303 Two new species of Oobius (Hymenoptera: Encyrtidae) and their phylogenetic relationship with other congeners from northeastern Asia Yan-Xia Yao,1 Jian J. Duan, Jason L. Mottern, Xiao-Yi Wang, Zhong-Qi Yang, Leah S. Bauer, Michael W. Gates Abstract—Two new species of egg parasitoids, Oobius saimaensis Yao and Mottern new species and Oobius fleischeri Yao and Duan new species (Hymenoptera: Encyrtidae), are described from eggs of Agrilus fleischeri Obenberger, 1925 (Coleoptera: Buprestidae). Agrilus fleischeri is a phloem- feeding woodborer of poplar (Populus Linnaeus; Salicaceae) in northeastern China. These two species can be distinguished morphologically as O. fleischeri has five tarsomeres and O. saimaensis has four tarsomeres. Although O. saimaensis is morphologically similar to its sympatric congener O. agrili Zhang and Hang, 2005, an important natural enemy of the invasive emerald ash borer, Agrilus planipennis Fairmaire, 1888, molecular phylogenetics and morphological data indicate that they are distinct species. Phylogenetic relationships among the new species and other closely related species are also inferred by using DNA sequence data from several ribosomal and mitochondrial genes. In addition, we expand the known distribution of Oobius primorskyensis Yao and Duan, 2016 to include South Korea. Introduction (Costa Rica). More recently, two new Oobius species were described from the Nearctic Region The genus Oobius Trjapitzin, 1963 (Hymenoptera: (United States of America) (Triapitsyn et al. 2015) Encyrtidae) was not well studied until recently. and one from the Palaearctic Region (Russia) Before 2010, only nine species were described (Yao et al. 2016). (Trjapitzin and Volkovitsh 2011), with six species Most species of Oobius are solitary egg from the Palaearctic Region (Nowicki 1928; parasitoids of wood-boring Coleoptera in the Trjapitzin 1963; Myartseva 1979; Guerrieri et al. families Buprestidae and Cerambycidae (Zhang 1989; Zhang et al. 2005), and three species from the et al. 2005; Triapitsyn et al. 2015). Among those, Afrotropical Region (Annecke 1967). Subsequently, O. longoi (Siscaro, 1992) was introduced from Noyes (2010) synonymised Avetianella Trjapitzin, Australia to the United States of America (California) 1968, Szelenyiola Trjapitzin, 1977, and Oophagus in the late 1990s, where it successfully established as Liao, 1987 under Oobius and described 20 new a biocontrol agent of the invasive eucalyptus species of Oobius from the Neotropical Region longhorned beetle, Phoracantha semipunctata Received 10 August 2017. Accepted 9 January 2018. First published online 19 April 2018. Y.-X. Yao,1 X.-Y. Wang, Z.-Q. Yang, Key Laboratory of Forest Protection, China State Forestry Administration, Research Institute of Forest Ecology, Environment and Protection, Chinese Academy of Forestry, Beijing, 100091, China J.J. Duan, United States Department of Agriculture, Agricultural Research Service – Beneficial Insects Introduction Research Unit, Newark, Delaware, 19731, United States of America J.L. Mottern, United States Department of Agriculture, Agricultural Research Service – Systematic Entomology Laboratory, Beltsville, Maryland, 20705, United States of America; and United States Department of Agriculture, Animal and Plant Health Inspection Service, Riverdale, Maryland, 20737, United States of America L.S. Bauer, United States Department of Agriculture, Forest Service, Northern Research Station, Lansing, Michigan, 48910, United States of America M.W. Gates, United States Department of Agriculture, Agricultural Research Service – Systematic Entomology Laboratory, Beltsville, Maryland, 20705, United States of America 1Corresponding author (e-mail: [email protected]) Subject editor: Derek Sikes doi:10.4039/tce.2018.17 http://zoobank.org/urn:lsid:zoobank.org:pub:C9491659-478C-4A5B-9D1AAC7B183D6E6B Can. Entomol. 150: 303–316 (2018) © 2018 Entomological Society of Canada. Parts of this are a work are that of the U.S. Government and therefore such parts are not subject to copyright protection in the United States. 304 Can. Entomol. Vol. 150, 2018 (Fabricius, 1775) (Coleoptera: Cerambycidae), (Salicaceae) and native poplar trees P. davidiana which feeds on ornamental Eucalyptus L’Héritier Dode in Saima Village and nearby Nanmiao (Myrtaceae) trees (Hanks et al. 1996; Paine and Village, Liaoning Province, China (40°54.658'N, Millar 2002). Also, O. agrili Zhang and Huang, 2005 124°16.305'E) from May to September 2014, and was introduced from China to the United States of 2–5 May 2016. The parasitised host eggs were America (Michigan) in 2007 for biocontrol of the placed in 1.5 × 7.5 cm glass vials and reared at the invasive emerald ash borer, Agrilus planipennis room temperature in the Key Laboratory of Forest Fairmaire, 1888 (Coleoptera: Buprestidae), which Protection, China State Forestry Administration, feeds on ash trees (Fraxinus Linnaeus; Oleaceae) and at the Research Institute of Forest Ecology, has become a devastating ash tree pest in North Environment and Protection, Chinese Academy America (Federal Register 2007; Liu et al. 2007; of Forestry (CAF), Beijing, China. Newly Duan et al. 2012, 2014; Abell et al. 2014; Bauer et al. emerged adult egg parasitoids were either killed 2015). Most recently, O. primorskyensis Yao and directly in 100% ethanol for molecular studies or Duan, 2016 from the Russian Far East, is being killed with carbon dioxide and critical point dried evaluated for possible use in biocontrol of the emer- for card or slide-mounting. Parasitoid wasps ald ash borer in North America (Larson and Duan that emerged from the A. fleischeri eggs collected 2016; Yao et al. 2016). in 2013 were also reared individually on Research in northeast China on Agrilus A. planipennis eggs freshly laid on coffee filter fleischeri Obenberger, 1925 (Coleoptera: paper (Larson and Duan 2016) for one generation Buprestidae), an emerging wood-boring pest of in the quarantine laboratory at the United States Populus Linnaeus (Salicaceae), led to the Department of Agriculture, Agricultural Research discovery of several species of insect natural Service, Beneficial Insects Introduction Research enemies. These included two new species of Unit, Newark, Delaware, United States of Oobius that parasitise eggs of A. fleischeri,with America. While some laboratory-reared (F1) combined egg parasitism rates on poplar trees parasitoids were designated as paratypes, most ranging from 7% to 48% (Zang et al. 2016). These were used for DNA sequencing for molecular two species are described here as O. saimaensis phylogenetic analysis at the Laboratories of Yao and Mottern, new species and O. fleischeri Analytical Biology (Washington, District Yao and Duan, new species. Herein, we describe of Columbia, United States of America), the morphological, biological, and molecular dif- Smithsonian Institution, National Museum of ferences between these two species, and contrast Natural History (NMNH). them to the morphologically similar congeners O. agrili (sympatric) and O. primorskyensis Taxonomic studies (allopatric), both of which parasitise emerald ash Selected parasitoid specimens were slide- borer eggs on ash trees. In addition, we sequenced mounted in Canada balsam (Platner et al. 1999) and analysed several ribosomal and mitochondrial for detailed examination. All specimens were genes to infer their phylogenetic relationships. examined under a ZEISS Stemi 508 stereomicro- scope (Bio-Research Scientific Cooperation, Agent Company, Beijing, China). Slide-mounted Materials and methods specimens were imaged using a Leica DM6000B microscope with a Leica DFC 300FX high Sample collection and rearing sensitivity digital camera (Leica Microsystems, Parasitised A. fleischeri eggs, which are darker Wetzlar, Gemany) according to the methods in colour than healthy eggs, were first collected described in Yao et al. (2016). Some specimens from European black poplar trees (Populus nigra were also examined in ethanol to determine the Linnaeus; Salicaceae) infested with A. fleischeri number of multiporous plate sensillae on the on 18 September 2013 in Saima Village, Liaoning antennae. Images of card-mounted specimens Province, China (40°57.473'N, 124°15.788'E). were generated using Helicon Focus software Additional parasitised A. fleischeri eggs were (Helicon Soft, Kharkiv, Ukraine) after generating collected from other infested exotic poplar an image stack with an Olympus CX31 micro- trees Populus nigra var. italica Münchhausen scope (Olympus, Tokyo, Japan) fitted with a © 2018 Entomological Society of Canada. Parts of this are a work are that of the U.S. Government and therefore such parts are not subject to copyright protection in the United States. Yao et al. 305 Canon EOS 700D digital camera. Scanning elec- (Tiangen Biotech), 10 μLddH2O and 1.5 μL DNA tron microscope images were produced with a template for a final volume of 25 μL. The amplifi- Hitachi/S4800 desktop unit (Hitachi, Tokyo, cationwasperformedin0.2mLtubesinEppendorf Japan). Some specimens were manually cleaned B Mastercycler (Eppendorf, Hamburg, Germany) of external debris with forceps or brushes and with the following profile: incubation for five affixed to 12.7 × 3.2 mm Leica/Cambridge alumi- minutes at 94 °C (initial denaturation step), then for num scanning electron microscope stubs with 35 cycles consisting of 94 °C for 45 seconds, 46 °C carbon adhesive tabs (Electron Microscopy for 45 seconds and 72 °C for one minute and a final Sciences, #77825-12). Stub-mounted specimens extension for five minutes at 72 °C. The sequencing