Molecular Ecology (2011)
doi: 10.1111/j.1365-294X.2011.05054.x
The importance of pre-mating barriers and the local
demographic context for contemporary mating patterns
in hybrid zones of Eucalyptus aggregata and Eucalyptus
rubida
D A V I D L . F I E L D , * D A V I D J . A Y R E , † R O B E R T J . W H E L A N † and A N D R E W G . Y O U N G ‡
*Department of Ecology and Evolutionary Biology, University of Toronto, 25 Willcocks Street, Toronto, ON M5S 3B2, Canada,
†Institute for Conservation Biology, School of Biological Sciences, University of Wollongong, NSW 2522, Australia, ‡CSIRO
Plant Industry, GPO Box 1600, ACT 2601, Australia
Abstract
The frequency of hybridization in plants is context dependent and can be influenced by
the local mating environment. We used progeny arrays and admixture and pollen
dispersal analyses to assess the relative importance of pre-mating reproductive barriers
and the local demographic environment as explanations of variation in hybrid frequency
in three mapped hybrid zones of Eucalyptus aggregata and E. rubida. A total of 731 openpollinated progeny from 36 E. aggregata maternal parents were genotyped using six
microsatellite markers. Admixture analysis identified substantial variation in hybrid
frequency among progeny arrays (0–76.9%). In one hybrid zone, hybrid frequency was
related to pre-mating barriers (degree of flowering synchrony) and demographic
components of the local mating environment (decreasing population size, closer
proximity to E. rubida and hybrid trees). At this site, average pollen dispersal distance
was less and almost half (46%) of the hybrid progeny were sired by local E. rubida and
hybrid trees. In contrast, at the other two sites, pre-mating and demographic factors were
not related to hybrid frequency. Compared to the first hybrid zone where most of the
E. rubida (76%) and all hybrids flowered, in the remaining sites fewer E. rubida (22–
41%) and hybrid trees (0–50%) flowered and their reproductive success was lower (sired
0–23% of hybrids). As a result, most hybrids were sired by external E. rubida ⁄ hybrids
located at least 2–3 km away. These results indicate that although pre-mating barriers
and local demography can influence patterns of hybridization, their importance can
depend upon the scale of pollen dispersal.
Keywords: hybrid zones, hybridization, mating patterns, paternity analysis, pollen dispersal,
reproductive isolation
Received 6 February 2010; revision received 24 January 2011; accepted 4 February 2011
Introduction
Natural hybridization is widespread among plant species (Mallet 2005). Many species pairs, however, exhibit
dramatic variation in hybrid frequency at both the individual and population level (e.g. Field et al. 2008). It is
important to understand the basis of this variation
Correspondence: David L. Field, Fax: +1 416-978-5878;
E-mail: [email protected]
Ó 2011 Blackwell Publishing Ltd
because the evolutionary outcome of hybridization will,
in part, depend on the frequency of hybridization
within populations (Rieseberg & Carney 1998). In addition, frequent hybridization among common and rare
species may increase the risk of local extinction of the
rare species because of pollen swamping and dilution
of the gene pool (Rhymer & Simberloff 1996). For many
species, pre-mating isolation and post-zygotic hybrid
inviability severely limit the incidence of hybridization
(Rieseberg & Carney 1998). The demographic context in
2 D. L. FIELD ET AL.
which reproduction occurs can also play a critical role
in determining the frequency of interspecific mating.
This is particularly important for plants because their
sessile nature results in mostly local mating and dispersal (Ellstrand 2003). The amount of interspecific pollen received by individuals is therefore expected to be
influenced by factors related to their local mating environment.
Hybrid zones provide an excellent opportunity to
study the factors responsible for reproductive isolation
(Hewitt 1988). Most studies of hybrid zones have used
the genetic structure of adult populations to infer the
factors likely to be responsible for reproductive isolation (Jiggins & Mallet 2000). However, genetic estimates
of mating patterns using progeny arrays may provide a
more accurate assessment of the factors responsible for
variation in hybrid production. The availability of
highly polymorphic markers (e.g. microsatellites) and
powerful statistical analyses to identify hybrids (e.g.
Bayesian clustering) and assign paternity to offspring
from known mothers, provide important methods for
describing mating patterns in hybrid zones. Reports of
hybrids in populations consisting of only one parental
species (Lepais et al. 2009) indicate that long-distance
pollen dispersal between populations can also shape
variation in hybrid production. Therefore, a spatially
explicit framework is required to assess both hybrid frequencies and pollen dispersal patterns within and
among populations. Surprisingly, few studies have
examined contemporary mating patterns within hybrid
zones (but see; Bacilieri et al. 1996; Hodges et al. 1996;
Valbuena-Carabana et al. 2005), particularly in relation
to characteristics of the local mating environment. As a
result, the relative importance of long-distance dispersal, pre-mating barriers, and local demography in
shaping observed patterns of hybridization is not well
understood.
We know little about mating patterns in hybrid zones
of animal-pollinated trees, for which the scale of pollen
dispersal and the relative importance of pre-mating barriers and local demography may differ from wind-pollinated systems (e.g. Quercus; Bacilieri et al. 1996). The
genus Eucalyptus (Myrtaceae), which contains 700 species (Potts & Wiltshire 1997), provides a good model
system for investigating contemporary patterns of
hybridization. Eucalyptus are long-lived, animal-pollinated trees that dominate forest vegetation communities
throughout Australia. On the basis of morphological
patterns, interspecific hybridization is considered widespread in Eucalyptus (50% of species hybridize; Griffin
et al. 1988), and several reports have found evidence of
substantial variation in hybrid frequency (Potts et al.
2003). For example, genetic analyses of hybrid zones
between Eucalyptus aggregata and E. rubida identified
striking levels of variation in hybrid frequencies among
progeny arrays (0–57%; Field et al. 2008). The study of
mating patterns in hybrid zones of Eucalyptus may
therefore be useful in developing our understanding of
the processes maintaining species barriers in long-lived,
animal-pollinated trees.
Here, we investigate mating patterns within hybrid
zones of E. aggregata and E. rubida located in southeastern Australia. These species are well differentiated morphologically (Field et al. 2009) and genetically on the
basis of microsatellite markers (mean FST = 0.21) (Field
et al. 2011). Moreover, there are strong differences in
habitat preference with E. aggregata found on poorly
drained flats and E. rubida on well-drained skeletal soils
of loams on clay subsoils (Cayzer 1993). Where the species co-occur, both F1- and later-generation hybrids
have been identified, particularly in transition zones
between the parental habitats (Field et al. 2011). Previous reports have indicated that hybrids are fertile and a
range of hybrid generations are present in seed cohorts
and in adult populations (Field et al. 2008, 2011). Partial
overlap in flowering times between the parental species
(October–January for E. rubida, December–February for
E. aggregata) likely provides opportunities for interspecific pollen flow. Given the considerable range in hybrid
production in open-pollinated progeny arrays of
E. aggregata, this system provides a great opportunity
to examine the influence of various pre-mating and
demographic variables on hybrid frequencies.
In this study, we address the importance of flowering
synchrony, local demography and long-distance pollen
dispersal in shaping mating patterns between E. aggregata, E. rubida and hybrids. We examine the relationship between a set of spatially explicit pre-mating and
demographic variables, and hybrid frequency among
progeny arrays from different maternal parents. To
describe mating patterns within the hybrid zones, we
use Bayesian approaches, paternity analysis and indirect estimates of pollen dispersal parameters. We
selected three hybrid zones differing in demographic
variables, which contain populations of E. aggregata,
E. rubida and their hybrids. Eucalyptus aggregata was
chosen as the seed parent because molecular evidence
suggests asymmetrical hybridization, with E. aggregata
more often the maternal parent (Field et al. 2011). This
asymmetry probably reflects differences in style lengths
(E. rubida 7 mm, E. aggregata 4 mm), which can
limit pollen tubes of smaller-flowered species from fertilizing larger-flowered species (Gore et al. 1990). Specifically, we address the following questions: (i) Is the
frequency of hybridization related to pre-mating barriers (flowering synchrony)? (ii) Is the frequency of
hybridization related to demographic variables of the
local mating environment (population size, relative
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MATING PATTERNS IN EUCALYPTUS HYBRID ZONES 3
abundance, proximity)? (iii) What is the relative importance of local compared to inter-population pollination
in hybrid zones?
Materials and methods
Study sites
This study was conducted within three separate hybrid
zones located 40 km east of Canberra on the Southern
Tablelands of New South Wales, Australia (Fig. 1). In
each of these sites, all reproductive Eucalyptus aggregata,
E. rubida and hybrids (total N = 421) were previously
mapped and genotyped to confirm ancestry (Field et al.
2011). Site 1: Bendoura—a relatively intact open woodland; site 2: Duck Flat—a fragmented population in
grazing land; and site 3: Norongo—a small disturbed
population along a roadside (Table 1). These Eucalyptus
species are mass-flowering, with up to thousands of
hermaphrodite flowers open simultaneously. Like most
Eucalyptus, they have mixed-mating systems and are
pollinated by a range of insects, small marsupials, and
birds. At these sites, only the introduced honeybees
(Apis mellifera) were seen foraging within the flowers of
the two study species (Field et al. 2008). However,
other animals such as birds could account for rare,
long-distance dispersal between populations. The only
other Eucalyptus species at these sites was E. pauciflora,
which is distantly related and not reported to hybridize
with either E. aggregata or E. rubida (Griffin et al.
1988).
Flowering synchrony
Flowering observations were conducted every 10–
12 days from December 2003 to February 2004. Using a
telescopic lens, trees were assessed for the presence or
absence of any opened flowers. At Duck Flat and Norongo, every tree within the population was observed
for flowering at each interval. At Bendoura, all E. aggregata trees sampled for seed, and an additional random
sample of 50 E. aggregata and 50 E. rubida trees, were
monitored as a sample of the flowering effort of these
species.
Fig. 1 Map showing the location of three study sites, each consisting of populations of Eucalyptus aggregata, E. rubida and their
hybrids. Individual maps of each site indicate the area covered by E. aggregata (dark grey areas) and E. rubida (light grey areas), the
location of E. aggregata trees sampled for open-pollinated seed arrays (filled symbols) and the location of hybrid trees (stars). Arrow
at site 3 indicates an E. aggregata tree sampled for seed that is located next to four hybrids.
Ó 2011 Blackwell Publishing Ltd
4 D. L. FIELD ET AL.
Table 1 Site characteristics of three hybrid zones of Eucalyptus
aggregata and E. rubida (Bendoura, Duck Flat, Norongo), and
the proportion of purebred and hybrid offspring within openpollinated progeny arrays collected from E. aggregata mothers
Parameter
Bendoura
Duck Flat
Norongo
Latitude
Longitude
Density (trees ⁄ ha)
N adults
(E. aggregata)
N adults (E. rubida)
N adults (hybrids)
N mothers
N progeny
E. aggregata (%)*
Hybrid (%)*
Hybrid range (%)†
35°30¢ S
142°42¢ E
33.7
171
33°28¢ S
150°02¢ E
13.1
18
32°42¢ S
149°25¢ E
6.3
10
119
47
22
467
89.9
10.1
0–76.9
24
17
11
211
81.5
18.5
0–57.2
9
6
3
53
64.2
35.8
23.5–56.3
N adults, number of adults sampled for genotyping; N
mothers, number of purebred E. aggregata adults sampled for
open-pollinated seed arrays; N progeny, total number of
progeny analysed; *percentage of purebred E. aggregata and
hybrid offspring detected in progeny arrays among mothers;
†range in the percentage of hybrids detected in progeny arrays
among mothers.
We used the following methods (modified from Augspurger 1983) for calculating an index of flowering synchrony, Xik, for individual i with species k, which is
defined as:
Xik ¼
X
1
1 n
ej6¼i
n ÿ 1 fi j¼1
where ej = the number of observation days both individual i and j are flowering synchronously, j „ i;
fi = the number of observation days individual i is flowering; n = the number of individuals in surveyed population of species k.
The flowering synchrony index was calculated for
each maternal tree (sampled for seed) with respect to
the E. aggregata (Xia) and E. rubida (Xir) population at
each site. When Xik = 1.0, perfect flowering synchrony
occurs, whereas for Xik = 0, there is no synchrony. This
was the best method for measuring flowering synchrony for Eucalyptus owing to the difficulty in obtaining flower counts in mass-flowering trees. We also
calculated a relative flowering synchrony Ri for each
individual i, defined as:
Ri ¼ Xi a=Xi r
This was used as a measure of the relative flowering
display of the two species during the period each individual tree was flowering.
Demographic context
Eight variables characterizing the maternal tree and its
local demographic context were measured: (i) the number of all compatible tree species (E. aggregata, E. rubida
and hybrids), (ii) the number of E. aggregata trees, (iii)
the number of E. rubida and hybrids, (iv) the relative
abundance of E. aggregata compared to E. rubida and
hybrids, and the average distance to the five nearest
neighbour trees for (v) all compatible tree species, (vi)
E. aggregata only, (vii) E. rubida and hybrids and (viii)
relative nearest neighbour distances between E. aggregata and the combined E. rubida and hybrids. Variables
(i)–(iv) were measured at a range of spatial scales
defined by incremental concentric circles around each
maternal tree from a radius of 20 to 160 m (i.e. 20,
40,...., 160 m). Simulated matings between purebred
E. aggregata mothers and either E. rubida or hybrid
fathers resulted in offspring genotypes with similar
admixture coefficients (0.1 £ Q1 £ 0.9; see genetic analysis) (Field et al. 2011). Given that F1 hybrids and backcrosses are difficult to distinguish with these markers,
but either E. rubida or hybrid fathers result in admixed
offspring, we combined these classes of individuals
(E. rubida and hybrid) for several of the demographic
variables.
Seed sampling
To examine variation in hybrid production among
maternal trees, open-pollinated progeny arrays (families) were collected from randomly selected flowering
E. aggregata from Bendoura (N = 22), Duck Flat
(N = 11) and Norongo (N = 3) (total N = 36 trees)
(Fig. 1) (Table. 1). Because Eucalyptus has a canopy
stored seed bank, seeds were only collected from new
capsules following the flowering observation period
(2003–2004) after a 12-month maturation period. Bulked
collections of up to 300 open-pollinated capsules were
collected from the canopy of each tree. For each tree,
up to 100 randomly selected seeds were germinated
under glasshouse conditions and 15–30 germinated
seedlings were randomly selected for genotyping. Previous work indicated that the majority of F1 hybrids
could be distinguished from purebred seedlings on the
basis of intermediate morphology for several leaf and
stem traits; however, identification of backcross hybrids
was less accurate (Field et al. 2009).
Genetic analysis
Seedling leaves were stored at )80 °C until ground to
powder using a Mixer Mill MM 300 (Qiagen). DNA
was extracted from 10 mg of ground material using
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MATING PATTERNS IN EUCALYPTUS HYBRID ZONES 5
DNeasy 96 Plant extraction kits (Qiagen) (as per the
manufacturer’s protocol), with 1–2 lL (2–15 ng) of DNA
used for polymerase chain reaction (PCR) in a final volume of 10 lL (Field et al. 2011). All seedlings were
genotyped for six microsatellites markers previously
used to genotype all of the adult trees at each of the
three sites. Amplified fragments were separated by capillary electrophoresis on an ABI 3130 Genetic Analyser
(Applied Biosystems), and fragment sizes were assessed
using GenemapperÒ version 4.0 with an internal lane
standard (GS-500 LIZ; Applied Biosystems) as described
in detail elsewhere (Field et al. 2011). Individuals with
missing peaks or for which mis-matching occurred
between progeny and known mother were amplified
and genotyped a second time. No evidence of null
alleles was detected at Duck Flat or Norongo using the
software MICROCHECKER (van Oosterhout et al. 2004). At
Bendoura, there was a low probability (0.05) of a null
allele at locus Fmrsa1. However, we used all loci, as the
removal of the Fmrsa1 locus had no effect on paternity
or STRUCTURE analyses.
Across the three populations, the mean number of
alleles per locus ranged from 4.7 to 9.3 and 6.7 to 12.8
per locus for E. aggregata and E. rubida, respectively.
Mean gene diversity (He) ranged from 0.65 to 0.7 and
0.75 to 0.79 in E. aggregata and E. rubida, respectively
(Field et al. 2011). Six loci were sufficient to distinguish
any single adult tree at each of the sites (i.e. each adult
had a unique multilocus genotype). The unique multilocus genotypes and the high cumulative paternity exclusion probability at each locus (>0.98) confirmed there
are no clones in the population and paternity could be
assigned to seedlings with high statistical confidence.
Frequency of hybridization
Hybrid seedlings were identified from admixture coefficients (Q) using Bayesian methods implemented in the
program STRUCTURE version 2.2 (Falush et al. 2003). For
all analyses, we used STRUCTURE with the admixture
model, no prior population information, correlated
allele frequencies, a burn-in period of 20 000 and
2 00 000 MCMC. We also assumed K = 2 (Q1 =
E. aggregata, Q2 = E. rubida) as this agrees with the
presence of two species and was the most likely number of genetic clusters (Field et al. 2011). To check the
performance of Bayesian assignment methods in the
detection of hybrids, a previous study generated simulated individuals (purebred, F1, backcross generations
in both parental directions) and reported Q = 0.9 had
the highest efficiency and accuracy for identifying
early-generation hybrids (Field et al. 2011). Therefore,
we classified individuals with Q1 > 0.9 as E. aggregata,
Q1 < 0.1 as E. rubida and 0.1 £ Q1 £ 0.9 as hybrids. This
Ó 2011 Blackwell Publishing Ltd
threshold is expected to be a conservative estimate of
hybrid frequency, as the simulations indicate a slight
underestimation of the true hybrid frequency (actual
hybrid frequency = 7.4%, detected hybrid frequency = 7.1%; Field et al. 2011). Thus, the frequency
of hybridization was calculated as the proportion of all
hybrid types (i.e. F1, backcrosses) in the open-pollinated
seed array of each maternal plant.
Separate linear regressions were performed using R
version 2.12.1 to examine the relationship between each
predictive variable (individual pre-mating traits and
demographic context) and the response variable (proportion of hybrid offspring per individual). The hybrid
proportions used in the regressions consisted of all
hybrid types. Norongo was excluded from each of these
analyses owing to a lack of data points (N = 3 maternal
trees). Bonferroni corrections were used to ensure conservative tests of significant relationships between predictive and response variable for multiple analyses (36
regression analyses performed). Corrected alpha (a) values resulted in no significant relationships for any premating or demographic variables, therefore following a
less conservative approach (Moran 2003); we report
uncorrected standard a = 0.05 for all tests.
Paternity analysis
To identify the fathers of purebred and hybrid progeny
(classified with STRUCTURE) as well as rates of inter-population pollen dispersal, we performed paternity analysis
using the maximum-likelihood methods implemented
in the program CERVUS (Marshall et al. 1998). Paternity
analysis (assignment of a father to a seedling from a
known mother) was conducted on the genotypes of
progeny collected from maternal trees, with all reproductive trees at the site acting as potential fathers. For
each site, we ran 10 000 simulations to determine confidence levels, and using the following settings: (i) a minimum number of matching loci = 6; (ii) mistyping error
rate = 0.1%; (iii) proportion of candidates sampled =
0.95 (considering the presence of nearby populations);
(iv) proportion of loci typed = 0.99. All trees genotyped
were made available to the analysis as potential fathers,
including maternal trees (selfing). Considering recommendations by Slate et al. (2000), we accepted the single
most likely father at an 80% confidence interval when
LOD scores were ‡3, and we did not allow mis-matching for any of the six loci.
On the basis of the paternity analysis and STRUCTURE
classification, each seedling was assigned to (i) a known
E. aggregata father within the site, (ii) a known E. rubida
father within the site, (iii) a known hybrid father within
the site, (iv) an unknown (external) E. aggregata father
(no father located within the site and seedling classified
6 D. L. FIELD ET AL.
as purebred E. aggregata using STRUCTURE) or (v) an
unknown (external) E. rubida or hybrid father (seedling
classified as hybrid using STRUCTURE). The contemporary
rate of intra- and interspecific pollen immigration was
deduced from the percentage of seedlings sired by
external fathers. Considering that adult sampling and
genotyping at each site was exhaustive, unknown (not
genotyped) paternal parents must be located in surrounding populations outside the boundary of each
site.
The pattern of intra- and interspecific pollen dispersal
was evaluated by using the paternity assignments and
determining the distance between the mother and identified father that sired each offspring. The number of
observed dispersal events was plotted as a function of
distance between the mother and father for each offspring (excluding offspring sired by trees external to
site). This analysis was conducted at the two larger
sites, Bendoura and Duck Flat, which had sufficient
sample sizes of both maternal trees and progeny
arrays.
Effective pollen dispersal
To estimate pollen dispersal parameters including the
scale (a), shape (b) and average pollen dispersal distance (d), we used the indirect estimations employed by
KINDIST in the software package POLDISP (Robledo-Arnuncio et al. 2006, 2007). Norongo was excluded from KINDIST analysis owing to insufficient maternal parents and
spatial genetic structure. A significant negative correlation between correlated paternity and distance between
sib-ship pairs at Bendoura (rs = )0.19, P < 0.01) and a
marginally significant and negative correlation at Duck
Flat (rs = )0.18, P < 0.08) suggests pollen dispersal
parameters can be estimated with these data (recommended rs < )0.10; Robledo-Arnuncio et al. 2007). As
variation in selfing rates among mothers may influence
(a)
(b)
levels of correlated paternity, as suggested by RobledoArnuncio et al. (2007), we removed selfed progeny
(identified with CERVUS) from the analyses. The probability of effective pollen dispersal with distance was modelled for parameter estimates of the dispersal curve and
fitted for three different probability density functions
including (i) a normal distribution curve, (ii) a oneparameter exponential curve and (iii) a two-parameter
exponential-power curve. A threshold distance of 200 m
at Bendoura and 380 m at Duck Flat was chosen on the
basis that these had the most consistently low leastsquared residuals. Analyses for each model were
repeated five times to test for the stability of parameter
estimates, and least-squared values were used to evaluate the best model fit.
Results
Hybridization among progeny arrays
Trees within the Bendoura hybrid zone exhibited the
greatest range in hybrid frequency (Table. 1); however,
fewer of the trees produced hybrids (72%) compared to
Duck Flat (81%) and Norongo (100%). The overall proportion of hybrids detected was lowest in the largest
population Bendoura with 10.1% hybrids, followed by
Duck Flat with 18.5% and Norongo with 35.8%
(Table 1). Overall, the majority of the admixture coefficients (Q1) of the assigned hybrids ranged from 0.90 to
0.23 (Fig. 2). Two individuals at Norongo with Q1 0.2
probably reflects the lower genetic differentiation of
parentals at this site, or crosses between Eucalyptus
aggregata and a more advanced generation hybrid.
Flowering synchrony and hybrid frequency
The majority of the trees at the two largest sites (Bendoura and Duck Flat) flowered during the course of the
(c)
Fig. 2 Ranked admixture coefficients (Q1) from Bayesian analysis of progeny arrays from three hybrid zones of Eucalyptus aggregata
and E. rubida. For each individual, the proportion of their multilocus genotype assigned to the E. aggregata genetic cluster (Q1, black
dots) and the 90% posterior probability intervals (grey bars) are shown for progeny (open-pollinated arrays) from purebred E. aggregata at each of three sites: (a) Bendoura, (b) Duck Flat, and (c) Norongo. Dashed lines indicate threshold Q1 > 0.9 for the assignment
of purebred E. aggregata, Q1 < 0.1 for E. rubida and 0.1 £ Q1 £ 0.9 for hybrids.
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MATING PATTERNS IN EUCALYPTUS HYBRID ZONES 7
season. Peak flowering for E. aggregata and E. rubida at
these sites was also separated by a similar amount of
time, 25 days at Bendoura and Duck Flat and
20 days at Norongo. At Bendoura, 65% of E. aggregata
adults, 41% of E. rubida, and 50% of the surveyed
hybrids flowered at some time during the observation
period. In comparison, at Duck Flat, only 66% of
E. aggregata, but 76% of E. rubida and 100% of hybrid
trees flowered. Norongo had the lowest flowering rates,
with 40% of E. aggregata, 22% of E. rubida and no
hybrids flowering during the survey.
Substantial overlap in flowering was observed
between purebred E. aggregata and both E. rubida and
hybrids at the two largest sites (Bendoura and Duck
Flat) (Fig. 3). However, at Norongo, there was limited
flowering overlap between E. aggregata and either
E. rubida or hybrids. At Duck Flat, the proportion of
hybrids in progeny arrays had a positive relationship
with the degree of flowering synchrony with the E. rubida ⁄ hybrid population (R2 = 0.51, F1,9 = 10.44, P = 0.01;
(a)
(b)
Fig. 4a). There were no significant relationships
between the proportion of hybrids and flowering variables at Bendoura and Norongo.
Demographic context and hybrid frequency
The demographic context of trees from which progeny
arrays were sampled varied substantially among individuals and sites owing to local differences in tree density and position in the hybrid zone. At Duck Flat, the
proportion of hybrids in progeny arrays had a strong
negative relationship (at a scale of 60 m) with the
number of all compatible trees (R2 = 0.53, F1,9 = 12.35,
P = 0.007; Fig. 4b), the number of E. aggregata
(R2 = 0.58, F1,9 = 13.34, P = 0.007) and the number of
E. rubida and hybrids combined (R2 = 0.53, F1,9 = 11.41,
P = 0.009). These relationships were also detected at
other spatial scales at Duck Flat (80–160 m) but those
detected at 60 m explained the most variation (data
not shown). At Duck Flat, the hybrid frequency in
(c)
Fig. 3 The percentage of trees flowering at each survey (observation day) for population samples of Eucalyptus aggregata, E. rubida
and hybrids at three sites: (a) Bendoura (N = 72), (b) Duck Flat (N = 59) and (c) Norongo (N = 25). For each observation day, the percentage of flowering E. aggregata (black circles), E. rubida (open triangle) and hybrids (grey squares and dashed line) is indicated.
(a)
(b)
(c)
Fig. 4 Relationship between the percentage of hybrid progeny (in open-pollinated seed arrays) of Eucalyptus aggregata and (a) flowering synchrony with the E. rubida population (Xir), (b) the local population size (total number of E. aggregata, E. rubida and hybrids
within 60 m), and (c) distance to the five nearest neighbour (NN) E. aggregata trees relative to nearest five E. rubida. Linear regressions were used for each site separately: with individuals from Duck Flat indicated with filled symbols [flowering synchrony
(R2 = 0.51, P = 0.01), local population size (R2 = 0.53, P = 0.007), relative nearest neighbour distance (R2 = 0.56, P = 0.008)] and at
Bendoura with open symbols (P > 0.05). Trend lines are indicated with solid lines for Duck Flat. Arrows indicate outliers at Bendoura (see Results).
Ó 2011 Blackwell Publishing Ltd
8 D. L. FIELD ET AL.
progeny arrays also had a strong positive relationship
with the distance of E. aggregata trees relative to
E. rubida and hybrids (R2 = 0.56, F1,9 = 12.38, P = 0.008;
Fig. 4c). This indicates that more hybrid offspring are
produced by trees where the distance to the nearest
five E. aggregata was greater than the nearest five
E. rubida.
At Bendoura, the proportion of hybrids was moderately and positively correlated (at a scale of 20 m) with
the number of all tree species (R2 = 0.34, F1,20 = 10.2,
P = 0.005) and the number of E. aggregata trees
(R2 = 0.38, F1,20 = 13.19, P = 0.002). There was also a
weak negative correlation with the proximity of all trees
(R2 = 0.24, F1,20 = 6.52, P = 0.02). However, at Bendoura,
these relationships were mostly driven by two outliers,
with substantially higher hybrid frequency (77% and
50%) compared to the remaining 20 trees (<25%).
Removal of these two trees resulted in no significant
relationships with any of the variables at this site. At
Norongo, no relationships were found between hybrid
frequency and any of the ecological variables (data not
shown).
Paternity analysis
Pollen parents could be assigned within the sites using
paternity analysis for 76.7% (Bendoura) and 75.8%
(Duck Flat) of offspring at the two largest sites, but to
only 52.8% of the offspring at the smallest site (Norongo)
(Fig. 5). Of the offspring where paternity could be
assigned, 97% exhibited LOD scores ‡3, indicating
strong assignment confidence in the first-candidate
father compared to a randomly selected father. Considering that adult sampling from within each site was
exhaustive, unknown pollen is likely from external
E. aggregata, E. rubida and hybrid trees located in nearby
populations. This shows that the overall proportion of
intra- and interspecific pollen immigration was 23.3% at
Bendoura, 24.2% at Duck Flat and 47.2% at Norongo
(Fig. 5). Therefore, a substantial proportion of pollen
moves large distances between populations as the
nearest external E. aggregata and E. rubida trees occur
2 km from Bendoura and 3 km from Duck Flat and
Norongo.
The importance of local versus external pollen sources
varied markedly for purebred and hybrid offspring.
Local E. aggregata pollen within the sites contributed to
82.6% (Bendoura), 82.5% (Duck Flat) and 82.4%
(Norongo) of the purebred progeny. In contrast, local
interspecific pollen (E. rubida and hybrid) contributed
to 23.4% (Bendoura), 46.2% (Duck Flat) and zero
(Norongo) of the hybrid progeny (Fig. 5). These results
indicate that compared to intraspecific pollen, relatively
more of the interspecific pollen is coming from
Fig. 5 Proportion of hybrid progeny sampled from Eucalyptus
aggregata mothers that were sired by local parents (E. rubida ⁄ hybrid father) and parents external to the three study sites
(paternity unknown). The proportion of purebred progeny
sired by local paternal parents (E. aggregata father), through
selfing, and sired by external E. aggregata paternal parents are
also shown.
distant sources, particularly for the smallest population
Norongo.
The frequency of pollen dispersal events inferred
from direct paternity showed a general decline with
distance between the mother and the assigned father
(Fig. 6). At Bendoura, interspecific pollen dispersal
events showed similar low frequencies (<4%) across
distance classes from 60 to 360 m (Fig. 6a). At this site,
E. rubida and hybrid trees sired a similar proportion of
the hybrid offspring (45% and 55%, respectively). In
contrast, the majority (81.0%) of interspecific pollen
flow at Duck Flat arrived from pollen parents within
the first 160 m (Fig. 6b). Furthermore, at Duck Flat,
local E. rubida were more successful fathers compared
to hybrid trees (sired 89.9% and 11.1% of assigned
hybrids, respectively).
Ó 2011 Blackwell Publishing Ltd
MATING PATTERNS IN EUCALYPTUS HYBRID ZONES 9
(b)
(a)
Fig. 6 The distribution of observed pollen dispersal events as a function of distance between the mother and the assigned father
(identified using paternity analysis) within two hybrid zones (Bendoura and Duck Flat) of Eucalyptus aggregata and E. rubida. Seedlings sired by fathers external to site are not indicated. Pollen dispersal events divided into intraspecific (E. aggregata father, indicated in black) and interspecific (E. rubida or hybrid father, indicated in grey) sources.
Effective pollen dispersal
Discussion
At Bendoura, both the normal and exponential dispersal functions exhibited similar dispersal parameters.
The exponential function had the best fit (Table. 2),
with a mean pollen dispersal (d) of 64.2 m and a long
extended tail of infrequent pollen movement (Fig. 7).
While the exponential-power function exhibited slightly
lower degree of error (least-squared residual), parameter estimates were highly variable between replicate
runs (data not shown), indicating an overall poor fit
with this model. At Duck Flat, all models exhibited similar dispersal parameters, but the best fit was with the
exponential model (Table. 2) where d = 21.6 m (Fig. 7).
Considering that d was less for all models at Duck Flat
compared to Bendoura, this suggests that pollen dispersal is more restricted in the smaller hybrid zone.
Determining which components of the local mating
environment influence patterns of hybridization among
species is important for understanding what maintains
distinct species identities. This study examined the
importance of pre-mating barriers, local demographic
context and long-distance pollen immigration in shaping mating patterns in three hybrid zones. At the
medium-sized site (Duck Flat), rates of hybridization
were related to both pre-mating barriers and the local
demographic context of the maternal parents (Fig. 4).
These variables, however, had little power to explain
patterns of hybridization in the smallest site Norongo
and the largest site Bendoura. The results of paternity
analysis showed substantial among-site variation in
the proportion of hybrids sired by local Eucalyptus rubida and hybrid trees. These combined results suggest
that for animal-pollinated tree species like Eucalyptus,
patterns of hybridization and associations with premating isolating barriers and demography may vary
markedly among hybrid zones within different landscapes.
Table 2 Effective pollen dispersal parameters (a, b, d) fitted
under three models (normal, exponential and exponentialpower), estimated for outcrossed seed arrays in two hybrid
zones of Eucalyptus aggregata and E. rubida (Bendoura, Duck
Flat) using the methods employed in KINDIST
Population Model
Bendoura
Duck Flat
a
Normal
17.46
Exponential
9.42
Exponential-power 0.00007
Normal
7.58
Exponential
4.00
Exponential-power 2.62
b
d
–
52.74
–
64.23
0.18 652.12
18.15
–
21.60
0.79 24.74
LS
54.98
52.77
49.63*
13.57
13.47
13.80
a, scale parameter homogenous to distance; b, shape parameter
affecting the extended tail of the dispersal curve; d, average
dispersal distance (m); LS, least-squared residual variance; The
best-fitted models (lowest LS and stable parameter estimates)
are indicated in bold.*indicates lowest LS but unreliable
parameter estimates (see Results).
Ó 2011 Blackwell Publishing Ltd
Pre-mating barriers and hybrid frequency
Hybrid frequency was strongly related to individual
flowering synchrony in one of the three E. aggregata
and E. rubida hybrid zones studied (Duck Flat). In this
site, individual E. aggregata tended to produce more
hybrid offspring when they exhibited greater flowering
overlap with the E. rubida population. This result is
likely because (i) greater flowering overlap would
increase the opportunity for interspecific transfer by
pollinators, and (ii) the dominant pollinator of these
species are generalist insects (honeybees) that likely
exhibit low floral constancy. Divergent flowering times
10 D . L . F I E L D E T A L .
(a)
(b)
can be a strong isolating barrier in both animal-pollinated (Marques et al. 2007) and wind-pollinated plant
species (Valbuena-Carabana et al. 2005). In Eucalyptus,
Adams et al. (1992) found that a divergence in peak
flowering time of only 2 weeks reduced inter-provenance crosses by up to 65%. Our results at one of the
hybrid zones also demonstrate that in some cases,
flowering synchrony can be an important predictor of
hybridization rates in natural Eucalyptus populations.
The lack of correlation between flowering synchrony
and hybridization at Bendoura is surprising considering E. aggregata individuals also exhibited a similar
range of flowering synchrony with the local E. rubida
population as observed at Duck Flat. However, almost
twice as many of the E. rubida population flowered at
Duck Flat compared to Bendoura and Norongo
(Fig. 3).
In studies of intraspecific pollen dispersal, both flowering intensity and tree size have been identified as
important factors related to male mating success in animal-pollinated tree species (Oddou-Muratorio et al.
2005). This is because pollinators are expected to alter
their foraging strategies to match changes in the distribution of floral resources. Optimal foraging theory predicts pollinators should concentrate their efforts on
denser patches of trees with higher floral rewards (Pyke
1984). Given that E. aggregata and E. rubida populations
are spatially segregated (Fig. 1), pollinators within a
site may be less inclined to forage outside dense floral
displays of E. aggregata when nearby E. rubida have
poor floral rewards. This scenario would enhance assortative mating among E. aggregata and reduce the siring
success of local E. rubida fathers, despite ample flowering synchrony between the species. In the case of Norongo, the failure of local E. rubida to sire hybrid
offspring is unsurprising considering their poor floral
display and asynchronous flowering with E. aggregata.
This is further supported by the paternity analysis,
which showed that local E. rubida fathers were much
less successful in siring hybrids at Bendoura and Norongo compared with Duck Flat.
Fig. 7 Effective pollen dispersal curves
within two hybrid zones (Bendoura and
Duck Flat) of Eucalyptus aggregata and
E. rubida. Each curve is derived from
parameter estimates for exponential
models given in Table 2, estimated
using KINDIST.
Local demographic context and hybrid frequency
In the case of E. aggregata, hybrid frequency in progeny
arrays was only strongly related to demographic variables at Duck Flat, which received high rates of withinpopulation pollen flow. This among-site variation may
reflect differences in plant density and the geometry of
the population, given that the spatial arrangement of
trees is well known to shape pollen dispersal patterns
(Oddou-Muratorio et al. 2005). In dense clusters of
plants, the pollen pool for individuals tends to be dominated by their nearest neighbours (Stacy et al. 1996)
because of restricted foraging range of pollinators. In
contrast, within sparse stands of plants, insect pollinators may be more likely to move between flowers of different plant species (Fenster 1991). Such changes in
pollinator behaviour may increase the probability of
interspecific pollen movement, particularly in populations with mosaic distributions of sympatric species.
There is also a growing body of evidence in both windand animal-pollinated plants that the relative abundance and proximity of sympatric species is important
in shaping the frequency of hybrids (Lepais et al. 2009).
For example, in the wind-pollinated Nothofagus obliqua,
isolated trees surrounded by the cross-compatible
N. nervosa produced more hybrid seed (Gallo et al.
1997). In our study, E. aggregata trees tended to
produce more hybrids when neighbouring E. rubida
were in closer proximity than E. aggregata (Fig. 4c). In
animal-pollinated trees such as Eucalyptus, these results
likely reflect differences in the local availability of interspecific pollen, combined with localized patterns of pollinator movement.
The relative importance of local population size, relative abundance and proximity can be difficult to distinguish in highly heterogeneous environments. This is
illustrated at Bendoura, where complex interactions
between local density, relative abundance, flowering
intensity and proximity may have affected the relationships observed. For example, in comparison with the
negative relationship between population size and
Ó 2011 Blackwell Publishing Ltd
M A T I N G P A T T E R N S I N E U C A L Y P T U S H Y B R I D Z O N E S 11
hybridization rate at Duck Flat, analyses at Bendoura
(with outliers included) indicated a weak positive trend
between these variables. However, in contrast to Duck
Flat, the paternity analysis results at Bendoura indicated
that the majority of hybrids were sired by fathers
located outside the site boundary. These findings illustrate that identifying the sources (fathers) of interspecific pollen may be vital for the correct interpretation of
the factors driving fine-scale patterns of hybridization.
Pollen dispersal
Our investigations using direct paternity analysis illustrate that the importance of local versus external pollen
parents can differ substantially among hybrid zones. At
the local scale, the paternity analysis confirmed that
E. rubida and hybrid trees sired hybrid offspring at
both Bendoura and Duck Flat. This result supports previous evidence of contemporary F1 and backcross
hybrid formation in progeny arrays at these sites (Field
et al. 2008, 2009), and the presence of hybrid swarms in
the adult cohorts (Field et al. 2011). At the landscape
scale, the considerable levels of immigrant pollen
detected in E. aggregata (23.3–47.2%) are similar to
reports in a hybrid zone of wind-pollinated Quercus
species (mean 36%; Valbuena-Carabana et al. 2005) and
intraspecific levels in animal-pollinated systems (e.g.
40%; Oddou-Muratorio et al. 2005). In our study,
intraspecific pollen immigration showed little variation
among sites (Fig. 5), but the contribution of external
interspecific pollen parents varied substantially for
hybrid offspring (53.8–100%). At Bendoura, the proportion of interspecific immigration was somewhat surprising, considering there was ample flowering synchrony
between E. aggregata and local E. rubida, although overall flowering display in E. rubida was low. In contrast,
the lack of assigned E. rubida and hybrid fathers located
within Norongo makes biological sense, as there was
limited flowering synchrony between E. aggregata and
local E. rubida or hybrid trees at this site. We are unaware of any studies that have reported such striking
differences in interspecific immigration rates among different hybrid zones. Our results could be explained by
differences in both the level of flowering synchrony and
the size of the floral display of E. rubida populations
external to the main study sites (2–3 km distant).
Indirect estimates of effective pollen kernels (exponential model) in E. aggregata confirmed that local pollen
parents sire a greater proportion of the offspring, with a
long tail of less-frequent and long-distant pollen dispersal events. The average pollen dispersal distances we
estimated (Duck Flat, d = 21.6m; Bendoura d = 64.2m)
were substantially less than reports in a number of windpollinated (e.g. d = 140–7599 m; Gerard et al. 2006;
Ó 2011 Blackwell Publishing Ltd
Slavov et al. 2009) and animal-pollinated trees (e.g.
d = 69–737 m; Klein et al. 2008; Mimura et al. 2009). In
addition to variation among pollination systems (i.e. animal versus wind), pollen dispersal curves can also differ
substantially among plant populations owing to differences in ecological and demographic characteristics (Oddou-Muratorio et al. 2005; Mimura et al. 2009). In the
case of E. aggregata, it was the lower density and most
fragmented site Duck Flat that had more restricted pollen
dispersal distances than the continuous site Bendoura.
The former site was also the one hybrid zone where local
factors were related to hybridization rates. The variable
nature of pollen dispersal curves reported among different plant populations (Ellstrand 2003), including our
study in Eucalyptus, illustrates that associations between
hybridization and the local mating environment may
depend on average dispersal distances and the broader
landscape context.
In animal-pollinated species, there is increasing evidence that habitat fragmentation may increase pollen
dispersal distances (e.g. Dick et al. 2003; Mimura et al.
2009). This likely reflects changes in the distribution of
floral rewards following habitat fragmentation, requiring pollinators to travel greater distances to forage
(Young et al. 1996). In contrast, we found evidence that
average pollen dispersal distances were greater in the
more continuously forested site (d = 64.2 m) compared
to the smaller fragmented site (d = 21.6 m). While some
pollinators such as bumblebees have been shown to forage extensively across different landscapes (Chapman
et al. 2003), others such as honeybees tend to move
among neighbouring plants (Monzon et al. 2004). Pollinator observations within each of the hybrid zones may
be required to determine whether isolated trees receive
fewer honeybees and their foraging behaviour differs in
continuous vs. fragmented populations. This information could also be vital for determining whether other
animals such as birds, rather than honeybees, are
responsible for less-frequent long-distance pollen dispersal between populations.
Evolutionary and practical implications
From an evolutionary point of view, the strong association between flowering synchrony and hybridization
may provide the selective pressure for reinforcement of
pre-zygotic isolating barriers. Hybrids often exhibit
poor fitness compared to parentals, especially as taxonomic distances between parental species increase and
recombination breaks up co-adapted gene complexes in
later-generation hybrids (Rieseberg & Carney 1998). In
our study, there are no obvious reductions in hybrid fitness for up to 12 months relative to purebreds (Field
2008), but in general Eucalyptus hybrids tend to exhibit
12 D . L . F I E L D E T A L .
poorer fitness at later life history stages (Lopez et al.
2000). As a consequence, frequent hybridization can significantly reduce female and male reproductive fitness,
driving the reinforcement of stronger isolating barriers
(Grant 1993). Given that hybrids are likely to exhibit
poor lifetime fitness, the strong genetic-based variation
in flowering time reported in Eucalyptus (see references
in Potts et al. 2003) could provide the heritable basis for
increased divergence in flowering times between
E. aggregata and E. rubida.
Our findings demonstrate that hybrid frequencies can
be highly variable both within and among populations.
It situations where hybridization is considered a threat
to species integrity, conservation efforts will depend, in
part, on identifying populations likely to produce high
numbers of hybrids. Our data suggest that hybrid seed
is more likely produced in small populations with
nearby congener populations. To reduce the potential
number of hybrid seed collected for forest restoration
programs, local-scale collection efforts should focus on
sampling from trees surrounded by same species neighbours and those that flower asynchronously with the
more common species. Despite the deleterious consequences of hybridization for rare species, the potential
adaptive benefits should not be ignored. Hybridization
and introgression can be an important process in adaptation to new environments (Rieseberg et al. 2003; De
Carvalho et al. 2010) and is increasingly recognized to
play a role in plant speciation (Rieseberg & Willis 2007).
Small populations in particular may benefit from the
influx of new genetic material, which may counteract
the deleterious effects of drift and inbreeding.
Long-distance pollen dispersal can be the major driver of hybridization dynamics in plants, and in some
cases, it may override the importance of local pre-mating variables and demographic factors. Our results suggest that local factors can be good predictors of mating
patterns in hybrid zones, particularly in populations
with restricted pollen dispersal and higher rates of
within-population pollen flow. More studies are
required in both animal- and wind-pollinated species
with similar life histories to confirm the trends observed
in E. aggregata and E. rubida hybrid zones. A few studies have also shown that mating patterns can vary substantially between years because of varying climatic
conditions (e.g. Oddou-Muratorio et al. 2005). Thus,
experiments that examine mating patterns over time,
within replicated sites, will be important in understanding whether the among-site variation reflects phenological or structural (e.g. tree density) differences among
hybrid zones. Further understanding of the factors
related to variation in male reproductive success and
relative importance of pre-mating and demographic
variables may also require assessments of flowering
synchrony and demography at larger spatial scales. This
approach will allow identification of the factors driving
hybridization from the local to the landscape level,
which will be valuable in understanding how reproductive isolation operates in heterogeneous landscapes.
Acknowledgements
Authors thank Linda Broadhurst, Judy Cassells and Bronwyn
Matheson for assistance. We thank Melinda Pickup for comments that significantly improved the quality of the manuscript.
This work was conducted while D.L.F was conducting his PhD
and receiving a University of Wollongong Postgraduate Research
Award and ‘top-up’ scholarship from CSIRO Plant Industry.
References
Adams WT, Griffin AR, Moran GF (1992) Using paternity
analysis to measure effective pollen dispersal in plant
populations. American Naturalist, 140, 762–780.
Augspurger CK (1983) Phenology, flowering synchrony, and
fruit-set of 6 neotropical shrubs. Biotropica, 15, 257–267.
Bacilieri R, Ducousso A, Petit R, Kremer A (1996) Mating
system and asymmetric hybridization in a mixed stand of
European oaks. Evolution, 50, 900–908.
Cayzer LW (1993) The Investigation of Phenotypic Variation
within and between Three Eucalypt taxa Occurring in the A.C.T.
Honours Thesis, Australian National University.
Chapman RE, Wang J, Bourke AFG (2003) Genetic analysis of
spatial foraging patterns and resource sharing in bumble bee
pollinators. Molecular Ecology, 12, 2801–2808.
De Carvalho D, Ingvarsson PK, Joseph J et al. (2010)
Admixture facilitates adaptation from standing variation in
the European aspen (Populus tremula L.), a widespread forest
tree. Molecular Ecology, 19, 1638–1650.
Dick CW, Etchelecu G, Austerlitz F (2003) Pollen dispersal of
tropical trees (Dinizia excelsa : Fabaceae) by native insects
and African honeybees in pristine and fragmented
Amazonian rainforest. Molecular Ecology, 12, 753–764.
Ellstrand N (2003) Current knowledge of gene flow in plants:
implications for transgene flow. Philosophical Transactions of
the Royal Society B: Biological Sciences, 358, 1163–1170.
Falush D, Stephens M, Pritchard JK (2003) Inference of
population structure using multilocus genotype data: linked
loci and correlated allele frequencies. Genetics, 164, 1567–1587.
Fenster CB (1991) Gene flow in Chamaecrista fasticulata
(Leguminosae) I. Gene dispersal. Evolution, 45, 398–409.
Field DL (2008) The Importance of Ecological Factors in Determining
the Pattern of Interspecific Hybridisation in Fragmented Landscapes
of Eucalyptus aggregata. PhD thesis, University of Wollongong.
Field DL, Ayre DJ, Whelan RJ, Young AG (2008) Relative
frequency of sympatric species influences rates of
interspecific hybridization, seed production, and seedling
performance in the uncommon Eucalyptus aggregata. Journal
of Ecology, 96, 1198–1210.
Field DL, Ayre DJ, Whelan RJ, Young AG (2009) Molecular
and morphological evidence of natural interspecific
hybridization between the uncommon Eucalyptus aggregata
and the widespread E. rubida and E. viminalis. Conservation
Genetics, 10, 881–896.
Ó 2011 Blackwell Publishing Ltd
M A T I N G P A T T E R N S I N E U C A L Y P T U S H Y B R I D Z O N E S 13
Field DL, Ayre DJ, Whelan RJ, Young AG (2011) Patterns of
hybridization and asymmetrical gene flow in hybrid zones
of the rare Eucalyptus aggregata and common E. rubida.
Heredity doi: 10.1038/hdy.2010.127.
Gallo LA, Marchelli P, Breitembucher A (1997) Morphological
and allozyme evidence of natural hybridization between
two southern beeches (Nothofagus spp.) and its relation
to heterozygosity and height growth. Forest Genetics, 4, 15–23.
Gerard PR, Klein EK, Austerlitz F, Fernandez-Manjarres JF,
Frascaria-Lacoste N (2006) Assortative mating and
differential male mating success in an ash hybrid zone
population. BMC Evolutionary Biology, 6, 14.
Gore P, Potts B, Volker P, Megalos J (1990) Unilateral crossincompatibility in Eucalyptus: the case of hybridisation between
E. globulus and E. nitens. Australian Journal of Botany, 38, 383–394.
Grant V (1993) Effects of hybridization and selection on floral
isolation. Proceedings of the National Academy of Science, 90,
990–993.
Griffin AR, Burgess IP, Wolf L (1988) Patterns of natural and
manipulated hybridization in the genus Eucalyptus.
Australian Journal of Botany, 36, 41–66.
Hewitt GM (1988) Hybrid zones-natural laboratories for evolutionary studies. Trends in Ecology and Evolution, 3, 158–167.
Hodges S, Burke J, Arnold M (1996) Natural formation of Iris
hybrids: experimental evidence on the establishment of
hybrid zones. Evolution, 50, 2504–2509.
Jiggins CD, Mallet J (2000) Bimodal hybrid zones and
speciation. Trends in Ecology & Evolution, 15, 250–255.
Klein EK, Desassis N, Oddou-Muratorio S (2008) Pollen flow in
the wildservice tree, Sorbus torminalis (L.) Crantz IV. Whole
interindividual variance of male fecundity estimated jointly
with the dispersal kernel. Molecular Ecology, 17, 3323–3336.
Lepais O, Petit RJ, Guichoux E et al. (2009) Species relative
abundance and direction of introgression in oaks. Molecular
Ecology, 18, 2228–2242.
Lopez GA, Potts BM, Tilyard PA (2000) F1 hybrid inviability in
Eucalyptus: the case of E. ovata x E. globulus. Heredity, 85,
242–250.
Mallet J (2005) Hybridization as an invasion of the genome.
Trends in Ecology and Evolution, 20, 229–237.
Marques I, Rossello-Graell A, Draper D, Iriondo JM (2007)
Pollination patterns limit hybridization between two
sympatric species of Narcissus (Amaryllidaceae). American
Journal of Botany, 94, 1352–1359.
Marshall TC, Slate J, Kruuk LEB, Pemberton JM (1998)
Statistical confidence for likelihood-based paternity inference
in natural populations. Molecular Ecology, 7, 639–655.
Mimura M, Barbour RC, Potts BM, Vaillancourt RE, Watanabe
KN (2009) Comparison of contemporary mating patterns in
continuous and fragmented Eucalyptus globulus native
forests. Molecular Ecology, 18, 4180–4192.
Monzon VH, Bosch J, Retana J (2004) Foraging behavior and
pollinating effectiveness of Osmia cornuta (Hymenoptera :
Megachilidae) and Apis mellifera (Hymenoptera : Apidae) on
‘‘Comice’’ pear. Apidologie, 35, 575–585.
Moran MD (2003) Arguments for rejecting the sequential
Bonferroni in ecological studies. Oikos, 100, 403–405.
Oddou-Muratorio S, Klein EK, Austerlitz F (2005) Pollen flow
in the wildservice tree, Sorbus torminalis (L.) Crantz II. Pollen
dispersal and heterogeneity in mating success inferred from
parent-offspring analysis. Molecular Ecology, 14, 4441–4452.
Ó 2011 Blackwell Publishing Ltd
van Oosterhout C, Hutchinson WF, Wills DPM, Shipley P
(2004) micro-checker: software for identifying and correcting
genotyping errors in microsatellite data. Molecular Ecology
Notes, 4, 535–538.
Potts BM, Wiltshire RJE (1997) Eucalypt genetics and
genecology. In:Eucalypt Ecology: Individuals to Ecosystems (eds
Williams J, Woinarski J). pp. 56–91, Cambridge University
Press, Cambridge.
Potts BM, Barbour RC, Hingston AB, Vaillancourt RE (2003)
Genetic pollution of native eucalypt gene pools–identifying
the risks. Australian Journal of Botany, 51, 1–25.
Pyke GH (1984) Optimal foraging theory: a critical review.
Annual Review of Ecology and Systematics, 15, 523–575.
Rhymer JM, Simberloff D (1996) Extinction by hybridization
and introgression. Annual Review of Ecology and Systematics,
27, 83–109.
Rieseberg LH, Carney SE (1998) Plant hybridization. New
Phytologist, 140, 599–624.
Rieseberg LH, Willis JH (2007) Plant speciation. Science, 317,
910–914.
Rieseberg LH, Raymond O, Rosenthal DM et al. (2003) Major
ecological transitions in wild sunflowers facilitated by
hybridization. Science, 301, 1211–1216.
Robledo-Arnuncio JJ, Austerlitz F, Smouse PE (2006) A new
method of estimating the pollen dispersal curve
independently of effective density. Genetics, 173, 1033–1045.
Robledo-Arnuncio JJ, Austerlitz F, Smouse PE (2007) POLDISP:
a software package for indirect estimation of contemporary
pollen dispersal. Molecular Ecology Notes, 7, 763–766.
Slate J, Marshall T, Pemberton JM (2000) A retrospective
assessment of the accuracy of the paternity inference
program CERVUS. Molecular Ecology, 9, 801–808.
Slavov GT, Leonardi S, Burczyk J, Adams WT, Strauss SH,
Difazio SP (2009) Extensive pollen flow in two ecologically
contrasting populations of Populus trichocarpa. Molecular
Ecology, 18, 357–373.
Stacy EA, Hamrick JL, Nason JD, Hubbell SP, Foster RB, Condit
R (1996) Pollen dispersal in low-density populations of three
neo-tropical tree species. American Naturalist, 148, 275–298.
Valbuena-Carabana M, Gonzalez-Martinez SC, Sork VL et al.
(2005) Gene flow and hybridisation in a mixed oak forest
(Quercus pyrenaica Willd. and Quercus petraea (Matts.) Liebl.)
in central Spain. Heredity, 95, 457.
Young A, Boyle T, Brown T (1996) The population genetic
consequences of habitat fragmentation for plants. Trends in
Ecology & Evolution, 11, 413–418.
David Field is interested in the importance of interspecific
hybridization for adaptive evolution and the conservation of
rare species, the role of polyploidy in plant speciation and
adaptation, and plant mating system evolution. David Ayre is
interested in the evolution of life histories and especially the
consequences of differing strategies of reproduction and dispersal. Rob Whelan is interested in how plants, animals and
ecological communities respond to disturbance, particularly
the effects of fire, population fragmentation by human development, and introduced pollinators. Andrew Young is interested in understanding the ecological and genetic aspects of
population viability in fragmented landscapes.