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ARTICLE / ARTICLE
Effects of invasive American signal crayfish
(Pacifastacus leniusculus) on the reproductive
behaviour of threespine stickleback (Gasterosteus
aculeatus) sympatric species pairs
G.J. Velema, J.S. Rosenfeld, and E.B. Taylor
Abstract: Environmental change, including that caused directly or indirectly by invasive species, presents a major threat to the
persistence of native freshwater biodiversity. The invasive American signal crayfish (Pacifastacus leniusculus (Dana, 1852)) has
recently been implicated in the collapse of a pair of threespine stickleback (Gasterosteus aculeatus L., 1758) species in Enos
Lake, British Columbia, through introgressive hybridization; yet there are few data describing potential interactions between
crayfish and these recently evolved stickleback species. We conducted a behavioural study, using an intact sympatric
G. aculeatus species pair from a nearby lake, to examine if and how interactions with P. leniusculus may influence the breeding
behaviour of sticklebacks. We found that the reproductive behaviour of limnetic males was disrupted to a greater degree than that
of benthic males, suggesting that crayfish may disproportionately impact limnetic male reproductive success and may have
contributed to biased hybridization between the Enos Lake species pair. Our study illustrates how newly differentiated taxa may
be especially susceptible to environmental perturbations, particularly those caused by invasive species.
Key words: Gasterosteus aculeatus, threespine stickleback, Pacifastacus leniusculus, American signal crayfish, invasive
species, conservation, hybridization, reverse speciation, reproduction, species at risk.
Résumé : Les changements environnementaux, dont ceux causés directement ou indirectement par des espèces envahissantes,
constituent une grande menace pour la persistance de la biodiversité d’eau douce indigène. L’écrevisse de Californie
(Pacifastacus leniusculus (Dana, 1852)), une espèce envahissante, a récemment été impliquée dans l’effondrement d’une paire
d’espèces d’épinoches à trois épines (Gasterosteus aculeatus L., 1758) dans le lac Enos, en Colombie-Britannique, découlant
d’une hybridation introgressive. Peu de données sont toutefois disponibles sur les interactions potentielles des écrevisses et de ces
espèces d’épinoches récemment formées. Nous avons réalisé une étude comportementale dans laquelle nous avons utilisé une
paire d’espèces sympatriques intacte de G. aculeatus provenant d’un lac à proximité pour vérifier si des interactions avec
P. leniusculus peuvent influencer le comportement reproducteur des épinoches et, le cas échéant, de quelle manière. Nous avons
observé que le comportement reproducteur des mâles limnétiques était plus perturbé que celui des mâles benthiques, ce qui
suggère que les écrevisses pourraient avoir une incidence disproportionnée sur le succès de reproduction des mâles limnétiques et
pourraient ainsi avoir contribué à une hybridation biaisée entre la paire d’espèces du lac Enos. L’étude illustre comment des
taxons nouvellement différenciés pourraient être particulièrement sensibles aux perturbations du milieu, notamment celles causées
par des espèces envahissantes.
Mots-clés : Gasterosteus aculeatus, épinoche à trois épines, Pacifastacus leniusculus, écrevisse de Californie, espèce
envahissante, conservation, hybridization, spéciation inversée, reproduction, espèces en péril.
[Traduit par la Rédaction]
Received 29 April 2012. Accepted 21 August 2012. Published at www.nrcresearchpress.com/cjz on 19 October 2012.
G.J. Velema.* Department of Zoology, Biodiversity Research Centre, The University of British Columbia, 6270 University Boulevard,
Vancouver, BC V6T 1Z4, Canada.
J.S. Rosenfeld. British Columbia Ministry of the Environment, The University of British Columbia, 2202 Main Mall, Vancouver,
BC V6T 1Z4, Canada.
E.B. Taylor. Department of Zoology, Biodiversity Research Centre and Beaty Biodiversity Museum, The University of British
Columbia, 6270 University Boulevard, Vancouver, BC V6T 1Z4, Canada.
Corresponding author: G.J. Velema (e-mail: [email protected]).
*Present address: Stantec Consulting Ltd., 4370 Dominion Street, 5th Floor, Burnaby, BC V5G 4L7, Canada.
Can. J. Zool. 90: 1328 –1338 (2012)
doi:10.1139/z2012-102
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Introduction
Repeated glaciation of temperate regions is associated with
lower alpha diversity in freshwater systems relative to those in
lower latitudes (Lomolino et al. 2006), but is also associated
with rapid evolutionary divergence of lineages, some of which
behave as distinct biological species in sympatry (reviewed in
Taylor 1999). This rapid evolution often takes the form of
ecologically divergent limnetic and benthic species (Robinson
and Wilson 1994). In fact, Weir and Schluter (2007) suggested
frequent and massive environmental perturbations such as
those experienced during repeated glaciations may have been
important in generating rapid speciation, extinction, and species turnover relative to tropical environments. Recent speciation events may also occur in nonglaciated environments such
as from recent refilling of desiccated lakes (Johnson et al.
1996; Seehausen 2002), recent formation of bluehole lakes on
subtropical–tropical islands (Langerhans et al. 2007), or when
contemporary populations establish new migratory routes
(Rolshausen et al. 2009). One consequence of such recent,
postglacial speciation events is that barriers to gene exchange
may be incomplete; in such cases, ecological conditions that
promote assortative mating or postzygotic reproductive isolation may serve as especially important reproductive barriers. A
corollary of this idea is that evolutionarily young reproductive
barriers may be more easily eroded during environmental
change, making such species vulnerable to collapse through
the failure of reproductive barriers, a process often referred to
as “reverse speciation” (e.g., Savvaitova 1980; Hendry et al.
2006; Taylor et al. 2006; Vonlanthen et al. 2012). For example, in a subset of coastal lakes in the southern Strait of
Georgia region of British Columbia, Canada, several populations of freshwater-adapted threespine sticklebacks (Gasterosteus aculeatus L., 1758), descendants of the ubiquitous marine
threespine stickleback, have independently formed sympatric
species pair complexes over the past 13 000 years (McPhail
1993; Taylor and McPhail 2000). These sympatric species
pairs are still differentiating and have thus become important
models of ecological speciation (McPhail 1993, 2007;
Schluter 2000).
In the small number of lakes that gave rise to sympatric
stickleback species pairs, hybrids are viable but various factors
constrain hybridization. Prezygotic isolating barriers such as
divergence in body size, breeding colouration (Boughman
2001; Boughman et al. 2005), and courtship behaviour
(Ridgway and McPhail 1984; Shaw et al. 2007) normally limit
hybridization by reducing the likelihood of cross-fertilization.
Extrinsic postzygotic selection may also limit hybridization in
a given adaptive landscape by reducing the fitness of interspecific hybrids relative to offspring from either benthic– benthic
or limnetic–limnetic matings (Schluter 1995; Hatfield and
Schluter 1999; Gow et al. 2007; Taylor et al. 2012). Both
processes are thought to have been critical to ecological speciation in sticklebacks (e.g., Schluter 2000). Nevertheless,
reproductive isolation between the bottom-feeding benthic and
planktonic-feeding limnetic species remains incomplete because intrinsic postzygotic isolation has not yet evolved (i,e.,
hybrids remain viable; McPhail 1994; Hatfield and Schluter
1999).
Of the seven sympatric stickleback species pairs discovered
within the last 36 years, two have already disappeared. The
1329
Hadley Lake (Lasqueti Island) species pair went extinct in the
1990s due to the introduction of an invasive predator (brown
bullhead (Ameiurus nebulosus (Lesueur, 1819)) (Hatfield
2001). More recently, the Enos Lake (Vancouver Island) species pair collapsed into a hybrid swarm due to introgressive
hybridization and have has been cited as a case of “reverse
speciation” (Kraak et al. 2001; Taylor et al. 2006).
The specific ecological processes involved in the collapse of
the Enos Lake species pair into a hybrid swarm have not been
definitively established, but may include changes in water
chemistry or clarity, back-flooding from construction of a
low-head dam at the lake outlet, habitat changes associated
with seasonal water level reductions for irrigation, or impacts
from the American signal crayfish (Pacifastacus leniusculus
(Dana, 1852), which appeared in Enos Lake sometime between 1984 and the early 1990s—at approximately the same
time as the collapse is thought to have commenced (Taylor et
al. 2006). It is well established that invasive species can be
important agents of environmental change and threaten native
biodiversity through a variety of mechanisms (Vitousek et al.
1996; Pyšek and Richardson 2010). In particular, non-native
crayfishes have a multitude of deleterious effects on native
freshwater biodiversity; crayfishes may act as direct predators,
strong disruptors of physical habitat, or competitiors for food.
Small-bodied benthic fishes appear to be particularly susceptible to effects from crayfishes (e.g., Light 2005; see reviews
by Lodge et al. 2000 and Snyder and Evans 2006).
Behm et al. (2010) suggested that postzygotic selection
against hybrid sticklebacks has been weakened in Enos Lake
due to perturbations in the adaptive landscape of the lake’s
ecology. Furthermore, the rapid collapse of the Enos Lake
species pair suggests that prezygotic isolation between the
species pair has also been weakened, i.e., both hybrid fitness
and frequency of hybridization have apparently increased
(Taylor et al. 2006; Behm et al. 2010).
Several hypotheses have been proposed to explain how
invasive crayfish may have contributed to the weakening of
prezygotic isolation between the Enos Lake species: the removal of aquatic vegetation important to nest-site selection in
Enos Lake is one notable example (Taylor et al. 2006; National Recovery Team for Stickleback Species Pairs 2007). No
existing hypothesis, however, accounts for the possibility of
direct behavioural impacts of crayfish on sticklebacks.
Stickleback males build elaborate nests and are known to
vigorously defend them from predators (Ridgway and McPhail
1987). As benthic omnivores, crayfish roam the littoral zone of
Enos Lake in search of food, and therefore move through
stickleback nesting territories. In sympatric species pair populations such as Enos Lake, limnetic males tend to be more
territorial than benthic males (Ridgway and McPhail 1987),
possibly due to the vulnerability of their nest sites (limnetics
construct nests over open substrate, benthics nest in dense
aquatic vegetation). We therefore suspected that limnetic
males might have a stronger reaction to crayfish than benthic
males. Assuming that a strong defensive reaction to crayfish
presence would lead to a reduction of reproductive behaviour
(i.e., courtship and nesting behaviour; Ridgway and McPhail
1984), crayfish may more severely impact reproductive success of limnetic males (see also COSEWIC 2002; Taylor et al.
2006; Behm et al. 2010). This inference is supported by Dorn
and Mittelbach (2004), who demonstrated that crayfish caused
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1330
delays in reproduction and reduced reproductive success in
sunfishes (Lepomis Rafinesque, 1819) nesting in exposed
habitats.
In our study, we conducted detailed reproductive behaviour
experiments on an “intact” (i.e., well-differentiated) species
pair of G. aculeatus to test two hypotheses: (1) that the
presence of crayfish disrupts the reproductive behaviour of
male threespine stickleback and (2) that this disruption is
greater for male limnetic sticklebacks than for benthics. If our
hypotheses are upheld, this would suggest that limnetic ⫻
limnetic mating success is reduced under these circumstances
(i.e., there is a reduction in limnetic nests), which may promote limnetic ⫻ benthic matings. Tests of these hypotheses
should help us better understand threats to persistence of
stickleback species pairs, and the role of behaviour and interspecific interactions, specifically threats from predation, as
influences on reproductive isolation in sticklebacks and other
nest builders.
Materials and methods
Selection of animals and animal care
Our hypotheses were tested using an intact sympatric
stickleback species pair from a nearby lake as a proxy for
the precollapse Enos Lake population. Benthic and limnetic
sticklebacks from Paxton Lake, Texada Island (southwestern British Columbia, Canada), were chosen because this
sympatric species pair remains fully differentiated, readily
exhibits reproductive behaviours in captivity (Nagel and
Schluter 1998; Boughman et al. 2005), and is evolutionarily
and ecologically naïve to crayfish. In studying this proxy
species pair, we assume that the relative vulnerability of limnetics and benthics to crayfish is similar between different
lakes. This assumption is based on the inference that vulnerability is a function of the selected ecological attributes of a
species (body size, nesting behaviour, etc.), which are highly
convergent among species pair lakes.
Benthic and limnetic sticklebacks in Paxton Lake are currently
listed as Endangered under Canada’s Species at Risk Act and
collections from the lake are strictly controlled (COSEWIC
2010). Adult benthic and limnetic sticklebacks were collected
from Paxton Lake from mid-April to early May 2009 using
unbaited minnow traps (nbenthic ⫽ 40; nlimnetic ⫽ 83). American
signal crayfish were collected from Enos Lake between June
2008 and May 2009, using prawn traps baited with dry cat
food. All collection activities were conducted under permits
issued by the BC Ministry of Environment (NA/SU 09-52085)
and Fisheries and Oceans Canada (SECT 08 SCI 027 and
SARA 121).
Sticklebacks and crayfish were transported in aerated coolers and held in glass aquaria in a temperature-controlled environmental chamber. Adult sticklebacks were separated by
sex and species and held at a late springtime temperature
(17 °C) and photoperiod regime (16 h light : 8 h dark) known
to accelerate the rate at which male stickleback become sexually mature (Wootton 1976). Crayfish and fish were visually
isolated from each other in the holding tanks. Water quality
was monitored and pH maintained at approximately 8.0 to
reflect conditions in Paxton Lake (Ormond 2010). Fish were
fed at least once every 2 days with frozen chironomid larvae
and Daphnia O.F. Müller, 1785. Crayfish were fed regularly
with wafers of dried algal Spirulina Turpin ex Gomont, 1893.
Can. J. Zool. Vol. 90, 2012
All fish were cared for according to the University of British
Columbia animal care protocols (permit No. A07-0378).
Experimental apparatus
Four behavioural observation arenas were set up in an environmental chamber (two for limnetic experiments, two for benthic experiments). Arenas were constructed from 189 L glass
aquaria, measuring 47 cm ⫻ 92 cm ⫻ 48 cm. Three sides of each
aquarium were covered with opaque plastic sheets to limit external stimuli while one side was left open for observation but was
shielded from external light by a black plastic canopy (observation blind). Each arena was illuminated from above with fluorescent lights. By lighting the arena only from above, the observer
was rendered virtually invisible to the experimental subjects.
Each arena was provided with a nesting dish (diameter ⫽
25 cm) filled with filter sand and topped with needles of Douglasfir (Pseudotsuga menziesii ((Mirb.) Franco) and Java the Hutt
moss (Taxiphyllum barbieri (Card. & Coppey) Iwats) for use as
stickleback nesting material; 10 pieces of limestone cobble (diameter ⫽ 5 cm) and a piece of PVC tube (diameter ⫽ 3 cm;
length ⫽ 5 cm) for shelter; and two 500 mL clear glass jars for
crayfish and female stickleback confinement (Fig. 1).
Confinement of female sticklebacks and crayfish during
specific treatments allowed for visual stimulation of each male
stickleback by either a potential mate or an intruder, without
allowing physical contact. Confinement jars were kept in fixed
positions, allowing the observer to clearly differentiate between behaviours directed toward the female, the nest, and the
crayfish. In each arena, the female confinement jar was suspended in the water column at the back of the arena, while the
crayfish confinement jar was placed on the floor of the arena
opposite the nesting dish. Each jar was covered with a fiberglass screen lid, held in place by a PVC ring. A length of string
tied to the lid of each jar allowed the observer to remotely
release the lid with minimal external disturbance.
Experimental protocol
Male stickleback reproductive behaviour was observed during a sequence of treatments (Table 1), beginning with an
initial baseline treatment, three experimental treatments, and a
final baseline treatment. The sequential design allowed for the
observation of reproductive behaviour over varying degrees of
exposure to crayfish and at different stages of courtship. To
create a gradient of increasing crayfish exposure and reproductive intensity, the treatments involved the sequential release of the female stickleback and the crayfish into the arena.
To minimize variable disturbance effects associated with
the recapture and confinement of sticklebacks or crayfish
during a given trial, the order in which baseline and experimental treatments were applied was not randomized (i.e., we
consistently proceeded from “confined” to “free” status for
both the female stickleback and the crayfish).
At the start of each trial, a sexually receptive stickleback
male (based on the expression of bright red and blue colouration and dominant territorial behaviour) was selected from a
holding tank. The male was added to an arena and given
sufficient time to begin tending a nest (which occurred for all
individuals used in the experiments, usually within 24 – 48 h).
To determine a baseline level of male stickleback reproductive
activity in response to visual stimuli alone (i.e., in a “confined”
courtship scenario), the male was given visual access to a
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1331
Fig. 1. Representative layout of the observation arenas used in
behavioural trials. The top view of the arena shows the orientation
of the observation blind where the observer was located during
observation periods, allowing for nondisruptive viewing of
threespine stickleback (Gasterosteus aculeatus) and American
signal crayfish (Pacifastacus leniusculus) in the arena.
Table 1. Summary of experimental treatments carried out to test
for impacts of American signal crayfish (Pacifastacus leniusculus)
on the reproductive behaviour of male threespine sticklebacks
(Gasterosteus aculeatus).
Treatment
Free-roaming
subjects
Confined
subjects
Initial baseline
1
Stickleback male
Stickleback male
2
Stickleback
Crayfish
Stickleback
Crayfish
Stickleback
Stickleback
Stickleback
male
Stickleback female
Stickleback female
Crayfish
Stickleback female
male
None
female
male
female
None
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Female
jar
48 cm
FRONT
VIEW
Nest box
3 (including 3.1 and 3.2)
Final baseline
jar
Note: Treatments were carried out sequentially on one male stickleback
at a time. Confined subjects were contained within a covered glass jar.
Air
92 cm
Female
jar
Shelter
47 cm
TOP
VIEW
Nest box
jar
Air
Observation Blind
confined, gravid conspecific female stickleback during the
initial baseline treatment, while the crayfish confinement jar
remained vacant (Table 1). To determine the effect of a visual
crayfish stimulus on male stickleback reproductive activity, a
medium-sized crayfish (18.1 ⫾ 0.6 g; mean ⫾ SE) was added
to the crayfish confinement jar during treatment 1, while the
female stickleback remained confined. To determine the
effect of a freely roaming crayfish on male stickleback
reproductive activity, the crayfish was released from its
confinement jar at the start of treatment 2. To determine the
effect of the freely roaming crayfish on male stickleback
reproductive activity during a “free” courtship scenario
(i.e., with physical access to a mate), the female stickleback
was released into the arena at the start of treatment 3.
Finally, to determine a baseline (unperturbed) level of male
reproductive activity in a “free” courtship scenario (final
baseline treatment), the crayfish was quickly removed from
the arena with a dip net, leaving the male and female
sticklebacks relatively undisturbed.
Behavioural observations
A total of 19 benthic (60.7 ⫾ 0.96 mm; mean ⫾ SE) and 16
limnetic (53.0 ⫾ 0.61 mm) stickleback males successfully
came into breeding condition and constructed nests in the
experimental arenas, allowing for a total of 35 courtship trials.
During each treatment, male reproductive behaviours, as described by Wootton (1976) and Ridgway and McPhail (1984),
were scored over a period of 10 min (Table 2). The 10 min
duration was selected to maximize the likelihood of observing
a variety of behaviours while minimizing the time that each
male was exposed to a crayfish.
Each 10 min period was divided into 120 five-second time
intervals. A positive score (⫹1) was assigned to each interval
during which a male stickleback exhibited reproductive behaviour(s), as described in Table 2. Positive scores were tallied
up for each treatment period and divided by 120, resulting in
a number indicating the proportion of the treatment devoted to
reproductive behaviour. This number is hereafter referred to as
the reproductive behaviour frequency (RF). The total length
(TL) of all 35 male sticklebacks was recorded as a measure of
size.
Between treatments, a minimum “settling down” time of
5 min was used to minimize carry-over effects from the
previous observational period (Liang and Carriere 2010). During these settling down periods, no changes were made to the
experimental apparatus and care was taken not to disrupt
experimental subjects in any way.
To correct for inherent species-specific and individual differences in RF between benthic and limnetic males, RF values
for each individual fish were subtracted from their respective
baseline (initial baseline treatment) RF values. These baselinecorrected RF values were used in all subsequent statistical
analyses in which benthic and limnetic RF values were directly compared (i.e., all two-sample Student’s t tests).
Effect of crayfish size
In a subset of the 35 trials (six benthic and six limnetic), two
additional treatments (3.1 and 3.2) were inserted between the
end of treatment 3 and the start of the final baseline treatment.
The additional treatments were designed to determine whether
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Table 2. Summary of key reproductive behaviours displayed by male threespine sticklebacks (Gasterosteus aculeatus) during nesting and
courtship (or sexual) phases, as described by Wootton (1976); Ridgway and McPhail (1984).
Behaviour
Reproductive phase
Digging
Nesting
Description
Repeatedly picking up and moving mouthfuls of sand away from the nest site, resulting in the
creation of a shallow pit
Vegetation collection Nesting
Sampling or carrying pieces of vegetation to the nest pit where they are deposited
Glueing
Nesting
Nesting material is glued with a mucus secretion of the male’s kidneys using a conspicuously
smooth motion over the nest with head and tail raised
Boring
Nesting
Creation of a tunnel through the nest, including a distinct entrance
Fanning
Nesting or courtship Driving a current of water through the nest by male’s pectoral fins in a head-down position
Creeping through
Nesting or courtship Male swimming though the nest, exiting at the back; regarded as the beginning of the
courtship phase
Zig-zag dance
Courtship
Swimming towards female in a series of lateral jumps: often away from or towards the
female, sometimes accompanied by biting or butting
Direct approach
Courtship
Swimming straight towards female
Leading
Courtship
Following zig-zag dance, male leading female to the nest; may be direct or meandering
Showing
Courtship
Female follows male to the nest; male poking head into the nest entrance
or not male sticklebacks respond differently to different sizes
of crayfish. Treatments 3.1 and 3.2 involved the separate
sequential introduction of two sizes of crayfish, respectively:
one that was smaller than usual (5.0 ⫾ 0.28 g; mean ⫾ SE)
and one that was larger than usual (36.1 ⫾ 1.89 g). A coin toss
was used to determine the order in which the treatments were
performed.
Statistical analyses
The assumptions of parametric statistics for our data sets
(normality and homogeneity of variance) were tested using the
Shapiro–Wilk test statistic and Fisher’s F test, respectively.
Data sets that did not conform to these assumptions were
analyzed using nonparametric statistics (Wilcoxon matched
pairs signed-rank test in the place of paired t tests; Wilcoxon
rank-sum test in the place of two-sample Student’s t tests).
Reproductive behaviour frequency values from all treatments
were transformed using an arcsine square-root transformation
(normalized datum ⫽ arcsine·公datum).
For within benthic or within limnetic comparisons, paired
t tests were used to test for differences in RF before and after
the original introduction of a crayfish into the arena, as well as
before and after the removal of the crayfish from the arena at
the end of each trial (see Table 1). For comparisons between
species, two-sample Student’s t tests were used to compare
baseline-corrected benthic and limnetic RF values from each
of the four treatments (initial baseline treatment excluded) to
determine whether or not changes in RF, if any, were the same
for limnetic and benthic males.
Limnetic males are generally smaller than benthic males
(McPhail 1984), and if benthic and limnetic RFs are affected
differently in the presence of crayfish, it is possible that this
difference may be attributable to body size rather than intrinsic
differences in behaviour between species. To test for a potential
stickleback size effect and to discriminate any species effect from
a size effect, baseline-corrected RF values from treatment 2 were
analyzed as a linear function of fish length (TL) and stickleback
species identity (benthic or limnetic) according to the formula
RF ⫽ slope (TL) ⫹ slope (species) ⫹ intercept.
A linear model was used to analyze data from the subset of
12 trials designed to test for a crayfish size effect: baseline-
corrected RF values collected during treatments 3, 3.1, and 3.2
of the 12 trials were analyzed as a linear function of crayfish
mass (CM) and stickleback species identity (benthic or limnetic) according to the formula RF ⫽ slope (CM) ⫹
slope (species) ⫹ intercept. The Shapiro–Wilk test statistic
was used to test the normality of the residuals of linear models
used in our analyses of crayfish size, while the homogeneity of
variance in linear models was tested using a regression of the
absolute value of residuals against the model’s independent
variable.
Inclusion of the 12 trials containing additional crayfish
treatments (3.1 and 3.2) in the general analyses implicitly
assumes that the delays and additional manipulations associated with these treatments did not significantly influence the
RF values exhibited in the subsequent final baseline treatment.
To test if this assumption was valid, male recovery (the change
in RF between treatment 3 and the final baseline treatment;
benthic and limnetic combined) was assessed, both including
(n ⫽ 35) and excluding (n ⫽ 23) the additional 12 trials, using
a paired t test.
Results
In most trials, male stickleback began performing alternating courtship and nesting behaviour almost immediately upon
addition of a confined female to the arena (initial baseline
treatment), and continued performing alternating courtship
and nesting behaviour after introduction of a confined crayfish
to the arena in treatment 1, while occasionally approaching
and inspecting the crayfish confinement jar. In the presence of
a freely roaming crayfish during treatment 2, however, some
stickleback males aggressively attacked the crayfish, particularly when they came within approximately 20 cm of the
male’s nest. Crayfish generally appeared to pay little attention
to male sticklebacks during the trials, aside from occasionally
defending themselves from attacks. Crayfish also did not exhibit any obvious attraction to stickleback nests.
Limnetic RF values were significantly reduced from baseline levels following the introduction of a crayfish to the arena
in treatment 1 (t[15] ⫽ 4.39, p ⬍ 0.001; Fig. 2), while benthic
RF values did not significantly change from baseline levels
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Change in Reproductive Behaviour Frequency
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Fig. 2. Mean (⫾SE) changes in reproductive behaviour frequency (RF) exhibited by male benthic (n ⫽ 19) and limnetic (n ⫽ 16)
threespine sticklebacks (Gasterosteus aculeatus) following the initial introduction (initial baseline RF value vs. treatment-1 RF value) and
initial removal (treatment-3 RF value vs. treatment-4 RF value) of American signal crayfish (Pacifastacus leniusculus) from the
observational arena. The RF value for a given 10 min treatment was calculated as the proportion of the treatment’s duration during which
the stickleback male exhibited reproductive behaviour, as described in Table 2.
Benthic
Limnetic
0.7
0.5
*
0.2
0.0
0.2
*
0.5
0.7
Introduced
Introduced
*
Removed
Removed
Introduced
Introduced
Removed
Removed
p < 0.05
(t[18] ⫽ 0.94, p ⫽ 0.36). Similarly, baseline-corrected limnetic
RF values increased significantly between treatment 3 and the
final baseline treatment when the crayfish was removed from
the arena (t[15] ⫽ – 4.67, p ⬍ 0.001; Fig. 2). Baseline-corrected
benthic RF values, however, did not change significantly (W ⫽
50, n ⫽ 19, p ⫽ 0.13), although they followed the same trend
as those of limnetic males.
Benthic and limnetic males reacted differently to the presence of crayfish. Although limnetic males exhibited higher
(⫹33%) baseline RF values than benthic males during (t[33] ⫽
–2.12, p ⫽ 0.04; Fig. 3), baseline-corrected limnetic RF values
were consistently more greatly reduced than those of benthics
in treatment 1 (–38% vs. –12%; t[33] ⫽ 2.46, p ⫽ 0.02),
treatment 2 (– 65% vs. – 40%; W ⫽ 221, p ⫽ 0.02), and
treatment 3 (–58% vs. –29%; t[33] ⫽ 3.12, p ⬍ 0.01) when
crayfish were present (Fig. 4). When crayfish were permanently removed from the arena for the final baseline treatment,
baseline-corrected RF values for both species recovered markedly and the difference between benthic and limnetic males
was no longer significant (–24% vs. –7%; t[33] ⫽ 1.21, p ⫽
0.23; Fig. 4).
According to our linear model, interspecific differences in
baseline-corrected RF reduction in the presence of crayfish
were not attributable to differences in body size between
benthic and limnetic males. In the model, species (benthic or
limnetic) was a significant predictor of baseline-corrected RF
reduction (F[1,32] ⫽ 10.57, p ⫽ 0.003), while male total length
(MTL) was not (F[1,32] ⫽ 0.81, p ⫽ 0.38).
Crayfish size did not significantly influence the magnitude
of baseline-corrected RF reduction for either stickleback species: our linear model showed that crayfish size was not a
significant predictor of baseline-corrected RF reduction for
benthic (r2 ⫽ 0.06, p ⫽ 0.31) or limnetic (r2 ⫽ 0.22, p ⫽ 0.06)
males. Moreover, the delays and additional manipulations
associated with this subset of 12 trials did not influence the
overall results: the paired t test comparing data from treatment 3 and the final baseline treatment yielded similar results
with (n ⫽ 35, p ⫽ 0.006) and without (n ⫽ 23, p ⫽ 0.007) the
inclusion of these 12 trials.
Discussion
Behavioural effects of crayfish on sticklebacks
Although crayfish are known to have more general negative
effects on several freshwater fishes and freshwater ecosystems
(Guan and Wiles 1998; Snyder and Evans 2006; Hirsch and
Fischer 2008), the evidence implicating American signal crayfish as a factor in the collapse of the Enos Lake species pair is
circumstantial (Taylor et al. 2006) owing, largely, to a lack of
knowledge of the extent and consequences of interactions
between sticklebacks and crayfish. Our results, however, suggest that crayfish generally depress male threespine stickleback reproductive behaviour. This is consistent with
observations by Candolin (1997) who demonstrated reduced
courtship behaviour in European male threespine sticklebacks
under threat of predation. Our data further showed that this
effect is disproportionately severe for male limnetic sticklebacks. In the presence of crayfish, limnetic males spent significantly less time engaged in nesting and courtship
behaviour, while benthic males were not significantly affected.
According to our linear model, differences in stickleback size
do not explain the greater vulnerability of limnetic males. This
Published by NRC Research Press
1334
Can. J. Zool. Vol. 90, 2012
Fig. 3. Mean (⫾SE) reproductive behaviour frequency (RF) exhibited by male benthic (n ⫽ 19) and limnetic (n ⫽ 16) threespine sticklebacks
(Gasterosteus aculeatus) during baseline and treatment observational periods. The RF value for a given 10 min treatment was calculated as the
proportion of the treatment’s duration during which the stickleback male exhibited reproductive behaviour, as described in Table 2.
1.0
Benthic
Reproductive Behaviour Frequency
0.8
*
0.6
0.4
0.2
0.0
Baseline
T1
T2
T3
F free
*
T4
Baseline(Final)
F free
p < 0.05
Fig. 4. Mean (⫾SE) changes in baseline-corrected reproductive behaviour frequency (RF) for male threespine sticklebacks (Gasterosteus
aculeatus) during four experimental treatments. Negative values indicate a reduction in RF values, relative to baseline. The RF value for a
given 10 min treatment was calculated as the proportion of the treatment’s duration during which the stickleback male exhibited
reproductive behaviour, as described in Table 2.
Change in
in Reproductive
Reproductive Behaviour
Change
BehaviourFrequency
Frequency
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For personal use only.
Limnetic
0.7
Benthic
Limnetic
0.5
0.2
0
0.2
*
0.5
*
0.7
*
0.9
T1
T2
T3
F free
*
T4
Baseline(Final)
F free
p < 0.05
suggests that there are intrinsic behavioural differences between benthic and limnetic males that influence their relative
vulnerability to disruption during breeding. The higher baseline RF values observed in limnetic trials suggest that limnetic
males are generally more active than benthic males, and may
thus be either more visible to potential intruders or more likely
to investigate and (or) defend against intrusions into their
breeding territories.
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Velema et al.
The greater impact of crayfish intrusion on male limnetic
behaviour is consistent with observations by Ridgway and
McPhail (1987), who demonstrated that limnetic males tend to
invest more of their time and resources in territorial defense
than benthic males when confronted with an intrusion by a
rival male. The defensive tendencies exhibited by limnetic
males may be a behavioural adaptation to compensate for their
open nesting sites, which are more vulnerable to predation and
intrusion by rival males (Ridgway and McPhail 1987). Under
normal circumstances (i.e., in the absence of crayfish), a
greater investment in territorial defense is therefore presumably adaptive.
Crayfish, unlike rival stickleback males, are not as easily
deterred by a territorial stickleback; in our behavioural trials,
crayfish appeared to take little notice of a male’s defensive
attacks. In the presence of crayfish, male stickleback territoriality may therefore be maladaptive because, as our trials
demonstrated, territorial behaviour can lead to at least a
short-term reduction in nesting and courtship behaviour (cf.
Candolin 1997).
Following the appearance of crayfish in the 1980s, it is
possible that the production of viable limnetic nests may have
been differentially reduced, leading to a shortage of conspecific nesting sites for limnetic females. Dorn and Mittelbach
(2004) reported such an effect in two species of substratenesting sunfishes, pumpkinseed (Lepomis gibbosus (L.,
1758)) and bluegill (Lepomis macrochirus Rafinesque,
1819), when exposed to a native crayfish. Interestingly,
Dorn and Mittelbach (2004) reported that this effect was
especially pronounced in ponds with lower densities of
aquatic vegetation—the preferred nesting habitat of limnetic sticklebacks.
Studies of the Enos Lake species pair collapse suggest that
hybridization between benthics and limnetics was asymmetric
and favoured the production of benthic-like sticklebacks during the crayfish invasion (Kraak et al. 2001; Gow et al. 2006;
Taylor et al. 2006). Morphological and genetic data show that
the resulting hybrid swarm contained a preponderance of
benthic phenotypes and was virtually devoid of pure limnetic
or benthic genotypes (Kraak et al. 2001; Gow et al. 2006;
Taylor et al. 2006). This suggests that limnetic males achieved
relatively low reproductive success during the collapse, leading to a greater relative abundance of benthics and a paucity of
limnetics and limnetic-like phenotypes in the resulting hybrid
swarm (Gow et al. 2006). The disproportionate impacts of
crayfish on limnetic reproductive behaviour demonstrated by
our study suggests one potential mechanism by which crayfish
may have contributed to the asymmetric collapse of the Enos
Lake species pair. For instance, it is possible that limnetic
males were less reproductively active or were more distracted
from nesting behaviours than benthic males following the
appearance of crayfish in Enos Lake. If so, the population of
available limnetic males would have been effectively reduced,
creating a situation in which hybridization would be more
likely to occur between limnetic females and benthic males,
and the resulting hybrids would be less likely to encounter and
backcross with limnetic males. In fact, Todd and Stedman
(1989) and Willis et al. (2004) demonstrated that, in general,
members of a pair of sympatric species are much more likely
to hybridize when either males or females of one species are in
short supply (see review by Wirtz 1999).
1335
Other potential impacts of crayfish on stickleback
breeding biology
Although our study has focused on direct effects of crayfish
on the reproductive behaviour of male stickleback, several
alternative hypotheses have been proposed whereby crayfish
may have facilitated the collapse of the Enos Lake stickleback
species pair.
First, colouration is important in assortative mating in
threespine stickleback (Boughman 2001; Kraak et al. 2001).
Colouration is also important in mate choice of cichlid fishes
(e.g., Seehausen and van Alphen 1998), and a decline in water
clarity in Lake Victoria has been linked to reduced colour
perception and increased hybridization (Seehausen et al.
1997). Interestingly, Enos Lake limnetic males have more
intense red colouration in the throat than benthic males, which
are black in nuptial colouration (Malek et al. 2012). Crayfish
in general have eyes that are well adapted to seeing red (Wald
1967; Nosaki 1969) and it is possible that red limnetic males
are more detectable by crayfish, especially because they build
nests and court females in more open areas, less concealed by
vegetation than benthic males (Ridgway and McPhail 1984).
Similarly, it has been suggested that the removal of aquatic
vegetation by crayfish in Enos Lake may have led to decreased
water clarity, affecting the accuracy of mate recognition and
thus disrupting fragile assortative mating cues (Taylor et al.
2006; National Recovery Team for Stickleback Species Pairs
2007; Rosenfeld et al. 2008). Unfortunately, there are no
pre-crayfish water clarity data with which to test this hypothesis, and there is little evidence of increased turbidity in Enos
Lake.
Second, benthic and limnetic stickleback males are known
to select different microhabitats as nesting territories: benthics
tend to nest in aquatic vegetation, while limnetics tend to nest
in relatively open areas (Ridgway and McPhail 1984). Consequently, removal of aquatic vegetation by crayfish could
have led to reduced spatial segregation between benthic and
limnetic nesting sites, thus increasing opportunities for
hybridization.
Finally, as voracious omnivores in the benthic zone of
aquatic environments, crayfish may be significant nest predators and also may significantly reduce the local abundance of
benthic invertebrates (Dorn and Mittelbach 2004; Gherardi
and Acquistapace 2007; Rosenfeld et al. 2008). It is possible
that limnetic nests, characteristically built in open littoral areas
(Ridgway and McPhail 1984), are more susceptible to crayfish
predation and increase the parental care costs for limnetic
males, thus potentially reducing their reproductive success
(Steinhart et al. 2005). Furthermore, reduction of available
food for benthic sticklebacks could lead to decreased size at
maturity. Given the importance of body size and shape as
assortative mating cues between benthic and limnetic stickleback (Nagel and Schluter 1998; Boughman et al. 2005), such
an effect could have eroded size-assortative mating between
benthics and limnetics.
Some of the potential impacts of crayfish described above
may also act disproportionately against male limnetics (e.g.,
nest-site selection, colouration, nest predation) which, combined with our demonstration of disproportionate effects of
crayfish on limnetic male behaviour, suggests that behavioural
disruption during reproduction may have contributed to the
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1336
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For personal use only.
collapse of the species pair, although mating disruption is not
mutually exclusive to the alternative mechanisms noted above.
Conservation implications
Our study suggests that crayfish disproportionately disrupt
nesting and courtship behaviour in male limnetics; however,
how crayfish may influence the behaviour of female sticklebacks, subsequent choice of males and their nests, and egg
survival remains unclear. It is, for instance, possible that
female limnetics may not only reduce their courtship with
limnetic males under threats of predation from crayfish, but
may actually reverse their preferences for limnetic males that
are more vulnerable to, and affected by, crayfish than benthics,
thus seeking out benthic matings. In fact, Candolin (1997)
demonstrated that European male threespine stickleback were
less attractive to females when the males were under predation
threat (and reduced their courtship behaviour). Furthermore,
Johnson and Basolo (2003) found that female green swordtails
(Xiphophorus hellerii Heckel, 1848) reversed their inherent
preferences for males with long swords to those with shorter
ones after they were exposed to video recording of long-tailed
males being preyed upon by cichlids. Impacts of crayfish on
nest-site selection also warrant investigation. Candolin and
Voigt (1998) showed that threats from predation can induce
male stickleback to show a greater preference for nesting
under cover. If this is true for limnetic and benthic stickleback,
then it could impact male limnetics to a greater extent, because
they nest in more open areas, and bring them into greater
contact with benthic females and promote hybridization. It
also remains unclear whether the collapse of the Enos Lake
species pair may be reversible by engaging in a crayfish
removal program, or if crayfish removal is even possible. Hein
et al. (2006) and Freeman et al. (2009) found that attempts at
crayfish extirpation by physical removal from an invaded lake
had limited success, suggesting that permanent crayfish eradication in Enos Lake may be difficult, if not impossible. Also,
the currently accepted model of stickleback species pair evolution involves character displacement between two temporally spaced colonist populations (McPhail 1993; Taylor and
McPhail 2000). Inherent in this model is a period of allopatry
and divergence between the initial and the subsequent colonist
populations—a situation that cannot be re-created under the
current hybrid swarm condition. Consequently, even if crayfish eradication in Enos Lake was successful, it is unlikely that
the current stickleback hybrid swarm would re-differentiate
into a benthic–limnetic species pair, particularly because
trophic resources in the lake appear to have shifted in favour
of morphologically intermediate hydrid phenotypes (Behm et
al. 2010). Finally, while the exact mechanism by which crayfish may have contributed to the collapse of the Enos Lake
species pair remains unknown, our results contribute to a
growing body of evidence highlighting the heightened sensitivity of recently evolved species pairs to environmental
change (cf. Seehausen 2006; Vonlanthen et al. 2012) and, in
particular, the susceptibility of freshwater fishes to the negative impacts of invasive species (Hatfield 2001; Dextrase and
Mandrak 2006; Ormond et al. 2011).
Acknowledgements
We thank D. Schluter and T. Vines for guidance with experimental design; R. FitzJohn for lending his expertise in data
Can. J. Zool. Vol. 90, 2012
analyses; and P. Tamkee and G. Conte for their immeasurable
help in the laboratory and field, respectively. We also appreciate
productive comments on earlier versions of this paper provided
by anonymous reviewers. Our research was supported by grants
awarded to E.B.T. and J.S.R. from the Natural Sciences and
Engineering Research Council of Canada (NSERC, Discovery
Grant Program), the British Columbia Forest Science Program,
and the British Columbia Ministry of Environment.
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