Molecular Ecology (2014) 23, 688–704
doi: 10.1111/mec.12635
Ecological adaptation and reproductive isolation in
sympatry: genetic and phenotypic evidence for native
host races of Rhagoletis pomonella
T H O M A S H . Q . P O W E L L , † A N D R E W A . F O R B E S , ‡ G L E N R . H O O D and J E F F R E Y L . F E D E R
Department of Biological Sciences, University of Notre Dame, Galvin Life Sciences Building, Notre Dame, IN 46556, USA
Abstract
Ecological speciation with gene flow may be an important mode of diversification for
phytophagous insects. The recent shift of Rhagoletis pomonella from its native host
downy hawthorn (Crataegus mollis) to introduced apple (Malus domestica) in the
northeastern United States is a classic example of sympatric host race formation. Here,
we test whether R. pomonella has similarly formed host races on four native Crataegus
species in the southern United States: western mayhaw (C. opaca), blueberry hawthorn
(C. brachyacantha), southern red hawthorn (C. mollis var. texana) and green hawthorn
(C. viridis). These four southern hosts differ from each other in their fruiting phenology and in the volatile compounds emitted from the surface of their fruits. These two
traits form the basis of ecological reproductive isolation between downy hawthorn and
apple flies in the north. We report evidence from microsatellite population surveys
and eclosion studies supporting the existence of genetically differentiated and partially
reproductively isolated host races of southern hawthorn flies. The results provide an
example of host shifting and ecological divergence involving native plants and imply
that speciation with gene flow may be commonly initiated in Rhagoletis when ecological opportunity presents itself.
Keywords: allochronic isolation, Crataegus, eclosion time, ecological divergence, speciation with
gene flow
Received 5 February 2013; revision received 4 December 2013; accepted 11 December 2013
Introduction
In the last few decades, there has been a growing
appreciation of the importance that ecology may play in
initiating speciation (Rundle & Nosil 2005). Traits
undergoing differential adaptation to ecologically dissimilar habitats could often serve as key initial barriers
to gene flow between nascent species. Such ecologically
based reproductive isolation may be particularly crucial
for speciation to occur in the face of gene flow. However, major theoretical constraints on this process are
thought to exist (Felsenstein 1981; Gavrilets 2003), and
Correspondence: Thomas H. Q. Powell, Fax: 352-392-0190;
E-mail: [email protected]
†Present address: Department of Entomology and Nematology,
University of Florida, Steinmetz Hall, Gainesville, FL 32611,
USA
‡Present address: Department of Biology, University of Iowa,
Biology Building, Iowa City, IA 52242, USA
many questions about ecological speciation with gene
flow remain open. For instance, how strong and what
types of divergent selection enable differentiation and
the formation of new ecological races and species. Even
more fundamental is the question of how common the
process is in nature; is it exceedingly rare that ecological opportunities arise such that divergent selection
results in speciation in the face of gene flow?
Several groups of organisms have contributed to our
understanding of the role of ecological adaptation in
population divergence including insects (e.g. Egan et al.
2008), fish (e.g. Rogers & Bernatchez 2007), birds (e.g.
Sorenson et al. 2003), plants (e.g. Schemske & Bradshaw
1999), and fungi (Fournier & Giraud 2008). One group
of particular significance is phytophagous insects due to
their great diversity and often close association between
feeding ecology and systems of mating. The host specificity of phytophagous insects has been argued to make
them generally more amenable than other organisms to
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N A T I V E H O S T R A C E S O F R H A G O L E T I S P O M O N E L L A 689
adaptive diversification (Bush 1993; Berlocher & Feder
2002). Indeed, speciation with gene flow was first proposed in regard to sympatric host shifting for phytophagous insects (Walsh 1864).
Our understanding of ecological speciation with gene
flow could benefit from additional study of three particular aspects of insect host shifting. First, several documented examples of host shifting and/or ecological
adaptation for phytophagous insects involve humanmediated introductions of either novel plants or insects
to new areas (e.g. Bush 1966; Carroll et al. 1997; Prowell
et al. 2004; Bourguet et al. 2014). Other putative cases
of host race formation or ecological speciation involve
native insects and hosts (e.g. Craig et al. 1993; Emelianov et al. 2001), but the original biogeographical context
of these shifts is often unknown. Thus, evidence demonstrating host shifting of endemic insect populations
between native plants that are directly complementary
to known cases of de novo divergence on introduced
hosts is important for evaluating the frequency that speciation with gene flow occurs ‘naturally’.
Second, many examples of host-associated differentiation in insects do not describe the evolutionary progression from races to sibling species, but just one stage in
the process. As a result, it can be difficult to ascertain
whether specific cases of ecologically diverged taxa will
eventually speciate. Instead, they may represent stalled
ecotypes at evolutionary equilibrium in which the
degree of ecological differentiation possible has run its
course (Nosil et al. 2009; Thibert-Plante & Hendry 2011).
Documentation of an evolutionary series spanning the
transition from races to species for a single taxonomic
group does not demonstrate that a given ecotype will
ever attain species status. However, it does imply that
divergent ecological selection pressures can be strong
enough in principle for gene flow to be reduced to levels for taxa to eventually constitute different species
given the appropriate genetic variation.
Third, it remains to be determined what ecological
dimensions of host plants are most important for generating reproductive isolation and initiating speciation.
Traits involved with (i) host plant recognition that influences oviposition and mate choice (e.g. Linn et al. 2004),
(ii) life history timing that synchronizes insect activity
with host plant availability (e.g. Horner et al. 1999), (iii)
adaptation to external conditions associated with host
plants (e.g. Nosil & Crespi 2006) and (iv) feeding adaptations specific to the nutritional and/or chemical characteristics of plants (e.g. Via 1991) are four likely factors
contributing to ecological specialization. However, it is
unclear whether any of these forms of host plant adaptation may be particularly critical for insect diversification, exerting the greatest divergent selection pressures
and strongest barriers to gene flow.
© 2013 John Wiley & Sons Ltd
Here, we address these three issues of speciation with
gene flow by testing for genetic differentiation among
populations of the tephritid fruit fly Rhagoletis pomonella
(Walsh) attacking different species of hawthorns (genus
Crataegus) in the southern United States. The apple
maggot fly, R. pomonella, is a member of a sibling species complex that is a model for ecological speciation
with gene flow (Funk et al. 2002). The complex is comprised of a number of taxa at varying stages of divergence from partially reproductively isolated host races,
to more fully isolated sibling species, to reciprocally
monophyletic species (Berlocher 2000; Xie et al. 2008).
An exceptional feature of the complex is that although
the populations and species that comprise the complex
have broadly overlapping geographical distributions
and close morphological similarities, their host affiliations are discrete. These considerations led Bush (1966)
to propose that the R. pomonella complex radiated via a
series of sympatric host shifts.
The most well-known example of sympatric host race
formation in the R. pomonella complex involves the
recent shift ~150 years ago of the species R. pomonella
from its native host, downy hawthorn, Crataegus mollis,
to introduced domesticated apple (Malus domestica) in
the northeastern United States. Previous work has demonstrated that apple and hawthorn flies are differentiated by two key traits: (i) olfactory behavioural
response differences to host fruit surface volatiles (Linn
et al. 2003) and (ii) differences in the timing of diapause
and adult eclosion, which correspond to difference in
fruiting phenology between the host plants (Feder et al.
1993). Divergent selection on these two traits has led to
partial extrinsic prezygotic and postzygotic isolation
between apple and downy hawthorn flies (Feder et al.
1994; Linn et al. 2004). Genetic differentiation between
apple and hawthorn flies is characterized by consistent
allele frequency differentiation at a subset of loci, superimposed on strong latitudinal effects on loci associated
with life history timing (Feder et al. 1988; Feder & Bush
1989; Michel et al. 2010). While the formation of the
apple race is one of the most well-characterized
instances of ecological speciation in sympatry (Funk
et al. 2002), it remains to be determined whether it represents a special case, made possible only by the unique
set of ecological conditions presented by the intercontinental introduction of apples.
Southern populations of R. pomonella attack a suite of
native hawthorn species, providing an opportunity to
address the issue of endemic host race formation
(Fig. 1; see Supporting Information for a full description
of these hawthorn species). In the north, where R. pomonella shifted to apple, the downy hawthorn is the
predominant native host for the fly. In addition,
feral and backyard domesticated apples suitable for
690 T . H . Q . P O W E L L E T A L .
A
Blueberry
Western mayhaw
hawthorn
C. opaca
C. brachyacantha
Southern red
hawthorn
C. mollis var.
texana
Green hawthorn
C. viridis
B
3-methylbutan-1-ol
44
0.6
0.4
5
Butyl acetate
–
50
9
–
DMNT
–
–
–
20.5
Butyl hexanoate
26
16.8
20
24
Fig. 1 (A) Ripe fruit of southern hawthorn infested by Rhagoletis pomonella. (B)
Percentages of key behaviourally active
compounds in fruit volatile blends (Cha
et al. 2011a,b, 2012; Powell et al. 2012).
See Table S1 (Supporting Information)
for complete volatile blends. (C) Host
fruiting times. Coloured areas indicate
range of fruiting times reported by Berlocher & Enquist (1993) and personal
observations from 2005 to 2010.
C
April
May
June
July
Aug.
Sep.
supporting R. pomonella thrive in the north. However,
this is not the case further south. Consequently, the
range of the apple race is limited to the northeast and
Midwest, with the exception of a small finger that
extends southward in higher elevations of Appalachia
(Bush 1966). But the distribution of hawthorn-infesting
R. pomonella extends well past that of the apple race,
into the southeastern United States (Fig. 2). Here, the
fly attacks several suitable native hawthorn hosts,
including the western mayhaw (Crataegus opaca Hook
and Arn.; ‘mayhaw’ hereafter), the blueberry hawthorn
(Crataegus brachyacantha Sarg. & Engelm), the green
hawthorn (Crataegus viridis L.) and the southern red
hawthorn (C. mollis var. texana Buckl.) (Bush 1966, 1969;
Berlocher & Enquist 1993). These southern hawthorns
vary from one another in characteristics such as fruiting
time (Fig. 1) and fruit volatiles (Table S1, Supporting
Information) that are involved in partially ecologically
isolating the apple and downy hawthorn fly races in
the north (Linn et al. 2003). The potential therefore
exists for ecological divergence among southern hawthorn flies. Consequently, documenting genetic differentiation among sympatric southern R. pomonella
populations would offer a natural historical example of
ecological race formation complementing the recent
shift to introduced apple in the north.
In regard to the second problem of taxonomic
analysis across the divergence continuum, the southern
hawthorn fly populations represent possible key transitionary stages between host races and more fully
formed sibling species. Southern hawthorn flies may
display varying levels of divergence representative of
the evolutionary transition from the recently formed
apple race to R. pomonella’s most closely related sister
taxon, the undescribed flowering dogwood fly that
attacks Cornus florida L.
Oct.
Nov.
Finally, the southern hawthorn-infesting populations
of R. pomonella provide a test of whether the two key
traits isolating the apple race are of general importance
for Rhagoletis flies. For apple flies in the north, olfactory
differences are important because R. pomonella flies
mate in trees on or near the fruit of their respective host
plants (Prokopy et al. 1971). Flies use the volatiles emitted off the surface of ripening fruit as the primary cues
for finding and discriminating among host plants (Roitberg & Prokopy 1984). Thus, differences in fruit volatile
preference result in differential habitat choice for flies,
directly translating into ecologically based prezygotic
isolation. Diapause-related life history differences are
important because R. pomonella is univoltine, having
one generation per year. In addition, adults live for only
4–6 weeks in the field (Boller & Prokopy 1976). As a
result, flies must eclose as adults at times matching the
phenology of their respective host plants to maximize
fruit availability for mating and oviposition. Apple varieties favoured by R. pomonella generally fruit 3–4 weeks
earlier than downy hawthorn (Bush 1969). Apple flies
consequently eclose earlier than downy hawthorn flies
in the field, generating allochronic mating isolation
(Feder et al. 1994). A fitness trade-off causing postmating isolation has also been found due to the interplay
between differences in the length of the prewinter period and the facultative nature of the pupal diapause
(Filchak et al. 2000).
The southern hawthorn hosts appear to differ along
the same two ecological axes in a manner analogous to
downy hawthorns and apples. The peak fruiting times
of southern hawthorns vary over a much greater time
period (total span of almost 8 months from late April to
early December) than the 3- to 4-week difference
between apple and downy hawthorn (Fig. 1; Table 1)
(Bush 1969; Berlocher & Enquist 1993). A previous
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Fig. 2 Range map and collection sites for
Rhagoletis pomonella in this study. Numbers refer to collecting sites (see Table 1
for designations).
study by Lyons-Sobaski & Berlocher (2009) demonstrated flies reared from different southern hawthorn
hosts differed in their eclosion timing, even when
reared through a laboratory generation on apple. While
this study did not address differentiation at sympatric
sites, it did provide strong support for the potential of
allochronic isolation among these populations. All four
southern hawthorn hosts have also been found to differ
in the composition of their fruit volatile profiles as
detected by R. pomonella antennae (Table S1, Supporting
Information) (Cha et al. 2011a,b, 2012). Moreover, flight
tunnel testing has shown that southern hawthorn flies
have strong preferences for their natal host volatiles
and may actively avoid the volatile blends of non-natal
hawthorn species (Powell et al. 2012). These differences
in olfactory behavioural response showed strong consistency within host associations across the region as well
as strong behavioural differentiation between different
host associations at sympatric sites. Both lines of evidence point towards the potential for divergent host
plant adaptation in this system to result directly in prezygotic isolation.
Here, we test for evidence of microsatellite genetic
differentiation among southern hawthorn flies through
parts of western Mississippi, Louisiana and eastern
Texas where the ranges of the four southern hawthorn
hosts of R. pomonella overlap in varying combinations
(Fig. 2). Assessing host-associated differentiation in this
system requires both local and regional analyses of
genetic variation to determine whether (i) flies infesting
alternative hosts at sympatric sites represent panmictic
populations and (ii) genetic variation is random with
respect to host plant across space. We also examine the
extent to which differences in diapause and host fruit
odour discrimination are associated with genetic
© 2013 John Wiley & Sons Ltd
differentiation to assess the relative importance of these
two traits for ecologically isolating southern hawthorn
flies.
Materials and methods
Sampling of flies
Flies were collected in the field as larvae in infested
blueberry hawthorn, green hawthorn, southern red
hawthorn and mayhaw fruit from a total of 16 different
sites in Texas, Louisiana and Mississippi from 2006 to
2010 (Fig. 2; Table 1). Infested fruit were transported to
the laboratory where larvae were reared to adulthood
using standard Rhagoletis husbandry techniques (Neilson & Mcallan 1965). Upon eclosion as adults, flies were
frozen at 80 °C.
mtDNA
To quantify matrilineal divergence among southern
hawthorn-infesting Rhagoletis pomonella, a maximum
parsimony mtDNA gene tree was constructed using
PHYLIP 3.69 (Felsenstein 2005) based on a 864-bp fragment covering a 3′ partial sequence of the cytochrome
oxidase subunit I gene (COI), the entire tRNA-Leu gene
and a 5′ partial sequence of the cytochrome oxidase
subunit II (COII) gene. Newly generated sequence data
from southern flies and two downy hawthorn flies from
the southern edge of its range were combined with previously published R. pomonella and outgroup data
(Feder et al. 2003) for this analysis (see Appendix S1,
Supporting Information for primer and analysis details
and Table S2, Supporting Information for sample details
and accession nos).
692 T . H . Q . P O W E L L E T A L .
Table 1 Host plant origin, location [latitude (N) and longitude (W) in degrees], year sampled and number of flies genotyped (n)
Host plant origin
No.
Location
Lat.
Long.
Year
n
Green hawthorn (Crataegus viridis)
1
2
3
4
5
6
7
8
4
9
10
3
9
11
1
2
Brazos Bend SP, Fort Bend Co., TX
Palmetto SP, Gonzalez Co., TX
L. Sam Rayburn, Angelina Co., TX
Dewey Wills WMA, La Salle Pr., LA
Fort Necessity, Caldwell Pr., LA
Rolling Fork, Sharkey Co., MS
HW Jackson Farm, Polk Co., TX
LSU Idlewild, E. Feliciana Pr, LA
Dewey Wills WMA, La Salle Pr., LA
SFA Exp. Forest, Nacogdoches Co., TX
Morris Ferris Park, Angelina Co., TX
L. Same Rayburn, Nacogdoches Co., TX
SFA Exp. Forest, Nacogdoches Co., TX
Hungerford, Wharton Co., TX
Brazos Bend Sp, Fort Bend Co., TX
Palmetto SP, Gonzalez Co., TX
29.22
29.35
31.24
31.27
32.04
32.51
30.52
30.49
31.27
31.31
31.21
31.30
31.31
29.22
29.22
29.35
95.36
97.35
94.31
92.07
91.55
90.47
94.7
90.57
92.07
94.46
94.45
94.22
94.46
96.05
95.36
97.35
2007
2009
2010
2009
2007
2008
2006
2007
2009
2006
2010
2007
2008
2010
2007
2010
98
56
30
50
48
55
94
34
40
94
48
36
47
46
35
87
Mayhaw (Crataegus opaca)
Blueberry hawthorn (Crataegus brachyacantha)
S. red hawthorn (Crataegus mollis v. texana)
LA, Louisiana; MS, Mississippi; TX, Texas.
Numbers correspond to site designations in figures.
Microsatellites
A total of 900 southern hawthorn flies (Table 1; Fig. 2)
were genotyped for 26 microsatellite loci developed by
Velez et al. (2006) for R. pomonella from Grant, MI.
These 26 microsatellites map to five of the six chromosomes comprising the R. pomonella genome (the small
dot sixth chromosome currently lacking any marker)
(Table S3, Supporting Information). Laboratory methods
follow Michel et al. (2010). See the Supporting Information methods for further details concerning loci and
genotyping methods.
Statistical analyses of microsatellite differentiation
To simplify the microsatellite analysis, we collapsed
variation present at each locus down to two major allele
classes, as previously described in Michel et al. (2010).
Past work on R. pomonella has indicated that major
allele classes exist for nuclear genes defined by patterns
of linkage disequilibrium among loci and latitudinal
geographical variation. These allele classes are the result
of three inter-related factors: (i) a biogeographical
history characterized by periodic episodes of isolation
and secondary contact between hawthorn-infesting populations in the United States and Mexico over the past
~1.5 My, (ii) inversion polymorphisms which appear to
have arisen under this biogeographical scenario and
(iii) adaptive latitudinal clines for diapause life history
traits that also likely established through past introgression from Mexican hawthorn fly populations
(Feder et al. 2005; Xie et al. 2008). Thus, not only do
R. pomonella display local host-related genetic differ-
ences in the timing of diapause but also geographical
variation due to latitudinal differences in fruiting time
among fly populations within a given host species
(Dambroski & Feder 2007). Highly polymorphic markers erode the signal of these important underlying features of the R. pomonella system. The allele pooling
method used here and elsewhere (Michel et al. 2010;
Powell et al. 2013) seeks to maximize the signal of the
strong clinal variation and linkage disequilibrium
known in this system. It is important to note that this
method is blind to host plant and does not maximize
host-associated differentiation. One important benefit of
this approach is that it allows the results from this
study to be compared to the patterns reported in classic
and recent Rhagoletis work that modelled allele
frequency differentiation between hosts across latitude
(Feder & Bush 1989; Berlocher & McPheron 1996;
Michel et al. 2010; Powell et al. 2013).
Major microsatellite allele classes were identified by
testing up to one million random combinations of
alleles at a locus to determine the combination of variants that maximized latitudinal variation and linkage
disequilibrium to flanking markers in the genome. Latitudinal variation was calculated as the variance
explained by the linear regression of allele frequencies
on latitude (R2) times the absolute value of the slope (b)
of the regression within each host association. The standard composite linkage disequilibrium coefficient (D) of
Weir (1979) was used to quantify nonrandom associations of alleles between the most closely linked pairs of
loci within populations. The overall metric assessed for
each combination of alleles was the product of the total
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D 9 R2 9 b. Statistical significance was determined by
nonparametric Monte Carlo simulations assessing the
proportion of 100 000 simulated runs with a higher
D 9 R2 9 b value than the actual estimate. Resulting
allele pools are presented in Table S4, Supporting Information. Pooled allele data were subsequently used in
the analyses based on generalized linear model (GLM)
and Nei’s D described below.
Individual microsatellite loci were analysed for hostassociated and latitudinal variation in a GLM framework in R 2.13.1 (R development core, Vienna, Austria
2011). Allele frequency was modelled as a quasibinomial variable with host association as a discrete factor and latitude as a continuous factor. This analysis
was performed separately for each of the four pairwise
comparisons of southern hawthorn populations that
overlap in their geographical ranges: (i) mayhaw vs.
green hawthorn, (ii) mayhaw vs. blueberry hawthorn,
(iii) green hawthorn vs. blueberry hawthorn and (iv)
green hawthorn vs. southern red hawthorn. Significance
of factors, host, latitude and host 9 latitude interaction
was determined by F-tests.
To examine overall patterns of microsatellite relatedness among fly populations, neighbour-joining networks
were constructed based on Nei’s D pairwise distance
measures (Nei 1972) using the program PowerMarker
(Liu & Muse 2005), with 10 000 bootstrap replicates
across loci. Networks were constructed for all host populations of R. pomonella considered together, as well as
for each of the four paired comparisons of geographically overlapping hosts listed above. For comparative
purposes, the network containing all host populations
also included two populations of the flowering dogwood fly from the Stephen F. Austin Experimental Forest, TX, and Kisatchie National Forest, LA, and two
populations of the northern downy hawthorn race of
R. pomonella from Urbana, IL, and Dowagiac, MI (data
from Powell et al. 2013).
To further examine how host plant adaptation affects
the genetic differentiation between populations, we conducted analyses on five ‘local’ field sites where flies
infested alternate southern hawthorn species in either
sympatry or close proximity: (i) SFA Experimental
Forest, TX (mayhaw and blueberry hawthorn; 700 m
apart); (ii) Dewey Wills WMA, LA (mayhaw and green
hawthorn; 1 m apart); (iii) Brazos Bend SP, TX (green
hawthorn and southern red hawthorn; 3 m apart); (iv)
Palmetto SP, TX (green hawthorn and southern red
hawthorn; 40 m apart); and (v) Lake Sam Rayburn, TX
(blueberry hawthorn and green hawthorn, 15 km apart).
Tests for local site differentiation were conducted using
unpooled allele frequencies. Significance of individual
microsatellite allele frequency differences between
hosts at local sites was determined by exact G-tests
© 2013 John Wiley & Sons Ltd
implemented in Genepop (Raymond & Rousset 1995a).
FST values between local fly populations were calculated in Genepop (Raymond & Rousset 1995b). Patterns
of individual-level clustering among host-associated
populations were also examined using the program
STRUCTURE 2.3.4 (Pritchard et al. 2000) (see the Appendix
S1, Supporting Information for details).
Eclosion time
To compare eclosion phenotypes among local populations, pupae were reared to adulthood under constant
26 °C; 14:10 L:D laboratory conditions. The considerable
phenological spread among alternative host plants coupled with sampling limitations made it impractical to
rear flies under a range of conditions mimicking nature.
Therefore, these eclosion data may not reflect temporal
overlap between populations. However, they do represent the difference in phenotypic response to a standard
set of environmental conditions. Eclosion curve differences between paired local populations were assessed
by Kolmogorov–Smirnov (KS) tests in R.
Genetic analysis of fruit volatile discrimination and
eclosion time
We also assessed the degree to which variation in fruit
volatile behavioural response and eclosion time was
associated with overall microsatellite divergence among
southern hawthorn flies at local sites, as measured by
the fixation index (FST). Our prediction was that if
either trait restricts gene flow between populations,
then it should show a pattern of ‘isolation by adaptation’ (Funk et al. 2006; Nosil et al. 2008) in which the
greater the difference in phenotype between flies, the
greater the overall FST value should be for genetic
markers. Data for fruit volatile response came from
Powell et al. (2012). Phenotypic differentiation in fruit
volatile response was calculated as 1Bxy, a measure of
behavioural overlap in the responses of a pair of local
host-associated populations x and y to each other’s fruit
blends according to the formula:
Bxy ¼
px þ py
2
where px is the proportion of responding flies from host
plant x which displayed upwind-directed flight to the
alternative blend y and py is the proportion of flies from
host plant y that responded to the alternative blend x
(Powell et al. 2012). Our metric for diapause-related differentiation was the difference in median eclosion time
for pupae reared under the conditions described above.
Eclosion data were lacking for the Sam Rayburn Lake
blueberry hawthorn population, so a composite eclosion
694 T . H . Q . P O W E L L E T A L .
curve of two other blueberry hawthorn populations at
similar latitude (Nacogdoches and Lufkin, TX) was
used in its place. FST values were log-transformed to
achieve normality (Shapiro–Wilk test; W = 0.9805,
P = 0.9375). Linear regression analysis of FST values as
a function of behavioural overlap and median eclosion
time was performed in R.
Results
Mitochondrial sequence variation
PHYLIP produced a single maximum parsimony mtDNA
gene tree displaying no homoplasy (Fig. 3). There was
no evidence of host-associated differentiation for
mtDNA; no synapomorphy existed that defined any
southern hawthorn fly population as a distinct matrilineage. This rules out any of the southern populations of
Rhagoletis pomonella representing long-established cryptic species. The southern hawthorn-infesting populations also did not form a distinct clade relative to
Fig. 3 Maximum parsimony gene tree for mtDNA. Red triangles = southern red hawthorn; green squares = green hawthorn;
blue circles = blueberry hawthorn; pink diamonds = mayhaw;
black inverted triangles = northern downy hawthorn. Two outgroup taxa from the Mexican Altiplano highlands and Rhagoletis
electromorpha from Michigan are included to root the network.
Numbers following terminal nodes indicate collecting sites for
haplotypes (see Table 1 for designations). Numbers in italics
indicate multiple individuals sequenced from a single collecting
site.
northern downy hawthorn flies. However, there may be
frequency differences in mitochondrial haplotypes
between these two regions. The two northernmost
downy hawthorn sequences shared a common mutation
with sequences from only a single southern hawthorn
fly from blueberry hawthorn in Texas (Fig. 3). Furthermore, the sequences from New Madrid, MO, near the
southern range limit of downy hawthorn, shared a common mutation with 55% of the 20 southern hawthorn
flies sequenced. The results suggest possible haplotype
frequency differences between southern and northern
hawthorn fly populations, requiring further sampling to
quantify.
Microsatellite differentiation
No fixed or uniquely private allele was found for any
of the 26 microsatellite loci scored in the study distinguishing R. pomonella flies infesting different hawthorn
host species. (Table S5, Supporting Information). Fly
populations shared all alleles in common except for singleton alleles (Table S5, Supporting Information). Differentiation among host-associated populations was
therefore due entirely to allele frequency divergence,
implying that gene flow is either ongoing or only very
recent ceased among flies.
Inbreeding coefficient (f) did not vary significantly
among host-associated populations (Table S6, Supporting Information; F = 2.75; P = 0.09; d.f. = 3). There was
considerable variation in f among loci within populations, however, such that in all cases the standard deviation around overall mean f values overlapped with
zero (Table S6, Supporting Information). The lack of
evidence for significant inbreeding implies that host
effects, rather than within-population demography or
biased sampling (i.e. unrepresentative scoring of a subset of related individuals or families), were the primary
drivers of the observed pattern of genetic differentiation
discussed below.
Generalized linear model analysis conducted between
the four pairs of geographically overlapping southern
hawthorn flies (mayhaw vs. green hawthorn, mayhaw
vs. blueberry hawthorn, green hawthorn vs. blueberry
hawthorn and green hawthorn vs. southern red hawthorn) revealed a number of significant host plant and
latitude effects on microsatellite frequencies (Table 2).
Overall, six of the 26 microsatellites located on four different chromosomes showed a significant host-related
differentiation (either a significant main effect or
interaction term with latitude) across all four southern
hawthorn pairwise comparisons (P3 [chr. 1]; P46 and
P73 [chr. 2]; P11 and P25 [chr. 4]; P18 [chr. 5]). Similarly, eight microsatellites located on four different
chromosomes showed a significant latitude-related
© 2013 John Wiley & Sons Ltd
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Table 2 Microsatellites showing significant host, latitude and host 9 latitude interaction effects in GLM analyses for four pairwise
host comparisons
Factor
LG
MH vs. GH
MH vs. BB
GH vs. BB
GH vs. SRH
Host
1
2
3
4
5
1
2
3
4
5
1
2
3
4
5
P3; P4
P46; P70
P23; P80
P11
P18
P3; P4
P46; P70; P73; P17; P54
P16; P23; P66; SR40
P25; P50
P9; P18; P5
P3; P4
P46; P73
P80
P11; P25; P50
P3; P39
P46; P70; P73
P23; SR40
P11; P25; P29
P18
P3; P4; P39
P46; P73
SR40
P11; P25; P29; P50
P3
P46; P73
P3; P4; P71
P17; P54
P23; P66; P80
P11; P25
P5
P3; P4
P46; P70; P73; P17; P54
P16; P23; P66; SR40
P11; P25; P50
P9; P18
P3; P4
P46; P73; P54
P66
P11; P25; P50
P9; P18; P5
Latitude
Interaction
P3
P46; P73
P11; P25
P18
P3; P4
P46; P70; P73; P54
P16; P23; P66P SR40
P11; P25; P50
P18
P3
P73
P60
P5
LG, linkage group for loci; MH, mayhaw; GH, green hawthorn; BB, blueberry hawthorn; SRH, southern red hawthorn; GLM, generalized linear model.
See Tables S7–S11 (Supporting Information) for full results.
differentiation across all four paired comparisons (P3
and P4 [chr. 1]; P46 and P73 [chr. 2]; P40 [chr. 3]; P11,
P25 and P50 [chr. 4]). In addition to these globally significant loci, many other microsatellites displayed host
and/or latitudinal effects for a subset of the paired host
comparisons (Table 2).
The results from the GLM analysis implied that not
only do host and latitude have major effects, but that
they also interact in a complex manner to shape the pattern of differentiation among southern hawthorn flies.
This was evident in (i) five of the globally significant
loci for all four southern hawthorn fly comparisons (P3,
P46, P73, P11 and P25) displaying both host and
latitude-related divergence and (ii) many loci showing
host x latitude interaction effects (Table 2). The complex
interaction between host and geography was underscored in the neighbour-joining network for all the collecting sites scored in the study (Fig. 4) that depicted
host-related differentiation overlaid upon a pattern of
clinal geographical variation across populations. Green
hawthorn flies had the widest distribution and displayed the greatest degree of geographical genetic differentiation, which was manifested in green hawthorn
sites being arrayed in a general north-to-south pattern
from top to bottom in the network (Fig. 4). A similar
pattern was evident for the other hawthorn flies, as
well, most notably among the southern red hawthorn
populations. This geographical variation complicated
interpretation of host-related effects because it resulted
in several populations of different hawthorn-infesting
flies intersecting with each other in the network (e.g.
green hawthorn with southern red hawthorn and
© 2013 John Wiley & Sons Ltd
mayhaw flies). The pattern was not just an artefact of
the network being based on overall genetic distance; as
discussed above, many individual microsatellite loci
showed not just latitudinal effects in the GLM analysis,
but also host x latitude interactions that generated
crossing patterns in allele frequencies for loci between
different hawthorn flies (Table 2).
Although the overall pattern of genetic differentiation
was complex and affected by geography, a signature of
host-related divergence was nevertheless evident in the
neighbour-joining network (Fig. 4). For example, the
three blueberry hawthorn fly populations formed a discrete cluster relative to the other flies. However, the
bootstrap support was lower (58%) and the branch
length between this cluster and the cluster to other
southern hawthorn populations was smaller (0.0021)
than those distinguishing R. pomonella from its undescribed sister species, the flowering dogwood fly (98%
and 0.0147, respectively). The four mayhaw fly populations also grouped closely together in the network, but
did not form a discrete cluster due to the inclusion of
the green hawthorn site from Dewey Wills, LA. Moreover, the two paired groupings of mayhaw populations
are clustered by latitudinal similarity rather than geographical distance. This pattern of differentiation is similar to those resolved for the apple and downy
hawthorn host races of R. pomonella in the northeastern
United States, where apple and hawthorn flies show
significant microsatellite allele frequency differences at
local sympatric sites, but populations of the two races
do not form discrete genetic clusters globally across
their respective ranges due to pronounced latitudinal
696 T . H . Q . P O W E L L E T A L .
A
C
Fig. 4 Neighbour-joining network based on genetic distance for
all 26 microsatellites. Green squares = green hawthorn; blue
circles = blueberry hawthorn; pink diamonds = mayhaw; red
triangles = southern red hawthorn; black inverted triangles = northern downy hawthorn; brown pentagons = flowering dogwood fly (brown pentagons). Numbers in symbols
indicated collecting site (see Table 1 for designations). Populations sharing a number in common represent local sympatric
sites. Green hawthorn sites are vertically oriented based on latitude from top (north) to bottom (south). Numbers on branches
indicate per cent bootstrap support for 10 000 replicates across
loci.
clines within the races (Michel et al. 2010; Powell et al.
2013).
To further clarify the extent of southern host-related
differentiation, (i) separate neighbour-joining networks
were constructed for each of the four pairwise host
plant comparisons based on loci showing host or
host 9 latitude interactions (Fig. 5) and (ii) FST and allelic differentiation were tested for significance between
populations of flies at local sites infesting alternate hawthorns. The results for the neighbour-joining networks
indicated that flies infesting different hawthorns form
discrete clusters when considered in the context of
pairwise host comparisons based on differentiation at a
subset of microsatellite loci (Fig. 5). Thus, while geographically distant populations of mayhaw, green hawthorn and southern red hawthorn do not group across
all loci when taken together, they can be distinguished
by subsets of markers when pairs of taxa are analysed
separately.
B
D
Fig. 5 Neighbour-joining networks based on microsatellites
displaying a significant host-related differentiation in generalized linear model analysis (Table 2) for paired population comparisons: (A) mayhaw vs. blueberry hawthorn, (B) mayhaw vs.
green hawthorn, (C) green hawthorn vs. blueberry hawthorn
and (D) southern red hawthorn vs. green hawthorn. Numbers
in symbols designate collecting sites (Table 1) with shared
numbers representing local sympatric populations. Numbers
on branches indicate per cent bootstrap support for 10 000 replicates across loci.
Analysis of local collecting sites provided further evidence for host-related differentiation, but also revealed
that levels of divergence varied among different hawthorn fly pairs. Specifically, 17 of the total of 26 microsatellites scored displayed significant allele frequency
differences between mayhaw and blueberry hawthorn
flies at the Stephen F. Austin Experimental Forest, TX
site; ten loci between green and southern red hawthorns
at the Brazos Bend State Park, TX site; 15 loci between
green and southern red hawthorn flies at the Palmetto
State Park, TX site; and all 26 between green hawthorn
and blueberry hawthorn flies at the Lake Sam Rayburn,
TX site (Table 3). In contrast, for the mayhaw/green
hawthorn fly comparison at Dewey Wills, LA, only a
single locus, P46, showed a significant differentiation
(Table 3). FST values reflected the number of loci differentiating flies at sites (linear regression; r2 = 0.9848,
P = 0.0008, d.f. = 4.), varying from 0.075 for the green
hawthorn/blueberry hawthorn comparison to 0.0004
between mayhaw and green hawthorn flies.
STRUCTURE analysis did not identify discrete clusters of
individual flies attacking different southern hawthorn
hosts (see Table S12 and Appendix S1, Supporting
Information for details). The apple and downy hawthorn host races of R. pomonella in the northeastern United States also similarly do not form discrete clusters in
STRUCTURE analysis (Powell et al. 2013). Host-associated
© 2013 John Wiley & Sons Ltd
N A T I V E H O S T R A C E S O F R H A G O L E T I S P O M O N E L L A 697
Table 3 Exact G-test for microsatellite allele frequency differences between host-associated populations at the five local paired sites.
Letter designations for hosts as in Table 2 and site designations as in Table 1
Chr.
Locus
1
P3
P4
P37
P71
P75
P39
P17
P46
P54
P70
P73
P7
P16
P23
P66
P80
P40
P11
P25
P29
P50
P60
P5
P9
P18
P27
2
3
4
5
MH vs. GH
(4)
GH vs. SRH
(1)
GH vs. SRH
(2)
*
**
****
****
*
*
*
****
***
****
*
**
**
**
*
*
*
*
*
**
**
****
***
*
**
*
MH vs. BB
(9)
*
**
*
**
*
**
***
****
****
****
***
***
*
*
****
*
*
*
**
*
GH vs. BB
(3)
****
****
****
**
**
****
***
****
****
****
****
****
**
****
****
****
****
****
****
****
****
****
**
*
***
****
*0.05 > P ≥ 0.01, **0.01 > P ≥ 0.001, ***0.001 > P ≥ 0.0001, ****0.0001 > P.
populations in sympatry do not appear to form discrete
clusters of individuals in Rhagoletis until more diverged
populations of flies are compared representative of sibling species (Powell et al. 2013).
Diapause differences among hawthorn fly populations
Laboratory eclosion curves were significantly different
between flies infesting alternative hawthorn hosts for
all five paired site comparisons (Fig. 6; KS tests;
P ≤ 0.00016 in all cases), indicating that R. pomonella
populations infesting different hosts displayed different life history phenotypes under the same set of
environmental stimuli. Understanding how these differences relate to phenological overlap in nature is an
important question requiring additional study. Interestingly, the magnitude of phenotypic differentiation in
eclosion timing was not directly a function of the phenological difference between host plants (Fig. 6). The
comparison with the greatest phenological difference
in host plants, mayhaw vs. green hawthorn (170 days
between collection dates), had the most similar eclosion curves.
© 2013 John Wiley & Sons Ltd
Phenotypic associations with genetic differentiation
Both olfactory behavioural response and eclosion timing
have been shown to significantly differ among southern
hawthorn fly populations (Lyons-Sobaski & Berlocher
2009; Powell et al. 2012; Fig. 6). To assess the degree to
which these two host-related adaptations were associated with the overall pattern of microsatellite differentiation among hawthorn flies, linear regression analyses
were performed of log-transformed FST values against
(i) the difference in median eclosion time and (ii) the
extent of behavioural cross-response to natal fruit odour
for the five local site comparisons (Fig. 7). A strong, significant relationship was found for phenotypic variation
in eclosion time explaining genetic divergence
(r2 = 0.979, P = 0.0013, d.f. = 4). Olfactory behavioural
response was not significantly related to overall genetic
divergence (r2 = 0.378, P = 0.2697, d.f. = 4.).
Discussion
Our results indicate that genetically differentiated populations of Rhagoletis pomonella exist on different native
698 T . H . Q . P O W E L L E T A L .
A
B
C
D
Fig. 6 Cumulative eclosion curves for
flies for paired host comparisons at five
local sympatric sites: (A) SFA forest (site
9), (B) Dewey Wills WMA (site 4), (C)
Brazos Bend SP (site 1), (D) Palmetto SP
(site 2) and (E) Sam Rayburn Lake (site
3). Significance was assessed by Kolmogorov–Smirnov tests.
E
hawthorn species in the southern United States, providing examples of endemic host shifting that complement
the classic downy hawthorn historical shift to introduced apple. The phenotypic and genetic patterns of
differentiation observed among southern hawthorn flies
are consistent with the operational definition of host
races put forward by Dres & Mallet (2002) including (i)
host association and fidelity, evidenced by the phenotypic
differentiation described by Powell et al. (2012); (ii)
sympatry, host plant distributions overlap at both a
broad geographical scale (Fig. 2) and within local sites;
(iii) genetic differentiation, each pair of host-associated
populations is distinguished by allele frequency differences at a subset of loci (Figs 4 and 5; Tables 2 and 3);
and (iv) some gene flow, evidenced by the complete
shared allelic variation among populations (Table S5,
Supporting Information in DRYAD). Evaluating southern hawthorn populations for additional criteria outlined by Dres & Mallet (2002) to further distinguish
between host races and less differentiated biotypes or
‘host forms’ (Funk 2012) would require additional
studies examining the patterns of hybrid fitness and the
stability of genetic differentiation through time. However, the results presented here are strikingly similar to
the patterns observed in the classic apple race system,
for which these additional criteria have been confirmed
(Feder et al. 1993, 1994).
Below, we discuss how these findings for southern
hawthorn flies touch on three important general questions: (i) How prevalent is ecological speciation with
gene flow? (ii) What ecological adaptations may be
most important for generating reproductive isolation in
phytophagous insects? and (iii) How do populations
transition from host races to species along the speciation continuum?
The prevalence of ecological speciation
The phenotypic and genetic differentiation observed
among southern R. pomonella implies that reproductive
isolation often evolves as a pleiotropic consequence of
divergent host plant selection in Rhagoletis flies. The
© 2013 John Wiley & Sons Ltd
N A T I V E H O S T R A C E S O F R H A G O L E T I S P O M O N E L L A 699
A
B
Fig. 7 Linear regression analysis between overall genetic divergence (FST) and differences in (A) median eclosion time and
(B) behavioural responses to fruit volatile blends (1-b) for the
five paired local host site comparisons.
recent host shift of R. pomonella into apple is not an isolated incident that came about through a set of entirely
unique ecological circumstances. Rather, the same processes that generated the apple race appear to apply to
the more complex community of hawthorn host plants
in the southern United States. Thus, when ecological
opportunity is present in the form of a suitable host
plant, R. pomonella seems to readily take advantage and
adapt to the new host. This conclusion is further bolstered by another emerging part of the overall Rhagoletis
story. A likely introduction of apple race R. pomonella
flies to the Pacific Northwest in the twentieth century
appears to have led to very rapid host race formation on
two additional hawthorn species, the native Crataegus
douglasii and the introduced Crataegus monogyna (Linn
et al. 2012; Sim et al. 2012; Hood et al. 2013). It remains
to be determined how general the results for Rhagoletis
are elsewhere in nature. However, the potential for
ecological divergence would appear great, at least for
phytophagous specialist insects, given the great diversity of this group of organisms.
© 2013 John Wiley & Sons Ltd
What traits are important for ecological speciation in
phytophagous insects?
The two major traits involved in the reproductive isolation of the apple race, diapause-mediated adult eclosion
phenology (Dambroski & Feder 2007) and olfactory
behavioural response to fruit volatiles (Linn et al. 2003),
also differentiate southern hawthorn flies. In cases
where insects find mates on or near their host plants
and have limited adult longevity, such as R. pomonella,
diapause timing and host choice both generate
prezygotic isolation. Moreover, evidence indicates that
these two traits can also generate postzygotic isolation
among Rhagoletis flies (Feder et al. 1997; Linn et al.
2004).
An important implication of the current study, however, is that the effects of host fruiting time and volatiles on generating reproductive isolation among
southern hawthorn flies, as measured by overall genetic
divergence, appears complex and is not strictly linear.
This is most clearly seen with respect to host phenology
and the allochronic isolation of flies. Mayhaw and green
hawthorn trees have the most distinct fruiting times of
the southern hawthorns, separated by over 6 months.
One would therefore expect that mayhaw and green
hawthorn flies should be the most allochronically isolated and thus display the greatest amount of genetic
divergence. Yet the reverse is true; these two flies had
the most similar eclosion characteristics and were the
least genetically differentiated. This suggests that there
can be limits on the extent to which host phenology
generates allochronic isolation. In the case of mayhaw
and green hawthorn, it may be that the difference in
fruiting time is great enough for (i) a portion of the
mayhaw fly population to eclose later in the same field
season, rather than overwintering, to be bivoltine and
utilize both hosts, and (ii) a portion of green hawthorn
fly population to eclose early postwinter and attack
mayhaw, reducing phenotypic and genetic differentiation. This does not mean that mayhaw and green
hawthorn flies form a single panmictic population.
Developmental trade-offs may still exist for optimal
eclosion in the spring (mayhaw) and fall (green hawthorn), as well as for host discrimination, that restrict
the two fly populations fusing into a single, bivoltine
population. Thus, diapause-related traits clearly play a
role in the differentiation of southern hawthorninfesting populations of R. pomonella. However, this
trait may be most effective in promoting reproductive
isolation in a range of host fruiting time differences
bounded at the low end by adult life expectancy and at
the upper end by the minimal developmental time to
complete the fly life cycle. As the difference in host
plant phenology becomes greater than the physiological
700 T . H . Q . P O W E L L E T A L .
limit of development time, the possibility of a bivoltine
life cycle erodes trade-offs in life history timing.
We did not find a clear relationship between host
fruit volatile response and genetic differentiation, as we
did for eclosion time. We do not know the reason for
the more muted relationship with odour discrimination.
One reason may simply be the limited statistical power
of only five comparisons. However, we hypothesize that
part of the answer may involve a constraint on the
upper limit of behavioural response difference possible
for Rhagoletis. We note that the four most diverged
responses among southern fly populations in the study
all show a similar degree of behavioural differentiation
to that observed for the flowering dogwood fly (73.7%;
Linn et al. 2005). Limits on the neural complexity of
insect brains (Bernays 2001) may result in an asymptote
of potential chemosensory differentiation. All of the volatile fruit blends of different Rhagoletis hosts overlap in
some key agonist compounds, which may preclude further differentiation in olfactory discrimination. Consequently, there is not a sufficient spread in the upper
end of the range of behavioural differences among fly
populations to detect a more pronounced relationship
with genetic divergence.
Genetic divergence along the speciation continuum
Mayhaw, blueberry hawthorn, southern red hawthorn
and green hawthorn flies display a range of genetic differentiation from each other bounded at the upper end
by blueberry hawthorn flies and at the lower end by
green hawthorn versus mayhaw flies. None of the southern hawthorn fly populations possess diagnostic or private alleles (see Supporting Information); they differ
from each other in frequencies of shared alleles. While
the same is also true for the flowering dogwood fly, the
putative sister taxon to R. pomonella, the level of differentiation between dogwood and hawthorn flies is ~2 times
greater than the highest level observed among southern
hawthorn flies (Fig. 4; Powell et al. 2013). It is interesting
to note that reciprocal rearing experiments of downy
hawthorn, apple and flowering dogwood flies have, to
date, not revealed substantial fruit-related performance
differences in survivorship between natal and non-natal
hosts. In comparison, performance, as well as diapause
and fruit volatile response differences, distinguishes
snowberry (Rhagoletis zephyria), blueberry (R. mendax)
and apple (R. pomonella) flies (Feder et al. 1989, 1999). In
these latter cases, private or near private alleles differentiate these three taxa for certain loci (Berlocher & Enquist
1993). Hence, it may be that the transition to more fully
differentiated taxa in Rhagoletis possessing unique, but
not diagnostically fixed, alleles may involve host fruits
that exert feeding-related selection pressures on flies.
Despite the lack of private alleles, blueberry hawthorn fly populations nevertheless still formed a distinct
cluster of populations from the other southern hawthorn populations across their range of geographical
overlap (Fig. 4). This has been argued to be a potential
criterion for distinguishing species from host races or
ecotypes, the latter taxonomic categories being characterized by significant local genetic differences between
sympatric populations, but a lack of global geographical
clustering into distinct groups (Feder et al. 2012; Powell
et al. 2013). This view of host races is consistent with
predictions of early stages of divergence along the speciation continuum where reproductive isolation is more
a property of specific loci rather than a characteristic of
the entire genome (Wu 2001; Feder et al. 2012). The
clustering of the blueberry haw populations, however,
was considerably weaker than that of the flowering
dogwood fly. Under this criterion, blueberry hawthorn
flies may be afforded marginal species status, near the
diffuse threshold between host races and sibling species. In contrast, latitudinal variation resulted in the
other southern hawthorn populations, much like downy
hawthorn and apple flies in the north, not forming distinct clusters and thus representing host races (Fig. 4).
In this regard, the southern hawthorn races displayed
levels of overall genetic divergence at local sites ranging
from approximately equivalent (southern red hawthorn
and green hawthorn) to less (mayhaw and green hawthorn) than those of downy hawthorn and apple flies
(Fig. 7). Consequently, when considered as a whole,
southern hawthorn flies appear to span a diversity of
phases of speciation from very weakly differentiated
host races to populations at the cusp of incipient sibling
species.
Conclusions
Genetically and phenotypically differentiated populations of Rhagoletis pomonella infest different native hawthorn species in the southern United States. These
populations appear to encompass critical phases of speciation with gene flow ranging from weakly diverged
host races to newly evolved sibling species. None of
these taxa differ by fixed diagnostic differences or even
distinct private alleles. Instead, they display varying
levels of allele frequency differences. Geographical variation in the form of latitudinal clines is present for all
the taxa. For mayhaw, green and southern red hawthorn flies, latitudinal variation is more pronounced
than host-related divergence. As a result, although they
displayed local differentiation in sympatry, these populations did not group into distinct clusters across their
respective geographical distributions and may be considered host races. In contrast, blueberry hawthorn
© 2013 John Wiley & Sons Ltd
N A T I V E H O S T R A C E S O F R H A G O L E T I S P O M O N E L L A 701
populations did cluster and may be tentatively assigned
species status. Eclosion timing and behavioural
response differences to fruit volatiles contribute to
reducing gene flow among southern hawthorn populations. Selection on diapause timing could help explain
why many loci displaying host-related divergence also
vary latitudinally. Substantial host-related trade-offs in
performance have not yet been detected for these flies,
potentially accounting for why they vary in frequencies
for shared alleles (e.g. represent quantitative taxa)
rather than possess diagnostic differences (e.g. represent
qualitative species such as Rhagoletis zephyria and
R. mendax).
It remains to be seen how general the results for the
R. pomonella group are elsewhere in nature. Rhagoletis
flies present an ideal group for study, providing a
diverse array of taxa at varying stages of speciation
with gene flow. Other groups either do not allow for
such a comparison or have not been adequately studied
to draw inferences (although see Nadeau et al. 2012;
Renault et al. 2012). However, with recent advances in
high-throughput genome-scale sequencing allowing for
intensive genomic scans of formerly nonmodel genetic
but model ecological systems, a wealth of data will
soon be available to perform meta-analysis comparing
whole groups of taxa across the speciation continuum
to test for general patterns and processes associated
with population divergence.
Acknowledgements
We would like to thank Stewart Berlocher, Scott Egan, Dan
Hahn, Kirsten Prior and Greg Ragland for helpful discussions
on the study. Help with field collections was provided by Dave
Costello, Andy Michel and Matt Michel. This research was
funded by a grant from the NSF to JLF (0614252). Additional
funding came from the University of Notre Dame’s GLOBES
IGERT program.
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© 2013 John Wiley & Sons Ltd
The study was designed by T.H.Q.P and J.L.F. Field collections and laboratory rearing were conducted by
T.H.Q.P., A.A.F., and G.R.H. Genotyping was performed by T.H.Q.P. Data analysis was conducted by
T.H.Q.P and J.L.F. The manuscript was written by
T.H.Q.P and J.L.F with input from A.A.F. and G.R.H.
Data accessibility
DNA sequence data were submitted to GenBank. Accession nos are presented in Table S2 (Supporting Information).
Microsatellite data, mtDNA alignment and eclosion
data were submitted to DRYAD doi:10.5061/drayd.
qk12c.
Supporting information
Additional supporting information may be found in the online version of this article.
Appendix S1 Methods.
Table S1 Relative percentages of chemical compounds comprising the fruit volatile blends of the four southern hawthorn
species (WMH = western mayhaw, GH = green hawthorn,
BB = blueberry hawthorn, SR = southern red hawthorn) and
the two northern R. pomonella hosts, apple (AP) and downy
hawthorn (DH).
Table S2 Information for mitochondrial sequence data used in
Fig. 3.
Table S3 List of 26 microsatellite markers used in this study.
Table S4 Alleles included in one of two groups generated by
Monte Carlo allele pooling method for each of 26 microsatellite
loci across five chromosomes (Chr.).
Table S5 Microsatellite allele frequencies for the 26 loci analysed in study for 16 field sites, including six green hawthorn,
four western mayhaw, three blueberry hawthorn and three
southern red hawthorn populations (uploaded to DRYAD).
Table S6 Mean demic inbreeding coefficient (f) and standard
deviation (r) across all 26 loci for each of the 16 populations
and sampling regime (S.R.) determined by host plant patch
size as described above.
Table S7 Results of GLM analyses of allele frequency for loci
on chromosome 1.
Table S8 Results of GLM analyses of allele frequency for loci
on chromosome 2.
Table S9 Results of GLM analyses of allele frequency for loci
on chromosome 3.
704 T . H . Q . P O W E L L E T A L .
Table S10 Results of GLM analyses of allele frequency for loci
on chromosome 4.
Table S11 Results of GLM analyses of allele frequency for loci
on chromosome 5.
of paired local populations for K = 1 and K = 2, using a
burn-in of 500 000 followed by 750 000 MCMC repetitions under a correlated allele frequency with admixture
model.
Table S12 Mean estimated Ln likelihood and standard
deviation across five replicates of STRUCTURE analysis
© 2013 John Wiley & Sons Ltd