Journal of Heredity 2014:105(3):365–380
doi:10.1093/jhered/est096
Advance Access publication January 7, 2014
© The American Genetic Association 2014. All rights reserved.
For permissions, please e-mail: [email protected]
Genetic Status and Timing of a Weevil
Introduction to Santa Cruz Island,
Galápagos
Hoi-Fei Mok, Courtney C. Stepien, Maryska Kaczmarek, Lázaro Roque Albelo, and
Andrea S. Sequeira
From the Department of Biological Sciences, Wellesley College, Wellesley (Mok, Stepien, Kaczmarek, and Sequeira);
Department of Terrestrial Invertebrates, Charles Darwin Research Station, Puerto Ayora, Galápagos, Ecuador (Roque
Albelo). Hoi-Fei Mok is now at the School of Land and Environment, University of Melbourne, Melbourne, Australia;
Courtney C. Stepien is now at the Graduate Program, Committee on Evolutionary Biology, University of Chicago, Chicago,
IL; Maryska Kaczmarek is now at the Department of Molecular Genetics and Microbiology, The University of Texas at
Austin, Austin, TX; Lázaro Roque Albelo is now at the Invertebrate Science Team, Ecologia Environment, Australia and
Curtin Institute for Biodiversity and Climate, Curtin University, Perth, Australia.
Address correspondence to Dr. Andrea S. Sequeira, Department of Biological Sciences, Wellesley College, 106 Central
Street, Wellesley, MA 02481, or e-mail: [email protected].
Data deposited at Dryad: http://dx.doi.org/doi:10.5061/dryad.3ks4v
Abstract
Successful invasive species can overcome or circumvent the potential genetic loss caused by an introduction bottleneck
through a rapid population expansion and admixture from multiple introductions. We explore the genetic makeup and the
timing of a species introduction to Santa Cruz Island in the Galápagos archipelago. We investigate the presence of processes
that can maintain genetic diversity in populations of the broad-nosed weevil Galapaganus howdenae howdenae. Analyses of combined genotypes for 8 microsatellite loci showed evidence of past population size reductions through moment and likelihoodbased estimators. No evidence of admixture through multiple introductions was found, but substantial current population
sizes (N0 298, 95% credible limits 50–2300), genetic diversity comparable with long-established endemics (Mean number of
alleles = 3.875), and lack of genetic structure across the introduced range (FST = 0.01359) could suggest that foundations are
in place for populations to rapidly recover any loss of genetic variability. The time estimates for the introduction into Santa
Cruz support an accidental transfer during the colonization period (1832–1959) predating the spurt in human population
growth. Our evaluation of the genetic status of G. h. howdenae suggests potential for population growth in addition to our field
observations of a concurrent expansion in range and feeding preferences towards protected areas and endemic host plants.
Key Words: bottlenecks, Galapaganus h. howdenae, microsatellite loci, Msvar, older introduction, range expansion
Several genetic events are possible on the introduction of
exotic species. Introduced species themselves can undergo
population bottlenecks, where a drastic reduction in population size causes the genetic makeup of the remaining few
to become the new gene pool (Nei et al. 1975). If severe
and long enough, bottleneck events can result in loss of
variants, decreased genetic flexibility, and loss of phenotypes
(Nei et al. 1975; Garza and Williamson 2001; Allendorf and
Lundquist 2003; Colautti et al. 2005)—all possibly detrimental to the success of the recently introduced population.
However, rapid demographic growth and/or range expansions after translocations have made the detection of bottlenecks impossible (Zeisset and Beebee 2003) and caused the
retention of genetic diversity (Zenger et al. 2003). Despite
the presumption that bottlenecks are disadvantageous on
invasiveness, the length of the population size reduction during the introduction event and the speed of the recovery after
the introduction event can effectively diminish or avoid those
detrimental effects. Additionally, one key to invasion success
could be the occurrence of multiple introductions that may
transform among-population variation in native ranges to
within-population variation in introduced areas (Kolbe et al.
2004; Williams et al. 2005). Alternatively, maintaining connectivity across the area of introduction could ensure the spread
of less frequent allelic variants, which could either arrive or
arise at the edges of the introduced range, throughout the
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Journal of Heredity
area of introduction. In summary, the substantial loss of
variability that can be associated with an introduction can
be mitigated or circumvented altogether through at least 3
processes: introductions from multiple genetically differentiated continental sources (Kolbe et al. 2004; Facon et al.
2008), rapid demographic and/or range expansions (Zeisset
and Beebee 2003; Zenger et al. 2003), and population connectivity through effective dispersal (Mishra et al. 2006). If
a population maintains or increases genetic variability after
introduction through any or all of these processes, chances
of deleterious alleles surfacing are decreased and overall fitness could be increased (Lindolm et al. 2006). Still, the underlying assumption that all types of genetic diversity are equally
important for the long-term survival of a population is challenged by the fact that some endangered populations have
maintained small census sizes and very low levels of neutral
genetic diversity more than hundreds or thousands of years
(Johnson et al. 2009) and that low levels of diversity may
not be accompanied by obvious reductions in fitness (Brodie
2007). Undeniably, newly introduced populations could
remain unaffected by a reduction in census size if they do
not experience a corresponding reduction in effective population size. Nonetheless, monitoring the genetic makeup of
introduced populations using neutral markers, such as microsatellite loci, still remains an informative mean of exploring
the underlying genetics of invasiveness (Alda et al. 2013;
Frisch et al. 2013; Ghabooli et al. 2013; McMahon et al. 2013;
Perdereau et al. 2013; Simpson et al. 2013).
The effects of introduced populations on the genetic
makeup of their native counterparts can range from
increased vigor to complete loss of fitness in the introduced–
native hybrids (Huxel 1999; Fitzpatrick and Shaffer 2007).
Additionally, hybridization may stimulate invasiveness in the
introduced populations (Ellstrand and Schierenbeck 2000). In
contrast, the effects of introduced species on the ecological
communities that harbor their native counterparts are harder
to unravel (Evans 2004). Species invasions of continental habitats are rarely accompanied by the total exclusion of native
competitors. Island systems, on the other hand, appear more
vulnerable because they seemingly are less saturated or the
native species are competitively inferior (Simberloff 1986);
this has caused at least one oceanic archipelago to become a
geographic hotspot of invasion for certain insect taxa (Beggs
et al. 2011). The vulnerability of native biotas to ecological
disruption may be especially great on oceanic islands invaded
by continental species with unique ecological traits (Wilson
and Holway 2010). An important correlate of the vulnerability to displacement by invading counterparts of insect species on islands is the provenance of the populations studied,
where populations of rare endemics appear to be more vulnerable to displacement by invasives than introduced species
sharing their habitat (Krushelnycky and Gillespie 2010).
Although oceanic islands such as those in the Galápagos
archipelago are natural models for studying the process of
generation and maintenance of species diversity (Emerson
and Kolm 2005), such endemic diversity may become threatened by the introduction of exotic species. Since human settlement on the Galápagos in 1535, the native biodiversity
366
has been altered through the addition of alien insect species (Desender et al. 1992; Klimaszewski and Peck 1998;
Desender et al. 2002). Although laws restricting transportation of live animals and plants in 1994 and 1998 have reduced
introductions, accidental establishment of insects still occurs
through importation of construction materials and food
supplies, especially on the large and central islands (Peck
et al. 1998; Causton et al. 2006). Furthermore, introduced
species from nearby continental areas have been identified
as the principal threat to the terrestrial ecosystems of the
Galápagos Islands, given that many later become established
and widespread (Snell et al. 2002a, 2002b). Since humans first
arrived to Galápagos the rate of unintentional insect introductions has been about one new insect species per year;
however since 1998 the rate has increased by 59% (Peck et al.
1998; Causton et al. 2006). A large proportion of these introduced insects are plant feeders (42%), threatening endemic
flora with disease transmission and herbivory (Causton et al.
2006). Evidence of regular ongoing human-assisted introductions suggests that the measures already in place are not
sufficient to stem the tide of incoming species (Bataille et al.
2009). Considering the susceptibility of Galápagos biota and
the environmental damage possible, identifying potential
invasive insects and monitoring their impact on native and
endemic species on the Galápagos archipelago remains a relevant conservation question.
The geologic age of the extant islands within the Galápagos
archipelago sits within the “middle age” range of the life
cycle of volcanic islands (Peck 2005); the maximum age of
the oldest islands is reported to be between 3 and 4 million
years (Bailey 1976; Hickman and Lipps 1985) or up to 6.3 million years (Geist 1996; White et al. 1993). However, the history of human–nature relationships in Galápagos is relatively
short and spans no more than 5 centuries (Gonzalez et al.
2008). The four periods within the historical profile of this
interaction determined by Gonzalez and coauthors (2008)
pinpoint time-frames where the rate of accidental introductions increased significantly. The four periods are described as
extractive exploitation (1535–1832), colonization (1832–1959),
wilderness conservation (1959–1998), and conservation–
development balance (1998–present)(Gonzalez et al. 2008).
Even though the first alien species are thought to have entered
Galápagos during the early period of extractive exploitation
(Tye et al. 2002), it was the establishment of the first human
settlements during the colonization period that brought a dramatic increase in the numbers of exotic plants and animals.
Interestingly, human population numbers increased significantly later, more markedly between 1950 and 2001.
The weevil genus Galapaganus Lanteri (Coleoptera:
Curculionidae) has presented a suitable system to study species radiations on islands (Lanteri 1992; Sequeira et al., 2000;
Sequeira, Lanteri et al., 2008; Sequeira, Sijapati et al., 2008;
Sequeira et al., 2012). We will now attempt to use introduced
island populations of a continental Galapaganus species to
explore the genetic signals of a presumed recent introduction in this ecosystem that is hailed as an example for conservation efforts while under increasing pressure from tourism
and development. The genus Galapaganus contains a total of
Mok et al. • Genetics of a Weevil Introduction to Galápagos
15 species; 13 within the darwini species group are flightless
and fairly heavy bodied. Ten of the species within the darwini group are endemic to the islands (Lanteri 1992), whereas
the remaining three, together with winged Galapaganus in the
femoratus group, reside on the Ecuadorian mainland and Perú
(Lanteri 2004). Lanteri (2004) described 2 subspecies for one
of the femoratus group species, Galapaganus howdenae howdenae
from the lowlands of Guayas, Manabí and Los Rios provinces, and G. howdenae pecki from the highlands of Pichincha
province in Ecuador.
The central island of Santa Cruz in Galápagos harbors
2 endemic and 1 introduced Galapaganus species (G. conwayensis and G. ashlocki: endemics; G. h. howdenae: introduced).
Galápagos introduced specimens are typically more densely
covered in scales and have broader bodies than those found
at higher altitudes of mainland Ecuador, fitting the description of coastal G. h. howdenae (Lanteri 2004). All collections
suggest that to date only G. h. howdenae has been introduced
into the focus area of this study—the Agricultural Zone
(AZ) of Santa Cruz Island—and that it has not been introduced elsewhere. Like its endemic relatives, G. h. howdenae
larvae are root feeders, whereas adult weevils feed on leaves
of a variety of plant hosts. Unlike its endemic counterparts,
G. h. howdenae have well-developed and flight-capable wings,
giving this introduced species more efficient dispersal abilities. Interestingly, this distinction in ecological traits between
the continental and island endemic species could contribute to the vulnerability of the populations of island natives.
Galapaganus h. howdenae was first reported on the island of
Santa Cruz in 1992 (Lanteri 2004). Given the location of the
main ports of entry for goods in Puerto Ayora, a likely introduction scenario is that G. h. howdenae could have been introduced either once or multiple times together with imported
plants, first into lowland areas of Santa Cruz and later either
passively transported with those plants or actively dispersed
into the AZ where it established populations. We have
now repeatedly found it beyond the boundaries of the disturbed AZ (in 2006, 2007), feeding on introduced as well as
endemic vegetation side by side with G. conwayensis—one of
its endemic close relatives. However, our most recent observations (in 2010, 2011) suggest that G. h. howdenae has also
further broadened its range into the moist highlands reaching the highest points in Santa Cruz. It now shares habitats
and plant hosts with a highland specialist and single island
endemic (G. ashlocki) (Sequeira et al. 2012). Analysis of mitochondrial sequences comparing populations of endemic and
introduced Galapaganus species indicated that time of establishment and dispersal abilities are important determinants
of genetic structure in weevil populations. In particular, the
3 introduced populations included in that study, though variable, lacked genetic structure and retained signals of a recent
change in population size (Sequeira et al. 2012).
Studying the genetic composition of populations of
G. h. howdenae across the introduced range using multiple
markers can continue to clarify the invasive potential of this
weevil introduction (Allendorf and Lundquist 2003; Kolbe
et al. 2004; Colautti et al. 2005). Many island introductions
show clear evidence of loss of variability (Puillandre et al.
2008); however, that loss is linked with differing effects on
the new arrival’s establishment success and its impact on
native species. In particular, the potential impacts of the
introduced populations of G. h. howdenae are compounded
by Santa Cruz’s central location in the Galápagos archipelago (Figure 1). Given the flight capabilities of G. h. howdenae,
the risk of it dispersing to the other islands from the central
island is increased (Roque Albelo et al. 2006). An assessment
of alien insect species in the Galápagos has already recognized G. h. howdenae as a potentially moderate invasive species
(Causton et al. 2006). As with other Galápagos insect introductions (Dudaniec et al. 2008), analyzing the genetic status of introduced populations can not only shed light on its
invasive potential but also aid identifying subsequent steps to
prevent further expansion. Considering the recent observations of expanding range of G. h. howdenae, and the potential
damages in consequence, such insight may prove important
for protection of the Galápagos’ endemic biodiversity.
This study will analyze the variation found at 8 microsatellite loci to assess the current genetic status of populations
of G. h. howdenae across the known introduced range in the
AZ and National Park (NP) areas of Santa Cruz. A second
objective is to explore the origin of that genetic variation by
reconstructing the timing and number of introduction events
into Santa Cruz.
The predictions associated with the genetic status of the
introduced G. h. howdenae populations in Santa Cruz are: 1) If
populations have undergone a bottleneck associated with
their introduction to Galápagos (or some other event) within
2–4 Ne generations, then we would observe i) Low ratio of
observed alleles to allele range (M ratio), ii) Excess heterozygosity with respect to the mutation-drift equilibrium, and iii)
Large differences between likelihood estimates of ancestral
and current population sizes. 2) If populations across the
introduced range are maintaining or recovering from the
potential depletion of variability caused by a bottleneck, then
we would find current amounts of variation comparable
with those of closely related long-established endemics. 3) If
introduced populations across the AZ and NP areas form
one cohesive unit, then we would find no significant genetic
structuring as demonstrated by lack of significant F statistic
values.
The predictions regarding the number and timing of
introduction events are the following: 1) If the current
genetic composition in the introduced range were the product of multiple introductions from already differentiated
populations in the source range, then we would detect signals of genetically differentiated clusters where either each
individual is itself the product of admixed origins or the
populations are composed of individuals that can be traced
as deriving from genetically differentiated populations. 2) If
the introduction of G. h. howdenae has been recent and aided
by humans, then we would observe a reduction in population size contemporaneous with either the establishment
of human settlements during the colonization period of
the islands between 1835 and 1959 or concurrent with the
rapid growth of human populations (between 1950s and
2001) (Gonzalez et al. 2008).
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Journal of Heredity
A. Galápagos Archipelago,
Ecuador
B.
0˚20’S
0˚30’S
Santa Cruz Island, Galápagos
SR01
SR11
SR12/SR17
SR09
SR08/SR18
SR06
SR07 Agricultural Zone
SR10
SR22
0˚40’S
Puerto Ayora
0
12 miles
0
0˚50’S
20 km
90˚30’W
90˚20’W
90˚10’W
Figure 1. Map of the island of Santa Cruz in the Galápagos Archipelago. Outline indicates the Agricultural Zone. Collecting
localities are labeled with population codes following Table 1. Inset A: Relative location of Santa Cruz within the archipelago.
Inset B: Relative location of the archipelago with respect to western South America.
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Mok et al. • Genetics of a Weevil Introduction to Galápagos
Materials and Methods
Sampling
We obtained samples of G. h. howdenae weevils from 9
localities across the AZ and NP areas of Santa Cruz in the
Galápagos archipelago (Figure 1, Table 1). Adult weevils
were collected by beating on introduced plant hosts such as
Erytrina, Guava and Aguacate with the exception of those
collected in El Chato (Figure 1, Table 1) which were found
feeding on Tournefortia rufoscericea, an endemic plant host,
alongside endemic congener G. conwayensis. Weevils were preserved in 100% ethanol until processed for DNA extraction.
In 2 cases, specimens from nearby sites or from the same
location collected in consecutive years were pooled and analyzed as a single locality (Media Luna and La Caseta, respectively, Figure 1, Table 1).
DNA Preparation and Genotyping
We used 3 legs from each specimen to isolate DNA according to the protocol of Normark (1996) or alternatively
used the DNeasy Tissue Kit (Qiagen, Valencia, CA). Two
microsatellite libraries were constructed using MseI and AseI
restriction enzymes, following the FIASCO (Fast Isolation
by AFLP of Sequences Containing repeats) protocol (Zane
et al. 2002; Stepien et al. 2010). Optimal PCR reaction and
cycling conditions differed across polymorphic microsatellite
loci (Stepien et al. 2010). Microsatellite variation was analyzed
for 8 polymorphic loci in 106 G. h. howdenae individuals from
multiple locations across Santa Cruz. Generally, amplification
reactions were performed in a final volume of 10 μl containing 1–2 μl DNA template, 1 μl 10× ThermoPol Reaction
Buffer containing 2 mM MgSO4 (New England Biolabs,
Ipswich, MA), Taq polymerase (1U) (New England Biolabs),
0–3.0 mM of MgCl2, 1 fluorescently tagged forward primer
(5 μM; HEX: hexachloro-fluoresceine, 6-FAM: 6-carboxyfluorescein, NED and PET: property of Applied Biosystems,
now Life Technologies, Foster City, CA) and reverse primers (5 μM), dNTPs (0.8 mM) (Invitrogen, Carlsbad, CA), and
high performance liquid chromatography water. Quartets
of polymerase chain reaction products were combined with
Table 1 Locality information: population codes, locality details,
and locality area designations for introduced Galapaganus h. howdenae
populations on Santa Cruz, Galápagos
Population
code
Locality
(altitude, in meters)
Zone
N
SR10
SR07
SR11
SR22
SR06
SR08/ SR18
SR09
SR12/SR17
SR01
Road to Cerro Croker (249)
Finca Steve Devine (351)
Above El Chato (422)
El Chato (106)
2 km north of El Cascajo (240)
Media Luna (469)
Miconia Zone (500)
La Caseta (613)
Cerro Croker trail (400–800)
AZ
AZ
AZ
AZ
AZ
NP
NP
NP
NP
15
15
4
9
15
13
6
14
15
N = number of individuals analyzed per locality.
LIZ600 or LIZ500 size standard (Applied Biosystems) and
run on an ABI3100 Genetic Analyzer. Genemapper version
4.3 was used to score positive reactions. In order to ensure
accuracy in allele calling, individuals from all localities were
randomly selected to be genotyped 2 or 3 times, and allele
sizes were verified through blind tests.
Genetic Analyses
Genetic Diversity, Population Differentiation, and Mixed
Ancestry
We determined microsatellite loci independence through the
linkage disequilibrium test in Genepop 4.0 (Raymond and
Rousset 1995). A probability test (G-test) was computed
for contingency tables of each loci pair generated using the
Markov chain algorithm with the following parameters: 10 000
dememorization steps, 100 batches, and 5000 iterations per
batch. Linkage disequilibrium tests in Arlequin version 3.11
(Excoffier et al. 2005) confirmed results from Genepop 4.0.
Microsatellite genetic diversity (Mean number of alleles,
A; expected and observed heterozygozity, HE and HO) and
deviation from Hardy–Weinberg equilibrium (Fis indices)
were determined per locus and per locality area (AZ, NP,
and all populations: Table 2) using Genepop 4.0. FIS indices
were calculated and tested for significance using exact tests
with the Markov chain algorithm with 10 000 dememorization steps, 20 batches, and 5000 iterations per batch (Haldane
1953; Guo and Thompson 1992) (Table 2). Molecular diversity indices obtained in Arlequin 3.11 (Excoffier et al. 2005)
confirmed results from Genepop 4.0. Statistical differences
in mean number of alleles and heterozygosity values between
the 2 locality areas were assessed using the Mann–Whitney
test (Mann and Whitney 1947).
We searched for signals of genetic structuring through
analyses of molecular variance (Amova) in Arlequin
3.11(Excoffier et al. 2005). The hierarchical distribution of
genetic variance was assessed for all localities as a single group.
To evaluate if there was any geographic pattern of genetic differentiation across the distribution range in Santa Cruz, we
grouped localities into AZ and NP. Additionally, we assessed
differentiation across pairs of localities computing pairwise
FST indices. All analyses consisted of 10 000 permutation
steps (Cockerham and Weir 1984; Cockerham and Weir 1987)
(Table 3) and were run in Arlequin 3.11(Excoffier et al. 2005).
We used the clustering approach implemented in
Structure 2.3 (Pritchard et al. 2000) to explore the likelihood of mixed ancestry in the introduced range of
G. h. howdenae in Galápagos. If we were to find evidence of
multiple population units within Santa Cruz, that could be
interpreted as multiple introductions from differentiated
continental sources. The natural logarithm of the probability
of the data set was calculated more than 10 trials per treatment using a burn-in of 10 000 and 90 000 replicates with
either independent or correlated allele frequencies for all
trials (Figure 2, Table 4). The admixture and no admixture
ancestry models were used in each case with and without the
addition of sample group information to aid in the clustering (LocPrior function)(Hubisz et al. 2009). For all ancestry
369
Journal of Heredity
Table 2 Genetic diversity in Galapaganus howdenae howdenae populations across the introduced range
Group
N
All loc
AZ
NP
106
58
48
#A
HO
HE
3.875 ± 1.536
3.375 ± 1.061
3.500 ± 1.309
0.4966 ± 0.266
0.4758 ± 0.269
0.5208 ± 0.271
0.5388 ± 0.146
0.5394 ± 0.126
0.5291 ± 0.182
FIS
0.0738*
0.1112*
0.0146*
Indices are calculated across all loci for all localities together and each locality area.
N = number of individuals; #A = mean number of alleles across all loci; HO and HE = observed and expected heterozygosity; All loc = FIS values for all
localities; AZ = localities in the Agricultural Zone; and NP = localities in the National Park.
*P < 0.05.
Table 3 Genetic structure across localities
Groups
Fixation indices
P values
All localities
AZ
NP
By locality area
(AZ and NP)
FST = 0.01359 (1.36%)
FST = 0.00060 (0.06%)
FST = 0.00834 (0.83%)
FSC = 0.00572 (0.56%)
FCT = 0.01390 (1.39%)
0.058
0.264
0.178
0.311
0.007
Hierarchical analyses of molecular variance (Amova) in introduced
Galapaganus populations, all grouped by locality (All Localities), only localities within the AZ, and only localities within the NP and all localities grouped
by area AZ and NP.
F statistics (FST, FSC, and FCT) among all localities, among localities within
areas, and among locality groups.
models, values of 1.0 and 10.0 were used for initial and maximum alpha, respectively, for all numbers of clusters (K) set
between 1 and 6. The locality groupings of AZ and NP as
designated populations were used as locality priors under
the LocPrior function, given the subtle genetic structure
detected between these 2 units (see Amova results below).
Following the recommendations for additional prior settings
(Pritchard et al., 2000), the probability that an individual is
an immigrant to the community or has a recent immigrant
ancestor, otherwise known as MIGPRIOR or v, was set at
0.05. To analyze results from multiple runs simultaneously,
we summarized the mean proportion of membership across
all trials and models either by measuring the deviation from
perfectly symmetrical assignments (1/k) for the runs without
a LocPrior or by calculating mean and standard deviation of
membership in the largest cluster across identical runs for
the LocPrior models (Table 4). Additionally, we explored the
possibility of multiple clusters both visually, with plots of
mean likelihood for increasing numbers of inferred clusters,
and analytically, through DeltaK values following the Evanno
method (Evanno et al. 2005). Structure results for all 8 sets
of runs (Admixture and No Admixture; correlated and independent allele frequencies; with and without addition of
LocPrior) were summarized and analyzed using Structure
Harvester (Earl and Vonholdt 2012) (Supplementary
Material Figure S1 and Figure 2).
Demographic History
Moment-based estimators. We used 2 lines of evidence to
search for genetic signals of a past reduction in population
370
size within the G. h. howdenae range in Santa Cruz potentially associated with its introduction to the archipelago. The
population bottleneck was explored through tests of excess
heterozygosity with respect to the mutation–drift equilibrium
and of a significantly reduced ratio of the number of microsatellite alleles to allele size range—known as the M ratio.
Bottleneck 1.2.02 (Piry et al. 1999) tested for heterozygosity excess with respect to mutation–drift equilibrium characteristic of population size reductions using the Sign test and
the Wilcoxon signed rank one-tailed test for excess (Table 5).
Three mutation models were used: infinite allele model
(IAM), stepwise mutation model (SMM), and 2-phase model
(TPM) with 1000 replicates for each model and parameters
derived from the G. h. howdenae data set implemented in the
TPM (36% stepwise mutations, variance = 130). As the variance in repeat number at a locus increases, the frequency or
magnitude of multi step mutations also increases (Di Rienzo
et al., 1994). Though low variance is recommended for
microsatellites (Di Rienzo et al., 1994; Piry et al. 1999), the
high percentage of large step mutations in the G. h. howdenae
data set prompted use of a higher variance value (Table 6).
In addition to the Sign and Wilcoxon tests, mode shifts
for allele distribution were calculated to detect any population bottleneck–related deviation from the normal allele distribution (with one most common allele size and other less
frequent allele sizes that differ by varying number of steps).
Because low-frequency alleles are lost during a population
bottleneck, this can be detected as a shift in the distribution
and frequency of allele sizes (Table 5).
During bottlenecks, the range of possible allele sizes stays
relatively constant, whereas the number of alleles decreases,
leading to a characteristic reduction in M ratio (Garza and
Williamson, 2001; Hawley et al., 2006). The software program
M (Garza and Williamson, 2001) was used to calculate the M
ratios and significant deviations from equilibrium situations
(Table 6). M ratios were calculated for all localities as a single
group, as well for the 2 locality areas: AZ and NP. The following
parameters were used in the analyses: 64–67% proportion of
nonstep mutations, 4.6–4.8 average base size of nonstep mutations, assumed marker mutation rate (μ) of 5 × 10−4 (Goldstein
and Schlotterer 1999), effective prebottleneck population size
of Ne = 2000 (Goldstein and Schlotterer 1999; Hawley et al.
2006), and 1000 simulations (Table 6). A recent study exploring the reliability of moment-based estimators—M ratio and
heterozygote excess—remarks that, in general, bottleneck
tests fail to detect declines when changes in population size
Mok et al. • Genetics of a Weevil Introduction to Galápagos
Figure 2. Structure v. 2.3 analysis of the G. h. howdenae populations in Santa cruz. (A) Plot of estimated ln likelihood against
number of inferred genetic clusters (K) and (B) Delta K values without (i) and with (v) the locality areas as locality priors, under
admixture with indepedent allele frequencies.
had been recent (Peery et al. 2012). Even when using the more
realistic 2-phased mutation model Peery and coauthors (2012)
suggest, among other things, increasing sample size and number of loci compared to add rigor to the inferences and warn
against underestimating the percentage of multi step mutation
in the TPM model. In line with those recommendations, in
this study we are including weevil samples spanning the entire
range of G. h. howdenae in Galápagos and using an elevated percentage of multi step mutations derived from our data set. The
number of polymorphic loci analyzed falls within the range of
most studies reviewed by Peery and coauthors (2012).
Likelihood-based methods. We inferred past population dynamics using a likelihood-based Bayesian method
(Beaumont 1999; Storz and Beaumont 2002) implemented in
Msvar 0.4 and Msvar 1.3. Simulations have shown that Msvar
is efficient at detecting population declines and expansions,
provided that the event is neither too weak nor too recent
(Girod et al. 2011). Additionally, simulations suggest that the
method is robust to moderate departures from a strict SMM
(Girod et al. 2011). The method implemented in Msvar 0.4
estimates the posterior probability distributions of the rate
of population size change (r = N0/N1), the time since the
population size change scaled by the current population size
(tf = ta/N0), and the mutation rate as θ = 2 N0μ, where μ is
the locus mutation rate. The population size changes inferred
in Msvar 0.4 were calculated under an exponential model
(Figure 3). To quantify and date the change in population size
we sampled from the posterior distribution of parameters r
and tf. We used rectangular prior distributions for log r, log tf
and log θ as well as combinations of starting values for each
parameter. Given that MSVAR 0.4 does not allow for the
direct calculations of the time of population change (ta ), this
was calculated from tf after independently determining the
current effective population size (N0) using the web-based
program ONeSAMP (Tallmon et al. 2008). This method
assumes that all loci are neutral and unlinked and is based on
simulations of a single closed population. The assumptions
detailed above seemed realistic in our study system, given the
nature of the markers themselves, the general lack of linkage
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Journal of Heredity
Table 4 Proportion of membership in clusters inferred from Structure v. 2.3 analyses of the introduced populations of Galapaganus
howdenae howdenae
No loc prior
Loc prior
Range [SSD (proportion
of membership − 1/k)/N]
Range [Mean and (SD)
proportion of membership]
Admixture
Independent
[7 × 10−4; 2 × 10−3]
Correlated
[5 × 10−6; 3 × 10−3]
No admixture
Independent
[0.9014 (0.006); 0.9279 (0.003)] (L)
[0.0186 [0.009]; 0.0721 [0.003] (S)
[0.7292 [0.005]; 0.8281 [0.004] (L)
[0.0092 [0.012]; 0.1719 [0.004] (S)
[5 × 10−5; 2 × 10−4]
Correlated
[2 × 10−5; 3 × 10−3]
[0.8699 [0.003]; 0.9388 [0.002] (L)
[0.025 [0.005]; 0.0612 [0.002] (S)
[0.5446 [0.124]; 0.775 [0.005] (L)
[0.091 [0.072]; 0.225 [0.005] (S)
Symmetry of assignments in the No loc prior runs illustrated through the range of sum of squared differences (SSD) between proportion of membership
and 1/k scaled by the number of values (N) calculated across 10 runs per set of conditions for different numbers of inferred clusters (k = 2–6).
Preponderance of assignments to a major cluster in the loc prior runs illustrated through the range of mean and SD values of proportion of membership
across runs for the cluster with the largest (L) and smallest (S) proportion of assignments calculated across 10 runs per set of conditions for different numbers of inferred clusters (k = 2–6).
Each run set was performed with different allele frequency models (independent and correlated), different ancestry models (admixture and no admixture)
and with or without the addition of locality prior information to aid in the clustering (Loc prior and No loc prior).
Table 5 Evidence for significant population size reduction through significant heterozygote excess
Sign test
All loc
AZ
NP
Wilcoxon test
Mutational model
P value
Mutational model
P value
Mode shift
IAM
SMM
TPM
IAM
SMM
TPM
IAM
SMM
TPM
0.0292*
0.4764
0.1561
0.0354*
0.5024
0.0431*
0.0374 *
0.4985
0.1810
IAM
SMM
TPM
IAM
SMM
TPM
IAM
SMM
TPM
0.0059*
0.4219
0.0098*
0.0039*
0.1914
0.0039*
0.0137*
0.4727
0.0195*
Normal (L shaped)
Normal (L shaped)
Normal (L shaped)
Reported P values for the Sign and Wilcoxon tests (one tailed for excess heterozygosity) for departure from mutation–drift equilibrium and occurrence of
allele frequency distribution mode shift across all loci for all localities (All loc), localities in the AZ, and localities in the NP.
Mutational models used in Bottleneck: IAM, strict (1-step) SMM, and TPM.
Table 6 Evidence for significant overall population size reduction through significant M ratio with chosen model parameters for Theta,
size, and percentage of nonstepwise mutation
Group
Theta
%Nonstep
Size nonstep
M
P value
All Loc
AZ
NP
4
4
4
0.67
0.64
0.64
4.6
4.8
4.7
0.414061
0.411157
0.410856
0.0224*
0.0923
0.0951
Ratio calculated for all localities (All Loc), localities in the Agricultural Zone (AZ), and localities in the National Park (NP).
*P < 0.05
detected among the loci studied, and the isolated state of
the island localities with respect to the continental range of
G. h. howdenae. We provided a broad prior distribution for
the values of Ne based on informal estimates of population
abundance over multiple field seasons (100 individuals for
372
the lower bound on the prior and either 1000 or 4000 for
the upper bound). Ne values have been reported as typically
much lower than census size (Frankham 1995). Empirical
studies have reported Ne/N to vary widely between populations depending on a variety of conditions, including sex
Mok et al. • Genetics of a Weevil Introduction to Galápagos
Figure 3. Population size change. Posterior distributions
of the demographic parameters on a logarithmic scale with
the Beaumont (1999) method. r = N0/N1 represents the ratio
of present to past populations size; (tf): (ta/N0) represents the
ratio between the time in generations of the population change
and the present population size.
ratios, variance in family sizes, and fluctuations in population
size (Falconer 1989; Mace and Lande 1991). An additional
factor in the calculation is the transformation of generations
into calendar years; given that there are no ecological studies
for G. h. howdenae, we incorporated generation time data from
closely related species (Lapointe 2000; Ju et al. 2011) alternatively using 1 or 0.5 years for all Msvar runs (either 1 or 2
generations per year).
Even though simulation studies have shown that the
scaled parameters are more precisely estimated than the
natural parameters and advocate drawing conclusions from
the scaled rather than the natural parameters (Girod et al.
2011), our interest in estimating the timing of the population size reduction and presumably the introduction of
G. h. howdenae into Galápagos, independently of the uncertainties described above, led us to implement the method
of Storz and Beaumont (2002) to directly estimate N0, Na
(or N1), and tf (Figure 4). Fourteen independent runs were
performed with different combination of priors, hyperpriors, starting values, and run lengths (Supplementary Material
Table 1). Runs with the same priors were repeated 3 times as
an initial test for convergence (Goossens et al. 2006; Bourke
et al. 2010).
For all Msvar runs (either in version 0.4 or 1.3) we discarded the first 10% of the updates. The outputs were analyzed using the Locfit and Coda packages in R (Loader 1996;
Plummer et al. 2006; R-Core-Team, 2012). Convergence and
stationarity of individual chains were checked visually and
using the Geweke (1992) and Heidelberger and Welch (HW)
(1983) diagnostic statistics. Results of individual runs were
Figure 4. Ancestral and present population sizes. Posterior
distributions on a logarithmic scale for past (N1) and current
(N0) effective population sizes from the Stortz and Beaumont
(2002) method. Results are shown using 2 alternative
generation times (g). Each line corresponds to a different run.
considered only if all variables (No, Na, tf, and θ) passed at
least 1 test (provided that the values for the Geweke statistic
Z were not too far beyond the 2.5% and 97.5% quantiles of
the null distribution [Normal 0,1] and displayed a P value
larger than 0.05 for the HW statistic). All runs were repeated
after the exclusion of 10 individuals consistently assigned to
smaller secondary clusters in the Structure 2.3 runs.
Results
Substantial Genetic Diversity
Linkage disequilibrium tests showed that the majority of
microsatellite loci pairs in G. h. howdenae localities (27 out of
the 28 pairwise comparisons) do not contain alleles that are
inherited together more often than expected by chance (P >
0.05) and that the remaining comparison rejects independence only marginally (P = 0.048).
Genetic diversity within assigned groups of localities
(AZ and NP) and within all localities grouped together was
assessed through calculation of average number of alleles
per locus and expected and observed heterozygosity values. The average expected heterozygote frequency across
all localities was 0.5388, and the number of alleles per locus
ranged from 2 to 7 (mean = 3.875, Table 2). A slightly larger
mean number of alleles across all loci was recorded for the
NP area compared with the AZ; however, those differences
are not significant (P = 0.8, Table 2). Similarly, neither the
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Journal of Heredity
mean observed nor the expected heterozygosity values differ significantly across the 2 areas (P = 0.5403, for both HO
and HE). As a comparison point, values of mean number
of alleles from the introduced localities of G. h. howdenae are
in line with those derived from 13 localities of G. conwayensis, a long-established endemic in the same island, (A = 3.3,
SD = 0.8, range [1.9–4.9], data available from the authors),
albeit with a different set of microsatellite loci. This suggests
that Galápagos populations of G. h. howdenae bear genetic
variability comparable with populations of long-established
endemic close relatives, despite their supposedly recent
establishment.
Overall, deviation from Hardy–Weinberg equilibrium
measured through FIS averaged across all the loci and all
localities resulted in significant deficiency of heterozygotes
(P < 0.01) (Table 2). Given the presumed recent introduction
of G. h. howdenae into Santa Cruz, this is not an unexpected
result because evolutionary forces such as genetic drift could
be acting within the introduced range (Stepien et al. 2010).
Genetic Homogeneity Across the Introduced Range
Results from the analyses of molecular variance (Amova)
indicate that the majority of the genetic variation present
lies within localities and that there is no marked structuring
among collecting sites. FST values calculated among all localities, AZ localities and NP localities, were all nonsignificant
(Table 3). In every case, the variance among populations
was a very small percentage of the total variance, ranging
from 0.06–1.36% (Table 3) and nonsignificant FST values.
Moreover, these results are in line with findings derived from
mitochondrial DNA sequence variation for a smaller number
of G. h. howdenae localities (Sequeira et al. 2012). The lack
of significant genetic differentiation among localities is also
consistent with expectations considering the flight capability
of G. h. howdenae and the potential homogenizing effects of
mobility in disturbed environments.
When localities are grouped according to their placement
in either the AZ or the NP, a significant P value was reported,
even though the variance between these 2 groups was a small
percentage of the total variance (Table 3). Additionally, significant pairwise FST values were only found between localities
in the AZ and the NP (6 among the 36 pairwise comparisons performed), not within either of the groups. Pairwise
FST values were also significant when we compared clusters
including all individuals from each locality area (FST = 0.015).
This suggests that despite a general trend indicating a genetically homogenous population across the introduced range of
G. h. howdenae in Santa Cruz, there is subtle genetic differentiation between the AZ and NP areas.
Contemporary Effective Population Size
Using ONeSAMP, our median estimate of Ne was 184.37
with 95% credible limits (CL) of 139.01–387.52 breeding individuals when using 100 and 4000 for the lower and
upper priors, respectively. However, in this particular case,
ONeSAMP values were not robust to changes in the priors
374
and produced a lower estimate when using 1000 for the
upper prior (median Ne = 60.09 with 95% CL of 51.98–81.78
breeding individuals). We chose to use the lower estimate,
Ne = 60.09, for quantifying the timing of population decline
(tf) from Msvar 0.4, given that the range of values seemed
more in line with our field observations of weevil abundance
over multiple field seasons.
No Clear Evidence of Multiple Introductions
Differences between values of ln likelihood of the data (Ln
PD) for increasing number of inferred clusters provide limited
information about the likeliest value of k and the existence
of structure across the introduced range for G. h. howdenae in
Galápagos; under some conditions indicating a single cluster as the most likely scenario while in others suggesting up
to four clusters (Figure 2A, Supplementary Materials Figure
S1A). Overall, the differences among the ln likelihood values
of runs with increasing k are modest, in some cases as small
as 50, and often associated with large variance within each set
of runs. Even in the light of the Delta K results suggesting
alternatively k = 2 or k = 3 as the uppermost hierarchical
level of structure (Figure 2B, Supplementary Materials Figure
S1B), we are skeptical to accept this as an indication of real
structure (and potentially of mixed origins of samples within
the introduced range) because no other indicators support
this result. In every case, random clustering models, assuming k populations = 2–6, showed symmetrical proportions of
membership in said number of clusters (Table 4). In the face
of real structure, proportions would be asymmetric and the
probability that a random individual had recent ancestry in a
particular population would be skewed. The correlated allele
frequency model showed slightly more asymmetric membership proportions than the independent allele frequency
model (Table 4), but there was still no strong asymmetric
assignment for 1 cluster, as demonstrated by an almost negligible deviations from complete symmetry in the assignments.
Without incorporating geographical information, we find no
strong support for the notion that the current populations of
G. h. howdenae in Santa Cruz could be the product of multiple
introductions from already differentiated source populations.
When incorporating the locality information (LocPrior
option) the proportion of membership within 1 cluster was
higher for both of the allele frequency models (Table 4).
A very large proportion of individuals from both predefined
zones (AZ and NP) were assigned to a single mixed cluster
(even with higher numbers of assumed clusters); whereas
a few, usually less than 10%, were assigned to one of the
other clusters (Table 4). The inferred ancestry of individuals
assigned to any cluster other than the larger cluster was above
0.6 only when the models incorporated correlated allele frequencies (either with or without admixture). The individuals
originate from localities within both areas: in most “admixture” runs from NP locality SR18 and AZ, SR11 and SR07,
whereas most “no-admixture” runs add 1 or 2 individuals from NP localities SR01 and SR17 and AZ, and SR22.
Although simulations indicate that the LocPrior model does
not detect structure when there is none (Hubisz et al., 2009),
Mok et al. • Genetics of a Weevil Introduction to Galápagos
the contribution of a second introduction pulse seems very
small compared with the one that originated the majority of
the introduced G. h. howdenae weevils now distributed across
the AZ and NP. An additional indicator of the importance
of location information under the LocPrior model is the
value of r, which parameterizes the amount of information
carried by the locations. High values of r, such as the ones
obtained in our analyses of the G. h. howdenae data set (from
0.1 to 16.3), suggest that either there is no population structure or the structure is independent of the localities. Given
the mixed composition of the smaller cluster with individuals
from both AZ and NP, it becomes clear that those a priori
locality areas we established (AZ and NP) bear no biological
or historical significance. The current composition across the
introduced range could be the combination of descendants
from a larger introduction pulse either preceded or followed
by a minor introduction event.
Evidence of a Past Reduction in Population Size
Tests in Bottleneck 1.2.02 for excess heterozygosity with
respect to the mutation–drift equilibrium gave differing
results, depending on the test and underlying mutational
model employed. For the IAM, a significant excess heterozygosity was found with both the Sign and Wilcoxon
one-tailed test for excess for all the locality configurations
tested (Table 5). Neither test found significant values under
the stepwise mutational model (SMM). Under the TPM, only
the Wilcoxon test found significantly excess heterozygosity
across all localities, localities in the AZ and NP, though the
Sign test under TPM was significant for the AZ. SMM and
TPM are generally more appropriate than IAM for microsatellite loci with TPM being the more suitable of the 2
(Di Rienzo et al. 1994; Luikart et al. 1998; Piry et al. 1999).
Bottleneck 1.2.02’s dependency on mutational model for
detection of excess heterozygosity should be taken into consideration. However, given that the Wilcoxon test is more
powerful than the other tests with a small number of loci
(<20), the significant P values found under TPM across all
the populations using the Wilcoxon one-tailed test for excess
should not be disregarded. Thus, the excess heterozygosity
could support the hypothesis of G. h. howdenae populations
having undergone a reduction in population size within 2–4
Ne generations (Luikart et al. 1998; Piry et al. 1999). The
mode shift test did not find significant deviations from the
normal L-shaped allele frequency distribution in the analyses for all localities, AZ and NP, which is contrary to our
expectations.
The significant M ratio found for all individuals as a single
group and its implication for population reduction is congruent with the excess heterozygosity results (Table 6). Although
the populations in the AZ and NP analyzed separately did
not show significantly reduced M ratios, all localities grouped
together showed a significant reduction (Table 6). Thus, both
moment-based estimator tests support the hypothesis that
G. h. howdenae populations as a whole have undergone a population bottleneck potentially due to a significant reduction
in population size during their introduction to Santa Cruz.
Likelihood-based estimators also support the proposed scenario of a population size reduction in the past (Figures 3 and
4). Using the method of Beaumont (1999), we found a clear
signal of past population decline (Figure 3). Under a model of
exponential change, the mode for the rate of size change was
estimated as log −3.62 (with a 95% highest probability density
interval, −4.41; −2.93). These estimates correspond to declines
larger than 1000-fold in effective population size. Using the
method of Stortz and Beaumont (2002) the signal for a past
reduction in population size is also quite clear under multiple
starting conditions (Figure 4). Interestingly, despite the sharp
decline, likelihood estimates of current population size indicate
substantial populations (Mode N0 298 individuals, 95% HPD
50–2300)—larger than anticipated from field observations.
Simulation studies have warned that even the full-likelihood Bayesian methods used here to detect population size
changes (Storz and Beaumont 2002) could be affected by the
presence of population structure in the sample and/or by the
lack of coverage in the sampling scheme (Chikhi et al. 2010).
However, we contend that the FST values reported here are
mostly nonsignificant or suggest very subtle structuring
across the introduced range and that our sampling covers the
entirety of the known distribution of G. h. howdenae in Santa
Cruz Island, making any population size changes detected
less likely to result from spurious signals.
Timing of the Population Size Change
Likelihood-based estimators provide differing assessments of
the timing of the reduction in population size, depending on
the value used in the calculations to incorporate the period
between reproductive cycles (generation time) and whether we
estimate the scaled parameters through Msvar 0.4 or the natural parameters through Msvar 1.3. When using 1 year as the
generation time, Msvar 1.3 estimates the mode of the density
distribution of the timing of population size change as 90 years
ago, whereas the mode is around 119 years ago when incorporating the shorter generation time in the initial conditions
(t = 0.5). The upper and lower limits of the 95% HPD interval
of the timing of the population size change under both sets
of conditions suggest a decline between 50 and 250 years ago.
Admittedly, this provides a wide time frame for the population
size change and presumably the introduction of G. h. howdenae
to Santa Cruz. The window for this change broadens further
and encompasses an earlier time frame when deriving the timing
from the scaled parameters (ta = tf × N0). Despite the narrower
range obtained for the scaled time parameter itself (90% HPD
interval log tf 0.48–0.82), the combination of the uncertainties
derived from 1) the time interval between reproductive cycles
included in the calculation to transform generations into years
since the population size change (1 vs. 2 generations per year),
2) the appropriate Ne/N ratio used for this particular situation
(0.8, 0.5, or 0.3), and 3) the 95% CL of the Ne values produced
by ONeSAMP generate a broader and less informative time
estimate for the introduction (107–800 years ago). In an attempt
to narrow the timing of the major introduction of G. h. howdenae, we excluded the individuals that were consistently assigned
to a smaller, distinct genetic cluster in the Structure v. 2.3 runs
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Journal of Heredity
(a total of 10 individuals) that presumably could represent a
separate smaller introduction pulse. With the culled data set, the
estimates of the timing of the population size change and, presumably, the predicted time frame for the larger introduction to
Santa Cruz Island narrow somewhat. Msvar 1.3 estimates the
mode of the timing of introduction to 65 years ago (95% HPD
interval 35–90) using 1 generation per year in the calculations.
Interpreting the results derived from both the culled and complete data sets under either set of run conditions, the reduction
in population size could be interpreted as a signature of an accidental introduction in historical times either contemporaneous
or at the end of Gonzalez’s et al. (2008) colonization period
(1862–1959) together with the establishment, rather than the
later growth, of human settlements.
Discussion
Range Expansion and Connectivity Maintaining Genetic
Diversity From a Single Introduction Pulse
One of the possible mechanisms proposed to mitigate the
allele loss that can be brought about by a population bottleneck is a rapid increase in population size once the new
population has been established (Zeisset and Beebee 2003;
Zenger et al. 2003). Similarly, to the situation posed by a natural colonization, the reduction in population size will contribute more substantially to the loss of genetic variability of
the newly founded population if the bottleneck is sustained
and even repeated (Clegg et al. 2002). Microsatellite genetic
variation in European rabbit populations (Oryctolagus cuniculus) in Australia showed that a rapid population expansion at
the time of establishment severely diminished the founder
effects normally associated with introduction (Zenger et al.
2003). Lack of reduced genetic variance was observed in the
introduced rabbit populations, whereas historical records of
the rabbit expansion across Australia gave sufficient support
for population growth as a mechanism for genetic diversity
retention (Zenger et al. 2003). Similarly, a study of the welldocumented marsh frog introduction in Britain revealed that
the rapid reproductive success of Rana ridibunda resulted in
effective colonization with little bottleneck effect on genetic
diversity (Zeisset and Beebee 2003). Indeed, the authors
concluded that significant bottlenecking was undetectable
despite the small number of founding individuals.
Similarly, G. h. howdenae populations in Galápagos, though
still displaying a signature of population size reduction,
may not have detectable signals of long-term genetic loss.
Measures of microsatellite genetic diversity in introduced
populations are in line with that of populations of longestablished endemic close relatives in the same island (in both
number of alleles and heterozygosity), even though the most
valuable contrast with G. h. howdenae populations in the native
rage is not available. Additionally, mitochondrial DNA variation found in some G. h. howdenae populations was comparable
with that of younger endemics close relatives (Sequeira et al.
2012). As for the processes underlying the retention of diversity, one possible explanation is that the observed expansion
in range might also be accompanied by an increase in census
376
numbers after a lag phase following the initial colonization
(Sakai et al. 2001; Holt et al. 2005). During the last 2 collecting trips to the Galápagos in 2010 and 2011, G. h. howdenae
individuals were found farther away from the AZ on the westward side of Santa Cruz as well as at high altitudes in the NP.
In the context of the flight capability of G. h. howdenae, it is
reasonable to predict that the range expansion will continue
across the island, possibly instigating a rapid revival of genetic
diversity.
In addition to an expansion in range, the lack of strong
genetic structuring found across G. h. howdenae localities supports the notion that gene flow could be a mechanism for
homogenizing the genetic diversity present. Though subtle but still significant structuring was detected between the
AZ and NP localities, the amount of variance between the
grouped populations was too small to ascribe any biological
significance to those groupings. Mishra and coauthors (2006)
suggested that frequent gene flow contributed to the high
levels of genetic diversity found within the localities of the
fungal pathogen Fusarium pseudograminearum. In the case of
the fungus, the novel genotypes resulting from regular gene
flow and random mating were suggested to contribute to
range expansion and heightened disease severity (Mishra et al.
2006). In G. h. howdenae populations, lack of genetic structuring suggests there is regular gene flow between relatively
distant localities, which would in turn increase the available
mating pool for individuals and could lead to eventual evolution of new genetic combinations. With observations supporting range expansion in G. h. howdenae, there is potential to
quickly overcome any allele loss caused by the population bottleneck and potentially adapt to new territory in Santa Cruz.
As noted by Keller and Taylor (2008), although it is tempting
to ascribe phenotypic divergence to adaptive evolution, evolution of phenotypic traits during dispersal, establishment, and
range expansion of introduced populations may reflect either
adaptive evolution or neutral phenotypic changes.
When disentangling the invasion history of island or continental introductions, multiple introduction pulses and admixture have been associated with maintenance of genetic diversity
(Kelager et al. 2013) and genetically diverse populations (Riva
Rossi et al., 2012)—the establishment of distinct strains (Lee
and Akimoto 2013), rapid evolution (Konečný et al. 2013), and
increase of invasive potential (Kolbe et al. 2004; Kolbe et al. 2007;
Roman and Darling 2007; Kolbe et al. 2008; Riva Rossi et al.
2012). Alternatively, admixture in the native range, long before
the introduction occurred, has been ascribed as the underlying
process generating exceptionally high genetic diversity in other
recently introduced island populations (Tonione et al. 2011).
However, it has also been suggested that the importance of
admixture to invasion success has been overemphasized, because
introductions are capable of being successful both in the presence and absence of admixture (Chapple et al. 2013).
With no evidence for mixed ancestry from substantial population admixture, there is no support for the idea that G. h. howdenae populations can rebuild genetic variability through 2 or
more equally large introduction pulses. Rather, the contribution
of admixture to the current genetic diversity levels seems modest at best, and the existing diversity needs to be attributed to in
Mok et al. • Genetics of a Weevil Introduction to Galápagos
situ demographic processes such as a rapid range expansion and
growth after the original introduction bottleneck.
Bottleneck of G. h. howdenae During Introduction
Microsatellite markers and moment-based estimators have
been successfully used to explore the introduction history of
species with diverse life histories and dispersal strategies such
as Chinese mitten crabs (Eriocheir sinensis) (Herborg et al. 2007)
and the common ragweed (Ambrosia artemisiifolia) in Europe
(Genton et al. 2005) and invasive house finches (Carpodacusm
mexicanus) in North America (Hawley et al. 2006). The sensitivity of microsatellite markers to reductions in population size up to 500 generations in the past provides a broad
time frame for the introductions to be studied (Garza and
Williamson 2001). Results from moment-based estimators in
G. h. howdenae support the hypothesis that introduced weevil populations on Santa Cruz have suffered a reduction in
population size in the past. These findings are consistent with
theoretical and empirical studies of recently introduced populations, where allelic richness was reduced to a greater extent
than heterozygosity, disturbing the mutation–drift equilibrium
(Cornuet and Luikart 1996; Allendorf and Lundquist 2003).
Although microsatellite markers continue to be widely
employed to study the introduction history of a wide array
of organisms (Garcia et al. 2012; Frisch et al. 2013; Ghabooli
et al. 2013; McMahon et al. 2013; Perdereau et al. 2013;
Simpson et al. 2013), more recently, moment-based estimators have been under scrutiny and their effectiveness in
detecting population declines has been questioned (Peery
et al. 2012). Bayesian coalescent methods have been adopted
to infer demographic histories (Jones et al. 2013; Paz-Vinas
et al. 2013), including the detection of severe population bottlenecks (Bourke et al. 2010). Several recent studies remark
that in order to avoid spurious signals of population decline,
details of the dispersal strategies and population structure of
the species under study cannot be overlooked (Girod et al.
2011; Paz-Vinas et al.2013). The results of the likelihoodbased estimators in island populations of G. h. howdenae support the notion of a reduction in population size possibly
linked to introduction. This result has 2 implications. The first
is that in G. h. howdenae the recovery from the genetic depletion
that should accompany the introduction can be accomplished
by mechanisms other than admixture and independent from
multiple introductions. The second implication is that along
with the substantial current population sizes estimated (even
though significantly smaller than the estimated pre-introduction ancestral sizes of putatively continental populations) we
have also observed a concurrent expansion in range that now
places endemic plants at an increased risk of herbivory.
An Older Introduction to Galápagos
The timing of other human-mediated plant and animal introductions to Galápagos (either confirmed or suspected) spans
all of Gonzalez’s (2008) time periods from when humans first
arrived in the archipelago, all the way to ongoing introductions (Bataille et al. 2009; Jager et al. 2009; Caetano et al. 2012).
Thorough reviews focusing on insect fauna in Galápagos
show that accidental insect introductions, among those beetles, have never been uncommon (Roque-Albelo and Causton
1999; Causton et al. 2000; Causton et al. 2006); humans have
accidentally carried more than 22% of the presently known
total beetle fauna (Peck 2005). The first records of an introduced beetle species date back to 1835: Dermestes maculatus
was presumably accidentally transported into the islands with
stored products. The most probable mode of accidental introduction proposed for G. h. howdenae is accompanying plant
products (Peck 2005). Our most conservative estimate of the
timing of the introduction of G. h. howdenae to Santa Cruz
predates the first collecting record in 1992 by at least four decades whereas our most extreme ones by almost a century. The
time estimates for the introduction of G. h. howdenae into Santa
Cruz supports our predictions of an accidental transfer during the colonization period (1832–1959). By most estimates,
the introduction of G. h. howdenae into the AZ predates the
spurt in human population growth that took place between
the 1950s and 2001. The timing of arrival of an introduced
species can feature in the estimation of its invasive potential,
given that it can inform the speed of dispersal and naturalization of the new arrival. Following scoring system of Causton
et al. (2006) for predicting invasiveness in the archipelago, an
older introduction would render a lower invasive ranking.
Conservation Implications
Fitness loss associated with introductions and population bottlenecks may occur due to diminished genetic variation and
exposure of detrimental alleles (Colautti et al. 2005). Sufficient
genetic variability and its presumed links to fitness are necessary to permit adaptation and expansion of an introduced
species in the introduced range (Genton et al. 2005). Despite
the intermediate invasive ranking attached, G. h. howdenae has
successfully adapted to at least 2 different environments in
Santa Cruz, and its flight capability suggests that it could
also spread further across the island and into other islands.
Given the results of this study, we must incorporate to our
current knowledge the combined effect of substantial population sizes and expansions in introduced range and feeding
preferences. Because this introduced weevil’s herbivorous
preferences include several endemic host plants (Scalesia and
Tournefortia), the ecological impact would not only be limited
to damage to endemic plants, but the forced resource sharing between introduced and endemic Galapaganus weevils,
specifically those with specialized highland habitats and with
restricted populations. Even though not of the highest conservation priority, the situation merits consideration when
bearing in mind the central geographical location and logistical relevance of Santa Cruz within the Galápagos archipelago.
Results from this study as well as of others evaluating transport of alien insect species within the archipelago (Roque
Albelo et al., 2006) can inform the design of measures that
attempt to control accidental interisland transfer.
It is unclear what effect, if any, interactions between
endemic and introduced Galapaganus species are having on
the genetic pool of both species. Given that the introduced
and endemic Galapaganus now share endemic plant hosts
377
Journal of Heredity
throughout part of the introduced range, future behavioral
and ecological studies should focus on the details of their
interactions and explore the possibility of interspecies hybridization. Furthermore, little is known about the host preferences and genetic status of source populations within the
native range in Ecuador, and samples from those locations are
currently unavailable. Direct comparisons between source and
introduced populations of G. h. howdenae may clarify whether
the genetic patterns observed in island populations are only
a function of the introduction history of these localities and
whether they are related to invasive potential of G. h. howdenae
. If genetic variability of the introduced populations proves
to be higher than in the native range, this could indicate that
introduced G. h. howdenae may be predisposed for successful establishment and expansion beyond their present range.
Thorough sampling of the native range would aid in identifying the precise geographic source of this island introduction.
Supplementary Material
Supplementary material can be found at http://www.jhered.
oxfordjournals.org/.
Funding
National Science Foundation (0817978 to A.S.S.); the
Brachman Hofmann Funds (to A.S.S); the Howard Hughes
Medical Institute (52006325 to C.C.S and A.S.S. through
Wellesley College).
Acknowledgments
Scientific research permits for the collecting trips were obtained from
the Parque Nacional Galápagos through the Department of Terrestrial
Invertebrates at the Charles Darwin Research Station (Santa Cruz). We
gratefully acknowledge A. Lanteri, S. Cárdenas, L. Cruz (and the crew from
“El Pirata”), H. Herrera, P. Lincagno, A. Mieles, A. M. Ortega, J. Rosado, and
station volunteers for invaluable assistance in the field. Field logistical support was provided by S. Cisneros, P. Couenberg, and R. Pépolas, Division of
Visiting Scientists at the Charles Darwin Research Station.
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Received July 17, 2013; First decision October 16, 2013;
Accepted December 3, 2013
Corresponding Editor: Christopher Smith