ORIGINAL ARTICLE

doi:10.1111/j.1558-5646.2009.00883.x

COMPONENTS OF REPRODUCTIVE ISOLATION BETWEEN NORTH AMERICAN PHEROMONE STRAINS OF THE EUROPEAN CORN BORER

Erik B. Dopman,1,2 Paul S. Robbins,3,4 and Abby Seaman5,6 1Department of Biology, Tufts University, 163 Packard Avenue, Medford, Massachusetts 02155 2E-mail: [email protected] 3Department of Entomology, Cornell University, Geneva, New York 14456 4E-mail: [email protected] 5Integrated Pest Management Program, Cornell University, Geneva, New York 14456 6E-mail: [email protected]

Received January 27, 2009 Accepted September 30, 2009

Of 12 potential reproductive isolating barriers between closely related Z- and E-pheromone strains of the European corn borer moth (Ostrinia nubilalis), seven significantly reduced gene flow but none were complete, suggesting that speciation in this lineage is a gradual process in which multiple barriers of intermediate strength accumulate. Estimation of the cumulative effect of all barriers resulted in nearly complete isolation (>99%), but geographic variation in seasonal isolation allowed as much as 10% gene flow. ∼ With the strongest barriers arising from mate-selection behavior or ecologically relevant traits, sexual and natural selection are the most likely evolutionary processes driving population divergence. A recent multilocus genealogical study corroborates the roles of selection and gene flow (Dopman et al. 2005), because introgression is supported at all loci besides Tpi,asex-linkedgene.Tpi reveals strains as exclusive groups, possesses signatures of selection, and is tightly linked to a QTL that contributes to seasonal isolation. With more than 98% of total cumulative isolation consisting of prezygotic barriers, Z and E strains of ECB join a growing list of taxa in which species boundaries are primarily maintained by the prevention of hybridization, possibly because premating barriers evolve during early stages of population divergence.

KEY WORDS: Behavioral isolation, European corn borer, gene flow, hybridization, Ostrinia,prematingisolation,reproductive isolation, speciation, temporal isolation.

The biological species concept (BSC) posits that speciation is second, etc.) (e.g., Coyne and Orr 1989, 1997; Presgraves 2002; caused by reproductive isolating barriers that prevent gene flow Mendelson 2003; Christianson et al. 2005), or to identify biolog- between diverging populations (Dobzhansky 1937; Mayr 1942; ical traits associated with enhanced diversification (e.g., Mitter Coyne and Orr 2004). Two methods are commonly used to inves- et al. 1988; Arnqvist et al. 2000). The comparative approach, al- tigate the causes of speciation. The comparative approach seeks though ideal for issues related to evolutionary rate, suffers from to understand how isolating barriers evolve. In practice, this is at least one major limitation. Namely, speciation may often re- achieved by measuring the strength of reproductive isolation for quire the evolution of numerous and diverse barriers before being several barriers across a large group of taxa that vary in divergence complete (Coyne and Orr 2004), but measuring isolation across time. This information can then be used to estimate the sequen- aphylogenyforevenasinglebarrierposesexceptionaltechni- tial order of isolating-barrier evolution (i.e., which evolved first, cal demands. Over 150 different Drosophila species were used in

C C ! 2009 The Author(s). Journal compilation ! 2009 The Society for the Study of Evolution. 881 Evolution 64-4: 881–902 ERIK B. DOPMAN ET AL.

what is perhaps the best-known comparative study on speciation, and contribute to total reproductive isolation by preventing gene but general conclusions about the initial causes of speciation in flow that has escaped earlier-acting barriers (Coyne and Orr 1989, this group remain out of reach because only three barriers were 1997; Ramsey et al. 2003). Therefore, a barrier will dispropor- considered (Coyne and Orr 1989, 1997). tionately restrict gene flow if it operates at early life stages, be- An alternative approach for understanding the causes of spe- fore other barriers have had their effect. A barrier’s relative or ciation emphasizes the question that comparative studies com- “sequential” strength is meaningful for understanding speciation monly neglect. What are all of the habitat, morphological, be- because it informs a disagreement about the major contemporary havioral, physiological, and other barriers that restrict gene flow causes for the maintenance of diverging populations, which is between diverging populations? Rather than quantifying a small typically couched in terms of the relative importance of premat- number of barriers across a large phylogeny, “case studies” of ing barriers (e.g., those that derive from behavioral, habitat, or speciation take a comprehensive view of individual speciation temporal differences) versus postzygotic barriers (e.g., those that events by evaluating numerous and diverse forms of reproduc- derive from hybrid inviability or infertility) (Ramsey et al. 2003; tive isolation that could prevent a small number of closely related Husband and Sabara 2003; Nosil et al. 2005; Martin and Willis populations from fusing (McMillan et al. 1997; Ramsey et al. 2007; Mendelson et al. 2007; Nosil 2007; Lowry et al. 2008; 2003; Husband and Sabara 2003; Hurt et al. 2005; Nosil et al. Schwander et al. 2008; Schluter 2009). Because premating barri- 2005; Martin and Willis 2007; Mendelson et al. 2007; Nosil 2007; ers operate first, they may often be the strongest impediments to Lowry et al. 2008; Schwander et al. 2008; Schluter 2009). Besides gene flow even if postzygotic barriers are stronger when acting providing the only means for understanding the genetic and evolu- alone. One implication for the sequential nature of isolation and a tionary mechanisms contributing to individual speciation events, speciation process in which diverse barriers accumulate is that the case studies have the potential to confront many outstanding is- barriers that are most important for maintaining species bound- sues in speciation research. aries may ultimately be early-acting forms (i.e., premating- or One open question that case studies can address is how the postmating-prezygotic [e.g., those stemming from differences in strength of isolation varies among all of the individual compo- gametic compatibility or reproductive structures]), regardless of nents. There is a current debate about the role of one or a few whether strong postzygotic barriers evolved first (Coyne and Orr strong barriers to gene flow versus many weaker ones (Nosil et al. 1989, 1997). However, the generality of this prediction hinges on 2005; Lowry et al. 2008; Schluter 2009). This distinction matters the relative rates in which early and late-acting barriers evolve, an for at least two reasons. First, it relates to the ease in which speci- issue of substantial controversy (Coyne and Orr 1997; Kirkpatrick ation evolves. All else being equal, speciation between population and Ravigne 2002; Mendelson 2003; Coyne and Orr 2004). pairs that are isolated by a few strong barriers will have a less com- A third outstanding issue that case studies can address is the plicated genetic architecture (e.g., in terms of number of loci) and relationship between patterns of hybridization and gene flow in evolutionary basis (e.g., in terms of genetic drift, natural or sexual the field, and the biological traits that create these patterns. Quan- selection, and sexual conflict), compared to speciation in which tifying the maintenance (or erosion) of genetic differences be- dozens of weaker barriers are involved. Second, if barrier strength tween natural populations has become relatively straightforward typically increases with time, a barrier cannot have played a major following the emergence of modern genetics tools and methods historical role during speciation unless its contemporary strength of analysis. However, a biological context for understanding these is large (Coyne and Orr 2004). Testing the order of barrier evo- patterns is conspicuously absent for a majority of population and lution commonly requires a comparative approach because case species pairs. In addition to establishing this linkage, estimates of studies lack phylogenetically independent contrasts (e.g., Coyne genetic exchange can be calculated from cumulative reproductive and Orr 1997; Mendelson 2003; but see Christianson et al. 2005). isolation (e.g., Husband and Sabara 2003; Schwander et al. 2008), Nevertheless, under this assumption explicit hypotheses about the which are independent of those derived from genetic markers (e.g., major historical causes of speciation can be formulated for case Dopman et al. 2005; Malausa et al. 2007a). Finally, measures of studies in which strong individual components are identified. The total cumulative isolation can be brought to bear on taxonomic geographic mode of divergence bears on the debate about the role issues because the BSC defines species in terms of reproduc- of a few strong or many weak barriers because gene flow can con- tive barriers. Between some taxa, biological species status may strain divergence, although opportunities for reinforcement can be warranted because the combined action of all isolating barri- only exist among nonallopatric populations. ers reduces gene flow to nearly zero (e.g., Ramsey et al. 2003; A related problem to the strength of individual components of Husband and Sabara 2003). reproductive isolation is concerned with the relative contribution Here, we apply a case study approach to evaluate multiple of those components to total cumulative reproductive isolation. forms of premating barriers, postmating-prezygotic barriers, and Isolating barriers operate sequentially over organisms’ lifetimes postzygotic barriers to gene exchange between North American

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Z- and E-pheromone strains of Ostrinia nubilalis, the European isolation between sympatric populations. In this region, strains corn borer (ECB) moth. Pheromone strains of ECB are an excep- often differ in breeding cycle (i.e., univoltine Z [UZ] and bivol- tional model system to investigate speciation because multiple tine E [BE]) but the Z strain shows geographic variation such that trait differences have been reported that could restrict gene flow, BE or UZ populations can be found with bivoltine Z (BZ) moths and because many of these differences have a clear genetic com- (Roelofs et al. 1985; Glover et al. 1991). Although differences in ponent. In sequential order of when they appear over the ECB life life cycle occur in NY,across most of its range voltinism is broadly history, we quantified the strength of reproductive isolation for overlapping between strains, and the number of generations in- seasonal temporal isolation, circadian temporal isolation, male creases to—three to four in southern latitudes (Sorenson et al. behavioral isolation, female behavioral isolation, mechanical iso- 1992; Showers 1993). Breeding cycle differences can be a con- lation, gametic isolation (oviposition and fertilization), F1 hybrid sequence of environmental variation, but inheritance studies sug- inviability, behavioral hybrid (male) infertility, physiological hy- gest a strong sex-linked genetic component for a trait difference brid infertility (oviposition and fertilization), and F2 and BC invi- (postdiapause development time) that determines breeding cycle ability. Our study has three main goals: (1) to confirm past (often variation in NY (McLeod 1978; Reed et al. 1981; Glover et al. anecdotal) data on reproductive barriers and formally quantify 1991, 1992; Dopman et al. 2005). A second potential form of tem- isolation strength; (2) to evaluate new potential reproductive bar- poral isolation was reported by Liebherr and Roelofs (1975), in riers; and (3) to address the three outstanding issues in speciation which differences in the 24-h rhythm of mating is responsible for research that case studies are well suited to confront. “circadian” temporal isolation between a UZ and a BE population. Common garden experiments were used by Liebherr and Roelofs, arguing for a strong genetic component for this difference. Materials and Methods Forms of postmating-prezygotic and postzygotic isolation THE STUDY SYSTEM have also been studied for North American populations of ECB. The ECB is native to Europe, North Africa, and Western Asia, but In addition to mating rhythm, Liebherr and Roelofs (1975) in- it is clear that three, and possibly more, introductions from Europe vestigated gametic barriers and found that only one of three E × occurred into multiple eastern North American locations during Z matings produced fertile eggs. Although this loss of fecundity the period 1909–1914 (Caffrey and Worthley 1927; Mutuura and was not significant (P > 0.5), their result suggests that a larger Munroe 1970). With their recent introduction, most phenotypic sample size might confirm the presence of genetic incompati- and genetic variation in North American ECB will have orig- bilities operating between copulation and zygote formation. As inated in Europe, with subsequent evolution associated with a early as the 1940s hybrid inviability was assessed in the ECB relatively “new” environment after introduction and expansion (Arbuthnot 1944), but heterosis rather than inviability was dis- from Massachusetts, New York (NY), and Ontario, to most of covered between a multigeneration and a single-generation pop- the United States east of the Rockies. Population surveys reveal ulation. Likewise, Liebherr and Roelofs (1975) found that of the that at many sites in Europe and North America only Z strain few F1 crosses that were successful, F1-hybrid larvae were more males and females exist, that Z- and E-strain moths exist sym- than 1.5 times more likely to survive to adults compared to Z and patrically at a number of sites, and that the E strain rarely exists E parentals. Finally, reduced viability of F2 and backcross off- alone (Klun and Cooperators 1975; Kochansky et al. 1975; Carde´ spring were reported by both Arbuthnot (1944) and Liebherr and et al. 1978; Anglade and Stockel 1984). ECBs often are found Roelofs (1975). Although these studies support F1 hybrid vigor in or near agricultural crops, including maize (Zea mays)(Hudon and second-generation “hybrid breakdown” as general phenom- and LeRoux 1986), but the species is polyphagous (>200 host ena, questions regarding strain identity or low cross replication species are known, review in Ponsard et al. 2004). Nevertheless, leave uncertainty about this interpretation. local host–plant specificity has been found in Europe (Bethenod Although numerous reproductive barriers exist, debate about et al. 2005; Malausa et al. 2008). the taxonomic status of ECB strains has been fueled by their ZandEstrainsofECBareanestablishedmodelforbehav- morphological and genetic similarities (e.g., Cardeetal.1978;´ ioral isolation, in which two major genes determine trait differ- Dopman et al. 2005; Frolov et al. 2007; Malausa et al. 2007a, ences in components of the pheromone communication system b), and by potential variation in reproductive barrier strength. (female pheromone production and long-distance male orienta- Beyond trait differences that are associated with reproductive iso- tion) that are responsible for behavioral isolation (Roelofs et al. lation, Z- and E-pheromone strains are difficult to distinguish 1987; Klun and Huettel 1988; Dopman et al. 2004). Populations in (Liebherr 1974; Carde´ et al. 1978), and genetic surveys suggest North America also show evidence for at least two forms of tem- that gene flow is ongoing or recent. Of the many loci that have poral isolation. In NY,differences in the number of generations per been screened, only a single locus reveals appreciable genetic year (bivoltine or univoltine) can contribute to seasonal temporal differentiation (between BE and UZ/BZ moths in North America),

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the gene encoding Triose phosphate isomerase (Tpi) (Dopman We estimated the consequences of genetic differences in PDD et al. 2005 and references therein). In addition to life-cycle varia- for seasonal temporal isolation by monitoring annual changes in tion, populations show geographic differences in host–plant use. abundance of Z- and E-strain moths in NY. Populations asso- In North America, where our study focuses, strains have overlap- ciated with sweet corn (Z. mays var. rugosa), a common host– ping diets (e.g., Roelofs et al. 1985; Klun and Huettel 1988; Glover plant species, were monitored over a 9-year period (1999–2007) et al. 1991), but there is potential for host-associated reproductive at 41 different localities (Fig. 1), but not all sites were moni- isolation in France where strains use different hosts (Bethenod tored across all years. The monitoring schedule was designed et al. 2005; Malausa et al. 2008). Differences in host use have to sample both bivoltine populations (in June and August) and prompted some to suggest that populations in France are in fact univoltine populations (in July) (Eckenrode et al. 1983; Roelofs distinct species (Frolov et al. 2007). Adding to the taxonomic et al. 1985), and typically began on the last week of May and debate is the discovery of a similar pheromone communication ended on the last week of September (19 weeks). A passive system in the ECB’s sister species, O. scapulalis (Kim et al. 1999; sampling method was used in which adult male moths were Huang et al. 2002) that casts doubt on the biological species status captured in Scentry (Billings, MO) Heliothis traps that were of this more geographically restricted relative (Frolov et al. 2007). baited with synthetic Z- or E-strain sex-pheromone lures (Trec´ e;´ As our most comprehensive knowledge about reproductive Adair, OK), which were replaced every two weeks. At each lo- isolation comes from North American populations of ECB, we cation, one Z and one E trap were placed 40 m apart in habitat focused our study using pheromone strains from this region. With where adult ECB commonly aggregate (wild herbaceous vege- the exception of seasonal and circadian temporal isolation, barri- tation, Showers et al. 1976). To maximize catches, traps were ers were measured using a laboratory colony initiated from a BE positioned near cornfields at “preferred” developmental stages population in Geneva, NY (hereafter BE colony), and a colony (Spangler and Calvin 2000), but when corn reached the less at- made from a UZ population in Bouckville, NY (hereafter UZ tractive, dry brown-silk stage, traps were moved to neighboring colony). Both colonies were initiated using 500 male and 500 cornfields that were at an earlier, more attractive developmental ∼ ∼ female ECB collected as larvae, pupae, and adults, and were mass stage (i.e., late whorl or green silk). Secondary fields were within reared using 100 males and 100 females each generation (see 1 km of the primary field, but most were less than 0.5 km away. ∼ ∼ Dopman et al. 2004 for further details). Seasonal isolation was Traps were checked once per week by a network of cooperators studied using field populations in NY, whereas circadian isolation (www.nysipm.cornell.edu/scouting/scnetwork/). To estimate the was measured using the results from Liebherr and Roelofs (1975). effect of breeding cycle on temporal isolation between strains, we Because seasonal isolation was quantified under field conditions, subsampled the complete dataset to identify those sites with E and environmental effects cannot be excluded. However, for all other Z populations in sympatry (defined as >100 Z-strain and >100 potential barriers a common garden rearing scheme was used such E-strain moths). Additionally, only years with at least 10 consecu- that detected differences likely have a strong genetic component, tive weekly measurements were included. Kolmogorov–Smirnov and for some barriers major QTL have been previously detected. tests were used to determine whether strains showed significant differences in breeding cycle. PREMATING BARRIERS For our purposes, we assume that male ECB collected using Seasonal temporal isolation Z-pheromone lures were from the Z-pheromone strain and those In NY, ECB larvae diapause over the winter, then pupate and collected using the E lure were from the E strain. As a result, emerge as adults in the spring and summer. Differences in postdi- all hybrids and those pure-strain moths that flew to the opposite apause development time, the time to pupation under temperature pheromone blend were misidentified. However, in the laboratory, and photoperiod conditions conducive to breaking diapause, in only a small fraction of E-strain males complete upwind long- large part determine number of generations per year (Glover et al. distance flight to the Z-pheromone blend, and no Z-strain males 1991). Glover et al. (1992) used the sex-linked marker Tpi to show fly to the E-pheromone source (E male flights to Z pheromone: that postdiapause development (hereafter PDD) time is affected 1%, n 110 males; Z male flights to E pheromone: 0%, n 112 ∼ = = primarily by a major gene (Pdd)orgenesonthesexchromosome. males) (Roelofs et al. 1987; Glover et al. 1990; Linn et al. 1997). Dopman et al. (2005) confirmed these findings and showed that Considering these results, few pure-strain moths will be misiden- Pdd is tightly linked with Tpi on the Z chromosome. Because the tified. Hybrids do show partial response to the Z-pheromone blend periods of flight of UZ and BZ or BE moths are displaced in time but the number of hybrid moths in NY is relatively low (mean = (Eckenrode et al. 1983), and because moths with different de- 5.9%, n 2 mixed localities) (Roelofs et al. 1985). Because F1 = velopmental programs may be incompatible, genetic differences hybrids show intermediate PDD times (Glover et al. 1992), any affecting voltinism can have important consequences for the ex- hybrids that do fly to Z- or E-blend pheromone traps would make tent of gene flow in natural populations. estimates of seasonal temporal isolation conservative.

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Figure 1. New York localities where local abundance of Z- and E-strain moths was monitored. White place marks indicate sym- patric localities where >100 E and >100 Z ECB males were captured in at least one year (see Table 1). Image from Google Earth (http://earth.google.com/).

Circadian temporal isolation and males preferentially orient to a 3:97 mixture of (E)and(Z)- We turned to investigate how differences in the 24-h rhythm of 11-tetradecenyl acetates (!11–14:OAc), whereas in the E strain, mating affects gene flow (via circadian temporal isolation) by females produce and males orient to a 99:1 (E)/(Z)blend(e.g., revisiting the data from Liebherr and Roelofs (1975), in which Roelofs et al. 1987; Glover et al. 1990; Linn et al. 1997; Dopman mating periodicity was investigated for a UZ strain from London, et al. 2004). Hybrid females produce an isomeric mixture that is Ontario, Canada and a BE strain from Aurora, NY. Moths were intermediate in composition ( 65:35 E:Z) and F1 males orient ∼ reared under a common environment (24◦Cand65–70%r.h.), to all sources except the E source. Pheromone production is con- and patterns of mating were observed under a 16:8 hour L:D trolled by a single autosomal locus with two main alleles (Klun (light:dark) photoperiod to simulate day length for months in and Maini 1979; Roelofs et al. 1987; Zhu et al. 1996; Dopman which active reproduction occurs (May and June). (Day lengths et al. 2004), whereas upwind orientation to sex pheromone stimuli for May and June are roughly equivalent at Aurora, NY and in a 1–2 m flight tunnel is controlled by a single sex-linked locus London, Ontario.) We used these data to calculate an index of (Roelofs et al. 1987; Glover et al. 1990; Dopman et al. 2004). Al- isolation. though there are clearly two sexual communication systems with limited cross attraction in the laboratory, a reduced frequency of Male and female behavioral isolation hybrids can be found in nature (e.g., Roelofs et al. 1985; Klun and Using a specialized pheromone gland, female ECB emit a spe- Huettel 1988; Glover et al. 1991). As differences in sexual com- cific pheromone blend that is used by males with the “appropriate” munication can lead to assortative mating or in reduced ability behavioral response for long-distance orientation and navigation of hybrids to obtain mates (via behavioral “dysfunction”), male toward the signaling female. In the Z strain, females produce orientation to female pheromones has important implications for

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patterns of gene flow in the field. We estimated the effect of for experiments in which single one-day-old females were paired long-distance ( 2 m) male behavioral isolation on gene flow by for life with single one-day-old males under controlled laboratory ∼ using the data from Roelofs et al. (1987), Glover et al. (1990), conditions (see Behavioral Isolation). Unlike behavioral trials, and the 30 ug experiment in Linn et al. (1997). These studies mating was not observed. Females were allowed to lay eggs on used the same UZ and BE colonies that we use here to measure wax paper until death. Spermatophores persist in females after long-distance orientation response for Z, E, and hybrid males to death; therefore, we dissected females to infer the number of arangeofsexpheromonesources. times that each female had mated. Fertilization was assessed by a As ECBs are known to aggregate in the field (Showers et al. change in egg color. Eggs that are unfertilized remain cream col- 1976; Malausa et al. 2005), measures of long-distance behavioral ored, whereas in fertilized eggs the developing larval head capsule isolation may not accurately reflect natural conditions. To assess visibly darkens. From replicates crosses, Mann–Whitney U-tests how mating behavior is affected by spatial scale, we made ob- were used to detect significant differences in fitness components servations under shorter distances (<20 cm) using the UZ colony that could result in reproductive isolation. Interstrain crosses were and the BE colony. Initial no-choice observations were made of compared to the within-strain cross that had the same maternal Efemale Emale(E E) and Z female Zmale(Z Z) strain (i.e., Z Zvs.Z EandE Evs.E Z). × × × × × × × × pairings to characterize major elements of the mating behavioral sequence. Following these observations, we quantified the success POSTZYGOTIC BARRIERS rate of each behavioral element in no-choice experiments using F1 inviability the four possible types of Z and E mating pairs (E E, E Z, In response to uncertainty about the generality of heterosis in × × Z E, and Z Z). To simulate mating patterns that result when ECB hybrids (i.e., Arbuthnot 1944; Liebherr and Roelofs 1975), × × ECB aggregate, each trial consisted of a male released into a one- we evaluated F1 survival. We tested for heterosis (or inviability) pint ventilated, disposable, and clear plastic tub that contained in F1-hybrids by comparing survival of F1 and pure-strain off- a resident female and a water source. One-day-old virgin males spring produced from the crosses that were used to characterize and females were used in all experiments. Moths were used only postmating-prezygotic isolation. Viability was quantified at the once. Humidity (65 10%) and temperature (22–24 C) were early zygote stage as the number of fertilized eggs that hatched. ± ◦ controlled. Data analysis followed that used for postmating-prezygotic Moth behavior was monitored following Liebherr and isolation. Roelofs (1975), in which a light source (Petzl headlamp) was filtered with 2 thicknesses of number 92 Kodak Wratten filter Behavioral and physiological infertility and 2 thickness of typing paper. Continuous observations were In addition to generating behavioral isolation, mating behavior made from 5.5–6.5 h of the dark cycle or until mating, which we differences between populations can cause postzygotic isolation defined as a state of genital clasping that lasted more than 20 min. if behaviors in hybrids are either intermediate to parentals or if Although both strains were reported to mate during the observa- they are disrupted (review in Servedio and Noor 2003). By taking tion period (Liebherr and Roelofs 1975), we tested each moth for advantage of published data on long-distance male orientation, mating motivation. Female motivation was assessed by observ- we were able to assess the importance of this potential reproduc- ing the exposure of the sex-pheromone gland, which is consistent tive barrier. In their laboratory experiment of long-distance flight with release of sex pheromone (Webster and Carde1982).Males´ orientation to synthetic pheromone blends, Roelofs et al. (1987) that were actively flying before the experiment were assumed demonstrated that F1 males have the highest level of orientation to to be adequately motivated. Indices of the strength of reproduc- the intermediate, hybrid blend, but at an intensity that is reduced tive isolation were calculated for behavioral elements that signif- compared to that of pure males to their blends (Fig. 2A). Hybrid icantly differed between experimental groups, as defined using males may experience infertility due to failures or delays in mating contingency-table tests. For these tests, we distinguish between compared to Z- or E-strain males because of these “dysfunctional” the reciprocal interstrain crosses (i.e., Z EandE Z) by the behaviors. However, an appreciable level of hybrid orientation to × × sex of the moth expressing the observed behavior. the Z pheromone (46%) suggests that relative fitness will depend on the frequency of females in the population (i.e., hybrid males POSTMATING-PREZYGOTIC BARRIERS would be expected to suffer most when hybrid and Z females Gametic isolation are rare). We explored the consequences of behavioral hybrid To confirm the results of Liebherr and Roelofs (1975), in which dysfunction by first applying contingency-table tests to confirm one of three BE UZ matings was successful, we estimated the significance of differences among ECB males (Roelofs et al. × gametic isolation by measuring egg production and fertilization 1987). We then calculated fitness for F1 and parental males as over the lifetime of female ECB. UZ and BE colonies were used the total probability of male orientation to a female. Fitness was

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(1988) for Beltsville, MD, which has BE, BZ, and hybrid moths, but with the Z strain predominating. In both studies, diagnostic differences in female sex-pheromone blend were used to identify strains. Asecondformofhybridinfertilitythatwewereinterested in has a physiological basis. We measured lifetime oviposition

and percent fertility in F1 hybrids. F1 moths (E/Z and Z/E) from

UZ and BE colonies were paired with F1 or parental adults to generate six cross types: two maternal backcrosses (BC) (Z/Z × Z/E and E/E E/Z), two paternal backcrosses (E/E E/Z and × × E/Z Z/Z), and two F2 intercrosses (E/Z E/Z and Z/E × × × Z/E). Fitness differences between F1 and parental moths were assessed by comparing each second-generation cross to fecun- dity and fertility values from parental crosses. Data collection and analysis methods followed those described for postmating- prezygotic isolation.

F2 and BC inviability We sought to confirm evidence for “hybrid breakdown” in second- generation ECB offspring (Arbuthnot 1944; Liebherr and Roelofs 1975), by measuring zygote viability. We used the approach de-

scribed for measuring fitness of F1-hybrid offspring.

STRENGTH OF REPRODUCTIVE ISOLATION We took the quantitative approach of Ramsey et al. (2003) and Coyne and Orr (1989, 1997) in calculating an index of the absolute strength (AS) of reproductive isolation for each individual barrier when it operates alone (“reproductive isolation” in Ramsey et al. 2003). AS for the nth component of reproductive isolation was generically defined from mean fitness components as: between strain fitness 1 . (1) − within strain fitness Values of AS typically ranged from 0, which indicated free gene flow, and 1, which indicated complete isolation. AS could also be negative, in which case hybridization and gene flow was fa- Figure 2. (A) Proportion of parental and F1 males attracted to vored. We restricted our analysis to isolating barriers that were parental and hybrid sex-pheromone blends (n 40 for each ≥ statistically significant in at least one comparison. strain/pheromone blend combination, modified from Roelofs et al. Next, we calculated the sequential strength (SS)ofeachbar- (1987)). Reciprocal F males showed similar orientation and were 1 rier (“absolute contribution” in Ramsey et al. 2003), total (T)or combined (G-test, P > 0.5). (B) Estimated frequency of parental stage-specific cumulative reproductive isolation, and the relative and hybrid female ECB at Geneva, NY (n 46 moths, Roelofs et al. = 1985) and Beltsville, MD (n 78 moths, Klun and Huettel 1988). (C) contribution of each barrier to total isolation. Isolating barriers = Estimated relative male fitness at Geneva, NY and Beltsville, MD, were ordered according to the life stage when they had their ef- th suggesting that behavioral hybrid dysfunction occurs at some lo- fect. For the n barrier, SSn depends on ASn and the amount of calities. Fitness was calculated as the total probability of male ori- gene flow that has escaped earlier-acting barriers. Hence, SSn was entation to a female, given estimated female frequencies at each defined as: location. n 1 − calculated using the estimated frequency of females from mixed SSn ASn 1 SSi . (2) = − ! i 1 # sites in the field. We used values from Roelofs et al. (1985) for "= Geneva, NY, which has BE, UZ, and hybrid moths with the E From these values, T (or stage-specific cumulative reproductive strain predominating. We also used values from Klun and Huettel isolation) was calculated by summing SS across barriers (e.g.,

EVOLUTION APRIL 2010 887 ERIK B. DOPMAN ET AL.

T SSi for i 1ton). Finally, we measured the relative the index reflected differences in proportional abundance between = = contribution of each barrier to T (i.e., SSn T). strains across the season. $ ÷ Of the barriers that were measured, seasonal temporal isola- When breeding cycles were completely overlapping tion and behavioral hybrid male dysfunction ought to show pre- ASseasonal 0 and when breeding cycles were completely sep- = dictable differences in AS between Z and E population pairs. In arated ASseasonal 1. High seasonal isolation (ASseasonal > 0.7) = NY, the strength of seasonal isolation will differ between Z and E commonly reflected the presence of sympatric UZ and BE popula- populations because of genetic differences (in PDD) within the Z tions (e.g., Farmington in 2000) (Fig. 3A). Similarly, low isolation strain contribute to BZ or UZ breeding cycles (Glover et al. 1991). (ASseasonal < 0.4) often revealed sympatric BZ and BE populations In contrast, male dysfunction will vary because of environmental or BZ, UZ, and BE moths in sympatry (e.g., Dresden in 2003) dependence (stemming from differences in female frequency). We (Fig. 3B). The relationship between breeding-cycle and isolation, used several estimated values of AS for these barriers to explore however, was not always clear and patterns were sometimes vari- the extent to which different Z and E populations in nature might able across years at the same locality (Table 1). be reproductively isolated. Circadian temporal isolation With respect to temporal isolation from differences in circadian Results time of mating (AScircadian), Liebherr and Roelofs (1975) showed that Z-strain moths mated, on average, 1.7 h earlier than E-strain PREMATING BARRIERS moths (mean E 6.8, n 18; mean Z 5.1, n 24; t-test, Seasonal temporal isolation = = = = P < 0.01; Fig. 3C). Using the data from Liebherr and Roelofs Across years, a total of 16,949 and 38,674 male ECB were trapped (1975), we estimated AS with equation (3), except that the using E- and Z-pheromone lures, respectively. We found evidence circadian time interval over which isolation was based was hours rather than for sympatry (>100 E and >100 Z moths) at 12 localities (Fig. 1). weeks. Without other barriers, the absolute strength of AS Nine localities showed evidence for sympatry across multiple circadian was estimated to be 0.525. years, resulting in a total sample size of 30. E and Z moths captured using E and Z lures differed significantly in time of seasonal Male and female behavioral isolation occurrence across all years and locations (Kolmogorov–Smirnov Observations from a total of 119 no-choice mating experiments test, P < 0.03). suggested that after the release of sex-pheromone by female ECB, We estimated the strength of seasonal temporal isolation successful mating under short distances consists of three addi- based on the number of hybrid and pure-strain offspring expected tional elements: male orientation and courtship, female accep- after one generation of random mating. By assuming that male and tance, and copulation. If a male is sexually receptive to the fe- female frequencies were the same, we generated this expectation male, orientation and courtship is initiated. This stage consists using Hardy–Weinberg proportions. For each week i,theexpected 2 2 of the male flying and then walking to the signaling female with frequency of hybrid (2piqi) and pure-strain (pi qi )offspring + modified abdominal scales (“hair pencils”) everted and wings was calculated from the fraction of the total number moths (ni) fanning (e.g., Nakano et al. 2006). The male then positions him- that were of the Z (pi)strainandtheE(qi)strain.Theexpected self perpendicular to the female and attempts copulation. Female number of hybrid and pure-strain moths each week was then cal- acceptance of a courtship attempt may then lead to clasping of culated by multiplying the number of moths (ni) by their expected genitalia and mating. frequencies. Summing across weeks (w)providedanestimateof Within-strain pairings indicated that mating failures could the total number of hybrid and parental offspring produced in the occur at any stage of the behavior sequence. The release of sex season. In this manner, we estimated absolute-seasonal isolation pheromone appeared to be required for initiation of the sequence (ASseasonal)as: as otherwise, males did court (n 20). However, pheromone re- = w lease did not always result in orientation and courtship (attempts (2pi qi )ni = 90%, n 41) and not all courtship attempts were accepted by fe- i 1 1 "= (3) = − w males (acceptance 86%, n 37). Female rejection consisted 2 2 = = p q ni i + i of walking or flying away from a courting male or of abdom- i 1 "= % & inal bending such that her genitalia were inaccessible (Royer To adjust for the effect of abundance asymmetry on the index and McNeil 1992; Nakano et al. 2006). Finally, mating was sus- (e.g., if one strain was rare the index indicated high isolation even pended when male copulation attempts failed to result in clasping if strains shared breeding cycles), we forced the total number of Z of genitalia (clasping 78%, n 32). Failures at the genital = = and E moths across the season to be equal. With this formulation, clasping stage might result from incompatibilities in reproductive

888 EVOLUTION APRIL 2010 COMPONENTS OF REPRODUCTIVE ISOLATION

A Table 1. Strength of seasonal temporal isolation at sympatric 125 New York sites. E strain Location Year ASseasonal Weeks Total E Total Z 100 Z strain Baldwinsville 2000 0.445 18 268 232 75 2006 0.601 16 109 144 Batavia 2002 0.451 14 106 443

Number 50 Dresden 2003 0.298 14 280 179 Farmington 2000 0.751 19 338 344 25 2002 0.626 18 215 243 2003 0.388 17 904 275 0 2004 0.855 19 326 230 1 2 3 4 5 6 7 8 9 10 12 14 16 18 Hall 2001 0.520 19 182 189 Week 2002 0.550 18 337 282 2003 0.420 17 765 530 B 2004 0.438 18 830 831 150 2006 0.625 19 101 111 LeRoy 2000 0.539 19 158 614 E strain 120 Lockport 1999 0.506 19 182 275 Z strain 2000 0.754 18 144 797 90 Medina 2000 0.461 18 261 468 2001 0.611 18 109 284

Number 60 Penn Yan 2000 0.731 19 209 343 2004 0.702 19 130 490 30 2006 0.624 19 175 824 2007 0.667 18 225 198 0 Rush 1999 0.603 19 126 112 1 2 3 4 5 6 7 8 9 10 11 12 13 14 2000 0.681 18 135 270 2002 0.658 18 476 541 Week Spencerport 2000 0.716 18 109 399 2002 0.658 15 116 195 C 9 Williamson 2000 0.636 19 173 345 2001 0.281 18 171 186 Z strain 2002 0.551 16 110 250 6 E strain

structure rather than behavior (i.e., mechanical isolation). There-

Number fore, we report on genital clasping as a potential form of 3 postmating-prezygotic isolation below. Compared to that seen within-strain, mating behaviors in interstrain pairings commonly failed. Males were likely to court 0 females of the same strain, but not females of the other strain 12345678 (Fisher’s test; E E[n 21] vs. Z E[n 46], P < 0.01; Z Z Hour × = × = × [n 20] vs. E Z[n 32], P < 10 5;Fig.4A).Similarly,females = × = − Figure 3. Premating, temporal isolation. Examples of strong (A) were likely to accept males of the same strain, but reject males of and weak (B) seasonal temporal isolation. (A) In 2000, strains at the other strain (Fisher’s test; E E[n 19] vs. E Z[n 8], × = × = Farmington showed strong seasonal isolation, with Z moths dis- P < 0.02; Z Z[n 18] vs. Z E[n 23], P < 0.03; Fig. 4B). × = × = playing a predominant univoltine breeding cycle and E moths dis- Behavioral isolation was calculated using equation (1). playing a predominant bivoltine breeding cycle. (B) Seasonal dif- For male behavioral isolation (AS orient.), we considered short- ferences in male flight activity at Dresden, NY, in 2003, in which distance attraction (<20 cm, this study; Fig. 4A) and long-distance males of both strains showed overlapping (bivoltine) breeding cy- ♂ orientation ( 2 m, [pooled from Roelofs et al. (1987), Glover cle patterns and weak seasonal isolation. (C) Circadian differences ∼ in time of reproduction between Z- and E-strain ECB during sco- et al. (1990), and the 30 ug experiment in Linn et al. (1997)]; tophase (from Liebherr and Roelofs (1975)). Fig. 4C). With short-distance attraction, AS orient. was 0.448 for ♂

EVOLUTION APRIL 2010 889 ERIK B. DOPMAN ET AL.

Figure 5. Postmating-prezygotic isolation. (A) Proportion of clasping successes in within and between strain pairings. (B) Mean oviposition and (C) mean percent fertility in within and between

Figure 4. Premating, behavioral isolation. (A) Male orientation to strain crosses. Cross-type designations list females first. Numbers live females over short distances (<20 cm). (B) Female acceptance in parentheses are sample sizes. of male courtship in short-distance trials. (C) Male orientation to POSTMATING-PREZYGOTIC BARRIERS synthetic pheromones over long distances ( 2m)(pooledfrom ∼ Roelofs et al. (1987), Glover et al. (1990), and Linn et al. (1997)). Mechanical isolation Cross type designations list females first. Numbers in parentheses In contrast to overt male and female behaviors, there were no are sample sizes. significant differences in the proportion of successful clasping attempts among the four types of mating pairs (Fisher’s test; E × Z E pairings and 0.722 for E Z pairings (Fig. 4A). With E[n 13], Z E[n 13], Z Z[n 12], E Z[n 2], × × = × = × = × = long-distance attraction, AS was 0.988 for Z E pairings P > 0.25; Fig. 5A). The limited sample size for E Z pairings at orient. × × and 1.0 for E Zpairings(Fig.4C).Forfemalediscrimination this last stage of the mating sequence, however, prohibits a fully × ♂ behavior, AS discrim. 0.712 in E Z pairings and 0.298 in Z informative test of this possible mechanical form of postmating- = × × Epairings(Fig.4B).♀ prezygotic isolation.

890 EVOLUTION APRIL 2010 COMPONENTS OF REPRODUCTIVE ISOLATION

Gametic isolation ECB, nor did hybrids experience significantly reduced survival. Gametic barriers were assessed by recording the fate of 8754 Mean viability for E-strain and Z-strain zygotes was 96.8% and eggs from 40 unobserved crosses. Pairings that failed to produce 94.2%, respectively (U-test, P > 0.27; Table 2). For the Z E × offspring were excluded; thus, our analysis reflects a minimum cross, mean viability was 95.5% (vs. Z Z, U-test, P > 0.65), × strength of gametic isolation. Interstrain pairings failed to produce whereas mean viability was 92% in the Z Ecross(vs.E E, × × offspring more often (P < 0.001, proportion successful, Z E U-test, P > 0.07). × = 0.34, Z E 0.27 vs. 0.71 for within-strain pairings), but this × = probably reflects behavioral incompatibilities rather than severe Behavioral and physiological infertility To test for behavioral dysfunction, we revisited the data from postmating-prezygotic problems (or complete F1 inviability, be- low). Females that laid live progeny mated only once, as indicated Roelofs et al. (1987) and confirmed that F1 males show inhibited by the presence of only a single dissected spermatophore. Over orientation towards all pheromone blends compared to Z- or E- strain males to their sex-pheromone blends (G-test, P < 1 10 5, their lifetime, Z females mated to Z males laid about 280 eggs × − n 40 for each strain/pheromone blend combination; Fig. 2A). and E females mated to E males laid about 248 eggs (U-test, P > ≥ 0.4). Our results confirm gametic isolation in the ECB, in that Similarly, as was expected (e.g., Glover et al. 1991; Linn et al. oviposition was reduced in an interstrain cross (Fig. 5B), but it 1997), differences in hybrid-male orientation towards the inter- is in the opposite direction to that seen by Liebherr and Roelofs mediate (hybrid) pheromone blend (56%) and the Z-pheromone (1975). Z females mated with E males lay on average 194 eggs, blend (46%) were not significantly different (G-test, P > 0.2), a significant reduction in lifetime productivity compared to when but hybrids showed limited response to the E-pheromone blend (4%) (G-test, P < 1 10 8). Using orientation values from they mated with Z males (U-test, P < 0.03). E females paired × − with Z males were only slightly less fecund than when mated to E Roelofs et al. (1987) (Fig. 2A) and the estimated frequency of females from Roelofs et al. (1985) (Geneva, NY; n 46 females) males (mean 227.1; U-test, P > 0.4). Isolation was computed = = and Klun and Huettel (1988) (Beltsville, MD; n 78 females) for lifetime oviposition differences using equation (1). The abso- = (Fig. 2B), we calculated relative fitness for F and parental males. lute strength of isolation (ASovipos.) was 0.309 for Z E crosses 1 × and 0.084 for E Zcrosses.Unlikeoviposition,percentfertil- As a result of their promiscuous, although less vigorous behavior, × ization did not significantly differ between within-strain crosses hybrid male fitness exceeded that of E males when hybrid and Z (Z strain 90.2%, E strain 98.5%, P > 0.05), or between these females were in the majority (e.g., Beltsville, MD, in Fig. 2C). In = = and interstrain crosses (Z Zvs.Z E, U-test, P > 0.2; E E contrast, when E females were in the majority (e.g., Geneva, NY × × × vs. E Z, U-test, P > 0.15; Fig. 5C). in Fig. 2C), F1 behavior conferred reduced mating success when × compared to both parental males. Using these fitness values, we POSTZYGOTIC BARRIERS defined an index of isolation for behavioral hybrid dysfunction

F1 inviability (ASF1 dys.)as: In contrast to previous studies (Arbuthnot 1944; Liebherr and ♂ F1 fitness 1 . (4) Roelofs 1975), we did not find evidence for heterosis in hybrid − parental fitness

Table 2. Physiological forms of postzygotic isolation. Viability of F 1 zygotes, fertility of F 1 adults, and viability of backcross and F 2 zygotes.

Cross type n Mean P Mean P Mean P × ♀ ♂ oviposition fertility (%) viability (%) Parental (pooled) E EandZ Z20267.3 N/A93.6 N/A95.3 N/A 1 × × F1 Z E10(Fig.5)–(Fig.5)–95.50.677 × E Z10(Fig.5)–(Fig.5)–92.00.070 × Maternal backcross2 Z Z/E 10 173.2 0.0083 95.0 0.198 97.8 0.082 × E E/Z 5 260.4 0.940 92.3 1.000 95.6 1.000 × Paternal backcross2 Z/E E10190.00.09387.40.49798.10.010 × E/Z Z10202.60.01297.20.35094.80.900 2 × F2 E/Z E/Z 10 289.9 0.451 96.4 0.327 93.3 0.940 × Z/E Z/E 10 231.9 0.248 97.5 0.498 98.3 0.152 ×

1Mean viability of Z EversusZ Z (mean 94.3%) and mean viability of E ZversusE E (mean 96.8%). × × = × × = 2Versus pooled parental values. 3Significant after Bonferroni correction (α 0.05 6 second-generation crosses). = ÷

EVOLUTION APRIL 2010 891 ERIK B. DOPMAN ET AL.

At the Geneva site, ASF1 dys. was 0.184 for the Z strain and dysfunction used the value calculated for Geneva, NY (Fig. 2B,C). 0.753 for the E strain. At Beltsville,♂ isolation was positive for the “Weak” reproductive isolation was modeled after sites in which Zstrain(0.345),butnegativefortheEstrain( 1.422), reflecting strains are both bivoltine (BZ and BE) and the Z strain predom- − the superior fitness of F1 males at this location. inates. Under this scenario, seasonal isolation was calculated as

We tested for a physiological form of F1 infertility by mea- the value for Dresden, NY (Table 1), and dysfunction was equal suring oviposition and fertility for 55 different second-generation to the estimate for Beltsville, MD (Fig. 2B,C). crosses (six cross types), which yielded a total of 12,178 eggs. As Violation of several assumptions regarding trait variation and in gametic isolation experiments, pairings that failed to produce environmental effects may result in inaccurate estimates of the any progeny were excluded and female dissections recovered a strength of reproductive isolation between Z- and E-pheromone single spermatophore in each female from every successful cross. strains. With the exception of seasonal isolation, barriers were The exclusion of unsuccessful pairings could not have influenced evaluated between UZ and BE moths. Therefore, an assumption measures of F1 infertility (or F2/BC inviability, below) because under the weak model is that barrier strength is identical for all the six second-generation crosses were as successful as within- UZ versus BE population pairs and BZ versus BE pairs. Trait strain crosses (P > 0.06). Mean oviposition of each second- covariation has not been extensively studied in the ECB. How- generation cross was compared to pooled parental crosses (pooled ever, the observation that genetic variation across many loci is parental cross mean 267.3). Oviposition was significantly re- shared between BZ and UZ populations (e.g., Coates et al. 2004; = duced in both backcrosses to the Z strain (Z Z/E 173.2; Dopman et al. 2005), coupled with the finding that both popula- × = E/Z Z 202.6) (Table 2), but only the maternal backcross re- tions are strongly differentiated with respect to BE moths (at Tpi) × = mained significant after Bonferroni correction (P < 0.008). When (Dopman et al. 2005), suggests that gene flow has been relatively divided by cross class, only BC pairings were less productive than recent between BZ and UZ populations. A second assumption parentals (mean 198.8, U-test, P < 0.01). Both parental and that applies to both models is that circadian temporal isolation, = F2 intercrosses (mean 260.9, U-test, P > 0.8) showed similar which was measured by Liebherr and Roelofs (1975) between an = levels of productivity. Reproductive isolation was estimated using Ontario (UZ) and a NY (BE) strain, is equivalent between UZ equation (4), with F1 fitness conservatively estimated using mean and BE populations in NY (which were studied here). Finally, we oviposition of all second-generation crosses (224.7). Thus, isola- estimate total isolation assuming that all barriers have a genetic tion stemming from reduced oviposition in the second-generation basis. However, a combination of genetic (PDD) and environmen-

(ASF1 ovipos) was 0.159. In contrast to patterns seen for F2 or BC tal effects account for seasonal isolation because breeding cycle oviposition, none of the second-generation crosses significantly can only be measured in the field. In comparison, phenotypic dif- differed in mean percent fertility compared to parentals (U-test, ferences contributing to other barriers will have a larger genetic P 0.198; Table 2). With 87.4% of eggs fertilized, the Z/E component because they were measured in the laboratory using a ≥ × E paternal backcross had the lowest fertility and an F2 intercross common garden regime. (Z/E Z/E) had the highest percent fertility (97.5%). Table 3 shows estimates of AS, SS,andT under the two × isolation models. In both models, we adjusted for uncertainty in

F2 and BC inviability the spatial scale in which male orientation occurs (i.e., aggre- We were not able to confirm “hybrid breakdown” in BC or F2 gated vs. dispersed females) by using the mean of AS for short- offspring (Arbuthnot 1944; Liebherr and Roelofs 1975). Mean distance (Fig. 4A) and long-distance orientation (Fig. 4C). Under viability for second-generation zygotes ranged from 93.6% to the “strong” model, T from the measured traits was 99.4% (using 98.1% (Table 2). The only cross that significantly differed from mean SS values of E ZandZ E pairings, Table 3). Under × × parental crosses showed higher survivorship (Z/E E 98.1%, the “weak” model, this value was 96.4%. Although this difference × = P < 0.01), but this value was not significant after correcting for may not be statistically significant because our estimates of repro- multiple tests. ductive isolation are based on means with unknown confidence intervals, the biological significance of increased gene flow could CUMULATIVE REPRODUCTIVE ISOLATION be dramatic. Given that barriers operate sequentially over the ECB We explored how differences in the AS of seasonal isolation and life history, earlier-acting barriers contributed more to T in both behavioral hybrid male dysfunction could affect cumulative iso- scenarios (Table 3; Fig. 6). As a result, differences between mod- lation by investigating two scenarios. “Strong” reproductive iso- els were largely attributed to different values of ASseasonal rather lation was modeled after locations in which strains differ in vol- than ASF1 dys. The relative contribution of seasonal isolation to tinism (BE and UZ) and the E strain is at high population density. total isolation was 66% (SSseasonal 0.655) and 31% (SSseasonal ♂ = = Under this scenario, seasonal isolation was calculated as the mean 0.298) under the “strong” and “weak” models (Fig. 6). In contrast, index across years for Farmington, NY (Table 1), and behavioral behavioral dysfunction contributed <1% in both (mean strong

892 EVOLUTION APRIL 2010 COMPONENTS OF REPRODUCTIVE ISOLATION ers (see 0.015 − EMean × 0.065 ) − 2 ZZ × 0.008, Table 2). < P EMeanE × )(Weak tion used the values calculated for Geneva (Fig. 2). In 2 ronni correction ( ille, MD (Fig. 2). ZZ × 0.001 0.001 0.001 0.001 0.018 0.007 0.996 0.995 0.994 0.993 0.907 0.964 4 0.159 0.539 0.001 0.017 0.006 0.004 − 4 EMeanE × 0.309 0.197 0.0010.159 0.010 0.003 0.001 0.020 0.007 1.422 − ) (strong 1 2 . 3 4 ZZ × 0.159 4 0.159 Ostrinia nubilalis 4 EMeanE × 0.309 0.197 0.084 0.159 00000000000 ) (weak 2 3 4 ZZ × Absolute strength(strong E Absolute strength Sequential strength Sequential strength 000000000000 000000000000 000000000000 0.05). > P )describestheintensitythatthebarrierpreventsgeneflowthathasescapedearlieractingbarriers.0,freegeneflow;1,nogeneflow.Cross-type SS )typicallydescribestheintensityofgene-flowreductionbetweenstrainsrelativetothatoccurringwithinstrainsandintheabsenceofotherbarri AS and BC Inviability 0 Inviability OvipositionFertilization 0.159 -male behavioral dysfunction 0.184 0.753 0.469 0.345 2 1 1 1 1 F F Circadian temporal isolationMale orientationFemale discrimination 0.525Oviposition 0.525 0.525 0.712 0.525 0.862 0.298F 0.525 0.717 0.505 0.525 0.790 0.712 0.181 0.084 0.862 0.298 0.181 0.717 0.505 0.181 0.790 0.369 0.016 0.369 0.141 0.014 0.369 0.117 0.017 0.129 0.033 0.287 0.028 0.239 0.035 0.263 F Total Isolation Fertilization F Reproductive isolating barriers between Z-, and E-pheromone strains of Postzygotic Premating Seasonal temporal isolation 0.655 0.655 0.655 0.298 0.298 0.298 0.655 0.655 0.655 0.298 0.298 0.298 Postmating-prezygotic Clasping 0 0 0 0 0 0 0 0 0 0 0 0 Class Isolating barrier Absolute strength of reproductive isolation ( In a scenario of strong isolation, seasonal isolation was calculated from theValues mean index of across absolute years forCalculated isolation Farmington using (Table mean 1) oviposition and were of behavioral not parental dysfunc and statistically second-generation crosses. Only one significant of six second-generation ( crosses was significant after Bonfe text for details). Sequential strengthdesignations of list isolation females first. ( 1 2 3 4 Table 3. a scenario of weak isolation, seasonal isolation equaled that estimated for Dresden, NY (Table 1), and dysfunction equaled that estimated for Beltsv

EVOLUTION APRIL 2010 893 ERIK B. DOPMAN ET AL.

[Cooley et al. 2003], flowering plants [Martin and Willis 2007], corals [Knowlton et al. 1997], algae [Clifton 1997]). Indeed, ob- serving mating activity across breeding seasons represents a major technical challenge. In spite of these difficulties, and in support of its prevalence, nearly 30 years ago Roelofs and colleagues uncovered ecological differences contributing to seasonal tempo- ral isolation in the ECB and proceeded to characterize sympatric Figure 6. Relative contribution of isolating barriers to total repro- UZ, BZ, and BE voltinism “races” in NY (Eckenrode et al. 1983; ductive isolation (T) under “strong” and “weak” isolation scenarios Roelofs et al. 1985). Since this pioneering work, however, the con- in which seasonal isolation and behavioral dysfunction varied in absolute strength. Values for each barrier were obtained using sequences of ecological differences in breeding cycle on patterns mean sequential strength (see Table 3). of gene flow have remained unclear. Our survey of 41 localities (Fig. 1) over a 9-year-period SS 0.006, mean weak SS 0.015). Because (1999–2007) records the persistence of sympatric bivoltine and F1 dys. = F1 dys. = − seasonal isolation operates before all other barriers (ASseasonal univoltine populations of ECB in NY. The degree of asynchrony ♂ ♂ = SSseasonal), weak values resulted in increased sequential strength in breeding cycle across many sites indicates that voltinism can for all later-acting barriers. Weak ASseasonal also contributed to severely restrict gene flow between pheromone strains (e.g., more asymmetry in T between the Z- and E-pheromone strains. Fig. 3A). Indeed, our results show that when BE and UZ moths This effect stemmed from an increase in sequential strength of are sympatric, as much as 80% of gene flow is prevented by asymmetric later-acting barriers, specifically male behavioral iso- seasonal isolation alone (Table 1). However, strains at several lation (Fig. 4A). Indeed, T showed strong asymmetry only under sites in NY (Fig. 3B; Table 1) and in other geographic areas the weak model (Z E 90.7%; E Z 99.3%) (Table 3). (e.g., North Carolina, Sorenson et al. 1992) have overlapping × = × = breeding cycles, or patterns of PDD times that are consistent with overlapping breeding cycles (NY, Glover et al. 1991; France, Discussion Thomas et al. 2003). These findings indicate that seasonal Reproductive isolating barriers are among the primary forces gen- temporal isolation is only a locally important barrier to gene erating and maintaining biodiversity in nature (Dobzhansky 1937; flow between Z and E strains, and that, overall, breeding cycle Mayr 1942; Coyne and Orr 2004). Here, we report on a case differences constitute a rather ineffective isolating barrier. Indeed, study of North American Z- and E-pheromone strains of ECB, at the same study site, we found that the strength of temporal in which three major classes of reproductive barriers–premating, isolation can vary dramatically between years. At Farmington, postmating-prezygotic, and postzygotic–are considered. Our eval- ASseasonal was above 0.6 in three of the four sampled years, but uation of 12 potential isolating barriers demonstrates that repro- in 2003, it fell to 0.388 (Table 1). The cause of these fluctuations ductive isolation arises from manifold sources whose effects are is unknown, but environmental effects or sampling artifacts are far from uniform. Consequently, the genetic and evolutionary two possible explanations. Changes in weather are known to basis for speciation will be complex. Although the cumulative affect the number and shape of ECB breeding cycles (e.g, via action of all barriers produces nearly complete reproductive iso- temperature-dependent development; Beck 1983). Alternatively, lation, polymorphism in strength of reproductive isolation allows changes in farming practices, including the use of BT corn for substantial opportunity for gene flow. Indeed, the ECB likely or insecticides, can affect the size of annual flights through exists as a mosaic of incompletely isolated populations that vary differential mortality (e.g., Burkness et al. 2001). Such changes both in the strength and form of reproductive isolation. Neverthe- could increase the breeding cycle overlap for populations that are less, of the three classes of barriers that were measured, premating genetically bivoltine and univoltine (e.g., at Pdd). Finally, as traps forms appear to be the most potent in maintaining the integrity of were positioned on 41 active farms across 9 years, the uniformity sympatric Z and E populations in nature, possibly because they of sampling in some years may have been compromised. evolve rapidly between diverging populations. Although breeding cycle differences can be a consequence of environmental variation, voltinism in NY can be at least par- PREMATING BARRIERS tially explained as a byproduct of genetic differences at the major, Seasonal temporal isolation Z-linked QTL affecting PDD time (McLeod 1978; Reed et al. Although accounts of temporal isolation may be numerically rare, 1981; Glover et al. 1991, 1992). Therefore, the origin of seasonal the phylogenetic breadth of taxa that experience temporal isola- isolation can be understood as the evolution of developmental tion suggests that this paucity reflects limited study rather than rate. It has been suggested that trait divergence resulted from low evolutionary frequency (e.g., fish [Quinn et al. 2000], insects selection, most likely from an ecological source, because Tpi

894 EVOLUTION APRIL 2010 COMPONENTS OF REPRODUCTIVE ISOLATION

shows evidence for selection and is tightly linked to the Pdd locus that adding sex pheromone fails to increase interstrain mating (Dopman et al. 2005). PDD time may have evolved because it in- (Pelozuelo et al. 2007), this result can be taken as prima facie fluences fitness through its effect on generation time, or because evidence that male orientation to female cues, most likely to fe- it helps coordinate the performance of life-cycle activities (e.g., male sex pheromones, causes behavioral isolation over a broad reproduction, growth, diapause) at appropriate times in the season range of spatial scales. However, isolation strength is reduced (Bradshaw et al. 2004; Bradshaw and Holzapfel 2007). when males and females are in close proximity (AS ,UZ orient. × BE: 0.448 [<20 cm] vs. 0.988 [ 2 m]; E Z: 0.722 [<20 cm] ∼ × ♂ Circadian temporal isolation vs. 1.0 [ 2 m]), suggesting that moth dispersion could shape the ∼ Besides seasonal temporal isolation, at least some ECB strains are extent that differences in male behavior reduce gene flow. Further temporally isolated through differences in 24-h, circadian time of studies are needed to determine the reasons for increased rates mating (Liebherr and Roelofs 1975). Under laboratory conditions, of male orientation, but two possible explanations are environ- Liebherr and Roelofs reported that BE-strain moths showed de- mental dependence (e.g., flying 1–2 m upwind to sex pheromone layed mating compared to UZ-strain moths, which mate soon stimuli vs. a combination of walking and short flights to signal- after darkness (Fig. 3C). Other interpretations for this pattern ing females), or the presence of an unidentified short-distance have been proposed (Webster and Carde1982),andpheromone´ female cue (other than known sex pheromones) that elicits male strains in Europe display different circadian rhythms (Karpati orientation and which is similar in both strains. et al. 2007). Nevertheless, a 50% reduction in gene flow would Previous research has attributed patterns of assortative mat- ∼ be expected if the asynchrony in mating time reported by Liebherr ing between Z and E moths to male orientation specificity alone and Roelofs persists in the field (Table 3). The basis for circadian (e.g., Carde´ et al. 1978; Klun and Huettel 1988; Glover et al. 1991). mating rhythms in the ECB is unclear, but a genetic component However, like many other animal species (Andersson 1994), we seems likely because differences were detected after more than find that ECB females display choosy mate-selection behavior eight generations in a common laboratory environment. At least in that manifests as active discrimination when confronted by court- Drosophila,circadianmatingperiodicityisgeneticallycontrolled ing moths of the opposite strain (Fig. 4B). When rejecting a by an endogenous clock (e.g., Sakai and Ishida 2001). male’s courtship attempt, many females contort their abdomen so that copulation is impossible, others simply walk or fly away. Male and female behavioral isolation As a result, female behavioral isolation in some pairings is as Considering the prevalence of hybridization between animal taxa strong as male behavioral or seasonal temporal isolation (E Z: × that lack strong differences in mate selection preferences or sig- AS discrim. 0.712) (Table 3). Recently, it has been demonstrated = nals, Mayr (1963) argued that behavioral isolation is the most that♀ a male pheromone, produced by the hairpencil, is critical important form of reproductive isolation. In ECB, the impor- for female acceptance of courting males (Lassance and Lofstedt¨ tance of behavioral isolation is unequivocal. When Z and E moths 2009). Combined with our results, this finding suggests a role for are dispersed (1–50 m), numerous authors have reported that the this male pheromone in female behavioral isolation. In addition to use of different preferences and signals (i.e., male orientation to helping explain the deficit of hybrids in nature, male and female volatile sex pheromones released by females) results in limited mate preferences shed light on biases in the type of hybrids that are cross attraction (e.g., Roelofs et al. 1987; Bontemps et al. 2004), produced. Both male and female behaviors are asymmetric, with a and nearly complete reproductive isolation (Fig. 4C). What is deficit of successes when E females pair with Z males (Fig. 4A,B). not clear from these studies, however, is the extent that these Although behavioral asymmetries such as these are common be- preferences and cues prevent hybridization when moths are not tween animal species (Kaneshiro 1980; Arnold et al. 1996), and it dispersed. In general, behavioral isolation is usually examined in was known that Z males were more selective (Roelofs et al. 1987; one context, for example, a single spatial scale or a single ecolog- Glover et al. 1991; Linn et al. 1997), it is unusual that both sexes ical or laboratory setting (Coyne et al. 2005; but see Craig et al. of the Z strain are more discerning. Regardless of the evolutionary 1993). In the ECB, the spatial context for mating is important explanation for this pattern, the proximate consequence is strong because moths of both strains can be found to aggregate in the asymmetry in hybridization potential. This potential seems to be field where mate location or attraction could conceivably occur realized in the field, as an extensive survey of ECB in NY failed without the use of long-distance olfactory cues (Showers et al. to uncover E female Z male hybrids (Glover et al. 1991). × 1976; Malausa et al. 2005). Male behavioral isolation stems from changes at an unchar- By observing mating behavior, our study confirms that over acterized, autosomal fatty-acid reductase that affects pheromone very short distances (<20 cm), males of both strains continue to blend ratios (Zhu et al. 1996; Roelofs et al. 1987; Dopman et al. show orientation specificity towards females of their own strain 2004), along with changes at a sex-linked, major QTL for ori- (Fig. 4A). Combined with a recent investigation demonstrating entation (Glover et al. 1990; Dopman et al. 2004). A candidate

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gene(s) affecting male orientation has yet to be identified; how- many other potential mechanisms remain. It is possible that fertil- ever, a gene(s) that functions after peripheral sensory processing ization in Z E pairings proceeds normally, but foreign seminal × of pheromone cues is likely (Willett and Harrison 1999; Linn proteins or unrecognized copulatory behavior fails to stimulate et al. 1999; but see Karpati et al. 2008). As noted by Coyne and egg laying (e.g., Herndon and Wolfner 1995). Alternatively, in Orr (2004), monogenic inheritance of male behavioral isolation in some interspecific crosses (e.g., Gregory and Howard 1993), re- ECB contrasts with polygenic inheritance in other species. With duced oviposition reflects poor fertilization and the retention of this simple genetic basis, it is difficult to understand how new pref- unfertilized eggs. Even with good demonstrations of cryptic iso- erences or signals could have evolved because most mutations are lation (e.g., Swanson and Vacquier 1997; Price et al. 2001; Nosil expected to have large and deleterious phenotypic effects (review and Crespi 2006; Mendelson et al. 2007), the processes driving in Lofstedt¨ 1993). The existence of rare ECB males that orient to- evolution are poorly understood, and thus represent a clear avenue wards females from both the ECB and the Asian corn borer (ACB, for further work. O. furnacalis) provides a partial solution (Reolofs et al. 2002)– some ECB males may have been preadapted to orient towards POSTZYGOTIC BARRIERS the first mutant Z- or E-strain female, mitigating initial fitness Behavioral and physiological infertility costs associated with pheromone blend evolution. Regarding fe- Behavioral hybrid infertility or dysfunction has two primary male behavioral isolation, both proximate and ultimate causes causes that are often difficult to distinguish (Stratton and Uetz will remain unknown until the sensory modalities involved have 1986; Frey and Bush 1996; Noor 1997; Servedio and Noor 2003; been elucidated. Acoustic communication occurs in the ACB, Linn et al. 2004; Coyne and Orr 2004). Failures or delays in mat- in which males produce an ultrasonic courtship “song” (Nakano ing can result when hybrid behaviors are intermediate compared et al. 2006), but an olfactory system is also possible (Royer and to parental individuals (extrinsic behavioral dysfunction) (e.g., McNeil 1992; Lassance and Lofstedt¨ 2009). Stratton and Uetz 1986). Alternatively, failures or delays can re- sult when otherwise “normal” hybrid behaviors are inhibited or POSTMATING-PREZYGOTIC BARRIERS disrupted by physiological or neurological abnormalities (intrin- Mechanical and gametic isolation sic behavioral dysfunction) (e.g., Noor 1997). Our reanalysis of “Cryptic” reproductive barriers, which include mechanical and Roelofs et al. (1987) suggests that both extrinsic and intrinsic dys- gametic forms of postmating-prezygotic isolation, although diffi- function contribute to reduced F1 male mating success in the ECB cult to observe have been documented across diverse organisms (Fig. 2A). Although F1 males show moderate response to the Z (e.g., in broadcast spawners [Swanson and Vacquier 1997], in in- pheromone and therefore do not exhibit exclusively intermediate sects [Gregory and Howard 1993; Herndon and Wolfner 1995; behavior, few hybrids respond to the E-strain blend. Similarly, al- Price et al. 2001; Nosil and Crespi 2006], in fish [Mendelson though hybrids show appreciable orientation to the Z and hybrid et al. 2007], review in Coyne and Orr 2004). We find that me- blends, they do not respond as readily as Z and E males to their chanical isolation is either limited or absent between Z and E own pheromones. strains (Fig. 5A), but our ability to test for clasping failures was Due to orientation patterns among Z, E, and hybrid males, restricted by a small sample size in E Zpairings.Limitedme- behavioral hybrid dysfunction becomes apparent only under re- × chanical isolation would corroborate the findings of Cardeetal.´ stricted circumstances. Specifically, dysfunction and intermedi- (1978), in which genitalic differences between strains that might ate levels of isolation occur when E females predominate (e.g., contribute to mechanical isolation were not detected. In contrast Geneva, NY in Fig. 2B,C; mean AS 0.469, Table 3). F1 dys. = to mechanical isolation, we find that ECB strains exhibit partial When Z and hybrid females are at high frequency♂ (e.g., Beltsville, gametic isolation, confirming preliminary results obtained over MD, in Fig. 2B,C), hybrids experience intermediate relative fit- 30 years ago (Liebherr and Roelofs 1975). Gametic incompati- ness and introgression is promoted (mean AS 0.539, F1 dys. = − bilities are moderate in intensity and are significant in only one Table 3). Two relevant points suggest that behavioral♂ dysfunction cross direction. When Z females mate with E males, lifetime egg could be weak in nature. First, our measure is based on long- production drops by 30% (Fig. 5B; ASovipos 0.309, Table 3), distance attraction, but males may orient to a wider spectrum of ∼ = but fertilization rate remains high (Fig. 5C). female genotypes when aggregated (Fig. 4A vs. C). Second, con- Postmating-prezygotic problems are often highly variable ditions favorable to hybrid male orientation are probably common and asymmetric between diverged populations or species, and in the field because Z females are typically the most numerous cross failures can have multiple causes (Price et al. 2001; Nosil genotype (Table 1; Klun and Cooperators 1975; Anglade and and Crespi 2006; Mendelson et al. 2007). In the ECB, explanations Stockel 1984). Whatever its impact on gene flow, the evolution involving reduced remating frequency can be rejected based on the of behavioral dysfunction is linked to male behavioral isolation presence of a single transferred spermatophore in all females, but because both barriers have a common genetic basis.

896 EVOLUTION APRIL 2010 COMPONENTS OF REPRODUCTIVE ISOLATION

In contrast to behavioral dysfunction, the fertility effects of in strength (e.g., behavioral isolation in Drosophila,Coyneand hybrid physiology or development is one of the most commonly Orr 2004), or else population fusion will occur. In contrast, geo- studied forms of reproductive isolation (review in Coyne and graphical separation facilitates the origin of trait differences and Orr 2004). In the ECB, Liebherr and Roelofs (1975) reported reproductive barriers by almost any evolutionary process (besides that F2 intercrosses yielded fertile eggs, whereas only 70% reinforcement), and restricted migration may facilitate the persis- ∼ of backcrosses were successful. Our measure of fertility (per- tence and accumulation of trait differences and barriers of vary- cent fertile eggs) indicates that both F2 and BC pairings proceed ing strength (Rice and Hosert 1993). Under this reasoning, the normally ( 90% fertile, Table 2), but BCs do suffer dispropor- complex phenotypic architecture of speciation that is observed ∼ tionately in terms of fecundity ( 25% fewer eggs than F2 or between ECB strains may have been promoted by an extended ∼ parental crosses). However, among all second-generation crosses, historical period(s) of restricted gene flow, such as that imposed oviposition drops by only 15% compared to crosses occurring by physical separation. ∼ within-strain. Thus, our results indicate that physiological hybrid Uncovering manifold causes of speciation suggests a high infertility is a rather weak isolating barrier (ASF1 ovipos. 0.159, level of complexity in the underlying genetic architecture. That = Table 3). Inheritance patterns of oviposition in first and second- is, the genetics of speciation will typically consist of more loci generation crosses (Fig. 5B; Table 3) might be consistent with with the potential for more elaborate control (e.g., epistasis, ad- an incompatibility between an autosomal recessive allele in the Z ditive), phenotypic effects (e.g., minor versus major), and chro- strain and a dominant E-strain allele, but more work is needed to mosomal distributions, as the number of barriers preventing gene explore the genetic and evolutionary basis of this phenotype. flow increases. Besides underscoring the obvious truth that disen- tangling the genetics of speciation in many (or most) species will THE PHENOTYPIC ARCHITECTURE OF SPECIATION require tremendous effort, a more profound implication is how The number and strength of isolating barriers that have evolved this genetic complexity will shape patterns of gene flow when before speciation is complete, which we refer to here as the “phe- populations are sympatric. notypic architecture” of speciation, provides insight into several The genetic architecture of speciation has consequences for outstanding problems in speciation research. One important issue genome-wide patterns of gene flow because chromosomal re- is whether this phenotypic architecture typically consists of one gions linked to reproductive barrier loci will introgress across or a few strong barriers to gene flow or whether many weaker species boundaries at lower rates than unlinked regions (Barton barriers are involved. The Z and E strains of ECB most clearly and Hewitt 1981; Harrison 1990; Wu 2001; Ting et al. 2000). As a fall into the latter category, given that seven of 12 potential forms result of this effect, species or racial boundaries are often thought of isolation restrict gene exchange, but all are less than 80% com- of as “semipermeable” or “porous,” with permeability depending plete (Table 3). Comparable studies have revealed a similar pat- on the genetic marker (Barton and Hewitt 1981; Harrison 1990; tern (e.g., Chamerioin angustifolium populations, Husband and Wu 2001; Via 2009). For a semipermeable genome, in regions that Sabara 2003; sister species of Mimulus, Ramsey et al. 2003; lin- are unlinked to barrier loci, gene flow will constrain local adap- eages of Pogonomyrmex ants, Schwander et al. 2008; Etheostoma tation but facilitate the spread of new, globally beneficial alleles. darters, Mendelson et al. 2007; review in Coyne and Orr 2004), Conversely, restricted gene flow near barrier loci will promote suggesting that speciation is commonly an extended and complex further population divergence. Thus, the phenotypic architecture process that requires an accumulation of barriers of intermediate of speciation will shape the genomic context in which diverging strength. Nevertheless, the role of one or a few strong barriers to populations change in response to spatial or temporal heterogene- gene flow has been documented between some diverging popula- ity. Speciation in which a large number of reproductive barriers tions (review in Funk et al. 2002; Nosil et al. 2005; Lowry et al. are involved is predicted to increase the size of the semipermeable 2008). fraction of diverging genomes, whereas the strength of each bar- An evolutionary explanation for these two contrasting phe- rier (and possibly when it operates over an organism’s life history) notypic architectures of speciation may be related to differences in may affect the degree of permeability at underlying loci. the geographic mode of divergence. A history that includes long Z and E strains of ECB support the semipermeable genome intervals of sympatry might result in a small number of barriers model of speciation. In upstate NY,even where they exist together because gene flow will constrain the evolution of new pheno- in the same fields, E and Z borers remain differentiated for Tpi typic differences (possibly to a subset driven by strong selection suggesting that gene flow is limited or absent for this locus. Pat- [Rice and Hosert 1993]), and impede some types of evolution- terns of variation at Tpi may be a consequence of close physical ary processes from promoting divergence (e.g., by genetic drift linkage to Pdd (Dopman et al. 2004, 2005). In contrast, other or “mutation-order” speciation [Schluter 2009]). Those few bar- nuclear genes (two of which, kettin and Ldh,arealsoonthesex riers that do evolve in sympatry must arise rapidly and be great chromosome) reveal no evidence of differentiation, because of

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recent or ongoing hybridization and introgression (Dopman et al. large and that the (in)activation of different paralogs could pro- 2005). With a relatively large number of reproductive barriers be- vide a major source of “raw material” for rapid sex-pheromone tween Z and E strains (Table 3), the ECB provides an important evolution. Although pheromone blend differences between ECB study system in which to investigate the consequences of the phe- strains seems to be from allelic changes rather than from alterna- notypic and genetic architectures of speciation for introgression tive paralog usage, mutational mechanisms could be responsible and local adaptation. for rapid behavioral isolation in the Ostrinia genus, and perhaps, A complex phenotypic architecture of speciation presents in other species. challenges for understanding the evolutionary forces driving speciation. However, only a subset of barriers and evolutionary THE IMPORTANCE OF PRE- VERSUS POSTZYGOTIC processes will have been important in initiating the speciation BARRIERS TO SPECIATION process. Testing hypotheses about these early-evolving barriers There has been long-standing debate about the relative contribu- typically requires a comparative approach (e.g., Coyne and Orr tions of prezygotic versus postzygotic barriers to speciation (e.g., 1997; Mendelson 2003; but see Christianson et al. 2005), but Coyne and Orr 1989, 1997; Kirkpatrick and Ravigne 2002). It Coyne and Orr (2004) noted that it is unlikely for a barrier to have is clear that the timing of the evolution of barriers during the played a major historical role unless its contemporary strength often-extended process of speciation is most clearly related to is large. Therefore, premating barriers may be among the most the historical or evolutionary causes of speciation. In this sense, historically important between strains of the ECB (Table 3). those barriers that evolved first (e.g., as identified by comparative There are at least two reasons why premating barriers might studies) are the most “important.” However, a barrier’s relative be predisposed to rapid evolution–they may be common targets or “sequential” strength over an organism’s life history informs of selection, or they could be more mutable. Comparative studies a related issue about the contemporary or ecological causes of suggest that behavioral isolation can rapidly evolve by reinforce- speciation in which the main question is how diverging popula- ment in sympatry or as a byproduct of sexual selection in allopatry tions remain distinct. Accumulating evidence supports the inter- (Coyne and Orr 1989, 1997; Mendelson 2003). For reinforce- pretation that premating barriers may be most important forms of ment to explain patterns in the ECB, hybrids must experience isolation between in both contemporary and historical population reduced fitness. Hybrid ECB are relatively fit in the laboratory pairs, but the debate is far from resolved (Ramsey et al. 2003; (Fig. 2C; Table 2; Calcagno et al. 2007), but reinforcement can- Husband and Sabara 2003; Nosil et al. 2005; Martin and Willis not be ruled out until ecologically dependent costs (e.g., Rundle 2007; Mendelson et al. 2007; Nosil 2007; Lowry et al. 2008; and Whitlock 2001) have been excluded. In contrast, conditions Schwander et al. 2008; Schluter 2009). favoring sexual conflict are known. Male ECB fitness increases For the ECB, with more than 98% of total cumulative repro- with more mating (Royer and McNeil 1993), but the act of mating ductive isolation consisting of premating barriers, our findings reduces female survival (Fadamiro and Baker 1999). In contrast broadly confirm early studies (e.g., Arbuthnot 1944; Liebherr to behavior, breeding cycle differences are unlikely to have been and Roelofs 1975), which implicate prezygotic barriers as the historically important because Z and E strains breed at similar most important for the maintenance of Z and E strains in nature times at most sympatric sites (Glover et al. 1991; Sorenson et al. (Table 3; Fig. 6). In the weak isolation scenario, which proba- 1992; Thomas et al. 2003). Nevertheless, circumstantial evidence bly represents a close approximation to that experienced by many suggests that PDD time might be prone to rapid adaptive evo- ECB strains in the field, temporal isolation (seasonal circadian) + lution in the ECB (Dopman et al. 2005; Malausa et al. 2007a). contributes nearly two-thirds to the total, whereas another third ∼ Empirical studies have documented rapid evolution of circannual is contributed by behavioral isolation (male female) (Fig. 6). + and circadian rhythms in both animals and in plants (e.g., Weber All other isolating barriers combined (postmating-prezygotic + and Schmid 1998; Tauber et al. 2007), with diapause traits being postzygotic) contribute a little more than 1% at most to total iso- among those phenotypes showing adaptive responses in insects lation. Granted, not all isolating barriers have been measured in (e.g, Tauber et al. 2007). the ECB. Patterns consistent with “hybrid breakdown” have been Premating barriers might also rapidly evolve because their documented in life stages other than those investigated here (i.e., genetic determinants are predisposed to mutation. In yeast, the in larvae, Arbuthnot 1944; Liebherr and Roelofs 1975), and strong spontaneous gene duplication/deletion rate is orders of magnitude extrinsic (postzygotic and prezygotic) barriers may have gone un- larger than the nucleotide substitution rate (Lynch et al. 2008), detected in our benign laboratory setting (e.g., environmentally and the genes determining pheromone differences between Os- dependent postzygotic incompatibilities at Pdd). Nevertheless, trinia are in a multigene family (Roelofs et al. 2002; Roelofs and some Z and E populations in Europe differ in traits associated Rooney 2003). Together, these results suggest that the number of with specialization to different host plants (Thomas et al. 2003; pheromone genes that are polymorphic in copy number might be Calcagno et al. 2007; Malausa et al. 2008). If these differences

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prevent gene flow then Z and E strains of ECB would almost due to voltinism [here] or host–plant use [Thomas et al. 2003; certainly join the growing list of incipient species that support Calcagno et al. 2007; Malausa et al. 2008]). A more dynamic ap- premating barriers as the strongest impediments to gene flow plication of the BSC that acknowledges this diversity is necessary (e.g., Ramsey et al. 2003; Husband and Sabara 2003; Nosil 2007; for the ECB. When combined with a comprehensive genetic sur- Martin and Willis 2007; Mendelson et al. 2007; Lowry et al. vey, such a perspective can lead to a better understanding of the 2008). process in which alleles at reproductive barrier loci spread across populations before ultimately giving rise to completely isolated TOTAL REPRODUCTIVE ISOLATION AND PATTERNS and distinct species. OF GENE FLOW IN NATURE For most species the biological context for the maintenance or erosion of genetic differences between populations in nature is Conclusion obscure, but we can begin to characterize this relationship for Z Our study determined that speciation between the closely related and E strains of the ECB. One expected pattern is a negative asso- Z and E strains of ECB has diverse phenotypic causes, which are ciation between estimates of cumulative isolation and estimates incomplete when acting alone but also when operating together. of genetic exchange in the field. Based on measures of cumulative The presence of numerous incomplete barriers between these par- isolation, sympatric populations of UZ and BE moths should pro- tially isolated populations suggests that speciation in this lineage duce fewer hybrid offspring and experience less gene flow than is a slow process relative to the rate in which isolating barriers sympatric BZ and BE moths (Table 3). In agreement with this pre- evolve. Along with recent investigations, our observations indi- diction, the proportion of hybrid females is smaller at the UZ/BE cate that premating barriers represent the main impediments to Geneva site than at the BZ/BE Beltsville site (Fig. 2B; Roelofs gene flow between species, potentially because premating barri- et al. 1985; Klun and Huettel 1988), and genetic differences have ers evolve at early stages of the speciation process. Testing for been maintained for 20 years between UZ and BE populations rapid evolution of premating barriers requires a comparative ap- ∼ at Geneva (Glover et al. 1991; Dopman et al. 2005). Presumably, proach, with numerous barriers measured across a large group more recent evidence for introgression would be found between of taxa that vary in levels of evolutionary divergence. One such strains at Beltsville and at other sites where seasonal isolation is study has been conducted in Lepidoptera (Presgraves 2002), but weak. with only postzygotic forms of isolation characterized, the gen- Although it is unlikely that measures of cumulative isolation eral principles governing the origin of new Lepidopteran species translate easily into quantitative assessments of hybridization or have yet to be discovered. Estimating the strength of behavioral, gene flow, cumulative isolation can inform taxonomic issues be- temporal, and other forms of reproductive isolation across the cause the BSC defines species by the presence of reproductive Ostrinia genus and among related species holds promise for re- barriers (Ramsey et al. 2003; Husband and Sabara 2003; Martin vealing these general principles. and Willis 2007). This is particularly useful for Z and E strains ACKNOWLEDGMENTS of ECB because their morphological and genetic similarity (e.g., We are grateful to R. Harrison, L. Hatch, D. Hartl, C. Jiggins, and three Liebherr 1974; Dopman et al. 2005) has led to regular taxonomic anonymous reviewers for helpful comments on this manuscript and to C. debate and the emergence of two opposing views. Thirty years Linn, W. Roelofs, and K. Poole for advice, suggestions, and materials. ago, Cardeandcolleagues(1978)arguedonthebasisofallozyme,´ This research was partially supported by a National Institutes of Health pheromone, and hybridization data that pheromone strains are dis- Kirschstein-NRSA Postdoctoral Fellowship (GM080090-01 to E.B.D.). tinct species. More recently, Frolov et al. (2007) asserted that Z LITERATURE CITED and E strains using maize, such as those investigated here, be- Andersson, M. 1994. Sexual selection. Princeton Univ. Press, Princeton, NJ. long to one species, whereas those using non-maize hosts (e.g., Anglade, P., and J. Stockel. 1984. Intraspecific sex-pheromone variability in mugwort) belong to another. With as much as 10% gene flow the European corn borer, Ostrinia nubilalis (Lepidoptera, Pyralidae). ∼ permitted (in Z Ecrosses,Table3)andmorethan15%hybrids Agronomie 4:183–187. × at some sites (Fig. 2B; Klun and Huettel 1988), claims of dis- Arnold, S. J., P. A. Verrell, and S. G. Tilley. 1996. The evolution of asymmetry in sexual isolation: a model and a test case. Evolution 50:1024–1033. tinct species seems unjustified. On the other hand, Frolov et al.’s Arnqvist, G., M. Edvardsson, U. Friberg, and T. Nilsson. 2000. Sexual conflict (2007) treatment of maize-feeding strains as conspecific appears promotes speciation in insects. Proc. Natl. Acad. Sci. USA 97:10460– to be at odds with our finding that total isolation approaches 10464. completion if seasonal isolation is strong (Table 3). These con- Arbuthnot, K. D. 1944. Strains of the European corn borer in the United States. USDA Tech. Bull. 869:1–20. trary interpretations can be reconciled by interpreting Z- and E- Barton, N. H., and G. M. Hewitt. 1981. Hybrid zones and speciation. Pp. 109– pheromone strains as a mosaic of incompletely isolated popu- 145 in W. R. Atchley and D. S. Woodruff, eds. Evolution and speciation. lations that differ in levels of total reproductive isolation (e.g., Cambridge Univ. Press, Cambridge, U.K.

EVOLUTION APRIL 2010 899 ERIK B. DOPMAN ET AL.

Beck, S. D. 1983. Thermal and thermoperiodic effects on larval development Eckenrode, C. J., P. S. Robbins, and J. T. Andaloro. 1983. Variations in flight and diapause in the European corn borer, Ostrinia nubilalis.J.Insect patterns of the European corn borer (Lepidoptera, Pyralidae) in New Physiol. 29:107–112. York. Environ. Entomol. 12:393–396. Bethenod, M. T., Y. Thomas, F. Rousset, B. Frerot, L. Pelozuelo, G. Genestier, Fadamiro, H. Y., and T. C. Baker. 1999. Reproductive performance and and D. Bourguet. 2005. Genetic isolation between two sympatric host longevity of female European corn borer, Ostrinia nubilalis:effects plant races of the European corn borer, Ostrinia nubilalis Hubner. II: of multiple mating, delay in mating, and adult feeding. J. Insect Physiol. assortative mating and host-plant preferences for oviposition. Heredity 45:385–392. 94:264–270. Frey, J. E., and G. L. Bush. 1996. Impaired host odor perception in hybrids Bontemps, A., D. Bourguet, L. Pelozuelo, M. T. Bethenod, and S. Ponsard. between the sibling species Rhagoletis pomonella and R. mendax.En- 2004. Managing the evolution of Bacillus thuringiensis resistance in tomol. Exp. Appl. 80:163–165. natural populations of the European corn borer, Ostrinia nubilalis: host Frolov, A. N., D. Bourguet, and S. Ponsard. 2007. Reconsidering the taxomony plant, host race and pherotype of adult males at aggregation sites. Proc. of several Ostrinia species in the light of reproductive isolation: a tale R. Soc. Lond. B 271:2179–2185. for Ernst Mayr. Biol. J. Linn. Soc. 91:49–72. Bradshaw, W. E., and C. M. Holzapfel. 2007. Evolution of animal photoperi- Funk, D. J., K. E. Filchak, and J. L. Feder. 2002. Herbivorous insects: odism. Annu. Rev. Ecol. Syst. 38:1–25. model systems for the comparative study of speciation ecology. Ge- Bradshaw, W. E., P. A. Zani, and C. M. Holzapfel. 2004. Adaptation to netica 116:251–267. temperate climates. Evolution 58:1748–1762. Glover, T., M. Campbell, P. Robbins, and W. Roelofs. 1990. Sex-linked con- Burkness, E. C., W. D. Hutchison, P. C. Bolin, D. W. Bartels, D. F. Warnock, trol of sex-pheromone behavioral responses in European corn borer and D. W. Davis. 2001. Field efficacy of sweet corn hybrids expressing a moths (Ostrinia nubilalis) confirmed with TPI marker gene. Arch. In- Bacillus thuringiensis toxin for management of Ostrinia nubilalis (Lep- sect Biochem. Physiol. 15:67–77. idoptera : Crambidae) and Helicoverpa zea (Lepidoptera : Noctuidae). Glover, T. J., J. J. Knodel, P. S. Robbins, C. J. Eckenrode, and W. L. Roelofs. J. Econ. Entomol. 94:197–203. 1991. Gene flow among 3 races of European corn borer (Lepidoptera, Caffrey, D. J., and L. H. Worthley. 1927. A progress report on the investiga- Pyralidae) in New York State. Environ. Entomol. 20:1356–1362. tions of the European corn borer. USDA Bull. No. 1476, Governmental Glover, T. J., P. S. Robbins, C. J. Eckenrode, and W. L. Roelofs. 1992. Genetic Printing Office, Washington, DC. control of voltinism characteristics in European corn borer races assessed Calcagno, V., Y. Thomas, and D. Bourguet. 2007. Sympatric host races of the with a marker gene. Arch. Insect Biochem. Physiol. 20:107–117. European corn borer: adaptation to host plants and hybrid performance. Gregory, P. G., and D. J. Howard. 1993. Laboratory hybridization studies J. Evol. Biol. 20:1720–1729. of Allonemobius fasciatus and A. socius (Orthoptera, Gryllidae). Ann. Carde,´ R. T., W. L. Roelofs, R. G. Harrison, A. T. Vawter, P. F. Brussard, Entomol. Soc. Am. 86:694–701. A. Mutuura, and E. Munroe. 1978. European corn borer: pheromone Harrison, R. G. 1990. Hybrid zones: windows on evolutionary process. Pp. polymorphism or sibling species. Science 199:555–556. 69–128 in D. J. Futuyma and J. Antonovics, eds. Oxford surveys in Christianson, S. J., J. G. Swallow, and G. S. Wilkinson. 2005. Rapid evolu- evolutionary biology, Vol. 7, Oxford University Press, Oxford, UK. tion of postzygotic reproductive isolation in stalk-eyed flies. Evolution Herndon, L. A., and M. F. Wolfner. 1995. A Drosophila seminal fluid protein, 59:849–857. ACP26AA,stimulatesegglayinginfemalesfor1dayaftermating.Proc. Clifton, K. E. 1997. Mass spawning by green algae on coral reefs. Science Natl. Acad. Sci. USA 92:10114–10118. 275:1116–1118. Huang, Y. P., T. Takanashi, S. Hoshizaki, S. Tatsuki, and Y. Ishikawa. 2002. Coates, B. S., D. V. Somerford, and R. L. Hellmich. 2004. Geographic and Female sex pheromone polymorphism in adzuki bean borer, Ostrinia voltinism differentiation among North American Ostrinia nubilalis (Eu- scapulalis,issimilartothatinEuropeancornborer,O. nubilalis.J. ropean corn borer) mitochondrial cytochrome c oxidase haplotypes. J Chem. Ecol. 28:533–539. Insect Sci. 308:258–260. Hudon, M., and E. J. Leroux. 1986. Biology and population dynamics of the Cooley, J. R., C. Simon, and D. C. Marshall. 2003. Temporal separation and European corn borer (Ostrinia nubilalis), with special reference to sweet speciation in periodical cicadas. Bioscience 53:151–157. corn in Quebec: systematics, morphology, geographical distribution, Coyne, J. A., and H. A. Orr. 1989. Patterns of speciation in Drosophila. host range, and economic importance. Phytoprotection 67:39–54. Evolution 43:362–381. Hurt, C. R., M. Farzin, and P. W. Hedrick. 2005. Premating, not postmating, ———. 1997. “Patterns of speciation in Drosophila” revisited. Evolution barriers drive genetic dynamics in experimental hybrid populations of 51:295–303. the endangered sonoran topminnow. Genetics 171:655–662. ———. 2004. Speciation. Sinauer Associates, Inc., Sunderland, MA. Husband, B. C., and H. A. Sabara. 2003. Reproductive isolation between Coyne, J. A., S. Elwyn, and E. Rolan-Alvarez. 2005. Impact of experimental autotetraploids and their diploid progenitors in fireweed, Chamerion design on Drosophila sexual isolation studies: direct effects and com- angustifolium (Onagraceae). New Phytol. 161:703–713. parison to field hybridization data. Evolution 59:2588–2601. Kaneshiro, K. Y. 1980. Sexual isolation, speciation, and the direction of evo- Craig, T. P., J. K. Itami, W. G. Abrahamson, and J. D. Horner. 1993. Behav- lution. Evolution 34:437–444. ioral evidence for host-race formation in Eurosta solidaginis. Evolution Karpati, Z., B. Molnar, and G. Szocs. 2007. Pheromone titer and mating fre- 47:1696–1710. quency of E- and Z-strains of the European corn borer, Ostrinia nubilalis: Dobzhansky, T. 1937. Genetics and the origin of species. Columbia Univ. fluctuations during scotophase and age dependence. Acta Phytopathol. Press, New York, NY. Entomol. Hungarica. 10:331–341. Dopman, E. B., S. M. Bogdanowicz, and R. G. Harrison. 2004. Genetic Karpati, Z., T. Dekker, and B. S. Hansson. 2008. Reversed functional topology mapping of sexual isolation between E and Z pheromone strains of the in the antennal lobe of the male European corn borer. J. Exp. Biol. European corn borer (Ostrinia nubilalis). Genetics 167:301–309. 211:2841. Dopman, E. B., L. Perez, S. M. Bogdanowicz, and R. G. Harrison. 2005. Kim, C., S. Hoshizaki, Y. Huang, S. Tatsuzki, and Y. Ishikawa. 1999. Use- Consequences of reproductive barriers for genealogical discordance in fulness of mitochondrial COII gene sequences in examining phyloge- the European corn borer. Proc. Natl. Acad. Sci. USA 102:14706–14711. netic relationships in the Asian corn borer, Ostrinia furnacalis,and

900 EVOLUTION APRIL 2010 COMPONENTS OF REPRODUCTIVE ISOLATION

allied species (Lepidoptera: Pyralidae). Appl. Entomol. Zool. 34:405– 2008. Differences in oviposition behaviour of two sympatric sibling 412. species of the genus Ostrinia. Bull. Entomol. Res. 98:193–201. Kirkpatrick, M., and V. Ravigne. 2002. Speciation by natural and sexual Martin, N. H., and J. H. Willis. 2007. Ecological divergence associated with selection: models and experiments. Am. Nat. 159:S22–S35. mating system causes nearly complete reproductive isolation between Klun, J. A., and Cooperators. 1975. Insect sex pheromones: intraspecific sympatric Mimulus species. Evolution 61:68–82. pheromonal variability of Ostrinia nubilalisi (Lepidoptera, Pyralidae) Mayr, E. 1942. Systematics and the origin of species. Columbia Univ. Press, in North America and Europe. Environ. Entomol. 4:891–894. New York, NY. Klun, J. A., and M. D. Huettel. 1988. Genetic regulation of sex pheromone ———. 1963. Animal species and evolution. Harvard Univ. Press, Cambridge, production and response: interaction of sympatric pheromonal types MA. of European corn borer, Ostrinia nubilalis (Lepidoptera, Pyralidae). J. McLeod, D. G. R. 1978. Genetics of diapause induction and termination in Chem. Ecol. 14:2047–2061. the European corn borer, Ostrinia nubilalis (Lepidoptera: pyralidae), in Klun, J. A., and S. Maini. 1979. Genetic basis of an insect chemical communi- Southwestern Ontario. Can. Ent.110:1351–1353. cation system: the European corn borer. Environ. Entomol. 8:423–426. McMillan, W. O., C. Jiggins, and J. Mallet. 1997. What initiates speciation in Knowlton, N., J. L. Mate, H. M. Guzman, R. Rowan, and J. Jara. 1997. passion-vine butterflies? Proc. Natl. Acad. Sci. USA. 94:8628–8633. Direct evidence for reproductive isolation among the three species of Mendelson, T. C. 2003. Sexual isolation evolves faster than hybrid inviability the Montastraea annularis complex in Central America (Panama and in a diverse and sexually dimorphic genus of fish (Percidae: Etheostoma). Honduras). Mar. Biol. 127:705–711. Evolution 57:317–327. Kochansky, J., R. T. Carde,´ J. Liebherr, and W. L. Roelofs. 1975. Sex Mendelson, T. C., V. E. Imhoff, and J. J. Venditti. 2007. The accumulation of pheromones of the European corn borer in New York. J. Chem. Ecol. reproductive barriers during speciation: postmating barriers in two be- 1:225–231. haviorally isolated species of darters (Percidae: Etheostoma). Evolution Lassance, J. M., and C. Lofstedt.¨ 2009. Concerted evolution of male and female 61:2595–2606. display traits in the European corn borer, Ostrinia nubilalis.BMCBiol. Mitter, C., B. Farrell, and B. Wiegmann. 1988. The phylogenetic study of 7:10. adaptive zones: has phytophagy promoted diversification? Am. Nat. Liebherr, J. 1974. Studies on two strains of the European corn borer, Ostrinia 132:107–128. nubilalis.MastersThesis,CornellUniv.,Ithaca,NY. Mutuura, A., and E. Munroe. 1970. Taxonomy and distribution of the European Liebherr, J., and W. Roelofs. 1975. Laboratory hybridization and mating corn borer and allied species: genus Ostrinia (Lepidoptera: Pyralidae). period studies using 2 pheromone strains of Ostrinia nubilalis.Ann. Pp. 1–112 in D. P. Pielou, ed. Memoirs of the entomological society of Entomol. Soc. Am. 68:305–309. Canada. Entomological Society of Canada, Ottawa. Linn, C. E., M. S. Young, M. Gendle, T. J. Glover, and W. L. Roelofs. Nakano, R., Y. Ishikawa, S. Tatsuki, A. Surlykke, N. Skals, and T. Takanashi. 1997. Sex pheromone blend discrimination in two races and hybrids 2006. Ultrasonic courtship song in the Asian corn borer moth, Ostrinia of the European corn borer moth, Ostrinia nubilalis.Physiol.Entomol. furnacalis. Naturwissenschaften 93:292–296. 22:212–223. Noor, M. A. F. 1997. Genetics of sexual isolation and courtship dysfunction in Linn, C., K. Poole, A. Zhang, and W. Roelofs. 1999. Pheromone-blend dis- male hybrids of Drosophila pseudoobscura and Drosophila persimilis. crimination by European corn borer moths with inter-race and inter-sex Evolution 51:809–815. antennal transplants. J. Comp. Physiol. Neuroethol. A Sens. Neural Be- Nosil, P. 2007. Divergent host plant adaptation and reproductive isolation be- hav. Physiol. 184:273–278. tween ecotypes of Timema cristinae walking sticks. Am. Nat. 169:151– Linn, C. E., H. R. Dambroski, J. L. Feder, S. H. Berlocher, S. Nojima, and W. 162. L. Roelofs. 2004. Postzygotic isolating factor in sympatric speciation Nosil, P., and B. J. Crespi. 2006. Ecological divergence promotes the evolution in Rhagoletis flies: reduced response of hybrids to parental host-fruit of cryptic reproductive isolation. Proc. R. Soc. Lond. B 273:991–997. odors. Proc. Natl. Acad. Sci. USA 101:17753–17758. Nosil, P., T. H. Vines, and D. J. Funk. 2005. Reproductive isolation caused by Lofstedt,¨ C. 1993. Moth pheromone genetics and evolution. Philos. Trans. R. natural selection against, immigrants from divergent habitats. Evolution. Soc. Lond. B 340:167–177. 59:705–719. Lowry, D. B., J. L. Modliszewski, K. M. Wright, C. A. Wu, and J. H. Willis. Pelozuelo, L., S. Meusnier, P. Audiot, D. Bourguet, and S. Ponsard. 2007. As- 2008. The strength and genetic basis of reproductive isolating barriers sortative mating between European corn borer pheromone races: beyond in flowering plants. Phil. Trans. R. Soc. B. 363:3009–3021. assortative meeting. PLoS One 2:e555. Lynch, M., W. Sung, K. Morris, N. Coffey, C. R. Landry, E. B. Dopman, Ponsard, S., M. T. Bethenod, A. Bontemps, L. Pelozuelo, M. C. Souqual, W. J. Dickinson, K. Okamoto, S. Kulkarni, D. L. Hartl, et al. 2008. A and D. Bourguet. 2004. Carbon stable isotopes: a tool for studying the genome-wide view of the spectrum of spontaneous mutations in yeast. mating, oviposition, and spatial distribution of races of European corn Proc. Natl. Acad. Sci. USA 105:9272–9277. borer, Ostrinia nubilalis, among host plants in the field. Can. J. Zool. Malausa, T., L. Leniaud, J. F. Martin, P. Audiot, D. Bourguet, S. Ponsard, S. F. 82:1177–1185. Lee, R. G. Harrison, and E. Dopman. 2007a. Molecular differentiation Presgraves, D. C. 2002. Patterns of postzygotic isolation in Lepidoptera. Evo- at nuclear loci in French host races of the European corn borer (Ostrinia lution 56:1168–1183. nubilalis). Genetics 176:2343–2355. Price, C. S. C., C. H. Kim, C. J. Gronlund, and J. A. Coyne. 2001. Cryp- Malausa, T., M. T. Bethenod, A. Bontemps, D. Bourguet, J. M. Cornuet, and tic reproductive isolation in the Drosophila simulans species complex. S. Ponsard. 2005. Assortative mating in sympatric host races of the Evolution. 55:81–92. European corn borer. Science 308:258–260. Quinn, T. P., M. J. Unwin, and M. T. Kinnison. 2000. Evolution of temporal iso- Malausa, T., A. Dalecky, S. Ponsard, P. Audiot, R. Streiff, Y. Chaval, and D. lation in the wild: genetic divergence in timing of migration and breeding Bourguet. 2007b. Genetic structure and gene flow in French populations by introduced chinook salmon populations. Evolution 54:1372–1385. of two Ostrinia taxa: host races or sibling species? Mol. Ecol. 16:4210– Ramsey, J., H. D. Bradshaw, and D. W. Schemske. 2003. Components of 4222. reproductive isolation between the monkeyflowers Mimulus lewisii and Malausa, T., B. Pelissie, V.Piveteau, C. Pelissier, D. Bourguet, and S. Ponsard. M. cardinalis (Phrymaceae). Evolution 57:1520–1534.

EVOLUTION APRIL 2010 901 ERIK B. DOPMAN ET AL.

Reed, G. L., W. D. Guthrie, W. B. Showers, B. D. Barry, and D. F. Cox. Sorenson, C. E., G. G. Kennedy, W. Vanduyn, J. R. Bradley, and J. F. 1981. Sex-linked inheritance of diapause in the European corn borer: its Walgenbach. 1992. Geographical variation in pheromone response of significance to diapause physiology and environmental response of the the European corn borer, Ostrinia nubilalis, in North Carolina. Ento- insect. Ann. Entomol. Soc. Am. 74:1–8. mol. Exp. Appl. 64:177–185. Rice, W. R., and E. E. Hosert. 1993. Experiments on speciation: what have Spangler, S. M., and D. D. Calvin. 2000. Influence of sweet corn growth we learned in 40 years? Evolution 47:1637–1653. stages on European corn borer (Lepidoptera: Crambidae) oviposition. Roelofs, W. L., and A. P. Rooney. 2003. Molecular genetics and evolution Environ. Entomol. 29:1226–1235. of pheromone biosynthesis in Lepidoptera. Proc. Natl. Acad. Sci. USA Stratton, G. E., and G. W. Uetz. 1986. The inheritance of courtship be- 100:9179–9184. havior and its role as a reproductive isolating mechanisms in two Roelofs, W. L., J. W. Du, X. H. Tang, P. S. Robbins, and C. J. Eckenrode. species of Schizocosa wolf spiders (Araneae, Lycosidae). Evolution 40: 1985. Three European corn borer populations based on sex-pheromones 129–141. and voltinism. J. Chem. Ecol. 11:829–836. Swanson, W. J., and V. D. Vacquier. 1997. The abalone egg vitelline envelope Roelofs, W., T. Glover, X. H. Tang, I. Sreng, P. Robbins, C. Eckenrode, C. receptor for sperm lysin is a giant multivalent molecule. Proc. Natl. Lofstedt, B. S. Hansson, and B. O. Bengtsson. 1987. Sex-pheromone Acad. Sci. 13:6724–6729. production and perception in European corn borer moths is determined Tauber, E., M. Zordan, F. Sandrelli, M. Pegoraro, N. Osterwalder, C. Breda, by both autosomal and sex-linked genes. Proc. Natl. Acad. Sci. USA A. Daga, A. Selmin, K. Monger, C. Benna, et al. 2007. Natural selec- 84:7585–7589. tion favors a newly derived timeless allele in Drosophila melanogaster. Roelofs, W. L., W. T. Liu, G. X. Hao, H. M. Jiao, A. P. Rooney, and C. E. Linn. Science 316:1895–1898. 2002. Evolution of moth sex pheromones via ancestral genes. Proc. Natl. Thomas, Y., M. T. Bethenod, L. Pelozuelo, B. Frerot, and D. Bourguet. 2003. Acad. Sci. USA 99:13621–13626. Genetic isolation between two sympatric host-plant races of the Eu- Royer, L., and J. N. McNeil. 1992. Evidence of a male sex pheromone in the ropean corn borer, Ostrinia nubilalis Hubner. I. sex pheromone, moth European corn borer, Ostrinia nubilalis (Lepidoptera: Pyralidae). Can. emergence timing, and parasitism. Evolution 57:261–273. Entomol. 124:113–116. Ting, C.-T., S.-C. Tsaur, and C.-I. Wu. 2000. The phylogeny of closely related ———. 1993. Male investment in the European corn borer, Ostrinia nubilalis species as revealed by the genealogy of a speciation gene, Odysseus. (Lepidoptera: Pyralidae): impact on female longevity and reproductive Proc. Natl. Acad. Sci. USA 97:5313–5316. performance. Funct. Ecol. 7:209–215. Via, S. 2009. Natural selection in action during speciation. Proc. Natl. Acad. Rundle, H. D., and M. C. Whitlock. 2001. A genetic interpretation of ecolog- Sci. USA 106:9939–9946. ically dependent isolation. Evolution 55:198–201. Weber, E., and B. Schmid. 1998. Latitudinal population differentiation in two Sakai, T., and N. Ishida. 2001. Circadian rhythms of female mating activity species of Solidago (Asteraceae) introduced into Europe. Am. J. Bot. governed by clock genes in Drosophila.Proc.Natl.Acad.Sci.USA 85:1110–1121. 98:9221–9225. Webster,R.P.,andR.T.Carde.´ 1982. Influence of relative humidity on calling Schluter, D. 2009. Evidence for ecological speciation and its alternative. Sci- behavioral of the female European corn borer moth (Ostrinia nubilalis). ence 323:737–741. Entomol. Exp. Appl. 32:181–185. Schwander, T., S. S. Suni, S. H. Cahan, and L. Keller. 2008. Mechanisms of Willett, C. S., and R. G. Harrison. 1999. Pheromone binding proteins in the reproductive isolation between an ant species of hybrid origin and one European and Asian corn borers: no protein change associated with of its parents. Evolution 62:1635–1643. pheromone differences. Entomol. Exp. Appl. 29:277–284. Servedio, M. R., and M. A. F. Noor. 2003. The role of reinforcement in Wu, C.-I. 2001. The genic view of the process of speciation. J. Evol. Biol. speciation: theory and data. Ann. Rev. Ecol. Evol. 34:339–364. 14:851–865. Showers, M. R. 1993. Diversity and variation of European corn borer popula- Zhu, J. W., C. H. Zhao, F. Lu, M. Bengtsson, and C. Lofstedt. 1996. Re- tions. Pp. 287–309 in K. C. Kim and B. A. McPherson, eds. Evolution ductase specificity and the ratio regulation of E/Z isomers in pheromone of Insect Pests. Wiley and Sons, Inc., New York, NY. biosynthesis of the European corn borer, Ostrinia nubilalis (Lepidoptera: Showers, W. B., G. L. Reed, J. F. Robinson, and M. B. Derozari. 1976. Flight Pyralidae). Insect Biochem. Mol. Biol. 26:171–176. and sexual activity of European corn borer, Lepidoptera: Pyralidae. En- viron. Entomol. 5:1099–1104. Associate Editor: C. Jiggins

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