Heritable pollution tolerance in a marine invader
Louise A. McKenzien, Rob Brooks, Emma L. Johnston
Evolution & Ecology Research Centre and School of Biological, Earth and Environmental Sciences, The University of New South Wales, Sydney 2052, Australia
a r t i c l e i n f o
Available online 3 February 2011
Heavy metal pollution
a b s t r a c t
The global spread of fouling invasive species is continuing despite the use of antifouling biocides.
Furthermore, previous evidence suggests that non-indigenous species introduced via hull fouling may
be capable of adapting to metal-polluted environments. Using a laboratory based toxicity assay, we
investigated tolerance to copper in the non-indigenous bryozoan Watersipora subtorquata from four
source populations. Individual colonies were collected from four sites within Port Hacking (Sydney,
Australia) and their offspring exposed to a range of copper concentrations. This approach, using a full-
sib, split-family design, tests for a genotype by environment (G?E) interaction. Settlement and
complete metamorphosis (recruitment) were measured as ecologically relevant endpoints. Larval sizes
were also measured for each colony. Successful recruitment was significantly reduced by the highest
copper concentration of 80 mg L?1. While there was no difference in pollution tolerance between sites,
there was a significant G?E interaction, with large variation in the response of colony offspring within
sites. Larval size differed significantly both between sites and between colonies and was positively
correlated with tolerance. The high level of variation in copper tolerance between colonies suggests
that there is considerable potential within populations to adapt to elevated copper levels, as tolerance
is a heritable trait. Also, colonies that produce large larvae are more tolerant to copper, suggesting that
tolerance may be a direct consequence of larger size.
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Pollution and the global spread of invasive species are both
major causes of biodiversity loss and environmental change
(Carlton and Geller, 1993; Cohen and Carlton, 1998), with sub-
stantial ecological implications. The marine environments most
susceptible to introduced species are harbours and estuaries
where many invasives are introduced by the shipping trade
(Floerl et al., 2009, 2004). Harbours and estuaries are also subject
to high levels of anthropogenic disturbance, particularly in the
form of heavy metal pollution which enters the system through
numerous sources including industrial waste (Apte and Day,
1998; Hall et al., 1998) and antifouling biocides (Weis and Weis,
1996). Copper, which is particularly toxic to marine invertebrates,
is one of the more prevalent heavy metals in these environments
(Hall et al., 1998). An essential element for metabolism in many
marine organisms, at higher concentrations copper often becomes
lethal (Bryan, 1971). Whilst metal contamination reduces diver-
sity in all marine habitats (Johnston and Roberts, 2009) there is
evidence that invasive species diversity is not affected in hard
substrate communities, with spatial dominance actually increas-
ing (Crooks et al., 2010; Piola and Johnston, 2008). It has been
hypothesised that a number of fouling non-indigenous species
have some form of metal tolerance (Dafforn et al., 2009; Piola and
Johnston, 2006a), yet very little is known about the evolution of
this trait and whether it is heritable.
Whilst invasive species have provided the opportunity to
study evolution (Huey et al., 2005; Lee and Gelembiuk, 2008;
Sakai et al., 2001), there has been little research on the evolu-
tionary response of marine or aquatic invasives to heavy metal
pollution. If invasive species have the capacity to rapidly adapt to
a commonly and increasingly used toxic agents, such as copper as
a biocide, (I.M.O., 2001; Piola et al., 2009) then even greater
concerns are raised regarding the impact that invasives have on
the ecology of native assemblages. The efficacy of antifoulant
paints may also be reduced if particular species or populations
acquire tolerance to the active biocides, resulting in greater
economic and ecological costs (Piola et al., 2009).
Tolerance to anthropogenic contaminants has evolved rapidly
in numerous species, most notably as pesticide and herbicide
resistance in agriculture (Scarabel et al., 2007). The occurrence of
site-specific heavy metal pollution in terrestrial, aquatic and
marine habitats can expose populations to intense selection for
multiple generations (Macnair, 1987; Medina et al., 2007), result-
ing in population-specific resistance (Klerks and Weis, 1987) such
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nCorresponding author. Present address: Marine Invasions Laboratory, Smith-
sonian Environmental Research Center, Edgewater, MD 21037, USA.
E-mail addresses: email@example.com (L.A. McKenzie),
firstname.lastname@example.org (R. Brooks), email@example.com (E.L. Johnston).
Environmental Research 111 (2011) 926–932
as cadmium resistance in exposed populations of the wolf spider
Pirata piraticus (Hendrickx et al., 2008) and aquatic oligochaete
Limnodrilus hoffmeisteri (Klerks and Levinton, 1989).
The increasing occurrence of heavy metal pollution in marine
environments (Hall et al., 1998) and the use of antifouling
biocides on the hulls of ships, which are a major vector for intro-
duced marine species (Piola et al., 2009), means heavy metals can
act as a selective pressure during the process of species introduc-
tions (Floerl et al., 2005). In fact a range of non-indigenous marine
species have been identified as having a positive affiliation with
heavy metal pollution (Dafforn et al., 2008). These include a
variety of bryozoans, ascidians, algae and crustaceans (Dafforn
et al., 2008, 2009). This affiliation may be due to several mecha-
nisms, first the capacity for adaptation, a previous history of
selection or most likely a combination of both. The inherent
ability to endure and adapt to a broad or fluctuating range of
environmental conditions is a notable trait in some successful
non-indigenous species (Lee and Gelembiuk, 2008; Sakai et al.,
2001). The combination of adaptive capacity and a history of
exposure may select for greater plasticity in novel environments
translating to tolerance of heavy metal pollution as a novel
stressor (Agosta and Klemens, 2008; Lee and Gelembiuk, 2008).
This may partially explain the propensity for tolerance in intro-
duced species while many indigenous species may lack the
capacity to respond (Byers, 2002; Macnair, 1987).
Phenotypic plasticity is increasingly recognised as an important
component influencing contemporary evolutionary responses to
anthropogenic change (Carroll et al., 2007; Crispo, 2008), particu-
larly in the form of parental effects which can impact upon devel-
oping phenotypes through manipulation of reproductive traits in
response to environmental conditions (Badyaev and Uller, 2009;
R¨ as¨ anen and Kruuk, 2007). Plasticity in reproductive traits such as
fecundity and offspring size is commonly seen in a range of taxa
(for example birds (Reed et al., 2009), plants (Galloway et al., 2009)
and insects (Fox et al., 1999)) and is increasingly being used as
a proxy for maternal and offspring fitness (Marshall and Uller,
2007), particularly for species for which other fitness traits may be
difficult to quantify. With variation in offspring size documented
both within and between individuals as well as populations
(Marshall and Keough, 2008) this variation is attributable to an
interaction between parental genotype and environmental condi-
tions (Bernardo, 1996). For example, offspring size in invertebrates
has been shown to increase when the mother is under stress, such
as competition (Allen et al., 2008), pollutants (Marshall, 2008) and
food availability (Newlon et al., 2003), as a result of maternal
In species that do not have a discrete reproductive output
period, fertility measurements can be difficult. In these systems
offspring size and tolerance are measurable traits that can be
directly linked to both maternal and offspring fitness (Marshall
and Uller, 2007). The encrusting bryozoan Watersipora subtorquata
is a trickle spawner, where larvae are released over time, with
each colony containing larvae that are at different stages of
maturation (personal observation) therefore making overall or
lifetime fecundity measures for an individual colony difficult.
W. subtorquata is relatively insensitive to copper but has only
been tested at a general species or single population level (Piola
and Johnston, 2006a, 2009; Wisely, 1958), with no knowledge
about the prevalence or underlying basis of this trait. It has also
been described as a ‘foundation species’ due to its ability to recruit
to surfaces coated in antifoulant paint where it creates a less
toxic secondary surface for other fouling organisms to settle on
(Floerl et al., 2004). It can also form large 3D structures which
provide habitat for epibiota (Floerl et al., 2004; Stachowicz and
Byrnes, 2006) and can dominate hard substrate with high den-
sities (Sellheim et al., 2010). This species has a cosmopolitan
distribution; its native range is uncertain but thought to be in
the Caribbean (Mackie et al., 2006). The similarities between
W. subtorquata and many fouling non-indigenous marine species,
such as method of introduction, comparable physiological traits to
other colonial organisms (such as brooded offspring and vegeta-
tive growth in ascidians) and a global distribution (Mackie et al.,
2006) make this species an appropriate test organism for examin-
ing heavy metal tolerance within and between introduced
Tolerance to contaminants has generally been assessed at
a species or population level (Johnston, 2011), including for
W. subtorquata (Piola and Johnston, 2006a), and rarely at the
individual level. A novel way to assess individual tolerance is to
test offspring, as full or half siblings, independently in multiple
environments in a genotype by environment (G?E) design
(Falconer and Mackay, 1996). This enables us to test whether
there is a genetic basis to tolerance and estimate the heritability
of the trait (Galletly et al., 2007; Pease et al., 2010). Detailed
studies, such as this one, are important because in order for
selection to cause evolutionary change there must be heritable
variation among individuals. This study aimed to determine
whether tolerance to copper is consistent within and between
populations of the non-indigenous species W. subtorquata, and
whether tolerance is correlated with offspring size.
2.Methods and materials
2.1. Sample collection and site descriptions
Colonies of W. subtorquata (d’Orbigny, 1852) were collected from four sites
within Port Hacking estuary (34170S, 1511100E) south of Sydney in New South
Wales, Australia (Fig. 1); Burraneer Marina, Dolans Marina, Private pontoon and
Cronulla swimming enclosure. Port Hacking estuary is a recreational estuary
with no commercial shipping activity, but is still impacted by anthropogenic
pollution. Copper accumulation in experimentally deployed oysters have pre-
viously found copper levels within this section of the estuary to be elevated
approximately three times beyond natural oceanic levels due to urbanisation
(Copper: 57.6 mg g?1dry weight of oyster tissue; Dafforn et al., 2009). Sites were
separated by a minimum of 0.6 km. Colonies at all sites were collected from
artificial structures such as floating pontoons, fixed pilings and netting. W.
subtorquata colonies are most fecund during the summer months, but can
still produce larvae throughout the year. They brood their larvae for two weeks
before spawning lecithotrophic larvae that are competent to settle immediately
(Marshall and Keough, 2008).
Colonies were collected during February and March 2008 and were main-
tained in individual containers at 20 1C for up to 3 d without light except during
spawning. Spawning was induced after 1–3 days of collection by exposing the
colonies to light and stopping aeration for approximately one hour, stimulating
the release of larvae. Larvae were collected using a pipette and preserved for larval
size measurements or used for the copper toxicity assay. This was repeated for
two consecutive days if insufficient numbers of larvae were produced on the first
attempt, as W. subtorquata is a trickle spawner releasing larvae slowly over
successive days. Colonies were maintained and spawned in individual containers
to ensure maternal parentage was known.
2.2. Copper tolerance
Copper tolerances of the larvae of individual W. subtorquata colonies from
each site were tested by measuring larval settlement and metamorphosis after a
3 d exposure to copper concentrations of 0, 40 or 80 mg L?1. These concentrations
represent relevant values in polluted aquatic environments, particularly near
surfaces painted with antifoulant biocides (Schiff et al., 2004; Valkirs et al., 2003).
Copper solutions were prepared by adding copper II sulphate anhydrous to Milli-Q
water to create a stock solution of 1 g L?1Cu in freshwater. This was then diluted
in filtered (0.2 mm) and autoclaved seawater to the two experimental concentra-
tions of 40 and 80 mg L?1Cu. Filtered and autoclaved seawater was used as the
control treatment (0 mg L?1Cu). All equipment was acid washed in 5% nitric acid
for a minimum of 24 h then thrice rinsed in Milli-Q water prior to use. The plastic
35 mm diameter Petri dishes used in the assay were pre-soaked for 24 h in the
appropriate copper solution prior to commencing the experiment.
Only five colonies from each site produced enough larvae for the copper assay.
A full-sib, split-family design (Becker, 1984) was used to detect variation among
L.A. McKenzie et al. / Environmental Research 111 (2011) 926–932
colonies, with colony as family/genotype and copper concentration as environ-
ment (Genotype?Environment interaction). From each of these colonies an
individual larva was placed in a plastic Petri dish containing one of the three
copper concentrations; 0, 40 and 80 mg L?1(control, low and high). There were six
replicate larvae per treatment. Exposure to treatments was maintained for three
days, with the water within the Petri dish changed on day two. Survival to
settlement and metamorphosis was assessed on day three and was defined as a
fully metamorphosed zooid, complete with orifice (Piola and Johnston, 2006a),
and is hereafter referred to as successful settlement (Keough and Downes, 1982).
2.3. Larval size
To compare offspring sizes between colonies an independent subset of larvae
from each of the colonies used in the copper assay were also preserved in 10%
formaldehyde with seawater. Once preserved, larvae were then photographed
with a digital camera (PixeLINK) through a dissecting microscope (Olympus).
Larvae were photographed with the oral pole (Wisely, 1958) oriented upwards to
maintain consistency of measurement. The total area was estimated to the nearest
0.01 mm2using the program Scion Image (Scion Corp., Fredrick, Maryland, USA).
Replicate numbers differed between colonies; each colony had at least 4 replicates
and up to a maximum of 8 replicates (A1). The statistical model used is robust to
unbalanced designs such as these (Quinn and Keough, 2002). Of all the colonies
used within the copper assay, five per site, two colonies (one each from Burraneer
and Cronulla swimming enclosure) only produced enough larvae for the copper
assay and are without larval size data.
2.4. Statistical analyses
Using settlement success as a measure of tolerance, tolerance to copper was
compared between sites and colonies. As settlement success was binomial, a partly
nested three-factor, mixed model PERMANOVA (Anderson, 2001) was used, with
Euclidean distance to calculate the similarity matrix (factors: Site and Colony(Site)
random, Copper: fixed) and was run for 9999 permutations. As PERMANOVA is
based on permutations it is more robust to the assumptions of ANOVA (Anderson,
2001). Because there was no significant effect of site or interaction between site and
copper treatment (P40.25) the final model does not contain these terms. Broad
sense heritability, H2, was calculated for successful settlement for each copper
concentration using the formula for a balanced full-sibling design (Becker, 1984).
Variance components were obtained from one-way ANOVA’s performed for each
copper concentration, using PERMANOVA (Anderson, 2001).
2.4.2. Larval sizes
Larval size was compared between site and colony using a two-factor nested
analysis of variance (site and colony were both random factors, with colony nested
in site). The number of fecund individuals differed between the sites, resulting in
an uneven number of colonies included in the analysis, as two colonies only
produced enough larvae for the copper assay. Data were analysed using a two-
factor nested PERMANOVA (Anderson, 2001) with Euclidean distance to calculate
the similarity matrix, and then run for 9999 permutations using the same design as
above. Post-hoc pair-wise comparisons were then performed using PERMANOVA to
compare between sites. Significance was set at ar0.05.
Settlement success at 80 mg L?1Cu was compared to average larval size
within each colony to determine if there was any correlation between size and
tolerance using a randomisation test (Monte Carlo) for significance. Only the high
copper treatment was compared as it was the most lethal, significantly reducing
settlement making it statistically comparable, as well as being a more extreme
indicator of a colony’s tolerance. Sites were pooled as tolerance was found not to
differ between them.
3.1. Copper assay
Copper significantly reduced successful settlement (F2,32¼
105.18, Po0.001) and increased mortality. The average settle-
ment within a colony ranged from 9372% and 8275% in the
control and low copper treatments respectively, to 1874% in
high copper (mean across sites7SE) (Fig. 2). There was a
significant G?E interaction, with colony interacting significantly
0 µgL-1 Cu
40 µgL-1 Cu
80 µgL-1 Cu
Fig. 2. Copper assay: effect of 0, 40 and 80 mg L?1Cu in seawater on successful
settlement and metamorphosis of W. subtorquata larvae at each site. Values are
mean settlement7SE for colony at each site (n¼5 colonies). Letters represent
significant differences between sites in post-hoc pair-wise comparisons (a¼0.05).
Fig. 1. Sampling locations within Port Hacking estuary. Mature W. subtorquata colonies were collected at the four sites: 1. Burraneer Marina; 2. Dolans Marina; 3. Private
pontoon; 4. Cronulla swimming enclosure.
L.A. McKenzie et al. / Environmental Research 111 (2011) 926–932
with copper treatment (F32,307¼1.60, P¼0.028). Settlement suc-
cess differed between colonies in both the low and high copper
treatments (Fig. 3(a)–(d)), ranging from 33% to 100% and from 0%
to 50%, respectively. High settlement success in 40 mg L?1Cu did
not predict settlement success in 80 mg L?1Cu, with no linear
response between the copper treatments. There was no difference
between sites nor an interaction between site and copper treat-
ment as P40.25. Broad sense heritability for successful settle-
ment was highest in the low copper treatment (H2¼0.38) but
reduced dramatically in the high copper (H2¼0.05) and control
3.2. Larval sizes
Larvae varied widely in size between sites within the estuary
(from 0.034 to 0.101 mm2) and within individual colonies, with the
greatest maximum difference between siblings being 0.066 mm2.
Average larval size for each colony covered a similar range from 0.054
to 0.084 mm2, with colonies differing significantly (F14,113¼3.211,
Po0.001) (Fig. 3(e)). Sites were significantly different (F3,14¼5.159
P¼0.016), with one site in particular driving the difference. Colonies
from Private pontoon had significantly larger larvae than colonies
from Burraneer Marina and Dolans Marina (Fig. 3(e)). The comparison
of average larval size to successful settlement in 80mg L?1Cu found
colonies which produced larger larvae were more tolerant of high
copper levels (R¼0.448, P¼0.036) (Fig. 3(f)).
The increasing prevalence of non-indigenous sessile inverte-
brates in metal-polluted estuaries, whilst many native species are
declining (Piola et al., 2009; Piola and Johnston, 2008) suggests
some form of heavy metal tolerance. We found that both offspring
size and tolerance varied greatly between individuals, with
colonies which produced larger offspring correlating with greater
tolerance to high levels of copper. The significant G?E interaction
Larval size mm2
Larval size mm2
0.060.07 0.08 0.09
0 µgL-1 Cu40 µgL-1 Cu 80 µgL-1 Cu
Fig. 3. Individual colony tolerance and larval size. Total successful settlement per colony at the four sites for each copper concentration (0, 40 and 80 mg L?1Cu)
at (a) Burraneer Marina, (b) Dolans Marina, (c) Private pontoon and (d) Cronulla swimming enclosure (n¼6 larvae), 0 represents no settlement, (e) mean larval size per
colony (7SE) at each site (n¼4–8 larvae) and (f) the correlation plot between mean larval size and settlement at 80 mg L?1Cu for each W. subtorquata colony, showing a
line of best fit. Letters represent significant differences between sites in post-hoc pair-wise comparisons (a¼0.05).
L.A. McKenzie et al. / Environmental Research 111 (2011) 926–932
between colony and copper implies a genetic basis to tolerance,
which is heritable. Our results found similar effects of copper on
W. subtorquata offspring to previous studies which investigated
tolerance only at a species/population level, where high levels of
copper (Z50 mg L?1) significantly reduced survival (Piola and
Johnston, 2006a) and successful metamorphosis (Ng and Keough,
2003). These findings are the first to demonstrate that tolerance
to copper in W. subtorquata’s is a variable and heritable trait,
indicating potential for further selection for this trait in polluted
Tolerance differed on an individual basis, with each colony
interacting differently with the various levels of copper. Interest-
ingly high settlement in clean and low copper conditions was
not indicative of tolerance to high copper treatments, as there
were no consistent settlement patterns across copper treatments.
The lack of differential tolerance between sites in this study
suggests that there are no large scale differences in contamination
between sites. Differential tolerance between populations has
been found in other species, but these experiments have com-
pared clean and heavily polluted sites (Durou et al., 2005;
Johnston, 2011; Piola and Johnston, 2006b) where selection
pressures are polarised. Instead it is more likely that small scale
temporal and spatial fluctuations in exposure (Addison et al.,
2008) would have a greater influence on selection for tolerance.
A number of mechanisms have been identified for handling
heavy metal pollution; such as decreased uptake (Gale et al.,
2003) and increased detoxification (Rainbow et al., 2009) which
may incorporate the non-enzymatic proteins metallothioneins
(Amiard et al., 2006). The exact processes utilised by bryozoans
have not been explored but many invertebrates employ at least
one tolerance mechanism, which can even differ between popula-
tions of the same species (Daka and Hawkins, 2004; Mouneyrac
et al., 2003). The lack of consistency that we observed between
colonies in their tolerance across copper levels not only precludes
predicting overall tolerance but also suggests that there may be
several mechanisms involved (Galletly et al., 2007). These may
not be consistent between individuals considering that various
pathways have been discerned for multiple populations of
the same species (Daka and Hawkins, 2004). It is possible that
these non-indigenous populations have been founded by indivi-
duals from many source areas that have experienced distinct
exposure and selection regimes, resulting in several mechanisms
One potential mechanism for tolerance could be the use of
matrotrophy. Numerous factors have been found to influence
offspring size through maternal provisioning, such as maternal
size, resource availability (Lampert, 1993), competition (Marshall
and Keough, 2009) and predation (Tollrian, 1995). Offspring size
is a strong predictor of settlement and post-settlement success in
many marine organisms (Bernardo, 1996; Marshall and Keough,
2007) including W. subtorquata (Marshall and Keough, 2004),
with variability in larval sizes anticipated due to the interaction
between parental genotypeand
(Bernardo, 1996). Bryozoans in particular have evolved to utilise
reproductive plasticity to deal with variable environmental con-
ditions, such as competition (Allen et al., 2008; Marshall and
Keough, 2008; Ostrovsky et al., 2009); therefore, it is likely that
maternal provisioning has become a strategy for dealing with
novel anthropogenic disturbances. In another bryozoan species,
Bugula neritia, colonies exposed to toxicants responded by produ-
cing larger, more tolerant offspring (Marshall, 2008), and con-
strengthening the relationship between pollutants and maternal
Within an impacted site, such as a marina where there are
multiple sources of copper pollution (Weis et al., 1998; Srinivasan
and Swain, 2007), exposure can differ at a spatial and temporal
scale. On a fine spatial scale proximity to pollution sources, such
as an antifoulant coated surface (Valkirs et al., 2003), will result in
heterogeneous exposure between individuals that may only be a
few metres apart (Addison et al., 2008). This small scale variation
would explain the maintenance of highly tolerant and intolerant
individuals, and therefore the variety of offspring sizes in
response to individual exposure as a result of maternal provision-
ing. Another way to deal with unpredictable environmental
conditions is to produce an array of offspring sizes within a
brood; bet-hedging (Crean and Marshall, 2009). Considering the
variability that was present within some broods, it is possible that
bet-hedging is a strategic way to deal with fluctuating heavy
metal pollution levels. As larger lecithotrophic larvae are capable
of longer swimming times, faster growth and quicker reproduc-
tive maturation rates post-settlement due to greater energy
reserves (Marshall and Keough, 2003, 2007), they would be at
an advantage in a stressful, polluted environment.
Although colonies that produce larger larvae appear to be
more tolerant to higher levels of contamination, continual pro-
duction of large offspring is costly, reducing overall fecundity
(Smith and Fretwell, 1974), and unnecessary if pollution levels are
inconsistent. Although matrotrophy no doubt evolved to deal
with other environmental and biological stressors, reproductive
plasticity has potentially contributed to the success of this species
in novel and anthropogenically polluted environments (R¨ as¨ anen
and Kruuk, 2007; Whitney and Gabler, 2008). Considering the
proposed vector for the translocation of W. subtorquata, via hull
fouling (Floerl and Inglis, 2005; Mackie et al., 2006), it is possible
that this species has been under selection not only for an overall
increased heavy metal tolerance (Floerl et al., 2004) compared
to most native species, but also ‘broad organismal plasticity’
(Lee and Gelembiuk, 2008). Inconsistent and fluctuating levels
of heavy metal pollution expose populations within a generation
to a heterogeneous selection regime (Lee and Gelembiuk, 2008;
Morgan et al., 2007), resulting in selection for individuals that can
utilise this phenotypic plasticity as a mechanism for tolerance.
Ultimately, the within-population variation suggests that heavy
metal tolerance is a heritable trait under selection which can
rapidly evolve in novel environments (Reznick and Ghalambor,
2001), such as a boat hull coated in copper based antifouling paint.
Whilst tolerance is not consistent in every individual, plasticity to
deal with fluctuating environmental conditions is a trait that
appears to be perpetually selected for (Morgan et al., 2007) and
it is likely that manipulation of maternal provisioning (Crean and
Marshall, 2009; Ostrovsky et al., 2009) may be ameliorating the
effect of heavy metal pollution. With heavy metal pollution an
increasing disturbance on a global scale (Birch et al., 2008) com-
bined with the ever increasing spread of non-indigenous species
(Vitousek et al., 1997) we can expect to see a greater prevalence of
tolerant populations of invasive species.
We are grateful to Subtidal Ecology and Ecotoxicology labora-
tory members for assistance in the field. The authors were
supported by the Australian Government and Australian Research
Council through Research Fellowships, Postgraduate Awards and
Grants. We thank Alistair Poore for comments which improved
earlier draughts of this article.
L.A. McKenzie et al. / Environmental Research 111 (2011) 926–932
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