Jeffrey L. Feder, Department of Biological Sciences, 290C Galvin Life Sciences Building, University of Notre Dame, Notre Dame, IN 46556, USA. Tel.: +1 574 6314159; fax: +1 574 6317413; e-mail: firstname.lastname@example.org
Prezygotic isolation due to habitat choice is important to many models of speciation-with-gene-flow. Habitat choice is usually thought to occur through positive preferences of organisms for particular environments. However, avoidance of non-natal environments may also play a role in choice and have repercussions for post-zygotic isolation that preference does not. The recent host shift of Rhagoletis pomonella (Diptera: Tephritidae) from downy hawthorn, Crataegus mollis, to introduced apple, Malus domestica, in the eastern United States is a model for speciation-with-gene-flow. However, the fly is also present in the western United States where it was likely introduced via infested apples ≤ 60 years ago. R. pomonella now attacks two additional hawthorns in the west, the native C. douglasii (black hawthorn) and the introduced C. monogyna (English ornamental hawthorn). Flight tunnel tests have shown that western apple-, C. douglasii- and C. monogyna-origin flies all positively orient to fruit volatile blends of their respective natal hosts in flight tunnel assays. Here, we show that these laboratory differences translate to nature through field-trapping studies of flies in the state of Washington. Moreover, western R. pomonella display both positive orientation to their respective natal fruit volatiles and avoidance behaviour (negative orientation) to non-natal volatiles. Our results are consistent with the existence of behaviourally differentiated host races of R. pomonella in the west. In addition, the rapid evolution of avoidance behaviour appears to be a general phenomenon for R. pomonella during host shifts, as the eastern apple and downy hawthorn host races also are antagonized by non-natal fruit volatiles.
Habitat choice is an important component of many models of ecologically based speciation, especially when population divergence occurs in the face of gene flow (Berlocher & Feder, 2002). When habitat choice affects mate choice, the result is an ecologically based, prezygotic barrier to gene flow (Feder et al., 1994; Berlocher & Fede, 2002). This most frequently occurs when individuals mate in their preferred habitats (e.g. if populations form leks or court in different habitats or at different times of the year). The resulting assortative mating can facilitate ecological speciation-with-gene-flow because it accentuates selection for performance traits that in turn increase selection for habitat fidelity (Diehl & Bush, 1989; Fry, 2003; Feder & Forbes, 2007). By mating in preferred habitats, individuals that possess traits increasing their survivorship in a particular habitat will tend to mate with other individuals possessing the same suite of ecological adaptations, increasingly favouring those that remain in their ‘natal’ habitats. Thus, positive correlations can evolve between traits affecting habitat choice (e.g. host plant quality decisions for oviposition and mating by phytophagous insects) and offspring survival in habitats (Gripenberg et al., 2010).
In general, it is thought that organisms decide where to reside based on positive habitat preferences (Mayr, 1947, 1963; Fry, 2003). However, habitat choice may not be solely determined by positive preference. It has also been contended that phytophagous insect specialists use both positive cues from hosts and negative cues from nonhost plants when evaluating their environment (Bernays & Chapman, 1987; Bernays et al., 2000; Bernays, 2001). Consequently, as well as possessing genes to prefer specific habitats, phytophagous insects may also have allelic variation that causes them to avoid non-natal, alternative habitats (Feder & Forbes, 2007, 2008). Most importantly, habitat avoidance may commonly have nonadditive phenotypic and fitness effects with important consequences for ecological speciation that go beyond the implications of preference (Linn et al., 2004; Feder & Forbes, 2007, 2008). In contrast to preference, if avoidance genes exist, then hybrids possessing alleles to avoid alternative parental habitats may be behaviourally conflicted and accept no habitat. Thus, hybrids could suffer a degree of post-zygotic reproductive isolation, being incapable of finding suitable habitats to feed and mate in (i.e. hybrids would incur a degree of behavioural inviability/sterility). Moreover, in contrast to preference genes, the more genes there are that strongly and independently affect avoidance, the stronger the barrier to gene flow between populations, as it would become harder and harder to segregate out a parental behavioural phenotype that would be willing to reside in any one habitat (Feder & Forbes, 2007, 2008). Thus, rather than the genetics of habitat choice providing a bridge, avoidance could create a greater reproductive chasm fostering ecological speciation-with-gene-flow. Consequently, when a component of habitat choice involves avoidance, there can be repercussions that can have consequences for enhancing the potential for specialization and post-zygotic reproductive isolation and, hence, for ecological speciation.
Here, we tested for both habitat preference and avoidance behaviours in populations of the fruit fly Rhagoletis pomonella (Walsh) (Diptera: Tephritidae) infesting apple (Malus domestica), black hawthorn (Crataegus douglasii) and ornamental hawthorn (C. monogyna) host fruit in the western United States. Specifically, we performed field trials investigating whether R. pomonella flies in the state of Washington are captured significantly more often on sticky traps baited with volatile compound blends of their respective natal host fruit of origin and less often on non-natal fruit volatiles compared to blank, odourless control traps. Finding the former result would indicate that the volatiles of native hosts (M. domestica fruit odour for apple-infesting flies, C. douglasii fruit odour for black hawthorn-infesting flies and C. monogyna fruit odour for ornamental hawthorn-infesting flies) serve as behavioural agonists that cause R. pomonella to positively orient to host fruit. Finding the latter would indicate that R. pomonella flies are also antagonized by non-natal volatiles and, thus, tend to avoid alternative host fruit. Because R. pomonella mate on or near the unabscized fruit on their respective host plants (Prokopy et al., 1971, 1972; Feder et al., 1994, 1998), differential preference and avoidance behaviours would generate ecological, prezygotic barriers to gene flow, facilitating speciation-with-gene-flow.
The geographical range of the apple maggot fly is not restricted to the eastern United States, however, as the fly now also exists in the western United States in the states of Washington, Oregon, California, Idaho and Utah (AliNiazee & Westcott 1986; Brunner, 1987; Tracewski et al., 1987; Dowell, 1988; Allred & Jorgensen, 1993; Yee, 2008; Yee & Goughnour, 2008), as well as in British Columbia, Canada (Canadian Food Inspection Agency, 2009). It is generally believed that R. pomonella was originally introduced into the Pacific Northwest via larval-infested apples within the last 60 years (AliNiazee & Penrose, 1981; Brunner, 1987; Tracewski et al., 1987; Yee, 2008). In addition to apple, R. pomonella now also attacks the native black hawthorn (C. douglasii) and the introduced English ornamental hawthorn (C. monogyna) in the Pacific Northwest (Tracewski et al., 1987; Yee & Goughnour, 2008). If R. pomonella was indeed introduced to the western United States via infested apples, then the current C. douglasii and C. monogyna attacking forms of the fly would represent recent sympatric host shifts of R. pomonella back to hawthorn. Although less likely, it is also possible that R. pomonella is native to the Pacific Northwest and shifted from black hawthorn to feral apple and C. monogyna when these latter two hosts were introduced into the region. Indeed, black hawthorn-infesting populations of R. pomonella are known from a geographically isolated region in Utah, where apple does not appear to be attacked (Allred & Jorgensen, 1993). Moreover, it is even conceivable that R. pomonella was independently introduced to the west via apple and ornamental hawthorn and shifted to black hawthorn from the latter host. Regardless, the presence of R. pomonella on a number of different alternative hosts in the west presents a potential example of how ecologically differentiated host races may exist in syntopy (i.e. as populations living together at the same locality).
We have previously found that like apple and downy hawthorn flies in the east (Linn et al., 2003, 2004, 2005a,b; Forbes et al., 2005), co-occurring black hawthorn-, ornamental hawthorn- and apple-origin flies collected from Washington state all preferred the volatile compounds emitted from their respective natal host fruits compared to those of the alternative, non-natal fruits in laboratory-based flight tunnel assays (C.E. Linn Jr, W.L. Yee, R.B. Goughnour, S. Sim & J.L. Feder, unpublished data). These behavioural differences are important for host race formation and incipient speciation because R. pomonella flies use fruit volatiles as their first long- to intermediate-range olfactory cues to orient to host plants for mating and oviposition (Prokopy & Roitberg, 1984; Prokopy et al., 1987; Feder et al., 1994; Forbes et al., 2005). However, trapping studies of eastern apple- and downy hawthorn-origin flies also showed that eastern flies were not only preferentially trapped on their respective natal fruit volatiles in the field, but also antagonized by non-natal volatiles (i.e. apple flies tended to avoid the odour of downy hawthorn fruit, and downy hawthorn flies were deterred by the odour of apple fruit) (Forbes et al., 2005; Linn et al., 2005a; Forbes & Feder, 2006). Avoidance behaviour is of potentially great significance to sympatric ecological speciation because F1 hybrids between eastern apple and downy hawthorn flies all failed to orient to either apple or hawthorn fruit volatiles in flight tunnel assays (Linn et al., 2004; Dambroski et al., 2005). Avoidance behaviour could explain the lack of response of apple × downy hawthorn hybrids in the flight tunnel if F1 flies possess alleles causing them to be antagonized by both parental fruit volatiles (Feder & Forbes, 2007, 2008). The lack of response to fruit volatiles implies that F1 hybrids may suffer a degree of behavioural sterility in nature due to a reduced ability to find host fruit for mating and oviposition. Thus, the evolution of host fruit odour discrimination can potentially generate post-zygotic, as well as prezygotic, ecological reproductive isolation among R. pomonella flies (Feder & Forbes, 2007, 2008).
Here, we report the results of field-trapping studies on western R. pomonella populations in stands of black hawthorn, ornamental hawthorn and apple trees in the state of Washington. Our aims were threefold: (i) to confirm whether the flight tunnel laboratory for western flies applies to nature, (ii) to test whether behaviourally differentiated apple and hawthorn host races exist in the western United States., as they do in the east and (iii) to determine whether western R. pomonella display both preference (positive orientation) to natal fruit volatiles and avoidance of (negative orientation to) non-natal volatiles, as eastern apple and hawthorn flies do.
Materials and methods
Overview of study
The basic experimental design in the field was similar to that of Forbes et al. (2005). Trials involved a series of replicated two-way choice experiments between dark red-coloured spheres (purchased from Michaels Craft Store) that were 3 cm in diameter and close in size to small apples that were coated with Tanglefoot™ (Contech Enterprises, Victoria, BC, Canada) and baited with a given fruit odour treatment vs. a blank, odourless control sphere. Field trials were conducted at four study sites (Table 1). Study site one consisted of a stand of 40 C. douglasii trees planted within the last 10 years on a nature trail along the Burnt Bridge Creek Greenway near the intersection of NE 65th Street and 18th Street in Vancouver, WA. Study site two was an old orchard of 12 apple trees located outside of Woodland, WA. Study sites three and four were stands of 15 ornamental hawthorn trees each at an extension of the Burnt Bridge Creek Greenway in Hazel Dell, WA, and on the Vancouver, WA, campus of Washington State University, respectively. At all four study sites, the nearest alternative host species was at least 100 m away, and usually more, from the stand of trees being trapped (Table 1). This spatial configuration of host trees at the study sites allowed confidence that the vast majority of flies trapped at each site originated from the host tree species of interest and not alternative hosts. This was especially true at the Woodland, WA site where there were no nearby alternative host populations of note at all. However, at the other sites, at least modest populations of flies were present on the other host species, raising the possibility of at least some movement. We examine the issue of inter-host movement further with reference to the black hawthorn-trapping data in the Discussion section.
Table 1. Site locations (latitude [N] and longitude [W] in degrees and minutes) trapped in the study, including information on trapping dates and blends tested.
Vancouver, WA (Burnt Bridge Creek Greenway)
WA, EA, BH, WA, OH
WA, BH, WA, OH
Hazel Dell, WA (Burnt Bridge Creek Greenway)
BH, WA, OH
Vancouver, WA (Wash. State Univ. Campus)
BH, WA, OH
Fruit odour lures and traps
Fruit odour lures used in the study were prepared by Suterra LLC (Bend, OR, USA), as described by Forbes et al. (2005). Lures have been found to retain potency for up to 1 month in the field. Five different odour treatments were tested at the black hawthorn site: the eastern apple (Zhang et al., 1999), western apple (Cha et al., in press), eastern downy hawthorn (Nojima et al., 2003), ornamental hawthorn (Cha et al., in press) and black hawthorn (Cha et al., in press) blends (see Table 2 for a list of the chemical composition of blends). Previous flight tunnel (Cha et al., in press; Linn et al., unpublished data) and field-trapping studies (Yee et al., 2005) of western apple flies showed that they were not overly attracted to the standard 5-component eastern apple blend, but required the additional compounds hexyl propionate and hexyl acetate in the western apple blend to achieve behavioural response levels typically seen for eastern apple flies to the eastern apple blend (Cha et al., in press; Linn et al., unpublished data). Due to space limitations on trees, four different odour treatments were tested at the apple site (western and eastern apple, and ornamental and black hawthorn), and three (western apple, and ornamental and black hawthorn) at the two ornamental hawthorn sites. Two lures were placed inside the opposite ends of a black binder clip that was attached to a tree branch by twist tie wire (Forbes et al., 2005). Traps were hung from the bottom of the clips to capture flies.
Table 2. Relative percentages of chemical compounds comprising the synthetic fruit volatile blends used in the study. BH, black hawthorn; OH, ornamental hawthorn; DH, downy hawthorn; WA, western apple; EA, eastern apple; DMNT indicates (3E)-4,8-dimethyl-1,3,7-nonatriene.
Field experimental design
A given two-way trial consisted of a red sphere trap baited with two lures for a particular fruit blend tested against an odourless control sphere baited with two wax-containing blank lures positioned 1 m apart in the tree canopy. Each odour treatment was replicated nine times at the black hawthorn study site, eight times at the apple study sites and four times each at the two ornamental hawthorn study sites. A maximum of three pairs of baited and blank control traps each spaced several metres apart were hung in any one tree to ensure that treatments did not interfere with one another and were independent. Traps were monitored for flies every other day at the black hawthorn study site over a 16-day period from 6 to 22 July 2010 and every day at the apple study site over an 8-day period from 14 to 22 August 2011. Because of other field commitments and relatively low fly numbers, the traps at the two ornamental hawthorn sites were only checked once at the end of a 1-month period from 2 September to 6 October 2011. Due to differences in fly population density within and between trees, as well as variation in the attractiveness of individual traps related to their specific positions in the tree canopy, we did not randomize the locations of two-way trials within odour treatment blocks after a given trial period at the black hawthorn and apple study sites. Instead, the positions of the odour-baited trap and blank trap constituting a given two-way replicate trial were exchanged, such that the pair of spheres resided in each of the two test positions in a tree for an approximately equal amount of time during the experiment. The positions of treatments within blocks and the initial assignment of treatments for traps in a given two-way trial within trees were determined by random draw.
Comparisons of field trials and flight tunnel assays
To determine the degree to which flies corresponded in their responses to host fruit volatile blends in the field based on predictions derived from their observed behaviour in the laboratory, we performed linear regressions between the proportions of flies at the black hawthorn, apple and ornamental hawthorn study sites that were captured on a given fruit-baited sphere against the proportions of flies of the same host origin that displayed upwind-directed flight to the blend in wind tunnel assays. Data for upwind-directed flight came from Linn et al. (unpublished data) and represented flies that were collected as larvae in infested fruit in the field and reared to adulthood in the laboratory. From previous studies (Linn et al., 2003, Linn et al., unpublished data), we have determined that an accurate measure of acceptance behaviour is whether or not a previously odour-naïve fly initiates ‘upwind-directed flight’ of 20 cm or more from a release point towards a fruit blend source placed upwind 1 m distant in the tunnel within a 1-min test period. We have yet to observe a fly displaying upwind-directed flight in control experiments with no synthetic fruit blend (Linn et al., 2003, Linn et al., unpublished data). Flight tunnel data for black hawthorn flies represented the pooled totals for two collecting years (2009 and 2010) from the same population at Burnt Bridge Creek where we performed the current field-trapping trials. For apple and ornamental hawthorn flies, we used pooled results across several different collecting sites and years available in a study described by Linn et al. (unpublished data). Regressions were tested for significance by nonparametric Monte Carlo simulations of the field data based on the mean proportions of flies captured at a site on blank vs. baited traps for all odour treatments as the null expectation to construct simulated field results (1 × 105 replicates) to compare with the actual regression value.
Black hawthorn-trapping data
At the Burnt Bridge Creek Greenway black hawthorn tree study site, a total of 408 flies were captured on the 45 traps across the eight different collecting periods from 6 to 22 July 2010 (complete trapping data for the entire study are archived on DRYAD). There was a significant difference in the proportion of black hawthorn flies captured among the five different fruit blend treatments from an even distribution ( = 43.59, P ≤1 × 10−5). The highest numbers of flies were trapped on the eastern downy hawthorn control (n = 82) and black hawthorn blend (n = 70) spheres, while the fewest numbers were trapped on the eastern apple blend (n = 11) and western apple blend (n = 22) spheres. A significantly higher proportion of black hawthorn fly males than females were captured on traps during each collecting period. Overall, 79.2% of the total of n = 408 captured flies were males **( = 138.8, P ≤1 × 10−5). These results indicated that males, which often reside on the under- and backside of fruit in wait of alighting females to mate with, were much more prone to be captured than females. There was no significant difference, however, in the ratio of black hawthorn fly males to females captured across the five fruit volatile treatments (G4d.f. = 8.20, P =0.104). There was also no significant difference between proportions of males and females caught on odour-baited vs. control traps for any of the five fruit volatile blend treatments (data not shown). Consequently, we present and discuss the combined black hawthorn male and female capture data for the remainder of the Results section.
Black hawthorn flies displayed significant differences in their fruit odour behavioural responses in the Burnt Bridge Creek trapping study (Table 3, Fig. 1a). There was no significant temporal heterogeneity across collecting periods in the proportion of flies captured on odour-baited vs. control traps for any of the five volatile treatments (P =0.775, 0.276, 0.373, 0.472 and 0.621 for black hawthorn, ornamental hawthorn, eastern downy hawthorn, western apple and eastern apple blends, respectively, as determined by G-heterogeneity tests). There were significant differences, however, in the total proportions of flies captured on odour-baited vs. control traps for four of the five fruit volatile treatments (Table 3, Fig. 1a). Significantly more flies were captured on the natal black hawthorn-baited traps (67.3% of n = 104 total flies captured) than the control traps ( = 12.46, P ≤0.00042, for deviation from 50 : 50 ratio; Table 3, Fig 1a). Thus, resident black hawthorn flies positively oriented to their natal fruit volatiles in the field, just as they did in previous laboratory flight tunnel assays (Linn et al., unpublished data). In contrast, significantly lower proportions of flies were captured on eastern apple (25.6% of n = 43 total flies; = 10.26, P ≤0.0014), western apple (34.4% of n = 64 total flies; = 6.25, P ≤0.0124) and eastern downy hawthorn-baited traps (29.9% of n = 117 total flies; = 18.88, P ≤0.000014) than on the blank control traps (Table 3, Fig. 1a). These results imply that just like the eastern apple and downy hawthorn host races of R. pomonella, black hawthorn flies in the western United States tend to avoid non-natal fruit volatiles. The exception was the English ornamental hawthorn blend; there was no significant difference in the proportion of resident black hawthorn flies captured on traps baited with the ornamental hawthorn volatile blend (47.5% of n = 80 total flies) vs. blank controls (53%; = 0.20, P =0.65; Table 3, Fig. 1a).
Table 3. Proportions of black hawthorn, apple and ornamental hawthorn flies captured at study sites on traps baited with the indicated fruit volatile blend out of the total number of flies captured (n = baited and blank controls) for a given treatment.
See Table 2 for blend designations and composition. Bolded frequencies indicate the natal blend at a site. *P ≤ 0.05; **P ≤ 0.01; ***P ≤ 0.001; ****P ≤ 0.0001, as determined by chi-square test from null 50 : 50 expectation of baited vs. blank control traps.
Apple field-trapping data
At the Woodland apple tree study site, a total of 569 flies were captured on the 32 traps across the eight different collecting periods from 14 to 22 August 2011. There was a significant difference in the proportion of apple flies captured between the five different fruit blend treatments from an even distribution ( = 55.94, P ≤1 × 10−5). The highest numbers of flies were trapped on the western apple blend (n = 123), western apple control (n = 87) and ornamental hawthorn control (n = 88) traps, while the fewest numbers were trapped on the eastern apple blend (n = 43) and ornamental hawthorn blend (n = 44) traps. A significantly higher proportion of apple fly males than females were captured on traps during each collecting period. Overall, 84.4% of the total of n = 569 captured apple flies were males ( = 166.4, P ≤1 × 10−5). There was no significant difference, however, in the ratio of apple fly males vs. females captured across the five fruit volatile treatments (G1d.f. = 4.06, P =0.260). There was also no significant difference between proportions of males and females caught on odour-baited vs. control traps for any of the five fruit volatile blend treatments (data not shown). Consequently, we present and discuss the combined apple male and female capture data for the remainder of the Results section.
Apple flies displayed significant differences in their fruit odour behavioural responses in the Woodland trapping study (Table 3, Fig. 1b). There was no significant temporal heterogeneity across collecting periods in the proportion of flies captured on odour-baited vs. control traps for any of the four volatile treatments (P =0.232, 0.099, 0.806, and 0.472 for western apple, eastern apple, black hawthorn, and ornamental hawthorn blends, respectively, as determined by G-heterogeneity tests). There were significant differences, however, in the total proportions of apple flies captured on odour-baited vs. control traps for three of the four fruit volatile treatments (Table 3, Fig. 1b). Significantly more flies were captured on the natal western apple blend traps (59.2% of n = 213 total flies captured) than on the control traps ( = 7.14, P ≤0.0075, for deviation from 50 : 50 ratio; Table 3, Fig. 1b). Thus, resident apple flies positively oriented to their natal fruit volatiles in the field, just as they did in previous laboratory flight tunnel assays (Linn et al., unpublished data). There was no significant difference in the proportion of apple flies captured on traps baited with the eastern apple blend (52.2% of n = 90 total flies) vs. blank controls ( = 0.178, P =0.673, 1 d.f.; Table 3, Fig. 1b). In contrast, significantly lower proportions of apple flies were captured on black hawthorn (36.3% of n = 138 total flies; = 10.46, P ≤0.0012) and ornamental hawthorn-baited traps (34.4% of n = 128 total flies; = 12.50, P ≤0.0004) compared to blank control traps (Table 3, Fig. 1b). These results imply that just like the eastern apple and downy hawthorn host races of R. pomonella, apple flies in the western United States tend to avoid non-natal fruit volatiles.
Ornamental hawthorn field-trapping data
A total of 33 ornamental hawthorn flies were captured at the Washington State University (n = 15 flies) and Burnt Bridge Creek Greenway (n = 18) sites in Vancouver, WA, on the 24 traps from 2 September to 2 October 2011. Unlike the black hawthorn and apple study sites, there was no significant difference in the proportion of ornamental hawthorn male (48.5%; n = 16) vs. female (51.1%; n = 17) flies trapped. There was also no significant difference in the number of ornamental hawthorn flies captured among the three different fruit blend treatments (ornamental hawthorn n = 9; black hawthorn blend n = 14; western apple blend n = 10) from an even distribution ( = 1.27, P ≤0.529). However, ornamental hawthorn flies displayed significant differences between the three different fruit blend treatments (see Table 3 and Fig. 1c for combined data across the Washington State University and Burnt Bridge Creek Greenway sites). A significantly greater number of ornamental hawthorn flies was captured on the blank traps than on the non-native western apple-baited ( = 6.4, P ≤0.0114) and black hawthorn-baited ( = 7.14, P ≤0.0075) traps (Table 3, Fig. 1c). Although the difference in the ratio of flies captured in the natal ornamental hawthorn to those in the blank control traps was not significant ( = 1.0, P ≤0.317) due to the overall small sample size (n = 9), the capture ratio for the natal ornamental hawthorn blend (66.7%) was significantly higher than those seen for the non-natal western apple (10.0%) and black hawthorn (14.3%) blends (P =0.0198 and 0.0228, respectively, as determined by Fisher’s exact tests). These results imply that just like the eastern apple and downy hawthorn host races of R. pomonella, ornamental hawthorn flies in the western United States tend to avoid non-natal fruit volatiles. However, the results from the ornamental hawthorn-trapping study, although showing behavioural differences between blend treatments, should be interpreted with some caution due to the smaller sample size.
Flight tunnel assays for laboratory-reared adults
In general, flight tunnel assays for laboratory-reared, odour-naïve flies were very good predictors of the field-trapping results (Fig. 2a–c). Indeed, the percentages of black hawthorn, apple and ornamental hawthorn flies captured on odour-baited vs. blank control traps in the field were significantly related to the percentages displaying upwind-directed flight in the flight tunnel (black hawthorn = 0.842, P ≤0.0088; apple = 0.909, P ≤0.0157; ornamental hawthorn = 0.987, P ≤0.0373; as determined by nonparametric Monte Carlo simulations). Only one flight tunnel test displayed any degree of difference from the field-trapping trials; black hawthorn flies reared from the Burnt Bridge Creek Greenway site showed limited upwind-directed flight to the ornamental hawthorn blend (17.3%, n = 110) in the laboratory akin to levels typical for other non-natal blends compared to the relative indifference implied by the 47.5% of flies captured on the ornamental hawthorn blend traps at the same site in the field (Table 3, Fig. 1a).
There are reasons to believe that ‘behavioural shifts have been involved in most evolutionary innovations, hence the saying that behaviour is the pacemaker of evolution’ (Mayr, 2001). For R. pomonella, this statement appears to be especially true. Rapid olfactory changes in the behavioural responses of R. pomonella to fruit volatiles appear to drive host plant shifts and the evolution of prezygotic reproductive isolation, ecological specialization and incipient sympatric speciation for the fly. Previous studies of R. pomonella in the eastern United States indicated that within the last 150 years, the newly formed apple fly race has quickly evolved a behavioural preference for the volatiles of its derived host fruit apple and avoidance to the volatiles emitted from its ancestral host fruit, downy hawthorn (Linn et al., 2003, 2005a, 2005b; Forbes et al., 2005). Western populations of R. pomonella appear to have similarly evolved comparable levels of behavioural discrimination for the volatiles of different apple and hawthorn host fruits that the fly attacks in the Pacific Northwest (Linn et al., unpublished data). In this regard, the results from the current study verify that behavioural preferences of western flies for their natal fruit volatiles observed in flight tunnel assays translate to the field. Indeed, if R. pomonella was originally introduced into the Pacific Northwest via larval-infested apples, as is generally believed to be the case (AliNiazee & Penrose, 1981; Brunner, 1987; Tracewski et al., 1987; Yee, 2008), then western fly populations in at most 60 years have evolved the ability to differentially recognize black and ornamental hawthorn fruit volatile cues for mating and oviposition. Moreover, discrimination for black hawthorn and ornamental hawthorn do not simply represent the reconstruction of an eastern downy hawthorn phenotype. Most black hawthorn flies avoided the downy hawthorn blend in the current trapping study and both black hawthorn and ornamental hawthorn flies in earlier flight tunnel tests (Linn et al., 2003) showed limited response to the downy hawthorn blend. Conversely, eastern downy hawthorn flies display low levels of upwind-directed flight to both the black hawthorn and ornamental hawthorn blends in the laboratory (Linn et al., 2003). Consequently, western R. pomonella have evolved new host odour discrimination behaviours in a relatively short period of time.
In addition to verifying positive behavioural orientation of western R. pomonella flies for their natal fruit volatiles, the current study also demonstrated that black hawthorn, apple and ornamental hawthorn flies were antagonized by non-natal fruit volatiles (Table 3, Fig. 1a–c). This finding is significant because it indicates that the evolution of avoidance behaviour may be a general phenomenon in R. pomonella and not restricted to only the eastern apple and downy hawthorn host races (Forbes et al., 2005; Linn et al., 2005a). In this regard, it will be intriguing to determine whether crosses between western apple, black hawthorn and ornamental hawthorn flies produce F1 hybrid offspring that fail to respond to parental fruit blends in the flight tunnel, mirroring the lack of behaviour seen for eastern apple × downy hawthorn hybrids (Linn et al., 2004). If so, then this would support a general role for fruit odour discrimination generating both post-zygotic and prezygotic barriers to gene flow during sympatric race formation (Feder & Forbes, 2007, 2008).
The only exception to the general antagonism displayed by western flies to non-natal volatiles was for black hawthorn flies captured on ornamental hawthorn blend traps. At the Burnt Bridge Creek Greenway black hawthorn site, 47.5% of flies were caught on ornamental hawthorn blend traps vs. 52.5% on blank control traps, which was not statistically different from a 50 : 50 ratio (Table 3, Fig. 1a). Chemical similarities between black hawthorn and ornamental hawthorn fruit volatiles could help to explain why black hawthorn flies in the trapping study were not averse to the ornamental hawthorn blend. Both the black hawthorn and ornamental hawthorn blends contain moderate amounts of 3-methylbutan-1-ol (Table 2). The eastern downy hawthorn blend also contains 3-methylbutan-1-ol, but at a lower level (Table 2), while both the eastern and western apple blends completely lack 3-methylbutan-1-ol. The compound 3-methylbutan-1-ol appears to be a universal volatile found in all hawthorn fruit and an attractant to all hawthorn flies (Cha et al., 2011a,b; Cha et al., in press). In addition, the black hawthorn and ornamental hawthorn blends also share moderate amounts of (3E)-4,8-dimethyl-1,3,7-nonatriene (DMNT), hexyl propionate and hexyl butanoate, which are either absent or found only in trace amounts in the downy hawthorn blend (Table 2). The presence of these three compounds in the ornamental, but not downy hawthorn blend, could therefore further help account for the higher behavioural receptivity shown by black hawthorn flies to the ornamental hawthorn vs. the downy hawthorn blend in the current study. In addition, the eastern and western apple blends lack DMNT, potentially contributing along with the absence of 3-methylbutan-1-ol, to the reduced response of black hawthorn flies, as well as ornamental hawthorn flies, to the apple blends.
The chemical compositions of the black and ornamental hawthorn blends also differ from one another, however. For example, the black hawthorn blend contains a relatively high amount of butyl hexanoate, a compound that is a key behavioural component of the eastern and western apple blend (Zhang et al., 1999; Cha et al., in press). Butyl hexanoate is not present in the ornamental hawthorn blend (Table 2). In addition, the ornamental hawthorn blend also lacks (Z)-3-hexenyl acetate, D-limonene and ethyl heptanoate. The absence of these compounds could therefore contribute to the lower level of response seen for black hawthorn flies to the ornamental than to the black hawthorn blend, at least in our field-trapping experiment (Table 3, Fig. 1a) and in previous flight tunnel tests of laboratory-reared flies (Fig. 2; Linn et al., unpublished data). Further chemical testing involving the addition and subtraction of compounds from the apple and hawthorn fruit blends will help resolve which volatiles are critical and which are less important for western hawthorn fly fruit odour discrimination.
Flight tunnel results of odour-naïve black hawthorn flies raised to adulthood from larval-infested black hawthorn fruit collected at the Burnt Bridge Creek Greenway site complicate the volatile story, however. In general, flight tunnel assays for the field-collected and laboratory-reared black hawthorn, apple and ornamental hawthorn flies were very similar to the field-trapping results (Fig. 2). However, there was an important difference. Rather than showing relative indifference to the ornamental hawthorn blend possibly due to the presence of shared volatiles with the black hawthorn blend, the behaviour of flight tunnel tested black hawthorn flies to the ornamental hawthorn blend was akin to the low level of response displayed by western flies in general to non-natal blends (Fig. 2).
There are three hypotheses that could alone or in combination help explain the difference between the field captured and laboratory-tested adult results, the first involving developmental irregularities, the second adult experience with volatiles, and the third inter-host movement. With respect to the first hypothesis, the decreased behavioural response of laboratory-reared flies to hawthorn volatiles could have been due to some aspect of fly husbandry that disrupted normal olfactory development and functioning for a portion of the laboratory-reared flies such that they do not respond to ornamental hawthorn volatiles. However, this scenario seems unlikely given that black hawthorn flies oriented normally to the black hawthorn blend in the flight tunnel and that the black and ornamental hawthorn blends share several compounds in common.
A second possibility is that repeated exposure of adults to black hawthorn fruit in the field subsequently increased the propensity of these flies to accept blends containing black hawthorn fruit volatiles compared to flies reared to adulthood in the laboratory. At least in the laboratory, however, there is no indication that prior adult experience affects fly behaviour (Linn et al., 2003); a given R. pomonella fly will behave similarly either responding or not responding to a specific volatile blend over the course of repeated flight tunnel trials. Moreover, individual black hawthorn flies tested in the flight tunnel were exposed to both the black hawthorn blend and the ornamental hawthorn blend in separate assays (Linn et al., unpublished data). However, this did not result in a high proportion of black hawthorn flies displaying upwind-directed flight to the ornamental hawthorn blend (only 17.3%, n = 110). It therefore seems unlikely, although not impossible, that flies should behave differently and become conditioned by certain volatiles to broaden their acceptance range in the field when they do not do so in the laboratory.
Finally, inter-host movement may also help explain the pattern. For example, movement of ornamental hawthorn flies onto black hawthorn trees could result in field flies showing higher levels of upwind-directed flight than laboratory-reared flies if (i) the migrants tended to be broad hawthorn volatile responders, liking the black hawthorn blend in addition to ornamental hawthorn and (ii) the migrants did not mate, oviposit and/or survive as well on black hawthorn as resident, natal flies. Further work is needed to resolve among these three possibilities.
In conclusion, the current study is consistent with the existence of behaviourally differentiated host races of R. pomonella being present on native black hawthorn, apple and ornamental hawthorn in the Pacific Northwest. Our results show that western black hawthorn flies are not particularly fond of the eastern downy hawthorn, eastern apple or western apple fruit blends. Thus, if the fly was introduced from the eastern United States via infested apple, it is clear that black hawthorn flies have rapidly evolved different behaviour preferences from eastern flies, as eastern apple and downy hawthorn flies are not attracted to the black hawthorn fruit blend (Linn et al., unpublished data). These behavioural changes in the black hawthorn population would presumably have accompanied a recent host shift of R. pomonella from apple to C. douglasii and C. monogyna in the west. Alternatively, if black hawthorn flies were native to the west and shifted to introduced apple, as well as ornamental hawthorn, in the region, then the fly evolved a new preference for these derived hosts independent of the eastern apple race. Such a scenario could account for why western apple flies prefer a slightly different volatile blend than eastern apple flies (Cha et al., in press; Linn et al., unpublished data). Finally, it is conceivable that apple flies were introduced and black hawthorn flies are native to the western United States. In this case, western apple and black hawthorn flies would still represent host races, but not an example of sympatric host shifting and incipient sympatric speciation, although one or the other likely formed a new race shifting to ornamental hawthorn. More detailed genetic surveys (McPheron, 1990) coupled with further analysis of host fruit odour discrimination and other host-related life-history traits, such as adult eclosion timing that is known to differentiate eastern apple and downy hawthorn flies (Feder et al., 1993, 1994; Filchak et al., 2000), will help resolve the intriguing evolutionary story of R. pomonella in the western United States.
The authors would like to thank the Clark County, Washington, 78th street Heritage Farm, the Washington State University Research and Extension Unit, Vancouver, Blair Wolfley, Doug Stienbarger, Terry Porter and Kathleen Rogers for their support and assistance on the project. We also appreciate the effort of Tom Glen and Scott Tracy at Suterra LLC (Bend, Oregon, USA) in making the wax fruit odour lures on short notice and Mike Chong at the University of Waterloo for the synthesis of DMNT. Special thanks also to Tom Powell, Glen Hood Tracy Arcella, Gilbert St. Jean and Scott Egan for helpful discussions, insight, advice and constructive criticism of the paper. This work was supported in part by grants to JLF from the NSF and the USDA, and to WLY by the Washington Tree Fruit Research Commission and Washington State Commission on Pesticide Registration.