Agricultural and Forest Entomology (2019), 21, 299–308 DOI: 10.1111/afe.12332 Hidden in plain sight: phylogeography of an overlooked parasitoid species Trioxys sunnysidensis Fulbright & Pike (Hymenoptera: Braconidae: Aphidiinae) ∗ ∗ ∗ † ‡ Jelisaveta Ckrkiˇ c´ , Andjeljko Petrovic´ , Korana Kocic´ , Zhengpei Ye , Ines M.G. Vollhardt , Paul D.N. Hebert§, † ∗ Michael Traugott and Željko Tomanovic´ ∗Institute of Zoology, Faculty of Biology, University of Belgrade, Studentski trg 16, Belgrade, 11000, Serbia, †Mountain Agriculture Research Unit, Institute of Ecology, University of Innsbruck, Technikerstrasse 25, Innsbruck, 6020, Austria, ‡Agroecology, Department of Crop Sciences, Georg-August-University Göttingen, Grisebachstrasse 6, Göttingen, 37077, Germany and §Centre for Biodiversity Genomics, Biodiversity Institute of Ontario, University of Guelph, 50 Stone Road East, Guelph, ON, N1G 2W1, Canada Abstract 1 The bird cherry-oat aphid Rhopalosiphum padi is a major cereal pest with an almost cosmopolitan distribution. As one of the main groups of biocontrol agents for aphids, numerous Aphidiinae are associated with R. padi, including the genera Binodoxys and Trioxys. 2 As a recently described species parasitizing R. padi, Trioxys sunnysidensis is recorded from Europe for the first time based on morphological and molecular data. Specimens from North America, Europe and New Zealand were used in the analysis of the cytochrome c oxidase subunit I to explore genetic variation among populations. The analysis revealed one of the highest haplotype diversities in Aphidiinae so far, with 25 haplotypes detected. The two most common haplotypes are shared across groups of populations, whereas all of the others are found either in North America or Europe. 3 Because the genetic structure of populations is an important factor to consider when choosing a biocontrol agent, the results obtained in the present study may be helpful in guiding potential biocontrol attempts. Keywords Aphidiinae, biological control, haplotype diversity, phylogeography, Trioxys. Introduction et al., 2008), with numerous taxonomical and ecological studies having been conducted to assess their effectiveness (Stary,´ 1981; Rhopalosiphum padi (Linnaeus 1758) (Hemiptera: Aphididae) Powell, 1982; Pike & Stary,´ 1995; Sigsgaard, 2002; Kavallieratos is a major pest of temperate cereal crops on a global scale et al., 2005; Traugott et al., 2008; Kos et al., 2011; Plecaš´ et al., reflecting damage caused both by feeding on plants, as well as 2014). According to Yu et al. (2012), there are currently 44 by its role in the transmission of plant viruses (Van Emden & known aphidiine parasitoids of R. padi, including species of Harrington, 2017). It is a vector of numerous viruses, including Binodoxys Mackauer, 1960 and Trioxys Haliday 1833. the Barley yellow dwarf virus group, cereal yellow dwarf virus, With 74 species described worldwide, Trioxys is one of the filaree red leaf virus, maize leaf fleck virus, rice giallume virus, most diverse genera in the subfamily Aphidiinae (Yu et al., oat yellow leaf disease and onion yellow dwarf virus (Van Emden 2012). It is classified into the Trioxina subtribe based on & Harrington, 2017). morphological characters (Mackauer, 1961; Yu et al., 2012). The Given the damage that cereal aphids inflict on economically most prominent morphological characters for differentiation of important plants, research on their natural enemies has been the genus are paired accessory prongs on the last abdominal extensive. As one of their most important groups of natural sternite, together with the absence of secondary tubercles on enemies, some species of Aphidiinae have been widely used as the petiole. Although the later characteristic apparently separates biocontrol agents against cereal aphids (Stary,´ 1981; Tomanovic´ Trioxys and Binodoxys, the relationship between these genera is not clear. Mackauer (1960) classified Binodoxys as a subgenus Correspondence: Jelisaveta Ckrkiˇ c.´ Te1.: +38164 169 3955; e-mail: of Trioxys but later raised it to a genus (Mackauer, 1961, 1965) [email protected] based on its possession of secondary tubercles on the petiole. © 2019 The Royal Entomological Society 300 J. Ckrkiˇ c´ et al. Stary´ (1981) treated them as subgenera based on the same trait of Life Database (BOLD) (http://www.boldsystems.org). Trioxys but noted that the grouping of species into Trioxys and Binodoxys species were chosen because of their morphological similarity to was more for identification purposes than a reflection of their T. sunnysidensis or because they parasitize cereal aphids. Bino- nomenclatural rank. Most of the later work did not consider doxys species were included in the analysis to examine relation- their taxonomic status or relationships but, instead, focused on ships with Trioxys and evolutionary divergence rates between the individual species or the position of the whole Trioxina subtribe two genera. within the subfamily. Based on past molecular analyses, Trioxys Sequences were edited using finchtv, version 1.4.0 (www has been placed in the separate tribe Trioxini (Belshaw & Quicke, .geospiza.com). Alignment of sequences was conducted using 1997) or viewed as a basal subtribe within the Aphidiini (Smith clustalw integrated in mega, version 5 (Tamura et al., 2011). et al., 1999; Kambhampati et al., 2000; Sanchis et al., 2000; Shi Sequences were trimmed to a length of 564 bp. A phylogenetic & Chen, 2005). tree was constructed using mrbayes, version 3.1.2 (Ronquist To date, 33 species of Trioxys from Europe (van Achter- & Huelsenbeck, 2003). The best fitting model of sequence berg, 2013) and 22 from North America (Fulbright & Pike, evolution based on the Akaike information criterion was the 2007) have been reported. Most parasitize arboricolous aphids, Hasegawa–Kishino–Yano model with a gamma distribution and although some parasitize aphids in steppe habitats (Tomanovic´ a fraction of invariable sites (HKY+G+I). Bayesian analysis was & Kavallieratos, 2002). Except for the three species employed conducted running two Markov chain Monte Carlo searches, as biocontrol agents of aphids [Trioxys complanatus Quillis each with one cold and three heated chains. The analysis ran 1931/Therioaphis trifolii (Monell 1882); Trioxys pallidus (Hal- for two million generations, sampling every 1000 generations, iday 1833)/Chromaphis juglandicola (Kaltenbach 1843) and and the first 250 trees were discarded as burn-in. Convergence Myzocallis coryli (Goeze 1778); Trioxys curvicaudus Mack- of the parameters was confirmed with tracer, version 1.5.0 auer 1967/Eucallipterus tilliae (Linnaeus 1758)], other Trioxys (Rambaut & Drummond, 2007), whereas figtree, version 1.3.1 species have only been collected sporadically (Stary,´ 1988; (Rambaut, 2006) was used to view the consensus tree with Tomanovic´ & Kavallieratos, 2002; Davidian, 2005). posterior probabilities. The North American species Trioxys sunnysidensis Fulbright Calculation of average genetic distances between sequences & Pike, 2007 was collected and described from R. padi in was performed in mega, version 5 (Tamura et al., 2011) using Washington State (Fulbright & Pike, 2007), which makes it just Kimura’s two-parameter method of base substitution (Kimura, the second known species in this genus that parasitizes cereal 1980). aphids, in addition to Trioxys auctus (Haliday 1833) on R. padi (Stary,´ 1976, 1981, 2006). In the present study, we report the first records of an overlooked Genetic diversity of T. sunnysidensis. In addition to the cereal aphid parasitoid T. sunnysidensis from Europe after a sequences for two T. sunnysidensis specimens from Germany long-term sampling campaign from cereal agroecosystems. We reared from R. padi that were also used in the morphological discuss its host range, the patterns of genetic variation among analysis (TRspA_331 and TRspA_333), 314 sequences of its populations across the world and its potential as a biocontrol T. sunnysidensis from BOLD were used to examine patterns agent. of genetic variation among populations (Hebert et al., 2016); sequences from BIN AAU8585 (https://dx.doi.org/10.5883/ BOLD:AAU8585) on BOLD are provided in the Supporting Materials and methods information (Table S1). These sequences were trimmed to a Adult parasitoids were dissected and slide-mounted for exami- length of 504 bp. The number of haplotypes was calculated nation. The external morphology of the specimens was studied usingdnasp,version6(Rozaset al., 2017). A median-joining using a Discovery V8 stereomicroscope (Carl Zeiss, Germany). network (Bandelt et al., 1999) was constructed using network, Species identification was performed in accordance with Ful- version 5.0.0.1 (http://www.fluxus-engineering.com/). bright and Pike (2007) and Fulbright et al. (2007). The material Haplotype and nucleotide diversity were determined in used in the morphological analysis and identification comprised: arlequin, version 3.5 (Excoffier & Lischer, 2010). Genetic Germany, Göttingen, reared from R. padi on Triticum aestivum, differentiation between populations was measured by comput- two females, two males (coll. Vesna Gagic);´ Canada, Ontario, ing pairwise ΦST statistics. To infer differentiation between Wellington County, Guelph, 5. IX 2013, one female; 27. IX 2013, groups of populations, we conducted an hierarchical analysis one male (coll. BIO Collections Staff); Walkerton, 4. X 2013, of molecular variance (amova) using genetic distances and one female (coll. Meridith White); Sudbury, 3. X 2014, one