Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

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Journal of Experimental Marine Biology and Ecology

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The importance of larval supply, larval habitat selection and post-settlement mortalityin determining intertidal adult abundance of the invasive gastropod Crepidula fornicata

Katrin Bohn ⁎, Christopher A. Richardson, Stuart R. JenkinsSchool of Ocean Sciences, Bangor University, LL595AB, Anglesey, Wales, United Kingdom

⁎ Corresponding author. Tel.: +44 1248 38 8196.E-mail addresses: Katrin.Bohn@bangor.ac.uk (K. Boh

C.A.Richardson@bangor.ac.uk (C.A. Richardson), S.Jenkin

0022-0981/$ – see front matter © 2012 Elsevier B.V. Alhttp://dx.doi.org/10.1016/j.jembe.2012.12.008

a b s t r a c t

a r t i c l e i n f o

Article history:Received 7 June 2012Received in revised form 3 December 2012Accepted 8 December 2012Available online 16 January 2013

Keywords:Crepidula fornicataLarval supplyPost-settlement mortalityRecruitmentSettlement

Understanding the processes that drive the recruitment of invasive non-native species is of critical importance inevaluating their potential to colonise previously unoccupied habitats. The slipper limpet Crepidula fornicata hasspread rapidly into most European waters since its first introduction from the North West Atlantic in the late19th century. Its invasion success is thought to have been aided by its long larval phase and its tolerance towardsa wide range of environmental conditions. The Milford Haven Waterway in Wales, U.K. supports a populationwith highly variable densities in the intertidal aswell as the subtidal zone. In the present study,we tested a seriesof existingmodels to investigate the roles of larval supply, larval habitat selection and post-settlement mortalityin determining the final distribution of C. fornicata in the intertidal zone of theMilford HavenWaterway. Duringthe main reproductive season of 2011, data on total settlement rates and recruitment were collected bydeploying slate panels for varying durations in the low intertidal zone, and data on larval abundances wereobtained by taking frequent plankton samples at two sites with contrasting adult densities: Beggars Reach(~15±13 ind m−2) and Pennar (~343±360 ind m−2). Total larval densities were much higher at Pennar,but densities of late-stage larvae (i.e. larval supply) were similar at both sites, indicating that local hydrodynam-ics may have resulted in the spatial homogenisation of supply of late-stage, metamorphically competent larvae,despite the higher larval production at the high adult abundance site. Settlement rates also did not differ betweensites. Seasonal recruitment was overall low, indicating that post-settlementmortality, likely as a consequence ofexposure to intertidal conditions, is very high. The lack of a relationship between adult abundance and settle-ment rates indicates that the final distribution of C. fornicata in the intertidal may be a result of differentialpost-settlement mortality. Understanding recruitment patterns in non-native species is essential for developingmanagement strategies for potentially harmful invaders such as C. fornicata.

© 2012 Elsevier B.V. All rights reserved.

1. Introduction

The recruitment success of invertebrates with complex life cyclesmay be determined by a variety of processes affecting the different lifecycle stages from the pelagic larval stage, through settlement and ulti-mately recruitment into the benthic population. The transition fromthe larval to the benthic stage during settlement is a critical period dur-ing which the larva must perform a number of complex behaviours,physiological adjustments and changes in morphology through meta-morphosis (Jenkins et al., 2009). It has long been recognised that thesupply of larvae is crucial to enable settlement at a site, and that levelsof larval supply may be strongly correlated with settlement rates andin some cases the distribution of adults (Carlon, 2002; Gaines et al.,1985; Grosberg, 1982; Hurlbut, 1992; Jeffery and Underwood, 2000;

n),s@bangor.ac.uk (S.R. Jenkins).

l rights reserved.

Minchinton and Scheibling, 1991). However, numerous studies alsofound that adult distribution may be determined through processesacting during and after settlement (Delany et al., 2003; Gosselin andQian, 1996; Jenkins, 2005; Jenkins and Hawkins, 2003; Jenkins et al.,2008; Kent et al., 2003; McGee and Targett, 1989; Power et al., 2006).Understanding how these processes interact in determining a species'final distribution can be highly challenging, but is nevertheless impor-tant in understanding its population dynamics.

The spread of non-native species (NNS) through natural or humanmediated vectors has received much attention in recent decades dueto their potentially severe ecological and economic impacts in the re-ceiving environments. Whilst much is known about the processes andfactors that enable successful transport, release, establishment andinitial spread of NNS in their non-native range, much less is knownabout how the species may interact with its environment once ithas become established. Studies on the processes affecting the sec-ondary spread of NNS have been under represented in research sofar. It is possible that once established, the recruitment success ofnon native species may be determined by ecological processes in asimilar manner to those operating on native species (Colautti and

133K. Bohn et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

MacIsaac, 2004; Davis et al., 2001). However, the patterns and pro-cesses that underlie the distribution of NNS need to be fully under-stood in order to predict their potential spread and impact.

The marine gastropod Crepidula fornicata (Linnaeus, 1758) is a NNSwith well established impacts on native biota, including commerciallyimportant shellfish species (Thieltges, 2005; Thouzeau et al., 2000). It isnative to the Atlantic coast of North America and has become acommon component of the coastal fauna along most European shoresshortly after itsfirst introduction in the late 19th century.C. fornicata's in-vasion success was greatly aided by its ability to colonise a wide range ofhard substrata, including shellfish species such as Crassostrea virginica,Crassostrea gigas and Mytilus edulis (Blanchard, 1997; Korringa, 1942;McMillan, 1938) and ship hulls (Cole, 1952; Cole and Baird, 1953). Fur-thermore, its relatively long larval phase of ~2–4 weeks (Pechenik,1980, 1984) suggests that larval transport is likely to be an importantmeans of dispersal in its non-native range (Adam and Leloup, 1934;Korringa, 1942; Orton, 1915).

The success of C. fornicata as an invasive species has also been in partdue to its high tolerance towards a variety of environmental conditionsand its ability to thrive in a range of habitat types (Blanchard, 1997;Loomis and VanNieuwenhuyze, 1985). Nevertheless, C. fornicata isknown to be habitat specific to some extent and although its initial es-tablishment and spread in most European coastal waters happenedwithin only ~70 years (Blanchard, 1997), its distribution and abundanceseems to be strongly controlled in some areas. It thrives best in the shal-low subtidal and low intertidal of sheltered bays, inlets or estuaries witha predominance of muddy–gravelly substrates, and it appears to be rarein the open sea (Blanchard, 1997; Loomis and VanNieuwenhuyze,1985), although Hinz et al. (2011) recorded high numbers in deep tidescoured areas of the English Channel. Although it may colonise nearlyany hard surfaces, C. fornicata is highly gregarious, and settlement hasbeen found to correlate with adult abundance (McGee and Targett,1989; Walne, 1956). Most likely this is in response to the presence ofwaterborne cues released by the adults that typically result in the forma-tion of permanent associations, commonly referred to as chains orstacks. Stack formation, and hence the ability of the larvae to detectthe presence of adults, is essential for the establishment of self-sustainingpopulations as adults reproduce through internal fertilisation.Recent studies have shown that larval supply may have a strong influ-ence on the regional distribution of C. fornicata through advection ofweakly swimming larvae away from the adult beds (Dupont et al.,2007; Rigal et al., 2010). It is likely that this may result in the transportof the larvae to areas with low adult abundance, hence minimising thechances of the larvae encountering already existing stacks for reproduc-tion and lowering the chances of the species to establish self-sustainingpopulations beyond its original location of introduction.

Although C. fornicata's relatively long pelagic larval phase togetherwith its gregarious behaviour have long been recognised as importantcharacteristics for controlling final adult distributions, the combinedimportance of these processes has not yet been studied. Field obser-vations on post-settlement processes are almost completely lacking.The focus of the present study is to investigate which processes deter-mine the final distributional patterns of the slipper limpet C. fornicatain the intertidal zone. In the first part of the study, we tested a seriesof three alternative, yet not necessarily mutually exclusive modelsdeveloped by Jenkins (2005) that aim at explaining if intertidaladult abundance of benthic invertebrates is controlled through pro-cesses acting prior to (larval supply), during (larval habitat selection)or after settlement (post-settlement mortality). Firstly, the distribu-tion of adults may be controlled at settlement as a result of differen-tial larval supply. For example, local and regional hydrographicregimes may cause the differential supply of propagules to settlementsites (Gaines et al., 1985; Grosberg, 1982; Jeffery and Underwood,2000; Minchinton and Scheibling, 1991). The first model hence pre-dicts that the magnitude of larval supply will be reflected in theresulting settlement densities, and be correlated with final adult

densities observed at the sites. Species may develop behaviour tocounteract such passive larval transport. For example, the larvae ofmany species are able to actively choose a site for settlement(Jenkins, 2005; Kent et al., 2003; McGee and Targett, 1989), thusavoiding habitats that may be unsuitable for later growth and surviv-al. A second model therefore predicts that the distribution of adults isdetermined at settlement, but as a result of larval choice instead oflarval supply. In the case of gregarious species, one would expect tofind higher settlement rates at sites with higher adult densities.Thirdly, adult distributional patterns may be determined after settle-ment has taken place, due to a variety of biological or physical factorscausing differential post-settlement mortality (Delany et al., 2003;Gosselin and Qian, 1996; Power et al., 2006). A third model thereforepredicts that settlement rates and adult abundance are not correlated,due to differing levels of post-settlement mortality between sites(Jenkins, 2005).

To test these models, we estimated the supply of late-stage larvaeas well as rates of settlement during the main larval and settlementseason (June/July–September, Bohn et al., 2012) at two study siteswith contrasting densities of adult C. fornicata. Previous work sug-gests that the supply of late-stage larvae and subsequent settlementrates do not differ between these two study sites, despite a differencein adult densities (Bohn et al., 2012). However, those data were col-lected at monthly intervals and did not allow for a distinction be-tween processes acting at various temporal scales. We thereforeinstigated a sampling programme during the main reproductive sea-son of C. fornicata during 2011 that involved frequent collection ofdata on larval supply, settlement rates and levels of successful recruit-ment within one season, to estimate the importance of these process-es in determining adult abundance more accurately. In a second partof the study, we quantified post-settlement mortality during the sum-mer months, a process that might be highly important in controllingadult C. fornicata abundance in intertidal conditions.

2. Methods

2.1. Study sites

Field experiments and collection of C. fornicata broodstock for juve-nile rearing were undertaken in the Milford Haven Waterway (MHW)in South West Wales, UK (Fig. 1). This natural ria holds the northern-most self-sustaining established population of C. fornicata since thefirst appearance of the species in Welsh coastal waters in the 1950s(Cole and Baird, 1953; Crothers, 1966) (Bohn unpubl.). The MHW ischaracterised by a rocky shore environment interspersed with muddyand gravelly banks (Nelson-Smith, 1965). Tides are semi-diurnal witha maximum range of nearly 8 m during spring tides. Low water duringspring tides occurs aroundmidday andmidnight, resulting in the expo-sure of the lower-shore flora and fauna to contrasting air temperatureextremes. All work was carried out along ~50 m transects running par-allel to the shore line at a tidal height of ~1.2 m or ~1.8 m above ChartDatum at two sites within the MHW, Pennar (PE) and Beggars Reach(BR) (Fig. 1). Both sites are situated in the area of the MHW whereC. fornicata is well established andwere chosen to cover the extremesof C. fornicata density at that tidal height in the MHW (mean±SD atBR: ~15±13 ind m−2, at PE: ~343±360 ind m−2, Bohn unpubl.).Freshwater input in theMHW is low (Nelson-Smith, 1965). At PE, salin-ities rarely fall below 30 psu and vertical mixing of salt and fresh watermasses is nearly complete. At BR, located in the upper stretches of theria, stratification of water masses may result in a maximum salinity dif-ference of 8 between surface and bottom waters. Surface salinity heremay be more variable than at PE, however marine conditions prevailuntil the upper stretches of theMHW, resulting in a diverse assemblageofmarine species in the intertidal zone (Nelson-Smith, 1965, 1967). Thelow intertidal zone of both study sites is characterised by a mix ofsoft-sediments and gravel. Hard substrata (i.e. stones with grain size

Fig. 1. Map of the study sites Beggars Reach (BR) and Pennar (PE) in the Milford Haven Waterway (MHW) in the south west of the United Kingdom (top left).

134 K. Bohn et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

of ~16–256 mm and shells) are more common at PE where ~45% of theintertidal seabed is covered by this substrata class, whilst only ~27% ofthe surface is covered by hard substrata at BR (Bohn unpubl.).

2.2. Field sampling to determine the processes controlling adult distribution

2.2.1. Larval supplyFrequent plankton samplingwas undertaken between June and Sep-

tember 2011 to estimate the densities of late-stage, metamorphicallycompetent larvae supplied to the intertidal zone at both study sites.This aimed at evaluating the role of larval supply in determining settle-ment rates and subsequent adult distributional patterns. Thirteen plank-ton samples were taken throughout the peak reproductive periodat ~weekly intervals from each shore by wading through chest-deepwater and towing a 250 μm plankton hand-net (diameter 35 cm)for ~2.5 min. A standard mechanical flowmeter (model 2030, GeneralOceanics Inc.) attached to the aperture of the net recorded that 1.3–5.0 m3 of seawater were filtered during each tow. All samples weretaken during the flood tide, up to 1.5 h after low water from the bodyof water above the transects where settlement panels were deployed(see below). All samples were preserved in 70% ethanol within 2 h ofcollection. They were analysed for total densities of C. fornicata larvae(i.e. counting all larvae across the whole size range), as well as for den-sities of only the late-stage, metamorphically competent larvae, using adissectingmicroscopewith anocularmicrometre (×63). Competent lar-vae were defined as those >650 μmwith morphological characteristicsthat indicate metamorphic competency (flattened shell geometry andpresence of a “brimmed shell”, see Pechenik, 1980; Pechenik and Lima,1984). Densities were calculated as numbers of larvae m−3 using esti-mates of the number of larvae and the flowmeter readings.

2.2.2. Settlement rates and seasonal recruitmentSpatfall densities accumulating over varying time periods (2 weeks,

1 month, 3 months) were monitored at 1.2 m above C.D. at both studysites between June and September 2011. This aimed at estimating themagnitude of total settlement rates and subsequent levels of successfulrecruitment over the reproductive season at sites with contrastingadult densities. Artificial settlement substrata made out of ordinaryroofing slate were used to enable the fast collection and subsequentmeasurements of settlement rates in the laboratory. The reliable estima-tion of settlement densities on natural substratawas found to be difficultdue to the prevalence of soft sediments amongst the mud–gravel sub-strata and the small minimum size of the early settlers (~0.7 mm, seePechenik, 1980). Previous work has demonstrated that spat densitieson slate panels equal those on natural substrata under intertidal condi-tions (Bohn et al., 2012). Panels were first deployed in the middle ofJune 2011, and changed at three different intervals until mid September2011. One set of twelve panels was deployed for two weeks at a time(“biweekly panels”), after which they were collected and replaced with

clean slate panels. This was repeated at biweekly intervals until thepanels were finally removed in mid September. Another set of twelvepanels was also deployed in mid June, and replaced with clean slatepanels each month until the end of the experiment (“monthly panels”).A third set of 12 panels was deployed for three months, i.e. the wholeduration of the experiment from mid June to mid September 2011(“seasonal panels”). Slate panels were cut to a size of 11 cm×11 cmand securely fastened to the intertidal zone by attaching them to metalframes (50 cm×50 cm). Each frame had four panels attached using asingle screw andwing nut per panel, andwas anchored into the soft sed-iments with four 30 cm rods. Each frame held at least one panel of eachof the three deployment durations. At the end of the deployment period,panels were transported to the laboratory by suspending them horizon-tally on metal stakes that rested in cooling boxes. Panels were separatedwith PVC rings (diameter 2 cm, height 1.5 cm) to avoid damage to thesettled spat on either side of the panel during transport. All C. fornicatapresent on both sides of each panel were counted and measured undera dissecting microscope with an ocular micrometre (×63) in thelaboratory.

Previous work has shown that the collection of monthly settlementrates may result in an underestimation of total settlement rates overthe deployment periods (Bohn et al., 2012). This is likely due to thehigh mortality of the newly settled spat during exposure to air duringspring low tide emersion or due to movement of the juveniles off thepanels onto the surrounding substrata some point after settlement. Inthe present study, biweekly settlement rates over the settlement seasonwere used as the best estimation of total settlement. Sampling wasundertaken on the last days of neap tides, i.e. before the settlementsubstrata were exposed to air at the beginning of a spring tide week.This minimised the aerial exposure of new settlers to a maximum ofthree days during spring low tide conditions immediately after deploy-ment. More frequent sampling of settlement panels was not possibledue to the limited accessibility of the low shore transect during neaptides. The collection of settlement densities on substrata that had beendeployed for longer time periods then allowed the comparison of totalsettlement rates (biweekly panels) with the magnitude of benthicrecruitment occurring within one month or the whole season.

2.3. Field monitoring of post-settlement mortality

To quantify the mortality of juvenile C. fornicata that occurred fol-lowing initial settlement, a known number of young juveniles weretransplanted into the low intertidal zone (~1.8 m above C.D.) at BR inJuly 2011. A tidal height of 1.8 m was chosen instead of 1.2 m to maxi-mise accessibility for frequent monitoring of juvenile survivorship. Toovercome the difficulty of finding and maintaining a sufficient numberof newly settled, and therefore very small, C. fornicata in the field, juve-niles were reared in the laboratory. Adult C. fornicata broodstock werecollected in March 2011 from the low intertidal zone in the MHW.

135K. Bohn et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

Adult stacks (~50 stacks per 25 L aquaria) were kept in constantlyflowing seawater in aerated, 120 μm filtered seawater at ~15–16 °Cand fed a diet of Isochrysis galbana (clone T-ISO). Larvae were collectedfrom the adult tanks within 12 h following release and transferred to1 μm-UV-filtered seawater at ~19–21 °C. Larvae were reared at densi-ties of ~0.25–0.5 larvae mL−1, and fed a diet of I. galbana (cloneT-ISO, cell density ~1.8∗105 cells mL−1). Seawater was changed andat the same time food added every other day. All larvae from a singlebatch were released on the same day, but not necessarily from thesame female. Larval growth was monitored to estimate larval develop-ment of each batch (data not shown). Once the larvae reached an aver-age size of 650 μm, they were either exposed to an artificial inducer(20 mM K+enriched filtered sea water, see Pechenik and Heyman,1987) or natural substrata (biofilmed shell fragments or stones) for6 h or 24 h to inducemetamorphosis. The newlymetamorphosed juve-nileswere kept under the same conditions as the larvae, but in ~150 mLcontainers at densities of b0.5 juvenile mL−1. Once juveniles attained asize>3 mm, theywere transplanted onto slate panels identical to thoseused to monitor settlement in the field. Using a glass pipette, sevenjuveniles from different batches were lifted onto each panel wherethey were left to re-position and re-attach. Prior to transplantationinto the field, the colonised panels were suspended horizontally in25 L tanks to minimise movement of the juveniles off the panels,and were kept under the same laboratory conditions as the adults.Juveniles were checked several times before transplantation into thefield, and any dead, damaged ormissing juveniles were replaced imme-diately. When the panels were deployed in the field, all the juvenileswere ~7–10 wk old, and 4–8 mm in size.

In July 2011, 15 panels each with 7 juveniles attached weretransported to the MHW in racks that were suspended in coolingboxes filled with aerated 1 μm- and UV-filtered seawater. Panels wereinstalled in the field on five metal bars (three panels per bar) thatwere anchored in the soft sediment ~2 m apart from each other. Tominimise the possibility of movement of the juveniles off the panelsonto the surrounding substrata, each panel was suspended on thebars by resting it on a PVC ring (diameter 2 cm, height 2.5 cm). Of the15 panels nine were caged to exclude potential predators of juveniles.Cages (14 cm×14 cm×10 cm in size; width×length×height) wereconstructed of plastic mesh (5 mm×5 mmmesh size). The experimentwas deployed during lowwater on 7th July 2011, the last day when the1.8 m tidal height was accessible during the spring tide that week.Panels were first sampled 5 days later on 12th July, when they wereuncovered for the first time again during the following spring tide.Thereafter, panels were visited at least once at the beginning and oncetowards the end of each spring tide period for ten weeks until the ex-periment ended in mid September. During each visit the cages wereopened carefully and the number of live juveniles attached to eitherside of the panels was noted in both treatments.

2.4. Statistical analyses

Most data were analysed using two-factorial ANOVA or the equiva-lent non-parametric Scheirer–Ray–Hare test if data transformationscould not establish homogeneous variances or normality (Dytham,2011). All data were tested for homogeneity of variances and normalityusing Levene's test and the Kolmogorov–Smirnov test, respectively.Total settlement rates, estimated by measuring biweekly settlementrates, were compared between study sites over the season using thetwo-factorial Scheirer–Ray–Hare test, using study site and samplingevents as factors. Spat densities recorded on biweekly,monthly and sea-sonal panels were compared to distinguish total settlement rates fromlevels of recruitment over varying time periods at both shores for eachsampling event separately (factors panel deployment duration andsite). For this, the two-factorial Scheirer–Ray–Hare test was used tocompare biweekly and monthly settlement rates recorded during themid July sampling event, and a two-factorial ANOVA for data recorded

during the mid-August sampling event. At the end of the experimentin mid September, when seasonal panels were sampled in addition,the Scheirer–Ray–Hare testwas used to compare data from all three de-ployment durations. One-way ANOVAwas used to compare total larvaldensities as well as densities of late-stage larvae between study sites.Calculated average densities obtained from duplicate samples for eachsampling event were used as replicates; comparisons between sam-pling events at the two study sites were not made as samples of bothstudy sites had to be taken on different days and sometimes at differentintervals. Total larval densities were log10 transformed to achievehomogeneity in variances. The log-rank test (Machin et al., 2006) wasused to compare differences in survivorship between caged and notcaged panel treatments in the field study on EPSMmonitoring.

3. Results

3.1. The processes controlling adult distribution

3.1.1. Larval supply and rates of settlementWhilst total larval densities were higher at the high adult abundance

site PE compared to the low adult abundance shore BR (One-wayANOVA on log10-transformed data, F1=24.57, pb0.001, Fig. 2a), densi-ties of late-stage larvae (i.e. larval supply) did not differ between the lowand high adult abundance sites (One-way ANOVA, F1=0.02, p=0.88,Fig. 2b). Late-stage, metamorphically competent larvae were rarethroughout the study and densities never exceeded 0.8±1.1 ind m−3

(mean±SD) (Fig. 2b). Two peaks in total larval density were observed,one at the beginning of July at PE and a secondpeak at both PE and BR ap-proximately fourweeks later towards the end of July (Fig. 2a). Both peaksin larvae were followed by peaks in settlement with a lag phase of ap-proximately fourweeks (Fig. 2c). Biweekly settlement rates varied great-ly throughout the season at both locations. Differences between the twostudy sites were not consistent throughout the season (interaction site xsampling event: pb0.001, Table 1).

3.1.2. Benthic recruitmentSpat densities recorded at the low adult abundance site (BR) and

the high adult abundance site (PE) differed significantly on each sam-pling occasion (factor site: pb0.05 for each sampling event, Table 2),irrespective of the duration of panel deployment (interaction site xduration: p>0.05 for each sampling event, Table 2). However differ-ences were not consistent throughout the season and spat densitieswere highest at BR in mid July and mid September, and at PE in midAugust (Table 2, Fig. 3a, b and c). Levels of recruitment were not sig-nificantly different amongst the different deployment durations(Table 2, Fig. 3a, b and c), whether comparisons were made betweentwo-week and one-month deployments in mid July and mid August,or between two-week, one-month and three-month deployments inmid September. This indicates that spat had not accumulated duringpanel deployment and that successful seasonal recruitment into theadult populations was negligible on both shores. This is supportedby the fact that the majority of the juveniles were small (b1650 μm,Fig. 4a and b), suggesting that they were young and had settled re-cently. Juveniles on panels deployed for various durations were ofsimilar sizes, suggesting that they were approximately the same age.

3.2. Post-settlement mortality during summer

Mortality of young C. fornicata spat during the summer of 2011was high and differed significantly between the caged and un-cagedtreatments (log-rank test, χ2=40.48, pb0.001). Almost no mortalityoccurred in both treatments during the first five days following paneldeployment (Fig. 5). This time corresponds with periods of neap tideconditions when the panels were continuously immersed. The major-ity of spat mortality occurred in the following three days, when thepanels first became emersed during low water of spring tides. By

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Fig. 2. Crepidula fornicata larval production (a), larval supply (b), and rates of settle-ment (c) in the intertidal zone (~1.2 m above C.D.) at Beggars Reach, a site with lowabundance of adult C. fornicata, and Pennar, a site with high adult abundance, in theMilford Haven Waterway between mid June and mid September 2011. Mean±SD.(a) Total larval densities as a measure of larval production were estimated from dupli-cate plankton samples, (b) Densities of late-stage, metamorphically competent larvaewere estimated from duplicate plankton samples, and (c) Biweekly settlement rateswere estimated from the deployment of 12 slate settlement panels.

136 K. Bohn et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

day 8, mortality was 100% on all uncaged panels, but only 41±28% onthe caged panels (mean±SD). Mortality of the caged juveniles in-creased steadily until it reached 78±18% at the end of the experi-ment. The largest increases in the percentages of dead juvenileswere usually observed during spring tide emersion.

Table 1Results of non-parametric 2-factorial Scheirer–Ray–Hare test on differences in biweeklysettlement densities at Pennar and Beggars Reach in the Milford Haven Waterway at 6sampling events during the settlement season of 2011. Densities were recorded on panelsdeployed for two weeks at Pennar and Beggars Reach. SS: Sum of squares, MS: Meansquares, df: degrees of freedom. Significant differences are shown in bold (pb0.05).

SS SS/MStotal df p

Site 60.06 4.57 1 0.03Sampling event 954.95 72.66 5 b0.01Site×sampling event 232.23 17.67 5 b0.01Residual – –

4. Discussion

In this study, we have simultaneously examined the importance oflarval supply, larval settlement and post larval settlement processes incontrolling successful recruitment of the American slipper limpetC. fornicata into the intertidal in the Milford Haven Waterway (MHW).Results demonstrate that adult abundance and distributionmay primar-ily be determined following settlement, rather than through larval sup-ply or settlement behaviour. At two sites with highly contrasting adultabundances in the low intertidal zone no obvious differences in thelevels of supply of late-stage larvae to the sites were found, and differ-ences in estimates of total settlement rates between both sites werenot consistent throughout the season. Post-settlement mortality wasvery high, resulting in negligible recruitment into the adult populationwithin one season. However, we did not observe any consistent differ-ences in mortality between both sites, which would have been neces-sary to unambiguously prove that differing levels of post-settlementmortality caused the observed differential patterns in adult abundance.Nevertheless, results from the present study clearly show that this pro-cess can be critical in limiting recruitment success in the intertidal and isa likely cause for the observed differential adult distribution.

Pelagic larval dispersal is a common mechanism through whichmarine non-native species disperse. This was early recognised as animportant vector for the spread of C. fornicata into European waters.For example, Orton (1915) mentioned that “Crepidula furnishes anexcellent example of the efficacy of a free-swimming larva inextending the domain of a sea-dwelling animal”. More recently how-ever it has been shown that the importance of larval supply in deter-mining the final distribution and spread of C. fornicata may greatlydepend on local and regional hydrodynamics, geographical featuresof the area, and the location of adult spawning grounds (Dupont etal., 2007; Rigal et al., 2010; Viard et al., 2006). We found that at thetwo sites monitored, larval supply was not important in determiningthe final distribution of C. fornicata in the intertidal zone. Althoughtotal larval densities were much higher at the high adult abundanceshore (PE), densities of large, late-stage larvae (i.e. larval supply)did not differ between the high and low adult abundance sites. Thissuggests that larval productionmight be higher at the site with higheradult densities (PE), but that the distribution of late-stage larvae maybe independent of the location of original larval release as a result oflarval export away from the adult beds. The majority of the larvae col-lected were small and hence likely sampled shortly after release inclose proximity to their parental stacks. This is in accordance withdata recorded in 2010 from the same sites (Bohn et al., 2012). Rigalet al. (2010) also found that metamorphically competent larvaewere rare, but when they were present they occurred at sites furthestaway from the adult beds, most likely due to the export of larvae intothe open ocean. Similarly, transport of larvae via the strong tidal andcurrent regime in the MHW (Nelson-Smith, 1965) may have causedthe spatial homogenisation of the pool of late-stage larvae in thepresent study, much as Jenkins (2005) observed in barnacle larvaeover similar scales. However, it is important to note that densities oflate-stage larvae were generally very low. Differential supply mayhave thus been difficult to detect.

Both peaks in total larval abundance (likely originating from peaksin larval production) were followed by peaks in settlement densitiesby approximately four weeks, corresponding well with the expectedduration of the larval phase from release to settlement (Conklin,1897; Pechenik, 1980, 1984; Werner, 1955). This indicates that thetiming of larval release may to some extent control settlement.Despite this potential relationship between the larval phase andsettlement, data of the present study suggest that neither larval sup-ply nor habitat selection explain the observed difference in adultabundance between sites, implying that the final distribution ofC. fornicata is determined following settlement of the larvae. No rela-tionship between late-stage larval densities and adult abundance was

Table 2Results of non-parametric 2-factorial Scheirer–Ray–Hare test on spat densities observed in mid July and mid September, and a parametric 2-way crossed ANOVA on spat densitiesrecorded in mid August. Densities were recorded on panels deployed for two weeks or one month (mid July and mid August) and two weeks, one month or three months (midSeptember) at Pennar and Beggars Reach. SS: Sum of squares, MS: Mean squares, df: degrees of freedom. Significant differences are shown in bold (pb0.05)

Mid July Mid August Mid September

SS SS/MStotal df p df MS F p SS SS/MStotal df p

Site 7.52 10.40 1 b0.001 1 370107.92 12.33 0.001 82.35 24.38 1 b0.001Duration 0.19 0.26 1 0.610 1 3557.36 0.12 0.732 7.58 2.25 2 0.325Site×duration 0.19 0.26 1 0.610 1 32016.26 1.07 0.307 1.03 0.30 2 0.859Residual – – 44 30024.13 – –

137K. Bohn et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

found, which is contrary to what might have been expected for a gre-garious species such as C. fornicata: it is possible that active larvalhabitat selection prior to settlement results in the aggregation oflarge, late-stage larvae above adult beds. Similarly, no consistent dif-ferences in settlement rates were observed between sites where adultabundances differed. The lack of a relationship between settlementrates and adult abundance is especially surprising as gregarioussettlement of C. fornicata has previously been observed in the field

0

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nile

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b)

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Fig. 3. Total settlement, monthly and seasonal recruitment of Crepidula fornicata in the inteSeptember 2011. Densities of juveniles b5250 μm found on artificial settlement substrata armid August) and (c) the third month (mid August–mid September) of the experiment. Settbiweekly intervals (black bars, sampled a total of 6 times during the experiment), changed ator only sampled once after three months at the end of the experiment (i.e. seasonally, dark gto the maximum size of juvenile slipper limpets found on any of the panels. N.B.: No settle

(McGee and Targett, 1989; Walne, 1956). Also, waterborne cues re-leased by conspecifics were found to successfully induce metamor-phosis of competent C. fornicata larvae (McGee and Targett, 1989;Pechenik, 1980; Pechenik and Heyman, 1987), which inevitably initi-ates settlement due to the associated loss of the velum, the swimmingorgan (Werner, 1955). We had postulated that the high adult densi-ties at PE should have more readily resulted in the induction of meta-morphosis and hence settlement of C. fornicata larvae compared to

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rtidal of Beggars Reach and Pennar in the Milford Haven Waterway between June ande shown for (a) the first month (mid June–mid July), (b) the second month (mid July–lement substrata were first deployed in the middle of June and then either changed atmonthly intervals (light grey bars, sampled mid July, mid August, and mid September),rey bars, sampled mid September). 5250 μmwas used as a size limit as this correspondsrs were observed at Pennar at the end of June. Mean±SD, n=12.

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biweekly (n=1)

monthly (n=1)

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biweekly (n=41)monthly (n=35)seasonal (n=31)

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biweekly (n=15)monthly (n=5)seasonal (n=9)

Fig. 4. Seasonal variation in size frequencies (%) of Crepidula fornicata juveniles b5250 μm on artificial settlement substrata at (a) Beggars Reach and (b) Pennar in the Milford HavenWaterway during summer 2011. Substrata were first deployed in mid June, and either sampled every two weeks (black bars), once a month (light grey bars) or after three months(dark grey bars) on the stated dates. Size frequencies from first collection (end of June) are not shown due to the very low numbers of juveniles present on the panels (BeggarsReach n=2, b1200 μm; Pennar absent).

138 K. Bohn et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

the location at BR where adult numbers are low and settlement cuesare likely to be scarce.

It is likely that differences in settlement rates between sites had beennullified by factors causing early post-settlement mortality (EPSM).EPSM is known to be high for most benthic invertebrates and maygreatly limit the distribution and abundance of species, especially inthe intertidal zone where environmental conditions fluctuate exten-sively over very short time periods (Gosselin and Qian, 1997; Huntand Scheibling, 1997). Settlement densities recorded in the presentstudy suggest that this may be the case for C. fornicata in the intertidal

zone of the MHW. No differences were found between densities andsize frequencies of spat onpanels thatwere deployed for varying lengthsof time at either of the study sites. Even at the end of the experimentbiweekly and seasonal spat densities did not differ, indicating that spathadnot accumulated on panels over the threemonths of the experimen-tal study. Large juveniles were rare, even on the seasonal panels. It istherefore more likely that spat had settled less than two weeks priorto sampling, independent of panel deployment duration. Further evi-dence for high EPSM acting on C. fornicata under intertidal conditionscomes from observations from the transplantation experiment where

0

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0 5 10 15 20 25 30 35 40 45 50 55 60 65 70

Mor

talit

y (%

)

Time after Installation (days)

CagedNot Caged

Fig. 5.Mortality (%) of juvenile Crepidula fornicata at Beggars Reach during summer 2011.Fifteen slate panels with 7 laboratory-reared juveniles attached were transplanted intothe low intertidal (~1.8 m above C.D.) in July 2011. Panels were either caged (n=9) oruncaged (n=6). The number of live juveniles found attached to each panel was recordedat least twice during each spring low tide until the end of the experiment on day 71. Datapoints are average mortality (%) calculated for each sampling event and treatment(mean±SD). Arrows show the periods of spring tides when juveniles were emersedtwice a day.

139K. Bohn et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

mortality of the juveniles was as high as 100% after emersion at lowwater for only three days. Almost no mortality occurred during neaptides, for example between days 1 and 5 of the experiment when thecolonised panels remained continuously immersed (Fig. 5). This alsosuggests that the settlement densities recorded from biweeklymonitor-ing of the panels are a good estimator of total settlement rates occurringat the site, as samplingwas always undertaken before exposure to inter-tidal conditions during the following spring tide, and therefore beforeexposure to potentially high levels of EPSM. Panels that were deployedfor one or even threemonths howeverwere exposed to intertidal condi-tions repeatedly for several days during spring tides. It is hence not sur-prising that spat did not accumulate on the monthly or seasonal panels.It is possible that patterns of juvenile distribution that were establishedat settlement have been affected by factors causing EPSM. Movement ofthe juveniles off the panels onto the surrounding substrates could alsoexplain why we did not record more, and larger, juveniles on panelsthatwere deployed for longer periods of time. However, successful juve-nile movement is likely to depend on the availability of hard substrata,which are rare at the predominantly soft-sedimentary study sites. It isthus unlikely that post-settlement movement caused the low juveniledensities observed at both sites. Also, previous work undertaken at thesame sites found no indication of an accumulation of juveniles on natu-ral substrata compared to slate panels over the duration of a month(Bohn et al., 2012).

Common causes of EPSM include biological factors such as preda-tion, competition for space and food, diseases and parasites, as well asabiotic factors such as desiccation and strongly fluctuating temperatureor salinity regimes (see Gosselin and Qian, 1997; Hunt and Scheibling,1997 for review). It is unclear the extent to which biotic factors areimportant in limiting the recruitment success of C. fornicata in the inter-tidal but it may be low. Field evidence for predation on juveniles isscarce, at least over its native range (Pechenik et al., 2010). Densitydependent factors are also unlikely limiting. The attachment to conge-ners during stack formation minimises intraspecific competition forspace and its generalist feeding behaviour with a dual feeding modeduring the juvenile stage enables feeding on a variety of food sources(Beninger et al., 2007; Decottignies et al., 2007; Pechenik et al., 1996).It is more likely that abiotic factors caused the high EPSM and resultedin the low recruitment observed in the intertidal of theMHW. The estu-arine conditions in theMHWmay present a highly stressful habitat dueto variable salinity regimes as a result of tidal and weather conditions(Nelson-Smith, 1965) which, especially when combined with tempera-ture stress may be a considerable threat to newly settled juveniles(Gosselin and Qian, 1997). Air temperatures may fluctuate by >22 °Cwithin less than 24 h in the low intertidal zone of the MHW duringspring tides (Bohn et al., 2012) as low water of spring tides in theMHW occurs during midday to early afternoon and midnight to early

morning. This may potentially make the MHW a “hot spot”, i.e. anareawhere the body temperatures of intertidal organismsmay beunex-pectedly high for their latitudinal distribution, hence increasing the riskfor increased physiological stress and juvenilemortality (Helmuth et al.,2006; Orton, 1915).

In considering the role of biotic or abiotic factors in determining thefinal distribution of C. fornicata, the results of the caging experiment areinformative. Our findings suggest that predation can be excluded as themain cause for juvenilemortality. Although this experimentwas lackingthe necessary procedural control treatments to unambiguously investi-gate the effects of predation on juvenile distribution, we found thatalmost no mortality occurred between days 1 and 5, i.e. during thefirst neap tide. This was the case even on the un-caged panels. If preda-tion howeverwas themain cause for EPSM, this should have resulted injuvenile mortality in this treatment from the start of the experiment.Differences in survivorship between caged and un-caged treatmentsonly became apparent after day 5 when survival was considerablyhigher in the caged panels. This is likely due to a microhabitat-effectof the cages, which may attenuate the effects of abiotic stressors, espe-cially by providing shading or protection from other physical distur-bances (Gosselin and Chia, 1995). This was not available on the bare,horizontal slate panels. Nevertheless, EPSM was generally high, also inthe caged treatments, suggesting that despite the provision of a micro-habitat, recruitment into the adult population remains low in the inter-tidal. Monthly settlement estimates recorded in 2010 at the same sitesindicate that spat densities on slate panels equal those on natural sub-strata (Bohn et al., 2012) offering a variety of microhabitat structures,indicating that EPSM may result in low recruitment to the intertidalirrespective of substratum type.

We expected to find that the differing distribution of adults at thetwo sites in the MHW had resulted from varying levels of larval sup-ply, the avoidance of larvae of certain habitat sites, or a combinationof both. Results of this study clearly show that C. fornicata is highlysusceptible to intertidal conditions during the juvenile phase andthat post-settlement mortality may be the prime limiting factor forthe recruitment success of C. fornicata in the intertidal zone. To unam-biguously prove that EPSM caused the observed differential adult pat-tern in the intertidal zone of the MHW, a difference in levels ofpost-settlement mortality between both study sites needed to be ev-ident. This was not the case, instead settlement rates were identical atboth sites, and seasonal recruitment was in fact negligible. The highEPSM and very low seasonal recruitment likely masked the varyinglevels of mortality factors responsible for the final distribution ofC. fornicata in the intertidal zone. It is likely that higher percentagesurface cover of hard substrata at PE (~45% gravel and shell) com-pared to BR (27%) result in higher levels of benthic recruitment dueto the provision of more microhabitat structures in the otherwisesoft-sedimentary habitats. A previous study from the same sitesshowed that the season during which settlement, and hence benthicrecruitment takes place may be shorter by several weeks than the pe-riod during which larvae are present in the water, potentially limitingits spread in the area (Bohn et al., 2012). This shows the importanceof including settlement and post-settlement processes into studiesthat are aimed at understanding the distribution and potential spreadof C. fornicata. Also, the environmental tolerances of C. fornicata havenot been sufficiently studied, especially with regards to the juvenilestage. Future research should be directed at investigating the process-es affecting the various life cycle stages of C. fornicata in both the in-tertidal as well as the subtidal zone to allow accurate predictionsabout its potential spread and impacts in its non-native range.

Acknowledgements

This study was funded by the Countryside Council for Wales andthe Bangor Mussel Producers Ltd via a PhD studentship to KB andwe thank both organisations for their assistance and advice during

140 K. Bohn et al. / Journal of Experimental Marine Biology and Ecology 440 (2013) 132–140

the study. We thank Ben Harvey and Jorge Dominguez for their assis-tance in the field. [ST]

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