M Vázquez-Luis

J M Guerra-García

P Sanchez-Jerez

J T Bayle-Sempere

M M Thiel

Vázquez-Luis

Caprellid assemblages (Crustacea: Amphipoda

Keywords: Amphipoda, Caprellidae, Caulerpa racemosa var. cylindracea, Southeastern Spain, Invasive species

Introduction

Caprellids are marine crustaceans, which inhabit algae, hydroids, ascidians, anthozoans, bryozoans, sponges and seagrasses [START_REF] Mccain | The Caprellidea (Crustacea: Amphipoda) of the Western North Atlantic[END_REF][START_REF] Guerra-García | Habitat use of the Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa[END_REF]. They feed on suspended materials, prey on other organisms or graze on epibiotic fauna and Xora (Guerra-García et al. 2002b;[START_REF] Thiel | The distribution of littoral caprellids (Crustacea: Amphipoda: Caprellidea) along the PaciWc coast of continental Chile[END_REF], and they are important prey for many coastal Wsh species [START_REF] Caine | Caprellid amphipods: fast food for the reproductively active[END_REF]. Recently, caprellids have also been found to be useful bioindicators of marine pollution and environmental stress (Guerra-García and García-Gómez 2001; [START_REF] Ohji | DiVerences in the acute toxicities of tributyltin between the Caprellidea and the Gammaridea (Crustacea: Amphipoda)[END_REF][START_REF] Takeuchi | Butylin concentrations along the Japanese coast from 1997 to 1999 monitored by Caprella spp. (Crustacea: Amphipoda)[END_REF].

During the last decade, an eVort has been undertaken to contribute to the knowledge of the Caprellidea from the Iberian Peninsula and nearby areas, especially in the Strait of Gibraltar [START_REF] Guerra-García | Habitat use of the Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa[END_REF]Guerra-García and García-Gómez 2001;[START_REF] Guerra-García | The Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa, with the description of three species of Caprella, a key to species of Caprella and biogeographical discussion[END_REF][START_REF] Guerra-García | Redescription of Caprella hirsuta Mayer, 1890 (Crustacea, Amphipoda, Caprellidea) from the Strait of Gibraltar[END_REF][START_REF] Guerra-García | Habitat use of the Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa[END_REF], 2002a, b). A recent contribution has dealt with the community structure of caprellids on seagrasses from southern Spain [START_REF] González | Community structure of caprellids (Crustacea: Amphipoda: Caprellidae) on seagrasses from southern Spain[END_REF]), but the ecological distribution of caprellids clinging to Caulerpa beds has been little studied. The only available studies, which report caprellids associated with Caulerpa species in Iberian Peninsula waters, consist of descriptions of the macrofaunal community associated with Caulerpa 123 prolifera from Algeciras Bay, southern Spain [START_REF] Sánchez-Moyano | EVect of the vegetative cycle of Caulerpa prolifera on the spatio-temporal variation of invertebrate macrofauna[END_REF], 2007) and the taxonomic description of the new species Caprella caulerpensis from this bay (Guerra-García et al. 2002a). Because of the lack of information about caprellids inhabiting Caulerpa beds, we conducted an ecological study to characterise the caprellid distribution in shallow habitats of southeastern Spain colonised by the invasive Caulerpa racemosa (Forsskål) J. Agardh var. cylindracea (Sonder) Verlaque, Huisman et Boudouresque [START_REF] Verlaque | On the identity and origin of the Mediterranean invasive Caulerpa racemosa (Caulerpales, Chlorophyta)[END_REF] (hereafter C. racemosa).

The presence of vegetation allows a greater species diversity and abundance of individuals than those found in unvegetated habitats [START_REF] Heck Kl | Seagrass habitats: the roles of habitat complexity, competition and predation in structuring associated Wsh and motile macroinvertebrate assemblages[END_REF][START_REF] Peterson | The inXuence of seagrass cover on population structure and individual growth rate of a suspension-feeding bivalve, Mercenaria mercenaria[END_REF][START_REF] Irlandi | Large-and small-scale eVects of habitats structure on rates of predation: how percent coverage of seagrass aVects rates of predation and siphon nipping on an infaunal bivalve[END_REF], which is usually correlated with an increase in habitat complexity [START_REF] Johnson | Variations in diversity within benthic marine communities[END_REF][START_REF] Stoner | Perception and choice of substratum by epifaunal amphipods associated with seagrasses[END_REF][START_REF] Dean | Marine invertebrates in algal succession II. Test of hypotheses to explain changes in diversity with succession[END_REF][START_REF] Edgar | Patterns of colonization of mobile epifauna in Western Australian seagrass bed[END_REF][START_REF] Taylor | Mobile epifauna on subtidal brown seaweeds in northeastern New Zealand[END_REF][START_REF] Ayala | Relaciones entre la comunidad de anfípodos y las macroalgas a las que están asociados, en una plataforma rocosa del litoral central de Venezuela[END_REF]. Therefore, changes in habitat structure of vegetated substrata resulting from the invasion of species such as C. racemosa could aVect the associated fauna, including caprellids. Along the Mediterranean coast, two seagrass species, Posidonia oceanica and Cymodocea nodosa, develop meadows on shallow sandy bottoms [START_REF] Buia | Reproductive phenology of the Mediterranean seagrasses Posidonia oceanica (L.) Delile, Cymodocea nodosa (Ucria) Aschers., and Zostera noltii Hornem[END_REF]. Like seagrasses, the alga Caulerpa prolifera usually forms dense beds on soft bottoms from 1 to 20 m depth, especially in sheltered areas, with low Xow, little water renewal and considerable input of organic matter [START_REF] Sánchez-Moyano | EVect of the vegetative cycle of Caulerpa prolifera on the spatio-temporal variation of invertebrate macrofauna[END_REF]. A remarkable spread of C. racemosa throughout the Mediterranean has been recorded during the last 15 years or so, during which the alga invaded diVerent habitats and most types of substrata located at water depths of 0 m to more than 60 m. The recorded rate of invasion has been much faster than that of other invasive species such as Caulerpa taxifolia [START_REF] Verlaque | On the identity and origin of the Mediterranean invasive Caulerpa racemosa (Caulerpales, Chlorophyta)[END_REF]. Since the Wrst records of C. racemosa spreading along Mediterranean coastal areas, several studies concerning the ecology of this species have been undertaken (see [START_REF] Ruitton | Seasonal changes of the introduced Caulerpa racemosa var. cylindracea (Caulerpales, Chlorophyta) at the northwest limit of its Mediterranean range[END_REF][START_REF] Cavas | Antiproliferative and newly attributed apopotic activities from an invasive marine alga: Caulerpa racemosa var cylindracea[END_REF]). However, information on changes in the associated fauna brought about by the invasive alga is lacking (but see [START_REF] Argyrou | Expansion of the macroalga Caulerpa racemosa and changes in softbottom macrofaunal assemblages in Moni Bay, Cyprus[END_REF][START_REF] Piazzi | The spread of Caulerpa racemosa var cylindracea in in the Mediterranean Sea: an example of how biological invasions can inXuence beta diversity[END_REF][START_REF] Vázquez-Luis | Changes in amphipod (Crustacea) assemblages associated with shallow-water algal habitats invaded by Caulerpa racemosa var cylindracea in the western Mediterranean Sea[END_REF].

The aim of the present study was to compare the caprellid assemblages among the most important shallow water environments (rocky and sandy bottoms) in Mediterranean coastal areas and to explore the eVect of the invasive alga C. racemosa on the caprellid community in comparison with natural habitats (algae, seagrasses and sand).

Methods

Study area

Fieldwork was carried out along the Cape of Santa Pola (Alicante, southeastern Spain; Fig. 1a). C. racemosa was Wrst recorded from Alicante in 2002, at a site located approximately 10 km north of our study area, where it colonised soft sediments and dead matte of Posidonia oceanica. Two months after its appearance in Alicante, C. racemosa was detected on a rocky platform in our study area [START_REF] Pena-Martín | Caulerpa racemosa (Forssk.) J Agardh (Caulerpaceae, Chlorophyceae), nueva para la Xora de Alicante[END_REF]. During the study period, C. racemosa occurred in extensive areas of ecologically important rocky bottom habitat, on sandy and muddy substrata and on dead matte of Posidonia oceanica. It was also found mixed patchily with Cymodocea nodosa in meadows of the seagrass (0.2-1.5 m). Depth in the study area varied between 0 and 5 m. The seabed present in the shallower parts (0-0.5 m) of the study area is characterised by a rocky platform that supports native seaweeds, namely Halopteris scoparia, Jania rubens, Padina pavonica, Dictyota fasciola, Cystoseira brachicarpa and C. racemosa. The seabed within the 0.5-5 m depth range comprised a sandy bottom that is colonised by C. racemosa and native macrophytes, namely Cymodocea nodosa, C. prolifera and P. oceanica. The two seabed types (rocky and soft bottom) are separated by a small rocky step (Fig. 1b). Seven diVerent shallow habitats were selected for the present study. Two habitats were studied on hard bottoms (0.2-0.5 m depth): natural habitats of native seaweeds (A) and similar habitats invaded by C. racemosa (CrHB). On soft bottoms, Wve habitats were studied (2-5 m depth): soft bottoms colonised by C. racemosa (CrSB), Caulerpa prolifera beds (Cp), Cymodocea nodosa meadows (Cym), Posidonia oceanica meadows (Po) and unvegetated substratum (Sand). C. racemosa was distributed across the study area in patches of around 100 m 2 .

The two species of Caulerpa distributed in the study area, C. racemosa and C. prolifera, are algae of tropical and subtropical aYnity, and both are strongly aVected by natural seasonal disturbances in the Mediterranean Sea. Their growth and reproductive cycles are dependent on water temperature, with considerable decrease in spatial extension during winter and notable growth when temperature rises [START_REF] Meinesz | Contribution a l'étude des Caulerpales (Chlorophytes). Avec une mention particulière aux espèces de la Méditerranée occidentale[END_REF][START_REF] Piazzi | Développement et dynamique saisonnière d'un peuplement méditerranéen de l'algue tropicale Caulerpa racemosa (Forsskål) J. Agardh[END_REF]. Therefore, the seven habitats were sampled in September 2004 (summer conditions with an average water temperature of 27.5°C), which coincides with the period of maximum vegetative growth of C. racemosa [START_REF] Piazzi | Développement et dynamique saisonnière d'un peuplement méditerranéen de l'algue tropicale Caulerpa racemosa (Forsskål) J. Agardh[END_REF], and again in March 2005 (winter conditions with an average water temperature of 13°C), which is the period of minimum growth of C. racemosa.

Within each habitat (Ha) and sampling time (Ti), three sites (Si) were randomly selected. Each site corresponded to a diVerent patch of habitat with similar conditions that were separated by hundreds of meters. At each site (patch), three random samples separated by tens of meters were taken using a 20 £ 20 cm 2 quadrat by scraping the whole surface using a trowel [START_REF] Edgar | The inXuence of plant structure on the species richness, biomass and secondary production of macrofaunal assemblages associated with Western Australian seagrass beds[END_REF]). In soft bottom areas, samples were taken by SCUBA diving. A 300-m mesh bag was attached to the quadrat to avoid loss of the motile fauna. Samples were preserved in a 4% solution of formaldehyde in seawater. Each replicate was sieved in sea-water through a 500-m mesh, retaining the Wne fraction of detritus. In the laboratory, the caprellids were separated, identiWed and counted. Algae were sorted and identiWed to species level, and detritus was also separated. The macrophytes and the detritus were dried for 24 h at 80°C and weighed. Habitat structure was characterised using three attributes: species richness of macrophytes, biomass of each species (g) and quantity of detritus (g).

Data analysis

The aYnities among sampling habitats based on vegetal species biomass, and among caprellids based on their abun-dance in the diVerent habitats, were established by cluster analysis using UPGMA method and the Bray Curtis similarity index. Data were fourth root transformed. Multivariate analyses were carried out using the PRIMER package [START_REF] Clarke | PRIMER (Plymouth Routines In Multivariate Ecological Research) v5: user manual/tutorial[END_REF].

Total abundance, species richness and abundance of each caprellid species were analysed using ANOVA. To test whether the abundance of caprellids was similar across habitats and times, we used an analysis of variance (ANOVA), which incorporated the following factors: 'Time of sampling', a Wxed factor and orthogonal, with two treatments: September 2004 and March 2005; 'habitat', a Wxed factor, with seven treatments: native seaweeds on hard bottoms (A), C. racemosa on hard bottoms (CrHB), C. racemosa on soft bottoms (CrSB), Caulerpa prolifera beds (Cp), Cymodocea nodosa meadows (Cym), Posidonia oceanica meadows (Po) and unvegetated sandy bottoms (Sand); 'Site', a random factor and nested within both main factors, with three random sampling sites.

Prior to ANOVA, heterogeneity of variance was tested with Cochran's C test. Data were qx + 1-transformed if variances were signiWcantly diVerent at P = 0.05. Where variances remained heterogeneous, untransformed data were analysed, as ANOVA is a robust statistical test and is relatively unaVected by heterogeneity of variances, particularly in a balanced design [START_REF] Underwood | Experiments in ecology: their logical design and interpretation using analysis of variance[END_REF]). In such cases, special care was taken in the interpretation of results, and to reduce type I errors, the level of signiWcance was reduced to <0.01. When ANOVA indicated a signiWcant diVerence for a given factor, the source of diVerence was identiWed by applying the Student-Newman-Keul (SNK) test [START_REF] Underwood | Techniques of analysis of variance in experimental biology and ecology[END_REF][START_REF] Underwood | Experiments in ecology: their logical design and interpretation using analysis of variance[END_REF].

Results

Habitat description

A total of 25 taxa of macroalgae and two seagrass species were identiWed (Table 1). Vegetal biomass on natural hard bottoms was higher in March than in September, mainly due to the proliferation of Corallina elongata, Jania rubens, Dyctiota fasciola, Halopteris scoparia and Cystoseira compressa (these species represented 79% of total biomass). Jania rubens was the dominant species, together with Corallina elongata, which was the species with highest biomass in March. During September, hard bottom habitats invaded by C. racemosa were characterised by a low species richness of associated algae (Wve species plus C. racemosa), whereas species richness increased in March (14 species plus C. racemosa). In the case of soft bottoms, the diVerent ) values (n = 9) for the diVerent macrophyte species, together with total biomass, total species richness and detritus weight for the two sampling periods and 1), except for Caulerpa racemosa, which decreased in biomass in March, allowing other algae to colonise the substrata. In general, the amount of detritus was higher in March than in September, and the highest amounts were found with signiWcant diVerences at C. prolifera and C. racemosa patches on hard bottoms (Ha, P < 0.01). The most diverse habitats in terms of algal composition were the native hard bottoms and those invaded by C. racemosa, both on hard and soft bottoms.

The classiWcation analysis showed segregation of sampling sites according to the habitat, regardless of the season (Fig. 2). Bottoms dominated by Caulerpa prolifera and Posidonia oceanica clustered together with the sand habitat. These three ecosystems were the poorest in terms of macrophyte species richness. Natural hard bottoms and invaded habitats of C. racemosa formed a second group, characterised by high species richness of associated algae (the highest species richness was found on soft bottoms with C. racemosa; 17 species in March). A third group including the habitats of Cymodocea nodosa could be observed, with an intermediate number of vegetal species associated (Wve species in September and six in March plus C. nodosa).

Caprellid assemblages

The native algae on hard bottoms and C. racemosa on both soft and hard bottoms had the highest abundance and species richness of caprellids, with a peak in March, reaching a maximum value of 3,747 § 1,022 individuals/m 2 in native seaweeds. These diVerences were signiWcant (Ti £ Ha, P < 0.01; Table 2, Fig. 3). In C. prolifera, C. nodosa, P. oceanica and on sand, abundances were very low at both sampling times (Fig. 3). Although abundances were very high on hard bottoms, the number of caprellid species was low in all habitats. Natural hard bottoms and habitats invaded by C. racemosa had signiWcantly more caprellid species than the rest of the habitats in March but not in September (Ti £ Ha, P < 0.05, Table 2, Fig. 3).

Seven caprellid species were found during the present study: Caprella acanthifera (Leach, 1814), Caprella grandimana (Mayer, 1882), Caprella hirsuta Mayer, 1890, Caprella santosrosai Sanchez-Moyano, Jimenez Martín and [START_REF] Sánchez-Moyano | Caprella santosrosai n. sp. (Amphipoda: Caprellidea) from the Strait of Gibraltar (southern Spain)[END_REF], Deutella schieckei Cavedini, 1982, Phtisica marina Slabber, 1769 and Pseudoprotella phasma (Montagu, 1804) (Fig. 4). C. grandimana and C. hirsuta were the dominant species in terms of abundance. The abundance of C. hirsuta on algae was signiWcantly higher in March (Ti £ Ha, P < 0.01, 4). The abundance of C. grandimana was higher in March on hard bottoms (both habitat types) and C. racemosa on soft bottoms, although no statistical diVerences were found (Table 2, Fig. 4). Caprella grandimana, C. hirsuta and C. acanthifera were mainly associated to hard bottoms, whereas the remaining species were only found in soft bottom habitats. Deutella schieckei were exclusively found associated with Posidonia beds, while C. santosrosai (Ha, P < 0.01, Table 2, Fig. 4), P. marina and P. phasma were associated to Caulerpa beds, as shown by the cluster analysis based on the caprellid assemblage (Fig. 5).

Discussion

This study shows that caprellid assemblages diVer markedly among habitats, with considerable seasonal variation.

The presence of Caulerpa racemosa inXuences the spatial distribution of caprellid assemblages on both hard and soft bottoms, probably due to changes in the composition of vegetation and the increase in detritus levels. C. racemosa held reduced abundances of Caprella hirsuta, but other species such as C. acanthifera, C. santosrosai, P. marina and P. phasma were more abundant in this alga. The 'positive eVect' of C. racemosa on the caprellid community has also been found in habitats of the Balearic Islands, where the presence of C. racemosa signiWcantly favoured C. acanthifera and also enabled other caprellid species to colonise (Box et al. 2006).

Some caprellid species found in the present study, such as P. marina, P. phasma or C. acanthifera, have been reported as common species on many diVerent substrata (see Guerra-García 2001) and can tolerate stressed habitats of low Xow and high rates of sedimentation and organic matter (Guerra-García and García-Gómez 2001). Deutella schieckei is a Mediterranean endemic described as interstitial [START_REF] Krapp-Schickel | Suborder Caprellidea. In: S. RuVo. The Amphipoda of the Mediterranean[END_REF], and along the Spanish coast, this species has been only reported from algae in shallow waters [START_REF] Jimeno | Gammaridea and Caprellidea of the northest coast of Spain: ecological distribution on diVerent types of substrata[END_REF]Box et al. 2006). Caprella santosrosai was described from bryozoans at Algeciras Bay, Cádiz [START_REF] Sánchez-Moyano | Caprella santosrosai n. sp. (Amphipoda: Caprellidea) from the Strait of Gibraltar (southern Spain)[END_REF], and later it was collected from hydroids, anthozoans and sponges in North Africa [START_REF] Guerra-García | Habitat use of the Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa[END_REF][START_REF] Guerra-García | The Caprellidea (Crustacea: Amphipoda) from Ceuta, North Africa, with the description of three species of Caprella, a key to species of Caprella and biogeographical discussion[END_REF] and the present study represents the third locality in which this species has been found, after Algeciras Bay and Ceuta, enlarging its Several studies include data related to caprellids associated to diVerent species of Caulerpa, demonstrating that some species seem to be associated to the habitat. In a study on the eVect of wave exposure on the amphipod fauna of Caulerpa brownie in New Zealand, [START_REF] Fenwick | The eVect of wave exposure on the amphipod fauna of the alga Caulerpa brownie[END_REF] reported the caprellid Caprellina longicollis as one of the dominant amphipod species. In Cyprus, [START_REF] Argyrou | Expansion of the macroalga Caulerpa racemosa and changes in softbottom macrofaunal assemblages in Moni Bay, Cyprus[END_REF] concluded that the expansion of C. racemosa imposed successional changes on macrofaunal communities: gastropods and crustaceans decreased while polychaetes increased and became the dominant group. However, no caprellids were reported in their study and gammarids had surprisingly low abundances. Most studies dealing with the eVect of Caulerpa on macrofaunal communities from the Iberian Peninsula have dealt with C. prolifera beds. [START_REF] Sánchez-Moyano | EVect of the vegetative cycle of Caulerpa prolifera on the spatio-temporal variation of invertebrate macrofauna[END_REF], 2007) studied the invertebrate macrofauna from a C. prolifera bed in Algeciras Bay and reported the presence of the caprellids Phtisica marina and Pariambus typicus in low abundances. These authors concluded that C. prolifera allowed establishment of important crustacean assemblages and, in some cases, showed richer communities than more structured habitats, such as seagrass beds. However, Bellan-Santini (1995) found a decrease in the amphipod community in a Caulerpa taxifolia bed located on the Mediterranean French coast compared with a reference station. These observations suggest that changes in the epifaunal community due to the expansion of Caulerpa species diVer greatly from one location to another.

Caprellids showed considerable seasonal variation in abundance, with a peak during the cold season, reaching maximum values of thousands of individuals per square meter in native seaweeds. The high abundances of caprellids in the shallow habitats on the rocky terrace is possibly due to limited access of Wsh predators to these habitats, compared to the habitats on the deeper sandy substrata. Seasonal variation of invertebrate abundance is common in many vegetated systems [START_REF] Edgar | The inXuence of plant structure on the species richness, biomass and secondary production of macrofaunal assemblages associated with Western Australian seagrass beds[END_REF]) and the inXuence of biomass [START_REF] Ansari | Seagrass habitat complexity and macroinvertebrate abundance in Lakshadweep coral reef lagoons, Arabian Sea[END_REF], surface area [START_REF] Lewis | The distribution of macrobenthic crustaceans associated with Thalassia, Halodule, and bare sand substrata[END_REF] and epiphyte biomass [START_REF] Schneider | Species speciWc relationships of invertebrates to vegetation in a seagrass bed. II. Experiments on the importance of macrophyte shape, epiphyte cover and predation[END_REF] on epifauna have been demonstrated. This reduction of abundance during summer is related to the increase of Wsh abundance due to the recruitment of juvenile Wsh and movement of adults into P. oceanica meadows, which increases the predation pressure on epifauna (Bell andHarmelin-Vivien 1982, 1983). It is necessary to consider the eVects of predators on epifaunal abundance to explain changes in the assemblage composition of fauna between habitats. The abundance of potential prey results from a balance between refuge against predation and the availability of favourable space for living [START_REF] Orth | A perspective on plant-animal interactions in seagrasses: physical and biological determinants inXuencing plant and animal abundance[END_REF]. Our results showed diVerent abundances of caprellids between the two sampling periods, which could be explained by natural population dynamics or by the diVerent feeding intensities of predators over a 1-year period [START_REF] Page | Trophic links and condition of a temperate reef Wsh: comparisons among oVshore oil platform and natural reef habitats[END_REF]. At low temperatures, Wsh may cease to feed, but as the temperature increases, the rate of consumption also increases up to a maximum. [START_REF] Wootton | Ecology of teleost Wshes. Fish and Wsheries series 1[END_REF] detected an immediate disappearance of epibenthic caprellid amphipods in Weld surveys as a result of the appearance of demersal Wsh species [START_REF] Caine | Caprellid amphipods: fast food for the reproductively active[END_REF]. However, [START_REF] Edgar | The inXuence of seagrass structure on the distribution and abundance of mobile epifauna: pattern and process in a Western Australian Amphibolis bed[END_REF] reported that while predators should not be the main cause for reduction of the epifauna in substrata with vegetation, they might play an important role in determining the spatial distribution of epifauna.

In conclusion, our study shows that C. racemosa stands promote changes in caprellid assemblages, namely by playing an important role as a new benthic habitat. The observed abundances of caprellids were relatively high, but the species richness was generally low. This may have resulted from the close relationship of certain caprellid species with particular habitats. For example, C. hirsuta and C. grandimana were present in high densities, while P. marina and P. phasma had low abundances. The results of the present study also detected an important eVect of the time of sampling, probably due the synergistic eVect of seasonal changes in algal composition and predation pressure. Further studies should be conducted to determine top-down and bottom-up ecological regulations of caprellid populations in habitats invaded by C. racemosa.

Fig. 1

 1 Fig. 1 Map showing the study area in Santa Pola Cape, southeastern Spain (a) and transverse proWle of biotopes present in the study area (b)

  C. racemosa C. prolifera C. nodosa P. oceanica Sand Algae C. racemosa C. racemosa C. prolifera C. nodosa P. oceanica

Fig. 3

 3 Fig. 3 Total caprellid abundance (number of individuals/ m 2 § SE) and species richness (number of species per replicates § SE) recorded from the seven habitats and the two sampling times

Fig. 5

 5 Fig. 5 Cluster analysis based on caprellid abundances estimated within each habitat and sampling time

Table 1

 1 Mean biomass (g dw m ¡2

Table 2 ,

 2

	Fig. 2 Cluster analysis con-
	ducted using the mean values of
	macrophyte biomass within each
	habitat and sampling time. A,
	native algae; CrHB, Caulerpa
	racemosa hard bottoms; CrSB,
	Caulerpa racemosa soft bot-
	toms; Cp, Caulerpa prolifera;
	Cym, Cymodocea nodosa; Po,
	Posidonia oceanica; Sand, un-
	vegetated substrate; M, March;
	S, September

Table 2

 2 Results of ANOVA (three-factor) for caprellid total abundance, species richness and abundance of the seven species MS mean square, F F value, P level of probability, df degrees of freedom, ns non-signiWcant * SigniWcant at P < 0.05; ** signiWcant at P < 0.01

	Source of variation	df	Total abundance		Species richness		Caprella acanthifera	F versus
			MS	F	P	MS	F	P	MS	F	P
	Sampling time = Ti	1	29384.64	10.93	0.0026**	1.2419	15.05	0.0006**	16.79	1.00	0.3259	Si(Ti £ Ha)
	Habitat = Ha	6	14525.08	5.41	0.0008**	0.3064	3.71	0.0077**	16.79	1.00	0.4448	Si(Ti £ Ha)
	Ti £ Ha	6	13038.35	4.86	0.0016**	0.2338	2.83	0.0277*	16.79	1.00	0.4448	Si(Ti £ Ha)
	Si (Ti £ Ha)	28	2683.97	2.54	0.0006	0.0825	3.84	0.0000	16.79	1.00	0.4794	Res
	Residual	84	1058.45			0.0215			16.79
	Cochran's C test		C = 0.3806 (P < 0.01)	C = 0.1493 (ns)		C = 1.0000 (P < 0.01)
	Transformation		None			Sqrt(x + 1)		None
	Source of variation	df	Caprella grandimana	Caprella hirsuta		Caprella santosrosai	F versus
			MS	F	P	MS	F	P	MS	F	P
	Sampling time = Ti	1	1843.84	5.30	0.0290	15533.34	15.31	0.0005**	0.0079	0.50	0.4853	Si(Ti £ Ha)
	Habitat = Ha	6	437.59	1.26	0.3081	11476.53	11.31	0.0000**	0.0714	4.50	0.0026**	Si(Ti £ Ha)
	Ti £ Ha	6	398.43	1.15	0.3628	9979.60	9.84	0.0000**	0.0079	0.50	0.8029	Si(Ti £ Ha)
	Si (Ti £ Ha)	28	347.88	1.17	0.2897	1014.31	1.60	0.0521	0.0159	0.67	0.8869	Res
	Residual	84	298.23			633.50			0.0238
	Cochran's C test		C = 0.7873 (P < 0.01)	C = 0.5620 (P < 0.01)	C = 0.3333 (P < 0.01)
	Transformation		None			None			None
	Source of variation	df	Deutella schieckei		Phtisica marina		Pseudoprotella phasma	F versus
			MS	F	P	MS	F	P	MS	F	P
	Sampling time = Ti	1	0.6429	1.53	0.2266	0.1270	2.00	0.1683	0.0317	1.00	0.3259	Si(Ti £ Ha)
	Habitat = Ha	6	1.3413	3.19	0.0164	0.0529	0.83	0.5545	0.0317	1.00	0.4448	Si(Ti £ Ha)
	Ti £ Ha	6	0.6429	1.53	0.2055	0.0529	0.83	0.5545	0.0317	1.00	0.4448	Si(Ti £ Ha)
	Si (Ti £ Ha)	28	0.4206	2.04	0.0068**	0.0635	4.00	0.0000**	0.0317	1.00	0.4749	Res
	Residual	84	0.2063			0.0159			0.0317
	Cochran's C test		C = 0.5000 (P < 0.01)	C = 0.5000 (P < 0.01)	C = 1.0000 (P < 0.01)
	Transformation		None			None			None

  Cluster analysis based on caprellid abundances estimated within each habitat and sampling time

										C	p a	r	l e	l	a	g	r	n a	d	i	m	n a	a
										C	p a	r	l e	l	a	h	i	r	u s	t	a	D R A H	S M O T T O B
										p a C	a l l e r	n a c a	h t	a r e f i
										h P	a c i s i t	n i r a m	a
										P	u e s	d	o	p	r	o	t	l e	a l	p	h	a m s a	T F O S	S M O T T O B A P R E L U A C	W	I	H T
										p a C	a l l e r	n a s	t	o	o r s	i a s
										u e D	t	a l l e	h c s	i e k e i	A I N O D I S O P	S D E B
	0	20	40	60				80	100
			l i m i S	i r a	t	y	(	%	)

Acknowledgments This work was supported by Instituto Alicantino de Cultura Juan Gil Albert, Excma. Diputación de Alicante. Additional support of this work was provided by the Ministerio de Educación y Ciencia (Project CGL2007-60044/BOS) coWnanced by FEDER funds, and by the Consejería de Innovación, Ciencia y Empresa, Junta de Andalucía (Project P07-RNM-02524). We are very grateful to Damián Fernández and Teresa Rubio for their assistance in the Weld, Alfonso A. Ramos for help with the identiWcation of the seaweeds and Tim Dempster for improving the English version and constructive comments. We would like to thank two anonymous reviewers and the editor for their valuable comments.


Fernando G Brun
email: fernando.brun@uca.es

Elleke Van Zetten

Eva Cacabelos

Tjeerd J Bouma

K F G Reise

E Brun

Van Zetten

Role of two contrasting ecosystem engineers (Zostera noltii and Cymodocea nodosa) on the food intake rate of Cerastoderma edule

Keywords: Ecosystem engineers, Seagrasses, Biodiversity, Filter feeding rate, Cockles abundance, Habitat modiWcation

Seagrasses are well known ecosystem engineers that can signiWcantly inXuence local hydrodynamics and the abundance and biodiversity of macrobenthic organisms. This study focuses on the potential role of the seagrass canopy structure in altering the abundance of Wlterfeeding organisms by modifying the hydrodynamic driven food supply. We quantiWed the eVect of two ecosystem engineers with contrasting canopy properties (i.e. Zostera noltii and Cymodocea nodosa) on the food intake rate of a suspension-feeding bivalve Cerastoderma edule living in these seagrass meadows. Field experiments were carried out in two seagrass beds (Z. noltii and C. nodosa) and bare sediment, located on sandXat characterised by a relatively high hydrodynamic energy from waves and currents. Results demonstrated that the Wlter-feeding rate was almost twofold increased when C. edule was inhabiting Z. noltii meadows (1.10 § 0.24 g Chl g Fresh Weight ¡1 ) when compared to cockles living on the bare sediment (0.65 § 0.14 g Chl g FW ¡1 ). Intermediate values were found within C. nodosa canopy (0.97 § 0.24 g Chl g FW ¡1 ), but Wlter feeding rate showed no signiWcant diVerences with values for Z. noltii meadows. There were no apparent correlations between canopy properties and Wlter-feeding rates.

Our results imply that food refreshment within the seagrass canopies was enough to avoid food depletion. We therefore expect that the ameliorated environmental conditions within vegetated areas (i.e. lower hydrodynamic conditions, higher sediment stability, lower predation pressure…) in combination with suYcient food supply to prevent depletion within both canopies are the main factors underlying our observations.

Introduction

Over the past decades there has been a growing interest on seagrass-dominated areas as indicated by a rapidly increasing number of studies (e.g. [START_REF] Duarte | Seagrass ecology at the turn of the millennium: challenges for the new century[END_REF]; see references therein Larkum et al. 2006a). This has resulted in a much better knowledge of biological and ecological processes, spanning from the individual to the whole ecosystem level (Larkum et al. 2006b). From these studies, it has become clear that seagrasses are highly valuable ecosystems that provide several important ecosystem services [START_REF] Costanza R, D'arge | The value of the world's ecosystem services and natural capital[END_REF]). Many of these ecosystem services are related to the ecosystem engineering (sensu [START_REF] Jones | Organisms as ecosystem engineers[END_REF]) characteristics of seagrasses, which are known to be able to strongly ameliorate hydrodynamic conditions within their meadows (e.g. see [START_REF] Abdelrhman | EVect of eelgrass Zostera marina canopies on Xow and transport[END_REF][START_REF] Bouma | Trade-oVs related to ecosystem engineering: A case study on stiVness of emerging macrophytes[END_REF][START_REF] Koch | Beyond light: physical, geological, and geochemical parameters as possible submersed aquatic vegetation habitat requirements[END_REF]; Verduin and Backhaus 2000 and references therein).

One of the most important ecosystem services of seagrass beds is the support of a high biodiversity of invertebrates and Wshes [START_REF] Heck Kl | The nursery role of seagrass beds[END_REF][START_REF] Beck | The identiWcation, conservation, and management of estuarine and marine nurseries for Wsh and invertebrates[END_REF][START_REF] Heck Kl | Critical evaluation of the nursery role hypothesis for seagrass meadows[END_REF], some of them with a high economical value (e.g. blue crab; [START_REF] Pile | Density-dependent settler-recruit-juvenile relationships in blue crabs[END_REF]. These biodiversity eVects have been ascribed to a number of diVerent possible indirect mechanisms: (1) altered plant-animal interactions 123 (settlement of new recruitments; [START_REF] Eckman | Hydrodynamic processes aVecting benthic recruitment[END_REF][START_REF] Boström | Zoobenthic community establishment and habitat complexity-the importance of seagrass shoot-density, 123 morphology and physical disturbance for faunal recruitment[END_REF], (2) changes on the intensity of biological interactions (mainly predation-prey interactions; [START_REF] Nelson | Fish predation and habitat complexity: Are complexity thresholds real?[END_REF][START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF], (3) decrease on the Xow [START_REF] Fonseca | InXuence of the seagrass, Zostera marina L., on current Xow[END_REF][START_REF] Gambi | Xume observations on Xow dynamics in Zostera marina (eelgrass) beds[END_REF]) causing (3a) an increase on the sediment stability [START_REF] Fonseca | A comparison of canopy friction and sediment movement between four species of seagrass with reference to their ecology and restoration[END_REF][START_REF] Irlandi | The eVects of seagrass patch size and energy regime on growth of a suspension-feeding bivalve[END_REF]) and (3b) alterations on the food supply to the organisms [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF][START_REF] Allen | Native eelgrass Zostera marina controls growth and reproduction of an invasive mussel through food limitation[END_REF]. Especially the latter mechanisms are closely related to the ecosystem engineering ability of seagrasses, and raise some intriguing questions with respect to Wlter feeding macrobenthic animals that live in seagrass meadows.

Filter feeders normally beneWt from strong hydrodynamics that cause a higher refreshment rate of the food and a higher horizontal and vertical transport of particles on the water column [START_REF] Fréchette | Energy Xow between the pelagic and benthic zones: factors controlling particulate organic matter available to an intertidal mussel bed[END_REF][START_REF] Emerson | InXuence of sediment disturbance and water Xow on the growth of the soft-shell clam, Mya arenaria L[END_REF]. Hence, a reduced water movement typical for inside seagrass canopies [START_REF] Gambi | Xume observations on Xow dynamics in Zostera marina (eelgrass) beds[END_REF]) would be expected to reduce food availability and consequently the growth of Wlter-feeding organisms. However, a careful analysis of literature reveals rather contrasting results. Whereas some studies demonstrated that the growth rate and/or survival of Wlter feeder organism living within seagrass beds are lower than those occurring in non-vegetated areas (Reusch andWilliams 1999, Allen and[START_REF] Allen | Native eelgrass Zostera marina controls growth and reproduction of an invasive mussel through food limitation[END_REF], others concluded that these organisms obtain beneWts living inside vegetations [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF][START_REF] Irlandi | The eVects of seagrass patch size and energy regime on growth of a suspension-feeding bivalve[END_REF][START_REF] Peterson | Positive interactions between suspension-feeding bivalves and seagrass -a facultative mutualism[END_REF]. Food availability within seagrass canopy was acknowledged as a main factor responsible of such observations in both negative [START_REF] Allen | Native eelgrass Zostera marina controls growth and reproduction of an invasive mussel through food limitation[END_REF] and positive [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF] interactions.

The extent to which hydrodynamic energy within a meadow (and thus the food supply to Wlter feeders) is mod-iWed, is largely dependent on the canopy characteristics of the seagrass meadow (e.g. density, stiVness, length, etc.…; see [START_REF] Bouma | Trade-oVs related to ecosystem engineering: A case study on stiVness of emerging macrophytes[END_REF][START_REF] Koch | Beyond light: physical, geological, and geochemical parameters as possible submersed aquatic vegetation habitat requirements[END_REF][START_REF] Koch | Water Xow in tide-and wave-dominated beds of the seagrass Thalassia testudinum[END_REF]references therein). Hence, we speculate that contrasting results between studies on the growth and/or survival of Wlter feeder in seagrass meadows might perhaps be partly explained by contrasting seagrass canopy properties by having diVerent eVects on food supply. In order to elucidate the eVect of canopy properties on the food supply to Wlter feeding organisms inhabiting vegetated areas, we compared the food uptake of Cerastoderma edule in two seagrass species with contrasting canopy properties (Zostera noltii Horneman vs. Cymodocea nodosa (Ucria) Ascherson) and in the non-vegetated surroundings. Previous studies demonstrated that diVerences in canopy properties (shoots density, height and stiVness) caused the water Xux through the canopy of C. nodosa to be approximately double of that through a Z. noltii canopy [START_REF] Morris | Interaction between hydrodynamics and seagrass canopy structure: spatially explicit aVects on ammonium uptake rates[END_REF]. Therefore, we hypothesised that (a) both seagrass species will decrease the food intake rate of C. edule compared to non-vegetated areas and, (b) that the Wltration rate will be the lowest for cockles inhabiting Z. noltii beds. Our hypotheses were tested by measuring the chlorophyll content of starved cockles after 24 h feeding within a Z. noltii meadow, a C. nodosa meadow, or in the non-vegetated surroundings. Chlorophyll content was used as proxy for the food intake rate of the cockles, which is the resultant of the combination of food availability and Wlter-feeding rate [START_REF] Hummel | Food intake of Macoma balthica (Mollusca) in relation to seasonal changes in its potential food on a tidal Xat in the Dutch Wadden Sea[END_REF][START_REF] Kamermans | Food limitation in cockles (Cerastoderma edule (L): InXuences of location on tidal Xat and of nearby presence of mussel beds[END_REF].

Materials and methods

Field site characterisation

Our study was conducted in an intertidal sandXat at Los Toruños (el Bajo de la Cabezuela), a salt marsh ecosystem of 773 Ha situated in the Cadiz Bay Natural Park (Southern Spain, latitude 36°31Ј35.37ЉN, longitude 6°14Ј4.15ЉW). This sandXat, located at the mouth of a tidal channel, undergoes strong tidal currents, waves and winds with recurrent episodes of sediment erosion and accretion (see [START_REF] Brun | Evidence for vertical growth in Zostera noltii Hornem[END_REF] for further information and [START_REF] Kagan | Weak wind-wave/tide interaction over Wxed and moveable bottoms: a formulation and some preliminary results[END_REF] for a detailed physical description of the location). Mean velocity module measured 10 cm above the sea Xoor during a tidal cycle showed a velocity range from 5 to 30 cm s ¡1 (Lara M. personal communication) while wave heights varies from 0 cm on calm days to 15-20 cm on windy days [START_REF] Kagan | Weak wind-wave/tide interaction over Wxed and moveable bottoms: a formulation and some preliminary results[END_REF]. This sandXat is colonized by patches of the seagrass Z. noltii Hornem. in the intertidal areas, whereas more continuous beds of Z. noltii and Cymodocea nodosa (Ucria) Ascherson thrive in the lower intertidal and shallow subtidal zones. In the study site, Z. noltii develops in denser stands but with a shorter canopy height compared to C. nodosa (see [START_REF] Brun | Clonal building, simple growth rules and phylloclimate as key steps to develop functional-structural seagrass models[END_REF] for further details).

To determine the background chlorophyll a concentrations in the water column, at some of the days, water samples were taken by collecting the water at 50 cm depth some metres far away from the main leading edge of the meadow. This was done in triplicate per day, and repeated three times over the experimental period. In the lab, the water samples were Wltered (1 l; whatman GF/C) at low pressure and analysed after soaking the Wlter for 24 h on acetone (kept in cold and darkness; 90% buVered acetone). Chlorophyll a concentration (including phaeophytin) was subsequently measured using a Turner Xuorometer according the methods described by [START_REF] Strickland | A practical handbook of seawater analysis[END_REF]. Data are expressed as g Chl a l ¡1 . The average chlorophyll a concentration in the water samples was found to be 0.65 § 0.73 g Chl a l ¡1 (n = 3).

123

C. edule sampling and preparation Adult specimens of Cerastoderma edule were collected from bare sediment, 500 m upstream from the experimental location and transported within the next hour to the laboratory. For all experimental days and treatments, the cockles had an average size of 24.56 § 2.42 mm and an average fresh weight of 1.78 § 0.54 g FW ¡1 (n = 336). To starve the animals, all cockles were kept for 24 h in Wve aquaria with 40 l natural seawater, which was continuously bubbled with air. After this starvation period, 30 cockles per treatments (4-5 plots per treatment; in total 360-450 cockles) were selected and allocated in a non-labelled bag and transported to the Weld. Another ten starved cockles were frozen to estimate the initial values of chlorophyll content on the individuals. This basal value was subtracted every day from the experimental cockles to assess the food intake rate. This procedure was repeated every sampling day.

Experimental plot selection and carrying out the experiment

Mono-speciWc stands of Z. noltii and C. nodosa were selected in the shallow subtidal (15-20 cm water depth on low tide) of the study site. The experimental plots were selected taking into account several conditionals. First, the seagrass meadows had to exceed a radius of at least 10 metres. Second, the three treatment plots (bare sediment, Z. noltii and C. nodosa meadows) had to be relatively near to each other in order to obtain comparable hydrodynamic conditions. Third, the place to put the cockles were selected to not contain natural populations of cockles and to be far away from the leading edge of the meadow (i.e. usually in the midpoint of the meadow) to avoid any enhanced mixing due to leading edge eVects (e.g. see [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF][START_REF] Gambi | Xume observations on Xow dynamics in Zostera marina (eelgrass) beds[END_REF][START_REF] Folkard | Hydrodynamics of model Posidonia oceanica patches in shallow water[END_REF][START_REF] Bouma | Spatial patterns in Xowand sedimentation within vegetation patches: comparing Weld, Xume and hydrodynamic modelling experiments[END_REF].

Every sampling day (see below), four to Wve plots within each treatment (bare sediment, Z. noltii and C. nodosa meadows) were selected and marked with small wooden sticks during low tide. In these experimental plots (0.25 £ 0.25 m), the starved C. edule individuals were haphazardly distributed on the top of the sediment, reaching a Wnal density of 480 cockles m ¡2 , which was in good agreement with natural cockle densities in the area where cockles were previously collected (F.G. Brun unpublished data). After a tidal cycle (24 h) cockles and seagrasses were recollected from the experimental plots by extracting the sediment to 20 cm depth, sieved in the Weld and transported in cool boxes with ice till laboratory (less than 1 h). Once in the laboratory, cockles were frozen (¡20°C) and analysed on the following days (see C. edule analysis below). Seagrass samples were analysed on the same day (see seagrass analysis below).

The whole experimental procedure was repeated Wve times during June-July corresponding with the lowest tides occurring in the morning (experiments started between 9:00 and 10:00 am).

Chlorophyll analysis in C. edule analysis

The foot and body part of the frozen C. edule individuals were cut away from the shell and weighed. Anteroposterior axis of the shell was also measured. The chlorophyll content of the tissues was analysed spectrophotometrically after grinding and extracting it overnight in acetone (3 ml, buVered acetone 90%). Equations by JeVrey et al. (1997) were used to determine the concentration of chlorophyll a and b of the extracts. Initial data from the starved cockles were also measured every sampling day and this value was subtracted from the experimental values (cockles after 24 h on the Weld). Data are expressed as g Chl g FW ¡1 .

Characterisation of the seagrass meadows

Seagrass material collected together with the cockles was split into above and below-ground tissues. The number of shoots in the experimental plots was measured and abovebelow-ground biomass weighed after 48 h in an oven (60°C). Above-ground and below-ground biomasses were expressed as g Dry Weight m ¡2 . Sub-samples (20 shoots per plot) were used to measure the number of leaves per shoot and the length and width of individual leaves. Leaf length was measured with a ruler (accuracy 1.0 mm), while leaf width was measured with a calliper (accuracy 0.01 mm). Leaf area index (i.e. LAI; expressed as m 2 leaf/m ¡2 sediment surface) was calculated by considering just one side of the leaf.

Statistical analysis

DiVerences between treatments were tested by using two factors blocked ANOVA design. The levels of variability were the sampling days (n = 5) and the experimental plots (n = 63). This statistical design where plots are considered as the experimental units is more conservative than other possible alternatives as for instance, treating individual cockles within plots as independent replicates. DiVerences on canopy properties between both seagrass species were analysed by a one-way ANOVA. Multiple correlations were conducted between seagrass canopy properties and the recorded food intake rate of the cockles. Normality of the data and homogeneity of variances were previously tested [START_REF] Zar | Biostatistical analysis[END_REF]) and data were log transformed when necessary. Multiple post hoc comparisons between means were assessed by the Tukey test procedure [START_REF] Zar | Biostatistical analysis[END_REF]. Level of signiWcance ( ) was set at 0.05. Data are presented as mean § SD.

123

Results

In all the experimental plots, we were able to re-collect more than 95% of all Cerastoderma edule individuals, indicating that there are no diVerences in the survival rate between days and experimental plots during these 24-h experiments (Table 1). Chlorophyll content on the cockles after 24 h of feeding (proxy for food intake rate), showed a great dependence on the treatments (bare sediment, Z. noltii and Cymodocea nodosa meadows; Table 1 and Fig. 1). That is, chlorophyll content (after subtracting initial values from starved cockles, Fig. 1 showed data without subtracting) increased almost twofold from those cockles occurring at the bare sediment (0.65 § 0.14 g Chl g FW ¡1 ) to those cockles placed on Z. noltii meadow (1.10 § 0.24 g Chl g FW ¡1 ). Cockles from C. nodosa stands showed intermediate values (0.97 § 0.22 g Chl g FW ¡1 ) and were just signiWcantly diVerent from those cockles thriving on bare sediment (Fig. 1). Sampling day had no eVect on chlorophyll content, and there were also no interactions between sampling day and treatments (Table 1).

As was expected, most of the characteristics measured in both seagrass species showed signiWcant diVerences (Tables 2,3). Cymodocea nodosa rendered the longest and widest leaves compared to Z. noltii (Table 3), while shoot density was four times higher for Z. noltii (1,072 § 239 and 3,842 § 838 shoots m ¡2 for C. nodosa and Z. noltii). However, neither above-ground biomass nor LAI was statistically diVerent between species (Tables 2,3). Although multiple correlation between seagrass canopy properties and chlorophyll content on C. edule individuals were conducted, the food intake rate of cockles did not correlate signiWcantly with shoot morphometry (leaf length or width; data not shown), shoot density (r = 0.27, p = 0.06, n = 42), above biomass (r = 0.28, p = 0.07, n = 42) or leaf area index (i.e. LAI; r = 0.11, p = 0.47, n = 42) (Fig. 2a-c).

Discussion

To our knowledge, this is the Wrst study to quantify the eVect of two ecosystem engineering seagrass species with strongly contrasting canopy properties on the food intake rate of an active suspension feeder. In contrast, previous studies focussed on patch size and energy regime [START_REF] Irlandi | The eVects of seagrass patch size and energy regime on growth of a suspension-feeding bivalve[END_REF], bentic infaunal recruitment [START_REF] Boström | Zoobenthic community establishment and habitat complexity-the importance of seagrass shoot-density, 123 morphology and physical disturbance for faunal recruitment[END_REF], density manipulation of seagrasses [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF] and/or bivalves [START_REF] Reusch | Variable responses of native eelgrass Zostera marina to a non-indigenous bivalve Musculista senhousia[END_REF][START_REF] Peterson | Positive interactions between suspension-feeding bivalves and seagrass -a facultative mutualism[END_REF] and food availability [START_REF] Allen | Native eelgrass Zostera marina controls growth and reproduction of an invasive mussel through food limitation[END_REF]. Our results clearly demonstrate for both seagrass species Cymodocea nodosa and Z. noltii that Cerastoderma edule living within seagrass beds in a zone with a relatively high hydrodynamic energy, is able to increase the food intake rate relative to that in non-vegetated areas. No signiWcant diVerences were found between both seagrass species, despite that earlier studies indicated that their contrasting canopy properties caused a factor two diVerence in canopy Xuxes [START_REF] Morris | Interaction between hydrodynamics and seagrass canopy structure: spatially explicit aVects on ammonium uptake rates[END_REF]. See "Statistical analysis" section for further details on statistical procedure. Note that in the plotted data the chlorophyll content of the starved cockles has not been subtracted from the experimental data, while this was done in the data showed in the "Results" section To our surprise, both our hypotheses were rejected. Our Wndings were however quite robust, as the Weld experiment was run Wve diVerent times over a 2-month period. Hence, our results should integrate both for temporal variability in environmental conditions (e.g. hydrodynamics, food availability) plus the physiological status of the C. edule individuals, and for spatial variability in canopy properties. Furthermore, statistical design was conservative using experimental plots as replicates in a blocked design. Nevertheless, we found no signiWcant diVerences between sampling days and a highly signiWcant eVect of the vegetated areas, which underlines the strength of the patterns recorded.

Previous studies showed contrasting results related to the response of Wlter-feeding organisms to living in vegetated areas. While some studies showed a positive eVect on growth and survival of clams [START_REF] Peterson | The inXuence of seagrass cover on population structure and individual growth rate of a suspension-feeding bivalve, Mercenaria mercenaria[END_REF][START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF][START_REF] Irlandi | The eVects of seagrass patch size and energy regime on growth of a suspension-feeding bivalve[END_REF][START_REF] Peterson | Positive interactions between suspension-feeding bivalves and seagrass -a facultative mutualism[END_REF] other authors found the opposite [START_REF] Reusch | Macrophyte canopy structure and the success of an invasive marine bivalve[END_REF][START_REF] Allen | Native eelgrass Zostera marina controls growth and reproduction of an invasive mussel through food limitation[END_REF]. The former authors concluded that a combination of indirect factors promoted by the vegetation might be the responsible of such positive interactions: (a) alteration of the biological interactions (mainly predation-prey), (b) increment of the sediment stability and (c) changes on the food supply, while the later authors demonstrated that food limitation was the responsible for the recorded negative eVects. Our study concluded that the food intake rate by Wlter-feeding C. edule was enhanced in those individuals feeding within the canopy, which may promote a higher growth rate and survival when the long-term is considered. Unfortunately, our study cannot explain whether the observed increment on the food intake rate was (a) due to an increment on the food availability within the canopy as recorded by [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF], as we only measured the food availability as Chl a in the water column outside the meadows (see site description methods), or (b) due to a relative reduction on Wlter-feeding activity of those cockles living on the nonvegetated areas [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF][START_REF] Irlandi | The eVects of seagrass patch size and energy regime on growth of a suspension-feeding bivalve[END_REF]. Reduction on the Wlter-feeding rate of benthic organisms living on bare sediment can be caused by a number of stresses. For example, an increment of predation disturbances may reduce the feeding time [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF]. Although C. edule within the canopy might be expected to proWt from lower predation interference [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF], this does not seem likely in our system, as recovery was over 95% for all experimental plots. Furthermore, the evaluation of siphon nipping and interference by predators in the absence of physical contact between predator and prey, as suggested by the same authors was not estimated in our experimental design. Alternatively, feeding time may be reduced by the continuous hitting of the siphons by particles transported on the water [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF] or due to a high dynamic sedimentary which may imply a high cost of maintenance of the feeding position [START_REF] Peterson | Bivalve growth and higher order interactions: importance of density, site, and time[END_REF]. The latter may seem a likely explanation, as the experiment was carried out in a relatively exposed area with strong hydrodynamics and high sedimentary dynamics (see [START_REF] Brun | Evidence for vertical growth in Zostera noltii Hornem[END_REF].

Individuals of C. edule that live within a canopy can only proWt from a local reduction of the Xow regime (i.e. higher sediment stability, lower disturbance on the feeding activity; [START_REF] Irlandi | ModiWcation of animal habitat by large plants: mechanisms by which seagrasses inXuence clam growth[END_REF][START_REF] Irlandi | The eVects of seagrass patch size and energy regime on growth of a suspension-feeding bivalve[END_REF][START_REF] Fonseca | A comparison of canopy friction and sediment movement between four species of seagrass with reference to their ecology and restoration[END_REF], provided that the rate of food refreshment within the canopy is high enough to prevent food depletion. Food availability/depletion within the canopy will depend on the balance between the consumption and refreshment rate. Consumption is mainly dependent on the density, and Wlter feeding capacity and activity of the cockles, while food refreshment will depend of food availability on the surroundings water and in the Xux of water through the canopy. It is obvious that food depletion within the seagrass meadows was not likely to occur in present study, regarding the high food intake rate of cockles present within the seagrass meadows. Also the lack of any negative correlations between the Wlter-feeding rate and canopy properties (shoots density, above biomass or LAI; Fig. 2) and the great increase in chlorophyll content between starved and experimental cockles (two to four times; Fig. 1) strongly suggests that in our experiment the food supply within both contrasting canopies was not limited.

Based on current Wndings, we speculate that some of the contradictory literature reports on the eVect of vegetations on Wlter feeding might be explained by contrasting hydrodynamic conditions. In areas with relative strong hydrodynamics, as was the case in our study, food refreshment is high enough to prevent food depletion, whereas in more sheltered area this need not be the case. This would suggest that outcomes of ecosystem engineering by seagrass meadows on Wlter feeder performance has a conditional outcome, as has also been shown for other ecosystems [START_REF] Norkko | Conditional outcomes of facilitation by a habitat-modifying subtidal bivalve[END_REF]. Flume studies would oVer an interesting approach for future studies to resolve these questions, since the use of a Xume allows the manipulation of individual factors (e.g. hydrodynamic conditions, canopy properties, etc.) while maintaining a great control over the other controlling factors (predators, sediment stability, food availability in the water column, etc.). The conditional outcome of ecosystem engineering on biodiversity remains a research topic with much scope for future research.

8 a 21 Fig. 2

 212 Fig. 2 Regression plots of the chlorophyll content on Cerastoderma edule and canopy properties of seagrasses. a Shoots density. b Aboveground Biomass. c Leaf area index (LAI). Statistical results of the regressions are showed in the "Results" section

Table 1

 1 Statistical results of the two factors blocked ANOVA design examining the eVect of treatments (bare sediment, Zostera noltii and Cymodocea nodosa meadows) on the chlorophyll content (proxy for food intake rate) and survival rate of Cerastoderma edule from the oneway ANOVA test examining the diVerences on biological properties between both seagrass species EVects of two seagrass species in the chlorophyll content (resembling the food intake) of Cerastoderma edule assayed under Weld conditions. Data are presented as mean § SD (n = 21). DiVerent letters indicate signiWcant diVerences between treatments determined by post hoc Tuckey's test.

	Source of variation	DF	SS	F	p
	Chlorophyll content
	Treatment	2	2.24	7.35	0.0017
	Days	4	0.17	0.28	0.887
	Treatment £ days	8	0.35	0.29	0.967
	Error	48	7.32
	Survival rate
	Treatment	2	1.31	0.22	0.81
	Days	4	6.72	0.57	0.68
	Treatment £ days	8	5.76	0.25	0.98
	Error	48	176.0
	SigniWcance level ( ) was set at 0.05 of probability

Table 2

 2 Statistical results of the one-way ANOVA test examining the diVerences on biological properties between both seagrass species

	Source of variation	DF	SS	F	p
	Seagrass properties
	Above-ground biomass	1	2386	0.81	0.373
	Below-ground biomass	1	216464	40.60	<0.0001
	Shoot density	1	789540406	80.63	<0.0001
	LAI	1	0.07	0.07	0.789
	Leaf length	1	2208	81.4	<0.0001
	Leaf width	1	62.63	421.9	<0.0001
	Leaf per shoot	1	4.47	20.57	<0.0001
	SigniWcance level ( ) was set at 0.05 of probability

Table 3

 3 Zostera noltii and Cymodocea nodosa canopy and population characteristics

	Seagrass characteristics	Zostera	Cymodocea	n
		noltii	nodosa
	Above-ground	134.4 § 30	117.3 § 26	21
	Biomass (g DW m ¡2 )
	Below-ground	183 § 40 b	38.9 § 9 a	21
	Biomass (g DW m ¡2 )
	Shoot density (shoots m ¡2 )	3,842 § 838 b	1,072 § 240 a	21
	LAI (m 2 m ¡2 )	2 . 1 9 § 0.48	2.31 § 0.52	21
	Leaf length (cm)	15.4 § 3.4 b	29.8 § 6.7 a	21
	Leaf width (mm)	1.38 § 0.3 b	3.80 § 0.9 a	21
	Leaf per shoot	2.7 § 0.6 b	3.4 § 0.
	Data are expressed as average § SD. DiVerent letters indicate diVer-
	ences between species derived from the one-way ANOVA analysis

Acknowledgments Brun FG is supported by an individual Marie

Curie fellowship (MEIF- CT-2005-515071) and by a European Reintegration Grant (MERG- CT-2007-205675). E Cacabelos is supported by a post-doctoral grant from the Xunta de Galicia. This work was also supported by the Spanish Projects CTM2005-00395/MAR, the Dutch grants SBP/JK/2006-15 and SBP/JK/2007-32 and conducted as part of the EU funded network of excellence Marine Biodiversity and Ecosystem Functioning (MarBEF) responsive mode proposal D-4-4.2-2 "The role of native and/or invasive ecosystem engineers in explaining biodiversity". This is the NIOO publication number 4304.

Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.


A C Smaal
email: aad.smaal@wur.nl

B J Kater

J Wijsman

K A C Reise

B J Smaal

Kater

Introduction, establishment and expansion of the PaciWc oyster Crassostrea gigas in the Oosterschelde (SW Netherlands)

Keywords: PaciWc oysters, Biomass, Bed area, Invasion, Adjustment

The PaciWc oyster Crassostrea gigas was Wrst introduced as an exotic species by oyster farmers in 1964 in the Oosterschelde estuary (SW Netherlands). The initial phase is not well documented but Wrst natural spatfall was recorded in 1975. Excessive spatfall occurred in 1976 and this is considered the start of the expansion phase of the wild oysters. Oyster beds in intertidal and subtidal areas of the Oosterschelde estuary have been growing since. The development in the intertidal area has been reconstructed by using aerial photography, validated by ground truth in 2000-2002. In the subtidal areas extensive oyster beds have been detected by using side scan sonar; on hard substrates along the dikes coverage with oysters up to 90% locally has been recorded by scuba diving surveys. Expansion has also occurred into adjacent water bodies including the Wadden Sea. By forming resistant reefs the oysters induce structural changes in the ecosystem. It is concluded that bed area is still expanding while decrease of the fraction live animals may indicate adjustment of the stock size to the local conditions.

Introduction

The PaciWc oyster Crassostrea gigas has been introduced in 1964 in the Oosterschelde estuary, SW Netherlands, for aquaculture purposes. In the severe winter 1962/1963 the existing culture stock of Xat oysters Ostrea edulis suVered from mass mortality and the extensive traditional Xat oyster culture in this area faced an economic disaster. Indeed out of 120 million oysters only 4 million survived and more than 90% of the oyster companies stopped their business [START_REF] Gmelich-Meyling-Van Hemert | Vissen op Zeeuws water[END_REF]. To maintain the culture activities PaciWc oysters were introduced at an experimental scale from British Columbia (Canada) (Drinkwaard 1999a;[START_REF] Wolv | Non-indigenous marine and estuarine species in the Netherlands[END_REF]. The experimental introduction of various strains of PaciWc oysters was considered successful, and some farmers started with paciWc oyster culture (Drinkwaard 1999a).The Wrst signs of successful recruitment of PaciWc oysters in the Oosterschelde are reported by Drinkwaard (1999a) for 1975, a year with relatively high summer temperatures. In the following summer of 1976, water temperatures over 20°C prolonged for over a month and high recruitment success was recorded. Warm summers of 1982 and 1986 were again followed by large PaciWc oyster spatfall. As shown by [START_REF] Diederich | Introduced paciWc oysters (Crassostrea gigas), in the Northern Wadden Sea: invasion accelerated by warm summers?[END_REF] warm summers are considered as the main factor for excessive PaciWc oyster spatfall. It is likely that proliferation of the PaciWc oyster along Dutch coastal waters started in the mid seventies, promoted by increasing use of the species for aquaculture in the Oosterschelde, and the more frequent occurrence of warm summers. [START_REF] Reise | Are aliens threatening European aquatic coastal ecosystems?[END_REF] describe the development of invasive populations in subsequent phases of invasion. It starts with (1) arrival, then there is (2) the establishment phase with a small-sized population of colonists, in (3) the expansion phase: population size increases rapidly till a maximum is 123 reached, followed by (4) the adjustment phase when the population decreases to an equilibrium size.

For the Oosterschelde the development is described on the basis of a reconstruction by aerial photographs and ground truth of contours and biomass [START_REF] Kater | The potential of aerial photography for estimating surface areas of intertidal paciWc oyster beds (Crassostrea gigas)[END_REF]; RIVO/Imares reports).

Further proliferation has been documented for the Dutch Wadden Sea [START_REF] Wolv | Non-indigenous marine and estuarine species in the Netherlands[END_REF][START_REF] Smaal | Does the introduction of the PaciWc oyster Crassostrea gigas lead to species shifts in the Wadden Sea?[END_REF], the Niedersachsen Wadden Sea [START_REF] Wehrmann | The distribution gap is closed-Wrst records of naturally settled PaciWc oysters Crassostrea gigas in the East Frisian Wadden Sea, North Sea[END_REF], and the Schleswig-Holstein parts of the German Wadden Sea [START_REF] Reise | PaciWc oysters invade mussel beds in the European Wadden Sea[END_REF][START_REF] Nehls | Wadden Sea mussel beds invaded by oysters and slipper limpets: competition or climate control?[END_REF].

The aim of this paper is (1) to document the arrival and the establishment of the oysters, (2) to describe the expansion phase, and (3) to quantify the actual size of the oyster population of the Oosterschelde. We hypothesize that the characteristics of the oysters as bivalve Wlter feeders in combination with the ability to form resistant structures result in a positive feedback for further expansion. The question is if and as to how far adjustment of the population will occur and how this can be estimated.

Materials and methods

Study area

The Oosterschelde estuary (SW Netherlands) is a tidal system of 350 km 2 with intertidal Xats (110 km 2 ), deep gullies, artiWcial rocky shores for dike defense, and shallow water areas (Fig. 1). A storm surge barrier between the estuary and the North Sea protects the area from Xooding but is normally open, allowing a tidal range varying from 2.5 m at the entrance to 4 m at the eastern boundaries. The system has an average freshwater load of 25 m 3 /s and is mesotrophic with an average salinity of 30 ppt; there are no untreated waste water discharges [START_REF] Nienhuis | The Oosterschelde estuary, a casestudy of a changing ecosystem: an introduction[END_REF].

The area is important as nature conservation area and of particular relevance for wader birds such as the Oystercatcher that overwinters in large numbers. The area is extensively used for shellWsh bottom culture and cockle Wshery. There are 1,550 ha oyster culture plots, all located in the eastern part (Fig. 1)

Estimation of oyster bed area

Intertidal beds

Oyster bed contours have been registered by walking around oyster beds at low tide and identifying the contours with GPS. At each bend in the contour a waypoint was registered. Contours were used to construct maps of oyster beds in GIS. An oyster bed was deWned in a similar way as mussel beds (CWSS 2007), as a cluster of oysters of at least 50 cm diameter located in the vicinity of other oyster clumps with a maximum distance of 10 m.

Main eVorts in the Oosterschelde were made in 1999-2002. In 2003 and 2005 additional contour surveys were carried out in the eastern and central areas, respectively. Reconstruction of the development of intertidal oyster beds was based on aerial photography of the Oosterschelde for the period 1980, 1989-1991 and 1999-2002. For the latter period, aerial photographs were combined with Weld data for ground truth. [START_REF] Kater | The potential of aerial photography for estimating surface areas of intertidal paciWc oyster beds (Crassostrea gigas)[END_REF].

Subtidal beds

In March 2002 a side scan sonar survey was carried out in the gullies of the Oosterschelde. Registrations were done in all gullies of the Oosterschelde. The registrations have been checked by scuba diving. In total 11 Weld observations by diving were carried out in 10 areas. For soft sediment, oyster beds could be identiWed with side scan sonar with suYcient accuracy, although no quantitative data could be derived from the Wrst explorative survey.

Coverage and biomass estimation

During contour mapping surveys coverage of the oyster bed with oysters was estimated by using a frame of 1 m 2 surface with a Wner grid of rope that has 100 crosslinks. The frame was placed at random on the oyster bed and the number of crosslinks that covered an oyster was counted, giving percentage cover per m 2 .

Total number of oysters covered by the frame was removed, separated in live oysters and shell remains and wet weight was measured of both fractions. Part of the live samples (15 L) were taken to the laboratory for cooked Xesh weight estimation, dry weight estimation after drying for 48 h at 70°C, and ashfree dry weight estimation after ashing for 4 h at 520°C.

Experimental Wshery sites

In the framework of a project to test the possibilities for the removal of excessive oyster stocks, a number of oyster beds were selected in 2006 for experimental Wshery and impact monitoring. In total 50 ha high density oyster beds were selected: two intertidal areas of 12.5 ha each and two subtidal areas (Fig. 1). On these beds biomass samples were taken, using a large 1.062 m 2 Van Veen type grab onboard a dredging vessel. On each bed ten samples were taken and sorted out per sample; oysters were separated into live oysters and dead shell remains, and wet weight was measured of both fractions. Dry Xesh weight and ash-free dry weight was estimated of the living oysters.

Results

Development of intertidal oyster bed area

Aerial photographs taken in 1980 covered about 60% of the Oosterschelde intertidal areas. In particular, areas in the eastern part were missing. The surface area of oyster beds reconstructed in the photographed area is 15 ha. The total surface area of oyster beds can be estimated by correcting for the areas not photographed, assuming a homogeneous scattering of oyster beds. On this basis the total estimated surface in 1980 is 15-35 ha. This is probably an underestimation as the eastern part has always shown the largest bed area, apparently due to the initial introduction in this area (Fig. 2; Table 1).

In 1989-1991, 89% of the intertidal areas were covered by aerial photographs. The surface area of reconstructed oyster beds in photographed areas is 210-236 ha. The oyster map 2005 is made up of contours measured in the period 2000-2005, and aerial photographs of 1999 and 2000. The total surface area of the Oosterschelde covered with oyster beds is estimated as 775 ha. Figures 2,3,4 show the distribution of oyster beds in 1980, 1990 and 2005. Table 1 shows the development for the various parts of the Oosterschelde. In 2005 the coverage of the estuary with oyster beds was 6.8% of the total intertidal area, in the eastern part 9.2%, north 7.6%, central 5.1% and west 4.8%. This spatial gradient from east to west corresponds with the temporal development, given Wrst introduction in the eastern part.

Biomass and standing stock

A pilot study in 1998 on intertidal plots in the Oosterschelde showed high density spots with a biomass up to 110 kg/m 2 and areas with moderate densities (10-27 kg/m 2 ). The fraction of live oysters was on average 60% (Table 2) [START_REF] Perdon | Het bestand aan Japanse oesters op de platen van de Oosterschelde[END_REF] A detailed study was carried out in 2003 to estimate coverage and biomass on a number of tidal Xats. In total ca 80 quadrats of 1 m 2 were sampled and analyzed [START_REF] Gelderman | Oestersurvey 2003: onderzoek naar de bestandsgrootte van de Japanse oester in de Oosterschelde[END_REF]. Table 3 shows average total weight of oysters of 32.5 § 24.1 kg/m 2 , of which 38.9 § 12.6% were live oysters. The live oysters had an average Xesh content of 5.3 § 1.6%.

In 2006, samples were taken with a 1-m 2 grab on sites, pre-selected for experimental Wshery [START_REF] Wijsman | Wegvisproef Japanse oesters in de Oosterschelde, tussentijdse rapportage[END_REF]. The average total oyster mass of these intertidal beds was 30.2 kg/m 2 of which 33% consisted of live oysters. For subtidal experimental sites total oyster mass was 24.4 kg/m 2 of which only 18.8% were live oysters (Table 4).

On the basis of contour surveys, the total wet weight in 2005 of live animals was estimated as 89.4 mln kg, out of a total weight of 216.6 mln kg live + dead oysters including shells. The total stock was estimated as 1.01 mln kg ashfree dry weight (Table 5).

Subtidal bed area

A Wrst estimate of the area of subtidal oyster beds in the gullies and along the slopes gives a Wgure of 700 ha where oyster beds occur. This is based on a side scan sonar survey in combination with scuba diving. In total, 1,900 ha were surveyed and oysters were observed over all water depths up to 42 m. The approach did not allow quantiWcation to the standing stock

Discussion

Arrival and establishment

Aerial photography of oyster beds in the Oosterschelde was evaluated by [START_REF] Kater | The potential of aerial photography for estimating surface areas of intertidal paciWc oyster beds (Crassostrea gigas)[END_REF] and considered useful in surveying and reconstructing the development of intertidal PaciWc oyster beds. They have shown that the chances of false positives (i.e., a spot located as an oyster bed without being an oyster bed, or surface areas being overestimated) were on average 2.4%. It should be noted that there is a chance of underestimation. The veriWcation with ground truth showed that in some cases more than half of the surface of oyster beds was missed on the photographs (false negatives average value 53%). Hence the estimates should be considered as conservative.

Based on the reconstruction, it was concluded at least 25 ha of wild intertidal oyster beds had developed already in 1980. In 1990 this had expanded almost tenfold to 236 ha. The present paper shows that this Wgure has more than tripled to 775 ha in 2005.

These Wgures are consistent with the development of subtidal oysters in high densities on the artiWcial rocky shores in the Oosterschelde. These habitats have been surveyed in the western, central and northern part since 1985 by scuba diving, and a fast colonization by the oysters is reported. The coverage of hard bottoms in 1985 the northern part was 10% and around 1% in the west and central parts (no data of east were reported). In 2002, 2003).

There is also a considerable wild oyster stock in shallow areas and gullies. This stock is exploited for spat collection, Wshery on juvenile oysters and recently also for the extraction of shell material. No complete quantitative survey has been accomplished yet. On the basis of a side scan sonar survey there are indications that the colonization is similar to the intertidal areas, and a preliminary estimate of 700 ha subtidal oyster beds were recorded.

The arrival phase of colonization is documented by Drinkwaard (1999a, b). He describes the introduction of two strains of spat of 1 cm size from British Columbia in 1964 by an oyster farmer that were in water channels of a lobster park in Yerseke, bordering the Oosterschelde (Fig. 1). After one growing season the Kumamoto (K) strain had a size of 5 cm and the Miyagi (M) strain 10 cm length. After the following winter 96 kg of the M strain and 54 kg of the K strain were brought to oyster culture plot nr 183 of the Wshery institute (RIVO), located in the eastern part of the Oosterschelde. The oysters have been harvested in October and November 1965 with a yield of 415 kg M strain and 130 kg K strain (RIVO data). In 1966 further experiments were carried out with a strain from the Hiroshima area (Drinkwaard 1999a). No quantitative data have been recorded of the follow-up of these experiments. Registration of oyster landings show a Wrst commercial harvest of PaciWc oysters of ca 1,000 kg in 1976 (Drinkwaard 1999a). The arrival deWnitely started in 1964, but it is not clear whether this was suYcient as an onset of the establishment phase. It is likely that farmers have introduced PaciWc oysters several times from various origins. In 1969 and 1970 spat from British Columbia was introduced in France followed by introduction of adult oysters as broodstock in 1971 in Marennes-Oleron and Arcachon bay (Drinkwaard 1999b). There are indications that these stocks were also a resource for Dutch oyster farmers [START_REF] Wolv | Non-indigenous marine and estuarine species in the Netherlands[END_REF][START_REF] Gmelich-Meyling-Van Hemert | Vissen op Zeeuws water[END_REF].

Meanwhile commercial exploitation developed further. In 1988 the harvest amounted up to 1,000 tons wet weight and evolved since to an annual average yield of appr. 3,000 tons over the period 2000-2003. As the average culture cycle is 3 years and the average biomass production per unit spat is 3-5, the stock on culture plots is estimated at 6,000 tons wet weight, based on 1,000 tons seed, 2,000 tons half-grown and 3,000 tons consumption sized oysters.

Expansion in the Oosterschelde

The Wrst indications of the establishment phase date from the mid seventies, as spatfall was Wrst registered in 1975 (Drinkwaard 1999a). The following year with a 50-day period of water temperature above 20°C showed excessive spatfall, and can be considered as the start of the expansion phase. There are indeed clear indications that colonization is driven by recruitment, and that recruitment success is higher after warm summers [START_REF] Diederich | Introduced paciWc oysters (Crassostrea gigas), in the Northern Wadden Sea: invasion accelerated by warm summers?[END_REF]). As mentioned by Drinkwaard (1999a) there was excessive recruitment in 1976, 1982, and 1989 and this became more frequent in the nineties. In these years, summer water temperatures indeed were over 20°C for prolonged periods

123

(http://www.waterbase.nl). The increase of the frequency of warm summers as a possible eVect of climate change will promote the further development of the PaciWc oysters in Dutch coastal waters. The total bed area of the oysters in the Oosterschelde is still increasing. Expansion of the oyster beds is limited to areas relatively low in the intertidal zone. Almost all oyster beds have developed below mid-tide level. It is furthermore noticed that oyster beds in some cases have developed in areas that have been in use as intertidal mussel culture plots until the early nineties. This is particularly observed on the tidal Xat area in the central part (Fig. 5). After 1994, intertidal plots were abandoned by the mussel farmers as the government issued permits for expansion of culture plot to the more preferred subtidal areas. It is likely that the intertidal areas that were appropriate for mussel farming also are favorable for oysters and that the end of intertidal mussel culture implied the end of maintenance of the culture plots, including removal of oysters. Therefore development after 1994 may be faster than before.

Considering the oyster development for ca 40 years no signs have been recorded of serious damage to the oyster development due to climatic conditions (severe winters, warm summers), predation, or infestations. Also excessive summer mortality as occurs in France [START_REF] Soletchnik | Summer mortality of Crassostrea gigas in relation to environmental rearing conditions[END_REF] has not manifested. No clear external limits to the development have been identiWed in the Oosterschelde so far, as well as in the other colonized areas. The lower biomass in dense beds might be an indication of self-thinning, hence of adjustment to local conditions and this phenomenon needs further documentation.

Meanwhile the impacts are manifold and measures are being prepared to reduce the stock by management measures. Novel observations show larviphagy of various shell-Wsh larvae by the oysters, while oyster larvae may escape from Wltration [START_REF] Troost | Can bivalve veligers escape feeding currents of adult bivalves?[END_REF]. As hypothesized by [START_REF] Smaal | Does the introduction of the PaciWc oyster Crassostrea gigas lead to species shifts in the Wadden Sea?[END_REF], reef-building species have more resistance in comparison to infaunal species that show a more resilient response. As a consequence an invasive ecoengineering species that is relatively resistant to environmental stress may induce structural changes in the ecosystem. The oyster as a reef builder typically shows this type of response.

It results in a strong competition between the wild oysters and the other Wlter feeders. Growth rates and Xesh content of commercially exploited mussels, cockles and oysters in the Oosterschelde estuary are decreasing and the major questions are as to how far the wild oysters are impacting the carrying capacity of the system, and as to how far largescale removal of the oysters is an eVective measure to maintain various nature and production functions of the estuary (Wijsman and Smaal, in prep).

Expansion to other areas

The expansion of PaciWc oysters in The Netherlands is not limited to the Oosterschelde (see [START_REF] Wolv | Non-indigenous marine and estuarine species in the Netherlands[END_REF]. Already in 1987, spatfall was observed in the saline lake Grevelingen, a former estuary just north of the Oosterschelde (Drinkwaard 1999a). Also PaciWc oysters have been recorded in the Westerschelde estuary and along the North Sea coast. Just recently the observation was reported that PaciWc oysters have settled in the ports of Amsterdam (Parool newspaper, 28 Dec 2007).

Introduction in the Wadden Sea has not yet been clearly documented. It could be the result of larval transport from the Oosterschelde with the residual current in northern direction (Drinkwaard 1999a;[START_REF] Smaal | Does the introduction of the PaciWc oyster Crassostrea gigas lead to species shifts in the Wadden Sea?[END_REF] or occasional transport by mussel boats that Wsh in both Oosterschelde and Wadden Sea. However, it is more likely that introduction occurred in 1978 when the RIVO facilities on Texel were used for the culture of Xat oyster spat from a French hatchery. This spat turned out to be mixed with Larger specimen were brought to the outlet basin of the desalinization plant at Oudeschild, where water temperatures were elevated (J. Bol, pers comm.). From this nursery system expansion in the Wadden Sea may have started. Indeed, in 1983 Wrst records of wild oysters were documented near Oudeschild, a spot in the Wadden Sea close to the outlet of the basin close [START_REF] Bruins | Crassostrea gigas op Texel[END_REF]Drinkwaard 1999a;[START_REF] Wolv | Non-indigenous marine and estuarine species in the Netherlands[END_REF].

Further expansion in the Dutch Wadden Sea is described in [START_REF] Tydeman | Japanse oesters in de Eemshaven[END_REF], [START_REF] Dankers | De verspreiding en uitbreiding van de Japanse oester in de Nederlandse Waddenzee[END_REF][START_REF] Wolv | Non-indigenous marine and estuarine species in the Netherlands[END_REF] and [START_REF] Smaal | Does the introduction of the PaciWc oyster Crassostrea gigas lead to species shifts in the Wadden Sea?[END_REF]. During the annual shellWsh survey in the Dutch Wadden Sea in 2006 oysters were observed in densities >0.2 kg/m 2 on 5% of 1,415 sampled intertidal stations. On the basis of this survey and an extrapolation of earlier survey results, the oyster bed area was estimated at 323 ha, while mixed beds of oysters with mussels occupied 376 ha. The total intertidal area with PaciWc oysters is thus about 800 ha. The standing stock was estimated at 20.5 mln kg fresh weight on the basis of weight estimates at the sampled stations [START_REF] Goudswaard | Het bestand aan Japanse Oesters in het litoraal van Nederlands deel van de Waddenzee in 2004-2006[END_REF]. Results of subtidal surveys for mussel seed also show occurrence of wild-oyster beds of several hundreds of hectares (Van Stralen, pers comm.) The 800 ha intertidal area covered with oysters in the Wadden Sea is 0.64% of the total tidal Xat area, compared to 7% in the Oosterschelde; the standing stock is more than 30 times lower per unit area than in the Oosterschelde. The Wadden Sea development shows arrival and establishment until the end of the 1990s and an expansion phase that may have started in the period 1995[START_REF] Gelderman | Oestersurvey 2003: onderzoek naar de bestandsgrootte van de Japanse oester in de Oosterschelde[END_REF][START_REF] Wolv | Non-indigenous marine and estuarine species in the Netherlands[END_REF].

Adjustment

Biomass data show a decrease in the fraction live oysters in the Oosterschelde. In 1998, up to 60% was alive while in 2003 this was 39%, and on the experimental sites in 2006 the fraction of live oysters was on average 33% on the intertidal sites. Total biomass was maintained at approximately 30 kg/m 2 .

The decrease in the fraction of live oysters might be an indication of adjustment of the oysters to the local conditions.

Conclusions

In conclusion, the introductions of the PaciWc oyster in 1964-1970 have resulted in invasion of the Oosterschelde estuary that is still in progress. From the Oosterschelde, proliferation has occurred to the adjacent water bodies and is now expanding to the North. Warmer summers will stimulate further expansion of the oysters in areas that have already been colonized, as well as in new areas. For existing functions of coastal waters such as for shellWsh culture and also as feeding areas for shellWsh-eating birds this can be a serious threat. However, oyster reefs can also provide an important habitat for speciWc species.

The fraction of live oysters in the Oosterschelde seems to decrease, and this may indicate adjustment of the oysters to local conditions. Meanwhile, total bed area is still increasing. We hypothesize that on high-density spots the oyster development now shows signs of adjustment to local conditions in the Oosterschelde, while the population is still able to expand.

Fig. 1

 1 Fig. 1 Oosterschelde estuary with diVerent parts (west, central, north, east) including tidal Xats and oyster culture plot area in the eastern part. In central and east the sites of experimental oyster Wshery are shown: intertidal sites: a and b, subtidal sites c and d

Fig. 2

 2 Fig. 2 Locations of littoral oyster beds in 1980 (black dots, not scaled) on the basis of aerial photography; estimated oyster bed area 15-25 ha

Fig. 3 Fig. 4

 34 Fig. 3 Littoral oyster bed (black spots) reconstruction in 1990, estimated surface 210 ha

Fig. 5

 5 Fig. 5 Oyster bed contours and former mussel bed locations (in grey) in the central area of the Oosterschelde

Table 1

 1 Estimated intertidal oyster bed area in the diVerent parts of the Oosterschelde

		Intertidal	Oyster bed area
		area (ha)	1980	1990	2002	2005
			ha % ha	% ha	% ha	%
	West	2,389	4.8 0.20 21.4 0.90 91.8 3.84 114.5 4.79
	Central 3,334	3.3 0.10 52.6 1.58 180.8 5.42 170.5 5.11
	North	1,642	6 0.37 67.6 4.12 97.3 5.93 125.3 7.63
	East	4,003	0.7 0.02 70.8 1.77 270.1 6.75 364.9 9.12
	Total	11,368	14.8 0.13 212.4 1.87 640 5.63 775.2 6.82

Table 2

 2 Biomass (kg/m 2 ), % live oysters and % Xesh in diVerent parts of the Oosterschelde in early spring 1998

	1998	Biomass (dead and alive)		Live %	Flesh %
		Avg	SD	n
	West	38.0	36.9	6	53.5	5.5
	Central	34.8	43.5	9	70	5.6
	North	ND	ND	3	ND	10.6
	East	35.6	44.2	6	58	5.8
	A v g	3 6 . 1	4 1 . 5	2 1	6 0 . 5	6 . 9

Table 4

 4 Oyster biomass on litoral and sublitoral experimental Wshing plots, before Wshery in February 2006

			Total weight		Live weight	Fraction live
			kg/m 2 SD	n	kg/m 2	SD	%
	Intertidal 1		23.7	10.2 10 11.9	5.7	50.4
	Intertidal 2		34.7	16.6 10	8.4	5.2	24.1
	Avg intertidal 29.2	14.6 20 10.2	5.6	34.8
	Subtidal 1		22	13.2 10	4.9	2.8	22.2
	Subtidal 2		26.9	19.6 11	4.1	3.6	15.0
	Avg subtidal	24.6	16.6 21	4.5	3.2	18.2
	Table 5 Estimated intertidal stock size in 2005; data based on oyster
	bed contour mapping in the period 2003-2005 and biomass data of
	Table 3
	2005	Total wet	Total live	Total wet	Ashfree
		weight	weight		Xesh weight	dry weight
		(mln kg)	(mln kg)	(mln kg)	(mln kg)
	West	50.49	22.02		1.30	0.32
	Central	45.86	14.58		0.76	0.19
	North	38.84	14.49		0.83	0.21
	East	81.37	36.29		1.16	0.29
	Total	216.57	87.38		4.04	1.01

Acknowledgments

The authors would like to thank Jack Perdon, Emiel Brummelhuis and the crew of the Schollevaar and the RIOS for assistance in the Weld, and TNO-NITG for the side scan sonar survey. Dr N. Dankers and three anonymous reviewers are acknowledged for valuable complementary remarks and critical comments on the manuscript.

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