Annals of Botany 114: 961– 971, 2014
doi:10.1093/aob/mcu167, available online at www.aob.oxfordjournals.org
Extensive long-distance pollen dispersal and highly outcrossed mating
in historically small and disjunct populations of Acacia woodmaniorum
(Fabaceae), a rare banded iron formation endemic
Melissa A. Millar*, David J. Coates and Margaret Byrne
Science and Conservation Division, Department of Parks and Wildlife, Locked Bag 104, Bentley Delivery Centre,
Bentley, WA 6983, Australia
* For correspondence. E-mail melissa.millar@dpaw.wa.gov.au
Received: 6 February 2014 Returned for revision: 12 May 2014 Accepted: 30 June 2014 Published electronically: 6 August 2014
† Background and Aims Understanding patterns of pollen dispersal and variation in mating systems provides
insights into the evolutionary potential of plant species and how historically rare species with small disjunct populations persist over long time frames. This study aims to quantify the role of pollen dispersal and the mating system in
maintaining contemporary levels of connectivity and facilitating persistence of small populations of the historically
rare Acacia woodmaniorum.
† Methods Progeny arrays of A. woodmaniorum were genotyped with nine polymorphic microsatellite markers. A
low number of fathers contributed to seed within single pods; therefore, sampling to remove bias of correlated paternity was implemented for further analysis. Pollen immigration and mating system parameters were then assessed in
eight populations of varying size and degree of isolation.
† Key Results Pollen immigration into small disjunct populations was extensive (mean minimum estimate 40 % and
mean maximum estimate 57 % of progeny) and dispersal occurred over large distances (≤1870m). Pollen immigration resulted in large effective population sizes and was sufficient to ensure adaptive and inbreeding connectivity in
small disjunct populations. High outcrossing (mean tm ¼ 0.975) and a lack of apparent inbreeding suggested that a
self-incompatibility mechanism is operating. Population parameters, including size and degree of geographic disjunction, were not useful predictors of pollen dispersal or components of the mating system.
† Conclusions Extensive long-distance pollen dispersal and a highly outcrossed mating system are likely to play a key
role in maintaining genetic diversity and limiting negative genetic effects of inbreeding and drift in small disjunct
populations of A. woodmaniorum. It is proposed that maintenance of genetic connectivity through habitat and pollinator conservation will be a key factor in the persistence of this and other historically rare species with similar
extensive long-distance pollen dispersal and highly outcrossed mating systems.
Key words: Acacia woodmaniorum, correlated paternity, disjunct populations, dispersal distance, entomophilous
pollination, gene flow, mating system, paternity analysis, pollen immigration.
IN T RO DU C T IO N
Patterns of pollen-mediated gene flow and variation in the mating
system directly influence levels of genetic diversity, levels of
genetic connectivity and genetic structure, and are key to the evolutionary potential of plant populations (Young et al., 1996;
Eckert et al., 2010). Population genetic theory predicts disruption of genetic connectivity when populations become small
and populations are fragmented or geographically isolated. A
loss of allelic diversity via increased levels of genetic drift is
expected to result in reduced levels of genetic diversity within
populations and increased genetic divergence among populations (Slatkin, 1987; Ellstrand, 1992; Young et al., 1996).
Reduced population size and increased isolation may also
disrupt the mating system, with reduced numbers of available
mates, increased levels of selfing for self-compatible species
and subsequent reduced levels of reproductive success and
fitness costs to progeny via inbreeding depression (Aguilar
et al., 2006; Eckert et al., 2010; Jacquemyn et al., 2012).
Historically rare species often have naturally (i.e. nonanthropogenically induced) small effective population size, and
geographically disjunct and patchily distributed populations with
geographically restricted ranges (Feidler and Ahouse, 1992). In
accordance with the predictions of population genetic theory, the
influence of these factors on genetic connectivity is expected to
be largely negative (Ellstrand, 1992; Ellstrand and Elam, 1993;
Gitzendanner and Soltis, 2000). Meta analyses have shown that
rare species are generally associated with low overall species
diversity, low levels of within-population genetic diversity and
increased levels of among-population genetic structure as a result
of the heightened impacts of genetic drift under conditions of
limited genetic connectivity and/or selection under a narrow
range of environmental conditions (Karron, 1987; Hamrick and
Godt, 1989; Gitzendanner and Soltis, 2000; Cole, 2003; Leimu
et al., 2006). The long-term impacts of restricted gene flow on
rare species and others with small disjunct populations and
geographically restricted ranges may be expected ultimately to
include increased risk of extinction (Ellstrand and Elam, 1993).
# The Author 2014. Published by Oxford University Press on behalf of the Annals of Botany Company.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/),
which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited.
961
962
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
Despite this, many historically rare plant species with small, patchily distributed populations and geographically restricted ranges
have persisted in changing environments over very long time
frames, and may not exhibit the classic effects of small population
theory. How well levels and patterns of genetic diversity within rare
species conform to population genetic predictions of small population size will depend on a range of life history traits and ecological
processes that affect pollen dispersal, and the spatial positioning of
individuals at the landscape scale (Ellstrand and Elam, 1993;
Hamrick and Godt, 1996; Cole, 2003; Hamrick, 2004). This
makes historically rare species valuable model systems for understanding how patterns of pollen-mediated gene flow and variation
in the mating system maintain genetic connectivity and genetic
variation and provide for long-term persistence of populations
and species. Such knowledge may also lead to increased consideration of the role of genetic connectivity in responses to more recent
anthropogenic habitat disturbance, long-term adaptation and conservation, and successful restoration of remnant vegetation (Young
et al., 1996; Broadhurst et al., 2008a; Eckert et al., 2010; Lankau
et al., 2011).
Investigation of pollen-mediated genetic connectivity and
variation in the mating system of rare species is readily achieved
in the ancient landscape of south west Western Australia (WA)
as it is rich in historically rare plant species with typically
small population size and disjunct and patchy population distributions. Within this landscape, banded iron formation (BIF)
inselbergs act as ecological islands that support an especially
large number of edaphically endemic and historically rare
species. Acacia woodmaniorum sect. Alatae is one historically
rare edaphic endemic of WA’s BIF outcrops that has a distribution suitable for investigation of the impacts of small disjunct
populations on patterns of pollen dispersal and mating system
parameters. Populations occur over two relatively large series
of BIF ranges, and a number of small populations are located
on small BIF outcrops at varying distances from the main
ranges. The taxon was only recently described (Maslin and
Buscumb, 2007), and little is known of the species biology or
ecology. Seed dispersal, specific pollinators and aspects of the
mating system have not been investigated.
Patterns of phylogeographic and genetic structure suggest that
the distribution of A. woodmaniorum has been historically
restricted, with long-term persistence of some small and disjunct
populations along with large populations in the main range.
Evidence from indirect F-statistic-based estimates has indicated
that seed and/or pollen dispersal in A. woodmaniorum is sufficient to maintain a moderate degree of genetic diversity with
some broad genetic structure among populations (global
FST ¼ 0.097; Millar et al., 2013), but limited negative genetic
effects of inbreeding and drift in the smallest and most spatially
isolated populations (Millar et al., 2013). These results led to a
hypothesis that genetic connectivity, diversity and long-term
persistence in this taxon are the result of extensive pollen dispersal among populations, coupled with a predominantly outcrossed mating system. Here we test this hypothesis by directly
assessing contemporary patterns of pollen dispersal and other
aspects of the mating system across the geographic range of
A. woodmaniorum. We used nuclear microsatellite genotyping
of progeny arrays from eight small disjunct populations for
which spatial and genotypic data are already available for all
adult plants. We then answered the following questions. (1)
How extensive is pollen immigration into spatially disjunct,
small populations? (2) Over what distances does pollen dispersal
occur? (3) What are the parameters of the mating system, including rates of outcrossing, inbreeding and correlated paternity
within pods? (4) Is pollen immigration, outcrossing rate or the
level of inbreeding in A. woodmaniorum affected by population
size or the degree of isolation?
M AT E R I A L S A N D M E T H O D S
Study species and site
Acacia woodmaniorum (Maslin and Buscomb) sect. Alatae
(Benth.) is a sprawling, prickly, woody shrub 1 – 2 m tall and
up to 2 m wide. Its small, globular, racemes are comprised of
many light golden flowers. Pods are dark brown, narrowly
oblong, curved, and sometimes twisted and 10– 45 mm long.
The pods bear small, hard coated seeds 3 – 4 mm long and dark
greyish brown to black in colour, that are released following
rapid dehiscence of the pods. Intensive surveillance over the
species range, which covers ,40 km2 in WA, has mapped
approx. 25 000 known plants. These are highly substrate specific
to the skeletal soils of the steep slopes, rock crevices and gullies
of low-altitude BIF outcrops and can be broken down into two
main geological and geographic regions (Fig. 1). Habitat is
more or less continuous across the main range on Mungada/
Windaning Ridge where the majority (approx. 18 000) of the
plants occur. The Jasper Hill region to the north covers a
smaller area, and populations in this region, which range in
size from tens of plants to just over 1000 plants, are located on
a number of smaller, discrete BIF ridges separated by intervening
unsuitable habitat. Several very small disjunct populations, on
which this study is focused, occur on ironstone breakaways
located off these main ranges. The largest at Blue Hill region
comprises 145 plants and the smallest at MDS comprises seven
plants.
The midwest region where the plants occur is located approx.
330 km north north east of Perth. The region experiences a semidesert Mediterranean climate (Beard, 1976) with annual rainfall of 300 – 400 mm mostly during winter. Off the BIF ranges,
the surrounding clay, silt and sand plains are dominated by
a low Mulga (Acacia aneura) woodland with other Acacia,
Allocasuarina, Melaleuca and eucalypt species, and surrounding low hills by Senna, Eremophila and Acacia shrubs (Beard,
1976). The area has been subject to previous mining activity,
and both the western end of Mungada Ridge and Blue Hill are
highly disturbed after mining activities in the 1960s and 1970s.
Population sampling and genotyping
In a previous study we genotyped plants from sampling locations (called populations from here on) across the species range at
15 nuclear microsatellite loci and collected detailed spatial
co-ordinates with a differential Global Positioning Satellite
system for each sampled plant (Millar et al., 2013). Eight of
these populations had varying degrees of geographic disjunction
from their nearest population and nearest large population
(Table 1, Fig. 1), and were treated as independent entities ( populations) to test pollen dispersal and immigration. The populations
were small enough such that all plants (ranging from seven to 145
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
963
Jasper Hill
JHBS
JHBSW
Mungada/Windaning Ridge
Terapod
MDSE
MASC
MA2
Blue Hill
WD
WE
0
N
0·5
1
2
km
F I G . 1. Map of the distribution (shading) and sampling locations (stars) of Acacia woodmaniorum. Grey lines show altitudinal contours. Population names
correspond to those in Table 1.
plants) could be genotyped, providing circumscribed study areas
for thorough paternity analysis and direct comparisons of pollen
immigration rates and distances for populations of varying size
and degree of isolation. For this study, we collected and genotyped progeny from the 2009 flowering season from mother
plants within these eight populations. Population parameters
including degree of isolation (linear distance to plants in the
next closest population), degree of isolation from a large population (linear distance to plants in the next closest population
containing .1000 plants) and size (the number of plants) were
determined for each population (Table 1).
We aimed to collect seed from ten evenly spaced maternal
plants (or all plants if less than ten) within each selected population. However, pod production and seed-set was low in
A. woodmaniorum, and in many populations few plants produced
pods. In line with best practice seed collection guidelines
(Mortlock, 1999), and so as not to affect population viability
negatively, we harvested no more than 20 % of pods within a
given population. This limited the number of maternal plants,
pods and seed available for harvest, so mother plants were
chosen opportunistically from those that had produced seed.
The number of suitable maternal plants per population ranged
from two to six (Table 1). Necessarily, mother plants in different
populations occurred at various locations within populations.
The mean distance between suitable mother plants within populations ranged from 3.6 to 102 m.
In Acacia species, the number of pollen grains in the polyad is
correlated with the number of ovules in the ovary so that a single
polyad pollinating a stigma is capable of fertilizing all ovules
within a flower (Kenrick and Knox, 1982; Tybirk, 2007).
Mating system parameters, calculated using allele frequency
estimates, and patterns of pollen dispersal may be biased in
964
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
TA B L E 1. Population size, isolation distances, number of mother plants sampled, size of progeny arrays and number of progeny used
for analysis of correlated paternity, other mating system parameters and paternity for 525 progeny of Acacia woodmaniorum
Population
JHBS
JHBSW
MASC
MDSE
Terapod
Blue Hill
WD
WE
MA2
Size
22
17
8
7
10
145
29
16
Total
Isolation
(m)
120
120
100
100
700
1870
910
180
Isolation from large
population (m)
1000
980
170
450
940
1870
970
180
No. of mother
plants sampled
3
2
4
4
5
6
3
4
10
41
cases where all seed within an Acacia pod are full siblings with
the same paternal parent as well as the same maternal parent
(Kenrick and Knox, 1982; Muona et al., 1991). We assessed correlated paternity within pods (rppm) at a site within the main
range (MA2; Fig. 1) so as to remove any bias from further assessment of paternity and mating system parameters. A correlated
matings model was used to characterize the extent that siblings
share the same father rp, the correlation of paternity. Pods were
collected from ten tagged maternal plants, and all seed from
two pods from each mother was sown for analysis.
Given the likelihood of a high degree of correlated paternity
within Acacia pods, we used a sampling design that removed
bias due to correlated paternity and selected a single seed from
each pod for further analysis. Seed were germinated on agar in
an incubator at 15 8C and then grown in potting mix under shadehouse conditions. Once seedlings had grown large enough to
survive harvest, DNA was extracted from phyllodes following
the methods of Millar (2009). Progeny were genotyped with a
sub-set of nine previously described nuclear microsatellite
markers (loci Aw124, Aw129, AwB008, AwB107, AwB108,
AwC001, AwD008, AwD012 and AwD116, Millar, 2009).
These loci are known to be in linkage equilibrium for adult
cohorts for all populations except Blue Hill, which has undergone recent anthropogenic disturbance (Millar et al., 2013).
Data analysis
Microsatellite variation. Samples that did not amplify clearly
were re-amplified at least once. Allele bins were manually
assigned and all bins and automatically assigned alleles were
manually checked and adjusted when necessary. The number
of alleles per locus was estimated for progeny cohorts using
the GenAlEx v 6.41 program (Peakall and Smouse, 2006).
Null allele frequencies were estimated for maternal plants and
progeny arrays combined using the CERVUS v 3.0.3 program
(Marshall et al., 1998). Means and standard errors of allelic diversity statistics including the percentage of polymorphic loci,
the number of alleles per locus, the number of effective alleles
per locus and expected and observed heterozygosities were
obtained for progeny cohorts using GenALEx.
Size of progeny arrays (¼ no.
of pods) per mother plant
9, 10, 10
6,12
7, 12, 14, 16
16, 18, 18, 18
1, 2, 6, 7, 9
12, 16, 18, 19, 19, 20
7, 15, 18
10, 16, 18, 26
2, 4, 4, 4, 5, 5, 5, 6, 6, 6, 7, 7, 8,
8, 8, 8, 9, 9, 9
525
Total no. of progeny used
Correlated
paternity analysis
Mating system and
paternity analysis
–
–
–
–
–
–
–
–
120
29
18
49
70
25
104
40
70
–
120
405
Correlated paternity within pods. Seed from one pod did not
survive to harvest; therefore, 19 progeny arrays comprising
120 progeny were genotyped for assessment of correlated paternity within pods. The number of progeny per pod ranged from
two to nine and averaged 6.3. We used genotype data for maternal
plants and progeny arrays to estimate multilocus correlated paternity, or the proportion of full sib progeny among a pair of siblings
within pods (rppm), using the sibling pair method in the MLTR v
3.4 program (Ritland and Jain, 1981; Ritland, 2002). We used the
expectation – maximization algorithm and obtained the standard
error with 1000 bootstraps. The effective number of pollen
donors per pod was determined as Ne ¼ 1/rppm.
Pollen immigration. A total of 405 progeny collected from the
eight small disjunct populations were genotyped for estimation
of pollen immigration via direct paternity analysis (Table 1).
The number of mother plants available for sampling (i.e. those
that produced sufficient pods) per population ranged from two
to six (Table 1). Given the likelihood of a high degree of correlated paternity within pods, only a single seed from a given
pod was genotyped so the sizes of progeny arrays are equal to
the number of pods collected from known maternal plants. The
number varied from one to 26 (Table 1). Genotypic data for
progeny arrays from known mother plants were combined with
known genotypes of all plants in each population for direct paternity analysis. We considered all plants within each of the eight
study populations as potential fathers since, within all populations, all plants were mature and presumably capable of producing pollen, excluding potentially ,10 less mature plants at
Blue Hill. However, inclusion of the genotypes of potentially immature plants at Blue Hill as potential parents will only act to
ensure a more conservative rather than a less conservative estimate of pollen immigration.
The probability that the set of loci will exclude an unrelated
candidate male parent from paternity for an arbitrary progeny
when the genotype of the mother is known (average PE2) was
determined for each population using CERVUS (Marshall
et al., 1998). Paternity analysis was conducted separately for
each population using two programs, NEWPATXL v 5.0 and
CERVUS, and the results compared. To account for the presence
of null alleles and scoring error, we conducted paternity analysis
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
for progeny with five or more loci genotyped. The NEWPATXL
program uses exclusion methods to detect matches between
progeny and potential paternal parents (Worthington et al.,
1999). The significance of matches between progeny and potential paternal parents is assessed by drawing alleles at random to
create pseudo-genotype files and examining levels of background paternity expected by chance. Given the degree of population isolation, the lack of data on allele frequencies in large
populations, such as that occurring across Mungada/Windaning
Ridge, and the likelihood of pollen immigration, we used sexspecific allele frequencies from each of the study populations
for analyses. We conducted paternity analysis allowing a single
mismatch between a progeny and a maternal parent, or between
a progeny and a potential paternal parent, a repeat unit difference
of one and a combined probability of 0.05 of a null match occurring. As the utility of the ‘repeat unit size’ function is not provided by the author, it was left at the default value. For each
progeny matching the analysis criteria, we obtained a most
likely father and calculated a conservative pollen immigration
rate as the percentage of progeny that could not be assigned a
father from within the population.
We also conducted paternity analysis with CERVUS. This
program finds optimal progeny – male parent pairs and uses
maximum likelihood for statistical evaluation of the matches
(Marshall et al., 1998). We ran the program for each population
with simulation of 10 000 progeny, a known number of potential male parents (i.e. the total number of plants within the
population), 100 % of potential male parents genotyped and
an error rate of 1 %. Critical Delta criteria [defined as the difference in LOD (the natural log of the overall likelihood ratio)
scores between the most likely paternal parent and the second
most likely paternal parent] were obtained from simulations
and used as a criterion for assignment of parentage. We compared trio Delta scores to assign most likely paternal parents at
a strict (95 %) confidence level, a relaxed (80 %) confidence
level and at ,80 % confidence, and trio LOD scores to
assess whether there was more than one equally likely potential
father within the population (equal positive LOD scores for
more than one most likely paternal parent) or whether there
was no potential paternal parent within the population (i.e. a
result of pollen immigration, negative LOD score). We did
not allow known mothers to be potential male parents due
to the high outcrossing rate. We considered all most likely
fathers assigned within a population as the true most likely
father, and calculated a conservative minimum pollen immigration rate as the percentage of progeny that could not be
assigned any father from within the population, at any confidence level. We also calculated a maximum pollen immigration rate as the percentage of progeny that could not be
assigned any father from within the population at a confidence
level of ,80 %.
We continued further analysis using results from CERVUS, as
it provided more interpretable degrees of confidence for paternity
assignments. We conducted regression analysis to test for correlation between minimum or maximum pollen immigration rates
and population parameters. Because of the large number of ungenotyped potential male parent plants located across the species
range, there was insufficient power to identify the specific
source of pollen immigration into the disjunct small populations.
To estimate pollen dispersal distances, we took a conservative
965
approach and assumed pollen immigrated into each study population originated from the next closest population.
Mating system. A total of 405 progeny were genotyped for further
assessment of the mating system. These were the same progeny
used for paternity analysis. The number of mother plants available for sampling (i.e. those that produced sufficient pods) per
population ranged from two to six (Table 1). Given the likelihood
of a high degree of correlated paternity within pods, only a single
seed from a given pod was genotyped so the sizes of progeny
arrays are equal to the number of pods collected from known maternal plants and varied from one to 26 (Table 1).
The use of nine highly variable microsatellite loci ensures that
progeny array sizes as small as two are informative in relation to
mating system estimates (see Ritland and Leblanc, 2004). We
used maximum likelihood methods based on the mixed mating
model of Brown and Allard (1970) in MLTR to estimate the following mating system parameters for each population simultaneously: the multilocus outcrossing rate (tm), the single locus
outcrossing rate (ts), the apparent level of selfing due to biparental inbreeding (tm – ts), the correlation of selfing among maternal
plants (rs) and the multilocus correlated paternity (rpm).
Parameters were estimated using the expectation – maximization
algorithm, with pollen and ovule gene frequencies estimated separately and standard errors obtained with 1000 bootstrap replicates, with families as the re-sampling units, and used to assess
whether values were significantly different from one or from
zero. Wright’s fixation index was estimated for the parental
(Fpar) and progeny (Fprog) generations using GenAlEx, and the
effective number of pollen donors per maternal tree was estimated as Ne ¼ 1/rpm. Regression analysis was conducted for
the outcrossing rate and the degree of biparental inbreeding,
and for the outcrossing rate and population parameters.
R E S ULT S
Population size was significantly positively correlated with
degree of population isolation and with degree of isolation
from large populations. The degree of population isolation was
also significantly correlated with the degree of isolation from a
large population (Table 2). The nine microsatellite markers
were moderately variable within A. woodmaniorum progeny
cohorts, with a total of 60 alleles detected in 525 seedlings. A
total of 98.77 % of loci were polymorphic, the mean number of
alleles per locus was 4.123 (+0.194), the mean number of effective alleles per locus was 2.070 (+0.094), and expected and
observed heterozygosities were 0.445 (+0.022) and 0.490
TA B L E 2. Statistics of correlation analysis between population
parameters for eight small disjunct populations of Acacia
woodmaniorum
Size
R
Degree of isolation
Isolation from a large
population
2
0.7845
0.6581
Degree of isolation
d.f.
P
R2
d.f.
P
7
7
0.0034
0.0145
–
0.8122
–
7
–
0.0143
966
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
(+0.029), respectively. Progeny allele frequencies per population are provided in Supplementary Data Table S1.
distances into disjunct small populations were considerable, up
to 1870m.
Correlated paternity within pods
Mating system
Multilocus correlated paternity within pods calculated using
the sibling pair method in MLTR was high, rpm ¼ 0.492
(+0.080), indicating that an average of two fathers sire all seed
within pods.
The mating system was assessed in 31 progeny arrays. The
number of progeny genotyped per population ranged from 18
for JHBSW to 102 for Blue Hill. Estimates of multilocus outcrossing rates and single locus outcrossing were high across all
populations (Table 5). Multilocus outcrossing rates were not statistically significantly correlated with population parameters of
size (R 2 ¼ 0.0372, d.f. ¼ 7, P ¼ 0.6472), degree of isolation
(R 2 ¼ 0.0009, d.f. ¼ 7, P ¼ 0.9411) or isolation from a large
population (R 2 ¼ 0.0546, d.f. ¼ 7, P ¼ 0.5775). Correlation of
outcrossing or selfing among maternal plants (rs) and mean
biparental inbreeding (tm – ts), were low for all populations.
There was no statistically significant correlation between the
degree of biparental inbreeding and population parameters of
size (R 2 ¼ 0.0111, d.f. ¼ 7, P ¼ 0.8047), degree of isolation
(R 2 ¼ 0.0008, d.f. ¼ 7, P ¼ 0.9442) or isolation from a large
population (R 2 ¼ 0.0007, d.f. ¼ 7, P ¼ 0.9505). Multilocus
correlated paternity was generally low and not statistically
Pollen immigration
The probability that the set of loci will exclude an unrelated
candidate male parent from paternity of an arbitrary progeny
when the genotype of the mother is known varied for the eight
populations and averaged 0.944 (Table 3). Rates of pollen immigration varied but were considerable for the eight populations
when analysis was conducted with either NEWPATXL
(ranging from 11.1 to 58.3 %) or CERVUS (ranging from 12.7
to 57.1 %; Table 3). Pollen immigration estimates obtained
using NEWPATXL were not correlated with minimum or
maximum estimates of pollen immigration obtained using
CERVUS. For four populations (MASC, MDSE, Terapod and
WE), there was a high level of discrimination among potential
fathers within the population and little variation between
minimum and maximum levels of pollen immigration determined using CERVUS. For the other four populations (JHBS,
JHBSW, Blue Hill and WD), there was a degree of ambiguity
regarding the most likely father and there was greater variation
between minimum and maximum levels of pollen immigration
for these populations. This was due to either a high proportion
of progeny assignment within the stand at confidence levels
,80 % (Blue Hill and WD; Table 3) or multiple potential
fathers identified within the stand (JHBS and JHBSW; Table 3).
Neither the minimum nor the maximum pollen immigration
rate (CERVUS) was significantly correlated with any population
parameter (Table 4). Minimum estimated pollen dispersal
TA B L E 4. Statistics of correlation analysis for minimum and
maximum pollen immigration rates (CERVUS) to population
parameters for eight small disjunct populations of Acacia
woodmaniorum
Population parameter
Size
Degree of isolation
Isolation from a large
population
Minimum pollen
immigration
Maximum pollen
immigration
R2
d.f.
P
R2
d.f.
P
0.4410
0.4008
0.3820
7
7
7
0.0725
0.0920
0.1024
0.0508
0.0099
0.1246
7
7
7
0.5913
0.8143
0.3912
TA B L E 3. Probability of exclusion (PE2) for nine loci and details of paternity assignments obtained using the NEWPATXL and
CERVUS programs as percentages of progeny, for eight small disjunct populations of Acacia woodmaniorum
Population
NEWPATXL
CERVUS
Within-population assignment
confidence
JHBS
JHBSW
MASC
MDSE
Terapod
Blue Hill
WD
WE
Mean
Pollen immigration
Pollen immigration
PE2
≥95 %
80–95 %
,80 %
Not identified
Minimum* pollen immigration
Maximum† pollen immigration
14.3
11.1
47.8
44.9
58.3
18.8
33.3
42.0
33.8
0.908
0.964
0.972
0.936
0.962
0.954
0.919
0.933
0.944
25.0
5.6
36.7
28.6
20.8
7.8
25.0
30.4
22.5
32.1
5.6
6.1
15.7
25.0
25.5
27.5
27.5
20.6
14.3
5.6
0.0
0.0
0.0
49.0
25.0
1.4
11.9
10.7
22.2
0.0
0.0
0.0
4.9
0.0
0.0
4.7
17.9
61.1
57.1
55.7
54.2
12.7
22.5
40.6
40.2
42.9
88.9
57.1
55.7
54.2
66.7
47.5
42.0
56.9
The percentage of progeny with pollen sources not identified within the population is presented for NEWPATXL. For CERVUS, most likely fathers were
assigned to progeny at confidence intervals of ≥95 % 80– 95 %, ,80 %, or, when listed as ‘not identified’, where a single most likely father was likely to exist
within the population but could not be identified, i.e. more than one potential father was identified within the population, both having equal likelihoods of being
the father. Values are expressed as percentages of the number of progeny analysed.
*Minimum pollen immigration estimate includes progeny which could not be assigned a most likely father within the population.
†
Maximum pollen immigration estimate includes progeny which could not be assigned any father from within the population, at a confidence level of ,80 %.
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
967
TA B L E 5. Estimates of mating system parameters for eight small disjunct populations of Acacia woodmaniorum including the
multilocus outcrossing rate (tm), the single locus outcrossing rate (ts), the apparent level of selfing due to biparental inbreeding (tm – ts),
the correlation of selfing among maternal plants (rs), the multilocus correlated paternity (rpm), the effective number of pollen donors
(Ne) and Wright’s fixation index for parental (Fpar) and progeny generations (Fprog)
Population
JHBS
JHBSW
MASC
MDSE
Terapod
Blue Hill
WD
WE
Total or mean
tm
ts
tm – ts
rs
rpm
Ne
Fpar
Fprog
0.911 (0.047)
1.000 (0.000)
1.000 (0.000)
1.000 (0.005)
1.000 (0.000)
0.995 (0.015)
0.900* (0.000)
1.000 (0.004)
0.975 (0.015)
0.921* (0.028)
0.996* (0.000)
0.999* (0.000)
0.940* (0.020)
0.991 (0.027)
0.968* (0.010)
0.900* (0.000)
1.000 (0.000)
0.964* (0.014)
–0.010 (0.034)
0.004* (0.001)
0.001* (0.000)
0.060* (0.020)
0.009 (0.027)
0.024 (0.015)
0.000 (0.000)
0.000 (0.004)
0.011 (0.008)
0.119 (0.088)
0.106* (0.000)
0.106 (0.085)
0.10 7 (0.078)
0.110 (0.130)
0.0940* (0.009)
0.100* (0.001)
0.109* (0.009)
0.106* (0.003)
0.022 (0.018)
0.000 (0.000)
0.052 (0.081)
0.104 (0.081)
0.003 (0.118)
0.179* (0.044)
0.100* (0.001)
0.035* (0.015)
0.062* (0.022)
45
1
19
10
333
6
10
16
16
0.000 (0.096)
–0.078 (0.074)*
–0.118 (0.062)*
–0.186 (0.098)*
0.043 (0.076)
0.108 (0.080)*
0.059 (0.107)
0.017 (0.097)
–0.088 (0.036)*
–0.123 (0.102)*
–0.142 (0.073)*
–0.099 (0.158)
0.043 (0.053)
0.062 (0.052)*
0.058 (0.060)
–0.293 (0.102)*
–0.182 (0.067)*
–0.070* (0.034)
Standard errors are in parentheses.
*Values are significantly different from 1 (tm and ts) or from zero (all other estimates).
significantly correlated with population parameters of size
(R 2 ¼ 0.2022, d.f. ¼ 7, P ¼ 0.2637), degree of isolation (R 2 ¼
0.0000, d.f. ¼ 7, P ¼ 0.9879) or isolation from a large population (R 2 ¼ 0.2196, d.f. ¼ 7, P ¼ 0.2416). Estimated numbers
of pollen donors among pods within maternal trees (Ne) varied
(Table 5), but were equal to or far greater than the census population size for most populations. Within-population fixation
indices were significantly less than zero for parental and
progeny generations (Table 5).
D IS C US S IO N
Contemporary estimates of pollen dispersal via paternity
analysis in Acacia woodmaniorum were high (mean minimum
estimate of 40.2 % immigration into eight small, disjunct populations) and occurred over large distances (≤1870m), confirming previous FST-based estimates of high levels of gene flow
across the species’ range, including between small disjunct
populations (Millar et al., 2013). Predominant outcrossing and
a lack of any inbreeding due to either self-pollination or mating
between close relatives was a feature of the small spatially disjunct populations. Population parameters, including the size
and degree of population isolation, were poor predictors of the
level of pollen immigration into populations and had no influence
on aspects of the mating system such as outcrossing rate, correlated paternity or biparental inbreeding, which were largely consistent across the species range. Our findings suggest that high
levels of long-distance gene flow and a predominantly outcrossed
mating system act to maintain large effective population sizes for
small disjunct populations of A. woodmaniorum.
Pollen dispersal
Estimates of pollen immigration rates and pollen dispersal
distances confirmed our hypothesis that high levels of pollenmediated gene flow maintain genetic diversity within populations and lead to marginal genetic differentiation among
populations of A. woodmaniorum (Millar et al., 2013). The proportion of pollen immigration into small, geographically disjunct populations, and the distances over which this occurs,
indicates that these populations are not genetically isolated
or static landscape elements and that they play an important
role in maintaining genetic connectivity across the landscape.
Pollen immigration rates and dispersal distances in A. woodmaniorum are remarkably similar to that detected for the common,
widespread Acacia saligna where 40 % of pollen immigration
into remnant patches occurred over a distance of 1650 m
(Millar et al., 2008, 2012). These findings contrast with
general population genetic predictions for rare endemic species
with small, patchily distributed populations and short ranges,
which might be expected to show more limited genetic connectivity (Karron, 1987; Gitzendanner and Soltis, 2000; Byrne et al.,
2007; Sampson et al., 2014). They are more consistent with the
general patterns observed in many studies of impacts of fragmentation in tree species where extensive pollen dispersal maintains
genetic connectivity (Hamrick, 2004; Kramer, 2008).
Life history traits such as large individual size and longevity
tend to promote extensive pollen production and dispersal
(Petit and Hampe, 2006). Individuals of A. woodmaniorum are
shrubs of small stature, and flowering is not prolific in this
species, which may be expected to limit pollen production.
However, as a woody perennial, individuals are presumed to
live for several decades, which typically provides more temporal
opportunity for outcrossed and long-distance pollination events
than in herbaceous or annual species. Patterns of extensive
pollen immigration and long-distance pollen dispersal in
A. woodmaniorum are, in fact, similar to those of a wide range
of typically common and widespread, large and long-lived, temperate, neotropical and tropical tree species that maintain extensive pollen dispersal over large distances of several kilometres
(Kaufman et al., 1998; Nason et al., 1998; White et al., 2002;
Dick et al., 2003; Bacles et al., 2005; Robledo-Arnuncio and
Gil, 2005; Ward et al., 2005). While extensive pollen dispersal
may be expected for large, wind-pollinated forest tree species
(Robledo-Arnuncio and Gil, 2005; Craft and Ashley, 2007), it
may be expected to vary more for those pollinated by animals.
Despite this, extensive pollen dispersal has also been identified
in a range of insect-pollinated tree species (White et al., 2002;
Bacles et al., 2005; Goto et al., 2006; Byrne et al., 2008;
Ahmed et al., 2009).
The exact mechanisms of pollen dispersal have not been
studied in A. woodmaniorum. Although Australian Acacia
968
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
display a range of pollen dispersal mechanisms, the nature of all
Acacia polyads, where pollen occurs as composite units comprised of 4 –32 pollen grains, means Acacia pollen is typically
thought to be too heavy for significant wind dispersal (Kress,
1981; Kenrick and Knox, 1982). Arid zone Acacia species including A. woodmaniorum also tend to lack extrafloral nectaries
that typically make those species adapted to pollen dispersal by
passerine birds (Ford and Forde, 1976; Glyphis et al., 1981;
Knox et al., 1985; Vanstone and Paton, 1988). As a result,
pollen of most Acacia is thought to be dispersed by a range of
generalist insect pollinators, including ants, moths, wasps,
beetles and bees (Stone et al., 2003). Generalist insects are
known to be capable of affecting fat-tailed dispersal curves and
long-distance pollen dispersal either directly or via pollen carryover when traversing intervening habitat matrices between plant
populations (Dick et al., 2003; Austerlitz et al., 2004; Lander
et al., 2010).
Pollinator foraging and movement are likely to be influenced
by a wide range of factors including the relative amount of
pollen, nectar or other reward available, and hence the relative fecundity of plant populations, as well as population shape and
other aspects of habitat quality (Leimu et al., 2010). Pollen immigration is typically expected to decrease with increasing geographic disjunction and as populations become smaller and
less dense (Aguilar et al., 2006; Leimu et al., 2006). Such a
pattern was not observed in A. woodmaniorum, with population
parameters being poor predictors of pollen immigration rates.
This may reflect a compounding effect of a significant positive
association between geographic disjunction and population
size in this species, or alternatively may indicate that geographic
distances between disjunct populations are not large enough to
have a significant impact on the behaviour of insect pollinators.
This finding may not be surprising given increasing evidence
that the degree of geographic disjunction required to produce a
significant level of genetic isolation between plant or tree populations may have been underestimated for a long time. An extensive literature has countered the previously held notion of
fragmentation driving genetic isolation in forest tree species
(Kramer, 2008; Bacles and Jump, 2011). In fact, a general
pattern of negative density-dependent gene flow has been
revealed for typically common, outcrossing, tree species that
occur at low densities across widespread ranges (Kramer,
2008). Comparison between undisturbed and fragmented forest
have revealed similar or increased levels of connectivity after
fragmentation in both wind-pollinated (Robledo-Arnuncio and
Gil, 2005; Craft and Ashley, 2007) and insect-pollinated
species (White et al., 2002; Hamrick, 2004; Bacles et al.,
2005; Goto et al., 2006; Bacles and Ennos, 2008; Byrne et al.,
2008; Jha and Dick, 2010; Rosas et al., 2011). This may be attributed to the characteristics of forest fragmentation that generally
lead to lower conspecific density and increasing geographic
extent of effective breeding units and of pollen dispersal distances (Nason et al., 1998; Kramer, 2008). Long-distance dispersal means that maximum pollinator dispersal distances are not
discovered in many empirical studies, and our findings suggest
that maximum pollinator dispersal distances exceed 1870m for
A. woodmaniorum.
Previous investigation of genetic structure in A. woodmaniorum also suggested a pattern of ( presumably pollen) dispersal
associated with prevailing wind conditions, indicating that
generalist insects carrying pollen loads may be conveyed over
long distances via thermal updrafts (Millar et al., 2013).
Wind-mediated and directional dispersal of small insect pollinators has been documented previously over distances of tens of
kilometres (Gardner and Early, 1996; Ahmed et al., 2009). The
potential role of wind in pollinator movement, and thus pollen
dispersal, would be an interesting area of investigation for
this and other endemics of terrestrial inselberg habitats, such as
the BIFs of WA, and other species with outcrossed or mixed
mating systems and generalist insect pollinators.
Our findings of extensive pollen immigration over large dispersal distances indicate that, like individuals of many
common and widespread tree species, the disjunct, small populations of A. woodmaniorum are not genetically isolated. The
degree of genetic connectivity produced by extensive pollen dispersal appears sufficient to provide a buffer against a low number
of plants in small populations. Effective population size was
greater than census population size in all but the two most isolated populations, Blue Hill and WD, which do not appear to
be experiencing limited diversity in available pollen. A lack of
true selfing, little evidence of biparental inbreeding and low to
negative values of the fixation index suggest that virtually all effective mating in A. woodmaniorum occurred between genetically unrelated plants and pollen dispersal is sufficient to produce
‘inbreeding connectivity’ (Lowe and Allendorf, 2010), largely
limiting any negative genetic effects of inbreeding due to
direct mate limitation, in even the smallest disjunct populations.
Mating system
Acacia display a wide range of both asexual (Coates, 1988;
NSWNPWS, 2003) and sexual mating systems, with sexual
mating systems that vary from predominantly outcrossing
(see Philip and Sherry, 1946; Moffet, 1956; Bernhardt et al.,
1984; Moran et al., 1989a; Muona et al., 1991; Broadhurst
et al., 2008b; George et al., 2008; Millar et al., 2008; Ng et al.,
2009), to substantial levels of selfing (Mandal et al.,
1994; Coates et al., 2006). All members of the predominant
Australian subgenus Phyllodineae, including A. woodmaniorum, have protogynous flowers however, a mechanism that
promotes outcrossing, although there can still be great variation
in outcrossing rates among populations of a single species
(Coates, 1988; Mandal and Ennos, 1995). The high outcrossing
rates and lack of true selfing found in A. woodmaniorum are comparable with those obtained from genetic studies of a number of
other Acacia species (Moran et al., 1989b; Casiva et al., 2004;
Broadhurst et al., 2008b; Millar et al., 2008), and demographic
studies of seed-set indicate that many Acacia are either highly
self-incompatible, or at least partially self-incompatible
(Kenrick and Knox, 1989; Morgan et al., 2002). Estimates of
mating system parameters were remarkably consistent across
all populations of A. woodmaniorum and did not vary with population parameters of size and isolation, providing further support
for a self-incompatible mating system.
Despite its rarity, short range and persistence in small populations, A woodmaniorum shows high outcrossing and a selfincompatible mating system. Long-lived woody perennial tree
and shrub species tend to have higher genetic loads, resulting
in strong inbreeding depression and, hence, tend to be selfincompatible (Petit and Hampe, 2006). Self-incompatibility
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
can be explained by pre-zygotic stylar incompatibility and/or
post-zygotic seed abortion mechanisms. Confirmation of preor post-zygotic self-incompatibility mechanisms in A. woodmaniorum would require assessment of the success of controlled
crosses. Field observations did indicate very low levels of podand seed-set over the species range despite high levels of
seed-set in many other sympatric species in the year of sampling
(M. Millar, DPAW, Perth, Australia, unpubl. res.). This observation suggests that poor pod-and seed-set in A. woodmaniorum
was not solely a result of adverse temporal environmental conditions and, combined with high levels of abortion of developing
pods and seed (M. Millar, DPAW, Perth, Australia, unpubl.
res.), may indicate post-zygotic seed abortion or the effect of
inbreeding depression following self-pollination or when pollination occurs between related individuals.
The realized outcrossing rate obtained here will be biased by
any inbreeding depression resulting in seed abortion after fertilization as well as that operating on young seedlings arising from
initially viable seed. Inbreeding depression could be further
quantified in this species with controlled crossing experiments.
The negative demographic impacts of mate limitation and
reduced connectivity have recently been shown to be especially
evident in self-incompatible species, although our findings
suggest that populations of A. woodmaniorum are not mate
limited due to extensive pollen dispersal (Aguilar et al., 2006;
Honnay and Jacquemyn, 2007; Leimu et al., 2010). Analysis
of levels of recruitment and long-term demographic response
in populations of A. woodmaniorum would also be valuable in
providing further insight into minimum seed production required
for population persistence.
Conclusions
Maintenance of genetic connectivity through significant
pollen-mediated gene flow over extensive dispersal distances,
and high levels of outcrossing, are important features of
A. woodmaniorum that may be critical for the persistence of
this species in a series of large and small disjunct populations
over a narrow geographic range. As long as this population
system remains intact, this species is likely to persist, even as
small populations, over significant historical time frames
(Millar et al., 2013). Acacia woodmaniorum is currently a
listed threatened species under the Western Australian Wildlife
Conservation Act 1950 (see http://florabase.dpaw.wa.gov.au),
due to its highly restricted distribution and the prospective
mineral exploration and active extraction activities that cover
its range. Future anthropogenic disturbance is also likely in this
landscape, and loss of populations may impact gene flow patterns
and thus influence population persistence. A number of conservation measures that aim to alleviate negative genetic and demographic impacts of reduced connectivity can be employed for the
long-term conservation of recently fragmented species and those
for which further or future population fragmentation is envisaged. Maintenance of gene flow can be achieved by the direct
augmentation of populations or establishment of populations at
previous or new sites with germplasm sourced from a number
of different populations. Genetic augmentation is likely to
improve mate availability and reproductive output, but must
also take into account the likelihood of any fitness reduction
via outbreeding depression in the resulting progeny (Byrne
969
et al., 2011; Weeks et al., 2011). Adaptation to different environmental conditions is unlikely for A. woodmaniorum given the
habitat specificity and limited geographic range, and this, in
combination with a highly outcrossed mating system, suggests
that outbreeding depression is unlikely to be an issue. Levels of
genetic diversity (Millar et al., 2013) and a lack of inbreeding
effects in small populations imply that direct genetic rescue is
not immediately required in this species as long as mate limitation or limitations to pollen dispersal remain minimal.
S U P PL E M E N TARY D ATA
Supplementary data are available online at www.aob.oxford
journals.org and consist of Table S1: Allele frequencies of nine
nuclear microsatellite loci in progeny cohorts from nine populations of Acacia woodmaniorum.
ACK NOW LED GE MENTS
The authors thank Karara Mining Limited for financial support,
H. Nistelberger for assistance in the field, and M. Williams for
assistance with data analysis. We also thank the Chief Editor,
Handling Editor and two anonymous reviewers for their suggestions in improving the manuscript. This work was supported by
Karara Mining Limited who played no role in the design, collection, analysis, interpretation of data, writing the manuscript or
the decision to submit the article for publication. The authors
have no actual or potential conflicts of interest that could inappropriately influence this work.
L IT E R AT U R E CI T E D
Aguilar R, Ashworth L, Geletto L, Aizen M. 2006. Plant reproductive susceptibility to habitat fragmentation: a review and synthesis through a
meta-analysis. Ecology Letters 9: 968–980.
Ahmed S, Compton S, Butlin R, Gilmartin P. 2009. Wind-borne insects
mediate directional pollen transfer between desert fig trees 160 kilometers
apart. Proceedings of the National Academy of Sciences, USA 106:
20342–20347.
Austerlitz F, Dick CW, Dutech C, et al. 2004. Using genetic markers to estimate
the pollen dispersal curve. Molecular Ecology 13: 937–954.
Bacles CFE, Ennos RA. 2008. Paternity analysis of pollen-mediated gene flow
for Fraxinus excelsior L. in a chronically fragmented landscape. Heredity
101: 358–380.
Bacles CFE, Jump A. 2011. Taking a trees perspective on forest fragmentation
genetics. Trends in Plant Science 16: 13– 18.
Bacles CFE, Burczyk J, Lowe AJ, Ennos RA. 2005. Historical and contemporary mating patterns in remnant populations of the forest tree Fraxinus excelsior L. Evolution 59: 979 –990.
Beard JS. 1976. Murchison, 1:1,000,000 vegetation series: the vegetation of the
Murchison region. Perth: University of Western Australia Press.
Bernhardt P, Kenrick J, Knox R. 1984. Pollination ecology and the breeding
system of Acacia retinoides (Leguminosae: Mimosoideae). Annals of the
Missouri Botanical Garden 71: 17– 29.
Broadhurst L, Lowe A, Coates D, et al. 2008a. Seed supply for broadscale restoration: maximising evolutionary potential. Evolutionary Applications 1:
587–597.
Broadhurst L, Young A, Forrester R. 2008b. Genetic and demographic
responses of fragmented Acacia dealbata (Mimosaceae) populations in
southeastern Australia. Biological Conservation 141: 2843–2856.
Brown A, Allard R. 1970. Estimation of the mating system in open pollinated
maize populations using allozyme polymorphisms. Genetics 66: 133– 145.
Byrne M, Elliott C, Yates C, Coates D. 2007. Extensive pollen dispersal in a bird
pollinated shrub, Calothamnus quadrifidus, in a fragmented landscape.
Molecular Ecology 16: 1303–1314.
970
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
Byrne M, Elliott CP, Yates C, Coates DJ. 2008. Maintenance of high pollen dispersal in Eucalyptus wandoo, a dominant tree of the fragmented agricultural
region in Western Australia. Conservation Genetics 9: 97–105.
Byrne M, Stone L, Millar MA. 2011. Assessing genetic risk in revegetation.
Journal of Applied Ecology 48: 1365– 1373.
Casiva P, Vilardi J, Cialdella A, Saidman B. 2004. Mating system and population structure of Acacia aroma and A. macracantha (Fabaceae). American
Journal of Botany 91: 58– 64.
Coates D. 1988. Genetic diversity and population genetic structure in the rare
Chittering Grass Wattle, Acacia anomala Court. Australian Journal of
Botany 36: 273–286.
Coates D, Tischler G, McComb JA. 2006. Genetic variation and the mating
system in the rare ghost wattle, Acacia sciophanes compared with its
common sister species Acacia anfractuosa (Mimoseaceae). Conservation
Genetics 7: 931– 944.
Cole T. 2003. Genetic variation in rare and common plants. Annual Review of
Ecology, Evolution, and Systematics 34: 213– 237.
Craft K, Ashley M. 2007. Landscape genetic structure of bur oak (Quercus
macrocarpa) savannas in Illinois. Forest Ecology and Management 239:
13– 20.
Dick C, 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.
Eckert C, Kalisz S, Geber M, et al. 2010. Plant mating systems in a changing
world. Trends in Ecology and Evolution 25: 35–43.
Ellstrand N. 1992. Gene flow by pollen: implications for plant conservation genetics. Oikos 63: 77–86.
Ellstrand NC, Elam D. 1993. Population genetic consequences of small population size: implications for plant conservation. Annual Review of Ecology and
Systematics 24: 217–242.
Feidler P, Ahouse J. 1992. Hierarchies of cause: towards an understanding of
rarity in vascular plant species. In: Feidler P, Jain S, eds. Conservation
biology. New York: Chapman and Hall, 23– 47.
Ford A, Forde N. 1976. Birds as possible pollinators of Acacia pycnantha.
Australian Journal of Botany 24: 793– 795.
Gardner R, Early J. 1996. The naturalisation of banyan figs (Ficus spp,
Moraceae) and their pollinating wasps (Hymenoptera: Agaoindae) in New
Zealand. New Zealand Journal of Botany 34: 103– 110.
George N, Byrne M, Yan G. 2008. Mixed mating with preferential outcrossing
in Acacia saligna (Labill.) H. Wendl. (Leguminosae: Mimosoideae). Silvae
Genetica 57: 139–145.
Gitzendanner MA, Soltis PS. 2000. Patterns of genetic variation in rare and
widespread plant congeners. American Journal of Botany 87: 783–792.
Glyphis J, Milton S, Siegfried W. 1981. Dispersal of Acacia cyclops by birds.
Oecologia 48: 138– 141.
Goto S, Shimatani K, Yoshimaru H, Takahashi Y. 2006. Fat-tailed gene flow in
the dioecious canopy tree species Fraxinus mandshurica var. japonic
revealed by microsatellites. Molecular Ecology 15: 2985–2996.
Hamrick J. 2004. Response of forest trees to global environmental changes.
Forest Ecology and Management 197: 323– 335.
Hamrick J, Godt M. 1989. Allozyme diversity in plant species. In: Brown AHD,
Clegg M, Kahler A, Weir B, eds. Plant population genetics, breeding and
genetic resources. Sunderland, MA: Sinauer Associates, 43– 63.
Hamrick J, Godt M. 1996. Effects of life history traits on genetic diversity in
plants. Philosophical Transactions of the Royal Society B: Biological
Sciences 351: 1291–1298.
Honnay O, Jacquemyn H. 2007. Susceptability of common and rare plant
species to the genetic consequences of habitat fragmentation.
Conservation Biology 21: 823–831.
Jacquemyn H, De Meester L, Jongejans E, Honnay O. 2012. Evolutionary
changes in plant reproductive traits following habitat fragmentation and
their consequences for population fitness. Journal of Ecology 100: 76–87.
Jha S, Dick CW. 2010. Native bees mediate long-distance pollen dispersal in a
shade coffee landscape mosaic. Proceedings of the National Academy of
Sciences, USA 107: 13760– 13764.
Karron JD. 1987. A comparison of levels of genetic polymorphism and selfcompatibility in geographically restricted and widespread plant congeners.
Evolutionary Ecology 1: 47–58.
Kaufman S, Smouse P, Alvarez-Buylla E. 1998. Pollen-mediated gene flow and
differential male reproductive success in a tropical pioneer tree, Cecropia
obtusifolia Bertol. (Moraceae): a paternity analysis. Heredity 81: 164–173.
Kenrick J, Knox B. 1982. Function of the polyad in reproduction of Acacia.
Annals of Botany 50: 721 –727.
Kenrick J, Knox B. 1989. Quantitative analysis of self incompatibility in trees of
seven species of Acacia. Journal of Heredity 80: 240 –245.
Knox B, Kenrick J, Bernhardt P, et al.1985. Extrafloral nectaries as adaptations
for bird pollination in Acacia terminalis. American Journal of Botany 72:
1185–1196.
Kramer A. 2008. The paradox of forest fragmentation genetics. Conservation
Biology 22: 878– 885.
Kress W. 1981. Sibling competition and evolution of pollen unit, ovule number,
and pollen vector in angiosperms. Systematic Botany 6: 101–112.
Lander T, Boshier D, Harris S. 2010. Fragmented but not isolated: contribution
of single trees, small patches and long-distance pollen flow to genetic connectivity for Gomortega keule, an endangered Chilean tree. Biological
Conservation 143: 2583– 2590.
Lankau R, Søgaard Jørgensen P, Harris D, Sih A. 2011. Incorporating evolutionary principles into environmental management and policy.
Evolutionary Applications 4: 315– 325.
Leimu R, Mutidainen P, Koricheva J, Fischer M. 2006. How general are positive relationships between plant population size, fitness and genetic variation? Journal of Ecology 94: 942–952.
Leimu R, Vergeer P, Angeloni F, Ouborg N. 2010. Habitat fragmentation,
climate change, and inbreeding in plants. Annals of the New York
Academy of Sciences 1195: 84– 85.
Lowe W, Allendorf FW. 2010. What can genetics tell us about population connectivity? Molecular Ecology 19: 3038–3051.
Mandal A, Ennos R. 1995. Mating system analysis in a natural population of
Acacia nilotica subspecies kraussiana. Forest Ecology and Management
79: 235–240.
Mandal A, Ennos RA, Fagg C. 1994. Mating system analysis in a natural population of Acacia nilotica subspecies leiocarpa. Theoretical and Applied
Genetics 89: 931– 935.
Marshall T, Slate J, Kruuk L, Pemberton J. 1998. Statistical confidence for
likelihood-based paternity inference in natural populations. Molecular
Ecology 7: 639– 655.
Maslin B, Buscumb C. 2007. Two new Acacia species (Leguminosae:
Mimosoideae) from banded ironstone ranges in the Midwest region.
Nuytsia 17: 263– 272.
Millar MA. 2009. Characterisation of microsatellite DNA markers for the rare
Acacia woodmaniorum (Leguminosae: Mimosaceae). Conservation
Genetics Resources 1: 441– 445.
Millar MA, Byrne M, Nuberg I, Sedgley M. 2008. High outcrossing and
random pollen dispersal in a planted stand of Acacia saligna subsp.
saligna revealed by paternity analysis using microsatellites. Tree Genetics
and Genomes 4: 367–377.
Millar MA, Byrne M, Nuberg IK, Sedgley M. 2012. High levels of genetic contamination in remnant populations of Acacia saligna from a genetically
divergent planted stand. Restoration Ecology 20: 260–267.
Millar MA, Coates D, Byrne M. 2013. Genetic connectivity and diversity in inselberg population of Acacia woodmaniorum, a rare endemic of the Yilgarn
Craton banded iron formations. Heredity 11: 437–444.
Moffet A. 1956. Genetical studies in acacias. I. The estimation of natural crossing
in black wattle. Heredity 10: 57–67.
Moran G, Muona O, Bell J. 1989a. Acacia mangium: a tropical forest tree of the
coastal lowlands with low genetic diversity. Evolution 43: 251 –235.
Moran G, Muona O, Bell J. 1989b. Breeding systems and genetic diversity in
Acacia auriculiformis and A. crassicarpa. Biotropica 21: 250– 256.
Morgan A, Carthew SM, Sedgley M. 2002. Breeding system, reproductive efficiency and weed potential of Acacia baileyana. Australian Journal of
Botany 50: 357– 364.
Mortlock W. 1999. Guidelines 5. Seed collection from woody plants for local revegetation. FloraBank. http://www.florabank.org.au/default.asp?V_DOC_
ID=877, accessed 11 June 214.
Muona O, Moran G, Bell J. 1991. Hierarchical patterns of correlated mating in
Acacia melanoxylon. Genetics 127: 619– 626.
Nason J, Herre E, Hamrick J. 1998. The breeding structure of a tropical keystone plant resource. Nature 391: 685–687.
Ng C, Lee S, Ng K, Muhammad N, Ratnam W. 2009. Mating system and seed
variation of Acacia hybrid (A. mangium × A. auriculiformis). Journal of
Genetics 88: 25–31.
NSWNPWS. 2003. Downy Wattle (Acacia pubescens) recovery plan. Hurtsville,
NSW: New South Wales National Parks and Wildlife Service.
Millar et al. — Pollen dispersal and mating in Acacia woodmaniorum
Peakall R, Smouse P. 2006. GenAlEx 6: genetic analysis in Excel. Population
genetic software for teaching and research. Molecular Ecology Notes 6:
288– 295.
Petit R, Hampe A. 2006. Some evolutionary consequences of being a tree.
Annual Review of Ecology, Evolution, and Systematics 37: 187– 214.
Philip J, Sherry S. 1946. The degree of natural crossing in green wattle, Acacia
decurrens Wild. and its bearing on wattle breeding. Journal of the South
African Forestry Association 14: 1– 28.
Ritland K. 2002. Extensions of models for the estimation of mating systems
using n independent loci. Heredity 88: 221–228.
Ritland K, Jain S. 1981. A model for the estimation of outcrossing rate and gene
frequencies using n independent loci. Heredity 47: 35–52.
Ritland K, Leblanc M. 2004. Mating system of four inbreeding monkeyflower
(Mimulus) species revealed using ‘progeny-pair’ analysis of highly informative microsatellite markers. Plant Species Biology 19: 149–157.
Robledo-Arnuncio J, Gil L. 2005. Patterns of pollen dispersal in a small population of Pinus sylvestris L. revealed by total-exclusion paterntiy analysis.
Heredity 94: 13–22.
Rosas F, Quesada M, Lobo J, Sork VL. 2011. Effects of habitat fragmentation
on pollen flow and genetic diversity of the endangered tropical
tree Swietenia humilis (Meliaceae). Biological Conservation 144:
3082–3088.
Sampson J, Byrne M, Yates C, et al. 2014. Contemporary pollen-mediated gene
immigration reflects the historical isolation of a rare, animal pollinated shrub
in a fragmented landscape. Heredity 112: 172–181.
971
Slatkin M. 1987. Gene flow and the geographic structure of natural populations.
Science 236: 787–792.
Stone GN, Raine NE, Prescott M, Willmer PG. 2003. Pollination ecology of
acacias (Fabaceae, Mimosoideae). Australian Systematic Botany 16:
103–118.
Tybirk K. 2007. Reproductive biology and evolution of the genus Acacia.
International Group for the Study of Mimosoideae.
Vanstone V, Paton D. 1988. Extrafloral nectaries and pollination of Acacia
pycnantha Benth. by birds. Australian Journal of Botany 36: 519– 531.
Ward M, Dick C, Gribel R, Lowe AJ. 2005. To self, or not to self . . . A review of
outcrossing and pollen-mediated gene flow in neotropical trees. Heredity
95: 246–254.
Weeks A, Sgró C, Young A, et al. 2011. Assessing the benefits and risks of translocations in changing environments: a genetic perspective. Evolutionary
Applications 4: 709– 725.
White G, Boshier D, Powell W. 2002. Increased pollen flow counteracts fragmentation in a tropical dry forest: an example from Swietenia humilis
Zuccarini. Proceedings of the National Academy of Sciences, USA 99:
2038– 2042.
Worthington WJ, Allen PJ, Pomeroy PP, Twiss SD, Amos W. 1999. Where
have all the fathers gone? An extensive microsatellite analysis of paternity
in the grey seal (Halichoerus grypus). Molecular Ecology 8: 1417–1429.
Young A, Boyle T, Brown A. 1996. The population genetic consequences of
habitat fragmentation for plants. Trends in Ecology and Evolution 11:
413–418.