Ecological Entomology (2012), 37, 521–528
DOI: 10.1111/j.1365-2311.2012.01392.x
Evidence for sexual isolation as a prezygotic barrier to
gene flow between morphologically divergent species
of Rhagoletis fruit flies
G L E N R . H O O D ,1 S C O T T P . E G A N 1,2 and J E F F R E Y L .
F E D E R 1 1 Department of Biological Sciences, University of Notre Dame, Notre Dame, Indiana, U.S.A. and 2 Advanced
Diagnostics and Therapeutics, University of Notre Dame, Notre Dame, Indiana, U.S.A.
Abstract. 1. Certain groups of fruit flies in the genus Rhagoletis (Diptera:
Tephritidae) are exemplars for sympatric speciation via host plant shifting. Flies
in these species groups are morphologically similar and overlap in their geographic
ranges, yet attack different, non-overlapping sets of host plants. Ecological adaptations
related to differences in host choice and preference have been shown to be important
prezygotic barriers to gene flow between these taxa, as Rhagoletis flies mate on or
near the fruit of their respective host plants. Non-host-related assortative mating is
generally absent or present at low levels between these sympatrically diverging fly
populations.
2. However, some Rhagoletis taxa occasionally migrate to ‘non-natal’ plants that
are the primary hosts of other, morphologically differentiated fly species in the
genus. These observations raise the question of whether sexual isolation may reduce
courtship and copulation between morphologically divergent species of Rhagoletis
flies, contributing to their prezygotic isolation along with host-specific mating.
3. Using reciprocal multiple-choice mating trials, we measured sexual isolation
among nine species pairs of morphologically differentiated Rhagoletis flies. Complete
sexual isolation was observed in eight of the nine comparisons, while partial sexual
isolation was observed in the remaining comparison.
4. We conclude that sexual isolation can be an effective prezygotic barrier to gene
flow contributing to substantial reproductive isolation between many morphologically
distinct Rhagoletis species, even in the absence of differential host plant choice and
host-associated mating.
Key words. Assortative mating, host-shifting, reproductive isolation, sympatric
speciation.
Introduction
Speciation, the ultimate source of biodiversity, occurs as
one formerly interbreeding population evolves into two or
more reproductively isolated taxa. In sexual reproducing
organisms, speciation is best understood by resolving the
basis for reproductive barriers to gene flow between taxa
(Mayr, 1963; Coyne & Orr, 2004). Reproductive isolation
can result from several factors, including (but not limited
Correspondence: Glen R. Hood, Department of Biological Sciences,
University of Notre Dame, Notre Dame, IN 46556, U.S.A. E-mail:
[email protected]
© 2012 The Royal Entomological Society
to): (i) prezygotic barriers that act before zygote formation by
reducing encounters, matings, sperm transfer, or fertilisation
between taxa (Dobzhansky, 1937; Feder et al., 1994), and (ii)
postzygotic barriers that act after zygote formation due to
‘intrinsic’ incompatibilities of the parental genomes expressed
in hybrids (Dobzhansky, 1937; reviewed in Coyne & Orr,
2004) or ‘extrinsic’ mismatches of the hybrid phenotype to
parental environments, which reduce hybrid survival (e.g. Egan
& Funk, 2009).
Different forms of prezygotic and postzygotic reproductive
isolation usually evolve and contribute to the evolution of
reproductive isolation during the course of speciation. An
important issue in speciation research involves determining
521
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Glen R. Hood, Scott P. Egan and Jeffrey L. Feder
the relative importance and timing that different prezygotic
and postzygotic barriers arise during population divergence.
It has been argued that for many taxa, prezygotic isolating
mechanisms are of greater importance because they act earlier
in the life cycle before hybrid zygotes are formed and
postzygotic barriers take effect. Thus, even when prezygotic
barriers evolve after postzygotic isolation, they may still have
the largest net effect on reducing current gene flow between
taxa (Ramsey et al., 2003). In addition, in cases of secondary
contact in which hybrids suffer reduced fertility or viability,
prezygotic barriers can evolve or be strengthened through a
process termed ‘reinforcement’ to lessen the adverse fitness
consequences of hybridisation (e.g. Noor, 1995).
Studies of phytophagous insects have made important
contributions to our understanding of speciation, especially
with respect to the role that ecological adaptation and host
plant specialisation play in initiating population divergence
(Funk et al., 2002). Among phytophagous specialists, hostspecific mating is often an important prezygotic barrier
to gene flow between populations (Berlocher & Feder,
2002; Funk et al., 2002). When insects chose to mate on
preferred host plants, a system of assortative mating is
established that can serve as an effective prezygotic barrier
to gene flow and facilitate the evolution of additional hostrelated performance (survivorship) traits between sympatrically
diverging populations (Thompson, 1988).
Host-specific mating may not represent the only prezygotic
barrier to gene flow between phytophagous insects, however.
For example, sexual isolation may also exist that reduces
maladaptive hybridisation between taxa in those instances
when individuals happen to migrate to ‘non-natal’ host plant
species because host fidelity is not always complete (e.g. Funk
& Bernays, 2001; Egan & Funk, 2006; Ferrari et al., 2007) and
individuals are sometimes observed on alternate, non-natal host
plants in the field (Clark et al., 2004) (Table 1). In addition,
studies have documented that sexual isolation exists between
at least some host-associated insect populations (Funk, 1998;
Nosil et al., 2002; Egan et al., 2012a,b). However, it remains to
be determined how general sexual isolation is between hostspecific phytophagous insects. Here, we test for evidence of
sexual isolation between morphologically divergent species
pairs of fruit flies belonging to the genus Rhagoletis (Diptera:
Tephritidae).
In North America, the genus Rhagoletis is comprised of
several different species groups that vary in their degree of
morphological divergence and that have been hypothesised
to undergo different modes of speciation (Bush, 1966, 1969;
Foote et al., 1993; Fig. 1). For example, the R. pomonella
group is comprised of a number of sibling species and host
races that broadly overlap in their geographic ranges and are
specialised on different sets of non-overlapping host plants.
These considerations led Bush (1966) to propose that the
group has radiated via sympatric host shifting (Filchak et al.,
2000; Coyne & Orr, 2004; Xie et al., 2008). The recent
shift of the species R. pomonella from its native, ancestral
host hawthorn (Crataegus spp.) to introduced, domesticated
apple about 150 years ago is often cited as an example of
ecological speciation-with-gene-flow in action (Berlocher &
Feder, 2002; Coyne & Orr, 2004). In contrast, the R. suavis
group is comprised of several morphologically distinct taxa that
either do not co-occur or are narrowly parapatric with other
members of the R. suavis species group in their geographic
ranges. All R. suavis group flies attack walnuts (Juglans spp.).
These considerations led Bush (1966) to propose that walnut
flies largely radiated by an allopatric mode of speciation.
Other species groups such as the R. cingulata complex of
cherry- and olive-attacking flies contain mixtures of taxa with
little to slight morphological divergence, sympatric to largely
allopatric geographic distributions, and similar to distinct host
associations (Bush, 1966). Members of these species groups
may have undergone both sympatric and allopatric modes of
speciation.
Analysis of reproductive isolation between Rhagoletis taxa
has primarily concentrated on traits related to host choice and
timing of the overwintering pupal diapause (Smith, 1988a;
Feder et al., 1994; Linn et al., 2003). This emphasis on
ecologically based barriers to gene flow is due to: (i) the
importance of these flies as models for sympatric speciation
via host shifting, and (ii) the predominant roles that host
discrimination and diapause timing play in isolating hostspecific populations of Rhagoletis, at least in the early stages
of divergence-with-gene-flow, as evidenced by the apple and
hawthorn host races of R. pomonella (Feder et al., 1994;
Linn et al., 2003). Studies of sexual isolation have only
been conducted between a few Rhagoletis species (Smith &
Prokopy, 1982; Smith, 1988b; Schwarz & McPheron, 2007;
Rull et al., 2010; Yee & Goughnour, 2011). Moreover, in cases
such as the recently formed apple and hawthorn host races
there is little evidence for prezygotic isolation aside from that
generated by host-associated mating.
Despite the emphasis of previous studies on host and
ecologically related forms of prezygotic reproductive isolation,
sexual isolation warrants further investigation in Rhagoletis
for several reasons. First, although sexual isolation may
not be the first trait to differentiate sympatrically diverging
Rhagoletis taxa, it could still contribute to speciation later in
the process, such as when flies begin to evolve morphological
differences that are more pronounced. For example, although
the R. pomonella group is comprised of morphologically and
genetically closely related sibling species, some taxa do show
slight, although not entirely diagnostic, differences in traits
such as overall body size (Fig. 1), shape and proportions of
the surstyli, shading of the posterior surface of femur, and
ratio between width of the medial and subapical cross bands
of the wing (Bush, 1966; Fig. 1). These slight morphological
differences could potentially influence mate choice. Moreover,
flies in the R. cingulata group show some differences in their
wing banding patterns. Rhagoletis cingulata (primary native
host: Prunus serotina, common name: black cherry) can be
distinguished from R. indifferens (primary native host: Prunus
emarginata, common name: pin cherry) by a largely diagnostic
difference in pigmentation on the apical wing band (Bush,
1966; Foote et al., 1993). Two other members of the cingulata
group, R. osmanthus (host: Osmanthus americanus, common
name: wild tea-olive or devilwood) and R. chionanthi (host:
Chionanthus virginicus, common name: fringe-tree), also differ
© 2012 The Royal Entomological Society, Ecological Entomology, 37, 521–528
Sexual isolation in Rhagoletis
523
Table 1. Field observations of alternative host use by Rhagoletis fruit flies.
Rhagoletis
species
Native host
Alternative host (natal species)
Observed behavior on
alternative host
R. mendax
R. suavis
R. cingulata
R. cingulata
R. striatella
R. juniperina
R. pomonella
R. indifferens
R. pomonella
R. completa
R. indifferens
R. completa
R. indifferens
Vaccinium
Juglans nigra
Prunus serotina
Prunus serotina
Physalis spp.
Juniperus spp.
Malus domestica
Prunus emarginata
Malus domestica
Juglans nigra
Prunus emarginata
Juglans nigra
Prunus emarginata
Malus domestica (R. pomonella)
Prunus serotina (R. cingulata)
Juglans nigra (R. suavis)
Creatagus mollis (R. pomonella)
Malus domestica (R. pomonella)
Juglans major (R. juglandis)
Juglans nigra (R. suavis)
Creatagus douglasii (R. pomonella)
Prunus emarginata R. indifferens
Creatagus monogyna (R. pomonella)
Juglans nigra (R. completa)
Malus domestica (R. pomonella)
Rosa sp. (R. basiola)
Foraging on fruits and/or
Foraging on fruits and/or
Foraging on fruits and/or
Foraging on fruits and/or
Foraging on fruits and/or
Foraging on fruits and/or
Foraging on fruits and/or
Reared from fruit
Reared from fruit
Reared from fruit
Resting on leaves
Foraging on leaves
Resting on leaves
Reference/observer(s)
leaves
leaves
leaves
leaves
leaves
leaves
leaves
Prokopy and Papaj (2000)
S. Berlocher, G. Hood (PO)
G. Hood (PO)
G. Hood, J. Smith (PO)
S. Berlocher (PO)
S. Berlocher (PO)
S. Berlocher (PO)
Yee and Goughnour (2008)
Yee and Goughnour (2008)
Yee and Goughnour (2008)
Yee and Goughnour (2008)
Yee and Goughnour (2008)
Yee and Goughnour (2008)
PO (pers. obs.).
Wing pattern
R. berberides
Body size (± S.E.)
Wing length
Head width
R. striatella
81
R. basiola
1.78 ± 0.036
4.80 ± 0.076
R. cingulata
1.55 ± 0.006
3.62 ± 0.021
1.48 ± 0.030
3.47 ± 0.059
R. suavis
1.65 ± 0.031
4.57 ± 0.122
R. berberis
1.36 ± 0.020
2.86 ± 0.069
R. pomonella
1.66 ± 0.027
4.10 ± 0.540
2.37 ± 0.030
5.13 ± 0.070
R. indifferens
100
R. osmanthi
96
R. chionanthi
67
100
53
R. zoqui
R. completa
56
R. juglandis
93
98
78
100
R. zephyria
R. mendax
51
R. cornivora
R. juniperina
100
R. tabellaria
R. electromorpha
R. fausta
Z. electa
Fig. 1. Rhagoletis phylogeny modified from Smith and Bush (1997) used to illustrate the evolutionary relationships and variation in wing pattern
morphology and body size (head width and wing length ± S.E.) for the seven species used in this study (modified from Bush 1965, 1966). Bootstrap
confidence limits >50% are indicated above each branch. Species groups used in this study are designated by shaded bars (from top to bottom:
Alternata, Cingulata, Suavis, Ribicola and Pomonella species groups). Body sizes were estimated from 10–20 females per species.
from the cherry-infesting flies by having a more forked apical
wing band. Finally, walnut-infesting taxa of the R. suavis
group show pronounced morphological differences in body
coloration and wing banding patterns from each other. In
addition, several walnut fly species display sexual dimorphism
in the colour patterns of the thorax and abdomen, consistent
with sexual selection.
Second, evidence indicates that sexually active Rhagoletis
adults belonging to the same and to different species groups
at least occasionally encounter one another in nature (see
© 2012 The Royal Entomological Society, Ecological Entomology, 37, 521–528
524
Glen R. Hood, Scott P. Egan and Jeffrey L. Feder
Table 1). At these times, sexual isolation could play a role in
restricting hybridisation between fly taxa. In this regard, many
of the host plants attacked by Rhagoletis overlap in both their
broad geographic ranges and the microhabitats they occur in
(Bush, 1966). In addition, although host fidelity is pronounced,
it is not always absolute for Rhagoletis flies. Observations
of flies frequenting alternative hosts have been reported
for Rhagoletis (Table 1). Consequently, sexual isolation may
constitute an important barrier to gene flow in these instances,
acting before postzygotic isolation due to divergent host
ecology and/or intrinsic genomic incompatibilities.
We test for possible sexual isolation in Rhagoletis in the
current study through pairwise reciprocal mating choice experiments between nine different species combinations of flies
spanning the R. pomonella, R. suavis, and R. cingulata groups,
as well as the taxa R. berberis (Ribicola group), R. basiola
(Alternata group), and outgroup species Zonosemata electa in
the sister genus Zonosemata (Smith & Bush, 1997). These
species provide comparisons across a wide range of morphological divergence from slight (the cherry flies R. cingulata and
R. indifferens within the R. cingulata group) to pronounced
(between different species groups and the sister genus Zonosemata) (Bush, 1966; Foote et al., 1993; Fig. 1). Thus, if sexual
isolation exists among Rhagoletis flies, it would be expected
to be revealed in the current study.
Methods
Rhagoletis biology
Flies in the genus Rhagoletis are host-specific frugivorous
insects that share several biological features in common (Foote
et al., 1993). Rhagoletis are univoltine, having one generation
per year. Flies overwinter as pupae and eclose in the summer
at times matching when fruit on their respective host plants
ripen (Bush, 1966). Because adult longevity is limited in the
field to ∼28 days, differences in adult eclosion times among
fly taxa result in partial allochronic mating isolation (Bush,
1966). Sexually mature flies use olfactory, visual, and tactile
cues to locate host fruit, which they use as rendezvous sites
for mating and oviposition (Prokopy et al., 1971; Prokopy &
Bush, 1972; Linn et al., 2003). Differences in host preference
therefore also generate a degree of prezygotic mating isolation
among fly taxa (Feder et al., 1994; Linn et al., 2003). Females
oviposit eggs into unabscised fruits. Eggs hatch within 48 h
and larvae undergo three instar stages as they feed internally
within host fruit for 2–5 weeks. When fruit abscise and fall to
the ground, larvae finish feeding, emerge from host fruit, and
burrow from 2 to 5 cm into the soil, where they pupate and
overwinter.
Specimen sampling
Larval-infested fruit were collected in the field from May
to October in 2010 (see Table 2 for list of collecting sites)
and transported back to laboratory at the University of Notre
Dame where they were reared to adulthood in a greenhouse
using standard Rhagoletis husbandry techniques (Filchak et al.,
2000). In the laboratory, fruit were held in wire racks above
plastic collecting trays. Flies were collected as they finished
feeding, emerged from fruit, and formed puparia in the trays.
Following collection, fly puparia were placed in petri dishes
containing moist vermiculite and held in an incubator at 22 ◦ C
for 10 days. After this 10 day prewinter period, the petri dishes
were placed in a refrigerator at 4 ◦ C for 16–18 weeks to
simulate winter. The petri dishes were then removed from the
cold and held at 22 ◦ C until adult eclosion. Upon eclosion,
flies were isolated by sex to obtain virgin males and females
for the sexual isolation mating assays. Male and female flies
were provided with food (a mixture of honey, brown sugar, and
brewers yeast) and water until they reached sexually maturity at
7 days post-eclosion (Prokopy & Bush, 1972). Sexually naïve
flies were used in sexual isolation assays and ranged from 7
to 40 days old.
Assays for sexual isolation
Reciprocal choice mating assays were performed to test for
sexual isolation for nine different pairwise combinations of
flies (see Fig. 1; Table 3), using the methodology described for
Rhagoletis in Smith (1988b), Schwarz and McPheron (2007)
and Yee and Goughnour (2011). For every pairwise assay,
5 males and 5 females of each fly species (20 flies total)
were placed in a 15 cm3 plexiglass mating cage that contained
several small plastic leaves, food, and water. Mating cages
were housed in a greenhouse at the University of Notre Dame
under natural light conditions at 28 ± 2 ◦ C. All experiments
were conducted between the 6th and 12th hour of the 16h light phase of the flies’ photoperiod, coinciding with the
timing of peak Rhagoletis mating activity (Prokopy & Bush,
1972; Schwarz & McPheron, 2007). For each mating trial, we
first placed female flies into the cages and allowed them to
acclimate for 5 min before adding males. In two instances
(R. pomonella × R. basiola and R. indifferens × R. basiola)
only females of the latter species were available for the
mating trials and, thus, sexual isolation was estimated from a
unidirectional cross with no choice between males. Each cage
was set up in non-consecutive days over a 50-day period from
9 March to 29 April 2011 and flies removed from each cage
at the end of the same day.
Flies in each mating cage were observed for copulation every
5 min. Copulations for Rhagoletis have been documented to
generally last ≥ 20 min (Smith & Prokopy, 1982; Schwarz
& McPheron, 2007). Our design therefore made it likely
that all copulations in a mating trial were observed. Once
copulation was initiated, it was only counted as a successful
mating event if the pair remained coupled for a period of
at least 5 min. Unsuccessful mating attempts (when a male
aggressively forced copulation but was soon thereafter rejected
by the female and repulsed) were also recorded. Successful
mating pairs were removed from each cage and replaced with
a virgin male and female of the appropriate species to ensure
equal sex ratios of taxa during the trials. Mated individuals
were not reused in later trials.
© 2012 The Royal Entomological Society, Ecological Entomology, 37, 521–528
Sexual isolation in Rhagoletis
525
Table 2. Host associations and sampling sites for Rhagoletis taxa used in the sexual isolation assays.
Host plant
Rhagoletis species
Scientific name
Common name
Sampling location
Latitude/longitude
R. pomonella
R. pomonella
R. indifferens
R. cingulata
R. suavis
R. berberis
R. basiola
Z. electa
Malus domesticus
Crataegus douglasii
Prunus avium
Prunus serotina
Juglans nigra
Mahonia spp.
Rosa spp.
Solanum carolinense
Apple
Black hawthorn
Sour cherry
Black cherry
Black walnut
Oregon grape
Rosehip
Nightshade
South Bend, IN
Vancouver, WA
Vancouver, WA
Fennville, MI
South Bend, IN
Devine, OR
Fargher Lake, WA
South Bend, IN
41◦ 36 0 N/86◦ 9 35.9 W
45◦ 37 48 N/122◦ 36 35.9 W
45◦ 38 59.9 N/122◦ 42 0 W
42◦ 36 0 N/86◦ 9 0 W
41◦ 41 23.9 N/86◦ 15 36 W
45◦ 38 24 N/122◦ 35 59.9 W
45◦ 54 23 N/122◦ 31 10 W
41◦ 44 24 N/86◦ 12 0 W
Table 3. Results for reciprocal sexual isolation choice assays between indicated pairs of Rhagoletis species.
Species 1
Species 2
Hours
observed
Sp.1♀ ×
Sp. 1♂
Sp.1♀ ×
Sp.2♂
Sp.2♀ ×
Sp.1♂
Sp.2♀ ×
Sp.2♂
IPSI ± SD
(cop. only)
R. indifferens
R. berberis
R. indifferens
R. indifferens
R. pomonella †
Z. electa
R. pomonella †
R. indifferens
R. indifferens
R. suavis
R. suavis
R. pomonella †
R. pomonella ‡
R. suavis
R. pomonella †
R. basiola §
R. basiola §
R. cingulata
18
16
28
18
18
20
17
17
18
15
12
12
9
17
7
18
23
11
0
0
0
0
0
0
0
0
8
0 (0)
0 (0)
0 (1)
0 (1)
0 (0)
0 (0)
n/a
n/a
7 (1)
3 (1)
0 (0)
9 (0)
11 (1)
2 (1)
12 (0)
n/a
n/a
15 (0)
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
0.27
(0)
(0)
(0)
(0)
(0)
(0)
(0)
(0)
(1)
(1)
(1)
(3)
(2)
(1)
(0)
(0)
(0)
(2)
±
±
±
±
±
±
±
±
±
0.01***
0.01***
0.01***
0.01***
0.01***
0.01***
0.01***
0.01***
0.16
IPSI ± SD
(cop. + att.)
0.91
0.35
0.91
0.83
0.90
1.0
1.0
1.0
0.22
±
±
±
±
±
±
±
±
±
0.11*
0.48
0.07***
0.12***
0.11*
0.01***
0.01***
0.01***
0.15
*P < 0.05; **P < 0.01; ***P < 0.001, as determined by non-parametric bootstrapping (100 000 reps.).
†
R. pomonella population from Malus domesticus (apple) from South Bend, IN.
‡
R. pomonella population from Crataegus douglasii (black hawthorn) from Vancouver, WA.
§
Index estimated from a unidirectional cross (only females present from species 2).
Given are the total number of observation hours for each mating assay, the number of copulations (and attempted copulations in parentheses) for
each of the four possible types of pairings in an assay, and the sexual isolation index (IPSI ) calculated based on successful copulations only (cop.
only) and successful copulations plus attempted copulations (cop. + att.).
Each fly species in a mating trial could be distinguishable
from its heterospecific by visually detectable differences in
body coloration and/or wing banding pattern (Fig. 1). This was
also true for the closely related sister species R. cingulata and
R. indifferens, which exhibit strong, but not fixed differences in
pigmentation on the apical wing band (R. indifferens = apical
wing band present, R. cingulata = apical wing spot and not
band present; Bush, 1966; Foote et al., 1993; Fig. 1). The
populations used for the present study were fixed for this wing
character, allowing for species diagnosis of these two cherry
fly taxa in the mating assay.
Statistical analysis of sexual isolation
We estimated the degree of sexual isolation for each pairwise
population comparison by using the standardised metric for
sexual isolation (IPSI ) devised by Rolan-Alvarez and Caballero
(2000) that ranged from −1 to 1 (−1 = complete disassortative mating; 0 = random mating, +1 = complete positive
assortative mating). We calculated IPSI using the program
JMATING 1.0.8 (Carvajal-Rodriguez & Rolan-Alvarez, 2006)
and tested the resulting value for statistical significance by
non-parametric bootstrapping (100 000 resampling runs). The
sexual isolation index IPSI was calculated in two different manners considering: (i) successful copulations only (cop. only),
and (ii) both successful and attempted but failed copulations
(cop. + att.), as measures of mating proficiency. We also compared the frequencies of attempted copulations across all mating trials between interspecific and intraspecific pairs of flies
using a binomial test.
Results
In the 170 h of observation performed for the nine different
pairwise mating assays, 191 successful copulations were
detected (Table 3). When considering only these successful
copulations, complete positive assortative mating (IPSI = 1.0)
was seen for eight of the nine heterospecific species pairs tested
(Table 3). The lone exception in which only partial sexual
isolation was observed (IPSI = 0.27) involved the closely
related sister species R. cingulata and R. indifferens. These two
flies both attack native and introduced cherries primarily in the
eastern and western U.S.A. respectively, and represented the
morphologically and phylogenetically most similar species pair
tested in the study (Bush, 1966; Foote et al., 1993; Fig. 1).
© 2012 The Royal Entomological Society, Ecological Entomology, 37, 521–528
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Glen R. Hood, Scott P. Egan and Jeffrey L. Feder
Seventeen attempted, but failed copulations were observed
in the study. Failed copulation attempts were detected in
the majority (six of nine, 66.7%) of the mating trails. As
predicted by the hypothesis of sexual isolation, a significantly
greater number of failed copulations involved interspecific
(n = 12) than intraspecific (n = 5) pairs of flies (P = 0.02;
one-tailed binomial test). Considering both successful and
failed copulations in estimates of sexual isolation did not
qualitatively alter the results and only substantially reduced
IPSI values below 1.0 for the mating assay between R. berberis
and R. suavis (Table 3). The reduced IPSI index in this
case was primarily due to the absence of intraspecific
mating for R. suavis in the trial (Table 3). Aside from
R. suavis, conspecific mating frequently occurred in the mating
trails, implying that experimental assay conditions were, in
general, appropriate for inducing mating behaviour in flies
(Schwarz & McPheron, 2007; Yee & Goughnour, 2011).
However, R. suavis flies displayed reduced intraspecific mating
propensity compared to the other taxa in all three mating assays
they were involved in (Table 3), although in contrast to the
trial with R. berberis, intraspecific copulations were observed
for R. suavis when they were paired with R. indifferens and
R. pomonella in mating cages. The result for R. suavis was
somewhat surprising, as in the absence of interspecific flies,
R. suavis flies readily mated with conspecifics in the laboratory
(G. Hood and S. Egan, pers. obs.). It would therefore appear
that the presence of interspecific flies in cages in some way
made R. suavis reticent to mating.
Discussion
Our study represents a first attempt to systematically document
the degree of sexual isolation between morphologically
divergent species pairs of Rhagoletis fruit flies. We report
finding that in the absence of host plant cues, eight of the
nine species pairs tested distributed across Rhagoletis and
the outgroup genus Zonosemata displayed complete sexual
isolation. The one exception was between the sister species
R. cingulata and R. indifferens, which are morphologically
similar and were the most closely related taxa tested in the
current study (Bush, 1966; Berlocher et al., 1993; Fig. 1).
Thus, it is clear that non-host-related sexual isolation could
represent a significant prezygotic barrier to gene flow if and
when morphologically divergent species of Rhagoletis flies
happened to encounter each other on the same host plant in
nature.
Previous results for flies within the R. pomonella sibling species complex have shown that sexual isolation
ranges from absent (R. pomonella apple race × R. pomonella
hawthorn race), to weak (R. pomonella × R. mendax ) to moderate (R. pomonella × R. cornivora) (Smith & Prokopy, 1982;
Smith, 1988b; Schwarz & McPheron, 2007; Rull et al., 2010;
Yee & Goughnour, 2011). Placed within a phylogenetic context (Berlocher et al., 1993; Smith & Bush, 1997), our present
findings combined with these earlier studies demonstrate a general pattern in which host-related pre- and postzygotic barriers
appear to evolve first, at least in cases of ecological speciationwith-gene-flow, followed later by sexual isolation and intrinsic
genomic incompatibilities, as flies evolve to become more
morphologically differentiated. Thus, differences in host plant
ecology (i.e. variation in fruit volatiles, size, texture, and shape
that affect host choice and differences in host fruiting time
that affect diapause life-history timing and the seasonal distribution of flies) contribute most significantly to reproductive isolation between closely related taxa early in the divergence process. However, through time, other non-host barriers
become important as well, as taxa increasingly evolve independently. Exceptions to this pattern may be the walnut flies in the
R. suavis group and possibly R. cingulata and R. indifferens.
Here, divergent sexual selection in allopatric populations may
play a more immediate role earlier in speciation, as these taxa
all attack similar host plants. Further work is needed on these
two Rhagoletis groups to fully resolve the importance of sexual
isolation for speciation for walnut and cherry flies. Regardless
of whether sexual isolation was involved in the speciation process itself, however, it is apparent that it currently can serve as
an effective prezygotic barrier between many extant and morphologically divergent species of Rhagoletis that occasionally
visit each other’s host plants.
Our current results also highlight that there are two
components of sexual isolation at work in Rhagoletis. The first
component involves the initiation of mating and the second
concerns whether copulation, once initiated, is successfully
completed. In our study, failed heterospecific matings always
involved a quick copulation attempt by the male, followed by
a short struggle initiated by the female, after which the female
escaped. Thus, species-specific recognition cues such as wing
banding patterns, body coloration, and cuticular hydrocarbons
may be important for initiating mating, while other aspects of
fly morphology (e.g. incompatibility of genitalia and/or other
secondary sexual characteristics) may also contribute to sexual
isolation by curtailing heterospecific couplings.
The exact mechanism(s) underlying non-host-related sexual isolation in Rhagoletis remain to be determined. However, male Rhagoletis commonly exhibit species-specific
wing movements and displays during courtship (Bush, 1969;
Prokopy & Papaj, 2000). For example, Alonso-Pimentel et al.
(2000) showed that R. juglandis successfully mate only following male wing displays. It therefore seems reasonable to
assume that differences in male wing display behaviours and
wing banding patterns (Fig. 1) likely affect the propensity
of heterospecific mating. Similar considerations would likely
apply to body coloration, as well. In addition, it is conceivable that differences in cuticular hydrocarbons, both genetically controlled and environmentally induced, could influence
Rhagoletis mate choice. In this regard, the abundance and composition of cuticular hydrocarbons found in adult stages can be
affected by the fruit fed on by the larval stage (Etges & Jackson, 2001), which in turn can affect mate choice and generate
sexual isolation (Brazner & Etges, 1993). The same could
also be true for Rhagoletis. However, the low level of sexual
isolation observed among sympatrically diverging taxa such
as those in the R. pomonella group that attack alternate host
plants, argues against a general role for the larval feeding environment affecting cuticular hydrocarbons in a manner having
a large impact on sexual isolation.
© 2012 The Royal Entomological Society, Ecological Entomology, 37, 521–528
Sexual isolation in Rhagoletis
The exact context under which sexual selection has evolved
to currently separate Rhagoletis species also remains to
be resolved. For example, it is possible that the observed
sexual isolation among taxa is the result of divergent sexual
selection acting independently in allopatric or ecologically
isolated demes. In this regard, the sexual dimorphism of
many taxa in the R. suavis group would appear to support
a role for intraspecific sexual selection in inadvertently
generating interspecific sexual isolation as a correlated byproduct. It is still possible, however, that genetic drift or
natural selection rather than sexual selection was responsible
for generating morphological differences between Rhagoletis
species, especially for taxa showing no differences between
males and females. In these cases, sexual isolation would result
from flies being too diverged to be recognised as conspecific.
Finally, it is also possible for sexual isolation to have been
directly selected for by reinforcement in situations where
postzygotically isolated fly taxa co-occur on hosts to reduce
maladaptive hybridisation. Comparisons of walnut flies in the
R. suavis group in areas where taxa overlap in parapatry versus
where they are separated in allopatry could help clarify the
issue of reinforcement if flies in contact zones are found to
show much higher levels of sexual isolation. These studies
will also require more detailed complementary analysis of
postzygotic isolation to verify reinforcement acting in cases
where gene flow is still ongoing between Rhagoletis taxa, as
opposed to reproductive character displacement where isolation
is already complete.
Our results also have ramifications for agriculture as many
Rhagoletis species are economic pests of important fruit
crops, including apples, blueberries, cherries, and walnuts
(Bush, 1966). In this regard, hybridisation could pose a
potential threat to agriculture by generating new fly pests.
Hybridisation between the blueberry-infesting fly, R. mendax,
and snowberry infesting fly, R. zephyria, has recently generated
a new homoploid hybrid species of fly on a novel host plant,
Lonicera (twinberry) (Schwarz & McPheron, 2007; Schwarz
et al. 2005). Although the Lonicera fly is not a pest, it does
demonstrate how hybridisation could generate a new race of
Rhagoletis fly that could attack an economically important
crop. Our data suggest that if this does occur, it would likely
have to involve closely related sibling species, such as those
in the R. pomonella group, as sexual isolation would appear
to greatly diminish the potential for hybridisation between
morphologically divergent taxa embodied by the species pairs
tested here.
In conclusion, in the present study we document that
among morphologically distinct species of Rhagoletis sexual
isolation is a potentially effective prezygotic barrier restricting
hybridisation. For Rhagoletis flies, sexual isolation may
generally evolve after other barriers to gene flow separate taxa,
although more work is needed to investigate this issue for
certain species groups (e.g. walnut and cherry flies). There
are examples in other phytophagous insects, however, where
strong sexual isolation appears to have evolved quite early
in the divergence process (e.g. Neochlamisus bebbianae host
forms; Funk, 1998) and be an important initial barrier to gene
flow (e.g. Timema cristinae ecotypes; Nosil et al., 2002). More
527
generally, variation in the strength of sexual isolation has
been documented between several pairs of phylogenetically
diverse taxa (reviewed in Nosil et al., 2005). Thus, further
study is required to assess whether any general rule or pattern
characterises the strength, timing, and conditions under which
sexual isolation evolves and contributes to speciation for hostspecific phytophagous insects.
Acknowledgements
The authors would like to thank R. Goughnour and W. Yee for
providing flies from the western U. S. GRH would like to thank
P. M. Morton and SPE would like to thank J. L. Greene for
assistance. Funding was provided by grants awarded to GRH
by the Entomological Society of America, Sigma Xi and the
Indiana Academy of Science, to SPE from the University of
Notre Dame Faculty Initiation Grants, and to JLF by NSF and
the USDA.
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Accepted 3 September 2012
First published online 17 October 2012
© 2012 The Royal Entomological Society, Ecological Entomology, 37, 521–528