Vol 436|28 July 2005|doi:10.1038/nature03800
LETTERS
Host shift to an invasive plant triggers rapid animal
hybrid speciation
Dietmar Schwarz1, Benjamin M. Matta1†, Nicole L. Shakir-Botteri1 & Bruce A. McPheron1
Speciation in animals is almost always envisioned as the split of an
existing lineage into an ancestral and a derived species. An
alternative speciation route is homoploid hybrid speciation1 in
which two ancestral taxa give rise to a third, derived, species by
hybridization without a change in chromosome number.
Although theoretically possible it has been regarded as rare1 and
hence of little importance in animals. On the basis of molecular
and chromosomal evidence, hybridization is the best explanation
for the origin of a handful of extant diploid bisexual animal
taxa2–6. Here we report the first case in which hybridization
between two host-specific animals (tephritid fruitflies) is clearly
associated with the shift to a new resource. Such a hybrid host shift
presents an ecologically robust scenario for animal hybrid speciation because it offers a potential mechanism for reproductive
isolation through differential adaptation to a new ecological
niche7. The necessary conditions for this mechanism of speciation7
are common in parasitic animals, which represent much of animal
diversity8. The frequency of homoploid hybrid speciation in
animals may therefore be higher than previously assumed.
Although uncommon, homoploid hybrid speciation has been well
described in plants9. In animals, potential examples of homoploid
hybrid speciation are even rarer, far less conclusive and the evolutionary mechanism remains elusive1. In general, homoploid hybrid
speciation faces two obstacles. Hybrids have to be both fit and
reproductively isolated in order to form an independent population10. Fitness not only includes the viability and fertility of hybrid
offspring but also its ability to avoid competition and backcrossing
with the parental taxa. By definition, homoploid hybrid speciation
has to occur in sympatry or parapatry. This requires that hybrids
need to occupy a hybrid-specific ecological niche10, otherwise hybrid
genotypes will either be outcompeted in parental habitats or lead to
introgression between the parental species without the formation of
an independent hybrid-origin lineage. The other major challenge is
the evolution of reproductive isolation in the face of gene flow from
the parental taxa in sympatry or parapatry. The host-specific life
history of many parasitic taxa offers a robust solution to the problem
of ecological and reproductive isolation, particularly if there is
habitat choice7. A shift to a new host could provide a hybrid
population with a separate resource that is free from competition
from the parental taxa. At the same time, a host shift offers a potential
mechanism of reproductive isolation, especially if mating occurs on
the host. Such an epistatic combination of mate and host choice
facilitates host-driven speciation in theoretical models7.
In 1997 we discovered the infestation of non-native, brushy
honeysuckle forms, Lonicera spp., by tephritid fruitflies within the
Rhagoletis pomonella species complex in the northeastern United
States (see Supplementary Information). All taxa within the
R. pomonella species complex are specialized fruit parasites—each
occupies only a very limited range of host plants11. The Lonicera
plants that were infested represent a mixture of parentals
(L. morrowii), described hybrids (L. £ bella and L. £ amoena) and
introgressed forms that originated from Asian introductions to
North America during the past 250 yr (ref. 12). These honeysuckle
forms are widely distributed and abundant invasive weeds throughout the northeastern United States13. Although the introduced
honeysuckle taxa serve as hosts for Rhagoletis in Asia and Europe14,
no infestation of introduced or native Lonicera by Rhagoletis has been
described in North America15. The new insect colonists, however,
belong to a monophyletic group of Rhagoletis that consists entirely of
native North American taxa, most of which overlap in distribution
with our newly discovered infestation of Lonicera11. This suggests that
the infestation of invasive honeysuckle forms is the result of a recent
Table 1 | Pooled allele frequencies of diagnostic alleles for the described species within the R. pomonella species complex and the Lonicera fly
N pop (N ind)
Had 100
Had 111
N pop (N ind)
Fum 158
N pop (N ind)
Dia-2100
N pop (N ind)
Aat-2100
R. pomonella
(all pop.)
R. pomonella
(central PA)
R. mendax
(all pop.)
R. mendax
(central PA)
Lonicera fly
(central PA)
R. zephyria
(all pop.)
R. zephyria
(central PA)
87 (7,581)
0.759
0.002*‡
26 (1,982)
0.001
77 (6,357)
0.721
79 (6,516)
0.353
2 (105)
0.772
0.000
1 (36)
0.000
2 (89)
0.826
2 (98)
0.470
26 (992)
0.094
0.000
29 (1,089)
0.849
26 (959)
0.001
26 (855)
0.011
1 (36)
0.125
0.000
1 (32)
0.923
1 (34)
0.000
1 (36)
0.000
3 (243)
0.157
0.673
2 (101)
0.178
3 (146)
0.000
3 (240)
0.000
11 (578)
0.000
0.992
3 (207)
0.013†
8 (503)
0.024*
8 (470)
0.000
2 (143)
0.000
0.986
2 (82)
0.000
2 (123)
0.000
2 (134)
0.000
R. cornivora, a member of the R. pomonella species complex, is excluded from the data. Central PA indicates populations surveyed in this study; all pop. indicates previously reported data from
populations throughout the ranges of the described taxa (for literature sources see Supplementary Table 1) except R. pomonella, for which only the northern populations are represented. N pop
indicates the number of surveyed populations; N ind indicates the number of surveyed individuals.
* Allele observed only in zones of described hybridization between R. pomonella and R. zephyria in the Pacific northwest and Minnesota.
†Allele observed only in the Pacific northwest.
‡ Had 111 has been observed at a frequency of 0.007 in one population (n ¼ 76) of the flowering dogwood fly, an undescribed species in the R. pomonella complex.
1
Department of Entomology, The Pennsylvania State University, 501 ASI Building, University Park, Pennsylvania 16803, USA. †Present address: Department of Surgery, University
of Pittsburgh, 200 Lothrop St, Pittsburgh, Pennsylvania 15213, USA.
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Figure 1 | Allele frequencies at hybrid diagnostic nuclear and mitochondrial
loci in Lonicera fly and parental populations from central Pennsylvania.
For sample sizes, see Supplementary Table 2 (for mitochondrial COII
(bottom row) n ¼ 31, 27, 19, 31, 17 left to right). There is a posterior
probability .0.05 that either one of the two Lonicera fly-specific haplotypes
was missed in at least one of the two parental species (see Supplementary
Information). Sny. Cty, Snyder County; SC, State College.
host shift, which we tested by analysing them for private alleles
known to characterize other Rhagoletis species.
Previous studies that sampled hundreds (sometimes thousands)
of individuals of the Rhagoletis species from multiple locations
within their range showed that these taxa are characterized by unique
high-frequency (private) alleles11. Given this previous information,
we expected to assign unequivocally this as-yet-undescribed population on Lonicera to a known Rhagoletis taxon. Instead we found that
the flies infesting Lonicera (subsequently referred to as the ‘Lonicera
fly’) showed a unique mixture of species-specific allozyme alleles that
indicate that the Lonicera fly formed through the hybridization of
the blueberry maggot (R. mendax) and the snowberry maggot
(R. zephyria). The Lonicera fly samples exhibit mixtures of private
alleles for both R. zephyria (Had 111) and R. mendax (Fum 158), and
lack two common R. pomonella alleles (Dia-2 100 and Aat-2 100;
Table 1). The hybrid origin of the Lonicera fly is further supported
by additional allozyme and sequence-based markers from five of the
six nuclear linkage groups in the R. pomonella species complex16 (see
Supplementary Information) and the mitochondrial genome (Fig. 1).
In addition to Had and Fum in linkage group III, the Lonicera fly
possesses alleles that are private in the parental species at both P1700
(ref. 16) (linkage group V) and the mitochondrial cytochrome
Figure 2 | Assignment of ancestry from two parental populations to
individuals of the Lonicera fly and its parents. Assignment was without any
prior information of population membership (as implemented in
STRUCTURE17). Data for R. mendax (black circles), Lonicera fly (white
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oxidase II (COII) gene. At Aat-2, Idh, Pgm (linkage group I), Mpi
(linkage group II) and P2963 (linkage group IV) the Lonicera fly
shows intermediate allele frequencies (Supplementary Fig. 2 and
Table 2). Our analysis of multilocus genotypes using a model-based
clustering approach17 shows that all Lonicera fly individuals are
consistently classified as recombinants of the two extremes of the
ancestry gradient between R. mendax and R. zephyria (Fig. 2). The
Lonicera fly individuals show a high variance in their ancestry
coefficient and span almost the entire spectrum between these
putative ancestors, with more individuals being similar to R. zephyria
than to R. mendax. Consistent intermediate allele frequencies across
independent linkage groups offer strong evidence for the hybrid
origin of the Lonicera fly against the competing hypothesis of
incomplete lineage sorting (see Supplementary Information). The
probability is very small (P , 0.0005) that such a pattern would have
arisen by drift or selection in an old, undescribed non-hybrid species
that shares a common ancestor with R. mendax and R. zephyria.
Incomplete lineage sorting is also contrary with the known history of
non-native Lonicera introduction to North America, which suggests
a recent Lonicera fly origin after a host shift. We considered the
alternative hypothesis of host shift followed by non-hybrid speciation
by either one of the two putative parental species. Given the sampling
of Rhagoletis (Table 1), an R. mendax/R. zephyria hybrid origin is a
much more parsimonious explanation than the drift- or selectioninduced increase of extremely rare or previously undetected alleles on
honeysuckle (see Supplementary Information). The 95% credible
intervals for the frequency of an unobserved R. zephyria-specific Had
allele in R. mendax and vice versa are 0–0.0029 and 0–0.0041,
respectively. Additional evidence is provided by the observed consistent intermediate pattern over all examined nuclear linkage groups
and the mitochondrial genome. It is unlikely that drift or selection in
a single parental origin population would have prompted the
consistent shift in the frequency of unlinked loci towards the
frequency of a second species that did not contribute to the host
shift (P , 0.008 considering only nuclear loci; see Supplementary
Information).
Do the flies on Lonicera comprise a hybrid zone18 that is maintained by continued, substantial immigration from both parental
species? If so, we would expect a high incidence of F1 hybrids (and
backcrosses) and strong deviations from Hardy–Weinberg and
linkage equilibrium18. We assigned individual multilocus genotypes
to six different classes of offspring that would result from two
generations of hybridization (pure parentals, F1 and F2 hybrids,
and first generation backcrosses). From this analysis we found no
evidence that any of 50 sampled Lonicera fly individuals represents an
F1 genotype. In contrast, we clearly identified simulated F1 hybrid
genotypes in both a hybrid swarm with continued immigration from
both parental species and a population consisting solely of F1 hybrid
genotypes (Fig. 3; see also Supplementary Fig. 1a–e for the assignment to other hybrid classes). In addition, no significant deviations
circles) and R. zephyria (grey circles) are shown. Collection sites: Snyder
County (Sny. Cty), State College (SC) and Munson (Mu.). Error bars
represent 95% credible intervals.
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from Hardy–Weinberg and linkage equilibrium in the observed
Lonicera fly populations were observed (Supplementary Tables 3
and 4). We only observed significant linkage between Had and Fum,
two genes that are known to be in the same linkage group19 and might
be part of a chromosomal inversion20. Given the postulated recent
origin of the Lonicera fly by hybridization and host shift, the overall
absence of detectable admixture linkage disequilibrium might be
surprising. However, even admixture linkage disequilibrium between
Aat and Idh (the two loci in our study separated by the shortest map
distance) is expected to decay below detectable levels in ,100 yr
under complete isolation (see Supplementary Information). Given
our results, it is unlikely that the Lonicera fly currently experiences
substantial immigration from both parental taxa. We cannot, however, exclude asymmetric immigration from R. zephyria. With
the current available genetic resolution it is not possible to distinguish between R. zephyria immigrants and Lonicera flies with
R. zephyria-like genotypes formed by random segregation in an
isolated population (Fig. 2).
The shift to honeysuckle probably freed the hybrid origin Lonicera
fly from parental species competition, and provided a potential
mechanism for reproductive isolation, as hypothesized above (our
ability to produce apparently fit R. mendax/R. zephyria F1 hybrids in
the laboratory demonstrates the potential for natural hybridization
in principle). Rhagoletis mates on the host, leading to an epistatic
interaction between mate and host choice7. In R. pomonella a recent
shift from native hawthorn to introduced apple has led to the
evolution of partially reproductively isolated host races7. Studies
on host choice, mate choice and host-specific fitness trade-offs of the
Lonicera fly and its parental species are needed to validate our
ecological assumptions, but the population genetic evidence presented above is consistent with the expected outcome of our
proposed model. Given that hybridization seems to be frequent in
animals21 and that host-specific lifestyles could represent as much as
50% of animal diversity8, the acquisition of hybrid-specific niches
through host shift may be a phenomenon in need of deeper
consideration. Anthropogenic changes offer new opportunities
for hybridization, because previously geographically separated
organisms come into contact due to human-mediated introductions22. At the same time, these community alterations provide access
to potential new hosts and thereby the opportunity for habitat shifts.
In this context it is important to note that the formation of the
Lonicera fly is a novel example of how invasive weeds can influence
the evolution of native fauna. Hybridization between parasites could
widen the spectrum of potential new hosts by generating new
phenotypes9,22,23, a mechanism akin to the idea that plant hybrids
Figure 3 | Posterior probability for the assignment of observed Lonicera fly
individuals as F1 hybrids between R. mendax and R. zephyria. Data for
observed Lonicera fly individuals (grey circles), simulated hybrid swarm
(white circles) and simulated population of F1 crosses between R. mendax
and R. zephyria (black circles; see Supplementary Information for details on
simulation) are shown.
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serve as a bridge for the acquisition of new hosts by herbivores24.
Hybridization can be difficult to detect, especially in morphologically
cryptic species, such as Rhagoletis, that may account for much of
insect diversity. The availability of extensive genetic data for the
R. pomonella species complex (including reports of local introgression between R. pomonella and R. zephyria25) is unusual compared to
many host-specific organisms. It is likely that specialized parasite
hybrid origin populations, such as the Lonicera fly, have gone
undetected because of methodological difficulties and the traditional
bias against hybridization as an evolutionary force in zoology2.
Hybridization should be considered as a viable hypothesis for the
origin of other host-specific animals, and we predict that future
studies will discover more populations with a Lonicera fly-like
evolutionary history.
METHODS
Sample collection. All samples were collected in central Pennsylvania, USA,
between 2000 and 2002. The Lonicera fly was collected at one location in State
College, Centre County (allozyme data from a second State College Lonicera fly
sample are included in Table 1) and one location in Munson, Clearfield County.
R. zephyria samples were taken from one location in State College and one
location in Munson. One R. mendax sample was collected near Middleburg,
Snyder County. Two local reference populations for R. pomonella were collected
from two different hawthorn trees in State College. The five sampling sites in
State College were separated by a maximum distance of 4 km from each other.
Munson is located 30 km northwest of State College and the Lonicera fly and
R. zephyria samples were separated by 0.7 km. The R. mendax sample location is
60 km east of State College. All samples consisted of larvae extracted from
infested fruit, or pupae or adults reared from fruit. Larvae, pupae and adult flies
were stored at 280 8C until further analysis.
Population genetic data collection. Total genomic DNA was extracted from the
head or one-third of each individual, whereas the remaining parts were used for
allozyme analysis. Seven allozyme loci were examined by standard starch gel
techniques11,26: b-hydroxyacid dehydrogenase (Had; E.C. 1.1.1.30), isocitrate
dehydrogenase (Idh; E.C. 1.1.1.42), NADH-dependent diaphorase-2 (Dia-2;
E.C. 1.6.2.2), aspartate aminotransferase-2 (Aat-2; E.C. 2.6.1.1), phosphoglucomutase (Pgm; E.C. 5.4.2.2), mannose-6-phosphate isomerase (Mpi; E.C. 5.3.1.8)
and fumarate hydratase (Fum; E.C. 4.2.1.2). Two additional nuclear loci,
developed from a complementary DNA library16, were amplified by polymerase
chain reaction (PCR) and scored for restriction length polymorphism. To
confirm heterozygote genotypes we cloned a limited number of heterozygote
individuals during the development of our protocol for the restriction digest.
We further used restriction enzymes in excess and added previously scored
individuals as positive controls to each new batch of digestions. P1700 (T3,
5 0 -ACATACATTCTGCATCTTGCGAAAG-3 0 ; T7, 5 0 -TTAAGCCGACTTCTTC
TTGAAACC-3 0 ) was polymorphic at one restriction site for Rsa1. P2963 (T3,
5 0 -AGTCAACGACCTGCTTATTT-3 0 ; T7, 5 0 -TGCACCTTAATTCACGAAAA
TC-3 0 ) was cut with Alu1, Ase1 and Tsp5091 at four restriction sites, and the
haplotype inference software HAPLOTYPER was used to determine genotypes27.
A 636-base pair (bp) piece of the mitochondrial COII coding region (C2-J-3136,
5 0 -CAAAATAGTGCCTCTCCC-3 0 ; TK-N-3772, 5 0 -GAGACCATTACTTGCTTT
CAGTCA-3 0 )28 was amplified and sequenced. The variation within a 423-bp
subsection of this sequence was used to characterize individual haplotypes.
Data analysis. Allele and haplotype frequencies were calculated using Arlequin
ver. 2000 (ref. 29). To compare the allelic composition of the Lonicera fly and its
parental species in central Pennsylvania, we collected all available published
allozyme data for the parental species of the Lonicera fly as well as for
R. pomonella (see references in Fig. 1). No population data were available for
the two nuclear sequence-based markers and the mitochondrial DNA. Published
allele frequencies at each locus were converted into allele counts, and a single
allele frequency was recalculated for the North American superpopulation of
each taxon. All samples gathered as part of this study were combined in a similar
fashion. Multilocus genotypes representing R. mendax (Snyder County, n ¼ 36),
Lonicera fly (State College, n ¼ 30; Munson, n ¼ 20) and R. zephyria (State
College, n ¼ 30; Munson, n ¼ 20) were analysed using STRUCTURE version 2
(ref. 17). All nuclear markers and mitochondrial DNA haplotypes were included
except for the monomorphic Dia-2 and Fum. Fum was excluded because it is in
strong linkage disequilibrium with Had (Supplementary Table 2), and no map
distance between these two markers is reported. The known map distances
between Aat-2, Pgm and Idh on linkage group I were incorporated into the
analysis by using the linkage model in STRUCTURE17 (1,000,000 Markov chain
Monte Carlo (MCMC) replicates and independent allele frequency model).
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NATURE|Vol 436|28 July 2005
Ancestry coefficients for membership in two populations were estimated without any prior knowledge of host-plant origin (that is, population membership).
The same data set, with the exception of the mtDNA haplotypes, was used in the
analysis with NewHybrids version 1.1 beta30. In the assignment of genotypes to
hybrid classes that result from two generations of hybridization, observed
R. mendax and R. zephyria genotypes were treated as being of known parental
origin (100,000 MCMC replicates). A simulated population of 50 F1 genotypes
and 50 genotypes from a simulated hybrid zone were generated from the
observed parental genotypes from R. mendax (Snyder County) and R. zephyria
(State College and Munson; see Supplementary Information). Hardy–Weinberg
and linkage equilibrium was tested using 100,000 permutations in Arlequin29.
We had sample sizes large enough to allow for a meaningful test of linkage
equilibrium between allozyme loci only in the Lonicera fly State College sample.
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank K. Shoemaker and A. Forbes for technical
assistance, G. Yatskievich for plant identifications, O. Bjørnstad for comments
on the manuscript and help with probability models, and J. Smith, S. Berlocher
and L. Rieseberg for comments on the manuscript. Partial funding for this study
came from the Pennsylvania Agricultural Experiment Station, the Herbert
E. Longenecker Student Research Endowment and the National Science
Foundation.
Author Information The mtDNA COII sequences from the populations of
R. mendax, R. zephyria and the Lonicera fly were submitted to GenBank with
accession numbers AY846885–AY847000 and AY847015–AY847031.
Reprints and permissions information is available at npg.nature.com/
reprintsandpermissions. The authors declare no competing financial interests.
Correspondence and requests for materials should be addressed to D.S.
(dxs332@psu.edu).
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