Reproductive isolating barriers are among the primary forces generating and maintaining biodiversity in nature (Dobzhansky 1937; Mayr 1942; Coyne and Orr 2004). Here, we report on a case study of North American Z- and E-pheromone strains of ECB, in which three major classes of reproductive barriers–premating, postmating-prezygotic, and postzygotic–are considered. Our evaluation of 12 potential isolating barriers demonstrates that reproductive isolation arises from manifold sources whose effects are far from uniform. Consequently, the genetic and evolutionary basis for speciation will be complex. Although the cumulative action of all barriers produces nearly complete reproductive isolation, polymorphism in strength of reproductive isolation allows for substantial opportunity for gene flow. Indeed, the ECB likely exists as a mosaic of incompletely isolated populations that vary both in the strength and form of reproductive isolation. Nevertheless, of the three classes of barriers that were measured, premating forms appear to be the most potent in maintaining the integrity of sympatric Z and E populations in nature, possibly because they evolve rapidly between diverging populations.
Seasonal temporal isolation
Although accounts of temporal isolation may be numerically rare, the phylogenetic breadth of taxa that experience temporal isolation suggests that this paucity reflects limited study rather than low evolutionary frequency (e.g., fish [Quinn et al. 2000], insects [Cooley et al. 2003], flowering plants [Martin and Willis 2007], corals [Knowlton et al. 1997], algae [Clifton 1997]). Indeed, observing 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 temporal isolation in the ECB and proceeded to characterize sympatric UZ, BZ, and BE voltinism “races” in NY (Eckenrode et al. 1983; Roelofs et al. 1985). Since this pioneering work, however, the consequences of ecological differences in breeding cycle on patterns of gene flow have remained unclear.
Our survey of 41 localities (Fig. 1) over a 9-year-period (1999–2007) records the persistence of sympatric bivoltine and univoltine populations of ECB in NY. The degree of asynchrony in breeding cycle across many sites indicates that voltinism can severely restrict gene flow between pheromone strains (e.g., Fig. 3A). Indeed, our results show that when BE and UZ moths are sympatric, as much as 80% of gene flow is prevented by seasonal isolation alone (Table 1). However, strains at several sites in NY (Fig. 3B; Table 1) and in other geographic areas (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, Thomas et al. 2003). These findings indicate that seasonal temporal isolation is only a locally important barrier to gene flow between Z and E strains, and that, overall, breeding cycle differences constitute a rather ineffective isolating barrier. Indeed, at the same study site, we found that the strength of temporal isolation can vary dramatically between years. At Farmington, ASseasonal was above 0.6 in three of the four sampled years, but in 2003, it fell to 0.388 (Table 1). The cause of these fluctuations is unknown, but environmental effects or sampling artifacts are two possible explanations. Changes in weather are known to affect the number and shape of ECB breeding cycles (e.g, via temperature-dependent development; Beck 1983). Alternatively, changes in farming practices, including the use of BT corn or insecticides, can affect the size of annual flights through differential mortality (e.g., Burkness et al. 2001). Such changes could increase the breeding cycle overlap for populations that are genetically bivoltine and univoltine (e.g., at Pdd). Finally, as traps were positioned on 41 active farms across 9 years, the uniformity of sampling in some years may have been compromised.
Although breeding cycle differences can be a consequence of environmental variation, voltinism in NY can be at least partially explained as a byproduct of genetic differences at the major, Z-linked QTL affecting PDD time (McLeod 1978; Reed et al. 1981; Glover et al. 1991, 1992). Therefore, the origin of seasonal isolation can be understood as the evolution of developmental rate. It has been suggested that trait divergence resulted from selection, most likely from an ecological source, because Tpi shows evidence for selection and is tightly linked to the Pdd locus (Dopman et al. 2005). PDD time may have evolved because it influences fitness through its effect on generation time, or because it helps coordinate the performance of life-cycle activities (e.g., reproduction, growth, diapause) at appropriate times in the season (Bradshaw et al. 2004; Bradshaw and Holzapfel 2007).
Circadian temporal isolation
Besides seasonal temporal isolation, at least some ECB strains are temporally isolated through differences in 24-h, circadian time of mating (Liebherr and Roelofs 1975). Under laboratory conditions, Liebherr and Roelofs reported that BE-strain moths showed delayed mating compared to UZ-strain moths, which mate soon after darkness (Fig. 3C). Other interpretations for this pattern have been proposed (Webster and Cardé 1982), and pheromone strains in Europe display different circadian rhythms (Karpati et al. 2007). Nevertheless, a ∼50% reduction in gene flow would be expected if the asynchrony in mating time reported by Liebherr and Roelofs persists in the field (Table 3). The basis for circadian mating rhythms in the ECB is unclear, but a genetic component seems likely because differences were detected after more than eight generations in a common laboratory environment. At least in Drosophila, circadian mating periodicity is genetically controlled by an endogenous clock (e.g., Sakai and Ishida 2001).
Male and female behavioral isolation
Considering the prevalence of hybridization between animal taxa that lack strong differences in mate selection preferences or signals, Mayr (1963) argued that behavioral isolation is the most important form of reproductive isolation. In ECB, the importance of behavioral isolation is unequivocal. When Z and E moths are dispersed (1–50 m), numerous authors have reported that the use of different preferences and signals (i.e., male orientation to volatile sex pheromones released by females) results in limited cross attraction (e.g., Roelofs et al. 1987; Bontemps et al. 2004), and nearly complete reproductive isolation (Fig. 4C). What is not clear from these studies, however, is the extent that these preferences and cues prevent hybridization when moths are not dispersed. In general, behavioral isolation is usually examined in one context, for example, a single spatial scale or a single ecological or laboratory setting (Coyne et al. 2005; but see Craig et al. 1993). In the ECB, the spatial context for mating is important because moths of both strains can be found to aggregate in the field where mate location or attraction could conceivably occur without the use of long-distance olfactory cues (Showers et al. 1976; Malausa et al. 2005).
By observing mating behavior, our study confirms that over very short distances (<20 cm), males of both strains continue to show orientation specificity towards females of their own strain (Fig. 4A). Combined with a recent investigation demonstrating that adding sex pheromone fails to increase interstrain mating (Pelozuelo et al. 2007), this result can be taken as prima facie evidence that male orientation to female cues, most likely to female sex pheromones, causes behavioral isolation over a broad range of spatial scales. However, isolation strength is reduced when males and females are in close proximity (AS♂ orient., UZ × BE: 0.448 [<20 cm] vs. 0.988 [∼2 m]; E × Z: 0.722 [<20 cm] vs. 1.0 [∼2 m]), suggesting that moth dispersion could shape the extent that differences in male behavior reduce gene flow. Further studies are needed to determine the reasons for increased rates of male orientation, but two possible explanations are environmental dependence (e.g., flying 1–2 m upwind to sex pheromone stimuli vs. a combination of walking and short flights to signaling females), or the presence of an unidentified short-distance female cue (other than known sex pheromones) that elicits male orientation and which is similar in both strains.
Previous research has attributed patterns of assortative mating between Z and E moths to male orientation specificity alone (e.g., Cardé et al. 1978; Klun and Huettel 1988; Glover et al. 1991). However, like many other animal species (Andersson 1994), we find that ECB females display choosy mate-selection behavior that manifests as active discrimination when confronted by courting moths of the opposite strain (Fig. 4B). When rejecting a male's courtship attempt, many females contort their abdomen so that copulation is impossible, others simply walk or fly away. As a result, female behavioral isolation in some pairings is as strong as male behavioral or seasonal temporal isolation (E × Z: AS♀ discrim.= 0.712) (Table 3). Recently, it has been demonstrated that a male pheromone, produced by the hairpencil, is critical for female acceptance of courting males (Lassance and Löfstedt 2009). Combined with our results, this finding suggests a role for this male pheromone in female behavioral isolation. In addition to helping explain the deficit of hybrids in nature, male and female mate preferences shed light on biases in the type of hybrids that are produced. Both male and female behaviors are asymmetric, with a deficit of successes when E females pair with Z males (Fig. 4A,B). Although behavioral asymmetries such as these are common between animal species (Kaneshiro 1980; Arnold et al. 1996), and it was known that Z males were more selective (Roelofs et al. 1987; Glover et al. 1991; Linn et al. 1997), it is unusual that both sexes of the Z strain are more discerning. Regardless of the evolutionary explanation for this pattern, the proximate consequence is strong asymmetry in hybridization potential. This potential seems to be realized in the field, as an extensive survey of ECB in NY failed to uncover E female × Z male hybrids (Glover et al. 1991).
Male behavioral isolation stems from changes at an uncharacterized, autosomal fatty-acid reductase that affects pheromone blend ratios (Zhu et al. 1996; Roelofs et al. 1987; Dopman et al. 2004), along with changes at a sex-linked, major QTL for orientation (Glover et al. 1990; Dopman et al. 2004). A candidate gene(s) affecting male orientation has yet to be identified; however, a gene(s) that functions after peripheral sensory processing of pheromone cues is likely (Willett and Harrison 1999; Linn et al. 1999; but see Karpati et al. 2008). As noted by Coyne and Orr (2004), monogenic inheritance of male behavioral isolation in ECB contrasts with polygenic inheritance in other species. With this simple genetic basis, it is difficult to understand how new preferences or signals could have evolved because most mutations are expected to have large and deleterious phenotypic effects (review in Löfstedt 1993). The existence of rare ECB males that orient towards females from both the ECB and the Asian corn borer (ACB, O. furnacalis) provides a partial solution (Reolofs et al. 2002)–some ECB males may have been preadapted to orient towards the first mutant Z- or E-strain female, mitigating initial fitness costs associated with pheromone blend evolution. Regarding female behavioral isolation, both proximate and ultimate causes will remain unknown until the sensory modalities involved have been elucidated. Acoustic communication occurs in the ACB, in which males produce an ultrasonic courtship “song” (Nakano et al. 2006), but an olfactory system is also possible (Royer and McNeil 1992; Lassance and Löfstedt 2009).
Mechanical and gametic isolation
“Cryptic” reproductive barriers, which include mechanical and gametic forms of postmating-prezygotic isolation, although difficult to observe have been documented across diverse organisms (e.g., in broadcast spawners [Swanson and Vacquier 1997], in insects [Gregory and Howard 1993; Herndon and Wolfner 1995; Price et al. 2001; Nosil and Crespi 2006], in fish [Mendelson et al. 2007], review in Coyne and Orr 2004). We find that mechanical isolation is either limited or absent between Z and E strains (Fig. 5A), but our ability to test for clasping failures was restricted by a small sample size in E × Z pairings. Limited mechanical isolation would corroborate the findings of Cardé et al. (1978), in which genitalic differences between strains that might contribute to mechanical isolation were not detected. In contrast to mechanical isolation, we find that ECB strains exhibit partial gametic isolation, confirming preliminary results obtained over 30 years ago (Liebherr and Roelofs 1975). Gametic incompatibilities are moderate in intensity and are significant in only one cross direction. When Z females mate with E males, lifetime egg production drops by ∼30% (Fig. 5B; ASovipos= 0.309, Table 3), but fertilization rate remains high (Fig. 5C).
Postmating-prezygotic problems are often highly variable and asymmetric between diverged populations or species, and cross failures can have multiple causes (Price et al. 2001; Nosil and Crespi 2006; Mendelson et al. 2007). In the ECB, explanations involving reduced remating frequency can be rejected based on the presence of a single transferred spermatophore in all females, but many other potential mechanisms remain. It is possible that fertilization in Z × E pairings proceeds normally, but foreign seminal proteins or unrecognized copulatory behavior fails to stimulate egg laying (e.g., Herndon and Wolfner 1995). Alternatively, in some interspecific crosses (e.g., Gregory and Howard 1993), reduced oviposition reflects poor fertilization and the retention of unfertilized eggs. Even with good demonstrations of cryptic isolation (e.g., Swanson and Vacquier 1997; Price et al. 2001; Nosil and Crespi 2006; Mendelson et al. 2007), the processes driving evolution are poorly understood, and thus represent a clear avenue for further work.
Behavioral and physiological infertility
Behavioral hybrid infertility or dysfunction has two primary causes that are often difficult to distinguish (Stratton and Uetz 1986; Frey and Bush 1996; Noor 1997; Servedio and Noor 2003; Linn et al. 2004; Coyne and Orr 2004). Failures or delays in mating can result when hybrid behaviors are intermediate compared to parental individuals (extrinsic behavioral dysfunction) (e.g., Stratton and Uetz 1986). Alternatively, failures or delays can result when otherwise “normal” hybrid behaviors are inhibited or disrupted by physiological or neurological abnormalities (intrinsic behavioral dysfunction) (e.g., Noor 1997). Our reanalysis of Roelofs et al. (1987) suggests that both extrinsic and intrinsic dysfunction contribute to reduced F1 male mating success in the ECB (Fig. 2A). Although F1 males show moderate response to the Z pheromone and therefore do not exhibit exclusively intermediate behavior, few hybrids respond to the E-strain blend. Similarly, although hybrids show appreciable orientation to the Z and hybrid blends, they do not respond as readily as Z and E males to their own pheromones.
Due to orientation patterns among Z, E, and hybrid males, behavioral hybrid dysfunction becomes apparent only under restricted circumstances. Specifically, dysfunction and intermediate levels of isolation occur when E females predominate (e.g., Geneva, NY in Fig. 2B,C; mean ASF1♂ dys.= 0.469, Table 3). When Z and hybrid females are at high frequency (e.g., Beltsville, MD, in Fig. 2B,C), hybrids experience intermediate relative fitness and introgression is promoted (mean ASF1♂ dys.=−0.539, Table 3). Two relevant points suggest that behavioral dysfunction could be weak in nature. First, our measure is based on long-distance attraction, but males may orient to a wider spectrum of female genotypes when aggregated (Fig. 4A vs. C). Second, conditions favorable to hybrid male orientation are probably common in the field because Z females are typically the most numerous genotype (Table 1; Klun and Cooperators 1975; Anglade and Stockel 1984). Whatever its impact on gene flow, the evolution of behavioral dysfunction is linked to male behavioral isolation because both barriers have a common genetic basis.
In contrast to behavioral dysfunction, the fertility effects of hybrid physiology or development is one of the most commonly studied forms of reproductive isolation (review in Coyne and Orr 2004). In the ECB, Liebherr and Roelofs (1975) reported that F2 intercrosses yielded fertile eggs, whereas only ∼70% of backcrosses were successful. Our measure of fertility (percent fertile eggs) indicates that both F2 and BC pairings proceed normally (∼90% fertile, Table 2), but BCs do suffer disproportionately in terms of fecundity (∼25% fewer eggs than F2 or parental crosses). However, among all second-generation crosses, oviposition drops by only ∼15% compared to crosses occurring within-strain. Thus, our results indicate that physiological hybrid infertility is a rather weak isolating barrier (ASF1 ovipos.= 0.159, Table 3). Inheritance patterns of oviposition in first and second-generation crosses (Fig. 5B; Table 3) might be consistent with an incompatibility between an autosomal recessive allele in the Z strain and a dominant E-strain allele, but more work is needed to explore the genetic and evolutionary basis of this phenotype.
THE PHENOTYPIC ARCHITECTURE OF SPECIATION
The number and strength of isolating barriers that have evolved before speciation is complete, which we refer to here as the “phenotypic architecture” of speciation, provides insight into several outstanding problems in speciation research. One important issue is whether this phenotypic architecture typically consists of one or a few strong barriers to gene flow or whether many weaker barriers are involved. The Z and E strains of ECB most clearly fall into the latter category, given that seven of 12 potential forms of isolation restrict gene exchange, but all are less than 80% complete (Table 3). Comparable studies have revealed a similar pattern (e.g., Chamerioin angustifolium populations, Husband and Sabara 2003; sister species of Mimulus, Ramsey et al. 2003; lineages of Pogonomyrmex ants, Schwander et al. 2008; Etheostoma darters, Mendelson et al. 2007; review in Coyne and Orr 2004), suggesting that speciation is commonly an extended and complex process that requires an accumulation of barriers of intermediate strength. Nevertheless, the role of one or a few strong barriers to gene flow has been documented between some diverging populations (review in Funk et al. 2002; Nosil et al. 2005; Lowry et al. 2008).
An evolutionary explanation for these two contrasting phenotypic architectures of speciation may be related to differences in the geographic mode of divergence. A history that includes long intervals of sympatry might result in a small number of barriers because gene flow will constrain the evolution of new phenotypic differences (possibly to a subset driven by strong selection [Rice and Hosert 1993]), and impede some types of evolutionary processes from promoting divergence (e.g., by genetic drift or “mutation-order” speciation [Schluter 2009]). Those few barriers that do evolve in sympatry must arise rapidly and be great in strength (e.g., behavioral isolation in Drosophila, Coyne and Orr 2004), or else population fusion will occur. In contrast, geographical separation facilitates the origin of trait differences and reproductive barriers by almost any evolutionary process (besides reinforcement), and restricted migration may facilitate the persistence and accumulation of trait differences and barriers of varying strength (Rice and Hosert 1993). Under this reasoning, the complex phenotypic architecture of speciation that is observed between ECB strains may have been promoted by an extended historical period(s) of restricted gene flow, such as that imposed by physical separation.
Uncovering manifold causes of speciation suggests a high level of complexity in the underlying genetic architecture. That is, the genetics of speciation will typically consist of more loci with the potential for more elaborate control (e.g., epistasis, additive), phenotypic effects (e.g., minor versus major), and chromosomal distributions, as the number of barriers preventing gene flow increases. Besides underscoring the obvious truth that disentangling the genetics of speciation in many (or most) species will require tremendous effort, a more profound implication is how this genetic complexity will shape patterns of gene flow when populations are sympatric.
The genetic architecture of speciation has consequences for genome-wide patterns of gene flow because chromosomal regions linked to reproductive barrier loci will introgress across species boundaries at lower rates than unlinked regions (Barton and Hewitt 1981; Harrison 1990; Wu 2001; Ting et al. 2000). As a result of this effect, species or racial boundaries are often thought of as “semipermeable” or “porous,” with permeability depending on the genetic marker (Barton and Hewitt 1981; Harrison 1990; Wu 2001; Via 2009). For a semipermeable genome, in regions that are unlinked to barrier loci, gene flow will constrain local adaptation but facilitate the spread of new, globally beneficial alleles. Conversely, restricted gene flow near barrier loci will promote further population divergence. Thus, the phenotypic architecture of speciation will shape the genomic context in which diverging populations change in response to spatial or temporal heterogeneity. Speciation in which a large number of reproductive barriers are involved is predicted to increase the size of the semipermeable fraction of diverging genomes, whereas the strength of each barrier (and possibly when it operates over an organism's life history) may affect the degree of permeability at underlying loci.
Z and E strains of ECB support the semipermeable genome model of speciation. In upstate NY, even where they exist together in the same fields, E and Z borers remain differentiated for Tpi suggesting that gene flow is limited or absent for this locus. Patterns of variation at Tpi may be a consequence of close physical linkage to Pdd (Dopman et al. 2004, 2005). In contrast, other nuclear genes (two of which, kettin and Ldh, are also on the sex chromosome) reveal no evidence of differentiation, because of recent or ongoing hybridization and introgression (Dopman et al. 2005). With a relatively large number of reproductive barriers between Z and E strains (Table 3), the ECB provides an important study system in which to investigate the consequences of the phenotypic and genetic architectures of speciation for introgression and local adaptation.
A complex phenotypic architecture of speciation presents challenges for understanding the evolutionary forces driving speciation. However, only a subset of barriers and evolutionary processes will have been important in initiating the speciation process. Testing hypotheses about these early-evolving barriers typically requires a comparative approach (e.g., Coyne and Orr 1997; Mendelson 2003; but see Christianson et al. 2005), but Coyne and Orr (2004) noted that it is unlikely for a barrier to have played a major historical role unless its contemporary strength is large. Therefore, premating barriers may be among the most historically important between strains of the ECB (Table 3).
There are at least two reasons why premating barriers might be predisposed to rapid evolution–they may be common targets of selection, or they could be more mutable. Comparative studies suggest that behavioral isolation can rapidly evolve by reinforcement in sympatry or as a byproduct of sexual selection in allopatry (Coyne and Orr 1989, 1997; Mendelson 2003). For reinforcement to explain patterns in the ECB, hybrids must experience reduced fitness. Hybrid ECB are relatively fit in the laboratory (Fig. 2C; Table 2; Calcagno et al. 2007), but reinforcement cannot be ruled out until ecologically dependent costs (e.g., Rundle and Whitlock 2001) have been excluded. In contrast, conditions favoring sexual conflict are known. Male ECB fitness increases with more mating (Royer and McNeil 1993), but the act of mating reduces female survival (Fadamiro and Baker 1999). In contrast to behavior, breeding cycle differences are unlikely to have been historically important because Z and E strains breed at similar times at most sympatric sites (Glover et al. 1991; Sorenson et al. 1992; Thomas et al. 2003). Nevertheless, circumstantial evidence suggests that PDD time might be prone to rapid adaptive evolution in the ECB (Dopman et al. 2005; Malausa et al. 2007a). Empirical studies have documented rapid evolution of circannual and circadian rhythms in both animals and in plants (e.g., Weber and Schmid 1998; Tauber et al. 2007), with diapause traits being among those phenotypes showing adaptive responses in insects (e.g, Tauber et al. 2007).
Premating barriers might also rapidly evolve because their genetic determinants are predisposed to mutation. In yeast, the spontaneous gene duplication/deletion rate is orders of magnitude larger than the nucleotide substitution rate (Lynch et al. 2008), and the genes determining pheromone differences between Ostrinia are in a multigene family (Roelofs et al. 2002; Roelofs and Rooney 2003). Together, these results suggest that the number of pheromone genes that are polymorphic in copy number might be large and that the (in)activation of different paralogs could provide a major source of “raw material” for rapid sex-pheromone evolution. Although pheromone blend differences between ECB strains seems to be from allelic changes rather than from alternative paralog usage, mutational mechanisms could be responsible for rapid behavioral isolation in the Ostrinia genus, and perhaps, in other species.
THE IMPORTANCE OF PRE- VERSUS POSTZYGOTIC BARRIERS TO SPECIATION
There has been long-standing debate about the relative contributions of prezygotic versus postzygotic barriers to speciation (e.g., Coyne and Orr 1989, 1997; Kirkpatrick and Ravigne 2002). It is clear that the timing of the evolution of barriers during the often-extended process of speciation is most clearly related to the historical or evolutionary causes of speciation. In this sense, those barriers that evolved first (e.g., as identified by comparative studies) are the most “important.” However, a barrier's relative or “sequential” strength over an organism's life history informs a related issue about the contemporary or ecological causes of speciation in which the main question is how diverging populations remain distinct. Accumulating evidence supports the interpretation that premating barriers may be most important forms of isolation between in both contemporary and historical population pairs, but the debate is far from resolved (Ramsey et al. 2003; Husband and Sabara 2003; Nosil et al. 2005; Martin and Willis 2007; Mendelson et al. 2007; Nosil 2007; Lowry et al. 2008; Schwander et al. 2008; Schluter 2009).
For the ECB, with more than 98% of total cumulative reproductive isolation consisting of premating barriers, our findings broadly confirm early studies (e.g., Arbuthnot 1944; Liebherr and Roelofs 1975), which implicate prezygotic barriers as the most important for the maintenance of Z and E strains in nature (Table 3; Fig. 6). In the weak isolation scenario, which probably represents a close approximation to that experienced by many ECB strains in the field, temporal isolation (seasonal + circadian) contributes nearly two-thirds to the total, whereas another ∼third is contributed by behavioral isolation (male + female) (Fig. 6). All other isolating barriers combined (postmating-prezygotic + postzygotic) contribute a little more than 1% at most to total isolation. Granted, not all isolating barriers have been measured in the ECB. Patterns consistent with “hybrid breakdown” have been documented in life stages other than those investigated here (i.e., in larvae, Arbuthnot 1944; Liebherr and Roelofs 1975), and strong extrinsic (postzygotic and prezygotic) barriers may have gone undetected in our benign laboratory setting (e.g., environmentally dependent postzygotic incompatibilities at Pdd). Nevertheless, some Z and E populations in Europe differ in traits associated with specialization to different host plants (Thomas et al. 2003; Calcagno et al. 2007; Malausa et al. 2008). If these differences prevent gene flow then Z and E strains of ECB would almost certainly join the growing list of incipient species that support premating barriers as the strongest impediments to gene flow (e.g., Ramsey et al. 2003; Husband and Sabara 2003; Nosil 2007; Martin and Willis 2007; Mendelson et al. 2007; Lowry et al. 2008).
TOTAL REPRODUCTIVE ISOLATION AND PATTERNS OF GENE FLOW IN NATURE
For most species the biological context for the maintenance or erosion of genetic differences between populations in nature is obscure, but we can begin to characterize this relationship for Z and E strains of the ECB. One expected pattern is a negative association between estimates of cumulative isolation and estimates of genetic exchange in the field. Based on measures of cumulative isolation, sympatric populations of UZ and BE moths should produce fewer hybrid offspring and experience less gene flow than sympatric BZ and BE moths (Table 3). In agreement with this prediction, the proportion of hybrid females is smaller at the UZ/BE Geneva site than at the BZ/BE Beltsville site (Fig. 2B; Roelofs et al. 1985; Klun and Huettel 1988), and genetic differences have been maintained for ∼20 years between UZ and BE populations at Geneva (Glover et al. 1991; Dopman et al. 2005). Presumably, more recent evidence for introgression would be found between strains at Beltsville and at other sites where seasonal isolation is weak.
Although it is unlikely that measures of cumulative isolation translate easily into quantitative assessments of hybridization or gene flow, cumulative isolation can inform taxonomic issues because the BSC defines species by the presence of reproductive barriers (Ramsey et al. 2003; Husband and Sabara 2003; Martin and Willis 2007). This is particularly useful for Z and E strains of ECB because their morphological and genetic similarity (e.g., Liebherr 1974; Dopman et al. 2005) has led to regular taxonomic debate and the emergence of two opposing views. Thirty years ago, Cardé and colleagues (1978) argued on the basis of allozyme, pheromone, and hybridization data that pheromone strains are distinct species. More recently, Frolov et al. (2007) asserted that Z and E strains using maize, such as those investigated here, belong to one species, whereas those using non-maize hosts (e.g., mugwort) belong to another. With as much as ∼10% gene flow permitted (in Z × E crosses, Table 3) and more than 15% hybrids at some sites (Fig. 2B; Klun and Huettel 1988), claims of distinct species seems unjustified. On the other hand, Frolov et al.'s (2007) treatment of maize-feeding strains as conspecific appears to be at odds with our finding that total isolation approaches completion if seasonal isolation is strong (Table 3). These contrary interpretations can be reconciled by interpreting Z- and E- pheromone strains as a mosaic of incompletely isolated populations that differ in levels of total reproductive isolation (e.g., due to voltinism [here] or host–plant use [Thomas et al. 2003; Calcagno et al. 2007; Malausa et al. 2008]). A more dynamic application of the BSC that acknowledges this diversity is necessary for the ECB. When combined with a comprehensive genetic survey, such a perspective can lead to a better understanding of the process in which alleles at reproductive barrier loci spread across populations before ultimately giving rise to completely isolated and distinct species.