Fertilization success and recruitment of dioecious and hermaphroditic fucoid seaweeds with contrasting distributions near their southern limit

MARINE ECOLOGY PROGRESS SERIESMar Ecol Prog Ser

Vol. 262: 173–183, 2003 Published November 7

INTRODUCTION

Marine species with external fertilization rely onthe bulk mixing of water to bring male and femalegametes together. If mixing is turbulent, gamete dilu-tion may occur rapidly, therefore reducing the proba-bility of egg fertilization (Denny 1988). Modelingapproaches and experimental field data (with inducedspawning) show that fertilization success of organismswith external fertilization is greatly reduced withincreasing water motion (e.g. Pennington 1985, Denny& Shibata 1989, Levitan et al. 1992, Oliver & Babcock1992). However, in dioecious organisms with separate

male and female individuals, some mixing may benecessary for gamete encounter and fertilization(Denny & Shibata 1989). Because of the greater proba-bility of gamete encounters when self-fertilization ispossible, both monoecious species, in which the maleand female gametes are released from different repro-ductive structures in the same individual, and her-maphroditic species, where eggs and sperm occur inthe same reproductive structure, may be at an advan-tage over dioecious species under turbulent conditions.Regardless of reproductive mode, wave swept organ-isms that reproduce via external fertilization oftenhave mechanisms to increase the probability of gamete

© Inter-Research 2003 · www.int-res.com*Corresponding author. Email: [email protected]

Fertilization success and recruitment of dioeciousand hermaphroditic fucoid seaweeds with

contrasting distributions near their southern limit

Lydia Ladah1, 2, Rafael Bermudez1, Gareth Pearson1, Ester Serrão1,*

1Centro de Ciências do Mar, FCMA, Universidade do Algarve, Campus de Gambelas, 8005-139 Faro, Portugal2Present address: Dept. de Oceanografía Biológica, CICESE, Apdo Postal 2732, Ensenada, Baja California CP22800, Mexico

ABSTRACT: Near its southern limit in the Northeastern Atlantic, the dioecious brown alga Fucusvesiculosus is absent from the exposed coast yet it is abundant in estuaries and coastal lagoons. Incontrast, the phylogenetically and ecologically related hermaphroditic species F. spiralis occurs alongthe open coast, though often in low abundance. We hypothesized that the absence of F. vesiculosusfrom exposed shores near its southern limit was due to reduced external fertilization success, as itsgametes may be diluted beyond the level required for successful fertilization, in contrast with its her-maphroditic, self-compatible congener. To test this hypothesis, individuals of both species weretransplanted to 3 exposed sites near their southern limit in the Northeastern Atlantic. Egg settlementand fertilization success (% of eggs fertilized) were evaluated daily during the main reproductiveseason. Recruitment was evaluated at the end of the reproductive season, and recruit mortality wasevaluated using outplants of laboratory-cultured embryos. On the exposed shores near their southernlimit, transplanted adults of both species survived and released eggs, and fertilization success wasunexpectedly high. However, recruitment and recruit survivorship of F. vesiculosus was significantlylower than F. spiralis. Our results suggest that F. vesiculosus is restricted to low water-motion envi-ronments because of recruitment failure and recruit mortality on exposed bare shores near its south-ern limit, and not because of inability to fertilize eggs in turbulent environments. This study does notsupport our hypothesis of a role for dioecy/hermaphroditism in explaining the distribution of exter-nally fertilizing marine organisms in high water-motion environments.

KEY WORDS: Reproductive ecology · Mating system · Gamete release · Post-settlement mortality ·Fucus · recruitment · External fertilization · Brown algae · Distributional limits

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encounters, such as synchronous spawning, release ofgametes under optimal conditions for encounters, mor-phological and physiological adaptations, and chemi-cal cues for gamete location (e.g. reviews by Giese &Kanatani 1987, Brawley & Johnson 1992, Levitan 1995,Levitan & Petersen 1995, Brawley et al. 1999, Yund2000, Santelices 2002).

Fucus spp. reproduce exclusively by external fertil-ization, with gamete release occurring synchronouslyduring calm periods and during the daytime (Pearson& Brawley 1996, Serrão et al. 1996). Fucoids use awater-motion sensing mechanism (Pearson & Brawley1998, Pearson et al. 1998) to minimize the negativeeffects of gamete dilution by restricting gamete releaseto calmer periods during the reproductive season.Gamete release often occurs on days with lower watermotion for subtidal populations (Serrão et al. 1996,1999a), during low-tide periods for tide-pool popula-tions when they would be isolated from waves (Pear-son & Brawley 1996), and at slack high-tide in inter-tidal populations (Berndt et al. 2002). Gamete releaseis restricted to daytime since photosynthetic signals arerequired for gamete release (Serrão et al. 1996b, Pear-son et al. 1998). In all natural fucoid populations stud-ied to date, fertilization success has been very high(Brawley 1992, Pearson & Brawley 1996, Serrão et al.1996, Berndt et al. 2002), except near the northern limitof distribution of Fucus vesiculosus in the Baltic Sea(Serrão et al. 1999a).

During reproduction, gametangia are released fromconceptacles in the reproductive tissue (the recepta-cles). Fucoid eggs are released from oogonia (8 eggsper oogonium in Fucus spp.) and the sperm fromantheridia (64 sperm per antheridium), either in thewater column or soon after reaching the substrate.Eggs, oogonia, and antheridia sink, and the sperm arenegatively phototactic. The sperm are attracted to theeggs by a pheromone (Müller & Gassman 1978). Eggscan reach the bottom (i.e. settle) either before or afterfertilization Because there is no planktonic stage,settlement in Fucus spp. is directly related to gameterelease and defined as the shower of eggs andzygotes that have fallen to the bottom. Adhesion tothe substrate is not simultaneous with settlement.Instead, settlement occurs within minutes of release,whereas attachment occurs a few hours after fertiliza-tion. Only zygotes attach; unfertilized eggs do notsecrete adhesive material. Attachment can thereforebe used as an assay to distinguish unfertilized eggsfrom zygotes. Another post-fertilization event that canalso be used to evaluate fertilization is germination,which results in the appearance of a rhizoid (an elon-gation of 1 zygote pole), approximately 1 d post-fertil-ization. Recruitment is defined as the appearance ofestablished individuals of a certain predefined size/

stage. In this study, any stage beyond a fertilized eggwas considered a recruit.

Fucoids can have either a hermaphroditic or dioe-cious reproductive mode. Fucus vesiculosus is dioe-cious, with each individual producing only 1 type ofgamete (i.e. separate males and females). F. spiralis,like several other hermaphroditic fucoids, has been,in our opinion, incorrectly referred to as monoecious,even in our own studies (e.g. Serrão et al. 1999b).Although the term monoecious has been defined forflowering plants, it refers to genets in which the maleand female function are separated (i.e. separate maleand female flowers in the same individual) (Richards1997), which is not the case for F. spiralis if a flowerand a conceptacle are considered to be analogous.F. spiralis is clearly a hermaphroditic, a genet withboth male and female function (Richards 1997). Thereproductive organs (e.g. flowers, or conceptacles inthe case of Fucus spp.) of hermaphroditic individualscan be either monoecious (each has a single sex) orhermaphrodite (both sexes in the same conceptacle,which is the case for F. spiralis).

There are advantages and disadvantages associatedwith the different reproductive modes of hermaphro-ditism versus dioecy (e.g. as reviewed by Bawa 1980,Thomson & Brunet 1990, Freeman et al. 1997, Barrett1998, Charlesworth 1999). Hermaphrodites that arecapable of self-fertilization can pass their entiregenome to the offspring, reduce the likelihood of dis-rupting locally co-adapted gene combinations throughrecombination, and guarantee the possibility of repro-duction at least by self-fertilization. This is particularlytrue when the likelihood of encounters with gametesfrom different individuals is very low, as is predicted tobe the case for externally fertilizing organisms on opencoasts (e.g. Denny 1988). Dioecious reproduction pro-vides the selective advantages of obligatory outcross-ing, including avoidance of inbreeding depression,and promoting genetic variability, which is importantfor adaptation to variable ecological conditions. Dioecyand hermaphroditism have evolved independentlyseveral times within the Fucaceae family (Serrão et al.1999), as well as in many different taxonomic groups,indicating that there may often be a thin and easilycrossed line dividing and balancing the selectiveadvantages of these contrasting reproductive modes.

The fucoid algae Fucus vesiculosus and F. spiralis aregood model species on which to test the role of matingsystems on fertilization success because the 2 speciesare similar in their ecology and morphology and, al-though evolutionarily very closely related (Serrão et al.1999b), they differ in their reproductive modes (dioe-cious vs. hermaphroditic). The dioecious F. vesiculosushas a much higher sperm-to-egg allocation than thehermaphroditic F. spiralis (Vernet & Harper 1980).

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F. spiralis allocates much more biomass to eggs thansperm, which is suggestive of a high degree of inbreed-ing (Vernet & Harper 1980). Lower investment intomale function by F. spiralis than for dioecious F. vesicu-losus (Vernet & Harper 1980) also suggests that it ismuch more efficient at achieving successful externalfertilization in the sea than dioecious species.

Both Fucus vesiculosus and F. spiralis are common inthe intertidal zone in cold temperate environmentsin the Northern Atlantic. Their southern limit in theNortheastern Atlantic is found in the biogeographictransition zone between Southwest Europe and NorthAfrica, between a cold temperate and a warm temper-ate environment (Lüning 1990). Near its southern limitalong the Portuguese and Moroccan coasts, F. vesicu-losus is restricted to growing in estuaries and coastallagoons (i.e. areas with minimal wave motion, where itoccurs in locally abundant stands) and it does not occuron the open coast as it does in more northern environ-ments. On the other hand, F. spiralis does occur onopen rocky shores near its southern limit, often at lowabundance in canopy-devoid rocky areas separatedby long stretches of sandy beaches.

This study addresses the question of whether the re-productive system, in particular hermaphroditism ver-sus dioecy, can be a factor determining the distributionof marine species with external fertilization, in particu-lar in exposed habitats where, once spawning takesplace, the probability of gamete encounters is predictedto be very low due to rapid gamete dilution. In compar-ing the role of reproductive mode in the reproductivesuccess of these 2 species, we hypothesized that (1) theabsence of Fucus vesiculosus (dioecious) on exposedshores near its southern limit is due to reduced fertil-ization success in the high water-motion environmentthere, and that (2) F. spiralis (hermaphroditic), by beingable to release both male and female gametes from thesame individual, increases the probability of sperm-eggencounters, and can thus achieve high fertilization suc-cess even on these exposed shores or in turbulent envi-ronments. In this study, our hypothesis was tested bymoving both species to the exposed shores near theirsouthern limit and determining fertilization successduring the reproductive season of 2001. The expectedoutcome was that F. vesiculosus would not be able tofertilize its eggs in the turbulent environment andwould have a much lower fertilization success than itshermaphroditic congener.

MATERIALS AND METHODS

Study sites. In Northern Portugal, Fucus vesiculosusand F. spiralis coexist in dense stands on the opencoast. One of the southernmost locations where both

species coexist on the open shore is Viana do Castelo(Fig. 1), where offshore rocky bluffs reduce waveaction in many inner sites where both Fucus speciesare abundant. This site was the source of all trans-plants. The 3 sites separated by 15 km to which bothFucus species were transplanted were approximately500 km south, in the area around Sines, and were rep-resentative of the exposed shores near the southernlimit (Fig. 1).

Viana do Castelo has a cool temperate climate; airtemperatures average 14.3 ± 2.9°C (SD) over the yearand annual precipitation averages 1444 mm (Insti-tuto de Meteorologia, Portugal, data available fromwww.meteo.pt). At this site, population density ofFucus spp. can exceed 600 ind. m–2 in some areaswith many small recruits, with other intertidal canopy-forming species such as Ascophyllum nodosum andPelvetia canaliculata. In most areas, canopy cover is100% (unpubl. data).

In Sines, average air temperature is approximately2°C warmer than in Viana do Castelo, with much loweraverage annual precipitation at 492 mm (Institutode Meteorologia, Portugal, data available fromwww.meteo.pt). Sines has long stretches of sandy

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Fig. 1. Map of study sites

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beach interspersed with rocky outcrops that areexposed to wave action. On rocks, stunted forms ofFucus spiralis exist in low-abundance stands; they arereproductive during spring/summer. F. vesiculosusis absent on the open coast, but occurs nearby in anestuary (Vila Nova de Milfontes, see Fig. 1).

Transplants. Reproductive individuals of Fucus vesi-culosus and F. spiralis, which were naturally attachedto small rocks, were collected in March 2001 in Vianado Castelo and transported to Sines (still attached torocks). The following day at low tide, half of the rockswere transplanted to the 3 wave-exposed sites nearSines (Porto de Sines, Porto Covo, and Praia de Serroda Águia), and attached to the bedrock using a smallamount of cement (Sika grout quick drying cement,Industrias Quimicas). The other half were extra boul-ders left in a nearby intertidal pool for replacements, asnecessary, when cement failed and rocks were lostthroughout the season. This way, replacements couldbe made with algae exposed to the same conditions asthe original transplants. Stands were transplanted tothe same shore levels occupied by the 2 species in theintertidal zone of Viana: 0.9 to 2.4 m above ELWS(extreme low water spring) for F. vesiculosus and 2.4 to3.0 m above ELWS for F. spiralis, and rocks withnumerous algae naturally attached were arrangedside-by-side so that algae were at the same density asin the source location, and so that males and females(of F. vesiculosus) were side-by-side in order to simu-late stands in ideal conditions for successful externalfertilization. Two stands of approximately 20 largereproductive individuals each were made for eachspecies in 2 of the 3 sites (in Porto Covo and in Praia deSerro da Águia). In the third site (Porto de Sines), only1 stand of each species was made, and the area wasnetted to exclude a grazing fish (Sarpa salpa) which iscapable of removing a large part of the adult Fucusbiomass (pers. obs.). Controls were performed bytransplanting algae back to their original site (as partof a student’s thesis, Monteiro unpubl.) and showed noeffects of transplantation.

Zygote adhesion assay. In order to determine fertil-ization success in the field, an inexpensive method todistinguish fertilized eggs from non-fertilized eggs wasdeveloped. Because only fertilized eggs attach to thesubstrate, the number of eggs that were attached ver-sus those that were not could be used as a proxy for thepercentage of eggs fertilized in the field. In order to dothis, the time necessary to give fertilized eggs a chanceto attach after collection, under laboratory conditions,needed to be established, since in field experimentsthe eggs could have been fertilized shortly before col-lection and still might not have had enough time toattach. Eggs of Fucus vesiculosus and F. spiralis fromViana do Castelo were released and fertilized at high

sperm:egg ratios in the laboratory in separate batchcultures of seawater at ambient temperature, withglass slides placed at the bottom of each container. Atvarious time intervals after fertilization, which usuallyoccurs immediately after gamete release in enclosedenvironments in culture, 3 glass slides for each specieswere collected, rinsed gently with seawater, and thenumbers of attached zygotes were counted with adissecting microscope (Zeiss). This was repeated atintervals for the first 10 h after fertilization, and a finalevaluation was performed again after 24 h. Thisexperiment was performed 3 times for F. spiralis andtwice for F. vesiculosus. The time after which no sig-nificant increase of attached zygotes occurred waschosen as the minimum time required for zygote adhe-sion. All field collected samples of settled eggs werethen allowed more than this amount of time to attach,after which time they were considered unfertilized ifthey were not attached or had not formed a polarrhizoid-thallus axis.

Egg settlement. Egg settlement was monitored usingcircular fiberglass collecting disks of 6 cm in diametermade from food-grade isophthalmic fiberglass resin(ALMO, Produtos Quimicos). In order to retain settledeggs, the surfaces of the disks were made rugose bycasting the fiberglass in a latex rubber mold with sand-paper (number 6 roughness) at the bottom. Once dry,isophthalmic resin is non-toxic; a preliminary experi-ment in which eggs were settled on the disks con-firmed this (they fertilized, attached, and developednormally). These disks were drilled in the center (5 mmdiameter drill bit). A larger hole was drilled into theintertidal bedrock and a stainless steel bolt (5 mmdiameter, 50 mm length) was fixed head-down in thehole with quick-drying cement (as used for the trans-plants). The perforated disks were placed onto thefixed bolt, and fastened with a nut. The disks were ableto withstand strong wave exposure, yet were quickand easy to remove during sampling at low-tideperiods.

At least 3 disks in each stand for each species werecollected daily at each low tide that occurred duringdaylight hours (twice a day during extreme low tides)in each of the 3 sites, from April to June 2001. In thesenatural stands some reproductive algae can be foundall year round, but there is a large peak when nearlyall individuals are reproductive during the spring, andthis period is designated as the reproductive season.Night sampling was not performed because fucoidsrequire light for potentiation of gamete release (Serrãoet al. 1996, Pearson & Brawley 1998, Pearson et al.1998), and have been shown not to release gametes atnight (Pearson & Brawley 1996). The number of eggsper disk was counted with a dissecting microscope(Leica MZ6).

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Fertilization success. Fertilization success was eval-uated on days when substantial egg settlement wasobserved (>50 eggs disk–1). Two assays were used todetermine fertilization: zygote attachment, and rhizoidformation, both of which only occur in fertilized eggs.Adhesion of fertilized eggs to the substrate takes placesome hours (see ‘Zygote adhesion assay’ above) afterfertilization. Unfertilized eggs settle and becometrapped in the disks’ crevices but do not adhere. Thenumber of eggs on the disk upon collection wasrecorded. Then the disks were rinsed gently with sea-water and loose eggs were kept in a Petri dish. After24 h, the number of eggs attached to the Petri dish plusthe number of eggs that were originally attached to thedisk were counted as fertilized. This number was thendivided by the number of eggs originally collected onthe disk to give the percentage of settled eggs thatwere fertilized, and to calculate fertilization success.The 24 h period was sufficient for all fertilized eggs toadhere. The attachment assay was confirmed with thegermination assay: formation of a rhizoid.

Recruitment. Recruitment during the reproductiveseason was estimated by placing permanent collectingdisks (as above) under the algae, interspersed with thedaily collecting disks. These were collected at the endof the sampling period. The number of recruits on thedisks was evaluated by counting under a dissectingmicroscope. In this study, any attached individual inthe field at the end of the study that had reached apost-settlement size/stage was considered a recruit.This number was divided by the total number of eggssettled on the disks (the sum of all the eggs on the3 disks over the entire collection period) in that siteduring the reproductive season to estimate the per-centage of settled eggs that recruited (i.e. attached andsurvived until the end of the reproductive season) andby the number of eggs that were fertilized on the disks(the sum of all the fertilized eggs on major days ofrelease on the 3 disks over the entire collection period).

Recruit mortality. Embryo mortality was estimatedby outplanting 1 mo old laboratory-cultured embryos tothe field sites. Disks were seeded with fertilized eggs inbatch culture in the laboratory and grown under nat-ural daylight at ambient temperature in natural sea-water which was replaced weekly. After 1 mo, the num-ber of recruits on the disks was evaluated and then thedisks were outplanted to 2 of the transplant sites (Portode Sines and Praia do Serro da Águia). The outplantexperiment followed a factorial design to test for theeffects of canopy (2 levels) and grazers (3 levels) onrecruit mortality (1 mo old embryos in this case). Theeffect of canopy was tested by placing outplants under-neath Fucus spiralis transplants which had had theirreproductive structures (receptacles) trimmed (to avoidinterference from egg release), versus placing the out-

plants on bare rock. The effect of grazers was tested byusing grazer exclusion cages, built from plastic mos-quito mesh sewn around a cage of plastic-coated wire.The cages were intended to exclude snails and limpets,which are abundant on the Portuguese open coast, andgrazers of Fucus spp. embryos. Sea urchins and largeamphipods were also excluded. This treatment wascompared with a cage control treatment (cage with thewire but no mesh) and a treatment where no cage orwire was used. Three replicate disks were used foreach treatment, for each site (Porto de Sines and Praiado Serro da Águia). The factors were: sites (n = 2),species (n = 2), cages (n = 3, cage, cage-control, and nocage), and canopy (n = 2, presence or absence). Thenumber of recruits surviving per disk was evaluatedafter 1 mo in the field, and percent survivorship wascalculated. The number of embryos outplanted per disk(28.3 cm2) ranged from 1000 to 1500.

Statistical analyses. ANOVA was used to comparemeans, followed by Student-Newman-Keuls (SNK)multiple comparisons, using α = 0.05 in both cases. Ifvariances were not homogeneous (Cochran’s test),they were log-transformed to comply with homoscedas-ticity. The percentage data were arcsine-transformedbefore analyses.

RESULTS

Zygote adhesion time

The time for zygotes to adhere to glass slides in cul-ture ranged from 7 to 8 h post fertilization (Fig. 2),showing that an overnight culture period for eggs thatwere not attached upon collection from the field wassufficient time for fertilized eggs to adhere.

Egg settlement

Egg settlement of both species occurred with a peri-odicity of ca. 2 wk (Fig. 3). The periodicity of eggrelease did not appear to be exclusively related tolunar phases. Few eggs were collected on days withhigh wave action, but wave activity was measured off-shore, and shore morphology and orientation obvi-ously strongly influenced the exposure observed at thetransplant sites. In 2 of the 3 sites, there was greateregg settlement for Fucus vesiculosus than for F. spiralis(Figs. 3 & 4a), and settlement of both species wasalways very patchy (see large SE in Fig. 3). Taking intoaccount only major settlement events (>500 eggs totalper site), 14 of the 16 events occurred on days whenthe low tide was later in the day (between 13:00 and21:00 h, Fig. 5).

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Fertilization success

Fertilization success, when taken as an average overthe whole reproductive season, was close to 100% forall 3 transplant sites for both species (Fig. 4b). Whenanalyzed on separate days, fertilization success variedbetween <1 and 100%, but was high (>80%) on mostdays (Fig. 6). Days with fertilization success below50% were only observed for Fucus vesiculosus(Fig. 6b). The lowest days of fertilization success were,in particular, on April 6 and 7 (major release ofF. vesiculosus only), and April 16, 18, and 19 for bothspecies. On April 7, one of the largest oceanic swellsoccurred during the sampling period, causing strongwave activity. Smaller wave events occurred on April18 and 19. Thus, low fertilization success usually coin-cided with days of high wave activity, although thewave height data are measured offshore and do notcorrelate precisely with local water motion at the sites.Fertilization success was higher for both species dur-ing the later half of the sampling period (Fig. 6).

Recruitment

Recruitment occurred in 2 of the 3 transplant sites.In the Porto de Sines site, recruitment could not beestimated as the stands were vandalized during theexperiment and recruitment disks were lost. Signifi-cantly more recruits were found at the end of thereproductive season for Fucus spiralis than for F.vesiculosus, and there was also a significant differ-ence between sites (ANOVA, p < 0.05, for the effectof species F1, 37 = 6.78, and for site F1, 37 = 7.80)(Fig. 7a). When recruitment was analyzed as a per-centage of the total number of eggs that had settled

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Fig. 2. Fucus vesiculosus and F. spiralis. Zygote adhesiontiming. Arrows mark time after which no significant increasein number of zygotes attached occurred (Student-Newman-Keuls [SNK] multiple comparisons, after ANOVA, p < 0.05,confirmed there was a significant difference among times).Last sample at 24 h post fertilization (not to scale). Values are

means ± SE. Panels represent experiment repetitions

Fig. 3. Fucus vesiculosus and F. spiralis. Daily egg settlement(mean ± SE, n = 3 to 5 disks per low tide) in 3 sites near Sines:(a) SA = Praia do Serro da Águia, (b) PC = Porto Covo, and (c)PS = Porto de Sines. (d) Significant wave height at a buoy di-rectly offshore of Sines. Note different scales for F. vesiculo-sus and F. spiralis. Lunar phases are shown above the graph

Ladah et al.: Fertilization and recruitment in Fucus populations

on disks in that site over the reproductive season, oras a percentage of the total number of eggs on daysof major release that were fertilized on disks over thereproductive season, the same pattern was observed(Fig. 7b,c).

Recruit mortality

In outplant experiments (Fig. 8), 1 mo old embryos ofFucus spiralis showed significantly greater survivor-ship than F. vesiculosus under all conditions, except inthe more wave-protected of the 2 sites (Porto de Sines)where outplants of F. vesiculosus outsurvived F. spi-ralis only if there was canopy present (ANOVA, inter-action between site, canopy, and species; p = 0.007,F1, 48 = 7.84). Regardless of the site, if there was nocanopy present, F. spiralis had significantly greatersurvivorship. There was no effect of caging and grazerexclusion.

DISCUSSION

The hypothesis that Fucus vesiculosus is absentfrom the exposed shores near its southern limitbecause it is unable to fertilize its eggs in environ-ments with high water motion was rejected. Highoverall levels of external fertilization success for bothF. spiralis and F. vesiculosus showed that dioeciousspecies of fucoids can achieve as high a fertilizationsuccess as hermaphroditic species, even in exposedenvironments. However, the absence of F. vesiculo-sus on exposed shores near the southern limit ofFucus spp. in the NE Atlantic, where rocks have nosignificant canopy-forming species, may be ex-plained by the reduced recruitment of F. vesiculosusin exposed sites, and the low survivorship of F.vesiculosus embryos (compared to F. spiralis) inexposed areas without canopy. Whether recruitmentand recruit survivorship of F. vesiculosus is greaterin less exposed areas is a question that will beaddressed in future experiments.

Gamete release during the experiments was oftenpatchy and was not well correlated with the lunarcycle. The patchiness found may represent either apatchy release pattern among different plants, or asmall scale hydrographic or hydrodynamic effect. Thistype of patchiness in fucoid egg settlement was previ-ously found in the Baltic Sea in a subtidal population(Serrão et al. 1996, 1999a), as well as in an intertidalpopulation on the western Atlantic (Pearson & Braw-ley, 1996). The correlation of Fucus gamete releasewith the lunar cycle has been found in natural shel-tered populations (Brawley 1992) and in the laboratory(Andersson et al. 1994), but is often obscured by otherenvironmental conditions in the field (Pearson & Braw-

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Fig. 4. Fucus vesiculosus and F. spiralis. (a) Total number ofeggs settled over reproductive season, and number that werefertilized (for days of major release) over reproductive seasonin each transplant site, and (b) average fertilization successover total sampling period (SA = Praia do Serro da Águia, PC= Porto Covo, PS = Porto de Sines). Values for (b) are means ±

SE, for all days with major release as defined in text

Fig. 5. Fucus vesiculosus and F. spiralis. Time (h) of low tideon days of substantial egg release (>500 eggs, released) andtotal number of eggs collected. SA = Praia do Serro da Águia,

PC = Porto Covo, PS = Porto de Sines

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ley 1996, Serrão et al. 1996, 1999a). This reflects theresponse of algae to factors other than tidal or lunarcycles. Laboratory and field experiments showedstrong inhibition of gamete release by water motion inall fucacean species tested: Fucus distichus, F. vesicu-losus (Pearson & Brawley 1996, Serrão et al. 1996,Pearson et al. 1998, Serrão et al. 1999a), Ascophyllumnodosum (Serrão 1996), and Pelvetia compressa (nowSilvetia compressa, Serrão et al. 1999b) (Pearson &Brawley 1998, Pearson et al. 1998). In our experiments,minimal gamete settlement was observed on days withhigh wave activity, even when the lunar cue of a full ornew moon occurred.

In this study, most eggs or zygotes settled on dayswhen the low tide was later in the day, similar to thatfound for Fucus distichus in tide pools (Pearson &Brawley 1996), and for F. vesiculosus in the Baltic Sea,where peaks of gamete release always occurredtowards the end of the day (Serrão et al. 1996). Thismay be due to the requirement of a period of photo-synthesis for potentiation of gamete release to occur inthe field. In the laboratory, a photosynthetic inhibitorreduced or eliminated gamete release in Silvetia com-pressa and F. vesiculosus (Serrão et al. 1996), and car-bon limitation during photosynthesis was shown to bea signal for gamete release in F. distichus and S. com-

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Fig. 7. Fucus vesiculosus and F. spiralis. Re-cruitment as (a) mean number of recruits perarea (n = 4 to 15 recruitment disks per site),(b) % of total eggs settled during the reproduc-tive season that recruited, and (c) % of totaleggs fertilized (measured on days of major re-lease) during reproductive season that re-cruited. Sites listed at the bottom refer to sitesof transplants: SA = Praia do Serro da Águia,

PC = Porto Covo

Fig. 6. Fucus vesiculosus and F. spiralis. Fertilization success ofsettled eggs in transplanted stands of (a) F. spiralis, and (b) F. vesicu-losus on days of higher egg settlement, in 3 sites near Sines: Praia doSerro da Águia (SA), Porto Covo (PC), and Porto de Sines (PS). Valuesare means ± SE, (data with no error bars had release on only 1 disk).Lines below graph represent days which had asignificant wave

height >100 cm measured offshore

Ladah et al.: Fertilization and recruitment in Fucus populations

pressa (Pearson et al. 1998). Because, overall, gameterelease was periodic, the data suggest that there is acombination of rhythmic cues (e.g. tidal or lunar) andlocal factors, such as periods of exposure to light andcalm conditions, necessary for the induction of gameterelease.

Fertilization success of Fucus vesiculosus at exposedsites was higher than we predicted. On only a few daysduring the reproductive season was fertilization suc-cess lower than 50%, and this often coincided withhigher wave activity. In other experiments on waveexposed shores, permanently submerged fucoids con-centrated gamete release on days or tidal phases withlow water motion, thereby achieving high fertilizationsuccess (Pearson & Brawley 1996, Serrão et al. 1996,and see Santelices 2002 for a review). Fucoids that areemersed at low tide on exposed shores can also adjusttheir timing of gamete release to take advantage ofperiods of limited water motion in order to release andfertilize their gametes, thereby increasing the chancesfor fertilization and recruitment (Berndt et al. 2002, thisstudy).

High fertilization success has been found in allfucacean populations studied to date (Brawley 1992,Serrão et al. 1996, Pearson et al. 1998, Berndt et al.2002), except for Fucus vesiculosus at their northerndistributional limit in the Baltic Sea (Serrão et al.1999a). However, studies of fertilization success inintertidal (non-tide pool) fucoid populations on ex-posed shores are rare (but see Berndt et al. 2002).Our discovery of high fertilization success in exposedshore intertidal populations of fucoids with contrastingreproductive modes raises new questions concerning

timing of gamete release during the tidal cycle. Furtherexperiments will address questions such as: (1) at whatphase of the tidal cycle does gamete release occur forself-compatible hermaphrodite versus dioecious spe-cies, and (2) when during the tidal cycle do signalscontrolling gamete release operate?

Recruitment was low in both species, though it wassignificantly lower in Fucus vesiculosus than in F.spiralis, in spite of greater egg settlement and agreater absolute number of eggs fertilized at thesites. This larger settlement of F. vesiculosus mightbe due to its larger egg production per individual atour sites (unpubl. data). This is due to adults of F.vesiculosus achieving large sizes with many recepta-cles, and does not necessarily represent higher bio-mass allocation to sex. In fact, Fucus spiralis hasbeen found to produce more eggs per weight than F.vesiculosus, although investment in sperm is muchlower than for dioecious fucoids like F. vesiculosus(Vernet & Harper 1980).

The low recruitment of Fucus vesiculosus measuredin this study is likely to have been caused by failure ofzygotes to survive after settlement and attachment,rather than dislodgment, since we found higher num-bers of zygotes of F. vesiculosus than F. spiralis on set-tlement disks. Recruits of F. vesiculosus may be moresensitive to temperature, wave motion, high light, UV,desiccation, or a combination of these factors, thanF. spiralis. Although air temperatures in the coastallagoons and estuaries where F. vesiculosus occurs atthe same latitude are similar to those on the exposedcoast, desiccation and photoinhibitory light stress mayactually be lower in estuarine and coastal lagoon envi-ronments because the fucoids in such habitats arepartially covered by a protective muddy film duringlow tide. However, soft sediment habitats have beenshown to be unfavourable for recruitment of Fucus(Albrecht 1998, Chapman & Fletcher 2002).

The survivorship of outplanted 1 mo old recruits wasalso significantly reduced for Fucus vesiculosus in thisstudy compared to F. spiralis, particularly where nocanopy was provided, which is the natural case on theexposed shores at the southern limit. It appears that, ingeneral, recruits of F. vesiculosus are less tolerant thanF. spiralis recruits. Recruitment has been found to be abottleneck stage for fucoid algae in other intertidalhabitats (e.g. Burrows 1964, McLachlan 1974, Gunnill1980, Vadas et al. 1990, Brawley & Johnson 1991, John-son & Brawley 1998). Canopy presence is reported tohave both beneficial and detrimental effects on survivalof early post-settlement stages of fucoid algae. Brawley& Johnson (1991) and Johnson & Brawley (1998)showed greater survivorship of outplanted zygotes andembryos of Silvetia compressa underneath canopy andin turf, which they attributed to protection from desic-

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Fig. 8. Fucus vesiculosus and F. spiralis. Survivorship of out-planted recruits (% per disk) when canopy was present vs ab-sent, and herbivores excluded vs controls, in 2 sites near Sines(SA = Praia do Serro da Águia, PS = Porto de Sines). Values

are means ± SE, n = 3 disks per treatment

Mar Ecol Prog Ser 262: 173–183, 2003

cation. In contrast, reduced survivorship of zygotes andrecruits of Ascophyllum nodosum and Fucus spp. un-derneath canopy has been reported by several authors(e.g. Chapman 1989, 1990, Vadas et al. 1992, Åberg &Pavia 1997, Jenkins et al. 1999), and is commonly at-tributed to whiplash and/or competition. In our study,both canopy presence and low water motion seemed tobe crucial for zygotes of F. vesiculosus to survive.

In summary, the experiments in this study haveshown high fertilization success in exposed environ-ments, synchronous gamete release during low water-motion periods, and surprisingly low recruitment andrecruit survivorship for intertidal populations of bothdioecious Fucus vesiculosus and hermaphroditic F. spi-ralis transplanted near their southern limit of distribu-tion. For both species, on days when settlement peaksoccurred, fertilization success was high; thus recruit-ment was not limited by gamete dilution and fertili-zation failure, even in exposed areas. Rather, theestablishment and survival of the early stages of bothspecies was very low on exposed shores, shown by lowrecruitment and embryo survivorship, and was sig-nificantly lower for F. vesiculosus, particularly in theabsence of canopy.

We conclude that the absence of Fucus vesiculosusfrom exposed shores where the hermaphroditic F. spi-ralis occurs, is not caused by fertilization failure ofdioecious species in exposed environments. The ab-sence of F. vesiculosus on exposed shores near itssouthern limit in the Northeast Atlantic, where rockshave no significant canopy-forming species, may beexplained by the reduced recruitment, and the lowsurvivorship of recruits in exposed areas withoutcanopy. We have shown that establishment (i.e. attach-ment and/or survival) of F. vesiculosus recruits is verylow or absent on shores that have both high waveexposure (on the open coast) and high desiccationstress (at their southern limit), particularly where nocanopy is present. Future studies should test thehypothesis that low establishment and/or survival ofearly post-settlement stages on the open coast deter-mine the confinement of F. vesiculosus to estuaries andcoastal lagoons at its southern limit.

Acknowledgements. We are thankful to the marine laboratoryof the Universidade de Évora, CIEMAR, in Sines, for hosting usduring the entire Fucus reproductive season of 2001. We areespecially grateful to J. Castro and T. Cruz at CIEMAR for in-teresting discussions on the experimental design and data,and for their great company, along with Sonia, Teresa, andEugenio during the project. We thank C. Engel for interestingdiscussions and information on mating systems in algae. L.B.L.and G.P. were supported by post-doctoral fellowships fromthe Portuguese Science Foundation (FCT) and the EuropeanSocial Fund (ESF). This research was funded by FCT pro-jects BIOPORT (PDCTM/P/MAR/15292/1999) and GAMETE(POCTI/BSE/39431/2001) to E.S. and G.P.

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Editorial responsibility: Otto Kinne (Editor), Oldendorf/Luhe, Germany

Submitted: February 28, 2002; Accepted: March 23, 2003Proofs received from author(s): October 22, 2003