Cymodocea nodosa vs. Caulerpa prolifera: Causes and consequences of a long term history of interaction in macrophyte meadows in the Mar Menor coastal lagoon (Spain, southwestern Mediterranean)

Estuarine, Coastal and Shelf Science 110 (2012) 101e115

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Cymodocea nodosa vs. Caulerpa prolifera: Causes and consequences of a long term history of interaction in macrophyte meadows in the Mar Menor coastal lagoon (Spain, southwestern Mediterranean) Angel Pérez-Ruzafa a, *, Concepción Marcos a, Carmen M. Bernal a, Victor Quintino c, Rosa Freitas c, Ana Maria Rodrigues c, Marta García-Sánchez a, Isabel M. Pérez-Ruzafa b a Departamento de Ecología e Hidrología, Facultad de Biología, Campus Espinardo, Regional Campus of International Excellence “Campus Mare Nostrum”, Universidad de Murcia, 30100 Murcia, Spain b Departamento de Biología Vegetal I, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain c Department of Biology and CESAM, University of Aveiro, 3810 193 Aveiro, Portugal

a r t i c l e i n f o

a b s t r a c t

Article history: Received 20 January 2012 Accepted 2 April 2012 Available online 12 April 2012

Seagrass communities are under direct threat as a result of anthropogenic impact and competition with macroalgae. Present day macrophyte meadows in the Mar Menor are the result of successive colonization events, associated with changes in the hydrographical conditions. In the last of these events, Caulerpa prolifera (Forsskål) J.V. Lamouroux colonized the lagoon in the early 1970’s following the widening and deepening of the El Estacio inlet into a navigation channel, which caused a substantial decrease in the salinity level and a lower annual water temperature range. The seaweed C. prolifera expanded rapidly and replaced the previous seagrass meadows with monospecific algae meadows or with meadows mixed with the seagrass Cymodocea nodosa (Ucria) Ascherson. This work analyzes the factors that explain the distribution of macrophyte meadows and their evolution, as well as subsequent sediment alterations. The mean biomass of C. prolifera (Ca) in the deeper areas of the Mar Menor increased from 1982 to 1987 and remained constant thereafter until 2008, while the mean biomass of C. nodosa (Cy) showed a continuous decrease during the same period. The evolution of the Ca/Cy ratio showed a progressive expansion of C. prolifera monospecific meadows from north to south. The decline in the C. nodosa meadows was more pronounced in the deeper areas and was especially verified in the inner or more confined areas. At present, monospecific C. nodosa meadows are restricted to the southern and western, shallowest areas of the lagoon. The negative relationship between Caulerpa prolifera and Cymodocea nodosa shown by multiple regressions suggests that there is a competition between the two species. A complex interaction of factors could be responsible for the equilibrium and evolution of the relationships between both species in the Mar Menor. In shallow waters, coarse sediments, high light levels and physiological adaptations could favour C. nodosa. In deeper areas, light scarcity might limit C. nodosa biomass, but a negative effect of C. prolifera through increasing the sediment silt content, sulphide production, organic matter and anoxia, cannot be ruled out. Ó 2012 Elsevier Ltd. All rights reserved.

Keywords: coastal lagoon transitional waters macrophytes seagrass meadows competition Caulerpa prolifera Cymodocea nodosa

1. Introduction Seagrass meadows are one of the main structuring assemblages in marine coastal habitats. They also constitute one of the world’s most valuable ecosystems (Costanza et al., 1997), providing important ecosystem services in the form of nutrient cycling and

* Corresponding author. E-mail address: [email protected] (A. Pérez-Ruzafa). 0272-7714/$ e see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.ecss.2012.04.004

habitats for many fish, bird, and invertebrate species (Orth et al., 2006; Hughes et al., 2009; Waycott et al., 2009). They are one of the nursery habitats for nekton and nekto-benthonic species, favouring the density, growth and survival of juvenile fish and decapod crustaceans (Minello et al., 2003). Given that coastal lagoons are dominated by soft substrates and that rocky reefs are very scarce, the role of seagrasses in these environments is even more fundamental: for example, they introduce habitat structure and complexity as benthic primary producers, contribute to sediment stabilization and act as a refuge for organisms, especially the

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larval and juvenile stages of many species, both lagoon and migratory, many of them supporting important fisheries. Seagrass communities are under threat from a range of anthropogenic activities (Orth et al., 2006). However, although impacts from coastal development, eutrophication, degraded water quality and climate change have been documented, very few quantitative assessments of seagrass loss at local or global scales have been attempted. Waycott et al. (2009) found that seagrasses have been disappearing globally at a rate of 110 km2 yr
1 since 1980 and that 29% of the known area has disappeared since seagrass areas were initially recorded at the end of the 19th century. Furthermore, rates of decline have accelerated from a median of 0.9% yr
1 before 1940 to 7% yr
1 since 1990. This places seagrass meadows and the communities that depend on them among the most threatened ecosystems on earth (Hughes et al., 2009; Waycott et al., 2009). The above explains why seagrass beds are special protection habitats under most international legislation and treaties and why there is a growing concern about the gradual regression of these communities (Orth et al., 2006). The regression of seagrass meadows and their replacement by algal beds usually introduces drastic changes in the associated faunal assemblages (Bachelet et al., 2000; Sfriso et al., 2001; Pérez-Ruzafa et al., 2006) and fisheries (Perez-Ruzafa and Marcos, 1987). The decline in seagrass beds has been attributed to various causes, mostly anthropogenic (Orth et al., 2006), such as increased water level, turbidity or nutrient enrichment and the consequent decrease in light availability (Best et al., 2001; Leoni et al., 2008), the instability produced by moorings (Hastings et al., 1995) or coastal works (Pérez-Ruzafa et al., 1991, 2006), changes in sediment grain size or organic matter content (Pergent-Martini et al., 2006; Calleja et al., 2007; Boudouresque et al., 2009), or competition with algae. However, the main cause for the replacement of seagrass beds by fast-growing seaweed beds is often attributed to eutrophication (Sand-Jensen and Borum, 1991; Duarte, 1995; Sfriso et al., 2003; Burkholder et al., 2007; Obrador and Pretus, 2010). An increase in the rate of organic matter supply or of nutrient availability in the system, which enhances primary production, leads to the gradual replacement of seagrasses and slow-growing macroalgae by fast-growing macroalgae, and phytoplankton, with a final dominance of phytoplankton at high nutrient loads (Likens, 1972; Nixon, 1995; European Environment Agency, 2001; Gamito et al., 2005). Nutrient over-enrichment leads to light reduction through the stimulation of algal overgrowth as epiphytes and macroalgae in shallow coastal areas, and as phytoplankton in deeper coastal waters, and also favours herbivore grazing (Pergent-Martini et al., 1996; Ruiz et al., 2001; Boudouresque et al., 2009). Direct physiological responses, induced by ammonium toxicity, water column nitrate inhibition through internal carbon limitation, salinity alterations (Charpentier et al., 2005), high temperature or low light levels may also contribute to exacerbating the adverse effects of nutrient over-enrichment and to accelerating seagrass disappearance, including sediment re-suspension from seagrass loss (Burkholder et al., 2007). In the 1960’s, the Mar Menor was extensively covered by several species of seagrass forming low density patches (Cymodocea nodosa (Ucria) Ascherson, Zostera marina Linnaeus, Zostera nana Roth, Ruppia maritima Linnaeus), as reported by many authors (Lozano, 1954; Simonneau, 1973). In the 1970’s, as a result of the widening and deepening of the El Estacio inlet communicating with the Mediterranean sea, the Mar Menor experienced drastic changes in the hydrographical conditions. These were associated with a rapid expansion of the seaweed Caulerpa prolifera (Forsskål) J.V. Lamouroux, leading to monospecific algal meadows or mixed meadows with the seagrass Cymodocea nodosa (Pérez-Ruzafa et al., 1987).

In the Mediterranean, mixed beds of Cymodocea nodosaeCaulerpa prolifera are considered a climax biocenosis (Meinesz pers. comm.) and Terrados and Ros (1991) questioned the idea that Cymodocea had been reduced at the expense of Caulerpa proliferation, suggesting that the seagrass had always been scarce in the deeper areas of the lagoon. Recent work has attributed this change to a eutrophication process due to the input of nutrients through the Albujón watercourse and to the increase of suspended solids in the water column, following alterations in agricultural practices in the Mar Menor watershed (Lloret et al., 2005). However, Terrados and Ros (1991), in previously unpublished data also showed that a rapid replacement of phanerogam meadows by mixed C. nodosaeC. prolifera and, later, monospecific C. prolifera beds occurred independently of changes in the trophic status of the lagoon associated with the Albujon watercourse. In the present study, we analyze the interaction between species and the factors that explain the distribution of macrophyte meadows and their evolution in the Mar Menor lagoon, as well as the subsequent alterations that followed in the sediment characteristics.

1.1. Study area The Mar Menor is a hypersaline Western Mediterranean coastal lagoon (Fig. 1). It has an area of 135 km2 with a mean and maximum depth of 3.6 m and 6.1 m, respectively. Five volcanic islands and small outcrops constitute the only natural rocky sustrata inside the lagoon. La Manga, a sandy bar 22 km long, acts as a barrier between the lagoon and the Mediterranean Sea. It is crossed by five inlets, called “golas”, of which the three most northern are natural and they were the only ones to communicate with the Mediterranean until the mid-nineteenth century. The other two, El Estacio, in the central area of La Manga, and Marchamalo or La Constanza in the south, were artificially constructed in 1860 for fisheries (Navarro, 1927). This lowered the highest salinity values from above 70 to about 55 and probably was the origin of the colonization of the lagoon by phanerogams, mainly Cymodocea nodosa (Butigieg, 1927; Navarro, 1927). In 1973 El Estacio was widened, up to 35 m wide in its minimum section, and deepened to 5 m depth, to make it a navigation channel. The lagoon has a restriction ratio (the ratio between the total width of the lagoon inlets and the parallel shore direction, Chubarenko et al., 2005) of 0.01 (Pérez-Ruzafa et al., 2005b), classifying the Mar Menor as a restricted lagoon (Kjerfve, 1994). The annual total inflow of freshwater through runoff and rainfall into the lagoon results in a hydric deficit ranging from 38 to 115 Hm3 a year (Pérez-Ruzafa et al., 2005b). Until recently, there were no permanent freshwater sources flowing into the lagoon. There are, however, more than twenty cataclinal watercourses in the watershed, most of them in the southern part of the lagoon, which collect rainfall from the surrounding mountains. This water is mostly lost through evaporation and infiltration before reaching the lagoon, except during torrential rainfall (Lillo, 1981). The single exception is the Albujón watercourse, which keeps a regular water flow into Mar Menor due to the changes in agricultural practices that have occurred since 1995 (Pérez-Ruzafa et al., 2002). The lagoon hydrodynamics is mainly driven by wind. The water exchange between the lagoon and the Mediterranean Sea is mainly through El Estacio inlet and is driven by phase differences in the sea level (Arévalo, 1988). Three main gyres can be identified in the general circulatory pattern along this axis, differentiating three basins in the Mar Menor: 1) the northern basin, with the lowest salinity level; 2) the southern basin, with the highest level as a consequence of the hydric deficit and the greatest degree of confinenment and 3) the central basin, showing intermediate

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Fig. 1. Location of the Mar Menor lagoon and organization of the sampling area in zones (Z1eZ5) for spatial analyses.

salinity values and corresponding to the mixing area of Mediterranean and lagoon waters. The water temperature distribution is relatively uniform over the whole Mar Menor with a few local exceptions, mainly related to the shallowest areas. Maxima of around 30  C are usually reached in August and minima in February (around 11  C). The southern basin is warmer in summer and colder in winter, but differences from the northern basin are usually less than 2  C at any time of the year (Pérez-Ruzafa et al., 2005a, 2005b). Before 1973, the salinity of the lagoon reached 53e54.7 (Navarro, 1927; Lozano, 1954; Arévalo and Aravio-Torre, 1969; Aravio-Torre and Arévalo, 1971; Moreno, 1975). Following the El Estacio inlet works, the water salinity range is now from 39 to 51 (Ros and Miracle, 1984; Pérez-Ruzafa et al., 1987, 2005a; unpublished data), and shows a north-south gradient (Pérez-Ruzafa et al., 2005b). Turbidity and suspended materials are highly variable, depending on distance to the coast, depth, the nature and slope of the bottom, planktonic productivity, wind and rainfall. Values range from 2 mg/l of suspended solids in calm water conditions on rocky bottoms to 6 g/l on muddy or sandy bottoms under the waves’ action (Pérez-Ruzafa et al., 2005b). Mean annual values for 2006e2009 were always lower than 0.08 g/l (unpublished data). Soft sediments dominate the Mar Menor sea-bottom. Muddy bottoms extend through the whole central area of the lagoon and the shallower parts of lower hydrodynamism. Sandy bottoms, with a sand content of up to 89%, are located at the margins of the basin and in small bays surrounding the islands (Pérez-Ruzafa et al., 2005b). Natural rocky bottoms are very scarce and only occur around the islands and in the form of some calcareous and volcanic outcrops. The widening and deepening the El Estacio inlet induced major changes in the lagoon dynamics. Modification of the water renewal rate had profound effects upon salinity and temperature, in turn permitting access to new coloniser species and altering the lagoon’s community structure with detrimental effects on fisheries (PérezRuzafa et al., 1991). Also contributing to the overall change, agriculture in the watershed has changed since 1986 from extensive dry crop farming to intensively irrigated crops, using water diverted

from the Tagus river, located 400 km north, to the Segura river. The introduction of irrigation water decreased the use of ground water and raised the phreatic levels (Pérez-Ruzafa and Aragón, 2003), in turn inducing a continuous flow of freshwater through the Albujón watercourse (about 24 L/s) into the Mar Menor since the 1990s, fed by ground water with high nitrate levels. This flow is at present the main source of nitrates into the lagoon and of pesticides into the trophic food web (Pérez-Ruzafa et al., 2000, 2005b). 2. Material and methods 2.1. Sampling and sample analyses The evolution of the spatial distribution of macrophyte meadows in the Mar Menor lagoon has been investigated using various data sources. A basic grid consisting of 20 sampling stations, evenly distributed across the lagoon at depths from 2.1 to 6.1 m, was sampled with a van Veen grab covering a sampling area of 400 cm2 (Fig. 2a). Quantitative samples were taken quarterly from summer 1982 to spring 1983 (see Pérez-Ruzafa et al., 1989) and in the spring of 1987 (33 sites in the 1987 survey). The most recent sampling campaign was in late springeearly summer 2008, coinciding with an acoustic survey (Quintino et al., 2010). The van Veen grab was weighted so as to ensure good penetration of the coarser substrates and the efficient sampling of both the relatively shallower stolons of Caulerpa and the deeper and stronger rhizomes of Cymodocea. Data from shallow stations (<2 m depth and <300 m from the coast) were obtained in different studies carried out from 1982 to 2007 (Terrados, 1986; Pérez-Ruzafa and Marcos, 1987; PérezRuzafa, 1989, 2003, 2007, 2008; Hegazi, 1999) (Fig. 2b). Sampling in the shallower sites was performed by diving and hand-collecting the vegetation in a square of 400 cm2 (20  20 cm). Most of the data corresponds to the spring-summer season, but some studies contain seasonal (Pérez-Ruzafa, 1989) or monthly (Terrados, 1986; Hegazi, 1999; Pérez-Ruzafa et al., 2008) data. Considering the whole study period, the results presented here were obtained for a total of 544 samples, 277 in shallow waters and 267 in deeper areas. In all the studies, the samples were washed in

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a

b

Fig. 2. Location of sampling sites: a) at depths from 2.1 to 6.1 m; and b) in shallow waters at depths <2 m. Isobaths for 2, 4 and 6 m are represented.

the laboratory using clean sea water to remove sediments, and the species Cymodocea nodosa and Caulerpa prolifera were separated. Biomass as dry weight (DW) was determined after removing epiphytes from the fronds or leaves, by drying to constant weight at 80  C, with a precision of 0.001 g. Hydrographic (hydrodynamism, nutrient concentration in the water column, pH, temperature and salinity) and sediment (grain size, organic matter, carbonates and heavy metals) characteristics were recorded for most of the samples during the collection of macrophytes or compiled from the literature for the respective sampling sites and dates. The water column is vertically well mixed, with no significant differences in temperature or salinity with depth (Pérez-Ruzafa et al., 2005a, 2005b). In the deeper sites, the water samples were taken at an approximate depth of 1 m with a Niskin bottle, or by pumping. At the shallower sites, all samples were taken by diving. Samples for nutrient analysis were kept in the dark at 4  C in the field and stored at
28  C. Nitrate (NO3eN), nitrite (NO2eN), ammonia (NH4eN) and phosphate (PO4eP) were determined following Parsons et al. (1984). Prior to 2005, salinity was determined with a Beckman RS 7B salinometer and pH was measured with a pre-calibrated meter. After 2006, in situ determinations were performed using a WTW Multiline F/Set3 multiple probe. Chlorophyll a was analysed with the spectrophotometric methods reported by Parsons et al. (1984). At the shallower sites, hydrodynamism, represented by wave exposure, was estimated according to Keddy (1983) and the value was corrected by depth and the expected wave effect on the bottom. According to the maximum period of waves in the Mar Menor, bottoms deeper than 2 m were not considered to experience a significant wave effect. The residence time was computed for each one of the 20 sampling areas constituting the basic grid, according to Umgiesser and Cucco (2010) at the end of a year’s simulation using a hydrodynamic finite element model (De Pascalis et al., in press). Sediment samples for grain size evaluation and other analyses were collected by diving. The samples were stored and transported in darkness and cold in polyurethane bags. In the laboratory they were dried at room temperature and sifted to a maximum size of 2 mm. Grain size was determined by the Bouyoucos hydrometer

method (Soil Conservation Service, 1973) after dispersion of clusters by mechanical stirring in a sodic-hexameta phosphate and Na2CO3 solution. Previously, salts were eliminated by washing and centrifugation, and organic matter by hydrogen peroxide treatment. Grain size classification followed the International Association for Soil Science (Duchaufour, 1975). Organic carbon was determined by the WalkleyeBlack method (Buchanan, 1984) and total nitrogen by the Kjeldahl method (Bremner, 1965). Heavy metals (total Pb, Zn, Cd, Cu, As and Fe) were analyzed in the <2 mm sediment fraction, after grinding to a fine powder and digested in Teflon vessels using concentrated HF and HNO3 acid solution, for 15 min at 1000 W in a Milestone ETHOS PLUS microwave. Zn, Fe and Mn were determined by flame atomic absorption spectrometry (FAAS), while Pb, Cd and Cu contents were determined by electrothermal atomization atomic absorption spectrometry (ETAAS). Arsenic was analysed by atomic fluorescence spectrometry using an automated continuous flow hydride generation (HG-AFS) spectrometer (Navarro et al., 2008). To represent the relative importance of Caulerpa prolifera (Ca) and Cymodocea nodosa (Cy) in the mixed meadows and to draw the border between these and the monospecific beds, the Ca/Cy ratio described by Pérez-Ruzafa et al. (1989) was used. This ratio uses quantitative (biomass) data and can be expressed as follows:

I ¼ log

ðCa þ 1Þ ðCy þ 1Þ

Ca and Cy being the mean biomass per site and species, in g (Dw) m
2. In the Mar Menor, this ratio can take values of between þ2 and
2. According to Pérez-Ruzafa et al. (1989), a meadow of CymodoceaeCaulerpa is mixed if the Ca/Cy ratio lies between þ1 and
1, and is monospecific for Caulerpa if I  þ1, and monospecific for Cymodocea if I 
1. 2.2. Data analysis To analyse the biomass evolution of the Caulerpa and Cymodocea meadows, sampling sites were grouped into 5 zones according to the main hydrological basins described for the Mar Menor and

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different terrestrial and marine influences (Fig. 1) (Pérez-Ruzafa et al., 2005a, 2005b). Each zone includes at least four sampling stations. The biomass of Caulerpa prolifera, of Cymodocea nodosa and the Ca/Cy ratio were analysed with a 2-factor ANOVA (Underwood, 1997), considering the orthogonal factors: Year (1982, 1983, 1987, 2008) and Zone (1e5). Both sources of variation were considered as fixed factors. To compare the behaviour in shallow and deep areas, samples from all depths were grouped into two periods, 1982e1988 and 2002e2008, representing, respectively, the situation of meadows before and after the eutrophication process started. Due to the heterogeneity in the number of samples at each area and period we performed a permutational analysis of the variance (PERMANOVA) on Euclidean distances (Anderson, 2001, 2005) using a random subset of 9999 permutations. By using permutations, the test requires no specific assumption concerning the number of variables or the nature of their individual distributions or correlations (Anderson, 2001). The experimental design consisted of three factors: Period (Pe) (fixed), with two levels (1982e1988 and 2002e2008); Zone (Zo), with five levels (Z1e5), considered fixed according to the main hydrographical basins and marine and runoff influence; and Depth (De), considered fixed with two levels (shallow <2 m and deep >2 m). Significant terms were investigated using a posteriori pairwise comparisons with the PERMANOVA tstatistic. Relationships between the biomass of Caulerpa prolifera and Cymodocea nodosa and the hydrographic, nutrients and sedimentological variables were tested for each independent variable and their quadratic and cubic terms using multiple linear regression models (GLM) with stepwise backward selection of variables (using p < 0.05 as the inclusion and/or rejection criterion). An additional multiple regression model considered the whole set of variables except heavy metal concentration in the sediments, which were excluded due to the lack of data in several sites. The relationship between the sediment and hydrological variables and the biomass of Caulerpa prolifera and Cymodocea nodosa meadows in deeper bottoms was studied by redundancy analysis (RDA) of the data collected in the 20 sites sampled in 2008 using Canoco 4.5 version. All calculations were performed on the covariance matrix, with square root transformed data. The relative contribution of each variable to the ordination established by the RDA was evaluated using a Monte Carlo permutation test after performing a forward selection of variables at a 0.1 level of signif icance (ter Braak and Smilauer, 2002). The analysis was repeated using only the 17 variables with highest conditional effects so that the number of explanatory variables was lower than the number of response variables, confirming the results. Furthermore,

105

environmental factors may respond to a merely spatial ordination due to confinement (marine influence gradients) or depth. Therefore, we separated both the effects according to the methodology proposed by Borcard et al. (1992) using the 20 sites sampled in May 2008. By performing two canonical ordinations, each one constrained by a set of explanatory variables, one achieves the overall species importance value for the effects imposed by: (1) the spatial conditions (x, y geographic coordinates of each sampling station) and (2) the hydrographic, trophic and sedimentological characteristics. In the aforementioned analyses, the amount of variation in biomass data due to the influence of space on environmental conditions was extracted by partial canonical ordination (RDA) of the species and space matrices while controlling for the effect of the environmental descriptors, and via the partial canonical ordination of the species biomass and environmental data sets, controlling for space, using, in both cases, the other matrix as covariate (Borcard et al., 1992). By using x and y coordinates in variation partitioning, only linear spatial gradients could be verified. Unfortunately, except salinity, temperature and dissolved oxygen, no other environmental data were sampled in the 1982e1987 samplings. This precludes us from analyzing the factors affecting the distribution of the meadows when the Mar Menor was oligotrophic and nutrient inputs were low. Therefore, to know the relative role played by non-trophic environmental variables we analyzed the effect of trophic variables discriminated from the other hydrographic and sedimentological factors, applying, to the 2008 data, the same partition of the variance methodology as described above but, instead of space coordinates, using each environmental matrix as covariable of the other in the respective canonical analyses. 3. Results 3.1. Evolution of patterns in the distribution of Caulerpa and Cymodocea meadows The mean biomass of Caulerpa prolifera in the deeper areas of the Mar Menor (sites located below 2 m depth), increased from 63.62 to 103.06 g (DW) * m
2 from 1982 to 1987 and presented that same value in 2008, while the mean biomass of Cymodocea nodosa showed a decrease from 49.61 to 2.69 g (DW) * m
2 in the same period (Fig. 3). The results of the analysis of variance (Table 1) point to significant differences in Cymodocea nodosa biomass between zones (p ¼ 0.039) and years (p ¼ 0.028). The analysis performed with the Caulerpa prolifera biomass data also shows a significant interaction term (zone  year) (p ¼ 0.031), indicating a different temporal

Fig. 3. Evolution of the mean biomass (g Dw * m
2  se) of C. prolifera and C. nodosa in the deeper areas of the Mar Menor lagoon from 1982 to 2008.

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Table 1 ANOVA results (F-values and associated significance) performed on C. nodosa and C. prolifera biomass data and Ca/Cy ratio in Mar Menor, considering the spatial factor zone (with five levels) and the temporal factor year (with four levels, 1982, 1983, 1987 and 2008). Significant values (p < 0.05) are in bold. Source

df

MS

F

p

C. nodosa biomass Zone 126.914 Year 114.778 Zone*year 216.147 Error 1394.314

SS

4 3 12 115

31.729 38.259 18.012 12.124

2.617 3.156 1.486

0.039 0.028 0.139

C. prolifera biomass Zone 145.196 Year 131.958 Zone*year 284.156 Error 1369.253

4 3 12 115

36.299 43.986 23.68 11.907

3.049 3.694 1.989

0.02 0.014 0.031

Ca/Cy ratio Zone Year Zone*year Error

4 3 12 115

3.262 2.942 1.271 0.902

3.615 3.26 1.408

0.008 0.024 0.172

13.05 8.826 15.247 103.787

dynamic at each zone. This is illustrated in Fig. 4. Zone 1 showed a nearly monospecific C. prolifera meadow throughout the period, with a rapid initial expansion, reaching 221.1 g (DW) * m
2 in 1983, and a reduction in subsequent years. The minimum, 12.91 g (DW) * m
2, was observed in 1987. This decrease in zone 1 was, however, accompanied by a strong expansion in zones 2, 3, 4 and 5 with a C. prolifera mean biomass of between 93.91 and 128.99 g (DW) * m
2. In 2008, the biomass of Caulerpa increased again in zone 1 and decreased slightly in zones 4 and 5. The evolution of the Ca/Cy ratio (shown in Fig. 5) indicates a gradual expansion, from north to south, of Caulerpa prolifera monospecific meadows, with mixed beds and monospecific Cymodocea nodosa meadows, mainly in the southern and western shallowest areas of the lagoon. Permanova analyses performed considering the factors Period (Pe), Zone (Zo) and Depth (De) for Cymodocea nodosa biomass found significant differences for the factors Depth (p ¼ 0.0105) and PexZo (p ¼ 0.0102) (Table 2). Cymodocea nodosa reached a mean 60.47 g (DW) m
2 in shallow areas and 18.67 g (DW) m
2 in the deeper zones. However, this difference was more evident in the 2002e2008 period than previously. Maximum biomass was reached in summer (51.73 g (DW) m
2 in 1982e1988 and 131.02 g (DW) m
2 in 2002e2008). Although no differences were detected between the periods, a slight decreasing tendency in mean biomass can be observed in the deep areas during spring, autumn and winter and an increase in mean biomass during summer, both in shallow and deep areas in the period 2002e2008 (Fig. 6). Pairwise comparisons showed that the differences between zones also changed between both periods. In 1982e1987 there were differences between zone 1 and zones 2 (p ¼ 0.0108) and 5 (p ¼ 0.0001), between zone 4 and zones 2 (p ¼ 0.0009), 3

(p ¼ 0.0213), and 5 (p ¼ 0.0001) and between zones 3 and 5 (p ¼ 0.0122). In 2002e2008 only zone 4 differed from zones 2 (p ¼ 0.0182) and 5 (p ¼ 0.008). Caulerpa prolifera showed significant differences between periods (p ¼ 0.0013) and for the interaction ZoxDe (p ¼ 0.0426). It significantly increased its mean biomass in all the areas between the periods considered (p ¼ 0.0013), from 76.16 g (DW) m
2 in 1982e1988 to 174.53 g (DW) m
2 in 2002e2008. Deep meadows showed significant differences between zone 5 and zones 3 (p ¼ 0.039) and 4 (p ¼ 0.0378) and a marginal difference from zone 2 (p ¼ 0.0517). Shallow areas only showed significant differences between zones 1 and 2 (p ¼ 0.0179). The Ca/Cy ratio showed significant differences between periods (p ¼ 0.0336) and the interaction ZoxDe (p ¼ 0.0455). An expansion of Caulerpa prolifera monospecific meadows occurred in deep areas and an increase in the C. proliferaeCymodocea nodosa mixed meadows occurred in shallow areas to the detriment of C. nodosa monospecific meadows, mainly in zones 2 and 3. Deep meadows differed only between zones 4 and 5 (p ¼ 0.0005) while the shallow meadows showed differences between zones 1 and 2 (p ¼ 0.0125), the former dominated by C. prolifera monospecific or mixed meadows and the latter by abundant C. nodosa monospecific patches.

3.2. Relationship between environmental variables and the biomass of Caulerpa and Cymodocea Table 3 shows the mean values and standard error of the mean for the variables considered in this work in the Cymodocea nodosa and Caulerpa prolifera monospecific meadows and in the CymodoceaeCaulerpa mixed meadow. Table 4 shows the results of the regression analyses. Most of the relationships between macrophyte biomass and explanatory variables are non-linear. Caulerpa prolifera showed the highest significant (p < 0.005) relationship with salinity (adjusted R2 ¼ 0.44), Temperature (adj. R2 ¼ 0.43), nutrients (adj. R2 ¼ 0.58) and percentage of saturation of dissolved oxygen (adj. R2 ¼ 0.86) in the water column and with depth (adj. R2 ¼ 0.32), gravel (adj. R2 ¼ 0.49), grain size (mainly coarse silt; adj. R2 ¼ 0.48), organic carbon (adj. R2 ¼ 0.50), total nitrogen (adj. R2 ¼ 0.44), carbonate (adj. R2 ¼ 0.39), copper (adj. R2 ¼ 0.38), and iron (adj. R2 ¼ 0.65) in the sediments. In general, C. prolifera showed a positive relationship with nitrate, nitrite, ammonia and silicate, but a negative relationship with high concentrations of phosphate (>0.7 mmol*L
1). Cymodocea nodosa biomass is related with grain size (with a strong negative relationship with coarse silt and a positive relationship with coarse sand and clay; adj. R2 ¼ 0.35), Chlorophyll a concentration (adj. R2 ¼ 0.68), nutrients (with a strong positive influence of phosphate; adj. R2 ¼ 0.32), but moderately positive influence at low concentrations (<25 ppm) of copper (R2 ¼ 0.24), and a strong negative influence of suspended solids and depth (adj.

Fig. 4. Mean biomass (g DW * m
2  se) of C. prolifera and C. nodosa in Mar Menor per zone (deeper areas only) and year.

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107

Fig. 5. Temporal changes in the spatial distribution of C. prolifera (red) C. nodosa (green) and mixed meadows (yellow), expressed as Ca/Cy ratio, in the deeper areas of the Mar Menor from 1982 to 2008.

R2 ¼ 0.44). Furthermore, it had weaker relationship than Caulerpa but significant with salinity (adj. R2 ¼ 0.18). The resulting models from the multiple regression analyses performed for all the variables together (except heavy metal concentration in the sediments) showed that Caulerpa prolifera meadow biomass was best explained by depth, fine sand and fine silt, while Cymodocea nodosa biomass increased with the coarse sand content in the sediment, and decreased with Caulerpa biomass and wave exposure (Table 5). The first two axes of the RDA analyses performed on the 20 stations sampled in the deep areas of the lagoon in 2008 explained 99.9% of the variance of the specieseenvironment relation. The first axis explained 63.3%. Fig. 7 represents tri-plot ordination diagrams

for species biomass and Ca/Cy ratio, samples and environmental descriptors. Only variables with a high and significant conditional effect are represented (p < 0.05). The analyses using the 17 variables with the highest conditional effects produced the same results with a very small (<0.1%) reduction in the explained variance. The variable with the highest marginal and conditional effect was organic matter content in the sediment (explained variance lambda A ¼ 0.24, p ¼ 0.008). The results of the analyses showed Cymodocea nodosa biomass to be associated with the positive part of axis II, that is, linked to the highest nitrate concentrations and salinity and low levels of organic matter in the sediments. Caulerpa prolifera biomass was associated with the negative part of axis I and linked to the maximum concentration of nitrite and phosphate, the

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Table 2 Results of PERMANOVA on Euclidean distances for C. nodosa and C. prolifera biomass and Ca/Cy ratio in the Mar Menor lagoon, showing the effects of the factors period (Pe: 1982e1988 and 2002e2008), zone (Zo: 1e5), depth (De: shallow, <2 m and deep, <2 m) and their interactions, indicating the factors for which significant variation (p < 0.05) exists (bold figures). Source

df

SS

MS

C. nodosa Pe Zo De PexZo PexDe ZoxDe pexZoxDe Res Total

1 4 1 4 1 4 3 345 363

3.5562 553.14 98.399 206.4 18.238 44.035 97.306 5238.5 6259.6

3.5562 138.29 98.399 51.599 18.238 11.009 32.435 15.184

C. prolifera Pe Zo De PexZo PexDe ZoxDe PexZoxDe Res Total

1 4 1 4 1 4 3 360 378

15.466 2.3811 42.195 8.6862 0.39136 15.081 3.9507 538.74 687.22

Ca/Cy ratio Pe Zo De PexZo PexDe ZoxDe PexZoxDe Res Total

1 4 1 4 1 4 3 360 378

6.494 7.3569 36.626 6.7942 4.6365 14.037 10.592 516.93 673.05

Pseudo-F

p (perm)

0.23421 9.1073 6.4804 3.3982 1.2011 0.72501 2.1362

0.6186 0.0001 0.0105 0.0102 0.2737 0.5652 0.1003

15.466 0.59528 42.195 2.1715 0.39136 3.7702 1.3169 1.4965

10.335 0.39778 28.196 1.4511 0.26152 2.5194 0.88

0.0013 0.8105 0.0001 0.2184 0.612 0.0426 0.4545

6.494 1.8392 36.626 1.6985 4.6365 3.5091 3.5307 1.4359

4.5226 1.2809 25.507 1.1829 3.229 2.4439 2.4588

0.0336 0.2763 0.0001 0.3196 0.0741 0.0455 0.0573

highest values of fine silt, organic matter content of sediments and the highest residence time. The reciprocal influence between sediment characteristics and the type of meadow is shown in Figs. 8e10. Fig. 8 shows the size grain characteristics of the sediments associated with the Cymodocea nodosa, Caulerpa prolifera and mixed CymodoceaeCaulerpa meadows in the Mar Menor and the evolution of these characteristics with time. Cymodocea meadows are restricted to bottoms with a high sand content. Fig. 9 shows the biomass distribution of C. nodosa meadows as a function of the organic matter and fine particles in the sediment. The presence of Cymodocea is very rare and the grass has very low biomass when the concentration of organic matter is greater than 7% and the percentage of fine particles (silt and clay) is greater than 40%. Fig. 10 shows the evolution of organic matter content in the sediment associated with the three types of meadow. The sediments associated with C. prolifera meadows have suffered a significant increase in organic matter content, exceeding 25% in some localities, while sediments with C. nodosa meadows maintain low values of organic matter. Partitions of the variance showed that the present distribution of grasslands depends more on the environmental conditions than on a defined linear spatial structure. Environmental variables explain 76.2% of total variance, while covariation of spaceeenvironment constitutes only 23.9%. There is no purely spatial structure in the macrophyte meadows distribution. The partition of variance between trophic conditions in the water column as a measure of eutrophication and other environmental variables in the water column and in the sediment shows that trophic conditions in the water column explain only 21.2% of the characteristics of the meadows, while the other environmental variables explain 56.5%. The covariation of both types of variables explains an additional 20.5%. Only 1.8% corresponds to unexplained variations and stochastic fluctuations.

Fig. 6. Seasonal variation of mean biomass (g DW *m
2  se) of C. prolifera and C. nodosa in the shallow and deep areas of the Mar Menor lagoon in the periods 1982e1988 and 2002e2008.

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Table 3 Mean values and standard error of the mean (se) for the variables considered in this work in the C. prolifera (CaM) and C. nodosa (CyM) monospecific meadows and in the CymodoceaeCaulerpa (CyeCaM) mixed meadow. Variable (units)

Abrev.

CaM

se

CyeCaM

se

CyM

se

Depth (m)

DEPTH

3.34

0.11

2.16

0.15

1.36

0.11

Gravel (%) Coarse sand (%) Fine sand (%) Coarse silt (%) Fine silt (%) Clay (%) Organic carbon (%) Organic matter (%) Total nitrogen (%) C/N ratio Carbonate (%) Salinity Temperature ( C) Suspended solids (g/L) Wave exposure Disolved oxigen (% saturation) Disolved oxigen conc. (mg/L) Chlorophyll a (mg/L) NO3 (mmol/L) NO2 (mmol/L) NH3 (mmol/L) PO4 (mmol/L) SiO4 (mmol/L) Cu (mg/Kg) Zn (mg/Kg) Cd (mg/Kg) Pb (mg/Kg) Mn (mg/Kg) Fe (mg/g)

GRAV C_SAND F_SAND C_SILT F_SILT CLAY OC OM N_TOT CN CO3 SALIN TEMP SS Wave DOS DOC CHL_A NO3 NO2 NH4 PO4 SIO4 CU ZN CD PB MN FE

23.71 35.44 23.34 7.99 15.10 18.09 4.88 8.39 1.64 3.69 44.52 44.99 21.68 0.04 5263.8 112.62 7.79 0.10 2.64 0.16 5.55 0.54 31.58 40.17 1215.72 8.91 959.53 440.00 27.82

2.22 2.34 1.55 0.55 1.21 1.33 0.27 0.62 0.29 0.99 1.99 0.23 0.60 0.00 568.3 1.93 0.20 0.01 0.65 0.05 0.82 0.05 3.70 7.83 224.21 1.27 207.33 0.00 4.74

16.85 51.70 18.66 3.40 6.39 10.47 1.06 1.82 0.97 2.29 40.94 43.96 19.18 0.04 11,748.8 110.20 8.98 0.21 59.48 1.52 17.54 1.28 26.74 24.48 236.88 2.83 192.38 246.00 14.88

5.66 5.48 2.53 0.65 1.65 2.38 0.20 0.60 0.18 0.70 3.49 0.38 0.97 0.00 1220.7 7.00 0.59 0.09 20.39 0.49 1.71 0.33 4.15 14.08 155.13 1.29 157.40 246.00 13.39

8.53 74.17 16.77 0.91 1.77 6.37 0.80 1.37 1.63 2.36 50.92 44.68 19.96 0.07 14,617.8 e 9.25 0.06 0.95 0.11 6.39 0.78 e 7.72 246.12 1.92 188.77 1000.00 16.40

1.82 1.90 1.22 0.14 0.32 0.71 0.12 0.23 0.46 1.13 4.18 0.37 0.90 0.01 2154.9

4. Discussion The macrophyte meadows that exist in the Mar Menor are the result of successive colonization events in the lagoon, associated with changes in hydrographical conditions. In the early 1860’s, several storms tore open the sandbar separating the lagoon from the Mediterranean. This, together with the opening of two new artificial inlets for fisheries (El Estacio, in the middle of the lagoon, and La Constanza or Marchamalo in the south), lowered the highest salinity values from above 70 to about 55. This allowed the colonization of several seagrasses, mainly Cymodocea nodosa (Navarro, 1927), which were, at the time, the only macrophytes in the deeper areas of the lagoon (Butigieg, 1927; Navarro, 1927; Lozano, 1954). Colonization by Caulerpa prolifera took place in the early 1970’s after the widening and deepening of El Estacio inlet into a navigation channel. This induced an important decrease in salinity and a smoothing of the temperature amplitude, mainly as a result of raising the lower values (Pérez-Ruzafa et al., 1989, 2005b). According to Meinesz (1979), C. prolifera cannot withstand temperatures below 10  C, which were common in winter, before the inlet works. In 1977 Garcia Carrascosa (1982) reported than most of Mar Menor bottom was covered by extensive Cymodocea nodosa meadows, with interspersed fronds of C. prolifera. In some depressions, in the deepest areas, on muddy substrate, the same author reported high densities of Caulerpa. Following its introduction into the Mar Menor in the early 1970’s, Caulerpa prolifera expanded rapidly. The alga spread from north to south, covering mainly the deepest areas of the lagoon (Pérez-Ruzafa et al., 1989). Some authors related this process to the nutrient inputs influenced by the Albujón watercourse mistaking a positive correlation with a causeeeffect relationship and ignoring the temporal framework of the Mar Menor’s recent history. In the present study, however, it is shown that the biomass of C. prolifera

0.35 0.01 0.26 0.01 1.43 0.10 3.28 184.89 1.64 186.40

in 2008 was similar to that of 1987, suggesting that the spread of Caulerpa took place at least a decade before changes in the agricultural practices in the Mar Menor watershed, which then altered the hydrological functioning of the Albujón watercourse. In contrast, the phanerogam Cymodocea nodosa is showing a gradual but continuous decrease in mean biomass throughout the lagoon. The present work shows that this decline is more pronounced in the deeper areas and has become more apparent in the inner or more confined areas of the lagoon, where, in 1987, it still grew in monospecific meadows or was still important in mixed meadows (Pérez-Ruzafa et al., 1987, 1989). The causes of this decrease may be diverse. Pérez-Ruzafa (1989) and Pérez-Ruzafa et al. (1989) cited as possible causes of the spread of Caulerpa prolifera beds at the expense of the pure stands of Cymodocea nodosa, the increasing demographic stress placed on the lagoon (mainly from domestic pollution), the construction of marine sports facilities, the dredging of parts of the lagoon bottoms, the filling-in of some beaches, the input of terrigenous allochthonous materials due to torrential rains and the enlargement of inlets. Some of these, such as the instability associated with the dredging and pumping of sediments, were later confirmed and the consequences for sediment characteristics (increase in silt-clay fractions and in organic matter content) and for faunal assemblages have been described (Pérez-Ruzafa et al., 1991, 2006). Other studies, performed in Southeast Asia, showed that seagrass species richness and community leaf biomass, including some species of the genus Cymodocea, declined sharply when the silt and clay content of the sediment exceeded 15% (Terrados et al., 1998). In the Mar Menor, Cymodocea nodosa predominates in sandy bottoms with a high coarse sand content. Furthermore, phosphate and chlorophyll a concentration in the water column enhance its biomass. In contrast, its growth seemed to be limited by coarse silt and by a high percentage of clay and organic matter in the

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Table 4 Results of regression analysis for mean values of biomass (g (Dw) m
2) for C. nodosa and C. prolifera. Only significant relationships are included. N: number of cases included in the analyses; see Table 3 for abbreviations of variable names. ^2 and ^3 represent quadratic and cubic terms, respectively. Shaded areas: the variables explaining more than 30% of the biomass variation of each species. C. nodosa

C. prolifera

N

Adj. R

Variable

Coefficient

p

N

Adj. R2

Variable

135

0.145

183

0.475

GRAV GRAV^2 GRAV^3 C_SAND C_SILT C_SAND^2 CLAY^3

180

0.089

183

0.495

51

0.271

51

0.441

141 171 121

0.180 0.148 0.437

0 0 0.003 0 0 0.004 0 0.023 0 0 0 0.001 0.02 0.034 0.009 0.007 0 0 0 0.009 0.003 0.007 0.045

0.491

0.351

10.322
0.292 0.002
46.298 0.019 4.445
0.167
0.123 10.475 48.6
9.35 0.405 27.26
32.549 9.545
0.554 0.001 0.101 1957.22
31,507.87 100.01
46.30 4.54

138

180

GRAV GRAV^2 GRAV^3 C_SILT C_SAND^2 C_SILT^2 CLAY^2 C_SILT^3 CLAY OC OC^2 OC^3 N_TOT CN CN^2 CN^3 SALIN^3 TEMP^2 SS SS^3 DEPTH DEPTH^2 DEPTH^3

144 174 489

86 86

0.077

DOC

81

0.681

56

0.211

CHL_A^2 CHL_A^3 CO3^2

114

0.323

NO3^2 PO4 NO2^2

nd 68

0.237

CU CU^2 CU^3

7

2

1

3.01 1284.77
611.95 0.022

0 0 0

0.008 138.62
40.38

0.024 0 0

1.67
0.034 0

0.026 0.002 0.001


2.74 0.009 0

MN MN^2 MN^3

0.009

Adjust. R2

C. prolifera 183 0.427

C. nodosa 175 0.310

Ca/Cy ratio 183 0.370

Effect

Coefficient

Std error

p

DEPTH F_SAND^2 F_SILT

45.661 0.068 0.514

10.717 0.019 1.932

0 0 0.791

C_SAND Ca_DW WAVE

1.661
0.128
0.001

0.211 0.033 0.001

0 0 0.156

0.149

0.014

0

OM

p

31.366
0.737 0.004 15.839 58.211
0.363
0.002

0 0 0 0.005 0.016 0.021 0.007

OC OC^2 OC^3 N_TOT^2 N_TOT^3

174.472
25.941 0.99 64.293
12.735

0 0 0 0.005 0.016

0.443 0.429 0.317

SALIN TEMP^2 DEPTH DEPTH^3

4.724 1.488 70.087
1.392

20 88

0.863 0.362

84

0.444

59

0.388

107

0.575

8 71

0.741 0.379

71

0.123

DOS^2 DOC DOC^2 CHL_A CHL_A^2 CO3 CO3^3 NO3 NO3^2 NH4 PO4 NO2^2 NH4^2 PO4^2 NH4^3 PO4^3 SIO4^2 CU CU^2 CU^3 CD CD^2 CD^3

0.008 40.226
2.277 1779.279
947.02 3.294 0.688 121.469
0.674
298.643 1045.098 534.499 54.528
1085.73
2.453 290.473 0.532 9.135
0.101 0 103.135
10.525 0.277

0 0 0.011 0 0 0 0.014 0 0 0 0 0 0 0 0 0 0.003 0 0 0.001 0.006 0.016 0.037

27

0.651

5.736
0.056

0 0.001

0 0 0 0

0 0 0

Table 5 Models resulting from the multiple regression analyses (GLM) performed for all the variables together, except heavy metal concentration in the sediments. N

Coefficient

FE FE^2

sediment. Suspended solids and depth, both of which would reduce light availability, also negatively affect C. nodosa biomass. Although there is a clear segregation of the meadows according to sediment grain size, C. nodosa was also present in muddy bottoms before the appearance of Caulerpa in the lagoon (Navarro, 1927; Garcia Carrascosa, 1982; Pérez-Ruzafa et al., 1987, 1989). As Terrados et al. (1998) pointed out, increased silting could be associated with reduced underwater light penetration, both through the direct shading effect of suspended sediments and through the promotion of phytoplankton and epiphyte growth by the associated increase in nutrients. Increased sediment deposition can also lead to seagrass loss through burial effects (Duarte et al., 1997). In the Mar Menor, this occurred when coastal works took place, including the dredging and pumping of sediments (Pérez-Ruzafa et al., 1991), but

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Fig. 7. Redundance analysis (RDA) tri-plot ordination diagrams for species biomass and Ca/Cy ratio, samples and environmental descriptors (for abbreviations see Table 1). Only the significant variables and with highest marginal and conditional effect were represented.

not necessarily in the deeper areas of the lagoon. In those areas, the diminishing light due to the new trophic conditions could have been a decisive factor, facilitating the development of Caulerpa and negatively affecting Cymodocea. However, the replacement of Cymodocea by Caulerpa preceded the deterioration of the water quality in the lagoon. At present, Cymodocea, although reduced in mean total biomass, actually shows the highest biomass during the growth season, the summer, both in shallow and deep areas. Pérez-Ruzafa et al. (2005b) proposed that the increase in organic matter in the bottoms colonized by Caulerpa prolifera meadows and the increase of nutrients in the water give a competitive advantage to the alga over the seagrass. Seagrasses are negatively affected by biogeochemical alterations such as

111

sediment anoxia as a result of increased hydrogen sulfide concentrations and internal nutrient loading via enhanced nutrient fluxes from sediments to the overlying water (Burkholder et al., 2007). A decreased flux of oxygen to the roots and rhizomes of seagrasses restricts their capacity to oxidize the sediment sulfide, a known phytotoxin (Hemminga, 1998). Competition between macroalgae and seagrass is difficult to demonstrate, it usually occurs at patch scale, and when the reaction to the interference between two species confirms that they are competing for space and/or resources, the underlying mechanisms are uncertain and it is difficult to determine the specific environmental and physiological processes involved (Ceccherelli and Campo, 2002; Schaffelke and Hewitt, 2007; Pergent et al., 2008; Thomsen et al., 2012). Terrados and Ros (1991) considered that there was no direct interaction between Caulerpa prolifera and Cymodocea nodosa and that the colonization of the Mar Menor by C. prolifera could have resulted from the occupation of an empty niche and not from a process of competitive exclusion. Terrados et al. (1999) also found that sulfide levels in pore water and changes in the vertical redox profile produced by sucrose additions on the sediment to simulate the role of organic matter, lead to a reduction in leaf growth and to increased shoot mortality in some tropical species of seagrasses, but that no such effects were experienced by the Mediterranean species C. nodosa. However, the negative relationship between C. prolifera and C. nodosa detected by multiple regressions suggests a degree of competition between the two species. The negative relationship between organic carbon content and C. nodosa biomass found by regression analyses showed a low but significant adjusted R2, and RDA indicated a significant negative influence from a high organic matter content in sediments on seagrass biomass. In fact, the biomass of C. nodosa was very low when organic carbon in the sediments was higher than 10%. The experiments performed by Terrados et al. (1999) limited the addition of organic carbon to the sediments to 2156 g C m
2 in the Cymodocea nodosa meadow, a concentration that would be insufficient to detect the effects due to increases in organic matter in the Mar Menor. The production dynamics of Caulerpa prolifera and the environmental conditions created in the sediment lead to a net accumulation of organic matter that has gradually been increasing in the bottoms dominated by C. prolifera monospecific meadows or

Fig. 8. Representation of the evolution of sediment characteristics of the bottoms associated with different macrophyte meadows in the texture triangle. a) C. nodosa and Ruppia cirrhosa monoespecific meadows, b) C. prolifera monospecific meadows and CymodoceaeCaulerpa mixed meadows. White symbols correspond to 1982e1988 samples, grey symbols to 1996e1997 samples and dark symbols to 2002e2008 samples.

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Fig. 9. Representation of the C. nodosa biomass distribution in the space defined by the organic carbon and fine particle content of the Mar Menor sediments. Circle diameters are proportional to seagrass biomass.

mixed CaulerpaeCymodocea meadows. According to our data, the organic carbon content in muddy sediments covered by C. prolifera meadows may reach 16.5% (28.38% of organic matter), which would be equivalent to around 7000 g C m
2 in the upper 10 cm sediment layer assuming a sediment density of 1.5 g*cm
3. Both species of macrophytes showed positive relationships with nitrate, phosphate and silicate, suggesting that nutrient inputs favour both species. However, Cymodocea nodosa showed a stronger relationship with phosphate, suggesting this nutrient, rather than N, could be the limiting nutrient in the Mar Menor for this species. This agrees with the fact that in the past 15 years, discharge of nitrogen compounds has increased while those of phosphorus have decreased (Pérez-Ruzafa et al., 1987, 2005a). Erftemeijer and Middelburg (1993) also indicated that a coarsegrained carbonate sediment maintains relatively high pore water phosphate concentrations, whereas fine-grained carbonate sediments show extremely low pore water phosphate concentrations, which could enhance the potential limiting action of P on C. nodosa in the muddy bottoms. Caulerpa prolifera does not suffer this

limitation by phosphorus so clearly, because when its nutrient content is compared with that of other macrophytes it contains a very low amount of phosphorus (Terrados and Ros, 1995). These authors give an average atomic C:N:P ratio of 1297:79:1 for C. prolifera in the Mar Menor, while, according to Atkinson and Smith (1983), the mean for benthic macrophytes is 550:30:1. Terrados and Ros (1995) also mentioned that, although more experimental testing is required to know which nutrient limits the vegetative growth of C. prolifera, experiments in aquaria suggest that it is nitrogen and not phosphorus that limits the growth of this species. Other works have considered the competition relationship between species of the genus Caulerpa and seagrasses. Caulerpa prolifera has recently colonized the lagoon habitats of the Suez canal where it is showing a very rapid progression (Gab-Alla, 2007). In these lagoons it also forms extended dense meadows with cover of nearly 100% in many sites, supplanting the seagrass Halophila stipulacea due to the competitive success attributed to it rapid growth, high efficiency in dim light conditions, high tolerance to

Fig. 10. Evolution of the mean, maximum and minimum content of organic matter in the sediments dominated by Caulerpa prolifera monospecific meadows (CaM), CymodoceaeCaulerpa mixed meadows (CyeCaM) and C. nodosa monospecific meadows (CyM).

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severe nutrient limitation and to the production of toxic secondary metabolites. The density of the seagrass Halodule wrightii is also negatively affected by the presence of C. prolifera in the Indian River Lagoon, FL, USA (Taplin et al., 2005). Other works show that Cymodocea nodosa is more vulnerable to competition with Caulerpa racemosa than other seagrasses probably due to its higher sensitivity to burial and siltation (Ceccherelli and Campo, 2002), In the Mar Menor, the lower biomass of Caulerpa prolifera in shallow areas and, in general, its positive relationship with depth and suspended solids concentration, suggests that excess light could also be a limiting factor for this species. This is confirmed by recent experiments on light adaptation performed by GarcíaSánchez et al. (in press), and also agrees with the faster initial spread of Caulerpa in the northern basin of the lagoon where it has traditionally shown higher light extinction coefficients than in southern areas (Gilabert et al., 1995; Pérez-Ruzafa et al., 1996), especially in the summer months (Gilabert, 1992) when excess light could limit C. prolifera growth. Mar Menor bottoms shows a high concentration of heavy metals due to ancient mining activities (Simonneau, 1973; Pérez-Ruzafa et al., 1987). Both Caulerpa and Cymodocea showed positive relationships with the concentration of some heavy metals in the sediment. Caulerpa prolifera showed positive relationships with Fe and both species with Cu at low concentrations. The positive role of iron additions in seagrass growth through reducing sulfide intrusion was demonstrated by Marba et al. (2008). Such a role could also be played by other metals, and the influence of heavy metals in general on sediment processes and their interaction with the development of macrophyte meadows would be a worthy topic of research. 5. Conclusion In conclusion, a complex interaction of factors could be responsible for the equilibrium and dynamic of the relationship between Caulerpa prolifera and Cymodocea nodosa in the Mar Menor. A synergic effect of multiple disturbances has been described for other seagrasses (Eklof et al., 2009). In shallow waters, coarse sediments and an excess of light, together with physiological adaptations (higher photosynthetic capacity and no photoinhibition) could favour C. nodosa. In deeper areas, according to Terrados and Ros (1991), light scarcity might limit C. nodosa biomass. However, a direct negative effect of C. prolifera on C. nodosa cannot be ruled out and, indeed, probably occurs through increasing sediment silt concentration, sulfide production, organic matter and anoxia. Acknowledgements This study was partly supported by the projects “Sistema de Monitorización Costera para el Mar Menor” (Plan de Ciencia y Tecnología de la Región de Murcia 2007e2010) (Consejería de Universidades, Empresa e Investigación) and ECOLIFE (Plan Nacional de I þ D þ I 2008e2011). Thanks are due to all colleagues who have participated in field work in the many projects developed throughout the years. We also thank the Puerto and Club Náutico de Lo Pagán for harbour facilities and to anonymous referees for their suggestions and comments. References Anderson, M.J., 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecology 26, 32e46. Anderson, M.J., 2005. PERMANOVA: A FORTRAN Computer Program for Permutational Multivariate Analysis of Variance. Department of Statistics, University of Auckland, New Zealand, 24 pp.

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