SYSTEMATICS Phylogeographic Diversity of the Winter brumata and O. bruceata (: Geometridae) in and North America

RODGER A. GWIAZDOWSKI,1,2,3 JOSEPH S. ELKINTON,1 JEREMY R. DEWAARD,2 1 AND MARINKO SREMAC

Ann. Entomol. Soc. Am. 106(2): 143Ð151 (2013); DOI: http://dx.doi.org/10.1603/AN12033 ABSTRACT The European winter , Operophtera brumata (L.), an invasive forest defoliator, is undergoing a rapid range expansion in northeastern North America. The source of this invasion, and phylogeographic diversity throughout its native range, has not been explored. To do this, we used samples from a pheromone-baited trap survey of O. brumata collected across its native range in Europe, and invasive range in North America. Traps in North America also attract a congeneric species, the Bruce spanworm O. bruceata (Hulst), and the western Bruce spanworm O. b. occidentalis (Hulst). From this sampling, we sequenced two regions of the cytochrome c oxidase subunit I mitochondrial gene; one region corresponds to the DNA ÔbarcodeÕ region, the other is a nonoverlapping section. We used these sequences, in combination with sequence data from a recent survey of the Geometridae in western North America, for phylogenetic and phylogeographic analyses to characterize genetic divergence and variation for O. brumata in North America and Europe, and O. bruceata and O. b. occidentalis in North America. We found O. brumata mtDNA diversity to be dominated by a single widespread, and common haplotype. In contrast, O. bruceata shows high haplotype diversity that is evenly distributed throughout North America. Phylogeographic patterns indicate an introduction of O. brumata in British Columbia likely originated from , and suggest the invasive population in northeastern North America may have its origins in the United Kingdom, and/or Germany. We found uncorrected pairwise sequence divergence between Operophtera species to be Ϸ7%. O. b. occidentalis is Ϸ 5% divergent from O. bruceata, has a restricted range in the PaciÞc Northwest, and has unique morphological characters. Together these lines of evidence suggest O. b. occidentalis may be deserving of species status. Additionally, a single morphologically unique Operophtera specimen, similar to O. bruceata, was collected in southern Arizona, far outside the known range of O. bruceata. This suggests that North America may contain further, unsampled, Operophtera diversity.

KEY WORDS DNA barcoding, , , Operophtera brumata, occidentalis

The winter moth, Operophtera brumata (L.) is a forest trolled by the introduction of two from defoliator native to Europe and Asia that has become Europe: the tachinid ßy, albicans (Falle´n), invasive in North America (Troubridge and Fitzpat- and the ichneumonid wasp, flaveolatum rick 1993). Despite its detection in Nova Scotia in the (Gravenhorst). Efforts are now underway to establish 1930s (Embree 1965) it is only in the past decade that C. albicans in New England (J.S.E. et al., unpublished this species has rapidly expanded its range in the data). northeastern United States Ñ causing persistent and In North America, winter moth co-occurs with its widespread defoliation in eastern and congener the Bruce spanworm, Operophtera bruceata nearby states (Elkinton et al. 2010). Outbreaks of (Hulst). Males of both species respond to the same winter moth have occurred in two other regions of pheromone compound (Roelofs et al. 1982), and are North America, namely Nova Scotia during the 1950s captured in the same pheromone-baited survey traps (Cuming 1961), and the PaciÞc Northwest during the (Elkinton et al. 2010). Males of both species are dif- 1970s (Kimberling et al. 1986). In each case, a decade- Þcult to tell apart based on wing patterns, but are long outbreak was successfully and permanently con- readily distinguished by their genitalia (Eidt et al. 1966). However, Elkinton et al. (2010) found some 1 Department of Plant Soil and Science, University of Mas- individuals with ÔintermediateÕ genitalia in central sachusetts, 270 Stockbridge Road, Fernald Hall, Amherst, MA 01003. Massachusetts, where the two species overlap, and this 2 Biodiversity Institute of Ontario, University of Guelph, Guelph, Ontario, Canada. observation is based on several thousand moths col- 3 Corresponding author, e-mail: [email protected]. lected over 3 yr as part of an extensive pheromone trap

0013-8746/13/0143Ð0151$04.00/0 ᭧ 2013 Entomological Society of America 144 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 106, no. 2

survey conducted across North America. Additionally, male moths was based on dissection of male genitalia Elkinton et al. (2010) used nuclear DNA analysis to as described by Elkinton et al. (2010) as well as hind demonstrate the existence of F1 hybrids in their pher- wing and abdominal characteristics as described by omone trap survey. While the hybrids formed a very Troubridge and Fitzpatrick (1993). We also included small percentage (1.9%) of the recovered moths, this specimens of O. rectipostmediana (Inoue) collected result agrees with earlier lab-based hybridizations from Japan. All specimens were collected in phero- (Hale 1989, Troubridge and Fitzpatrick 1993). Al- mone traps and were transferred to individual vials though the Bruce spanworm and winter moth appear and stored at Ϫ80ЊC upon arrival at the University of to share the same pheromone and hybridize, they Massachusetts, Amherst, with the exception of O. bru- differ in their North American distributions. The El- mata from Norway, which were collected by J.S.E., kinton et al. (2010) pheromone trap survey revealed and frozen as larvae. that Bruce spanworm is widespread, occupying inte- DNA Sequence Collection. Mitochondrial (mtDNA) rior regions of and New Brunswick, where sequence variation has been shown to correspond to winter moth is absent despite its long-term presence both morphological and nuclear DNA species-speciÞc nearby in Nova Scotia. Elkinton et al. (2010) proposed differences between O. brumata and O. bruceata (El- that these distributions arise because Bruce spanworm kinton et al. 2010). We used mtDNA sequences from is more cold tolerant than winter moth, and this pat- several sources: novel sequences generated for this tern agrees with a recent study demonstrating that study (described below), those reported from Elkin- cold-temperature limits the distribution of winter ton et al. (2010), those from a DNA barcode survey of moth in Norway (Jepsen et al. 2008). In addition, geometrids in western Canada (deWaard et al. 2011), Bruce spanworm only rarely causes outbreaks ( those from a DNA barcode survey of geometrids in and Lindquist 1982), whereas winter moth popula- Bavaria, Germany (Hausmann et al. 2011), as well as tions in New England are currently in perpetual out- sequences from other species of Operophtera and break phase (J.S.E. et al., unpublished data). These other geometrids from GenBank. Several geometrid outbreaks are similar to those that occurred in Nova species were used as outgroups for phylogenetic anal- Scotia before the establishment of C. albicans (Em- ysis (described below), and these species were chosen bree 1965). based on relatively close, and more distant relation- In western North America a subspecies of Bruce ships as inferred in a recent phylogeny of the spanworm, Operophtera bruceata occidentalis (Hulst), Geometridae (Yamamoto and Sota 2007). Sequences occurs mainly west of the Cascade mountains in Or- generated for this study were obtained as described egon, WA, and British Columbia (Troubridge and below. Fitzpatrick 1993). O. b. occidentalis was originally de- Genomic DNA was extracted from individual moth scribed as the species O. occidentalis (Hulst 1896) but heads using the DNEasy Blood and Tissue Kit (Qia- was subsumed as a subspecies of O. bruceata by Trou- gen, Valencia, CA) according to the manufacturerÕs bridge and Fitzpatrick (1993), despite possessing dis- instructions. The remainder of the body was retained tinct morphological traits. at Ϫ80ЊC. We used polymerase chain reaction (PCR) While the history of O. brumata outbreaks in North to amplify two fragments of the mitochondrial gene America is well documented (see Troubridge and cytochrome c oxidase subunit I (COI). One fragment Fitzpatrick [1993]), the sources and number of inva- corresponds to the mtDNA barcode region (Hebert et sions are unknown. To explore this situation, we pro- al. 2003), and the other fragment begins following the vided pheromone traps to colleagues throughout the 3Ј end of the barcode region; these fragments do not range of O. brumata in Europe, Japan, and throughout overlap. The barcode fragment used for analysis is a the ranges of O. brumata, and O. bruceata in North 615 bp fragment, and the additional COI fragment has America. Here we used specimens from this extensive 736 bp. Respectively, these fragments correspond to sampling effort to describe phylogeographic diversity bases 11,886 through 12,501, and bases 12,616 through within both species and infer their phylogenetic re- 13,352 in the Bombix mori (L.) mitochondrial genome lationships relative to other Operophtera species by (NCBI Reference Sequence NC_002355.1). sequencing two regions of cytochrome c oxidase sub- For the ampliÞcation of the barcode region, a 20 ␮l unit I (COI) mitochondrial DNA. We used our results reaction mixture contained 10 ␮l of Qiagen HotStar- to infer the potential number, and origin of invasions Taq Master Mix (Cat. No 203446, Qiagen GmbH of O. brumata to North America, and explore the D-40724 Hilden), 2 ␮lof10␮M forward primer LCO- possibility for cryptic diversity in both O. brumata and 1490 (Folmer et al. 1994) and 2 ␮lof10␮M reverse O. bruceata. primer HCO-2198 (Folmer et al. 1994), 0.8 ␮lof25mM ␮ ␮ MgCl2,3 l of template DNA and 2.2 l of sterile dH O. AmpliÞcation of the second gene fragment Materials and Methods 2 used the same 20 ␮l reaction mixture with the forward Sampling. Specimen Collection. The majority of primer ÔJerryÕ (Simon et al. 1994) and the reverse specimens are the species O. brumata, O. bruceata, and primer ÔPatÕ (Simon et al. 1994). All ampliÞcations the subspecies O. b. occidentalis, that were collected as were performed with a GeneAmp PCR System 9700 part of a recent, geographically extensive pheromone thermal cycler (Applied Biosystems, Foster City, CA) trap survey across North American and Europe re- using the following conditions: 15 min at 95ЊC fol- ported in Elkinton et al. (2010). Initial identiÞcation of lowed by 30 cycles of 30 s at 94ЊC,30sat56ЊC for the March 2013 GWIAZDOWSKI ET AL.: WINTER MOTH PHYLOGEOGRAPHY 145 barcode region, and 52ЊC for the second fragment, 1 outgroup taxa and divergent Operophtera specimens. min at 72ЊC, and a Þnal extension for 10 min at 72ЊC. Pairwise comparisons were calculated using PAUP* PCR products were cleaned using Exo SAP-IT en- 4.0b10 (Swofford 2003). zymatic digestion (USB Corporation, Cleveland, mtDNA Haplotype Networks. We diagramed haplo- OH). Sequencing of PCR products in both direc- type networks for O. brumata collected from North tions was performed on a 3730 DNA Analyzer (Ap- America, and throughout the native range in Europe. plied Biosystems) by Laragen, Inc. (Los Angeles, We also diagramed networks for O. bruceata through- CA). Sequences were edited using Sequencher 4.2 out North America, and O. b. occidentalis from the (Gene Codes Corporation, Ann Arbor, MI). Align- PaciÞc Northwest. Haplotype distances for both bar- ment of the barcode and additional COI fragments code and barcodeϩ sequences were calculated using was unambiguous. Arlequin 3.5 (ExcofÞer and Lischer 2010) and initial Molecular Analyses. mtDNA Phylogeny. For all anal- networks were calculated using HapStar (Teacher and yses, each unique haplotype is represented by a single GrifÞths 2011). Networks were edited so the area of a sequence. Haplotypes are given a number designation circle representing a haplotype is directly propor- relative to the individuals in that haplotype (e.g., hap- tional to the number of individuals with that haplo- lotype 1 [H1] has the most individuals, haplotype 2 type. The maps used in Fig. 1 are available from Wi- [H2] has the second most, etc.). The majority of kimedia commons (http://en.wikipedia.org/wiki/ specimens in our dataset are only represented by the File:North_america98.svg and http://en.wikipedia. barcode locus. To take advantage of any additional org/wiki/File:Location_European_nation_states.svg). information provided by specimens for which we have The specimens for which we have an additional frag- the additional COI sequence, we include haplotypes ment of COI (barcodeϩ), offer a complimentary da- based on the concatenated barcode plus additional taset that may provide additional resolution for a lo- COI sequence. We refer to these concatenated se- cation of origin, and we calculated separate quences as barcodeϩ, which have the same base networks for the barcode and the concatenated haplotype designation as the barcode haplotype (e.g., barcodeϩ datasets. H1). We conducted parsimony and Bayesian analyses to Results estimate phylogenetic relationships among several Operophtera species. Parsimony phylogenies were We used 632 barcode fragments of COI comprising conducted with PAUP 4.0b10 (Swofford 2003) using a 327 O. brumata specimens collected from 10 European heuristic search with 10 random-addition-sequence countries, and 10 North American states and prov- starting trees per step and treeÐbisectionÐreconnec- inces, along with 305 O. bruceata specimens collected tion branch swapping, and the maxtrees option equal from 19 North American states and provinces. Spec- to 500. Bayesian phylogenies were estimated using imens that we had both barcode and the additional BEAST 1.6.1 (Drummond and Rambaut 2007). In this COI fragment comprised 177 O. brumata from 8 Eu- analysis, the loci were concatenated and partitioned ropean countries as well as 14 states and provinces in by codon. All partitions were analyzed using a HKY North America, and 80 O. bruceata from 16 states and substitution model (four gamma categories), with a provinces in North America. All novel sequences gen- constant size coalescent tree prior under the assump- erated for this study have been deposited in GenBank tion of a strict clock, randomly generated starting tree, and the Barcode of Life Data System (BOLD: dx. and a piecewise linear and constant root population doi.org/10.5883/DS-PDOTWM, www.barcodinglife. size model. All other priors and operators were left at org). The GenBank accession numbers for the bar- default values. All analyses were run on Mac Pro quad code locus are JQ615968 - JQ616489, and the core computers of the Biology Computer Resource additional COI locus are JQ616490ÐJQ616772. Indi- Center at the University of Massachusetts. Samples vidual GenBank and BOLD accession numbers, along from the posterior were taken every 1 ϫ 103 steps and with all locality and ancillary information, are pro- chains were run for 5 ϫ 107 steps. Posterior distribu- vided in supplementary Table 1 (available online tions for each analysis were summed from three in- only). dependent chains after removing the Þrst 25% of sam- Phylogeographic Diversity. The overall topology of ples as burn-in from each chain. Convergence for our mtDNA phylogeny (Fig. 1) is consistent with the individual runs was visually inspected using the pro- recent geometrid phylogeny by Yamamoto and Sota gram Tracer v1.5 (Rambaut and Drummond 2007). (2007) where almost all specimens identiÞed as Oper- Posterior probabilities were also evaluated in Tracer, ophtera are placed within a monophyletic . based on the effective sample size (ESS) where an ESS A single moth (specimen 263) tentatively identiÞed as of Ͼ200 suggests sufÞcient sampling from the poste- “Operophtera sp.” was placed among the outgroup rior (Ho and Lanfear 2010). taxa. Subsequent querying of specimen 263Õs COI bar- mtDNA Sequence Divergence. We characterized code region on BOLD resulted in a perfect match to phylogenetic diversity in terms of potential species specimens of aceraria Denis & Schiffermu¨ l- diversity based on sequence divergence within Oper- ler 1775, a distantly related geometrid moth that like- ophtera. To do this, we used COI sequences to calcu- wise ßies late in the season. The parsimony analysis late pairwise uncorrected P values between the Þve produced an overall topology that is consistent across nominate species of Operophtera, as well as several all most parsimonious trees, but conßicts in several 146 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 106, no. 2 March 2013 GWIAZDOWSKI ET AL.: WINTER MOTH PHYLOGEOGRAPHY 147

Fig. 2. Haplotype networks for Operophtera brumata collected in North America and Europe. The two sets of networks display the proportion of the same genotypes collected on the respective continent. Each circle is a unique haplotype, and the area of each circle is directly proportional to the number of specimens sharing that haplotype. Colors in each circle correspond to the proportion and geographic location of specimens sharing that haplotype. Each line segment connecting haplotypes is proportional to single character state changes (mutations) between haplotypes. Haplotype designations (e.g., H1) and the number of specimens in that haplotype are placed adjacent to the haplotype. Haplotype designations occurring in both the United States and Europe are indicated in red. Networks for each continent are divided into networks for the barcode region, as well as the combined barcode and barcodeϩ regions, to visualize any additional information added by the barcodeϩ region. (Online Þgure in color.) places with a Bayesian consensus topology. The par- Europe and the United States, including H1, which simony nodes supporting the Hagnagora/Epirrita– was present in all sampled localities except for Nothoporinia split are reversed in the Bayesian con- Norway. The geographic distribution of all O. bru- sensus topology that also places O. danbyi (Hulst) as mata haplotypes is presented in Fig. 1. The wide- basal to the rest of the Operophtera, and shows poor spread H1 (clade B) was also present in Sweden, resolution for the O. fagata (Scharfenberg)/O. bru- Scotland, and Germany along with haplotypes from mataÐO. bruceata split. Nodes that are well supported clade A. by both parsimony and Bayesian analysis distinguish Within clade B, we found a haplotype (H4), which two clades within O. brumata, a distinction between was only recovered from British Columbia and Ger- specimens recognized as O. bruceata and O. bruceata many. Several unique O. brumata haplotypes occur occidentalis, and a distinction between O. b. occiden- only in North America (H6, 490, PFC 2007, and 587). talis, and a single specimen collected (in Arizona) These samples are phylogenetically closely related to from far outside the known range of O. b. occidentalis H1 (Fig. 2), suggesting they could also occur through- (Troubridge and Fitzpatrick 1993). out most of southern Europe. The population structure within our sampling of The species O. bruceata and subspecies O. b. occi- European O. brumata roughly corresponds to a geo- dentalis both show extensive haplotype diversity rel- graphic division where most haplotypes of clade A ative to O. brumata (Figs. 1Ð4), and are phylogeneti- (Fig. 2) are predominantly from Norway, Scotland, cally well supported as distinct clades by both and Sweden. However, three individuals of H2, from Bayesian and parsimony analyses. Within O. bruceata, the study of Hausmann et al. (2011) are recovered some haplotypes are unique to the PaciÞc Northwest from Bavaria, Germany. The individuals from clade and Alaska, but the overall lack of phylogeographic A that occur in North America were only collected structure in our sampling (Fig. 2) is consistent with a in Nova Scotia, and Long Island, NY. Clade B con- large population occurring across most of North tains the remainder of the diversity recovered from America (but see Bazin et al. 2006). Within O. b.

Fig. 1. Mitochondrial phylogeny of Operophtera and outgroup taxa. Shown is a maximum parsimony topology with node labels indicating presence and support of those nodes in a Bayesian analysis. NA indicates a node not present in the Bayesian analysis. Haplotypes are designated by haplotype number, and followed by the number of specimens in that haplotype (e.g., WM H1 109). Haplotypes and specimens marked with an asterisk comprise the barcode and barcodeϩ regions, those without an asterisk comprise just the barcode region. Branch lengths are directly proportional to the number of changes along that branch, and a scale bar is provided below. 148 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 106, no. 2

Fig. 3. Haplotype networks for Operophtera brumata collected throughout North America. Haplotype presentation is as described for the haplotypes in Fig. 2. (Online Þgure in color.) occidentalis the phylogeographic pattern of haplo- Discussion types also suggests a large range-wide population (Fig. The vast majority (Ϸ89%) of O. brumata in our 4). Although our sampling for this subspecies is biased samples from North America, and Europe belong to a by a high density of specimens collected in , the pattern of haplotype diversity using the barcode single haplotype (H1). In North America, H1 com- prises 93% of specimens, whereas in Europe, which has region (Fig. 4) suggests this subspecies shows similar Ϸ phylogeographic structure to O. bruceata. O. b. occi- higher haplotype diversity, H1 comprises only 71% dentalis does not appear to have a level of intraspeciÞc of specimens. The extreme prevalence of this haplo- mtDNA variation similar to O. brumata or O. bruceata. type on both continents reduces the available mtDNA This pattern highlights the closely related, but phylo- diversity to infer the number and origin of introduc- genetically distinct single specimen, 09ÐJDWGEOÐ tions. Additionally, while H2 was found in the original 463. This specimen, deposited in the National Museum introduction site in Nova Scotia and New York, and of Natural History (USNM ENT 00718320), is geo- predominates in Norway, Scotland, and Sweden. It is graphically disjunct from the coastal range (Alaska to also recovered in southern Germany that suggests this southern California) of O. b. occidentalis. It also has a haplotype may have a distribution similar to H1, albeit much larger body and wing size than all other native at lower proportions in the population. Winter moths North American Operophtera (J.R.W., unpublished are known to have been established in Nova Scotia in data). the 1930s (Embree 1965), and given the large amount Interspecific Divergence. Mitochondrial uncor- of commerce and trade between Nova Scotia and rected pairwise percent divergences between Oper- Britain, the latter is a likely source of North American ophtera species are Ϸ7Ð8% (Fig. 5). Notably the sub- winter moths. We found a unique haplotype match species O. b. occidentalis is Ͼ7% divergent from other between three moths from the city of Vancouver, a Operophtera, and Ϸ5% divergent from the nominate major port in British Columbia, and a single moth species O. bruceata. The unidentiÞed specimen 09Ð collected in Germany (Fig. 1) that all belong to H4. JDWGEOÐ463 is Ϸ5% divergent from O. bruceata, and This association probably represents a more recent, 2.4% divergent from O. b occidentalis. and separate introduction to Vancouver from Europe. March 2013 GWIAZDOWSKI ET AL.: WINTER MOTH PHYLOGEOGRAPHY 149

Fig. 4. Haplotype networks for Operophtera brumata occidentalis collected in North Western North America. Haplotype presentation is as described for the haplotypes in Fig. 2. (Online Þgure in color.)

And so, as H1 and H2 are both present in Germany, it While the mtDNA loci used here may not have is also possible this country may be a source of the enough resolution to pinpoint the origins of introduc- North American invasion. An additional, albeit small, tion, the phylogeographic patterns suggest new infer- inßuence on O. brumata haplotype diversity may ences about the biology and diversity of winter moths come from hybridization with O. bruceata, as these in Europe and North America. Mitochondrial DNA species hybridize in the lab (Troubridge and Fitzpat- variation in O. brumata is lower than in O. bruceata rick 1993), and in the Þeld (Elkinton et al. 2010). (Figs. 1 and 2), and this agrees with an earlier study

Fig. 5. Matrix of pairwise percent divergences between selected outgroup taxa (Lambdina fiscellaria (Guenee) and Epirrita pulchraria (Taylor)), and species of Operophtera. Haplotypes are labeled with their designation (e.g., H1); the Undet, AZ, haplotype is from specimen 09ÐTDWGEOÐ463. Cells are colored from light to dark according to a scale of increasing divergence in increments of 0Ð1, 1Ð3, 3Ð6, 6Ð10, and Ͼ10%. 150 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 106, no. 2

Þnding low O. brumata allozyme variation in Belgium most extensive sampling conducted for Operophtera in (Van Dongen et al. 1998). Despite this low variation, North America, pheromone traps were not deployed our sampling of O. brumata shows relatively higher in this region because it is considered to be outside the phylogeographic structure than O. bruceata. In Eu- range of O. bruceata (Troubridge and Fitzpatrick rope, O. brumata H1 predominates in the southern 1993). This individual is most closely allied to O. b. part of the continent, and H2 is only found in northern occidentalis (Fig. 1), but its COI sequence is Ϸ2.5% Europe (Germany, Norway, Scotland, and Sweden), divergent from O. occidentalis, whereas the largest whereas our sampling of O. bruceata haplotypes shows intraspeciÞc variation within O. bruceata is Ϸ1.6%, and very little structure across North America. It is possi- much less in O. b. occidentalis (Fig. 5). In addition, this ble these species-speciÞc differences are because of specimen has a considerably larger wing span, body disparities in population biology. O. brumata popula- size, and slightly lighter wing color than either O. b. tions have extensive outbreaks of approximately de- bruceata or O. brumata. This specimen possibly rep- cade-long cycles (Varley and Gradwell 1968), resents the Ôlight formÕ of O. occidentalis, described as whereas O. bruceata populations outbreaks usually last Rachela (now Operophtera) latipennis (Hulst 1896). 2Ð3 yr and tend to occur on local scales, particularly in Canada (Rose and Lindquist 1982). It is possible that However, Hulst only recorded this as from ÔCaliforniaÕ, selective sweeps occur more readily in O. brumata and as only a single type exists (as a dark morph) populations during these widespread outbreak phases, Troubridge and Fitzpatrick (1993) have synonymized reducing overall mtDNA variation (Galtier et al. latipennis with O. b. occidentalis. Despite the unclear 2009). In addition, the complex glacial history of Eu- species assignment of our specimen, its unique geo- rope has inßuenced the phylogeographic structure for graphic, morphological, and genetic variation merits many organisms (Hewitt 2000). The distribution of further taxonomic scrutiny for similar specimens in northern and southern O. brumata H1 and H2, could collections, and suggests further undiscovered Oper- be because of isolation in glacial refugia. ophtera diversity may be present throughout western Cryptic Diversity Within O. bruceata. O. bruceata North America. has several groups that can be identiÞed by congruent morphological, molecular, and geographic patterns, suggesting O. bruceata may be a complex of several Acknowledgments species. The nominate O. bruceata (Fig. 2), has low phylogeographic structure, suggesting a large inter- We thank Nathan Havill, Andrew Liebhold, and George breeding population spread across most of northern “Jeff” Boettner, who provided valuable comments on early North America (Fig. 3), possessing a relatively low versions of this manuscript. For help in the Þeld, lab, and with range of within-group mtDNA divergence (0.3Ð1.8%; data preparation we thank Roy Hunkins and George “Jeff” Fig. 5). O. b. occidentalis has unique morphological Boettner, Gloria Witkus, Suzanne Lyon, and Annie Paradis. features of the wing and abdomen color (Troubridge We thank Vic Mastro, Ashot Khrimian, Dave Lance, Julie and Fitzpatrick 1993), which are correlated with a Calahan, Natialie Leva, Nichole Campbell, and Patricia restricted distribution along the PaciÞc coast. For Douglas for helping to organize the pheromone trap survey. these reasons this subspecies has sometimes been And we also thank the following people for assisting with the pheromone trap setup, deployment and collection of spec- treated as a separate species (Miller and Cronhardt imens: In The UNITED STATES: F. Stephen: AR; V. Smith, 1982). We found specimens collected in the range of N. Campbell, P. Trenchard, D. Ellis: CT; V. DÕAmico: DE; D O. b. occidentalis form a monophyletic group (Fig. 1), & C Adams, C. Burnham, K. Gooch: MA; K. Coluzzi, C. and mtDNA divergence between O. b. occidentalis and Donahue, J. Crowe: ME; D. Zumeta, A. Jones: MN; R. Kim- O. bruceata is Ϸ 5% (Fig. 5). This divergence is similar mich, C. Elkinton, J. Miller: OR; J. Tatum, J. Weaver, J. to the Ϸ7% we observed in other species of Operoph- Wiener: NH; S. Vaiciunas: NJ; K. Carnes, P. Jentsch, D. tera (Fig. 5), and is higher than Ϸ3% divergence cor- Gilrein, H. McGinnis: NY; J. Simmons, S. Ga, J. Stimmel: PA; responding to species-level divergence in other S. Baxter, C. Sparks, H. Faubert: RI; R. Fine, B. Burns, P. geometrids from the PaciÞc Northwest (deWaard et Hansen: VT; E. La Gasa: WA; A. DissÐTorrance: WI; A. al. 2011). O. b. occidentalis has been considered a full Liebhold: WV; D. McCullogh: MI. In CANADA: L. Humble, species (O. occidentalis (Hulst)) until lowered to G. ZlahiÐBalogh, V. Nealis: British Columbia; R. Webster: subspeciÞc status by Troubridge and Fitzpatrick New Brunswick; B. Lyons: Ontario; J. Regniere, P. Duval: (1993) in a reasonable attempt to organize the ge- Quebec. In EUROPE: G. Hoch: ; F. Herard, C. Bonnet: nus. However, our data, combined with unique mor- France; F. Kruger: Germany; A. Battisti: ; J. Jepson, T. phology noted by Troubridge and Fitzpatrick Scott: Norway; L. Sukovata: Poland; Y. Baranchikov: Russia; (1993), suggests that O. b. occidentalis could be M. Galendekic: Serbia; M. Lombadero: Spain; H. Bylund: Sweden; M. Kenis: Switzerland; A. Watt, H. Evans, C. Tilbery, elevated back to full species. A. Vanbergen: UK. In JAPAN: Y. Higashiura. Lastly, we apol- Additionally, we found a single morphologically dis- ogize to anyone we failed to list. This work was supported by tinct, and phylogenetically divergent (Fig. 1) speci- a grant from the Massachusetts State Legislature, USDA men (09-JDWGEO-463 image available at http:// Forest Service and USDAÐAPHIS (Animal and Plant www.barcodinglife.com/, public data:GNAU163Ð10) Health Inspection Service). J.R.D. was funded by The collected near Mount Lemmon, in the Sky Islands of Natural Sciences and Engineering Research Council of southwestern Arizona, which was not collected at a Canada and by Genome Canada through the Ontario pheromone trap. Despite our trap survey being the Genomics Institute. March 2013 GWIAZDOWSKI ET AL.: WINTER MOTH PHYLOGEOGRAPHY 151

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