Guy L. Bush.
Host race formation and sympatric speciation in Rhagoletis fruit flies (Diptera: Tephritidae).
Psyche 99:335-357, 1992.

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HOST RACE FORMATION AND SYMPATRIC SPECIATION IN RHAGOLETZS FRUIT FLIES (DIPTERA: TEPHRITIDAE) Department of Zoology
Michigan State University
East Lansing, MI 48824-1 115
Dedication
I dedicate this contribution to my mentor, Frank M. Carpenter. His encouragement and support during my tenure as a graduate student made it possible for me to embark on a 30-year odyssey to study the process of speciation in a fascinating group of insects that do things in an extraordinary way.
In 1866, some 200 years after the introduction of apples to North America, a local newspaper reported that larvae of an unknown fly were infesting apples (Malus pumila) in the Hudson River valley of New York (Illingworth, 1912). Shortly after the maggots were found in apples, Benjamin Walsh (1867) described the fly as Trypeta pomonella (later recognized as Rhagoletis pomonella (Snow, 1894)). Walsh's description was based on speci- mens reared from apples grown in eastern United States, as well as from hawthorn fruit (Crataegus spp.) gathered in Illinois where, he reported, the fly did not attack apples. Walsh suggested that the flies infesting apples represented a newly established local host race biologically distinct from the flies associated with native hawthorn. He had proposed earlier (Walsh, 1864) that in phytophagous insects a host shift by individ- uals bearing a heritable change in host preference could give rise to new host races in the absence of geographic isolation. He rea- soned that there would be sufficient "phytophagic isolation" (host specificity) to allow the adaptive evolution of new species. Not only was Walsh the first biologist to propose that insects could speciate sympatrically via a host shift, but he also was quite clear Manuscript received 2 March 1993
Pu&e 99:3J5-357 t 1992). http:llpsyche cnlclub oreflW9-M html



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about what he meant by the term species. As an early and vocal advocate of Darwin in North America (Shepard, 1989), he was the first biologist to formulate what we now regard as the biological species concept (Walsh, 1863).
Although sympatric speciation remains a controversial topic, recent studies on a variety of organisms support Walsh's view that many organisms with a parasitic life style speciate sympatrically (Bush, 1993). One of the best understood examples is R. pomonella. A great deal of information on the ecology, behavior and genetics of this fly and its close relatives has accumulated over the years. Also, for the apple infesting population, the time of the original host shift and the pattern of dispersal throughout eastern North America are known in some detail. It is one of the few cases where the historical aspects of host race formation and its role in speciation can be studied with a considerable degree of accuracy. Here, I present an overview of the evidence, much of it resulting from research over the past thirty years, supporting the view that many Rhagoletis species have evolved in the absence of geograph- ical isolation.
HOST RELATIONSHIPS, HOST RACES, AND CRYPTIC SPECIES Infestations of R. pornonella on apples rapidly spread through- out the apple growing regions of eastern United States and south- eastern Canada, reaching western Iowa around 1890, the higher elevations of North Carolina in 1894, Nova Scotia by 1910, and southeastern Manitoba in 1916 (Bush, 1969a). Crataegus, repre- sented by numerous species in eastern North America, is found throughout the range of the apple-associated populations. Hawthorn, extending further south into central Florida and west to central Texas, is also infested by R. pomonella (Berlocher and Enquist, 1993; Paine and Berlocher, in prep.), but apples grown in the southern U.S.A. are not attacked. Attempts to rear R. pornonella from fruits of the five native North American crabap- pies (Malus spp.) have failed. Our native crabapples may not be congeneric with the Eurasian representatives of Malus (Robertson et al., 1991). Their high acidity, late maturation (O'Kane, 1914), and high phenolic content (Pree, 1977) appear to render them unsuitable for larval development. Therefore, they cannot be the source of the original apple population. For this and several other



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reasons detailed elsewhere (Bush et al., 1989; Feder and Bush, 1989), R. pomonella on apples came from native North American Crataegus.
Over the years, R. pomonella-like flies were found infesting the fruits of other plants such as blueberries (Vaccinium spp. and Gay- lussacia spp.) (Britton, 1906), dogwood (Cornus spp.) (Hall, 1934), snowberries (Symphoricarpus spp.) (Snow, 1894), native plums (Prunus spp.) (Benjamin, 1934), tart cherries (Prunus cera- sus L.) (Shervis et al., 1970; Messina and Jones, 1990), pears (Pyrus cornmunus L.) (Prokopy and Bush, 1972), rose hips (Rosa rugosa Thumb.) (Prokopy and Berlocher, 1980), and apricots (Prunus arrneniaca (L.)) (Lienk, 1970). Some of these host associ- ated populations, such as R. mendax on blueberries (Curran, 1932), R. zephyria on snowberries (Snow, 1894), and R. cornivora on shrubby dogwood (Bush, 1966), are now recognized as cryptic species. Others appear to represent undescribed species, for exam- ple, populations infesting flowering dogwood (C. florida L.) (Smith, 1988a; Berlocher et al., submitted), southern populations whose larvae feed in wild plums, and populations that infest sev- eral species of blueberry in the southeastern U.S.A. (S. Berlocher, pers. comm.).
The reports of R. pornonella attacking the fruits of pear and apricot probably represent transient infestations since this fly has failed to establish permanent populations on these important intro- duced commercial fruits that are constantly scrutinized for destruc- tive pests. However, extensive studies of R. pomonella, which I will discuss more fully, indicate that infestations on cultivated and feral apple represent well established populations that are distin- guishable from native haw populations by several important genet- ically biased biological traits. The apple and haw populations represent host races that have evolved different mate recognition systems sufficient to maintain these genetic distinctions (Diehl and Bush, 1984). These differences evolved as a direct outcome of adaptation of each race to a new host in the absence of any physi- cal barriers to gene exchange. Preliminary observations of estab- lished populations of R. pomonella on introduced tart cherry and rose hips suggest that they also are host races that have developed sympatrically within a very short period of time on introduced hosts.




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Boller and Prokopy (1976) reviewed early research on the biol- ogy of Rhagoletis. Details of the systematics and phylogenetic relationships of this genus have been summarized by Bush (1966) and Berlocher and Bush (1982). Only those aspects pertinent to host race formation and speciation are presented here. Egg and larval development: Eggs, inserted by the female's ovipositor under the skin of the host fruit, hatch within a few days. First instar larvae work their way into the interior of the fruit and in so doing reduce their exposure to parasitism (Feder et al., in prep. b). Larval development takes as little as two weeks under optimum conditions in fruits with high sugar content and low acid- ity. Conversely, eggs and larvae may experience up to 100% mor- tality in certain varieties and species of apple and haws (Dean and Chapman, 1973; Bush et al., 1989). In temperate climate Rhago- letis species, the majority of third instar larvae leave the fruit in two to four weeks and burrow into the soil under the host tree. Pupation occurs about four inches below the soil's surface where pupae overwinter for several months in diapause (Boller and Prokopy, 1976).
Diapause and eclosion: Although most temperate climate Rhagoletis have only one generation per year, diapause is gener- ally facultative (Prokopy, 1968). Because larvae of the apple race of R. pornonella are exposed to longer days than the haw race, from 1-5% of the pupae may not diapause, and a small second adult generation is produced usually too late in the season to repro- duce. The apple race apparently has not completely adjusted to the shift in day length and warmer temperatures to which they are exposed. Most temperate climate Rhagoletis undergo a winter dia- pause which is terminated after exposure to low temperatures. Adult development in the pupa is initiated by rising temperatures in spring when soil temperature extends above a minimum thresh- old level. Depending on temperature and moisture conditions in the soil, about 10% of the pupae may remain in the soil for 2-5 years before completing development. This "pupa banking" insures a reserve of adults from year to year in areas where masting of the host species may cause cyclic or sporadic drastic reductions in fruit or complete crop failure in a region.



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Each species or host race responds somewhat differently to moisture, temperature and other environmental conditions (e.g., day length) to which larvae and pupae are exposed (Prokopy, 1968; Feder et al., 1993). The physiological mechanism for con- trolling the rate of post-diapause development and the timing of eclosion in R. pomonella and other Rhagoletis species has not been determined, but it is highly correlated with the accumulation of thermal units over a developmental threshold temperature which varies from 4.4-8.7OC depending on locality, species, and method of measuring temperature (soil or air) (Reissig et al., 1979; Feder et al., 1993).
Food foraging, dispersal and longevity: Upon emergence, adults begin foraging on the surface of leaves of host and non-host plants for food such as insect honeydew, bird feces, yeast and bac- teria. Both males and females in R. pomonella require carbohy- drates, certain vitamins and amino acids for sexual maturation. Mating and oviposition begins on about the 10th to 12th day after eclosion (Webster et al., 1979). Adults begin to suffer high mortal- ity if deprived of food for more than one day (Malavasi and Prokopy, 1992).
Rhagoletis locate vegetation primarily by vision. Before sexual maturity, adults of both sexes are strongly attracted to surfaces reflecting in the yellow portion of the spectrum (500-600 nm). This color, which is a major component of light reflected from green vegetation, appears to be a foliar attractant used by the flies to locate food resources (Prokopy, 1976). Both males and females orient positively to tree models of specific shape, size and color, particularly ones with shifting patterns of individual leaves (Moer- icke et al., 1975). Odor of food resources on the leaves may also promote the movement of flies to particular trees (Prokopy, 1976), although the odors of host or non-host foliage and twigs alone are not particularly attractive (Prokopy et al., 1973). During this pre-copulatory period of feeding, adult Rhagoletis move continuously and can disperse some distance (Roitberg, 1982). Dispersal is influenced more by the availability of food than the search for mates and oviposition sites. Just how far the average adult is capable of moving during the feeding phase is not known, but field and laboratory flight studies indicate that they are capable of traveling several kilometers (Prokopy, 1976).



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How long Rhagoletis adults survive in nature has not been accu- rately established. Estimates of average longevity vary from two weeks for R. cerasi, three weeks for R. rnendax, three to six weeks for R. cornpleta Cresson, and up to four weeks for R. pornonella (Opp and Prokopy, 1987). It is clear that light, moisture, tempera- ture, and the availability of food and oviposition sites have pro- found effects on adult survival rates (Boiler and Prokopy, 1976). Host plant and fruit foraging behavior: Motivation for dis- persal, initially driven by the need for food, changes as flies become sexually mature. Adult R. pornonella continue to use the same physical cues of shape and color to locate host trees, but in addition become strongly attracted to specific odors emanating from ripening host fruit (Prokopy et al., 1973). Certain esters, identified by electroantennogram bioassays and behavioral obser- vations, are important components eliciting response to host fruit odor (Fein et al., 1982; Averill et al., 1988; Frey et al., 1992). Once on the host plant, both sexes begin visually searching for, and are strongly attracted to, dark colored spheres approximately the size of their natural host fruit (Prokopy et al., 1971 ; Prokopy, 1977). Other species, such as R. cerasi, R. mendax and R. corn- pleta, prefer fruits of varying sizes, colors, or combinations of fruit volatiles (Guerin et al., 1982; Riedel and Hilslop, 1985; Frey et al., 1992). In the absence of fruit, flies rapidly abandon a tree and dis- perse in search of mates and oviposition sites on other trees (Aluja and Prokopy, 1992). Host selection is, therefore, mediated by a combination of visual, tactile, and olfactory cues, with the latter providing a crucial chemically based signal for host specificity. Mating and oviposition behavior: Rhagoletis males and females use the host as a rendezvous for courtship and mating, a mode of mating behavior typical of many parasites (Bush, 1975; Zwolfer and Bush, 1984). Thus, the search for mates by both sexes of Rhagoletis is inexorably linked to the search for their host plant (Bush, 1969a, 1974). Therefore, the cues used to locate a mate are the same visual, tactile and chemical cues involved in the search for a host tree and fruit. At the beginning of the mating period, when most females of R. pornonella are unmated, matings may occur on leaves of the host plant following a courtship duet involv- ing wing waving, posturing, and short range male pheromones (Prokopy et al., 1973; Prokopy, 1975; Smith and Prokopy, 1980).



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Once mated, females ignore males and begin to oviposit on host fruits.
Female flies inspect the surface and reject or accept a fruit for oviposition on the basis of its size, surface structure, stage of ripeness and other stimuli (Boller and Prokopy, 1976). Although most Rhagoletis species lay only one egg at a time in a maturing fruit, members of the walnut infesting R. suavis (Loew) species group are batch egg layers (Boyce, 1934). After egg laying, a Rhagoletis female drags her ovipositor over the surface of the fruit depositing at least two water soluble pheromones (Prokopy, 1972). In R. cerasi one of these pheromones has been identified as a com- plex molecule detected through special tarsal chemosensilla (Stadler and Katsoyannos, 1979; Crnjar and Prokopy, 1982). This pheromone deters other females from laying eggs in the same fruit, and thus reduces intraspecific larval competition at least initially (Averill and Prokopy, 1989; Aluja and Boller, 1992). At the time of ovipositor dragging, a second, short acting sub- stance is deposited which arrests males on the fruit and informs them that a female has recently been in the vicinity (Prokopy and Bush, 1972b). This unidentified substance tends to aggregate males on the fruits in trees where females are actively ovipositing. Males of R. pornonella spend 89% of their time patrolling apples (Prokopy and Bush, 1973), aggressively driving away other males and attempting to forcibly copulate with arriving females (Prokopy, 1981). This enhances the chance for multiple matings, which are necessary to maintain maximum fertility (Neilson and McAllen, 1965; Opp and Prokopy, 1986). When females cease oviposition in a particular tree it is abandoned by males. Host race criteria: There have been several attempts to estab- lish criteria for distinguishing host races from populations of polyphagous generalists. Mayr (1970) regarded host races as "non- interbreeding sympatric populations that differ in biological char- acteristics but not, or scarcely, in morphology." They are "prevented from interbreeding by a preference for different food plants or other hosts." However, reproductive isolation is the same criterion used by Mayr to define species in his biological species concept. Others have proposed alternative host race definitions



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(Bush, 1969; Futuyma and Mayer, 1980; Jaenike, 1981; Bush and Diehl, 1982). Based on our current knowledge of recently estab- lished host specialists, host races are partially reproductively iso- lated, conspecific populations specializing on alternative hosts (Diehl and Bush, 1984). As discussed by Jaenike (1981) and Diehl and Bush (1984), to verify the existence of host races requires that: Individuals in breeding condition of different host-associ- ated populations must be sympatric.
Statistically significant genetic differences exist between these sympatric populations over several generations that are not directly related to host selection or solely the result of natural selection acting on a single generation. Males and females exhibit genetic variation in host prefer- ence that results in assortative mating, i.e., mating occurs on the preferred host plant and host preference is under genetic control.
Males and females show host-associated tradeoffs in fitness.
There is no evidence of post-mating reproductive incompat- ibility. Hybrid incompatibility between host-associated pop- ulations indicates they are sibling species, not host races. There is now sufficient biological and genetic evidence to show that apple and haw populations of R. pornonella meet all the above host race criteria.
1. Sympatry: Populations of R. pornonella on apple have been sympatric with the original hawthorn race from the moment this fly colonized apples in the Hudson River valley about 150 years ago. The original shift must have occurred in an area where apple trees were growing within the dispersal range of flies infesting hawthorns. Adults of Rhagoletis species are capable of colonizing stands of host plants isolated some distance from an infested tree. In a careful study of dispersal, marked R. cerasi flew at least 3.5 km in direct point-to-point flights over cleared land (Boller, 1979) and were able to reach adjacent orchards separated by 3 km of forests (Boller, 1987) when host fruit was unavailable for oviposi- tion. Comparable dispersal distances were recorded in earlier stud- ies for R. cornpleta (Barnes, 1959) and R. pomonella (Maxwell and Parsons, 1968). Because the recovery of marked flies is low, it is likely that individual Rhagoletis adults may disperse considerably further than the recorded 3.5 km. It is therefore unlikely that apple



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or Crataegus hosts with fruit suitable for larval development would long remain uninfested even if isolated by considerable dis- tance from infested apples or hawthorns. 2. Genetic differences over space and time: There are several recognized genetic differences between the apple and haw races. We (Feder et al., 1988; Feder et al., 1989a; Feder et al., 1990a; Feder et al., 1990b) and our co-workers (McPheron et al., 1988) have docu- mented allele frequency differences in six allozyme loci between paired sympatric samples of the apple and haw races throughout the northeastern United States. These frequency differences are maintained across different developmental stages of the fly as well as from year to year. Clinal allelic variation also exists at these six loci, and is highly correlated with local seasonal ambient tempera- tures experienced by each host race during larval development and adult activity.
While the allozyme alleles respond to clinal and seasonal tem- perature differences, their pattern of divergence indicates that they are genetically linked to other loci involved with adaptation to apple and haw. These adaptations include eclosion time (Smith, 1988b), response to host odors (Frey and Bush, 1990) and host fidelity (Feder et al., in prep. a). Divergent selection on these traits, discussed more fully below, contributes to the maintenance of the observed allozyme differences under sympatric conditions by reducing the effects of recombination and gene flow between the races. However, such host associated traits alone cannot be the only factors maintaining genetic differences between the host races. The allozyme results suggest that host specific assortative mating also occurs in R. pomonella.
3. Host preferences: Although the physical cues (color, shape, texture, etc.) used by both host races to locate a plant for feeding are the same, the electrophysiological response of R. pomonella to specific apple and haw odors differs significantly (Frey and Bush, 1990). This suggests they already discriminate between the two host fruits to some extent at the chemosensory level (Frey and Bush, 1990). Support for effective host fidelity based on geneti- cally constrained host selection behavior was obtained in a recent mark-release-recapture study (Feder et al., in prep. a). Apple and haw flies showed strong preference (around 94-95% host fidelity) for their respective host plants under natural conditions.



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Evidence for genetically based differences in host acceptance behavior has also been found (Prokopy et al., 1988). Although apple and haw fruit are equally accepted for oviposition by naive females from apple and hawthorn origin, apples are accepted sig- nificantly more often by females from apple than females from hawthorn. Males of the apple and haw races exhibit similar differ- ences in their pattern of host acceptance. Residence times spent on haws by naive males from apple and haw are the same, while males of apple origin tend to reside substantially longer on apples than males of hawthorn origin.
Another important feature of host fruit choice in R. pornonella, and possibly all Rhagoletis species, is the contribution that learn- ing (i.e., a change in behavior with experience) plays in host acceptance behavior. Both Rhagoletis apple and haw flies have the ability to learn to accept or reject novel fruit species and to accept familiar fruit species (Prokopy et al., 1982; Papaj and Prokopy, 1986; Papaj and Prokopy, 1988; Prokopy et al., 1989). Adult R. pornonella from apple can learn to reject unfamiliar physical and chemical stimuli of different host fruit species more readily than those from haws. They spend a significantly longer time on the same species of fruit with which they have had prior experience, and females prefer to oviposit on fruits in which they have already oviposited. Prior host fruit experience by both males and females of the apple race not only has a strong influence on what species of host fruit is preferred by males for courting females and by females for subsequent oviposition, but females (and presumably males) can also learn to discriminate between apple cultivars (Prokopy and Papaj, 1988). This discrimination, based on differ- ences in chemical stimuli among haws and apple cultivars, should increase oviposition preference and habitat fidelity and promote genetic subdivision of the races (Papaj and Prokopy, 1988). A further indication of host specialization is indicated by racial differences in seasonal distribution patterns. Adult eclosion in R. pornonella is synchronized with the ripening pattern of host fruit and differs between the host races. Under field conditions the apple race emerges about ten days to two weeks before the hawthorn race (Reissig and Smith, 1978; Feder et al., 1993). This seasonal asynchrony is the result of heritable genetic differences in development (Smith, 1988b; Feder et al, 1993). Furthermore, field studies suggest that adults of the haw race may reach sexual



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maturity somewhat slower than the apple race7 further reducing the opportunity for hybridization between the races (Stanek et ala7 1987; Feder et al., 1993). Although seasonal asynchrony con- tributes to allochronic isolation of the host races7 it alone is unlikely to be sufficient to sustain racial differences in the absence of host fidelity.
4. Host associated fitness tradeoffs: Careful experiments involv- ing reciprocal egg transplants found no evidence for host race- associated differences in larval survivorship on either apple or haw (Prokopy et al., 1988). Furthermore, haw appears to be a more suitable larval host than apple. Larvae of both races had equally higher rates of survival on haws than on apple. This suggests that the races have not evolved larval feeding specializations for either host. But factors other than host fruit chemistry do cause host- associated fitness tradeoffs. These tradeoffs deal with differences in the environmental conditions experienced by larvae and pupae. A major factor affecting fitness that imposes important selective constraints on larvae and pupae of each race is host fruit phenol- ogy. Apple flies that develop too soon fail to diapause, emerging the same season7 but too late to produce a second generation. Hawthorn flies that develop too slowly also fail to diapause before the onset of winter. Adaptation of each race to their respective hosts has thus required adjustments in those genes responsible for coping with differing ontogenies and timing of adult eclosion (Feder et al.? 1993).
The activity of each host race is synchronized with the phenol- ogy of its host fruit. Adults of the haw race therefore eclose about ten days to two weeks after members of the apple race (Reissig and Smith, 1978; Feder et al., 1993). Genetic and mark-release-recap- ture studies on local Michigan populations indicate that haw flies may also reach sexual maturity more slowly than apple flies (Feder et alm7 1993). Their eggs are thus deposited later in haw fruit. As a result eggs7 larvae and pupae of the haw race are subjected to lower average daytime temperatures, cooler nights and progres- sively shorter day lengths than members of the apple race (Feder et al., 1993). Early developmental stages of both races must therefore adapt to quite different environmental conditions. Adaptation to these temporal differences probably also involved the six allozyme loci that display allele frequency differences between local sympatric apple and haw populations (Feder et al.,