Genetic Structure in the Seabuckthorn Carpenter Moth (Holcocerus hippophaecolus) in China: The Role of Outbreak Events, Geographical and Host Factors
Geographical and Host Factors. PLoS ONE 7(1): e30544. doi:10.1371/journal.pone.0030544
Genetic Structure in the Seabuckthorn Carpenter Moth (Holcocerus hippophaecolus ) in China: The Role of Outbreak Events, Geographical and Host Factors
Jing Tao 0
Min Chen 0
Shi-Xiang Zong 0
You-Qing Luo 0
Joao Pinto, Instituto de Higiene e Medicina Tropical, Portugal
0 1 Beijing Forestry University , Beijing , People's Republic of China, 2 Silviculture and Conservation of Ministry of Education, Beijing Forestry University , Beijing , China
Understanding factors responsible for structuring genetic diversity is of fundamental importance in evolutionary biology. The seabuckthorn carpenter moth (Holcocerus hippophaecolus Hua) is a native species throughout the north of China and is considered the main threat to seabuckthorn, Hippophae rhamnoides L. We assessed the influence of outbreaks, environmental factors and host species in shaping the genetic variation and structure of H. hippophaecolus by using Amplified Fragment Length Polymorphism (AFLP) markers. We rejected the hypothesis that outbreak-associated genetic divergence exist, as evidenced by genetic clusters containing a combination of populations from historical outbreak areas, as well as non-outbreak areas. Although a small number of markers (4 of 933 loci) were identified as candidates under selection in response to population densities. H. hippophaecolus also did not follow an isolation-by-distance pattern. We rejected the hypothesis that outbreak and drought events were driving the genetic structure of H. hippophaecolus. Rather, the genetic structure appears to be influenced by various confounding bio-geographical factors. There were detectable genetic differences between H. hippophaecolus occupying different host trees from within the same geographic location. Host-associated genetic divergence should be confirmed by further investigation.
Pests with fluctuating population size are of major concern for
forest security. Knowledge of a pests population dynamics and
associated influential factors is crucial for forest management.
Habitat, weather, natural enemies and heritable traits are
considered to play roles in insect population dynamics . Despite
many studies, the factors involved in the origin of insect outbreaks
remain poorly understood. Multiple explanations have been
proposed including: escape from natural enemies , favorable
weather , changes in host quality and quantity , and
genetic variation of pests .
The seabuckthorn carpenter moth, Holcocerus hippophaecolus Hua
(Lepidoptera: Cossidae) is the main pest of seabuckthorn,
Hippophae rhamnoides L. (Elaeagnaceae). It usually occurs on
seabuckthorn, but can also occur on Ulmus pumila L. (Urticales:
Ulmaceae) as well as a couple of species of Rosaceae . The
larvae seriously obstruct water transportation of seabuckthorn by
boring into the trunk and roots. It has one generation every 34
years and larval stages occupy most of its life history. It is widely
distributed throughout its hosts range, with most damage being
caused to trees more than 5 years old. The adult females have
limited dispersal and lay their eggs in masses on nearby plants
where the larvae feed gregariously. Berryman  has
demonstrated that pests with low dispersal properties have short, intense,
restricted outbreaks whereas those with high vagility have long,
extended outbreaks. Consistent with the former pattern, the
seabuckthorn carpenter moth has limited dispersal ability and
exhibits short but intense outbreaks that are geographically
restricted . Zhou reported that outbreaks of H. hippophaecolus
can lead to more than 70% mortality of seabuckthorn in
plantations in the Inner Mongolia Autonomous Region .
Limited mobility appears to play a role in the spatial restriction of
the seabuckthorn carpenter moth. The outbreaks usually
continue for one or two years before pest numbers decline
Seabuckthorn is native to western and northern China, the
northern Himalayas and northwestern Europe, through to central
Asia and the Altai Mountains . It is a native in 11 provinces
(autonomous region, municipalities) in China, with less than 500
thousand hectares of natural forest in the 1950s . Because of
seabuckthorns nitrogen-fixing symbionts, this plant serves to
enrich and protect soils . It has been promoted widely in
western and northern China to prevent soil erosion and
desertification. There are now 2,000,000 ha of seabuckthorn
throughout 22 provinces in China, two-thirds of which are
monoculture plantations. H. hippophaecolus was firstly reported as a
pest of seabuckthorn in 1990 . Today H. hippophaecolus is
considered to be the main threat to seabuckthorn in China. It
infests 133,000 ha of seabuckthorn and killed 67,000 ha during
the 1990s. Most of the outbreak events occurred in Seabuckthorn
monoculture plantations . Prior to the spread of H. rhamnoides
plantations in western and northern China, no outbreak events of
H. hippophaecolus had been recorded. H. rhamnoides was introduced
as a novel plant to Jianping County (Liaoning Province) in the
1950s. Prior to that, H. hippophaecolus mainly fed on U. pumila
[16,19]. Jianping has been cultivating seabuckthorn widely since
the 1970s and used to have the largest area of seabuckthorn
plantation. However, by 2001, the forests were heavily disturbed
by the seabuckthorn carpenter moth.
Molecular markers are widely used for insect population genetic
research and are a useful tool to study population structure.
Correlation with ecological factors, to identify causes of the
observed genetic structuring, is often possible using these
techniques [21,22]. The Amplified Fragment Length
Polymorphism (AFLP) technique generates a large number of fragments
that are distributed throughout the genome, without requiring
background knowledge of the genome . In the present study,
the AFLP technique was used to examine population genetic
patterns of H. hippophaecolus to address the questions below.
Are genetic patterns a factor in causing outbreaks?
Natural selection, favoring certain genotypes at low densities
and others at high densities, may contribute to the regulation of
animal numbers . Many studies have demonstrated that the
genetic patterns may play a role in population dynamics. For
example, changes of allelic frequencies of different loci have been
shown to correlate with population fluctuations in Microtrous
ochgaster . Simchuk et al  suggested that esterase (Est-4)
and protease (Pts-4) loci in Tortrix viridana L. were directly related
to its population dynamics.Mormon crickets (Anabrus simplex)
were found to consist of genetically distinct clusters that
correspond with gregarious (outbreak) populations and solitary
(non-outbreak) populations, respectively . These genetic
clades provided evidence that the differences of propensities to
outbreak are likely due to genetic polymorphism. The
seabuckthorn carpenter moth is a very destructive pest, with outbreaks
reported in many areas within its range (outbreak areas), but it
exists at low densities in other parts of its range (non-outbreak
areas). H. hippophaecolus populations may therefore consist of
genetically distinct clusters with different propensities for
outbreak. Here we describe the genetic variation of H.
hippophaecolus populations from 10 areas across its range with
contrasting historical patterns of outbreak events [1517,26,27]
and assess whether the observed genetic patterns can be
explained by outbreak history.
Do geographical distance, outbreaks and drought affect
genetic population structure?
Identifying the factors responsible for structuring genetic
diversity is important for a better understanding of the insects
evolutionary history. Such fundamental studies help to infer
ecological characteristics that are crucial for establishing
management strategies. Geographical distance, outbreaks, and drought
were considered in this study. First, in the case of species with
restricted dispersal abilities, we expect to observe a positive
correlation between the genetic and geographical distance.
Moreover, in the historical outbreak regions, H. hippophaecolus
undergoes outbreak events that are followed by population decline
when food plants become unavailable. Such population
fluctuations may have large effects on genetic diversity within
populations. Furthermore, non-genetic studies indicate recent
outbreak events were related to a lack of rainfall . Heavy
periodic rainfalls could restrict H. hippophaecolus movement and
genetic exchange within populations. Therefore, we might also
expect lower genetic differentiation within populations during
Does host-associated diversity exist in a common
Herbivores and their host plants maintain an intimate
relationship in feeding, oviposition, mate finding and predator
avoidance. Distribution, availability, longevity and chemistry of
host plants are major factors that affect the genetic differentiation
of herbivorous populations . Seabuckthorn was a novel host
plant for insects following its introduction into Jianping. Herbivore
populations can suffer from disruptive selection following shifts to
novel host plants . How did they adapt to the changes? Is there
detectable genetic diversity among H. hippophaecolus populations
feeding on different host trees in a common location? To answer
these questions, we sampled larvae from sympatric populations of
four host trees, sebuckthorn, U. pumila, Prunus armeniaca L, (Rosales:
Rosaceae), Pyrus pyrifolia (Burman) Nakai (Rosales: Rosaceae), in
Materials and Methods
Sample collection and DNA extraction
Individuals (n = 217) were collected from 10 locations across the
carpenter moth range during the summer of 2008 (Table 1) by
directly sampling under the bark of infested trees and byusing light
and pheromone traps. Sampling locations represented two
contrasting patterns of historical outbreak events, based on a
literature survey and unpublished data (J. Zong, personal
communication) (Figure 1). Populations from some areas have
experienced outbreaks while in others population densities have
been consistently low. In Jianping, a further 24 insects were
collected from different hosts (U. pumila (JPY, n = 7), Prunus
armeniaca (JPX, n = 8), Pyrus pyrifolia (JPL, n = 9)). Individuals were
transported alive to the laboratory, and then kept at 280uC. Prior
to DNA extraction, insects were washed in 80% ethanol. Total
genomic DNA was isolated using the SDS-method of Zhang and
Hewitt . After extraction, DNA was dissolved in TE buffer and
stored at 220uC until further use.
Amplified fragment length polymorphism (AFLP) analysis was
used to assess genetic diversity among sampled populations of H.
hippophaecolus. The AFLP procedure followed Vos et al.  with
minor modifications. Genomic DNA was digested with EcoRI and
MseI restriction enzymes (New England Biolabs) and double
stranded adapters were ligated to the sticky ends of the fragments.
After 4 h incubation at 37uC, each sample was diluted 1:9 with
H2O and a two-step amplification strategy was used. Pre-selective
amplification was performed for 3 min at 94uC, then 30 cycles of
30 s at 94uC, 30 s at 56uC and 1 min 72uC. A 20-ml Pre-selective
amplification PCR mixture consisted of 30 mM MgCl2, 4.5 mM
dNTP, 0.6 U Taq DNA polymerase, 30 ng EcoRI-C and MseI-A
primer. In the selective amplification, we used the following nine
primer combinations selected from 100 tested combinations :
EcoRI-AAC/MseI-CAA, EcoRI-AAC/MseI-CAC, EcoRI-AAC/
MseI-CCT, EcoRI-AAC/MseI-CTT, EcoRI-AAG/MseI-CCA,
EcoRI-CA/MseICAC, EcoRI-CA/MseI-CCT. The EcoRI primers were labeled
with IRD-700. Selective amplification was performed with the
following touchdown thermal profile: 3 min at 94uC; 12
touchdown cycles at 94uC for 30 s, 65uC for 30 s (decreasing
the temperature by 0.7uC per cycle), and 72uC for 60 s; 30 cycles
at 94uC for 30 s, 56uC for 30 s, 72uC for 1 min; 5 min at 72uC.
The 10 ml PCR mixture contained 15 MgCl2, 1.5 ng MseI and
EcoRI primer, 2 mM dNTP), 2 ml diluted (1:9) Pre-amplified
DNA. All PCRs were conducted on a GeneAmp PCR System
9700 (USA Applied Biosystems).
Amplification products were separated on 6% polyacrylamide
gels for 2.5 h on a LI-COR 4300 DNA Analyzer (LI-COR
Biosciences, USA), using LI-COR 50700 bp (Labeled with
IRD700) as a size standard. Fragments from 100700 bp in size were
scored as present (1) or absent (0) using SAGA MX (LI-COR
Biosciences, USA), and exported for data analysis.
A blank control was carried out along with each set of DNA
extractions and PCR amplifications to monitor any possible cross
contamination. Poor-quality DNA samples that did not amplify
were excluded from further analysis.
Genetic variation and structure of H. hippophaecolus
populations. The diversity of geographic populations was
assessed by estimating the percentage of polymorphic loci (%P)
and Neis heterozygosity. Percentage of polymorphic loci estimates
were based on 99% criteria and heterozygosity estimates were
made using the software TFPGA .
The genetic structure was examined by an analysis of molecular
variance (AMOVA) performed by the software ARLEQUIN 3.1
. This method was used to partition the genotypic variance
among and within populations. Two separate analyses were
performed to test the hypotheses of genetic structure attributable
to variation: among individuals across the different localities
feeding on H. rhamnoides and among individuals across different
host plants in Jianping. An additional analysis of individuals
feeding on H. rhamnoides compared to the group combining three
other host plants in Jianping was also performed. Genetic
differentiation coefficients between populations (both geographic
and host-associated) were calculated as FST, with 95% confidence
intervals (CI) obtained by bootstrapping 1000 replicates over loci.
The TFPGA software was also applied to calculate Neis genetic
distance (D) . Neighbor-joining (NJ) trees were constructed
based on D using MEGA4 .
Identification of candidate outbreak loci under
selection. Outlier loci were identified using the Dfdist
approach [36,37] in Mcheza program  (available at http://
popgen.eu/soft/mcheza/). Allele frequencies are estimated in
Dfdist based on Zhivotovskys  Bayesian approach.
Because of our particular interest in outbreak-associated
divergence, the Dfdist was run for two groups of populations
(outbreaking population vs non-outbreaking population). A total of
50000 realizations were performed and maximum allowable allele
frequency was 0.99. We chose the 0.995 confidence interval and
set the significance level at 99%. The Benjamini and Hochberg
false discovery rate (FDR) correction method was used to correct
for the occurrence of false positives in loci identified as under
selection . Loci with significant P-values at FDR threshold of
50% were identified using the Benjamini and Hochberg method.
Testing outbreaks and environmental factors driving
genetic structure. The following analysis tested outbreaks and
environmental factors that potentially influenced genetic population
structure. The effect of geographical distance was assessed using
linear map distances between H. hippophaecolus populations. Secondly,
outbreak patterns were scored with 1 indicating populations from
areas where outbreaks had occurred and 0 representing populations
in non-outbreaking areas. Finally, an index for the degree of
drought, represented by the average annual rainfall collected over
50 years was obtained (19552007, China meteorological data
sharing service system http://cdc.cma.gov.cn/). Mantel tests were
conducted with the software TFPGA to test the correlation between
Euclidean distances for all the factors and genetic distances.
The general linear models (GLM) method was also used to test
the effect of outbreak and drought on the genetic differentiation
between populations. In this analysis the factor drought was
defined as locations with less than 400 mm average annual
rainfall. Values of 1 were used for drought locations (YY, YL, YC,
LX) and 0 for other locations (PY, JP, WQ, DS, WZ, FN). The
outbreak factor was standardized, as previously, for an outbreak
area of 1 and a non-outbreak area of 0. We performed a GLM
analysis of the heterozygosity with outbreak and drought as fixed
factors. A P-value of ,0.05 was used to indicate statistical
significance. GLM was implemented using SPSS 16.0.
Genetic variation and structure of H. hippophaecolus
The nine primer combinations produced a total of 933 bands.
The global among the 10 sites was 0.2106 (95% CI 0.1981
0.2230). Neis heterozygosity for each geographical population was
moderate and ranged from 0.1505,0.2042 (Table 2).
AMOVA conducted on AFLP markers confirmed the presence
of moderate genetic differentiation showing that 22.54% of total
variability was due to the variation among geographic populations
%Polymorphic loci (p)
Source of variation
Individuals within localities
Among host plants in Jianping
Individuals within host plants in Jianping
Among two host groups in Jianping
Individuals within groups in Jianping
Percentage of variation (%) P
(FST = 0.2254, P,0.0001) (Table 3). The pair-wise comparisons
between populations were characterized by values of FST ranging
from 0.04240.3663 (Table 3). Most of the populations showed
highly significant differences (P,0.0001) with the exception of the
YY and LX populations (P = 0.0182). This result indicates that
most of the 10 sampled populations represent differentiated
The Neighbor Joining phenogram (Figure 2) indicates that the
clusters comprised populations with a mixture of outbreak
patterns. For instance, populations from Dongsheng and Youyu
were in two distinct NJ genetic clusters, although they have the
same intensity of outbreak events.
Examination of the AFLP data using Dfdist in Mcheza sought
to determine whether there was evidence of any highly
differentiated loci. FST is plotted against heterozygosity in
Figure 3. The outbreak and non-outbreak population comparison
performed with Dfist resulted in four markers out of 993 (loci 93,
188, 223, 390) showing more differentiation than expected at the
99.5% confidence level. All these loci were detected as potential
positive outliers at the 50% FDR threshold (Figure 3).
Testing outbreaks and drought as factors driving H.
hippophaecolus genetic structure
The Mantel test based on the 10 localities gave an r value of
0.0554 (P = 0.3460, for 10000 randomizations), indicating no
correlation between geographic and genetic differences. The Neis
genetic distances between populations were not significantly
correlated to outbreak differences in the Mantel test (r = 0.2516,
P = 0.0740). The interaction between Euclidean distances for
average annual rainfall and genetic distances was also not
significant (Mantel test r = 0.1271, P = 0.2070). GLM analysis
showed that the factors of outbreak and drought, and their
interaction, did not have a significant effect on heterozygosity
(F1,10 = 0.053, P = 0.826, F1,10 = 1.329, P = 0.293 and
F1,10 = 2.904, P = 0.139 respectively).
The host plant was found to have a larger effect on the genetic
structure among populations than geographic location. The global
value among different hosts was 0.2785 (95% CI 0.2548
0.3024), higher than the value among 10 sites (0.2106). AMOVA
with ARLEQUIN found greater variation among populations in
host-plant groupings (31.73%) than populations in geographical
groupings (22.54%) (Table 3). Pairwise FST statistics between JPS
and each other location population ranged from 0.0856 to 0.2978
(Table 4), while the genetic divergences were all highly significant
0.3510,0.3773 in the host-associated analysis (Table 5).
In Jianping, individuals feeding on H. rhamnoides had a great
separation from individuals feeding on other host plants. When
combined individuals feeding on U. pumila, P. armeniaca and P.
pyrifolia as a group, compared to individuals feeding on H.
rhamnoide, the variation among two groups rose up to 34.82% by
AMOVA with ARLEQUIN. Pairwise comparisons of FST values
between all host plant combinations further supported the pattern
of genetic structure. FST values were much greater in comparisons
between the H. rhamnoides feeders (0.35100.3773) and each other
host-plant feeders (0.05270.1180) (Table 5). H. rhamnoides feeders
showed strongly significant differences (P,0.0001) with the moth
on other host plants (Table 5).
Genetic patterns associated with outbreak events of H.
Genetic clustering did not support distinct outbreak-associated
genetic clades in H. hippophaecolus. NJ genetic population clusters
contained a combination of populations from historical outbreak
areas as well as non-outbreak areas (Figure 2). The outbreak effect
may have been difficult to detect among different geographical
populations due to various confounding biogeographical factors
that also shape genetic structure in H. hippophaecolus. In addition,
one cannot exclude the possibility that the outbreak and
nonoutbreak patterns are associated with a single genotype, but
depend on the expression of different phenotypes in different
Indeed, our results support the notion that outbreak events were
likely to be endemic population changes from latent to epidemic
rather than being due to insects with an outbreak-associated
Neis genetic distances are below the diagonal. FST value and their significance level are above the diagonal. Significance level of associated FST value are shown as:
*0.01,P,0.05, unmarked mean P,0.0001.
genotype spreading to outbreak areas. This conclusion is also
consistent with the poor dispersal ability of H. hippophaecolus, which
has been observed in non-genetic studies. Zong et al.  indicated
female moths usually choose a nearby tree for mating after
emergence, and that the body weight of fertilized female moths is
too heavy for long-distance migration. Males are not attracted to sex
pheromone traps located too far away from the infested forests
(,100 m) . Furthermore, adults live for only several days, which
limits the degree of dispersal. Young seedlings (12 years) and seeds
of seabuckthorn are often used for its introduction. However, H.
hippophaecolus only harms seabuckthorn plants that are more than 5
years old. Therefore, H. hippophaecolus would not be dispersed
longdistances by artificial movement of host plants.
We rejected the hypothesis of genetic difference associated with
outbreaks in the seabuckthorn carpenter moth. However, habitat,
weather, natural enemies are also considered as main factors
affecting insect population dynamics. An outbreak occurs when the
physiological state of the plant permits a herbivore phenotype with a
high reproductive capacity to become dominant. Agricultural and
forest monocultures consisting of extensive plantings of hosts with
narrow genetic variability are havens for pest outbreaks .
Seabuckthorn monoculture plantations are optimal sites for survival
of H. hippophaecolus, especially for those that were introduced as an
exotic species growing under unfavorable conditions . Weather
and climatic conditions significantly affect population fluctuation.
Unusual weather is known to have strong effects on the dynamics of
insect populations [42,43]. Several authors associated the initiation
of outbreaks in Jianping and Dongsheng with consecutive dry years
before outbreak [15,19,]. Seabuckthorn plantations have plenty of
nutrients, relatively few natural enemies and are highly vulnerable to
drought or human disturbance, which may explain why outbreak
events happen there.
The outlier analysis revealed that although differentiation for the
majority of markers did not significantly deviate from neutral
expectations, a small number of markers (n = 4) were identified as
outlier loci. The false discovery rate test supported the conclusion
that all identified outlier loci are under selection. These results do not
allow us to reject the hypothesis that specific genome regions or
genes are associated with outbreak events. Having indentified outlier
loci in H. hippophaecolus, it will be necessary to try to find candidate
genes that could correspond to these AFLP markers. Then we can
characterize these genes with functional genomics analyses.
Factors influencing the population structure of H.
We found evidence of limited gene flow among samples
collected from 10 locations. The poor correlation between genetic
and geographical distances is an unexpected result given the
previous assumption that populations are isolated-by-distance.
These results suggest that multiple factors other than simple
geographic distance are influencing the genetic composition of
populations. The Mantel analysis failed to support the idea that
outbreak and drought are pertinent factors underlying genetic
structure in H. hippophaecolus. Compared to other observations in
insects, Neis heterozygosity values for each population are
moderate [44,45,46]. We found similar genetic diversity within
outbreak and non-outbreak populations of the seabuckthorn
carpenter moth. Similarly, GLM analysis showed that outbreaks
had no significant effects on heterozygosity. Genetic diversity
within outbreak populations was not strongly affected by increases
in population size during outbreak periods. This result could be
consistent with the theoretical prediction that long-term
fluctuating populations correspond to the harmonic mean size over time,
and should thus be closer in size to that during the remission
period than during an outbreak [47,48]. We rejected the
hypothesis that drought populations have lower genetic variation.
GLM analysis showed that drought had no significant effects on
Comparative studies of population structure in phytophagous
insects show that genetic structure is mostly determined by the
ability of the species to disperse . The probability of successful
dispersal is largely determined by habitat availability [50,51]. Louy
et al.  have shown in experiments with three skipper species
that dispersal ability and habitat availability determine the genetic
structure of species. Whether a habitat is available for
phytophagous insects strongly depends on the existence of host trees.
Several forest insects have pronounced geographical structure that
follows the distribution of their host tree species [46,53].
Louy et al.  suggest that limited habitat availability in
combination with low dispersal capacity result in independent
genetic structure with relative high genetic differentiations and low
gene flow among populations. H. hippophaecolus is a specialist,
which only feeds on a few plants (U. pumila, P.armeniaca, P.pyrifolia).
Moreover, most of the principal host in China, H. rhamnoides, is in
single species plantations which are suffering from human
interference. Could this independent population structure of H.
hippophaecolus be explained by low dispersal capacity and habitat
fragmentation? Combining habitat landscape and population
genetic analysis might answer this question in the future.
The role of the plant
Host races are genetically differentiated sympatric populations
of parasites that use different hosts and between which there is
limited gene flow . Our analyses uncovered very high FST
values (0.35100.3773) between JPS and other non-seabuckthorn
populations. It is indicated that H. rhamnoides constitutes a barrier
to gene flow between H. hippophaecolus populations from other host
plants in Jianping. H. hippophaecolus feeding on H. rhamnoides in
Jianping are more genetically differentiated than those from other
hosts in sympatric rather than other geographically distant
populations of seabuckthorn. Host races might therefore exist in
seabuckthorn and other host plant used by H. hippophaecolus.
Factors favoring host race formation include correlations between
host choice and mate choice. Although host fidelity and assortative
mating has not been fully explored in H. hippophaecolus, tests using
both artificial and natural methods suggest female host preferences
may exist. Adult emergence from the seabuckthorn roots
confirmed oviposition preference on H. rhamnoides, rather than
on U. pumila and P. armeniaca .
Seabuckthorn was an endemic perennial, sporadically growing
in Inner Mongolia, Shanxi and areas of Liaoning province before
it was widely promoted. The timing of host shifting of H.
hippophaecolus in Jianping is likely due to the introduction of H.
rhamnoides. However, how did host shifting occur in H.
hippophaecolus in Jianping? When did host-associated genetic divergence
initially occur in H. hippophaecolus? Data from many host utilization
systems gave rise to a possible scenario that host shifts occur as a
result of host plants increased abundance and availability as a
potential resource following human-mediated plant community
changes [56,57]. If this is the case, our data suggests a local host
shift and genetic differentiation of H. hippophaecolus following the
introduction of seabuckthorn in Jianping. Though a rapid range
expansion of H. hippophaecolus following human-mediated changes
is possible, it does seem unlikely given the wide extent of genetic
divergence observed during such a brief time. This scenario was
also rejected by Sword et al in the Hesperotettix viridis host utilization
system . Another possibility is a genetic divergence of moth
between H. rhamnoides and other hosts, prior to the host shift. Feder
et al.  found genetic divergence between apple and hawthorne
host races of Rhagoletis pomonella L. pre-dating the introduction of
the apple to North America. Given the long life history of H.
hippophaecolus and brief planting history of H. rhamnoides in Jianping,
we suppose the latter scenario is the case. Seabuckthorn is native
to parts of western and northern China although records for the
historical host plant use by H. hippophaecolus are lacking. Our results
indicate that an H. hippophaecolus lineage might have adapted to
utilize H. rhamnoides in China prior its spread. The possibilities of
an ancestral host shift and stable host-associated genetic
divergence in seabuckthorn carpenter moth are suggested.
We found no fixed diagnostic differences in AFLP data between
the different host-associated forms. Host-associated genetic
divergence should also be further demonstrated by sampling
additional populations feeding on different host plants in more
locations. In future studies, more different genetic markers are
recommended in this system. They should include co-dominant
markers such as microsatellites (not currently available for this
species) and incorporation of variable regions of the mitochondrial
genome. Microsatellites are highly polymorphic, locus-specific and
can show co-dominant inheritance. They may recover higher
levels of variability than other markers, particularly if following a
population bottleneck associated with host shift. Mitochondrial
sequences can be analyzed to determine patterns of evolutionary
relationships between different haplotypes. This may provide
information on the historical evolution of host-associated forms in
the seabuckthorn moth.
We are grateful to Jianwei Wang, Rong Wang, Zhizheng Wang for sample
collection. We would also to thank Mark P. Miller who helped with data
analysis using TFPGA software. We thank Michael Klein, Katie Robinson,
Tamara Pulpitel, Julie-Anne Popple for English editing of manuscript.
Conceived and designed the experiments: JT MC S-XZ Y-QL. Performed
the experiments: JT MC. Analyzed the data: JT MC S-XZ Y-QL.
Contributed reagents/materials/analysis tools: JT MC S-XZ Y-QL. Wrote
the paper: JT. Collected samples: JT S-XZ.
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