 2003, Noyes 2016. E. set parasitizes the crypt gall wasp Bassettia pallida Ashmead, 1896, which forms galls on American live oaks in the genus Quercus and the subsection Virentes across the southeastern United States (Ashmead 1896, Melika and Abrahamson 2007, Egan et al. 2013. There are six to eight different cynipid gall wasps that are highly specialized and form galls on this same live oak complex (Egan et al. 2013). We have reared out many of the parasitoids from this large community, including clearly documenting the community from another gall former, Belonocnema treatae (Forbes et al. 2016), but this is the first time we have observed a member of the genus Euderus in this system after two decades of work (Lund et al. 1996, Egan and Ott 2007, Egan et al. 2012, Egan et al. 2013. Published records almost certainly underestimate the diversity of subgenus Neoeuderus in North America, and many other species in the subgenus may also be specialist parasitoids of oak galling cynipids. Yoshimoto (1971) noted that the biological records of the Nearctic Euderus indicate that most species are host specific, while also cautioning that host records for Euderus are patchy and the result of field observation, which only represent the most common species where there are substantial rearing records. While Yoshimoto noted just four members of subgenus Neoeuderus, the current work adds a fifth, and we have recently reared another from the honey comb leaf gall wasp, Callirhytis favosa, on pin oak in Iowa. While this undescribed Euderus parasitoid of C. favosa has not yet been extensively studied, it is most similar in appearance to E. set, E. crawfordi, and E. multilineatus. If this truly is a different species, then three of the six species in subgenus Neoeuderus are known parasitoids of the oakassociated Cynipidae.
Emergence of E. set in the lab from field-collected B. pallida galls was concentrated from February to March coincident with new leaf growth of the host plants and adult maturation and emergence of the asexual generation Bassettia pallida (Melika and Abrahamoson 2007, Egan, unpubl. data). We also observed a smaller pulse in September and October, which could have been a natural occurrence, or induced by harvesting galled tissue and bringing it into a controlled environment. Regardless of harvest time (August or October), a similar emergence window was observed in February and March.
Euderus set is strongly associated with a behavioral phenotype in its host, the crypt gall wasp, Bassettia pallida, where infected gall wasps cut an emergence hole through the gall tissue as an adult, but then die and remain partially in the crypt to plug the emergence hole with its head (Weinersmith et al., in revision). When E. set emerges, it cuts an emergence hole directly through the head capsule plugging the hole (Weinersmith et al., in revision). The host's behavioral phenotype may benefit E. set by making it easier for the adult stage to emerge from the crypt (as it now only has to emerge through the parasitoid's head capsule, rather than through the tree stem itself; Weiner-smith et al., in review). This putative behavioral manipulation of the host by its parasitoid E. set is the first time this has been described by the species-rich and economically important Chalcidoidea and is also the inspiration behind both the scientific name, E. set, and the common name, the crypt-keeper wasp.
In addition to E. set, we have also reared eleven additional natural enemy species from Bassettia pallida galls on live oaks (Quercus; subsection Virentes), including two inquilines (genera Synergus and Ceroptres) and nine parasitoids including three species from the genus Sycophila, two species from genus Ormyrus, one each from the genera Eurytoma, Acaenacis, and Brasema, as well as a parasitoid from the platygastrid subfamily Platygastrinae that we have not yet been able to key to genus. The natural enemy community requires further description.