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Dispersal limitation and post-settlement survival of an introduced ascidian (Botrylloides violaceus) in San Juan
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Abstract
Distributions of invasive species are often patchy; however, the mechanisms regulating these patchy
distributions are poorly understood. Organisms with short-lived larvae provide an opportunity to test whether these
distributions are dispersal-limited or mediated through post-metamorphic processes. Here we used the invasive
colonial tunicate Botrylloides violaceus to examine the roles of dispersal and predation in determining its
distribution through field outplants of recently settled juveniles to locations with and without adult B. violaceus
colonies. Survival and growth were not different between caged and uncaged treatments, suggesting that predation
is not controlling the distribution of this species. However, survival and growth were different among sites. One site
without established colonies had significantly lower growth and survival than all others, indicating the importance of
post-settlement factors at this site. The other site where adults were absent had similar growth and survival to sites
with established colonies, indicating dispersal limitation at this site. Our study suggests that the distribution of B.
violaceus is limited both by dispersal and environmental conditions that affect juveniles after settlement.
Keywords: colonial tunicate, Botrylloides violaceus, San Juan Islands, dispersal, juveniles
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Introduction
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Colonization success of invasive species and interactions between native and non-native species can vary
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spatially and lead to different patterns of species richness and diversity (Fridley et al. 2007; Sax and Gaines 2008).
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Although the known range of a species may encompass a wide span of latitudes, its actual distribution can be patchy
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(MacArthur 1972; Pennington 1996). Understanding the factors that create these mosaic distributions is critical to
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predicting the population dynamics of invasive species.
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A number of explanations have been proposed to explain the success of invasive species, such as the
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evolution of increased competitive ability (Blossey and Nötzold 1995), the empty niche model (Elton 1958) and the
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enemy release hypothesis (Elton 1958). However, for a species to colonize a new area, propagules must first
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disperse and subsequently survive ambient biotic and abiotic stresses. The idea that dispersal to new locations is
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fundamental to the spread of invasive species has been widely recognized (Williamson and Fitter 1996; Blackburn
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and Duncan 2001; Drake and Lodge 2004; Lockwood et al. 2005, Lockwood et al. 2009); however, few studies have
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directly measured whether dispersal limitation or environmental factors are more important in limiting invasive
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species distributions (but see Pierson and Mack 1990; Von Holle and Simberloff 2005).
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In many marine species, propagules are pelagic larvae that are capable of greater dispersal than the adults.
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These larvae not only have to survive their planktonic larval stage, but then make the transition to a benthic juvenile.
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Furthermore, their small size at metamorphosis increases their susceptibility to predation. For a non-native species,
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establishment in an area may be inhibited by both native and introduced predators. A number of studies have
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examined the preferences of native predators for invaders (Shinen et al. 2009; Simoncini and Miller 2007), and
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others have demonstrated the role native predators may play in setting the range limits of invasive species (deRivera
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et al. 2005).
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The invasive colonial tunicate Botrylloides violaceus is native to the coasts of Asia in the NW Pacific
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Ocean (Cohen 2005) and was first documented in the San Juan Island Archipelago (Washington, USA) in 1998 (A.
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Cohen et al. 1998). Since that time B. violaceus has spread to a number of other locations around the San Juan
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Islands, while remaining absent from others. The factors that limit the spread of this non-native species are
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currently unknown. In an attempt to predict the distributions of B. violaceus based on abiotic factors, Epelbaum and
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others (2009a) found very few sites unsuitable for its survival in the waters of British Columbia, yet the species
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remains patchily distributed. Native predators are known to consume B. violaceus, but have not been demonstrated
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to control B. violaceus and prefer native prey (Epelbaum et al. 2009b; Simoncini and Miller 2007; Whitlatch and
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Osman 2009). At sites in the San Juan Islands where the species is already a resident member of the fouling
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community, caged field experiments with B. violaceus also suggest little influence of predation on recruitment
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(Jacobs 2006). However, there is some evidence that predation can be important in the first week after settlement
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(Osman & Whitlatch 2004).
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Here, we examine whether the distribution of B. violaceus is determined by dispersal limitation or post-
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settlement factors in the San Juan Islands. We artificially increased dispersal by outplanting newly settled juveniles
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and measured their survival and growth at sites with and without established adult colonies. We hypothesize that if
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the distribution of B. violaceus is limited by dispersal, then juveniles will grow and survive equally well at all sites
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in both caged and uncaged treatments. Alternatively, if colonies are limited by post-settlement factors we predict
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that juveniles will experience reduced growth or survival at the sites where adults are absent. Post-settlement
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limitation may occur through biotic or abiotic factors. If higher mortality occurs in uncaged treatments at sites
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where adults are absent, then predation is likely restricting its distribution. Equal mortality across caging treatments
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would suggest abiotic factors play an important role.
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Methods
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Study organism and study sites
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Botrylloides violaceus (Botryllidae) is a colonial ascidian that forms large sheets over many hard substrata
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and commonly overgrows other native species. It is native to the northwest Pacific along the coasts of Siberia,
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China and Japan (Cohen 2005). B. violaceus has been introduced to both coasts of the United States and a number
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of other locations worldwide including the Netherlands, Italy and Australia. Along the west coast of the United
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States it was first reported from San Francisco Bay in 1973. However, due to some confusion with identification it
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may have been present elsewhere on the west coast of the United States, including Willapa Bay and Puget Sound, in
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the 1970s. Subsequently, B. violaceus was reported in a variety of bays along the coasts of California, Oregon and
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Washington in the 1990s and 2000s (Cohen 2005). B. violaceus was first seen in the San Juan Islands, Washington,
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USA in 1998 (A. Cohen et al. 1998).
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In the San Juan Islands, B. violaceus is abundant at both Fisherman’s Bay (FB) and Roche Harbor (RH),
while it is absent at Friday Harbor Laboratories (FHL) and Jensen’s Shipyard (JS) in Friday Harbor (Fig. 1). All
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sites except FHL are public or private marinas, with heavy boat traffic; the FHL dock has a lower level of research
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vessel traffic.
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Larval collection and settlement
Adult colonies of B. violaceus were collected on 24 July 2009 from the floating docks at RH and placed in
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a dark flow-through seawater tank at FHL for 36 h prior to spawning. To induce spawning, all colonies were placed
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in a common tank, exposed to bright light, and gently torn. All actively swimming tadpole larvae were collected
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and placed on settlement plates (roughened Petri dishes; method from Marshall et al. 2006). Within 4 h,
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approximately 95% settlement had occurred. All larvae that were not firmly attached to settlement plates at this
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time were removed from the experiment. In the case of pairs of larvae that were in close proximity with each other,
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one was randomly selected for removal to eliminate the effects of space competition and colony fusion among
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settlers; both phenomena have been observed in B. violaceus (C. Cohen et al. 1998; Marshall et al. 2006). After
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thinning, each plate had 2-7 settled larvae, with a mean of 3.825 (± 1.18 SD).
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Settlers were marked and photographed using a Micropublisher 3.3 RTV camera with QCapture 3.1.1
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(Leeds Precision Instruments, Inc.). Plates were then attached to a Plexiglas array (approx. 10 cm x 1m) and
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deployed in the field. Each array contained ten settling plates with two small holes so that the plates could be
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attached to the arrays using plastic cables. Half of the plates were randomly selected to be covered with Vexar®
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mesh serving as a cage treatment to exclude predators larger than the 4 mm mesh size. Plates were randomly
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assigned to an array within treatments, ensuring equal numbers of caged and uncaged plates on each array, and then
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randomly assigned to a position on the array. Arrays were placed in the field on 27 July 2009, suspended on ropes
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approximately 1 m below the underside of floating docks with two arrays at each of four sites: FB, RH, FHL and JS.
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Growth, survival, and physical data
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For the first week of the experiment, the sites were surveyed for temperature and salinity using a YSI probe
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every two days. After six days the arrays were returned to the lab, where each colony was photographed for analysis
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of survival, growth, and number of zooids per colony. They were subsequently returned to the field for fifteen
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additional days, and final survival, growth, and zooid number were assessed as described above on 16 August 2009
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after 21 days in the field.
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Feeding experiment
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To estimate the potential amount of predation on newly settled larvae, a predation experiment was
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conducted with B. violaceus and Pandalus danae (dock shrimp or coon shrimp), a mobile predator that is present at
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the FHL and JS docks in large numbers and in much smaller numbers or absent at RH and FB (authors’ pers. obs.).
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Fifteen individuals of P. danae were collected at the FHL dock using a net and starved for approximately 72 h in
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individual cages. B. violaceus individuals were collected from RH, kept in the dark overnight, and spawned
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according to the above procedure. 12 to 15 larvae were settled in each of 15 pre-roughened Petri dishes as described
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above. After settling for approximately 24 h, settlers were thinned so that each plate contained 10 individuals.
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Fifteen separate feeding trials were run by placing one starved shrimp in a Tupperware® container with one
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plate containing 10 B. violaceus settlers in a sea table. The plates were glued to the lids of the container so that the
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settlers were presented to the shrimp in the same orientation as in the field experiment. After 24 h the number of
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settlers remaining was counted. The cephalothorax length of each shrimp was measured with digital calipers as an
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estimate of size.
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Data analysis
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Physical data (temperature and salinity) were analyzed graphically for differences among sites and dates.
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Photographs of the settlers, six-day-old colonies, and 21-day-old colonies were analyzed with ImageJ. The zooid
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area of each individual was measured three times and replicates were averaged to minimize measurement error. To
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measure the effect of initial settler size on final size, we ran an ANCOVA on final zooid area by site, with initial
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zooid area as a covariate at both six and 21 days.
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We calculated size-specific growth rates using the following formula:
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G = [(Af) – (Ai)] / [Ai*(tf-ti)]
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where Af is area (mm2) at time f (final), Ai is area (mm2) at time i (initial), and t is time. Data were arcsine square-
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root transformed, followed by a log transformation for normality. These transformed data were averaged per plate
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and then analyzed using a two-way factorial ANOVA with site and caging as fixed factors.
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was analyzed using a two-way factorial ANOVA with site and caging as fixed factors as above.
The number of zooids
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Differences in survivorship after 6 and 21 days were compared within sites between caged and uncaged
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treatments using Fisher’s exact tests. Differences in survivorship among sites were compared using contingency
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analysis via permutation testing (an extension of Fisher’s exact test to a 2x4 table). The permutation test was run
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with 100,000 iterations using a program developed by W.R. Rice, University of California, Santa Barbara. Pairwise
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comparisons among sites were made using Fisher’s exact tests. Logistic regressions were used to relate final
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survivorship to initial size for data pooled from all sites.
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We calculated the mean number and standard error of settlers eaten per individual shrimp and correlated
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cephalothorax length of each P. danae with the number of settlers eaten (Spearman’s rho, analysis done in R ver.
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2.8.1). All analyses were run in JMP ver. 7, unless otherwise indicated.
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Results
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Growth
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After 6 days, growth was similar among all sites (Fig 2A) and between both caged and uncaged treatments.
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The transformed relative growth data were both normal (Shapiro-Wilk test, W = 0.964, p = 0.09) and homoscedastic
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(Levene’s test, p = 0.90). The two-way factorial ANOVA showed no significant effect of site, cage, or site*cage
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interaction (Fig. 2A, Table 1). There was a significant effect of initial zooid size on final zooid size as determined
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by ANCOVA, but no effect of site on final zooid size (Table 2, Fig. 3A).
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The number of zooids per colony showed a significant effect of site (Table 3). The number of zooids per
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colony showed neither a site*cage interaction nor a cage effect. A one-way ANOVA among sites (pooling
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caged/uncaged data) followed by Tukey tests showed that there were significantly more zooids per colony at FB
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than at any of the other sites (Fig. 4A). The temperature at FB was consistently higher (15.4-19.0°C) than at the
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other three sites, which clustered together around 13ºC, while salinity measurements were consistent across sites.
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After 21 days, growth was significantly different among sites (Fig. 2B). Low survival at JS excluded this
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site from all growth analyses due to lack of power. The transformed relative growth data were both normal (Shapiro-
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Wilk test, W = 0.981, p = 0.58) and homoscedastic (Levene’s test, p = 0.75). The two-way factorial ANOVA
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showed a significant effect of site, but no significant effect of cage or site*cage interaction (Fig. 2B, Table 1). A
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one-way ANOVA by site on pooled caged/uncaged data followed by a post-hoc Tukey test showed that FB had the
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largest growth rate (Fig. 2B). There was a significant effect of site on final zooid size as determined by ANCOVA.
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However, there was no effect of initial settler size or site*size interaction (Table 2, Fig. 3B).
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The number of zooids per colony showed a significant effect of site (Table 3) with neither a site*cage
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interaction nor a cage effect. A one-way ANOVA among sites (pooling caged/uncaged data) followed by Tukey
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tests showed that there were significantly more zooids per colony at FB than at any of the other sites; RH and FHL
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were not different (Fig. 4B).
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Survival
Early survival (Fig. 5A) was similar at all sites and between both caged and uncaged treatments. Survival
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was not significantly different between caged and uncaged treatments at all sites (p > 0.25 in all comparisons).
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Caged and uncaged treatments were subsequently pooled and survivorship was not significantly different among
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sites (p = 0.08). Low mortality limited sample sizes, preventing the use of logisitic regression to examine the effect
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of initial settler size on early survival.
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After 21 days, survivorship continued to be similar between caged and uncaged treatments (all p > 0.09).
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Caged and uncaged treatments were again pooled. However, survival was significantly different among sites (p <
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0.001, Fig. 5B) at this later time point. Pairwise comparisons revealed that JS had the lowest survivorship (all p <
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0.001). FB and FHL did not have significantly different survivorship (p = 0.63) while RH had the highest
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survivorship (all p < 0.03). Logistic regression showed no significant effect of initial settler size on final survival
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among sites or when all data were pooled.
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Feeding experiment
P. danae were found to consume recently settled B. violaceus. Each shrimp consumed an average of 3.7
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colonies (± 1.17 SE) out of the 10 colonies offered. However, variability was high with some individuals
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consuming all colonies and others consuming no colonies. There was no correlation between shrimp size and the
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number of colonies consumed (Spearman’s rho = -0.235, p = 0.40). The mean cephalothorax length of the P. danae
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individuals was 29.28 mm (± 0.80 mm SE).
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Discussion
Our results suggest that the factors determining the distribution of B. violaceus in the San Juan Islands vary
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spatially. Initially, post-metamorphic growth and survival were similar at all sites (Figs. 2A & 5A), with no
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difference between caged and uncaged treatments in the first six days of the experiment. After 21 days growth and
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survival was still similar between caged and uncaged treatments, indicating predation is not an important factor in
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determining the distribution of juvenile colonies of B. violaceus. However, we observed site-specific differences in
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growth (Fig. 2B) and mortality (Fig. 5B). JS had significantly lower growth and higher mortality than the other
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three sites, suggesting post-settlement factors prevent B. violaceus from colonizing this marina. Dispersal limitation
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may also explain why adult colonies are not present here, but propagules that do disperse to JS will not be able to
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persist The second site where adults were absent (FHL) had the same survivorship as FB and the same growth rates
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as RH, both sites where adults are abundant. This suggests that the limited dispersal ability of B. violaceus prevents
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establishment at FHL. The highest growth rates were found at FB and the highest survivorship was found at RH,
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indicating site-specific differences in the mechanisms regulating the establishment of new B. violaceus populations.
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At RH persistence of B. violaceus may rely on high survival, while at FB B. violaceus may be successful through
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increased growth.
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Offspring size can strongly influence the success of subsequent life-history stages (Pechenik 2006). The
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relationship between offspring size and fitness can vary considerably with environmental quality where there is an
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advantage to size at intermediate levels of environmental quality, while this advantage is lost in both benign and
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extremely harsh environments (Moran and Emlet 2001; Allen et al. 2008). At six days overall initial settler size was
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correlated with colony size at all sites with no differences between sites (Fig. 3A). However, this relationship
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disappeared by 21 days (Fig. 3B). Additionally, no relationship was found between initial settler size and survival.
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This suggests larger initial sizes do not confer an advantage in growth or survival of B. violaceus at our study sites.
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Elevated temperatures likely caused the high growth rates in colony size and zooid number observed at FB.
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FB is a relatively shallow, artificially enclosed bay with limited connectivity to other water sources causing water
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temperatures to be elevated. Other studies have also found elevated growth rates of B. violacues at higher
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temperatures (Yamaguchi 1975; McCarthy et al. 2006). Epelbaum et al. (2009a) found B. violaceus zooid number
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increases the fastest in temperatures ranging from 20 to 25ºC, while only some growth was observed between 5 and
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15ºC. Temperatures in FB remained near optimal temperatures for growth during this experiment, which likely
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contributed to the increased growth rates of B. violaceus at this site. The degree to which new colonies allocated
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energy to growth of individual colonies or zooid production also varied by site. Zooid number was significantly
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greater at FB compared to all other sites at six days and at 21 days (Fig. 4). At six days, zooid number was greater
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at FB even though colonies were of similar size at all sites. This differential resource allocation to generating
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greater numbers of zooids could increase both feeding ability and future fecundity at FB.
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Although there was substantial mortality at several of our sites after 21 days in the field, there were no
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significant differences in survival between caged and uncaged treatments after six or 21 days (Fig. 5), indicating that
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predation did not limit early post-metamorphic survival in B. violaceus. Whitlach and Osman (2009) also found low
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predation on B. violaceus by a gastropod predator in Long Island Sound. However, our arrays were hung 1m below
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the surface of the water and may not have been exposed to the full suite of predators that are present in a natural
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fouling community that could limit recruitment, settlement, and subsequent survival of B. violaceus. Osman and
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Whitlatch (2004) tested for this effect and did find higher mortality in one week old colonies for plates closer to the
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floating docks compared to suspended plates. Despite this potential limitation, significant differences in mortality
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were not observed until colonies were older than one week and Osman and Whitlach (2004) did not find differences
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in predation on two week or three week old colonies deploying plates in different ways.
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Our feeding trials with P. danae showed that, when starved, some shrimp ate all available settlers of B.
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violaceus, while others did not eat any settlers within the 24 h trial. This indicates that although shrimp are able to
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consume B. violaceus, they are unlikely to control a newly introduced population. Given that P. danae is not the
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only species present in the area that could eat juvenile ascidians, further studies could test the feeding rates of
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different predators on B. violaceus (as in Epelbaum et al. 2009b), as well as the preference of these predators for B.
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violaceus or other prey.
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Abiotic factors are likely more important than predation in determining the growth and survival of juvenile
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colonies of B. violaceus. A number of environmental factors are known to influence reproduction and development
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in ascidians including temperature, salinity, food, light, UV exposure, and anthropogenic disturbance (Bates 2005;
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Lambert 2005). Environmental characteristics of the water column may have played a role in the decreased growth
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and survival at JS.
Future field measurements of food availability and water quality, and additional experimental
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tests of the effects of compounds present in the water on B. violaceus could help distinguish among abiotic factors
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influencing post-settlement survival of B. violaceus, especially the reduced survival at JS.
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Several lines of evidence suggest that the spread of B. violaceus is dispersal-limited at FHL and potentially
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JS as well. First, the larval behavior and rapidity of settlement of this species are consistent with short-distance
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dispersal. Botrylloides spp. have lecithotrophic tadpole larvae that settle within minutes to hours once they begin
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swimming, reaching distances as short as 0.6m from the parent colony (Worcester 1994), but the larvae can also
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disperse longer distances (Grosberg 1987; Jacobs 2006). Furthermore, evidence of small-scale variation in genetic
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population structure and population dynamics of colonial ascidians over scales as small as 20 m suggests
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populations are dispersal-limited and effectively closed to immigration (Ayre et al. 1997; Yund and O’Neil 2000;
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Grosholz 2001; Yund and Stires 2002). Within the San Juan Islands, sites without B. violaceus colonies are
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separated by straight-line distances of more than 5 km, suggesting that populations within the islands may also be
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dispersal-limited. A study modeling dispersal in the San Juan Islands found shorter dispersal distances within the
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archipelago relative to the more open areas of the Strait of Juan de Fuca south and east of the islands (Engie and
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Klinger 2007). Thus, B. violaceus populations within the San Juan Islands may be particularly dispersal-limited
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relative to other sites within Puget Sound. As a result, populations of B. violaceus may slowly spread within areas
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of suitable habitat until reaching a habitat barrier across which the planktonic duration is too short to cross (Forrest
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et al. 2009). This could lead to the patchy distribution observed within the San Juan Islands (Fig. 1).
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Due to the limited larval dispersal of B. violaceus, its range expansion may depend more on long-distance
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dispersal events caused by transport of adults (Kinlan et al. 2005) via shipping (Lambert and Lambert 2003),
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aquaculture (Dijikstra et al. 2007) or rafting on macrophytes (Worcester 1994). It has been suggested that B.
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violaceus was originally transported from Asia to North America in the early 20 th century during a boom in trans-
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Pacific shipping (Lambert and Lambert 2003). The tunicate is thought to have been subsequently introduced into
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local sites in the Puget Sound primarily as a hitchhiker on aquaculture oysters (Fuller 2009). Man-made structures
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at aquaculture facilities and marinas provide extensive substrate for local expansion, and many invasive ascidians
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are found exclusively on these substrates (Lambert & Lambert 2003). Moreover, the mobility of many of these
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structures provides the means to transport colonies to new areas. Once at the new location, adult colonies can
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reattach to new substrates (Edland and Koehl 1998; Bullard et al. 2007) or larvae can establish new colonies nearby.
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While the short larval duration of B. violaceus limits larval dispersal to new locations, this life history characteristic
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is favorable for larval retention and may promote self-recruitment and rapid expansion in a newly established site
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(Dunstan and Bax 2007). Site-specific oceanographic features that increase the residence time within an area can
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increase larval retention and allow faster population growth rates (Dunstan and Bax 2007), which should cause the
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rate of expansion within an area to vary with location.
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Acknowledgements
We would like to thank R. Strathmann, R. Emlet, M. Jacobs, and K. Chan for invaluable advice and
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guidance as well as G. Lambert and C. Lambert for sharing their ascidian expertise. We would also like to thank the
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staff at Roche Harbor Marina, Lopez Islander Resort and Marina, Albert Jensen and Sons Boatyard, and Friday
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Harbor Labs for dock access.
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435
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436
18
437
438
439
Table legend:
440
Botrylloides violaceus at six and 21 days using caging and site as fixed effects.
Table 1: Two-way Model I ANOVA for arcsine square root transformed data of relative zooid growth of
441
442
Table 2. ANCOVA examining the effect of site on final zooid size of Botrylloides violaceus with initial size as a
443
covariate at day six and day 21.
444
445
Table 3. Two-way Model I ANOVAon the number of zooids of Botrylloides violaceus at six and 21 days using
446
caging and site as fixed effects.
447
448
Table 4. Temperature (°C) and salinity (psu) measured at all four sites included in this study; FB: Fisherman’s Bay,
449
FHL: Friday Harbor Labs, RH: Roche Harbor, JS: Jensen’s Shipyard.
450
451
19
452
453
Table 1
Source
Day 6
Cage
Site
Cage*Site
Error
Total
Day 21
Cage
Site
Cage*Site
Error
Total
454
455
DF
SS
F
p
1
3
3
49
56
0.0422
0.010
0.046
1.444
1.561
1.432
0.112
0.521
0.237
0.953
0.6698
1
3
3
49
56
0.0001
0.2886
0.0228
0.3521
0.6856
0.0122
11.476
0.9067
0.91
< 0.001*
0.45
20
456
457
Table 2
Source
Day 6
Initial size
Site
Initial size*Site
Error
Total
Day 21
Initial size
Site
Initial size*Site
Error
Total
458
459
DF
SS
F
p
1
3
3
68
75
0.0596
40.671
1.2050
141.36
188.83
0.0287
6.5213
0.1932
0.87
< 0.01*
0.90
1
3
3
93
100
183.122
9.079
17.033
252.402
493.618
67.473
1.115
2.092
< 0.001*
0.347
0.107
21
460
461
Table 3
Source
Day 6
Cage
Site
Cage*Site
Error
Total
Day 21
Cage
Site
Cage*Site
Error
Total
462
463
464
DF
SS
F
p
1
2
2
40
45
0.1975
4.8775
0.1327
19.216
24.709
0.411
5.076
0.138
0.53
0.01*
0.87
1
3
3
152
159
2.778
67.387
3.452
156.145
231.944
2.704
21.866
1.120
0.102
< 0.001*
0.343
22
465
466
Table 4
Temp. (°C)
29-Jul
31-Jul
2-Aug
29-Jul
31-Jul
2-Aug
FB
19
15.4
17
30.15
30.35
30.4
RH
12.8
13
13
29.6
29.8
30
FHL
12.25
13.5
12.3
29.75
29.45
30.55
14
12
12.1
30
29.8
30.3
JS
467
468
469
470
Salinity (psu)
23
471
472
473
Figure captions:
474
Figure 1. A map of the study sites in the San Juan Islands, Washington, USA. Filled symbols indicate sites where
475
Botrylloides violaceus colonies are present; open symbols indicate sites with no known adult colonies (data from A.
476
Cohen et al. 1998; G. Lambert, pers. comm). Stars represent study sites.
477
Figure 2. Mean relative growth (± SE) of zooids at each site after 6 d (A) and 21 d (B) where adults are currently
478
present (FB and RH) and absent (FHL and RH). A two-way factorial Model I ANOVA of relative growth rates
479
showed no significant effect of site, cage, or site*cage after 6 d. After 21 d FB showed significantly higher growth
480
rates than RH and FHL. JS was excluded form the analysis in (B) due to lack of data. Letters represent significant
481
differences among sites based on post-hoc Tukey tests.
482
483
Figure 3. Linear regression of final zooid size by initial zooid size after 6 d (A) and 21 d (B) where adults are
484
currently present (FB and RH) and absent (FHL and JS)). ANCOVA results of final zooid size (mm2) by site, with
485
initial zooid size as a covariate showed a significant effect of initial zooid size, but no effect of site on final zooid
486
size at day 6. A significant effect of site, but not of initial size, was observed after 21 days. JS was excluded from
487
(B) due to lack of data.
488
489
Figure 4. Mean number of zooids per colony (±SE) at each site after 6 d (A) and 21 d (B) where adults are currently
490
present (FB and RH) and absent (FHL and RH). A two-way factorial Model I ANOVA of number of zooids by site
491
and cage showed no effect of cage or site*cage; letters represent significant differences among sites based on post-
492
hoc Tukey tests. JS was excluded from the analysis in (B) due to lack of data.
493
494
Figure 5. Final percent survivorship of B. violaceus juveniles after 6 d (A) and 21 d (B) where adults are currently
495
present . A Fisher’s exact test showed significant differences between sites but no differences between caged and
496
uncaged plates. Letters based on significance of pairwise comparisons of pooled caged and uncaged data.
497
24
498
499
500
501
502
503
Figure 1
25
Relative zooid growth (day)-1
A
Relative zooid growth (day)-1
B
0.5
Caged
Uncaged
0.4
0.3
0.2
0.1
0.0
0.18
0.16
a
0.14
0.12
0.10
0.08
b
0.06
b
0.04
0.02
n.d.
0.00
FB
505
506
507
509
FHL
Location
504
508
RH
Figure 2
JS
26
A
14
FB
RH
FHL
JS
FB
RH
FHL
JS
12
Day 6 size (mm2)
10
8
6
4
2
0
B
10
Day 21 size (mm2)
8
6
4
2
0
0.4
0.8
1.0
1.2
Initial size (mm2)
510
511
512
513
0.6
Figure 3
1.4
1.6
1.8
27
514
515
516
Figure 4
28
517
518
Figure 5