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2-2009 Mitochondrial DNA Variation and Range Expansion in (: ): No Evidence for a Recent Population Bottleneck Nicholas J. Miller Iowa State University

David L. Dorhout Iowa State University

Marlin E. Rice Iowa State University, [email protected]

Thomas W. Sappington Iowa State University, [email protected] Follow this and additional works at: http://lib.dr.iastate.edu/ent_pubs Part of the Agronomy and Crop Sciences Commons, Cell Biology Commons, Entomology Commons, Genetics Commons, and the Systems Biology Commons The ompc lete bibliographic information for this item can be found at http://lib.dr.iastate.edu/ ent_pubs/213. For information on how to cite this item, please visit http://lib.dr.iastate.edu/ howtocite.html.

This Article is brought to you for free and open access by the Entomology at Iowa State University Digital Repository. It has been accepted for inclusion in Entomology Publications by an authorized administrator of Iowa State University Digital Repository. For more information, please contact [email protected]. Mitochondrial DNA Variation and Range Expansion in Western Bean Cutworm (Lepidoptera: Noctuidae): No Evidence for a Recent Population Bottleneck

Abstract The ew stern bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is a pest of both corn and dry bean crops. At the beginning of the 21st century, the species began to extend its range out of the Great Plains, eastward through the Corn Belt. This rapid range expansion is remarkable because the species distribution had been stable for at least the previous half century, despite the apparent abundance of suitable habitat (i.e., cornfields) immediately to the east. We hypothesized that if the western bean cutworm had to overcome a stable barrier to movement before starting the current range expansion, it probably experienced a genetic bottleneck in doing so. To test this hypothesis, variation in the mitochondrial NADH dehydrogenase one (ND1) gene was studied in populations from Wyoming, Nebraska, and Iowa. No differences in overall genetic diversity or haplotype frequencies indicative of a bottleneck were observed between the recently founded populations in Iowa and the established populations in Wyoming and Nebraska. This result suggests that the sudden loss of an ecological exclusion mechanism, allowing the species to move east in appreciable numbers, is more likely to have triggered the range expansion than the surmounting of an extrinsic barrier to movement. The an ture of this mechanism is unknown but might be related to recent changes in corn farming practices and technology.

Keywords NADH dehydrogenase 1, Striacosta albicosta, Zea mays, biological invasion

Disciplines Agronomy and Crop Sciences | Cell Biology | Entomology | Genetics | Systems Biology

Comments This article is from Environmental Entomology 38 (2009): 274, doi:10.1603/022.038.0134

Rights Works produced by employees of the U.S. Government as part of their official duties are not copyrighted within the U.S. The onc tent of this document is not copyrighted.

This article is available at Iowa State University Digital Repository: http://lib.dr.iastate.edu/ent_pubs/213 MOLECULAR ECOLOGY AND EVOLUTION Mitochondrial DNA Variation and Range Expansion in Western Bean Cutworm (Lepidoptera: Noctuidae): No Evidence for a Recent Population Bottleneck

1,2 3,4 3 NICHOLAS J. MILLER, DAVID L. DORHOUT, MARLIN E. RICE, AND THOMAS W. SAPPINGTON1

Environ. Entomol. 38(1): 274Ð280 (2009) ABSTRACT The western bean cutworm, Striacosta albicosta (Smith) (Lepidoptera: Noctuidae), is a pest of both corn and dry bean crops. At the beginning of the 21st century, the species began to extend its range out of the Great Plains, eastward through the Corn Belt. This rapid range expansion is remarkable because the species distribution had been stable for at least the previous half century, despite the apparent abundance of suitable habitat (i.e., cornÞelds) immediately to the east. We hypothesized that if the western bean cutworm had to overcome a stable barrier to movement before starting the current range expansion, it probably experienced a genetic bottleneck in doing so. To test this hypothesis, variation in the mitochondrial NADH dehydrogenase one (ND1) gene was studied in populations from Wyoming, Nebraska, and Iowa. No differences in overall genetic diversity or haplotype frequencies indicative of a bottleneck were observed between the recently founded populations in Iowa and the established populations in Wyoming and Nebraska. This result suggests that the sudden loss of an ecological exclusion mechanism, allowing the species to move east in appreciable numbers, is more likely to have triggered the range expansion than the surmounting of an extrinsic barrier to movement. The nature of this mechanism is unknown but might be related to recent changes in corn farming practices and technology.

KEY WORDS Striacosta albicosta, NADH dehydrogenase 1, biological invasion, Zea mays

The western bean cutworm, Striacosta albicosta hosts to P. vulgaris and corn and speculated that S. (Smith) (Lepidoptera: Noctuidae), is a pest of corn albicosta may have evolved on mixed crops of corn and (Zea mays L.) and dry beans (Phaseolus vulgaris L.) beans grown by Native Americans. that is currently expanding its distribution eastward Before the 1950s, S. albicosta was an occasional pest through the Corn Belt of the United States. The spe- of dry beans, largely conÞned to Colorado, Kansas, cies is univoltine and overwinters as a Þfth-stage larva Nebraska, and Idaho (Hoerner 1948). From 1950 on- below the soil surface (Hoerner 1948, Douglass et al. ward, the species became an increasingly destructive 1957). Adults emerge in early July (Hagen 1962) and pest of beans in western Nebraska (Hagen 1963, 1976). oviposit on the leaves of the host plants. On beans, the At about the same time, it was found attacking corn larvae initially feed on the leaves and buds and later crops in southern Idaho (Douglass et al. 1957) and make feeding holes in the young pods (Hoerner 1948). western Nebraska (Hagen 1962). By 1970, S. albicosta On corn, young larvae feed over pollen, anthers, and was established throughout most of Nebraska but re- upper leaf tissue before burrowing into the ears to mained rare east of the Missouri river (Blickenstaff feed on young kernels as fourth and Þfth instars (Ha- and Jolley 1982). The species distribution remained gen 1962). Despite exploiting two very different crop stable for the remainder of the 20th century before species, S. albicosta does not seem to be particularly undergoing a second, more rapid eastward expansion. polyphagous. Blickenstaff and Jolley (1982) studied In 1999, western bean cutworms were found attacking larval development on several bean species, teosinte, experimental plots of corn throughout southwestern and several Solanaceae thought to be alternative hosts Minnesota (OÕRourke and Hutchinson 2000). Al- of S. albicosta. They concluded that all were inferior though the species had been present in South Dakota for many years, it did not begin to cause economic 1 USDAÐARS, CICGRU, Genetics Laboratory, Iowa State Univer- damage until 2000 (Catangui and Berg 2006). Also in sity, Ames, IA 50011. 2000, S. albicosta caused widespread and unexpected 2 Corresponding author, [email protected]. damage to corn crops throughout western Iowa (Rice 3 Department of Entomology, Iowa State University, Ames IA 2000). The species spread into Illinois and Missouri by 50011. 4 Present address: Pioneer Hi-Bred International, Johnston, IA 2004 (Dorhout and Rice 2004, Rice et al. 2004) and 50131. Wisconsin in 2005 (Cullen and Jyuotika 2008), and February 2009 MILLER ET AL.: WESTERN BEAN CUTWORM GENETIC VARIATION 275

Indiana, Michigan, and Ohio by 2006 (Pope 2007, Rice and Pilcher 2007, DiFonzo and Hammond 2008). It is striking that S. albicosta apparently remained largely conÞned to the west of the Missouri River for the last three decades of the 20th century despite an abundance of corn immediately to the east. This sug- gests the existence of some environmental factor that prevented further eastward movement during this pe- riod. Although the nature of such an environmental barrier is unknown, two general categories can be Fig. 1. Locations of western bean cutworm population envisaged. The barrier might be durable and still in samples. The course of the Missouri River is indicated by a existence, as would be expected for a physical land- dashed line. scape feature. Alternatively, the barrier might be tem- porary and have recently disappeared, allowing the present range expansion to begin. A temporary barrier from Wyoming, Nebraska, and Iowa as a Þrst step might consist of, for example, farming practices that toward understanding the processes that have re- make the agricultural environment unsuitable as a sulted in the current, dramatic eastward range expan- habitat but that can change rapidly over wide areas in sion. response to economic or technological developments. Although the identity of any barrier to movement Materials and Methods by S. albicosta is unknown, some clues as to its nature may be obtained by comparing genetic diversity in Sampling and DNA Extraction. Male S. albicosta populations that predate the current range expansion were collected in July 2006 from Goshen County, WY (i.e., west of the Missouri River) and new populations (42.246Њ N, 104.486Њ W); Dawson County, NE (40.972Њ further east. If S. albicosta had to cross a durable N, 100.361Њ W); Audubon County, IA (41.697Њ N, barrier before starting the current range expansion, it 95.214Њ W), and Iowa County, IA (41.866Њ N, 92.337Њ is unlikely that it did so spontaneously in large num- W) (Fig. 1). At each location, adult males were cap- bers. Thus, we may expect that there was a population tured in two traps placed 25 m apart. Traps were bottleneck at the beginning of the range expansion fashioned from 3.8-liter plastic milk containers into resulting in genetic founder effects associated with a which four Ϸ60-cm2 holes had been cut leaving a 6-cm small number of colonizers. If so, younger eastern deep reservoir that was Þlled with a 4:1 mixture of populations are expected to be less genetically diverse water and ethylene glycol (Seymour et al. 1998, Dor- than older populations. In addition, signiÞcant allele hout and Rice 2008). Each trap was mounted on a pole, frequency differences would be expected between 1.2 m above ground level, and was baited with an S. younger and older populations as a result of acceler- albicosta sex pheromone lure (Scentry Biologicals, ated genetic drift during the bottleneck. However, if Billings, MT). Traps were left in the Þeld for 10 d a temporary barrier recently dissipated, allowing large during the Þrst 2 wk of July, after which dead males numbers of S. albicosta to migrate east, a bottleneck were removed from the ethylene glycol solution and would not be expected. Under this scenario, younger stored at Ϫ20ЊC. populations would exhibit similar levels of genetic DNA was extracted from the thoraxes of individual diversity to older populations, and no unusually high S. albicosta using a modiÞed CTAB (Cetyl trimethyl- allele frequency differences would be expected be- ammonium bromide) method (Marcon et al. 1999). tween young and old populations. PuriÞed DNA was resuspended in 50 ␮l TE buffer and An obvious prerequisite for studying genetic vari- stored at Ϫ20ЊC. Up to 24 DNA extractions were pre- ation is some type of genetic marker. Where no such pared from each sample location. markers have been previously characterized, as is the Preliminary Sequencing Using Universal Primers. case for S. albicosta, sequencing mitochondrial genes Universal primers (Simon et al. 2006) were used in is convenient because catalogs are available of “uni- initial attempts to PCR amplify and sequence a region versal” polymerase chain reaction (PCR) primers that of the mitochondrial NADH dehydrogenase 1 gene can be used across a range of taxa (Simon et al. 1994, (ND1). PCR reactions were done in 20 ␮lof1ϫ 2006). Mitochondrial genes show varying rates of evo- GoTaq Flexi buffer (Promega, Madison, WI) contain- lution. RNA genes are more conserved than protein ing 5 pmol of primers N1-J11876 and N1-N12595, each coding genes, among which evolutionary rates also dNTP at 187.5 nM, 2 mM MgCl2, 0.5 U GoTaq Flexi vary. NADH dehydrogenases generally show higher DNA polymerase (Promega), and 2 ␮l DNA extract. A amino acid substitution rates than cytochrome oxi- touchdown thermal cycling program was used starting dases, for example (Simon et al. 1994). Thus, rapidly with 95ЊC for 2 min, followed by seven cycles of 94ЊC evolving mitochondrial genes can be used to study for 1 min, an annealing temperature decreasing from variation within species, whereas slower-evolving 65ЊCby2ЊC per cycle for 1 min and 72ЊC for 1.5 min, genes are better suited to studies concerned with followed by 35 cycles of 94ЊC for 1 min, 50ЊC for 1 min, longer evolutionary time scales. In this paper, we re- and 72ЊC for 1.5 min, with a Þnal incubation at 72ЊC for port a study of mitochondrial NADH dehydrogenase 10 min. Four to six PCRs were done for each individ- 1 gene sequence diversity in S. albicosta populations ual, which were pooled, puriÞed with Qiaquick PCR 276 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 1 puriÞcation kits (Qiagen, Valencia, CA), and recov- conÞrm that the unique sequences were not caused by ered into a Þnal volume of 30 ␮l. PCR or sequencing errors. PCR products were sequenced using Genome Lab Sequencing of Black Cutworm ND1. To obtain an DTCS Quick Start dye terminator cycle sequencing outgroup to the S. albicosta sequences, the same region kits (Beckman Coulter, Fullerton, CA) according to of the mitochondrial ND1 gene was sequenced from the manufacturerÕs instructions with N1-J11876, N1- black cutworm, Agrotis ipsilon (Hufnagel) (Lepidop- J12261, and N1-N12595 used as sequencing primers. tera: Noctuidae). A. ipsilon DNA was used as template Sequencing reaction products were separated by cap- in a PCR with universal primers N1-J11876 and N1- illary electrophoresis using a CEQ 8000 system (Beck- N12595. The PCR product was puriÞed using a Qia- man Coulter). Raw data from the CEQ 8000 were quick kit and cloned into pGEM-T vector (Promega) analyzed with CEQ 8000 Genetic Analysis software according to the manufacturerÕs instructions, which (Beckman Coulter) and exported in SCF format. was used to transform E. coli strain XL1-Blue (Strat- Phred (Ewing and Green 1998, Ewing et al. 1998) was agene, La Jolla, CA). Plasmid DNA was prepared from used to call base sequences and calculate associated eight clones using a Zyppy miniprep kit (Zymo Re- quality scores from the exported sequencer data. Se- search, Orange, CA) and sequenced using a DTCS quence reads were assembled into contiguous se- Quick Start kit and T7 and SP6 sequencing primers. quence using GAP4 (BonÞeld et al. 1995). Phred and Gap4 were used to analyze sequence reads Sequencing with Species-specific Primers. Efforts to generated by the CEQ 8000 and assemble them into a amplify and sequence a portion of the S. albicosta ND1 single contiguous sequence, from which a consensus gene using universal primers proved problematic. In sequence was calculated. particular, the target amplicon failed to amplify in Data Analysis. Distinct sequence haplotypes were numerous individuals as a result of degraded template identiÞed from the complete set of sequences using DNA, and many sequencing reads were of poor qual- standard UNIX command line tools. DNASP (Rozas et ity. However, enough high-quality data were obtained al. 2003) was used to identify and calculate the num- to assemble a 723-bp consensus sequence. Two pairs bers of synonymous and nonsynonymous (i.e., amino acid changing) polymorphisms. The nucleotide diver- of PCR primers that ampliÞed two overlapping ampli- ␲ cons were designed from the consensus sequence using sities ( ) in each of the four samples were computed using the ape package within R (R Development the program Primer 3 (Rozen and Skaletsky 2000). The “ ” Core Team 2007). The boot package within R was upstream 413-bp amplicon was ampliÞed using primers “ ” used to estimate 95% conÞdence intervals for ␲, based L-16 (TTGGAGCGTAAAGTATTAGGTT) and R-429 on 10,000 bootstrap resamplings of sequences within (AAAATCCATGATTATAACAATTC), and the down- samples. A minimum-spanning network of the haplo- stream 403-bp amplicon was ampliÞed using primers types was computed using TCS (Clement et al. 2000). L-302 (TTGCTGGTTGATCTTCTAATTCT) and R-705 The homogeneity of haplotype frequencies among the (AAAAATAAACCCCAAATTAAACC). The two am- samples was tested by a Fisher exact test using R. An plicons overlapped by 81 bp. analysis of molecular variance (AMOVA) was per- The concentrations of PCR components for the formed using Arlequin (ExcofÞer et al. 2005) to com- species-speciÞc primers were the same as those for pute FST, the fraction of the total genetic variance universal primers except that the amount of template partitioned between populations and to test its signif- DNA was adjusted to 40 ng/reaction. The temperature icance by permutation. The AMOVA approach con- Њ cycling program was 95 C for 2 min followed by 35 siders not only the frequencies of haplotypes in each Њ ϩ Њ cycles of 94 C for 1 min, 52 (L-16 R-429) or 55 C sample but also the genetic distances between the ϩ Њ (L-302 R-705) for 1 min, and 72 C for 1 min, with haplotypes themselves. a Þnal incubation at 72ЊC for 10 min. For each ampli- con, four PCRs were done for each individual, which were pooled, puriÞed using Qiaquick kits, and recov- Results ered into 50 ␮l. Each puriÞed amplicon was sequenced Six hundred forty-Þve base pairs of the ND1 gene in both directions using the PCR primers and DTCS were sequenced from each of 87 S. albicosta. Nine Quick Start kits. Sequencing reaction products were polymorphic nucleotide sites were identiÞed, produc- separated by capillary electrophoresis, and the data ing nine distinct sequence haplotypes. The sequence were analyzed with CEQ 8000 software and Phred in of each haplotype, labeled IÐIX, was deposited with the same manner as for the universal primers. Gap4 GenBank (accession numbers EU805541ÐEU805549), was used to assemble the reads from each individual as was the corresponding ND1 sequence from A. ip- into a single contiguous sequence and to manually silon (accession number EU805550). Two of the poly- remove sequences at the end of each read that were morphic sites were predicted to produce polymor- complimentary to the ampliconÕs PCR primer. Addi- phisms in the amino acid sequence of NADH tional sequencing was performed in cases where one dehydrogenase 1. A polymorphism at nucleotide po- or more low-quality reads meant that portions of the sition 40 predicted a methionine/threonine polymor- sequence from an individual were not reliable. In cases phism, and a polymorphism at nucleotide position 634 where the Þnished sequence was unique (i.e., not predicted a phenylalanine/tyrosine polymorphism. shared with any other individual), the entire species- Construction of a minimum-spanning network (Fig. speciÞc PCR and sequencing process was repeated to 2) showed that haplotype V was the closest haplotype February 2009 MILLER ET AL.: WESTERN BEAN CUTWORM GENETIC VARIATION 277

Fig. 2. Minimum spanning network of nine western bean cutworm mitochondrial ND1 haplotypes plus the orthologous sequence from Agrotis ipsilon. Branch labels indicate the nucleotide position at which the haplotypes differ. Nucleotide differences producing amino acid differences in the ND1 gene product are indicated in bold. to the sequence from A. ipsilon, separated by 52 nu- Discussion cleotide differences. Single nucleotide changes were Population bottlenecks have a characteristic effect responsible for most of the differences between hap- on genetic diversity, reducing variation and causing lotypes in the network with the exception of the dif- rapid changes in gene frequencies (Miura 2007, ferences between haplotype V and II (three changes), Dlugosch and Parker 2008). In the context of the and V and III (two changes). Homoplasies, or recur- western bean cutworm, a bottleneck associated with rent mutations, were identiÞed at two nucleotide sites. the start of the current range expansion would result Mutations at position 248 separated haplotypes I and in new populations, east of the Missouri river, being IX and also haplotypes III and IV. Mutations at position less diverse than established populations to the west, 518 separated haplotypes I and VIII and also haplo- and in divergent gene frequencies between the two types III and V. population groups. This study did not Þnd evidence for The frequencies of the nine haplotypes in each of a reduction in diversity in younger western bean cut- the four samples are shown in Table 1. A Fisher exact worm populations or divergent gene frequencies. test did not show any signiÞcant heterogeneity in Thus, the data from the mitochondrial DNA region haplotype frequencies among the samples (P ϭ 0.347). studied here do not support the hypothesis of a sub- In contrast, AMOVA did detect a modest (F ϭ 0.061) ST stantial and recent bottleneck. but signiÞcant (P ϭ 0.039) genetic heterogeneity The apparent lack of a genetic bottleneck could among the samples. Performing AMOVAs for each imply that western bean cutworm populations have pair of samples in turn (Table 2) showed that this been established in Iowa for some time but that they heterogeneity was caused by differences between the have only recently increased to sufÞcient densities to samples from Audubon and Goshen counties and Au- become noticeable. This, however, would mean that dubon and Iowa counties. Although the AMOVA for populations have been increasing in a wave from Audubon and Dawson counties did not indicate sig- “ ” west to east, which would be difÞcult to explain. A niÞcant heterogeneity, the value of F was the highest ST simpler explanation is that the species has begun to of any of the nonsigniÞcant analyses. Nucleotide di- spread eastwards in numbers sufÞcient to avoid sub- versities were comparable between the four samples, stantial genetic bottlenecks. This being the case, the with overlapping 95% conÞdence intervals (Fig. 3) question arises as to why western bean cutworm was and ranged from 0.0017 (Audubon) to 0.0051 (Iowa). largely conÞned to the west of the Missouri river throughout the latter half of the 20th century. It is Table 1. Frequencies of mitochondrial ND1 haplotypes in four western bean cutworm populations Table 2. AMOVA results for pairs of western bean cutworm Haplotype Goshen Dawson Audubon Iowa populations based on mitochondrial ND1 sequence data I1011167 II 2 6 4 6 Goshen Dawson Audubon Iowa III 3 2 3 Goshen 0.472 0.021 0.613 IV 2 1 1 3 Dawson Ϫ0.015 0.095 0.227 V22 1Audubon 0.134 0.057 0.005 VI 1 1 Iowa Ϫ0.027 0.019 0.198 VII 1 VIII 1 IX 1 Lower triangle, estimates of FST, upper triangle: P values based on permutation. 278 ENVIRONMENTAL ENTOMOLOGY Vol. 38, no. 1

able to compete with the aggressive larvae of Helicov- erpa zea (Boddie) when they are feeding together on Cry1Ab-expressing corn silks compared with the silks of nontransgenic isoline (Dorhout 2007). The results reported here are compatible with hypotheses that invoke a role of Bt-transgenic corn in promoting the western bean cutwormÕs current range expansion. Clearly, further studies of the interspeciÞc interac- tions between Bt corn, western bean cutworm larvae, and those of other corn-feeding Lepidoptera will be important in clarifying their role in the western bean cutwormÕs present range expansion. Although exact tests did not identify any signiÞcant differences in haplotype frequencies among the sam- ples considered in this study, AMOVA detected a weak but signiÞcant genetic differentiation between the sample from Audubon county, IA, and those from Fig. 3. Nucleotide diversity (␲) and its 95% bootstrap Iowa county, IA, and Goshen county, WY. A key conÞdence interval for part of the mitochondrial ND1 gene difference between the two analyses is that AMOVA in four western bean cutworm populations. incorporates information about the genetic distances between haplotypes in addition to variation in haplo- striking that the range expansion began only a few type frequencies between samples (ExcofÞer et al. years after the introduction of transgenic corn hybrids 1992). It is unclear whether the signiÞcance of the expressing insecticidal proteins derived from Bacillus AMOVA result is meaningful given that the population thuringiensis (Bt), suggesting the possibility of a causal in Audubon county was Þrst detected in 2003 (Rice link between the two events. Catangui and Berg 2003), and genetic divergence caused by the accumu- (2006) reported that western bean cutworms in South lation of new mutations in the population is unlikely Dakota were able to infest transgenic corn hybrids over such a short time scale. Indeed, this result may be expressing the Bt toxin Cry1Ab. These hybrids were an artifact of the modest sample sizes (Յ23 individuals Þrst commercialized in 1996, primarily for the control per sample). of European corn borer, and have been widely Nevertheless, the AMOVA result may indicate that adopted by U.S. corn farmers (Brookes and Barfoot there is some weak genetic structuring of the western 2006, Sivasupramaniam et al. 2007). Thus, the intro- bean cutworm population in the Corn Belt that cannot duction of these hybrids could have opened up corn be detected easily using mitochondrial DNA haplo- habitat to the western bean cutworm from which it type frequencies. The results of monitoring the spread was previously excluded by the use of synthetic in- of western bean cutworm with pheromone traps secticides applied to control European corn borer. (M.E.R., unpublished data, available at http://www. This seems unlikely to be the primary factor, however, ent.iastate.edu/trap/westernbeancutworm/) suggest because only 26Ð27% of corn growers applied insec- that the range expansion often involves isolated out- ticides for European corn borer control before the break populations establishing to the east of the main introduction of Bt corn (Pilcher and Rice 1998, Pilcher species distribution. This pattern of “stratiÞed dis- et al. 2002). persal” is often seen during range expansions and can It is also possible that the introduction of Bt-trans- greatly increase the rate at which new territory is genic hybrids that are not toxic to western bean cut- occupied (Liebhold and Tobin 2008). It is possible worm has allowed the species to escape from inter- that founder effects associated with the establishment speciÞc competition. Catangui and Berg (2006) of the disconnected outbreaks may produce transient reported that, on nontransgenic corn in South Dakota, genetic heterogeneity that is rapidly eroded by sub- western bean cutworms and European corn borers sequent migration as the outbreak populations rejoin rarely infested the same plant and that European corn with the main population. Determining whether this borer was the dominant species. They hypothesized is the case will require careful sampling, particularly that the recent spread of western bean cutworm and directed toward new outbreaks. its growing impact as a corn pest may be the result of Mitochondrial DNA is undoubtedly a useful tool for reduced interspeciÞc competition with European studying variation in uncharacterized genomes be- corn borer because of the widespread adoption of cause there are many universal PCR primers available transgenic hybrids that express Cry1Ab. In contrast, it that work with a wide range of taxa (Simon et al. 1994, should be noted that OÕRourke and Hutchinson 2006). However, the mitochondrial genome consti- (2000) found western bean cutworms infesting tutes a single genetic locus and may not be represen- Cry1Ab-expressing and nontransgenic corn hybrids at tative of the genome as a whole. Multiple markers from similar rates in Minnesota despite European corn bor- the nuclear genome would be an important resource ers being present on the nontransgenic plants. Exper- for obtaining a broader picture of the population ge- imental studies of intraguild competition in the labo- netics of western bean cutworm. Obtaining large num- ratory have shown that S. albicosta larvae are better bers of highly variable nuclear markers may prove February 2009 MILLER ET AL.: WESTERN BEAN CUTWORM GENETIC VARIATION 279 challenging in the case of this species. Microsatellites and Missouri. Crop management. DOI: 10.1094/CM- have become the mostly widely used DNA-based ge- 2004-1229-01-BR. netic marker system for studies of population and Dorhout, D. L., and M. E. Rice. 2008. An evaluation of ecological genetics in nonmodel organisms (Behura western bean cutworm pheromone trapping techniques 2006, Selkoe and Toonen 2006). This popularity is (Lepidoptera: Noctuidae) in a corn and soybean agro- largely because microsatellites are typically highly ecosystem. J. Econ. Entomol. 101: 404Ð408. variable and easy to assay by PCR. However, devel- Douglass, J. R., J. W. Ingram, K. E. Gibson, and W. E. Peay. 1957. The western bean cutworm as a pest of corn in oping microsatellites from Lepidoptera is notoriously Idaho. J. Econ. Entomol. 50: 543Ð545. problematic because loci with highly similar ßanking Ewing, B., and P. Green. 1998. Base-calling of automated regions are often duplicated throughout the genome, sequencer traces using phred. II. Error probabilities. Ge- possibly because of the movement of mobile genetic nome Res. 8: 186Ð94. elements (Meglecz et al. 2004, VanÕt Hof et al. 2007). Ewing, B., L. Hillier, M. C. Wendl, and P. Green. 1998. A more reliable source of genetic markers in Lepi- Base-calling of automated sequencer traces using phred. doptera is likely to be nucleotide polymorphisms I. Accuracy assessment. Genome Res. 8: 175Ð85. within expressed gene sequences (Coates et al. 2008). Excoffier, L., P. E. Smouse, and J. M. Quattro. 1992. Analysis of molecular variance inferred from metric distances among DNA haplotypes: application to human mitochon- Acknowledgments drial DNA restriction data. Genetics 131: 479Ð491. Excoffier, L., G. Laval, and S. Schneider. 2005. Arlequin We thank M. Minner and E. Testroet for providing tech- (version 3.0): an integrated software package for popu- nical assistance in the laboratory. Two anonymous reviewers lation genetics data analysis. Evol. Bioinform. 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