J. ENTOMOL. SOC. BRIT. COLUMBIA 107, DECEMBER 2010 25 DNA barcoding identifies the first North American records of the Eurasian moth, Eupithecia pusillata (Lepidoptera: Geometridae) JEREM Y R. deW AARD1,2, LELAND M . HUM BLE1,3 and B. CHRISTIAN SCHM IDT4 ABSTRACT The first North American records of the juniper pug moth, Eupithecia pusillata (Denis & Schiffermüller, 1775) (Lepidoptera: Geometridae), brought to our attention using DNA barcoding, are presented. Documentation and collection localities suggest it was introduced, established, and likely has persisted, at least in the Greater Vancouver area of British Columbia since the mid-1970s. W e discuss the integration of DNA barcoding into routine biosurveillance and forest insect surveys to prevent such delay in recogni- tion of non-indigenous species–in this case, 34 years. Key W ords: Eupithecia pusillata, Eupithecia interruptofasciata, Eupithecia niphado- philata, juniper pug moth, Juniperus, non-indigenous species, invasive species, DNA barcoding INTRODUCTION DNA barcoding of biological specimens and Budinoff 2005), agromyzid leafminers has demonstrated repeatedly its utility as a (Scheffer et al. 2006), tephritid fruit flies molecular diagnostic technique that merits (Armstrong and Ball 2005; Barr 2009), integration into biosurveillance programs. siricid wasps (W ilson and Schiff 2010), true In contrast to other molecular tools com- bugs (Nadel et al. 2010), and numerous taxa monly employed for species identification of moths (Ball and Armstrong 2006; Si- of intercepted organisms, DNA barcoding monsen et al. 2008; Humble et al. 2009; is a generic and standardized approach that deW aard et al. 2009; Gilligan and Epstein meets international standards of data quality 2009; Armstrong 2010). Here we report the and transparency (Floyd et al. 2010). Sev- first North American records of the juniper eral studies have demonstrated the efficacy pug moth, Eupithecia pusillata (Denis & of this technique for detecting non- Schiffermüller, 1775) revealed by DNA indigenous species and determining native barcoding. provenance, for example in leeches (Siddall M ATERIALS AND M ETHODS W hile compiling a DNA barcode library (deW aard et al., submitted), the cytochrome for the Geometridae of British Columbia c oxidase subunit I (COI) sequences de- 1 University of British Columbia, Department of Forest Sciences, Forestry Sciences Centre, Vancouver, BC, Canada V6T 1Z4 2 Royal British Columbia Museum, Entomology, 675 Belleville Street, Victoria, BC, Canada V8W 9W 2 (email: jdewaard@ interchange.ubc.ca) 3 Natural Resources Canada, Canadian Forest Service, Pacific Forestry Centre, 506 W est Burnside Road, Victoria, BC, Canada V8Z 1M5 (email: Leland.Humble@ nrcan-rncan.gc.ca) 4 Canadian Food Inspection Agency, Canadian National Collection of Insects, Arachnids and Nematodes, K.W . Neatby Building, 960 Carling Avenue, Ottawa, ON, Canada K1A 0C6 (email: Chris.Schmidt@ inspection.gc.ca) 26 J. ENTOMOL. SOC. BRIT. COLUMBIA 107, DECEMBER 2010 rived from two Eupithecia specimens were Natural Resources Canada, Canadian Forest found to be divergent from known native Service, Pacific Forestry Centre, Victoria, Eupthecia. The two sequences were com- BC), were dissected to examine the genita- pared to a reference barcode database of lia following the methods given by Lafon- Lepidoptera barcodes using the identifica- taine (2004). Images of genitalia were taken tion engine (BOLD-ID) of the Barcode of using a Leica M205C microscope equipped Life Data Systems (BOLD) (Ratnasingham with a Leica DFC490 camera kit and Leica and Hebert 2007), and tentatively identified LAS Montage system that assembles multi- as Eupithecia pusillata, a Eurasian species ple images in successive planes of focus not known to occur in North America. The into a single image with a large depth of reference barcode database for Geometridae field. The specimens were verified by com- used by BOLD-ID is continually validated parison of the structure of genitalia with by specialists to ensure accurate identifica- specimens held in the CNC (Canadian Na- tions, and is particularly well parameterized tional Collection of Insects, Arachnids and due to a global campaign to barcode the Nematodes, Ottawa, ON), and figures of E. nearly 23,000 species of the family (see pusillata in Skou (1986) and Mironov http://www.lepbarcoding.org/geometridae/ (2003). Related species in the E. niphado- index.php). The nine sequences with identi- philata Dyar, 1904 group (Bolte 1990) were cal and near-identical matches from Europe ruled out by genitalic comparison to speci- were obtained from Axel Hausmann mens in the CNC, as were other North (Zoological State Collection, Munich, Ger- American species. many) and Marko Mutanen (University of Historical data associated with the Oulu, Oulu, Finland) and combined with specimens were compiled from specimen related North American specimens (sensu labels and Forest Insect and Disease Survey Bolte 1990). A neighbour-joining tree was (FIDS) records (Van Sickle et al. 2001). constructed on BOLD using the Kimura-2- The single specimen from PFCA, collected parameter distance method (Fig. 1). by FIDS, is uniquely identified by a regis- To pursue confirmation of the identity tration number (e.g. 76-9-0019-01) that of the specimens, the two putative E. pusil- links the specimen to a FIDS sampling lata specimens obtained from the RBCM form, completed at the time of sample col- (Royal British Columbia Museum, Victoria, lection, as well as a rearing record docu- BC) and PFCA (Arthropod reference col- menting the status of laboratory rearings. lection, Pacific Forestry Centre (PFC), These records are held on file at PFC. RESULTS Specimens examined: 1I œ label data 1976. The host recorded was common juni- (handwritten information in italics, individ- per (Juniperus communis L.); Remarks & ual lines separated by comma, multiple Symptoms state —Attacking several orna- labels separated by ”|‘): mentals with moderate damage“. The date No. 76-9-0019-01, Date 19 vii, F.I.[D.] recorded on the specimen label is the date S.1976 | c. juniper, Port, Coquitlam BC | of adult eclosion. W hile the Rearing Record Ac. No. PFC, 2007-0271. indicates a second adult eclosed on 8.vii.76 The specimen was initially identified as and was subsequently spread, the specimen Eupithecia unicolor (Hulst). The FIDS re- could not be found in the PFCA reference cords document that this specimen was one collection. of two adults reared from five larvae and 1R œ label data: five pupae (10 individuals in total) col- BC, N. Vancouver, 5 AUG 1986, C.S. lected by the B.C. Forest Service on Mt. Guppy | ROYAL BRITISH, COLUMBIA Burke, Port Coquitlam (UTM 10 53 546 MUSEUM, ENT991-12573 |. [49.3, -122.7], Elevation 900 ft), on 15 May This specimen was identified as J. ENTOMOL. SOC. BRIT. COLUMBIA 107, DECEMBER 2010 27 Figure 1. Neighbour-joining tree of Eupithecia pusillata and two closely related species, E. niphadophilata and E. interruptofasciata. Tree was reconstructed with the barcode fragment of the cytochrome oxidase I (COI) gene. Sequences shaded in grey are from two individuals col- lected in Vancouver, Canada. Abbreviations: DE œ Germany, FI œ Finland, IT œ Italy, CA œ Canada, BC œ British Columbia, AB œ Alberta. Eupithecia sp. in the collection before ten- Description: A small moth with a wing- tative assignment to Eupithecia intricata span of 16œ22 mm (Mironov 2003) (Figs. taylorata Swett by JRD. 2a, 2e). Forewing narrow, mostly shades of Diagnosis: Eupithecia pusillata is most light brown with black transverse lines and similar to E. niphadophilata and particu- oblong discal spot. Hindwing pale grey- larly E. interruptofasciata, but a number of brown with weakly marked transverse lines Eupithecia species are superficially very and variable discal spot. Abdomen pale similar and identification should be based grayish brown with narrow black lateral on examination of genitalia. Compared to stripes. Male genitalia (Fig. 2d) composed E. interruptofasciata, which is structurally of broad valva with small ventral process, most similar, the male 8th sternite apical heavily sclerotized sacculus, vesica with prongs are narrower, more blunt and the three horn-like cornuti, simple aedeagus apical cleft is shallower; the base of the (Fig. 2c) and elongated 8th sternite with two sternite is also narrower overall with a shal- narrow apical processes (Fig. 2b). Female lower medial invagination. The basal half genitalia composed of elongate and scle- of the male vesica is armed with one spine, rotized bursa copulatrix (Fig. 2h) with small not two as in E. interruptofasciata. In the spines at base and larger spines at margin. female genitalia, the large spines on the left Ovipositor is simple with long setae (Fig. side of the ductus bursae do not extend be- 2f). Terminal segment of pupal case is stout yond the mid-point of the ductus, but ex- with prominent lateral lobes and cremaster tend beyond the midpoint in both E. inter- bearing four pairs of hook-like setae (Figs. ruptofasciata and E. niphadophilata. 2h, 2i). 28 J. ENTOMOL. SOC. BRIT. COLUMBIA 107, DECEMBER 2010 Figure 2. Morphology of Eupithecia pusillata. a) male, dorsal view, b) male, 8th sternite, c) male, aedeagus, d) male, genital capsule e) female, dorsal view, f) female, ovipositor, g) fe- male, bursa copulatrix, h) pupa, terminal segment, dorsal view, i) pupa, terminal segment, lat- eral view. Scale bars: a, e = 5 mm; bœd, fœi = 0.5 mm. A colour version of this figure is available from Dr. Lee Humble. Distribution and Habitat: In its native E. pusillata is present in the Caucasus European range, the nominate subspecies is Mountains (Mironov 2003). In Asia, its widely distributed from southern Europe, range extends across Russia from Sahkalin its range extends to the Mediterranean from through Siberia, the Altai and Caucasus eastern Spain to mainland Greece and Ro- regions (Skou 1986). The subspecies E. mania, then extends north and west across pusillata scoriata Staudinger, 1857 has northern Ukraine into western Russia. W ith been recorded only from Iceland and south- the exception of Corsica, it has not been western Greenland (Mironov 2003).