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Behavioural, ecological and genetic evidence confirm the
occurrence of host-associated differentiation in goldenrod
gall-midges
N. DORCHIN,*E.R.SCOTT,,àC. E. CLARKIN,* M. P. LUONGO,* S. JORDAN*
&W.G.ABRAHAMSON*
*Department of Biology, Bucknell University, Lewisburg, PA, USA
Whitman College, Walla Walla, WA, USA
Introduction
One of the mechanisms thought to promote speciation in
phytophagous insects is shifts to new hosts that lead to
the establishment of new species via an intermediate step
of host-race formation (Bush, 1969; Jaenike, 1981; Dre
`s
& Mallet, 2002). The role of host adaptation in speciation,
especially in sympatry, has been one of the most debated
topics among evolutionary ecologists over the last four
decades (reviews in Tauber & Tauber, 1989; Via, 2001;
Dre
`s & Mallet, 2002), since Bush (1969) first argued the
occurrence of sympatric host-race formation in the fruit
fly Rhagoletis pomonella. Populations that experience host-
associated differentiation (HAD – speciation that is driven
by the adaptation to hosts) can be categorized as
constituting a heterogeneous panmictic species, host-
races or established sister species based on the amount of
gene flow between them (Dre
`s & Mallet, 2002), with F
ST
estimates for host-races, for example, typically ranging
from 0.01 to 0.21 (e.g. McPheron et al., 1988; Via, 1999;
Nason et al., 2002; Blair et al., 2005; Stireman et al.,
2005). In order for HAD to occur, the host-associated
populations must exhibit certain premating and post-
mating mechanisms that promote reproductive isolation
and thus lead to genetic divergence.
Possible premating mechanisms include assortative
mating, wherein individuals show preference for mating
within the same host-associated population (Berlocher &
Feder, 2002; Rundle & Nosil, 2005), and phenological
differences exist in emergence or activity times between
host-associated populations (e.g. Bush, 1969; Smith,
1988; Craig et al., 1993; Nason et al., 2002; Thomas et al.,
2003). A major post-mating isolating mechanism is
host fidelity by ovipositing females, which has been
Correspondence: Netta Dorchin, Museum Koenig, Adenauerallee 160,
Bonn 53113, Germany.
Tel.: +49 228 9122 292; fax: +49 228 9122 212;
e-mail: n.dorchin.zfmk@uni-bonn.de
àPresent address: E. R. Scott, School of Integrative Biology, University
of Illinois, Urbana, IL 61801, USA.
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY 729
Keywords:
assortative mating;
Cecidomyiidae;
enemy-reduced space;
host races;
reproductive isolation;
speciation.
Abstract
Host-associated differentiation (HAD) is considered a step towards ecological
speciation and an important mechanism promoting diversification in phyto-
phagous insects. Although the number of documented cases of HAD is
increasing, these still represent only a small fraction of species and feeding
guilds among phytophagous insects, and most reports are based on a single
type of evidence. Here we employ a comprehensive approach to present
behavioural, morphological, ecological and genetic evidence for the occur-
rence of HAD in the gall midge Dasineura folliculi (Diptera: Cecidomyiidae) on
two sympatric species of goldenrods (Solidago rugosa and S.gigantea). Con-
trolled experiments revealed assortative mating and strong oviposition fidelity
for the natal-host species. Analysis of mitochondrial DNA showed an amount
of genetic divergence between the two host-associated populations compatible
with cryptic species rather than host races. Lower levels of within-host genetic
divergence, gall development and natural-enemy attack in the S. gigantea
population suggest this is the derived host.
doi:10.1111/j.1420-9101.2009.01696.x
documented in many case studies of phytophagous insects
(summary in Dre
`s & Mallet, 2002). Once a shift to a new
host has occurred, escape from natural enemies may be a
factor that offsets the cost of lower fitness resulting from
physiological maladaptations to this host (Price et al., 1980;
Jeffries & Lawton, 1984; Jaenike, 1990).
Despite the accumulation in recent years of empirical
evidence for HAD in herbivorous insects, the data
currently available represent only a tiny fraction of
potential cases within this extremely speciose and diverse
group. Most documented case studies are based on a
single type of evidence (usually genetic), and many
involve man-made rather than natural scenarios [e.g. the
fruit fly R. pomonella on hawthorn and apple (Bush,
1969), the European corn borer Ostrinia nubilalis on corn
and mugwort (Bethenod et al., 2005; Malausa et al.,
2005) and the aphid Acyrthosiphon pisum on alfalfa and
red clover (Via, 1999; Via et al., 2000)]. Difficulty or
reluctance to publish negative results, for cases in which
HAD has not been found, further contribute to the
difficulty of estimating the prevalence of this process.
Many more examples are therefore needed to establish
how prevalent HAD is under natural conditions, and
under what circumstances it is likely to occur. In
providing such empirical evidence it would be most
informative to explore HAD in unrelated groups of
herbivores that use the same set of hosts (Nason et al.,
2002; Blair et al., 2005; Stireman et al., 2005). If HAD has
indeed occurred independently in such cases, it would
imply that this process constitutes an important source of
biodiversity in nature.
An exceptional model system in this context is the rich
insect community associated with goldenrods (Solidago
spp., Asteraceae) in North America, many members of
which are highly specialized herbivores (McEvoy, 1988).
Among nine cases of goldenrod-feeding insects in which
HAD has been studied to date, four proved to have
radiated on the sympatric S. altissima and S. gigantea
(Abrahamson & Weis, 1997; Nason et al., 2002; Eubanks
et al., 2003; Blair et al., 2005; Stireman et al., 2005). In
the present study, we employ a multiple-evidence
approach to obtain genetic, behavioural, morphological
and ecological indications for HAD in the gall midge
Dasineura folliculi (Diptera: Cecidomyiidae), which
induces galls on Solidago rugosa and S. gigantea. We rely
on multiple rather than on a single type of evidence as
we consider it to be a more powerful approach for
establishing the existence of HAD, while also inherently
providing insight into premating and post-mating mech-
anisms that lead to and ⁄or maintain HAD in a studied
organism (see Via, 2001; Berlocher & Feder, 2002).
Specifically we asked whether: (1) adults exhibit host-
related morphological differences, (2) midges prefer
mates that originate from the same host (is there
assortative mating), (3) females prefer to oviposit on
their natal host (is there host fidelity), (4) the host-
associated populations are genetically differentiated and
(5) one of the hosts offers enemy-reduced space to the
population associated with it. We then discuss the
significance of parallel host shifts in phylogenetically
unrelated organisms as a means for estimating the
prevalence of HAD in phytophagous insects.
Materials and methods
The system
The gall midge D. folliculi (Diptera: Cecidomyiidae)
induces bud galls on the common and sympatric gold-
enrods S. rugosa and S. gigantea (Dorchin et al., 2007).
Adults of this species live for 1–3 days, mate on or under
the plants, and lay eggs between leaves that surround the
apical buds. The galls do not have defined chambers, and
the gregarious larvae develop among widened and
thickened leaves that form loose clusters on growing
shoot tips, with 5–80 larvae per gall. Larvae mature
within 3–4 weeks and then leave the gall to pupate in the
soil. Dasineura folliculi is multivoltine, completing at least
four generations a year between early May and October
(Dorchin et al., 2007).
Collecting and rearing of insects
We collected galls in 13 field sites within a 90-km radius
of Lewisburg, Pennsylvania (N4057¢W7653¢) between
late April and October in 2005 and 2006. The collection
sites included roadsides, natural areas and old fields that
supported large, sympatric populations of S. rugosa and
S. gigantea; although in some localities one of the plant
species was much more abundant than the other. We
collected all the galls we encountered that appeared
mature (i.e. containing third-instar larvae) and dissected
each of them under a stereomicroscope on the same or
on the next day. The numbers of larvae of gall inducers
and of all types of natural enemies were recorded for
each gall, and mature larvae were transferred into 25-mL
plastic cups filled with ProMix BX
TM
(Premier Horticul-
ture, Dorval, QC, Canada) potting mix to allow pupation.
Each soil cup contained all viable larvae originating from
a single gall. The cups were individually covered with
ventilated caps and kept moist in the laboratory, at room
temperature, until the end of emergence (up to 4 weeks).
The percentage of galls attacked by different types of
natural enemies was compared between the two host
plants via likelihood-ratio tests using
JMPJMP
version 5.1.2
(SAS Institute, Cary, NC, USA).
Morphology
Emerging adults were preserved in 70% ethyl alcohol for
morphological study, and 50–60 individuals from each
host-associated population were later mounted on per-
manent microscope slides in euparal according to the
method outlined in Gagne
´(1989). To detect possible
730 N. DORCHIN ET AL.
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
morphological differences between adults from the two
host-associated populations, we measured wing length
(which reflects body size) and the length of tergite 8
relative to that of tergite 6 of the abdomen in both males
and females. The relative lengths of the distal abdominal
tergites are one of the few morphological traits that may
vary among species in the otherwise rather morpholog-
ically uniform adults in the genus Dasineura (Gagne
´,
1989). The wing and tergite measurements were analy-
sed with a two-way
ANOVAANOVA
(with host plant and sex as
main effects). Tergite measurements were analysed after
)X
)0.5
transformation to better meet the distributional
assumptions of
ANOVAANOVA
. Analyses were carried out in
JMPJMP
version 5.1.2 (SAS Institute).
Mate-choice experiments
Adults of both sexes reared from field-collected galls as
described above were paired in small glass vials in the
four possible host-associated combinations (gigantea
female with rugosa male, gigantea female with gigantea
male, rugosa female with gigantea male and rugosa female
with rugosa male). Because D. folliculi galls yield mostly
single sex progeny (Dorchin et al., 2007), all females used
in the experiments were known to be virgins. In order for
mating to occur, females had to exhibit ‘calling behav-
iour’, during which they stood still and extended and
slightly waved their ovipositor, probably emitting pher-
omones. Mating never occurred when females were not
calling. We conducted the mating experiments in a no-
choice design, using a single male and a single female in a
vial at a time. Each couple was given 5 min to mate as
long as the female was calling. If a female stopped calling
during the observation, we stopped the clock and
resumed the time measurement once she started calling
again. The great majority of mating events occurred
within 10–120 s from the time a male was introduced
into the vial. Once a female had mated she retracted her
ovipositor and did not call or mate again. Overall, we
conducted 220 mating trials between individuals reared
from the same host (controls), and 139 mating trials
between individuals from different hosts (crosses). The
individuals used in these trials originated from at least 50
different galls collected at different localities and times.
Mating frequencies were compared with likelihood-ratio
tests using
JMPJMP
version 5.1.2 (SAS Institute).
Oviposition-choice and performance experiments
We tested female oviposition preference in a choice
experiment in which females were offered both host
plants in a greenhouse setting. We introduced individual
females immediately after they had mated with males
from the same host plant into ca. 0.5-L mesh cages
covering 20-cm standard pots that each contained one
S. rugosa and one S. gigantea ramet that were propagated
from rhizomes. Rhizomes for these experiments were
collected from numerous plants in two field sites and,
although we did not verify their genetic identity, each of
the host species must have been represented by at least
six different genets. This design assumed that variation
among the plant species would be of greater significance
to the gall midges than variation between individual
plants within the same species. The ramets were 4–
10 weeks old at the time of the experiment and were
matched for age and height in each pot. Females
remained in the cage until death (1–3 days later), after
which the cage was removed and the ramets were
inspected for eggs and then daily for gall development.
Like other gall midges, D. folliculi adults are very short-
lived [males live for up to 24 h, females for up to 3 days
(Dorchin et al., 2007)], and therefore females were
expected to engage in host searching and oviposition
soon after they had mated. Because Dasinuera females do
not insert their eggs into the plant tissues, the eggs are
visible on the shoot tips following oviposition. Overall,
we tested 137 females reared from S. gigantea and 87
females reared from S. rugosa. The differences in host
choice (i.e. presence of eggs on the shoot tip) and
progeny performance (i.e. gall induction and develop-
ment of midges to maturity) between females from the
two host-associated populations were compared via
goodness of fit tests using
JMPJMP
version 5.1.2 (SAS
Institute).
Genetic methods
We extracted genomic DNA from 35 whole adult midges
with the DNeasy Blood & Tissue extraction kit (QIAGEN
Inc. Valencia, CA, USA) and PCR-amplified a fragment
of ca. 650 bp of the mitochondrial cytochrome oxidase
subunit I (COI) for individuals from each host-associ-
ated population across our collecting localities. PCR
reactions contained 1–2 lL genomic DNA, 2.5 lL
(10 m
MM
)10·PCR Buffer, 2 lL (10 m
MM
) DNTP solution,
2.5 lL(1m
MM
) MgCl
2
, 2.5 lL (10 l
MM
) of forward and
reverse primers, 0.2 lL(5UlL
)1
) AmpliTaq Gold
polymerase (Applied Biosystems, Foster City, CA,
USA), and dH
2
Oto25lL. The primers we used were
LCO1490 (F) and HCO2198 (R) (Folmer et al., 1994).
The PCR conditions consisted of an initial 10-min
denaturation at 95 C, 35 cycles of 95 C for 30 s,
50 C for 1 min, 72 C for 1 min and a final 72 C
elongation phase of 4 min. PCR products were purified
using the MinElute PCR Purification Kit (QIAGEN Inc.).
Sequencing was conducted on an automated ABI
Hitachi 3730XL DNA Analyzer (Applied Biosystems) at
the Pennsylvania State University nucleic acid facility.
Sequences were initially aligned using CodonCode
Aligner version 1.5.2 (CondonCode Corporation,
Dedham, MA, USA). GenBank accession numbers are
given in Appendix 1 (http://www.ncbi.nlm.nih.gov/).
We carried out a maximum-likelihood (ML) phylo-
genetic analysis of 21 unique ingroup and four unique
Host-associated speciation in goldenrod gall-midges 731
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
outgroup haplotypes. First, we identified an appropriate
model of molecular evolution using
PAUPPAUP
* (Swofford,
1998) and the methods of Frati et al. (1997) and Buckley
et al. (2002). Both likelihood-ratio tests and the Akaike
information criterion supported the GTR+ Gmodel, and it
was used in all phylogenetic analyses. A heuristic search
was then carried out using 10 random addition sequence
replicates and Tree Bisection-Reconnection (TBR) branch
swapping. ML model parameters were initially estimated
using a neighbour-joining topology and fixed for the
first heuristic search. Model parameters were then
re-estimated using the resulting ML tree, and fixed for
a second heuristic search to confirm the results. In order
to assess nodal support, we performed 1000 bootstrap
pseudoreplicates using model parameters estimated from
the ML tree, one addition sequence replicate, retaining
one tree per replicate and TBR branch swapping.
The mean genetic distance for the ingroup sequences,
mean genetic distances within each of the host-plant
groups and net mean genetic distances between the
groups were calculated using
MEGAMEGA
4.0 (Tamura et al.,
2007) and the Tamura-Nei + Gmodel, with alpha fixed at
0.25 based on estimates from the phylogenetic analysis
(Tamura & Nei, 1993).
MEGAMEGA
does not include the GTR
model, so we chose a model of similar complexity.
Standard errors of these values were calculated using
1000 bootstrap pseudoreplicates. We then calculated
nucleotide and haplotype (gene) diversity using A
RLE-RLE-
QUINQUIN
2.0 (Schneider et al., 2000). Finally, we used
A
RLEQUINRLEQUIN
2.0 to infer the population genetic structure
with an analysis of molecular variance (
AMOVAAMOVA
). Here,
we defined two populations corresponding to the host-
associated populations. We used the Tamura and Nei
model with a gamma correction, alpha = 0.25 (Tamura &
Nei, 1993) and significance tests based on 1023
permutations.
Results
Morphology
We found significant morphological differences between
adult gall midges reared from S. rugosa and S. gigantea
(Table 1). Based on wing-length measurements, individ-
uals reared from S. rugosa were larger than individuals
from S. gigantea,(F
1,106
= 16.0, P= 0.0001), and males
were larger than females regardless of host and locality
(F
1,106
= 135.7, P< 0.0001). The interactions between
host and sex and between host and locality were not
significant (P= 0.28 and 0.33 respectively). The ratio in
length of tergite 8 to tergite 6 of the abdomen
was allometrically higher in both males and females
from S. rugosa than in those from S. gigantea (females:
F
1,44
= 19.3, P< 0.001; males: F
1,31
= 7.4, P= 0.01),
hence male and female post-abdomens are longer in
the population associated with S. rugosa. Because seg-
ment 8 of the female abdomen constitutes the basal
part of the ovipositor, this observation means that
females associated with S. rugosa have relatively longer
ovipositors.
Mate-choice experiments
The percentage of mating events within the same host-
associated populations was significantly higher than
between host-associated populations (Table 2), indicat-
ing assortative mating in D. folliculi based on host
association. Because individuals reared from S. gigantea
mated more readily than those from S. rugosa, we also
found significant differences in mating percentages
between the control groups (i.e. within host-plant
species) (v
2
= 14.78, P= 0.0001). No difference was
found between the two combinations that represented
crosses between individuals from different hosts.
Oviposition-choice and offspring survival
experiments
Forty-five of 87 (52%) mated females reared from
S. rugosa (‘rugosa females’) and 42 of 137 (31%) mated
females reared from S. gigantea (‘gigantea females’) ovi-
posited on potted plants. All of the rugosa females that
oviposited and 38 of the 42 gigantea females (90%) chose
their natal host species for oviposition, thus indicating
significant preference of females for oviposition on their
natal host (v
2
= 71.7, P< 0.0001, Fig. 1).
Larvae had higher performance on S. rugosa than on
S. gigantea (v
2
= 51.1, P< 0.0001, Fig. 1). Oviposition by
Table 1 Wing length (mm, mean ± 1 SE) and the length ratio of
abdominal tergites 8 to 6 in males and females of Dasineura folliculi
from Solidago rugosa and S. gigantea. Individuals from the two host-
associated populations differed significantly (P< 0.01) in all mea-
sured characters.
S. rugosa S. gigantea
Male Wing length 2.77 ± 0.03 (N= 31) 2.58 ± 0.04 (N= 28)
Tergite ratio 0.47 (N= 14) 0.41 (N= 19)
Female Wing length 2.29 ± 0.04 (N= 27) 2.19 ± 0.04 (N= 24)
Tergite ratio 1.70 (N= 25) 1.47 (N= 21)
Table 2 Percentages of matings out of total number of trials (N)in
the four possible crosses between Dasineura folliculi individuals from
the two host-associated populations.
Cross N% Mating
gigantea $·gigantea #129 87.6
a
rugosa $·rugosa #91 65.9
b
gigantea $·rugosa #71 31.0
c
rugosa $·gigantea #68 38.2
c
Different letters indicate significant statistical differences (P< 0.05)
according to likelihood-ratio tests.
732 N. DORCHIN ET AL.
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
all 45 (100%) rugosa females resulted in gall develop-
ment, whereas only 10 of the 38 (26%) oviposition
events among gigantea females resulted in gall develop-
ment, one of which was on an S. rugosa plant. All but
four of the galls produced adults (93%).
Genetic results
Gall-midge populations associated with S. rugosa and
S. gigantea were genetically divergent. We identified 22
unique ingroup haplotypes from 35 individuals, and the
alignment of 646 bp was unambiguous and contained no
indels. The overall mean mtDNA-corrected genetic dis-
tance was 0.148 ± 0.024 and the net mean genetic
distance between the S. gigantea and S. rugosa groups
was 0.188 ± 0.041. ML phylogenetic analysis found
genetic differentiation between the host-associated pop-
ulations with strong bootstrap support (Fig. 2). Within-
host mtDNA distances showed a higher level of variation
in the population associated with S. rugosa than in the
population associated with S. gigantea [0.020 ± 0.003
(N= 14) and 0.015 ± 0.004 (N= 21) respectively]. Sim-
ilarly, both haplotype (gene) and nucleotide diversity
were higher among S. rugosa individuals (0.96 ± 0.036
and 0.042 ± 0.022 respectively) than among S. gigantea
individuals (0.89 ± 0.057 and 0.0129 ± 0.007). The
AMOVAAMOVA
showed that 78.9% of the genetic variation was
between host-associated populations. The corresponding
F
ST
value of 0.789 was found to be highly significant
(P< 0.0001), suggesting low levels of gene flow between
host-associated populations, although increased sample
sizes would be needed to confirm this.
Occurrence of natural enemies
Natural enemies found in D. folliculi galls included
parasitic wasps from the families Pteromalidae and
50
45
30
35
40
15
20
25
Number of females
0
5
10
Eggs on
rugosa rugosa
Gall on Eggs on
gigantea
Gall on
gigantea
Fig. 1 Oviposition choice and larval performance on Solidago rugosa
and S. gigantea plants in a greenhouse experiment in which
individual Dasineura folliculi females were offered simultaneously
their natal host and the alternative host for oviposition. Oviposition
choice is represented by the presence of eggs. Larval performance is
represented by the development of galls. Filled bars represent
females reared from S. rugosa; open bars represent females reared
from S. gigantea.
88
gigH2
gigH1
gigSt8
gig2542
gigNA267
gigLB137,rugNA1
rugNA197, rugH2
rugNA2, rugNA3, rugRick
rugLair2, rugH1
rugLB1
rugLB3
rugLair226
Dasineura n.sp.1(Syd)
Dasineura n.sp.2(Vic)
rugM1
rugM2
rugSN1
rugSN2
gigV1, gigV2
gigNA216, gigCr, gigNA98, gig2541, gigM1, gigM2
gigMa, rugV
gigLair1, gigLair2
gigFR1, gigFR2
55
54
99
74
100
100
100
54 92
61
Dasineura carbonaria
0.1
Mayetiola destructor
Fig. 2 A phylogenetic tree inferred by max-
imum likelihood for Dasineura folliculi based
on 646 bp of the mtCOI gene. Numbers
indicate bootstrap support. The epithets
‘rug’ and ‘gig’ refer to Solidago rugosa and
S. gigantea, respectively, as the host from
which individuals were reared. The letters
and numbers following the host refer to
collecting sites (details in Appendix 1). Indi-
viduals that yielded identical haplotypes are
listed on the same terminal branch.
Host-associated speciation in goldenrod gall-midges 733
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Torymidae, the inquilinous gall midge Macrolabis ameri-
cana and larvae of an unidentified lepidopteran that fed
on the gall tissue (Dorchin et al., 2007). Each of these
natural enemies can have a devastating effect on larvae
of the gall inducer. For instance, a single parasitoid larva
usually killed many of the gall-midge larvae, and if more
than one parasitoid was present, the majority of the gall
midges were killed. Larvae of the inquiline M. americana
were often found in large numbers in a gall, where they
caused indirect mortality of the gall inducer, most
probably due to competition for gall resources. Feeding
by a single lepidopteran caterpillar typically consisted of
tunnelling through the centre of the gall and physically
destroying it. The percentage of galls attacked by each of
the types of natural enemies was significantly higher on
S. rugosa than on S. gigantea (wasps: v
2
= 9.2, P= 0.0024;
inquilines: v
2
= 50.1, P< 0.001; others: v
2
= 22.7,
P< 0.001), and overall attack by natural enemies was
about three times higher in galls on S. rugosa (v
2
= 60.1,
P< 0.001, Fig. 3).
Discussion
Evidence for host-associated differentiation
The data we present provide multiple types of evidence
showing D. folliculi has differentiated into two distinct
populations on its two hosts, S. rugosa and S. gigantea,
which represent cryptic species or well-established host
races. This is one of a handful of cases for which a full
array of genetic, behavioural, morphological and ecolog-
ical evidence for HAD is available (e.g. R. pomonella,
Eurosta solidaginis,Zeiraphera diniana). Dasineura folliculi
exhibited assortative mating and host choice for ovipo-
sition (i.e. host fidelity), and mtDNA sequence data
showed the host-associated populations are genetically
divergent.
Even in the absence of host plants, the gall midges
showed preference to mate within the same host-associ-
ated population, a phenomenon that was not found in
E. solidaginis, for example, where no assortative mating
was observed in the absence of hosts (Craig et al., 1993).
Difficulties stemming from the tiny size, ephemeral adult
longevity and other aspects of the biology of gall midges
precluded experiments in the presence of hosts. We
assumed that differences in mate choice and host fidelity
would characterize the host-associated populations in
general despite possible variations among different field
sites; hence we pooled all individuals from the same host
from all field sites for the analyses. Although this set-up
precludes a statistical estimation of inter-population var-
iation, our data suggest very limited variation at least with
regard to mate choice (N. Dorchin, unpublished data).
The ML tree based on our mtDNA data placed two
individuals that were reared from S. rugosa in the clade
otherwise representing the population associated with
S. gigantea. The occurrence of these exceptions, together
with the trend found in the mating experiments, implies
that the host-associated populations of D. folliculi expe-
rience a certain amount of gene flow. This amount is
considerably lower than that found between host races of
E. solidaginis (Itami et al., 1998) and of the gall moth
Gnorimoschema gallaesolidaginis (Stireman et al., 2005),
and is similar to the amount of gene flow between the
sister gall-midge species Rhopalomyia solidaginis and
R. capitata (Stireman et al., 2005). However, it is note-
worthy that in cases of rapid divergence, an incomplete
lineage sorting may occur, thereby sorting of genes into
monophyletic trees lags behind a speciation event that is
already manifested by adaptive traits (Forister et al.,
2008).
Allochronic emergence or activity times of adults from
different host-associated populations were proposed as
important premating factors that prevent gene flow in
some species (e.g. Bush, 1969; Smith, 1988; Craig et al.,
1993; Nason et al., 2002; Thomas et al., 2003). However,
these do not seem to play a major role in the case of
D. folliculi, because we did not find appreciable differ-
ences in larval development and adult emergence times
between the hosts (Dorchin et al., 2007). Although peaks
of gall development and adult emergence on S. rugosa
usually lag about a week behind those on S. gigantea
(N. Dorchin, personal observation), there is considerable
overlap in adult emergence between the populations,
which means midges from different hosts could poten-
tially interbreed.
A primary post-mating cause for reproductive isolation
in D. folliculi appears to be host choice by ovipositing
females. If adults prefer to mate on the same host species
in which they developed, then an oviposition choice
made by a female predisposes her offspring to choose the
same host for mating. Coupled with assortative mating,
host fidelity by ovipositing females can result in repro-
ductive isolation leading to genetic differentiation
35
40
45
50
15
20
25
30
0
5
10
Galls attacked (% )
Parasitoids Inquilines Others Overall
Fig. 3 Percentages of Dasineura folliculi galls on Solidago rugosa and
S. gigantea that were attacked by different types of natural enemies.
The term ‘others’ refers to inquilinous caterpillars and predatory gall
midges. Filled bars represent galls on S. rugosa (n= 645); open bars
represent galls on S. gigantea (n= 518).
734 N. DORCHIN ET AL.
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
between host-associated populations. In D. folliculi, galls
developing in the greenhouse contained much larger
numbers of larvae than field-collected galls (Dorchin
et al., 2007), suggesting that, under field conditions,
females spread their eggs among several plants. However,
in our greenhouse experiments, females deposited their
eggs in a single plant despite the availability of an
individual of the alternate host species. This behaviour
indicates that females not only choose their natal host
species for oviposition but also specifically avoid non-
natal hosts (see Forbes et al., 2005).
A second potential source of post-mating isolation is
reduced fitness in hybrids (Craig et al., 1997; Feder &
Filchak, 1999; Via et al., 2000). Although hybrids
between the S. rugosa and the S. gigantea populations of
D. folliculi are viable (N. Dorchin, unpublished data), we
currently do not know whether their fitness differs from
that of the pure populations.
We found both strong host fidelity and morphological
differences between host-associated populations in
D. folliculi. The differences in body size between the
populations could result from variable environmental
factors, but may be genetically based, given that these
differences were consistent in time and space. Morpho-
logical differences between host-associated populations
have been documented in several systems (Wood, 1980;
Rossi et al., 1999; Nosil et al., 2002; Pappers et al., 2002;
Emelianov et al., 2003; Ikonnen et al., 2003; Tabuchi &
Amano, 2003; Tokuda et al., 2004; Blair et al., 2005), and
when directly associated with mate attraction (Brown &
Cooper, 2006) or host use (Carroll et al., 1997; Diegisser
et al., 2003), they contribute to the establishment of
genetic differentiation. In D. folliculi, we found that
females from S. rugosa are larger and have longer ovipos-
itors than those from S. gigantea. A possible explanation is
that a longer ovipositor allows better manoeuvrability
and thus better access to the pubescent S. rugosa buds,
whereas the smooth surface of S. gigantea plants does not
require such an adaptation. Indeed, longer ovipositors
among Dasineura species are correlated with less accessi-
ble oviposition sites, and when accompanied by a divided
tergite 8, as in the case of D. folliculi, they are assumed to
permit more flexibility of the post-abdomen (Sylve
´n&
Tasta
´s-Duque, 1993). Despite the overall morphometric
differences, there may be some overlap between the
host-associated populations, especially among males;
hence we are reluctant to use these differences alone for
distinguishing between the populations.
Direction of the shift
At least four lines of evidence suggest that the direction
of the shift in D. folliculi was from S. rugosa to S. gigantea.
First, our genetic data show that the average within-host
mtDNA distance and nucleotide and gene diversity are
lower in the S. gigantea population than in the S. rugosa
population. Lower genetic diversity can be expected in a
derived lineage due to founder effects. Second, ‘oviposi-
tion mistakes’ were observed only among gigantea
females in our greenhouse oviposition trials, whereas
all 45 females from S. rugosa oviposited on their natal
host. The weaker host fidelity exhibited by gigantea
females could result from the fact that the development
of host preference and recognition mechanisms occurred
more recently in this population. Third, populations
shifting to a new host are expected to be less adapted to
utilizing it and will therefore have reduced fitness on this
host compared with populations on the original host
(Prokopy et al., 1988; Jaenike, 1990; Brown et al., 1995).
In our greenhouse experiments, only 9 of 38 oviposition
events (24%) by S. gigantea females on S. gigantea
resulted in successful gall induction and development,
compared to a success rate of 100% among S. rugosa
females on S. rugosa. In cases where oviposition by
S. gigantea females did not lead to gall formation, larvae
hatched from the eggs and fed on the buds for several
days, but died before moulting into second instars. This
observation suggests that S. gigantea gall midges are less
adapted to their host than those from S. rugosa.
A fourth type of evidence is the level of natural-enemy
attack on galls of the two host-associated populations.
Enemy-reduced space on a derived host has been
suggested as a factor facilitating host shifts or maintaining
isolation between conspecific populations in several
systems (e.g. Abrahamson et al., 1994; Brown et al.,
1995; Feder, 1995; Thomas et al., 2003; Blair et al.,
2005). Our results show that for D. folliculi,S. gigantea
offers enemy-reduced space with regard to the two main
enemy taxa we documented (parasitic wasps and gall-
midge inquilines). Although the percentage of galls
affected by parasitic wasps and by inquilines varied
among study sites (N. Dorchin & G. Lee, unpublished)
and generations (Dorchin et al., 2007), it was almost
always higher on S. rugosa regardless of the relative local
abundance of the two hosts.
Mitochondrial DNA analyses showed that S. gigantea
was the derived host in three other case studies of HAD
among goldenrod insects, namely, E. solidaginis,G. gal-
laesolidaginis and R. solidaginis +R. capitata, all of which
shifted to S. gigantea from S. altissima (Waring et al., 1990;
Brown et al., 1996; Stireman et al., 2005). In all three
cases the gigantea population exhibited less genetic
variation than the altissima population. This parallel
direction of the shift may reflect the fact that S. gigantea
is simply less abundant than the other two species due to
its narrower ecological requirements (Abrahamson et al.,
2005). Alternatively, it is possible that S. gigantea is a
younger species, or that it has expanded its distribution
into habitats where S. altissima and S. rugosa were already
established, thus offering an underused niche to the
insects that were associated with these hosts (see Rice,
1987; Berlocher & Feder, 2002). Given the complex and
yet unresolved classification of the Solidago species in this
group (Zhang, 1996), and the lack of knowledge on their
Host-associated speciation in goldenrod gall-midges 735
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
historic distribution in North America (Marks, 1983),
these explanations remain speculative. However, it is
noteworthy that a shift to S. gigantea has now been
documented in four different systems that show varying
amounts of gene flow.
All three cases of goldenrod gall-inducing insects
mentioned above, as well as the host races of the beetle
Mordellistena convicta (Mordellidae) that attack E. solidag-
inis galls (Blair et al., 2005), involved populations only
from S. altissima and S. gigantea, although each of these
insects is also found occasionally on S.rugosa. Several
other highly specialized herbivores that are shared by the
three largely sympatric host plants include the gall-
inducing fruit fly Procecidochares atra (Tephritidae) and at
least five additional species of gall midges (N. Dorchin,
unpublished). These observations imply some involve-
ment of S. rugosa in host shifts and speciation processes in
these insects. It appears that despite their presumed
distant phylogenetic relations (Zhang, 1996), S. rugosa
and the S. altissima species complex (subsection Triplin-
erviae of Semple & Cook, 2006) are at least physiolog-
ically similar with respect to the conditions they offer for
the development of gall inducers.
Conclusion
Dasineura folliculi joins three species of gall inducers and
one inquiline species from goldenrods that show HAD,
thus further establishing the goldenrod insect fauna as a
model system for the study of ecological diversification in
phytophagous insects. Gall inducers in this community
provide an excellent system for investigating possible
factors that promote HAD, which is particularly true for
gall midges, being by far the most numerous and
biologically diverse group among goldenrod gallers. Until
further molecular study, we cannot say whether the
host-associated populations of D. folliculi represent well-
established host races or whether they are cryptic species.
The high genetic differentiation is indicative of cryptic
species, but interspecific copulation, production of
hybrids and possible oviposition mistakes suggest a lower
degree of speciation. Major reproductive isolation factors
found in this study are assortative mating and host
fidelity by ovipositing females. We also found that
S. gigantea provided enemy-reduced space to the gall
midges, thus possibly counterbalancing their poor per-
formance on this host.
Acknowledgments
We thank Alan Snyder, Brian Lucas, Catherine Blair,
Anna Latimer, Patti Scarff and Jeff Williams for help with
field and laboratory work, Michael J. Wise for assistance
in statistical analyses, and Catherine Blair and Michael J.
Wise for discussions and comments on earlier versions of
the manuscript. This study was supported by Bucknell’s
David Burpee Plant Genetics Chair endowment and by
National Science Foundation Grant DEB-0343633 to
W.G.A. and Jason T. Irwin.
References
Abrahamson, W.G. & Weis, A.E. 1997. Evolutionary Ecology across
Three Trophic Levels: Goldenrods, Gallmakers, and Natural Ene-
mies. Princeton University Press, Princeton, NJ.
Abrahamson, W.G., Brown, J.M., Roth, S.K., Sumerford, D.V.,
Horner, J.D., Hess, M.D., How, S.T., Craig, T.P., Packer, R.A. &
Itami, J.K. 1994. Gallmaker speciation: an assessment of the
roles of host-plant characters and phenology, gallmaker
competition, and natural enemies. In: Gall-forming Insects
(P. Price, W. Mattson & Y. Baranchikov, eds), pp. 208–222.
USDA Forest Service, North Central Experiment Station,
St Paul, MN General Technical Report NC-174.
Abrahamson, W.G., Ball Dobley, K., Houseknecht, H.R. &
Pecone, C.A. 2005. Ecological divergence among five
co-occurring species of old-field goldenrods. Plant Ecol. 177:
43–56.
Berlocher, S.H. & Feder, J.L. 2002. Sympatric speciation in
phytophagous insects: moving beyond controversy? Annu.
Rev. Entomol. 47: 773–815.
Bethenod, M.T., Thomas, Y., Rousset, F., Fre
´not, B., Pe
´lozuelo,
L., Genestier, G. & Bourguet, D. 2005. Genetic isolation
between two sympatric host plant races of the European corn
borer, Ostrinia nubilalis Hu
¨bner. II: assortative mating and
host-plant preferences for oviposition. Heredity 94: 264–270.
Blair, C.P., Abrahamson, W.G., Jackman, J.A. & Tyrell, L. 2005.
Cryptic speciation and host-race formation in a purportedly
generalist tumbling flower beetle. Evolution 59: 304–316.
Brown, J.M. & Cooper, I. 2006. Evolution of wing pigmentation
patterns in a tephritid gallmaker: divergence and hybridiza-
tion. In: Galling Arthropods and their Associates (K. Ozaki,
J. Yukawa, T. Ohgushi & P.W. Price, eds), pp. 253–263.
Springer-Verlag, Tokyo, Japan.
Brown, J.M., Abrahamson, W.G., Packer, R.A. & Way, P.A.
1995. The role of natural-enemy escape in a gallmaker host-
plant shift. Oecologia 104: 52–60.
Brown, J.M., Abrahamson, W.G. & Way, P.A. 1996. Mitochon-
drial DNA phylogeography of host races of the goldenrod ball
gallmaker, Eurosta solidaginis (Diptera: Tephritidae). Evolution
50: 777–786.
Buckley, T.R., Arensburger, P., Simon, C. & Chambers, G.K.
2002. Combined data, Bayesian phylogenetics, and the origin
of the New Zealand cicada genera. Syst. Biol. 51: 4–18.
Bush, G.L. 1969. Sympatric host race formation and speciation
in frugivorous flies of the genus Rhagoletis (Diptera: Tephriti-
dae). Evolution 23: 237–251.
Carroll, S.P., Dingle, H. & Klassen, S.P. 1997. Genetic differen-
tiation of fitness-associated traits among rapidly evolving
populations of the soapberry bug. Evolution 51: 1182–1188.
Craig, T.P., Itami, J.K. & Abrahamson, W.G. 1993. Behavioral
evidence for host-race formation in Eurosta solidaginis.Evolu-
tion 47: 1696–1710.
Craig, T.P., Horner, J.D. & Itami, J.K. 1997. Hybridization studies
on the host races of Eurosta solidaginis: implications for
sympatric speciation. Evolution 51: 1552–1560.
Diegisser, T., Johannesen, J., Lehr, C. & Seitz, A. 2003. Genetic
and morphological differentiation in Tephritis bardanae (Dip-
tera: Tephritidae): evidence for host-race formation. J. Evol.
Biol. 17: 83–93.
736 N. DORCHIN ET AL.
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Dorchin, N., Clarkin, C.E., Scott, E.R., Luongo, M.P. & Abra-
hamson, W.G. 2007. Taxonomy, life history, and population
sex ratios of North American Dasineura (Diptera: Cecidomyii-
dae) on goldenrods (Asteraceae). Ann. Entomol. Soc. Am. 100:
539–548.
Dre
`s, M. & Mallet, J. 2002. Host races in plant-feeding insects
and their importance in sympatric speciation. Philos. Trans. R.
Soc. Lond. B Biol. Sci. 357: 471–492.
Emelianov, I., Simpson, F., Narang, P. & Mallet, J. 2003. Host
choice promotes reproductive isolation between host races of
the larch budmoth Zeiraphera diniana.J. Evol. Biol. 16: 208–
218.
Eubanks, M.D., Blair, C.P. & Abrahamson, W.G. 2003. One host
shift leads to another? Evidence of host-race formation in a
predaceous gall-boring beetle. Evolution 57: 168–172.
Feder, J.L. 1995. The effects of parasitoids on sympatric host
races of Rhagoletis pomonella (Diptera: Tephritidae). Ecology 76:
801–813.
Feder, J.L. & Filchak, K.E. 1999. It’s about time: the evidence
for host plant-mediated selection in the apple maggot fly,
Rhagoletis pomonella, and its implications for fitness trade-
offs in phytophagous insects. Entomol. Exp. Appl. 91: 211–
225.
Folmer, O., Black, M., Hoeh, W., Lutz, R. & Vrijenhoek, R. 1994.
DNA primers for amplification of mitochondrial cytochrome c
oxidase subunit I from diverse metazoan invertebrates. Mol.
Mar. Biol. Biotechnol. 3: 294–299.
Forbes, A.A., Fisher, J. & Feder, J.L. 2005. Habitat avoidance:
overlooking an aspect of host-specific mating and sympatric
speciation? Evolution 59: 1552–1559.
Forister, M.L., Nice, C.C., Fordyce, J.A., Gompert, Z. & Shapiro,
A.M. 2008. Considering evolutionary processes in the use of
single-locus genetic data for conservation, with examples from
the Lepidoptera. J. Insect Conserv. 12: 37–51.
Frati, F., Simon, C., Sullivan, J. & Swofford, D.L. 1997.
Evolution of the mitochondrial cytochrome oxidase II gene
in Collembola. J. Mol. Evol. 44: 145–158.
Gagne
´, R.J. 1989. The Plant Feeding Gall Midges of North America.
Cornell University Press, Ithaca, NY.
Ikonnen, A., Sipura, M., Miettinen, S. & Tahvanainen, J. 2003.
Evidence for host race formation in the leaf beetle Galerucella
lineola.Entomol. Exp. Appl. 108: 179–185.
Itami, J.K., Craig, T.P. & Horner, J.D. 1998. Factors affecting
gene flow between the host races of Eurosta solidaginis. In:
Genetic Structure and Local Adaptation in Natural Insect Popula-
tions: Effect of Ecology, Life History, and Behavior (S. Mopper &
S.Y. Strauss, eds), pp. 375–407. Chapman & Hall, New York.
Jaenike, J. 1981. Criteria for ascertaining the existence of host
races. Am. Nat. 117: 830–834.
Jaenike, J. 1990. Host specialization in phytophagous insects.
Annu. Rev. Ecol. Syst. 21: 243–273.
Jeffries, M.J. & Lawton, J.H. 1984. Enemy free space and the
structure of ecological communities. Biol. J. Linn. Soc. 23: 269–
286.
Malausa, T., Bethenod, M.T., Bontemps, A., Bourguet, D.,
Cornuet, J.M. & Ponsard, S. 2005. Assortative mating in
sympatric host races of the European corn borer. Science 308:
258–260.
Marks, P.L. 1983. On the origin of the field plants of the
Northeastern United States. Am. Nat. 122: 210–228.
McEvoy, M.V. 1988. The gall insects of goldenrods (Compositae:
Solidago) with a revision of the species of Rhopalomyia
(Diptera: Cecidomyiidae). MS Thesis. Cornell University,
Ithaca, NY.
McPheron, B.A., Smith, D.C. & Berlocher, S.H. 1988. Genetic
differences between host races of Rhagoletis pomonella.Nature
336: 64–66.
Nason, J.D., Heard, S.B. & Williams, F.R. 2002. Host-associated
genetic differentiation in the goldenrod elliptical-gall moth,
Gnorimoschema gallaesolidaginis (Lepidoptera: Gelechiidae).
Evolution 56: 1475–1488.
Nosil, P., Crespi, B.J. & Sandoval, C.P. 2002. Host-plant adap-
tation drives the parallel evolution of reproductive isolation.
Nature 417: 440–443.
Pappers, S.M., van der Velde, G., Joop Oborg, N. & van
Groenendael, J.M. 2002. Genetically based polymorphisms
in morphology and life history associated with putative host
races of the water lily leaf beetle, Galerucella nymphaeae.
Evolution 56: 1610–1621.
Price, P.W., Bouton, C.E., Gross, P., McPheron, B.A., Thompson,
J.N. & Weis, A.E. 1980. Interactions among three trophic
levels: influence of plants on interactions between insect
herbivores and natural enemies. Annu. Rev. Ecol. Syst. 11: 41–
65.
Prokopy, R.J., Diehl, S.R. & Cooley, S.S. 1988. Behavioral
evidence for host races in Rhagoletis pomonella flies. Oecologia
76: 138–147.
Rice, W.R. 1987. Speciation via habitat specialization: the
evolution of reproductive isolation as a correlated character.
Evol. Ecol. 1: 301–314.
Rossi, A.M., Stiling, P., Cattell, M.V. & Bowdish, T.I. 1999.
Evidence for host-associated races in a gall-forming
midge: trade-offs in potential fecundity. Ecol. Entomol. 24:
95–102.
Rundle, H.D. & Nosil, P. 2005. Ecological speciation. Ecol. Lett. 8:
336–352.
Schneider, S., Roessli, D. & Excoffier, L. 2000. A
RLEQUINRLEQUIN
ver.
2.000: A Software for Population Genetics Data Analysis.
Genetics and Biometry Laboratory, University of Geneva,
Geneva, Switzerland.
Semple, J.C. & Cook, R.E. 2006. Solidago. In: Flora of North
America, Vol. 20 (Flora of North America Editorial Committee,
eds), pp. 107–166. Oxford University Press, New York, NY.
Smith, D.C. 1988. Heritable divergence of Rhagoletis pomonella
host races by seasonal asynchrony. Nature 336: 66–67.
Stireman, J.O., Nason, J.D. & Heard, S.B. 2005. Host-associated
genetic differentiation in phytophagous insects: general phe-
nomenon or isolated exceptions? Evidence from a goldenrod-
insect community. Evolution 59: 2573–2587.
Swofford, D.L. 1998.
PAUPPAUP
*. Phylogenetic Analysis Using Parsi-
mony (*and Other Methods), Version 4b10. Sinauer Associ-
ates, Sunderland, MA.
Sylve
´n, E. & Tasta
´s-Duque, R. 1993. Adaptive, taxonomic,
and phylogenetic aspects of female abdominal features in
Oligotrophini (Diptera: Cecidomyiidae), and four new
Dasineura species fro the western Palearctic. Zool. Scr. 22:
277–298.
Tabuchi, K. & Amano, H. 2003. Host-associated differences in
emergence pattern, reproductive behavior and life history of
Asteralobia sasakii (Monzen) (Diptera: Cecidomyiidae) between
populations on Ilex crenaa and I. integra (Aquifoliaceae). Appl.
Entomol. Zool. 38: 501–508.
Tamura, K. & Nei, M. 1993. Estimation of the number of
nucleotide substitutions in the control region of mitochondrial
Host-associated speciation in goldenrod gall-midges 737
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
DNA in humans and chimpanzees. Mol. Biol. Evol. 10: 512–
526.
Tamura, K., Dudley, J., Nei, M. & Kumar, S. 2007.
MEGAMEGA
4:
Molecular Evolutionary Genetics Analysis (
MEGAMEGA
) software
version 4.0. Mol. Biol. Evol. 24: 1596–1599.
Tauber, C.A. & Tauber, M.J. 1989. Sympatric speciation in
insects: perception and perspective. In: Speciation and its
Consequences (D. Otte & J.A. Endler, eds), pp. 307–344. Sinauer
Associates, Sunderland, MA.
Thomas, Y., Bethenod, M.T., Pelozuelo, L., Fre
´rot, B. & Bourg-
uet, D. 2003. Genetic isolation between two sympatric host-
plant races of the European corn borer, Ostrinia nubilalis
Hu
¨bner. I. sex pheromone, moth emergence timing, and
parasitism. Evolution 57: 261–273.
Tokuda, M., Tabuchi, K., Yukawa, J. & Amano, H. 2004. Inter-
and intraspecific comparisons between Asteralobia gall midges
(Diptera: Cecidomyiidae) causing axillary bud galls on Ilex
species (Aquifoliaceae): species identification, host range, and
mode of speciation. Ann. Entomol. Soc. Am. 97: 957–970.
Via, S. 1999. Reproductive isolation between sympatric races of
pea aphids. I. Gene flow restriction and habitat choice.
Evolution 53: 1446–1457.
Via, S. 2001. Sympatric speciation in animals: the ugly duckling
grows up. Trends Ecol. Evol. 16: 381–390.
Via, S., Bouck, A.C. & Skillman, S. 2000. Reproductive isolation
between divergent races of pea aphids on two hosts. II.
Selection against migrants and hybrids in the parental envi-
ronments. Evolution 54: 1626–1637.
Waring, G.L., Abrahamson, W.G. & Howard, D.J. 1990. Genetic
differentiation in the gallformer Eurosta solidaginis (Diptera:
Tephritidae) along host plant lines. Evolution 44: 1648–1655.
Wood, T.K. 1980. Divergence in the Enchenopa binotata Say
complex (Homoptera: Membracidae) effected by host plant
adaptation. Evolution 34: 147–160.
Zhang, J. 1996. A molecular biosystematic study on North
American Solidago and related genera (Asteraceae: Astereae)
based on chloroplast DNA RFLP analysis. PhD Dissertation.
University of Waterloo, ON, Canada.
Appendix 1
Samples used for mtDNA analysis with host plants, collecting sites, dates, and GenBank accession numbers. All
specimens were collected in Pennsylvania, USA unless otherwise noted.
Sample name Host plant Site Date Accession no.
gig2541 Solidago gigantea Turbot 1 July 2006 EU375690
gig2542 Solidago gigantea Turbot 1 July 2006 EU375683
gigLB137 Solidago gigantea Stein Ln. Lewisburg 13 July 2005 EU375666
gigCr Solidago gigantea Pottsgrove 9 August 2005 EU375667
gigFR1 Solidago gigantea Furnace Rd, Lewisburg 30 May 2006 EU375682
gigFR2 Solidago gigantea Furnace Rd, Lewisburg 30 May 2006 EU375677
gigH1 Solidago gigantea Hughesville 8 June 2006 EU375688
gigH2 Solidago gigantea Hughesville 8 June 2006 EU375689
gigLair1 Solidago gigantea Lairdsville 22 June 2005 EU375668
gigLair2 Solidago gigantea Lairdsville 22 June 2005 EU375669
gigM1 Solidago gigantea Montour Preserve 1 July 2006 EU375695
gigM2 Solidago gigantea Montour Preserve 1 July 2006 EU375696
gigMa Solidago gigantea Mauses Creek 19 July 2005 EU375681
gigNA216 Solidago gigantea Bucknell University Chillisquaque Creek Natural Area 21 June 2005 EU375665
gigNA67 Solidago gigantea Bucknell University Chillisquaque Creek Natural Area 26 July 2005 EU375679
gigNA98 Solidago gigantea Bucknell University Chillisquaque Creek Natural Area 9 September 2005 EU375684
gigSt8 Solidago gigantea Stein Ln, Lewisburg 8 August 2005 EU375664
gigV1 Solidago gigantea Mauses Creek 10 July 2006 EU375691
gigV2 Solidago gigantea Mauses Creek 10 July 2006 EU375678
rugLB1 Solidago rugosa Lewisburg 20 June 2005 EU375670
rugLB3 Solidago rugosa Lewisburg 20 June 2205 EU375674
rugH1 Solidago rugosa Hughesville 8 June 2006 EU375699
rugH2 Solidago rugosa Hughesville 8 June 2006 EU375700
rugLair2 Solidago rugosa Lairdsville 22 June 2005 EU375685
rugLair226 Solidago rugosa Lairdsville 22 June 2005 EU375686
rugM1 Solidago rugosa Montour Preserve 4 June 2006 EU375675
rugM2 Solidago rugosa Montour Preserve 4 June 2006 EU375692
rugNA1 Solidago rugosa Bucknell University Chillisquaque Creek Natural Area 27 June 2005 EU375671
rugNA197 Solidago rugosa Bucknell University Chillisquaque Creek Natural Area 19 July 2005 EU375680
rugNA2 Solidago rugosa Bucknell University Chillisquaque Creek Natural Area 27 June 2005 EU375672
rugNA3 Solidago rugosa Bucknell University Chillisquaque Creek Natural Area 27 June 2005 EU375673
rugRick Solidago rugosa Ricketts Glen 29 June 2006 EU375701
rugSN1 Solidago rugosa Selinsgrove 14 June 2006 EU375698
738 N. DORCHIN ET AL.
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
Appendix 1 (Continued)
Sample name Host plant Site Date Accession no.
rugSN2 Solidago rugosa Selinsgrove 14 June 2006 EU375702
rugV Solidago rugosa Mauses Creek 17 July 2006 EU375693
Dasineura carbonaria Euthamia graminifolia Bucknell University Chillisquaque Creek Natural Area 24 May 2006 EU375703
Dasineura n.sp.1(Syd) Leptospermum laevigatum Sydney , NSW, Australia 21 August 2004 EU375687
Dasineura n.sp.2(Vic) Leptospermum laevigatum Pearcedale, Victoria, Australia 23 September 2004 EU375694
Mayetiola destructor Triticum Mishmar Hanegev, Israel 23 February 2005 EU375697
Received 30 June 2008; accepted 30 December 2008
Host-associated speciation in goldenrod gall-midges 739
ª2009 THE AUTHORS. J. EVOL. BIOL. 22 (2009) 729–739
JOURNAL COMPILATION ª2009 EUROPEAN SOCIETY FOR EVOLUTIONARY BIOLOGY
