Grazing of the invasive alga Codium fragile ssp. tomentosoides by the common periwinkle Littorina littorea: Effects of thallus size, age and condition

Journal of Experimental Marine Biology and Ecology 355 (2008) 103 – 113 www.elsevier.com/locate/jembe

Grazing of the invasive alga Codium fragile ssp. tomentosoides by the common periwinkle Littorina littorea: Effects of thallus size, age and condition Robert E. Scheibling ⁎, Devin A. Lyons, Catherine B.T. Sumi Department of Biology, Dalhousie University, Halifax, Nova Scotia, B3H 4J1, Canada Received 14 September 2007; received in revised form 30 November 2007; accepted 4 December 2007

Abstract We examined the potential of herbivory by the common periwinkle Littorina littorea to limit recruitment and vegetative re-growth of the invasive green alga Codium fragile ssp. tomentosoides in a series of manipulative field experiments in tidepools on a wave-exposed rocky shore in Nova Scotia, Canada. Snails were excluded or included from circular plots (14 to 20 cm diameter) with cages to compare growth and survival of C. fragile against procedural (partial cages) or natural (uncaged) control plots. Our results show that L. littorea may restrict growth and survival of C. fragile by grazing new recruits (b 2 cm thallus length), fronds of adult thalli that are bleached and necrotic, and residual holdfasts (b 2 mm thickness) of detached thalli (artificially severed to mimic wave dislodgement). Once recruits grow beyond a critical size (~ 3 cm), or damaged tissues or holdfasts regenerate, grazing of C. fragile by L. littorea appears to be limited and ineffectual. Our experimental results corroborate correlative evidence from previous studies that herbivory by L. littorea limits the abundance of C. fragile in tidepools on the Atlantic coast of Nova Scotia, particularly pools in the high intertidal zone where these snails are abundant. Lower on the shore, littorinid grazing and physical stressors that render algae more vulnerable to grazers (e.g. UV radiation and freezing) are less intensive, and probably have less of a regulatory effect on populations of C. fragile. © 2007 Elsevier B.V. All rights reserved. Keywords: Codium fragile ssp. tomentosoides; Herbivory; Invasive species; Littorina littorea; Rocky intertidal zone; Tidepools

1. Introduction The role of native species in mediating the success of introduced species is of considerable interest to ecologists, as these interactions can be both complex and variable. Disease, parasitism, predation, competition, and mutualism all may influence the invasiveness of introduced species, although the relative magnitude and direction of effects of these factors can differ among ecological systems (Naeem et al., 2000, Richardson et al., 2000, Levine et al., 2004). For introduced plants and macroalgae, herbivory can limit establishment, persistence, and population growth of an invader, provided that the native herbivores are adapted to feed on the novel species (Parker et al., 2006). Otherwise, escape from herbivory can

⁎ Corresponding author. E-mail address: [email protected] (R.E. Scheibling). 0022-0981/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jembe.2007.12.002

enable an introduced species to flourish and spread (Enemy Release Hypothesis, Keane and Crawley, 2002). Environmental stresses can interact with herbivory to reduce plant performance and survival. For example, damage caused by sea urchin grazing increases the likelihood that seaweeds are dislodged by wave action (Koehl and Wainwright, 1977). Desiccation, nutrient stress, and exposure to UV radiation can also influence the susceptibility of a plant or alga to herbivory by altering their nutritional value or anti-grazing defenses (Renaud et al., 1990, Yates and Peckol, 1993, Cronin and Hay, 1996, Heaven and Scrosati, 2004). In just over a century, the Asian green alga Codium fragile (Suringar) ssp. tomentosoides (van Goor) Silva has established populations along temperate shores worldwide, including the northwest Atlantic (Trowbridge, 1998a). In Nova Scotia, Canada, C. fragile is found in eelgrass (Zostera marina) beds on soft sediment (Garbary et al., 2004) and in shallow rocky subtidal (Bird et al., 1993, Scheibling and Gagnon, 2006,

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Schmidt and Scheibling, 2006) and intertidal (Bégin and Scheibling, 2003, Schmidt and Scheibling, 2005) habitats. The alga survives year round in Nova Scotia, although population density and thallus size of C. fragile are reduced by wave dislodgement and fragmentation during the stormy autumn and winter months (Bégin and Scheibling, 2003, D'Amours and Scheibling, 2007). In the intertidal zone in Nova Scotia, C. fragile occurs primarily in tidepools, where thalli are periodically damaged by harsh physical conditions (e.g., high UV radiation, freezing) in winter and early spring (Schmidt and Scheibling, 2005). Tidepools also harbour high densities of molluscan herbivores, mainly the common periwinkle L. littorea (L) (Schmidt and Scheibling, 2005). The importance of grazing by L. littorea in determining the structure of intertidal communities on rocky and soft sediment shores of the northwest Atlantic is well established (Lubchenco, 1978, 1980, 1983, Petraitis, 1983, 1987, Chapman, 1989, Bertness, 1984), although the effect of littorinid grazing in limiting the establishment, persistence and spread of C. fragile on these shores, and the potential interaction between herbivory and environmental stressors, are unknown. In this study we experimentally investigate condition- and stage-specific grazing of C. fragile by L. littorea in tidepools on a wave-exposed rocky shore in Nova Scotia. We show that L. littorea can potentially limit the survival and vegetative regrowth of C. fragile when snails graze new recruits, and naturally damaged fronds or residual holdfasts of detached adult thalli. We also present evidence of a size refuge for healthy thalli of C. fragile, above which recruits may escape littorinid grazing.

width. For MP2, which approximates an isosceles triangle in shape, surface area was estimated by multiplying the width (along the shortest of 3 sides) by the length (from the midpoint of the shortest side) and dividing by ½. HP was the largest pool spanning 22 m in length, with a surface area of 231 m2. MP2 and MP1 were 4.6 and 12 m in length, and 6.1 and 23.7 m2 in surface area, respectively. Maximum depth (when tidally isolated) ranged from 0.4 m (MP2) to 0.7 m (HP). The experimental areas in each pool ranged from 0.2 to 0.4 m in depth in each pool. The dominant grazer in the tidepools was L. littorea. A congeneric species (L. obtusata) and a limpet (Tectura testudinalis) were much less abundant, and sea urchins (Strongylocentrotus droebachiensis) were rare. Small crustacean grazers (gammarid amphipods and isopods) were also present in these pools and occasionally were observed on C. fragile. The sacoglossan sea slug Placida dendrictica, a specialist grazer of Codium spp. (Trowbridge, 1998b, 2002), was not found in tidepools during this study, although it occurs on C. fragile in the subtidal zone at Cranberry Cove (Schmidt and Scheibling, 2006). The predominant macroalgae at HP were C. fragile, filamentous green (Enteromorpha intestinalis) and brown algae (Scytosiphon lomentaria), and a fleshy red algal crust (Hildenbrandia rubra). Filamentous (E. intestinalis) and foliose (Ulva lactuca) green algae were predominant in MP2 and red crustose algae (H. rubra) dominated MP1; C. fragile was moderately abundant in the mid pools. Both mid pools were bounded by a dense cover of fucoid brown algae (Fucus vesiculosus and Ascophyllum nodosum) on the emergent rock.

2. Materials and methods

2.2. Experiment 1: grazing on damaged thalli

2.1. Study site

Thalli of C. fragile that were bleached and necrotic were evident in many of the tidepools at Cranberry Cove in May 2001. The incidence of this damage was particularly high (99% of thalli) at HP (Schmidt and Scheibling, 2005). To determine the effect of grazing by L. littorea on damaged C. fragile, a 12week grazer manipulation experiment was conducted at HP (12 June to 17 September 2001). Thalli of C. fragile selected for this experiment had numerous fronds originating from a single holdfast and ranging from 3 to 8 cm in length. These thalli had ~ 50% tissue damage, measured as the proportion of frond length. The completely randomized design consisted of 3 treatments, each with 5 replicates: 1) snail exclusion (within cages), 2) procedural control (partial cages), and 3) natural control (open plots). The treatments were randomly allocated to the selected damaged thalli. All cages were bottomless, conical in shape (20.5 cm diameter, 30 cm height), and constructed from plastic mesh (Vexar ®, aperture size: 1 cm2) supported by a wire frame. For partial cages, the mesh was cut ~ 5 cm from the bottom of the frame. The cages were sealed to the substrate with marine epoxy (Z-spar®, A-788 Splash Zone compound) to enclose a plot area of 0.033 m2. Natural control plots were marked with circles of marine epoxy to enclose the same area. The top of each cage was cut to create a removable lid that

Experiments were conducted in tidepools at Cranberry Cove (44° 28′ N, 63° 56′ W), near Halifax, Nova Scotia, Canada. The shore consists of large granite outcrops and boulders, with numerous tidepools of varying size on the south-facing shore. Three tidepools were selected for this study: one in the high intertidal zone (HP) and two (MP1, MP2) in the mid zone. The height of the pools relative to Chart Datum, measured during periods of calm sea state, was 1.2 and 1.6 m for MP1 and MP2 respectively, and 1.7 m for HP (Schmidt and Scheibling, 2005). Despite similar tidal heights, MP2 is flushed much more frequently than HP, placing it clearly in the mid zone. MP1 is within 3 m of a vertical rock wall extending from the low zone, and waves smashing into this wall usually spill into the pool within 2 to 4 h of low tide (depending on sea state and stage of the lunar tidal cycle). In contrast, HP is separated from the low zone by a broad (~ 20 m) rock platform that protects this pool from all but the largest waves. Consequently, HP can be isolated for days during calm periods and neap tides. The length of each pool was measured along the maximum dimension during tidal isolation. For HP and MP1, pool surface area was estimated by measuring pool width at 2-m intervals across the length, and multiplying the length by the average

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allowed access for measurements and removal of snails (in the exclusion treatment). Lids were fastened to the cages with plastic cable ties. The thallus length, defined as the average length of the 5 longest fronds (3 longest in the first 3 weeks) arising from a holdfast, was measured at weekly intervals. Snail densities (per cage or open plot) were recorded twice a week, and any small snails that penetrated the mesh were removed from exclusion cages. On 17 August, ~ 1 week after all damaged tissue had regenerated in all treatments, control and grazer exclusion treatments were transposed, i.e. natural control plots were caged to exclude snails and exclusion cages were removed to allow snails access. Thallus lengths were compared between grazing treatments by repeated measures analysis of variance (ANOVA). Separate analyses were done for the 1-month interval before (25 July, 9 and 17 August) and after (17 and 29 August, 17 September) the partial transposition of treatments. The Greenhouse–Geisser adjustment was used where departures from the sphericity assumption were significant (α = 0.05). To compare grazer treatments (between-subject effects), planned comparisons were conducted by t-test: grazer exclusion treatment and procedural control were compared (1-tailed test) to assess grazer effects, and the natural and procedural controls were compared (2-tailed test) to assess caging artifacts. All analyses were performed on untransformed data and satisfied the assumption of homogeneity of variance, as indicated by Levene's test (α = 0.05). 2.3. Experiment 2: grazing on recruits To determine the effect of littorinid grazing on new recruits of C. fragile, snail abundance was experimentally manipulated in three pools: HP, MP1 and MP2. There were 3 treatments applied in each pool: 1) snail exclusion, 2) snail inclusion, and 3) a natural control. Cages were used to exclude or include snails; controls were unmanipulated plots of comparable bottom area. The cages were similar to those used in the damaged tissue experiment but smaller (14 cm diameter, 20 cm height) making a frame unnecessary. Slits cut in the mesh allowed access for measurements, and were closed by plastic cable ties. In HP and MP1, the treatments were interspersed using a randomized block design: 2 replicates each of the snail exclusion and control treatments (10 replicates in total), and 1 replicate of the snail inclusion treatment, were randomly allocated to each of 5 blocks in areas naturally colonized by C. fragile. (Preliminary experiments suggested less replication was needed in the snail inclusion treatment for the desired degree of precision.) In MP2, 5 replicates of each treatment were allocated in a completely randomized design throughout this smaller pool. Snail density in the inclusion treatment approximated the natural density of L. littorea in each pool prior to the experiment: 2 snails per cage (33 snails 0.25 m− 2) in HP and MP1, and 3 snails per cage (50 snails 0.25 m− 2) in MP2. Small recruits of C. fragile used in this experiment were collected from a large tidepool in the low zone at Cranberry Cove (“low pool” in Schmidt and Scheibling, 2005). They were b 2 cm in thallus length and generally unbifurcated (although a

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few were in early stages of bifurcation) with a basal frond diameter of 1 to 2 mm. These minute recruits were extremely abundant (~ 125 individuals 0.25 m− 2) in July/August 2001 on a turf of coralline red algae (Corallina officinalis) around the seaward rim of this pool (Schmidt and Scheibling, 2005). Clusters of recruits were obtained by scraping sections of coralline turf, to which small thalli of C. fragile were attached, from the rock substratum. Small sections of turf, containing 3 to 5 recruits of C. fragile, were affixed to inverted plastic vial caps (2.5 cm diameter) with marine epoxy. After the epoxy had hardened (~ 30 min), the caps with their clusters of recruits were transplanted to the experimental tidepools and secured to the substratum within cages or control plots (1 cap per replicate) with marine epoxy. The experiment was initiated on 21 August 2001, when the recruitment of C. fragile in tidepools at Cranberry Cove had begun (Schmidt and Scheibling, 2005). The number of recruits in each replicate was recorded at the start of the experiment and at intervals of 3 to 7 days until the proportion of remaining thalli dropped below 0.1 in any treatment: 10, 23 and 44 days for MP2, MP1 and HP (where final measurement interval was 20 days), respectively. The length of recruits was measured at 10- to 24-day intervals and averaged for all thalli in a replicate at each measurement interval. Density of L. littorea in each tidepool was measured at the start of each experiment and then at about 1- to 2-week intervals (4 weeks for the last interval at HP) in 0.25 m2 quadrats (n = 10) that were haphazardly placed throughout the pool at 0.2 to 0.4 m depth. Snail inclusion and exclusion cages were inspected weekly to maintain snail densities, although corrections were rarely required. At HP, the size structure of the natural population of C. fragile was measured around the beginning (19 August) and end (3 October) of the experiment to examine size-specific patterns of survival. Thalli were counted and measured in 10 contiguous 0.25 m− 2 quadrats along the seaward rim of the pool at 0.2– 0.5 m depth. Because of the high density of small recruits in this area, thalli ≤ 2 mm were counted and measured in a 0.125 m− 2 inset quadrat and their numbers were doubled to combine counts and measures with larger thalli that were sampled throughout the 0.25 m− 2 quadrat. Differences among treatments and tidepools in the proportion of remaining thalli (relative to the initial number), or their average length, were compared by 2-factor ANOVA, with treatment as a fixed factor and pool as a random factor. Preliminary analysis showed no effect of block and it was excluded from our model. Because of the loss of all thalli in the control treatment at MP2, one ANOVA was used to compare thallus length between the two caged treatments (snail inclusion and exclusion) in all pools and a second ANOVA was used to compare the snail inclusion and the control in HP and MP1, where some thalli remained in the control. Post-hoc comparisons of means were done using Tukey's test. The per capita grazing rate of L. littorea (thalli snail− 1 day− 1) in the control and snail inclusion treatments for each pool was estimated as the change in the number of thalli in the first 7 to 10 days of the experiment divided by the density of snails, which was scaled to the cage area (0.015 m2) for control treatments.

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To estimate asymptotic survival (Sa) of recruits, the proportion of remaining thalli (Pr), averaged for all replicates of a treatment at each measurement time (t), was fit to a negative exponential function: Pr = Ae− Bt + Sa (where A and B are constants estimated by the model). When no plants survived at the end of the experiment (Sa = 0), or the model gave estimates of Sa that were not significantly different from zero (α = 0.05), Sa was constrained to zero in order to estimate the other parameters. The negative exponential function also was used to estimate the time (t) to 10% or 50% survival (Pr = 0.1 and 0.5 respectively) for each treatment. These estimates and Sa were then related to the average density of snails (D) in each treatment using a reciprocal [t = A × D − 1 ] or a negative exponential function (Sa = Ae− BD) respectively. 2.4. Experiment 3: grazing on holdfasts Large adult thalli of C. fragile that are dislodged by wave action (D'Amours and Scheibling, 2007) can regenerate from residual filaments of holdfast tissue, which adhere to the substratum (Scheibling and Gagnon, 2006). To determine the effect of littorinid grazing on residual holdfast tissue of C. fragile, a 5-month (October 2001 to March 2002) manipulative experiment was conducted at HP, during the period when C. fragile is naturally dislodged by waves (Bégin and Scheibling, 2003). Fifteen thalli of C. fragile, each with a single branching frond originating from the holdfast (mean basal diameter = 9 mm), were selected for this experiment. To mimic residual holdfasts of dislodged individuals, fronds of the experimental thalli were cut with a scalpel 1 to 2 mm above the substratum. Three treatments, with 5 replicates each, were randomly allocated to the 15 holdfasts: 1) snail exclusion, 2) snail inclusion, and 3) a natural control. Cages used in exclusion and inclusion treatments were of the same design and construction as those in the recruit experiment, and controls were open plots of the same bottom area. Snail density in the inclusion treatment was adjusted to approximate the mean density of L. littorea measured at monthly intervals in 0.25 m2 quadrats (n = 10) haphazardly placed in the tidepool [October to November 2001: 2 snails per cage (33 snails 0.25 m− 2); December 2001 to February 2002: 1 snail per cage (16 snails 0.25 m− 2)]. The presence or absence of macroscopic holdfasts was recorded at monthly intervals. The survival time of a holdfast was ranked according to the time elapsed (in months) until it was first recorded as absent. Holdfasts that survived the entire (5 months) experimental period were arbitrarily assigned a highest rank of 6. Survival times of the three treatments were compared using the log–rank test, which tests the null hypothesis that survival functions do not differ between groups (Peto and Peto, 1972). This test is capable of dealing with rightcensored data, such as ours, where some holdfasts survived to the end of the experiment. The log–rank test was also used for planned comparisons of the exclusion and inclusion treatments, and the inclusion and control treatments: grazer exclusion and inclusion treatments were compared (1-tailed test) to assess grazer effects, and control and inclusion treatments were compared (2-tailed test) to assess caging artifacts.

At the end of the experiment, snails were removed from the inclusion cages and open plots of the control treatment were caged. The caged plots were monitored for another 6 months (March to September 2002) to determine if microscopic filaments could re-grow in replicates where holdfast tissue was lost during the previous 5 months. 3. Results 3.1. Grazing on damaged thalli Thalli in all treatments recovered during the first 2 months of Experiment 1 as fronds grew and damaged tissue either regenerated or was grazed. By 9 August, all thalli appeared healthy. Before treatments were transposed on 17 August, mean thallus length in the natural and procedural control treatments had decreased by 3.2 and 0.6 cm respectively, while the mean in the exclusion treatment had increased by 3.9 cm (Fig. 1a). Over the 1-month period prior to transposition, repeated measures ANOVA showed a significant interaction between date and treatment (F4,18 = 10.39, P b 0.001). Within treatments, the change in thallus length over time was significant in both the natural control (F2,6 = 8.65, P = 0.017) and exclusion treatment (F2,6 = 12.8, P = 0.037), but not in the procedural control (F2,6 = 3.62, P = 0.093). At each date, thallus length did not differ significantly (P ≥ 0.07) between the natural control and procedural control, indicating that there was no detectable caging artifact. Fronds were significantly longer in the exclusion treatment than in the procedural control on the last 2 dates (9 August, t6 = 2.46, P = 0.025; 17 August, t6 = 2.32, P = 0.030). After transposing the natural control and exclusion

Fig. 1. Change in mean length (cm) of thalli of C. fragile with tissue damage (a) and density of L. littorea (b) in 3 treatments (natural control, procedural control, and snail exclusion) at HP. Data are mean + SE. Arrow indicates the timing of transposition of the control and exclusion treatments.

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treatment, thallus length increased in all treatments over the next month (Fig. 1a). Repeated measures ANOVA showed that the change over time was significant (F2,18 = 25.6, P b 0.001), as was the difference between treatments (F2,9 = 11.7, P = 0.003), but there was no significant interaction between these factors (F4,18 = 1.74, P = 0.186). Density of L. littorea in natural and procedural control treatments fluctuated in a similar manner over the 12-week experiment, with mean densities generally ranging between 20 and 60 snails 0.25 m− 2 (Fig. 1b). Snails were observed on the fronds and holdfasts of thalli, both in damaged and regenerated states, throughout this period. Snail density in the exclusion treatment was generally less than 11 snails 0.25 m− 2, and these were typically small juveniles. 3.2. Grazing on recruits In each tidepool, L. littorea in the control and snail inclusion treatments in Experiment 2 grazed on recruits of C. fragile, reducing the number of thalli remaining (Fig. 2a). The number of thalli in the exclusion treatment generally remained the same; a slight decrease in the first half of the experiment in HP was balanced with a slight increase in the second half due to the emergence of a few new recruits (Fig. 2a). Two-way ANOVA showed that proportion of remaining thalli at day 10 (once nearly all thalli had been lost in the control treatment at MP2) differed significantly among treatments in each tidepool (F2,4.2 = 20.24, P = 0.007) but not among pools (F2,56 = 2.42, P = 0.099), and there was no interaction between treatment and

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pool (F4,56 = 1.57, P = 0.20). Post-hoc comparisons using Tukey's test showed that the proportion of thalli remaining in the control and snail inclusion treatments, which did not differ (P N 0.05), was significantly lower than that in the snail exclusion treatment (P b 0.05). In MP1, however, where snail density in the tidepool (10 snails 0.25 m− 2) was about 1/3 of the fixed density in cages (equivalent to 33 snails 0.25 m− 2), the loss of thalli was significantly greater in the snail inclusion treatment than in the control by day 16 (t11.2 = 2.79, P = 0.02). In the snail inclusion treatment, the rate of thallus loss per snail at day 10 (day 7 in MP2) ranged from 0.15 (MP1, MP2) to 0.19 (HP) thalli snail− 1 day− 1, and did not differ significantly among pools (ANOVA, P = 0.78). The overall rate of thallus loss in this treatment, based on replicates averaged over all tidepools, was 0.17 (±0.02) thalli snail− 1 day− 1. The decrease in the proportion of remaining thalli (Pr) over time fits a negative exponential function for most treatments (Table 1). For the snail exclusion treatment at MP1, where there was no change in the number of thalli by the end of the experiment, we did not fit this model but set Sa = 1. In MP2, where the experiment lasted only 10 days, the small number of observations (n = 3) precluded fitting reliable models for the snail inclusion and exclusion treatments, although the control was fit. Asymptotic survival (Sa), estimated from these models, was strongly related to snail density (D, snails 0.25 m− 2) according to a negative exponential function: Sa = 0.84e− 0.096D (r2 = 0.97, P = 0.03). This shows that effectively no recruits of C. fragile survive at densities N50 snails 0.25 m− 2 (Fig. 3). The time to 10% and 50% survival (t0.1 and t0.5 respectively) for the

Fig. 2. Change in the number (a) and length (b) of recruits of C. fragile, and density of L. littorea (c) in 3 treatments (natural control, snail exclusion, and snail inclusion) at HP, MP1 and MP2. Data are mean + 1 SE.

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Table 1 Parameters of an exponential decay model (Pr = Ae− Bt + Sa) relating the proportion of surviving recruits of C. fragile (Pr) to elapsed time (t) for each combination of tidepool (HP, MP1, MP2) and treatment (C = control, I = inclusion, E = exclusion; with corresponding density of L. littorea) in Experiment 2 that yielded a significant fit to the model Treatment

HP control HP inclusion HP exclusion MP1 control MP1 inclusion MP1 exclusion MP2 control MP2 inclusion MP2 exclusion

Snails ⁎ m− 2

24 33 0 10 33 0 50 100 0

r2

Parameter estimate (SE) A

B

Sa

0.91 (0.03) 0.99 (0.07) 0.37 (0.16) 0.65 (0.05) 1.04 (0.06)

−0.13 (0.01) −0.19 (0.02) −0.05 (0.05) −0.08 (0.02) −0.14 (0.01)

1.00 (0.03)

−0.33 (0.03)

0.08 (0.02) 0 0.69 (0.17) 0.35 (0.05) 0 1 0

P

0.99 0.97 0.71 0.99 0.99

b0.001 b0.001 0.047 b0.001 b0.001

1.00

b0.001 n/a ns

The asymptote (Sa) is survival at t = infinity. Parameter estimates are given with standard error (SE, in parentheses); all but B for HP exclusion are significantly different from 0 (P b 0.05). r2 and P-value are given for the overall model in each case. The model could not be fit to data for MP2 inclusion (n/a, not applicable) and the model for MP2 exclusion was non-significant (ns, P = 0.77).

various treatments was inversely related to snail density according to a reciprocal function: t0.1 = 583D− 1 (r2 = 0.96, P = 0.003); t0.5 = 162D− 1 (r2 = 0.99, P b 0.001). This shows that at least 10% of recruits of C. fragile would be expected to survive for 30 days where densities of L. littorea were b20 snails 0.25 m− 2, and at least 50% would survive for 30 days where densities were b 6 snails 0.25 m− 2. At 60 days, however, these levels of survival could only be expected at densities b10 and b3 snails 0.25 m− 2 respectively (Fig. 4). Thallus length at day 10 was significantly greater in the snail exclusion than the inclusion treatment (F1,5.7 = 93.75, P b 0.001; Fig. 2b) but there was no difference between the 3 pools (F1,27 = 1.38, P = 0.27) and no interaction between treatment and pool (F2,27 = 0.14, P = 0.87). Thalli in the control treatment were significantly longer than those in the snail inclusion treatment at HP and MP1 (F1,1 = 272.3, P = 0.039); again there was no effect of pool (F1,13 = 7.21 × 10− 3 , P = 0.93) and no interaction between treatment and pool (F1,13 = 7.21 × 10− 3, P = 0.93). Thallus length increased in the control and snail exclusion treatment throughout the 44-day experiment at HP, although the rate of increase in the exclusion treatment was double that of the control (Fig. 2b). The growth rate of recruits of C. fragile was estimated as the change in mean thallus length in the exclusion treatment at HP,

Fig. 3. The relationship between the asymptotic survival of recruits of C. fragile and the density of L. littorea in tidepools.

where we had the longest growth record. Small buds that emerged in the latter half of the experiment (6 observations of 0.2 to 0.7 cm at day 44) were excluded for the purposes of these growth rate calculations. Mean length (h, cm) increased linearly with time (t, day) over the 44-day experiment: h = 0.077t + 0.800 (r2 = 0.981). This gives an average growth rate of 2.3 cm mo− 1, which is comparable to the average growth of undisturbed recruits of the same initial size (0.2 to 2 cm) at HP between June and September 2000 (2.1 cm mo− 1, Schmidt and Scheibling, 2005). The size–frequency distributions of protected thalli in the snail exclusion treatment were approximately normal, excluding the small buds on 4 October (day 44), and show a modal progression that is consistent with this growth rate (Fig. 5). The lengths of the 2 largest thalli (of 4 remaining) in the control treatment on day 44 at HP were 3.0 and 4.0 cm; these had grown ~ 1 cm from 2.0 and 2.7 cm respectively on day 24. The persistence and growth of these larger recruits over the 20-day interval suggest that C. fragile may approach a growth refuge from grazing by L. littorea at about 3 cm in length. Interestingly, this is also around the size at which the first bifurcation of the thallus occurs (personal observations).

Fig. 4. The relationship between the time to 50% and 10% survival of recruits of C. fragile and the density of L. littorea in tidepools. Dotted and dashed lines indicate snail densities at each level of survival at 30 days (when recruits exceed 3 cm in thallus length) and 60 days (when recruits exceed 5 cm in thallus length) respectively.

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Fig. 5. Size–frequency distributions of thalli of C. fragile in the snail exclusion treatment and in the natural population at HP. The number in parentheses above the 1-cm size class in the natural population on 19 August indicates the total count for this class (which exceeded the scale limit on the Y-axis, set for graphical clarity).

A growth refuge around 3 cm also is evidenced by changes in the size–frequency distribution of the natural population of C. fragile at HP over the experimental period (Fig. 5). At the start of the experiment, new recruits in the 1-cm size class dominated the population in the tidepool, accounting for 159 out of 263 individuals (60%) sampled on 19 August. Only 13 thalli in this sample (5%) were N 3 cm. If these larger recruits were immune to further grazing we would expect them to have grown to at least 6 cm by the end of the 44-day experiment on 4 October (assuming a growth rate of 2.3 cm month− 1 as estimated above). On 3 October, there were 14 thalli larger than 6 cm and 10 larger than 7 cm of 108 individuals remaining in the same sampling area, indicating little or no mortality of thalli that were N 3 cm in length on 19 August. In contrast, 93% of new recruits sampled on 19 August (n = 159) were assumed to be lost over this interval, based on 11 thalli remaining in the 4and 5-cm size classes combined (and assuming the same growth rate).

(χ22 = 11.12, P = 0.004). Planned comparisons showed that the rate at which holdfasts were lost was significantly greater in the snail inclusion than the exclusion treatment (χ 2 1 = 8.22, P = 0.004), but there was no difference between the inclusion treatment and control (χ21 = 1.42, P = 0.234). Fronds grew from two of the holdfasts in the exclusion treatment, one in December 2001 and a second in February 2002. These replicates had 23 and 3 fronds growing from their respective residual holdfasts by September 2002. After the removal of snails, 1 to 2 new fronds re-grew in the inclusion and control treatments in September and July 2002 respectively.

3.3. Grazing on holdfasts All holdfast tissue disappeared in 4 to 5 months in the snail inclusion and control treatments, whereas only one holdfast disappeared (in month 4) in the exclusion treatment (Fig. 6). The survival times differed significantly between treatments

Fig. 6. Change in the number of residual holdfasts of C. fragile in 3 treatments (natural control, snail exclusion, and snail inclusion) at HP.

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4. Discussion The importance of herbivores in determining the outcome of plant and algal introductions is a matter of much debate among ecologists. The predictions of ecological theory (e.g., Biotic Resistance Hypothesis, Elton, 1958; Enemy Release Hypothesis, Keane and Crawley, 2002) and experimental studies (Levine et al., 2004, Parker et al., 2006) often provide simple and sometimes contradictory generalizations. The effect of herbivory on introduced plants and algae may depend on the stage of the invasion process, interactions with other biotic and abiotic factors, and the life history stage of the invader (Mitchell et al., 2006), although many studies have disregarded these contingencies. In this study we examined the stage- and condition-specific susceptibilities of C. fragile to herbivory by the snail L. littorea. Our results show that L. littorea may restrict growth and survival of C. fragile in tidepools by grazing newly recruited C. fragile, adult thalli that are bleached and necrotic, and residual holdfasts of detached plants. Once recruits grow beyond a critical size, or damaged tissues or holdfasts regenerate, grazing by L. littorea appears to be limited and ineffectual. 4.1. Size- and condition-specific vulnerability to littorinid grazing Intertidal seaweeds are subjected to multiple stressors, including temperature extremes, the hydrodynamic forces of crashing waves, osmotic shock, desiccation, and high levels of photosynthetically active and UV radiation. Stressful environmental conditions, in combination with herbivory and competition, are known to play an important role in determining the zonation patterns and community structure of intertidal algal beds (Lubchenco and Menge, 1978, Lubchenco, 1980, Chapman and Johnson, 1990). In addition to lethal effects that limit algal abundance, environmental stress causes sublethal chemical and structural changes that reduce photosynthetic rate, growth and reproduction (Davison et al., 1993, Pena et al., 1999, Dethier et al., 2005, Schaffelke and Deane, 2005), and increase the likelihood of dislodgement due to wave action (Haring et al., 2002, Dethier et al., 2005). Some of these stressinduced structural and chemical changes may explain the variable susceptibility of C. fragile to littorinid grazing observed in our study. Desiccation stress, for example, mediates interactions between other species of Codium and specialist sacoglossan herbivores: P. dendritica feeds selectively on desiccation-prone C. setchellii in Oregon, while P. dendritica and Elysia maoria display a similar affinity for desiccationprone C. convolutum in New Zealand (Trowbridge, 1998b). Desiccation, freezing, and high irradiance are believed to cause bleaching in intertidal seaweeds (Lubchenco, 1980, Dudgeon et al., 1990, Scrosati and DeWreede, 1998). The tissue damage observed in C. fragile at HP may have been due to these sorts of stressors, given that damaged thalli were exposed at the water surface during the protracted isolation period of this high-zone pool. These damaged tissues appear to be more vulnerable to grazing by L. littorea than healthy tissue.

Similarly, Heaven and Scrosati, (2004) found that littorinids preferred bleached to photosynthetic tissues of Mazzaella parksii. Release of cellular fluid from damaged tissue may provide a chemical signal that attracts grazers, and a reduction in toughness may facilitate consumption of damaged tissue (Watson and Norton, 1985a), or make it more prone to breakage during grazing. Environmental stressors, such as desiccation and UV radiation, also may lower chemical defenses of algae (Hay and Fenical 1988, Renaud et al. 1990, Cronin and Hay 1996), thus increasing their vulnerability to herbivores. Trowbridge (1995) suggested that C. fragile contains unstable, chemical grazing deterrents. She found that freshly collected thalli emit a strong odor and are avoided by the herbivorous snail Turbo smaragdus, while less pungent specimens that were freezedried or maintained in the laboratory for an extended time were consumed. Dimethylsulfonopropionate (DMSP), a secondary metabolite that is broken down by enzymatic cleavage into acrylic acid and dimethylsulfide (a particularly odorous compound), is produced when C. fragile is physically damaged (Van Alstyne et al. 2001a, Lyons et al. 2007). Both acrylic acid and dimethylsulfide are effective grazing deterrents against the urchin S. droebachiensis (Van Alstyne et al. 2001a, Van Alstyne and Houser 2003, Lyons et al. 2007), Interestingly, L. littorea prefers algal species, such as Ulva sp. and Enteromorpha sp. (Lubchenco 1978, Watson and Norton 1985b), which produce significantly more DMSP than C. fragile (Van Alstyne et al. 2001a). Thus, if large, healthy individuals of C. fragile are chemically defended against littorinid grazing, it is likely by some other type of secondary metabolite. Juvenile and adult stages of algae can be structurally and chemically different from one another, and often vary in their attractiveness to herbivores (Van Alstyne et al., 1999, 2001b), including L. littorea (Watson and Norton, 1985b). The inability of L. littorea to effectively graze adult thalli of C. fragile may be related to thallus size. Littorina littorea typically grazes epilithic microalgae, such as diatoms and propagules of macroalgae, using its ventrally-positioned mouth and taenioglossan radula to scrape the surface of the substratum as it moves across it (Steneck and Watling, 1982). The erect branching form of C. fragile is difficult for L. littorea to pin down or climb upon, particularly when thalli are moving in wave surge. Periwinkles found on adult thalli of C. fragile are likely grazing on filamentous epiphytes, such as Polysiphonia harveyi, as there were no signs of grazing on the thallus (see also Bégin and Scheibling, 2003). The results of Experiment 2, coupled with measures of the natural population of C. fragile at HP, suggest that recently recruited thalli approach a size refuge from littorinid grazing at ~3 cm in length. Assuming a growth rate of 2.3 cm month− 1 for thalli that are not grazed by snails, the average length of C. fragile would exceed 3 cm after 30 days for thalli that are ~0.8 cm at the start of this interval (the average size of recruits at the start of Experiment 2). After 30 days, however, b 10% of new recruits are predicted to survive in tidepools where the density of L. littorea exceeds 20 snails 0.25 m− 2 (Fig. 4). Thus, few recruits are likely to escape grazing long enough to reach a possible growth refuge at

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3 cm, except in areas where snails are in relatively low abundance. After 60 days, b 10% of new recruits are predicted to survive where density of L. littorea exceeds 10 snails 0.25 m− 2 (Fig. 4). After 60 days, any non-grazed recruits would exceed 5 cm in thallus length. If a growth refuge from snail grazing is closer to 5 cm, very few recruits would escape in size except in pools where snails are in very low abundance (Fig. 4). Similarly, Lubchenco (1983) found that while L. littorea readily grazes small germlings of F. vesiculosus, individuals larger than 3 to 5 cm were much less susceptible to snail grazing. The small size of recruits of C. fragile (~ 0.2 cm branch diameter, ≤2 cm thallus length) may facilitate grazing by L. littorea because snails are able to topple and pin down the minute thalli. While surviving recruits in the exclusion treatment tended to grow throughout the experiment, recruits in the control and inclusion treatments became noticeably smaller, except in the control treatment at MP2 where snail density and recruit loss were low. It is possible that L. littorea also destroys new recruits by bulldozing, i.e. dislodging thalli while crawling over the substrate (Watson and Norton, 1985b). One or two dislodged thalli were observed in the inclusion treatment at MP1 on a few occasions. Changes in mean length of thalli in the different treatments in Experiment 1 at HP also suggest a growth refuge around 3 to 4 cm for thalli recovering from physiological damage (Fig. 1). Damaged thalli in control plots that had been reduced to ~ 2 cm by the time they had fully recovered on 9 August, began to increase in length only after snails were excluded on 17 August. However, in procedural control plots (which retained this treatment throughout the experiment), thalli that had been reduced to ~ 4 cm by 9 August continuously increased in length throughout the remainder of the experiment, amid moderately high densities of L. littorea (15 to 47 snails 0.25 m− 2). In exclusion plots, thalli that had grown to a mean length of 9.6 cm by 17 August, continued to grow after exposure to comparable densities of snails over the next month. Although this evidence for a growth refuge is indirect and preliminary, it serves as a basis for the design of further experiments to explicitly test this hypothesis and directly determine the size threshold. Residual holdfasts of C. fragile were grazed by L. littorea in the open plots and snail inclusion cages in Experiment 3. One or two fronds re-grew from holdfasts in these treatments once snails were excluded, suggesting that some filaments of C. fragile survived even where macroscopic tissues were no longer evident. In contrast, as many as 23 fronds re-grew from a holdfast in the snail exclusion treatment. The effectiveness of snail grazing on holdfasts, and its impact on population density of C. fragile, may be dependent on the substrate type. The coenocytic filaments of C. fragile can occupy any solid surface, including rocks, macroalgae, and even the shells of snails (Trowbridge, 1998a). Periwinkles graze most effectively on smooth substrata, and cracks and crevices may act as spatial refuges from snail herbivory (Lubchenco, 1980). In Scotland, Trowbridge and Todd (1999a) found that L. littorina commonly grazes the undifferentiated mat-forming (vaucherioid) stage of another invasive subspecies, C. fragile ssp. atlanticum, in tidepools on wave-sheltered shores.

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4.2. Role of littorinids in regulating intertidal populations of C. fragile Collectively, our experiments indicate the potential of herbivory by L. littorea in regulating populations of C. fragile in the intertidal zone of the Nova Scotia. The low survival of recruits of C. fragile exposed to snail grazing in Experiment 2, coupled with records of abundance of these two species from a concurrent study by Schmidt and Scheibling (2005), suggests that littorinids may substantially limit recruitment to adult populations of C. fragile within tidepools, particularly in the mid to high zone where snails are numerous. Schmidt and Scheibling (2005) observed similar seasonal patterns of abundance in L. littorea and C. fragile at HP between 2001 and 2003, which may reflect the concentration of snails in areas of high recruitment of C. fragile in summer. In each year, recruit density was maximal in July/August (100 to 150 thalli 0.25 m− 2) when snails also were most abundant (15 to 45 snails 0.25 m− 2). By December, however, densities of C. fragile and L. littorea had declined to low levels (1 to 10 thalli 0.25 m− 2 and 10 to 15 snails 0.25 m− 2). This strong seasonal pattern was not seen in a nearby low-zone pool and shallow inlet, where snails were much less abundant (1 to 8 snails 0.25 m− 2) and C. fragile persisted into early winter at densities of 20 to 180 thalli 0.25 m− 2 (Schmidt and Scheibling, 2005). More turbulent conditions, or the occurrence of recruits of C. fragile within extensive and dense turfs of coralline algae, may restrict the foraging ability of L. littorea lower on the shore. In high pools C. fragile is also subjected to heavier levels of tissue damage by environmental stressors than individuals in pools lower on the shore or in the shallow subtidal zone (Schmidt and Scheibling, 2005). In Experiment 1 we showed that although L. littorea can temporarily inhibit the vegetative regeneration of damaged thalli, the snails were incapable of entirely consuming damaged thalli at HP, which gradually recovered and grew into a size refuge from snail grazing. Moreover, major bleaching events, such as that observed in 2001, are infrequent: only one other comparable event occurred since then, in spring 2006 (personal observations). Dislodgement by wave action during large storm events in fall and winter can be a significant source of loss of C. fragile, particularly for larger thalli (Bégin and Scheibling, 2003, D'Amours and Scheibling, 2007). In Experiment 3, which mimicked wave dislodgement of adult thalli, we showed that there is little chance of regeneration of these individuals from residual holdfast tissues, provided littorinids are present even at relatively low densities. We selected HP as a natural mesocosm for Experiments 1 and 3 because of its large size, which enabled us to spatially segregate blocks of replicates within the same pool. Although HP is similar to other pools in the high and splash zones at Cranberry Cove, in terms of biotic composition and littorine abundance (Metaxas et al., 1994), our use of a single pool restricts generalization of our experimental results within the intertidal zone. Nonetheless, HP provided a natural arena that enabled us to draw inferences about the susceptibility of C. fragile to snail grazing that are ecologically relevant.

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We conclude that L. littorea plays a limited role in regulating populations of C. fragile in tidepools in Nova Scotia by reducing rates of recruitment via propagules or vegetative regeneration from residual holdfasts or damaged tissues. Once healthy adult thalli are established however, L. littorea and other mesograzers appear to have little effect on C. fragile (see also Bégin and Scheibling, 2003, Schmidt and Scheibling, 2005). Similarly, Trowbridge and Todd (1999b) found little evidence of grazing damage to C. fragile ssp. tomentosoides by generalist herbivores, including L. littorina, in the intertidal zone in Scotland. Since C. fragile was first recorded at Cranberry Cove in 1996, the invasive alga has persisted (Schmidt and Scheibling, 2005); it is currently a common and conspicuous component of the macroalgal assemblage of tidepools in this area. Acknowledgements We thank A. Schmidt, M. Wagstaff and C. Theriault for assistance in the field, and J-S Lauzon-Guay, P. Gagnon and A. Metaxas for comments on the manuscript. The research was funded by a Discovery Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to RES. [SS] References Begin, C., Scheibling, R.E., 2003. Growth and survival of the invasive green alga Codium fragile ssp. tomentosoides in tide pool on a rocky shore in Nova Scotia. Bot. Mar. 46, 404–412. Bertness, M.D., 1984. Habitat and community modification by an introduced herbivorous snail. Ecology 65, 370–381. Bird, C., Dadswell, M., Grund, D., 1993. First record of the potential nuisance alga Codium fragile ssp. tomentosoides (Chlorophyta, Caulerpales) in Atlantic Canada. Proc. N.S. Inst. Sci. 40, 11–17. Chapman, A.R.O., 1989. Abundance of Fucus spiralis and ephemeral seaweeds in a high eulittoral zone: effects of grazers, canopy and substratum type. Mar. Biol. 102, 565–572. Chapman, A.R.O., Johnson, C.R., 1990. Disturbance and organization of macroalgal assemblages in the northwest Atlantic. Hydrobiologia 192, 77–121. Cronin, G., Hay, M.E., 1996. Susceptibility to herbivores depends on recent history of both the plant animal. Ecology 77, 1531–1543. D'Amours, O., Scheibling, R., 2007. Effect of wave exposure on morphology, attachment strength and survival of the invasive green alga Codium fragile ssp. tomentosoides. J. Exp. Mar. Biol. Ecol. 351, 129–142. Davison, I.R., Johnson, L.E., Brawley, S.H., 1993. Sublethal stress in the intertidal zone: tidal emersion inhibits photosynthesis and retards development in embryos of the brown alga Pelvetia fastigiata. Oecologia 96, 483–492. Dethier, M.N., Williams, S.L., Freeman, A., 2005. Seaweeds under stress: manipulated stress and herbivory affect critical life-history functions. Ecol. Monogr. 75, 403–418. Dudgeon, S.R., Davison, I.R., Vadas, R.L., 1990. Freezing tolerance in the intertidal red algae Chondrus crispus and Mastocarpus stellatus: relative importance of acclimation and adaptation. Mar. Biol. 106, 427–436. Elton, C., 1958. The Ecology of Invasions by Plants and Animals. London, Methuen. Garbary, D., Fraser, S., Hubbard, C., Kim, K., 2004. Codium fragile: rhizomatous growth in the Zostera thief of eastern Canada. Helgol. Mar. Res. 58, 141–146. Haring, R.N., Dethier, M.N., Williams, S.L., 2002. Desiccation facilitates waveinduced mortality of the intertidal alga Fucus gardneri. Mar. Ecol. Prog. Ser. Vol. 232, 75–82.

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