Journal of Experimental Marine Biology and Ecology 454 (2014) 32–41

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Journal of Experimental Marine Biology and Ecology

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Neighbourhood competition in coexisting species: The native Cystoseirahumilis vs the invasive Sargassum muticum

Fátima Vaz-Pinto a,⁎, Brezo Martínez b, Celia Olabarria c, Francisco Arenas a

a Laboratory of Coastal Biodiversity, CIIMAR— Centro Interdisciplinar de Investigação Marinha e Ambiental, Rua dos Bragas, no 289, Universidade do Porto, 4050-123 Porto, Portugalb Área de Biodiversidad y Conservación, Universidad Rey Juan Carlos, c/Tulipán s/n, 28933 Móstoles, Madrid, Spainc Departamento de Ecología y Biología Animal, Facultad de Ciencias del Mar, Universidad de Vigo, 36310 Vigo, Spain

⁎ Corresponding author. Tel.: +351 223401835; fax: +E-mail address: f_vazpinto@yahoo.com (F. Vaz-Pinto)

http://dx.doi.org/10.1016/j.jembe.2014.02.0010022-0981/© 2014 Elsevier B.V. All rights reserved.

a b s t r a c t

a r t i c l e i n f o

Article history:Received 27 September 2013Received in revised form 4 February 2014Accepted 5 February 2014Available online xxxx

Keywords:Cystoseira humilisMacroalgaeNeighbourhood competitionNISNutrient uptake ratesSargassum muticum

The introduction of non-indigenous species (NIS) is expected to have negative effects on native competitors,particularly between functionally similar species. Nevertheless, the mechanisms underlying competitive rela-tionships remain poorly studied. Here, a substitutive neighbourhood approach was used to examine the role ofassemblage density and neighbour identity in the growth and nutritional strategy of a focal individual. Wequantified intra- and interspecific competition between two similar adult macroalgae (Fucales: Sargassaceae),the invasive Sargassum muticum and the native Cystoseira humilis, known to co-occur on intertidal rocky shoresfrom NE Atlantic. Using either S. muticum or C. humilis as the focal species, we monitored the focal individualgrowth responses and nutrient content in the field, with two different densities of neighbours. Additionally,we quantified the nutrient uptake rates for both species in the laboratory. Our results showed that C. humilisgrew at a significantly faster rate showing N accumulationwhen surrounded by S. muticum, whereas the invaderhas only showed growth at low density conditions. In addition, C. humilis presented greater uptake rates ofnitrate compared to S. muticum suggesting better competitive potential to exploit nitrogen transient pulses ofhigh concentration. Our results suggest a dominant native alga vs the invasive, which is not supported by fieldobservations. This research gives evidence that competition between adult macroalgal individuals may not bethe key mechanism linked to the dominance of NIS in introduced habitats.

© 2014 Elsevier B.V. All rights reserved.

1. Introduction

Biological introductions in marine ecosystems are widely reportedand have been increasing all around the world, mainly due to humanactivities such as international shipping, aquaculture and aquariumactivity (Bax et al., 2001; Carlton and Geller, 1993). Understandingchanges in biodiversity as an impact of non-indigenous species (NIS)introductions is a major challenge in ecology, particularly in coastal wa-ters which experience high susceptibility to such events and are amongthemost invaded ecosystems (Grosholz, 2002). A review of themecha-nisms underlying the impacts of non-indigenous plant invasions re-vealed that competition is, in most of the cases, suggested to be theprocess responsible for the registered impact in native communities(Levine et al., 2003; Thomsen et al., 2009). Particularly, strong competi-tion is expected to arise between ecologically similar native and inva-sive species (Dudgeon et al., 1999). Nonetheless, the mechanismunderlying competitive relationships remains poorly studied.

An important aspect of successful invasion is the existence of certainlife traits or fitness traits (e.g. ecophysiological characteristics), whichare frequently associated with invasive species. Introduced invasive

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species are considered highly efficient organisms using resources, and,as a result, have amore vigorous and faster growth than native or intro-duced non-invasive species (Pyšek and Richardson, 2007). For example,differences in physiological traits related to C acquisition and N alloca-tion may have direct consequences in growth rates and may indicate astrategy for competition and invasion (Funk et al., 2013; McAlpineet al., 2008; Nyberg and Wallentinus, 2005).

In the marine environment, macroalgal assemblages depend on re-sources such as space, nutrients and light (Carpenter, 1990). The intro-duction of NIS will be more successful either in habitats wherecompetition for these resources is reduced, and thus the invader has lit-tle or no competition from residents for resources (Davis et al., 2000), orin habitats where competition is very high, whenever an invader haslower maintenance requirements than resident species (Shea andChesson, 2002). In sessile organisms, differences in morphology andecophysiology traits of competing individuals, biomass, population den-sity and neighbour distance affect competition for resources (Carpenter,1990; Kim, 2002). Negative effects of density on survival and growth arecommon in algal populations (Ang and De Wreede, 1992; Steen andScrosati, 2004). In some cases, denso-dependent responses includechanges in architectures as reported for Sargassum muticum (Arenaset al., 2002; Strong and Dring, 2011). Furthermore, nutrients are thelimiting resource for seasonal macroalgal growth in most temperate

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marine systems (Hanisak, 1979), and are an important bottom-upfactor controlling the performance and structure of coastal macroalgalassemblages (Pedersen and Borum, 1996; Pedersen et al., 2010).Macroalgal species have developed different ecological strategies tocompete for nutrients intimately related to their life-cycle. Perennialsare typically slow-growing organisms and present low uptake rates.Nonetheless, perennials develop large nutrient stored pools in winter,particularly N, and use them for growing during spring (Martínezet al., 2012; Pedersen and Borum, 1996). This fact allows them to berelatively tolerant to nutrient seasonal limitation in summer whenlight levels increase, outcompeting other species in pristine temperategeographic areas (Pedersen et al., 2010). Opportunistic algal forms, onthe other hand, have a growth strategy to exploit a pulsed increase innutrient availability, escaping grazing control and achieving great bio-masses and dominance in a short time (Pedersen and Borum, 1996).High nutrient uptake rates drive their dominance in sites or seasons,of high nutrient and light availability to sustain their fast growth andreproductivematuration (Phillips andHurd, 2003). However, these spe-cies become rapidly limited if the supply decreases since they do not de-velop significant storage pools. There are species with strategies thatdiffered from this contrasting response, as for example summer-annuals,which sustain active growth during late spring to summer, achievingtheir maximum size in these periods of low nutrient supply (Martínezet al., 2012; Sears andWilce, 1975). Information of how invasive speciescopewith nutrient limitations is scarce and ambiguous. For instance, re-search on the brown macroalga Undaria pinnatifida showed nitrogenand light ecophysiological parameters which closely resembled oppor-tunistic algae (Dean and Hurd, 2007). On the other hand, the persis-tence and dominance of other invasive species were not affected bynutrient enrichment (e.g. Nejrup and Pedersen, 2010; Radford et al.,2010).

The brown macroalga S. muticum (Yendo) Fensholt 1955 is one ofthemost studied invasive seaweeds. Native to East Asia, it is consideredan invasive species almost all around theworld (Norton, 1976). Popula-tions of this species are distributedmainly in sheltered or semi-exposedrocky shores, although it may also attach to hard substrates on soft-bottoms, such as stones or shells (Strong et al., 2006). Commonly dis-tributed throughout the intertidal, S. muticum regularly invades thehabitats of algal species from the genus Cystoseira (Engelen andSantos, 2009; Fletcher and Fletcher, 1975; Otero-Schmitt and Pérez-Cirera, 2002). Previous studies showed varied results regarding the im-pact of the invasive S. muticum on native assemblages. In northernSpain, S. muticum showed limited impact on native assemblages(Olabarria et al., 2009; Sánchez and Fernández, 2005), whereas inNorthern Ireland competitive effects were not apparent betweenS. muticum and Saccharina latissima (Strong and Dring, 2011). In SanJuan Island, USA, however, the presence of S. muticum had a significantimpact on native communities at multiple trophic levels (Britton-Simmons, 2004). Overall, despite specific traits of the species, other fac-tors such as space availability and propagule supply, among others, playimportant roles in the invasion success of this species (Andrew andViejo, 1998; Vaz-Pinto et al., 2012).

Cystoseira C. Agardh and Sargassum C. Agardh are considered two ofthe most widely distributed fucacean genera across both hemispheres.Despite their common canopy morphology, these species differ intheir life-cycle and nutritional strategy. The native Cystoseira are peren-nial brown algae, known to dominate complex and diverse assemblagesof Mediterranean (Ballesteros et al., 2009) and Atlantic communities(Tuya and Haroun, 2006). The invasive S. muticum is considered apseudo-perennial species, and its invasive success has been related tothe combination of opportunistic traits and perennial persistence(Norton, 1976; Wernberg et al., 2000). In particular, it exhibits fastgrowth rates under a short favourable season (Norton, 1976), afterwhich senescence is observed. However, little is known about thesespecies nutritional strategy. Moreover, it has been suggested that indig-enous Cystoseira can be displaced by S. muticum (Engelen and Santos,

2009; Fletcher and Fletcher, 1975; Viejo, 1997). On the semi-exposedrocky shores of southern Portugal, S. muticum co-occurs with Cystoseirahumilis Schousboe ex Kützing 1860 in intertidal rock pools with seasonalnutrient limitation. The native C. humilis (Sargassaceae, Fucales) is thedominant alga in most intertidal rocky pools along the Atlantic south-west coast of Portugal (Engelen and Santos, 2009) and can grow up to150 cmhigh. It is described to occur in pools and in shallow standingwa-ters in the eulittoral zone, in moderately wave-exposed sites (GómezGarreta, 2001). In SW Portugal, observational results suggested lowerpopulation growth rates of S. muticum in pools where the nativeC. humiliswas dominant, compared to pools where S. muticumwas dom-inant (Engelen and Santos, 2009). These authors assumed an early phaseof invasion in pools dominated by Cystoseira,with slower growth rates ofthe invader compared to assemblages dominated by the invader i.e., latephase of invasion (Engelen and Santos, 2009). Underlying mechanismsof these competitive interactions, however, remain unknown.

The present study investigated the underlying mechanisms that de-termine the relative competitive abilities of S. muticum and C. humilis bycomparing the species' growth responses and total nutrient content infield experiments, and nutrient uptake rates in a laboratory experiment.The specific objectives were to (1) compare the relative effects of intra-and inter-specific competition on growth and nutritional content in thefield at relevant ecological conditions, 2) determine whether densityeffects influence in situ individual growth and nutritional state, and(3) determine the characteristic nutrient uptake kinetics from each spe-cies, as the first step to determine the species nutritional strategy. Wehypothesize that higher density assemblages will have reduced growthand survivorship compared with those at lower densities. In addition,the perennial C. humilis is expected to show slow-growing strategywith reduced uptake rates and richer nutrient tissues whereasS. muticummay present higher nutrient uptake rates and lower storagepotential.Wepropose a novel contribution on themechanisms involvedin neighbouring competition among invasive and native species atrelevant ecological conditions.

2. Materials and methods

2.1. Field study site

This study was carried out in the intertidal of Praia de Moledo(Northern Portugal, 41°50′22″ N, 8°52′30″W) from February to August2011. Moledo is an exposed site with a semi-diurnal tidal regime, withthe largest tidal range of 3.5–4 m during spring tides. Sea SurfaceTemperatures vary between 13 °C and 20 °C during the year. The exper-imentwas performed in 10mid-intertidal and low-intertidal rock-pools(~1–1.5 m above chart datum), and the experimental units were ran-domly displayed among pools. Pools selected were large pools (severalsquare metres) with enough depth to accommodate our assemblages(around 50 cm in the area where the plates were placed). Those poolshave a diverse seaweed flora dominated by encrusting and articulateCorallinaceae including species from genus Lithophyllum and Corallina,and non-calcified species like Chondrus crispus, Bifurcaria bifurcata,S. muticum and Cystoseira spp. We placed the plates away from largecanopy individuals from these species.

Individuals of S. muticum and C. humilis were collected in the rockyintertidal of Viana do Castelo (41°42′25″ N, 8°51′42″ W, Northwestcoast of Portugal) and Sines (37°53′12″ N, 8°47′43″ W, Southwestcoast of Portugal), respectively, and taken to the laboratory. Literaturedata described a gradual north/south trend along the Portuguese conti-nental shelf, with colder and nutrient-richer waters in the north andwarmer, nutrient-poorer waters in the south (Peliz and Fiuza, 1999).A recent study in the Portuguese coast (between Viana do Castelo andSines) also described a year-round latitudinal gradient in several ocean-ographic patterns (Tuya et al., 2012). In particular, monthly Sea SurfaceTemperature decreased ~1 to 2 °C with increasing latitude, while chl aincreased (Tuya et al., 2012).

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2.2. Macroalgal assemblage plates and experimental design

Experimental plates made of PVC were set with two different sizescontaining either individuals of S.muticum, C. humilis or both, at two dif-ferent final densities.We set 25 individuals per 500 cm−2 in plates of 27× 27 cm and 25 individuals per 250 cm−2 in plates of 20 × 20 cm, forlow and high density treatments, respectively. This gave final densitiesof 20 and 40 individuals per 400 cm2, respectively. These valuesmimic those observed in field conditions for the native species (16 to49 holdfasts per 400 cm−2 with a mean (±SD) of 31 (±12), fromthree scraped quadrates of 20 × 20 cm). Experiments comparing speciescompetition or performance in monospecific and mixed assemblagesuse additive or substitutive designs depending on whether the densityis kept constant or not in the experimental plots (Snaydon, 1991).Here, we used a substitutive neighbourhood approach to analyse sea-weed species competition by relating theperformance of a focal individ-ual to the identity and density of the neighbouring individuals. Usingplates with similar size, but different density, changes the overall bio-mass of each plate. Thus, to avoid confounding effect among overallplate biomass and density (Underwood, 1997), we changed plate sizeto achieve different densities but using the same number of individualsper plate. Thus,monospecific assemblages consisted of 25 individuals ofthe same species and the one in the centre was considered the focalindividual, whereas mixed assemblages had 1 focal individual of onespecies in the centre, surrounded by 24 individuals of the other species(see Fig. 1A). Individuals were fastened to the plates using a small stringthat wrapped the holdfast, keeping the main axis vertical in a similarposition to natural seaweeds. To do so we made 4 holes in the platearound the holdfast of every single individual and use the string to se-cure the holdfast in between those holes (Fig. 1B, C). This procedure isan adaption of the one described by Correa et al. (2006) for seaweedswith smaller holdfasts. Most of the individuals transplanted survived.The area of individuals' distributionwas inferior to the PVC area because

Fig. 1. Example of amixed assemblage plate with Sargassummuticum as the focal individual; it shof specimen attachment to the plates; B) 4 holes per specimen, and C) use of a small string to a

a 2 cm border was left intact to allow for plates deployment in the field.A total of 64 assemblage plates were built (n = 8).

Algal length, recorded at the time of transplanting, was 14.96 ±0.70 cm for S. muticum individuals and 8.46 ± 0.40 cm for C. humilis in-dividuals (mean ± SE, n = 32). Dry mass estimations of both specieswere made from maximum length (L) and maximum circumference(C) of each alga individual, following Åberg (1990). We used data ofdry weight and size from 40 individuals previously collected fromeach species to construct a regression model that allowed ourestimations.

S:muticum : DW ¼ 0:4092þ 0:006 V R2 ¼ 0:98;Pb0:001� �

C:humilis : DW ¼ 0:2636þ 0:007 V R2 ¼ 0:82;Pb0:001� �

where DW is dry weight and V is the specimen volume (LC2).After the laboratory manipulation, experimental assemblages were

transported to Praia de Moledo where they remained for 6 months inintertidal rock pools. Assemblage plates were randomly placed andscrewed to the bottom of the rock pools. We measured short-termchanges in biomass to estimate growth during two sampling events,over a 6-month period. Algal growth rate (per month) was estimatedfrom February to June 2011, i.e., during spring when maximum growthrates have been found in S. muticum and other Cystoseira species in thesame geographical area (Arenas et al., 1995). No net growth wasobserved from June to August, suggesting senescence of S. muticum inAugust.

For biochemical analysis of in situ C andN tissue content,we collectedapproximately 2–5 cm of non-reproductive tissue from the focal individ-ual at each assemblage plate at the end of the experimental period. Algalmaterial was dried at 60 °C for 48 h and total C and N determined using a

ows 1 individual of S. muticum surrounded by 24 individuals of Cystoseira humilis (A). Detailttach the holdfast.

35F. Vaz-Pinto et al. / Journal of Experimental Marine Biology and Ecology 454 (2014) 32–41

Carlo Erba CHNS-O Elemental Analyser (Model EA1108). Total C tissuecontent and N tissue content were standardised to algal dry weight (g).

2.3. Nutrient concentrations in the field

To assess the variation of nutrient availability in intertidal rock poolsover a tidal cycle, seawater sampleswere collected in August from threerock pools over a tidal cycle (5 h) in Praia de Moledo. The first watersample was taken as soon as the tide was low enough and water ex-change was no longer observed. Then, duplicate water samples weretaken every 30 min until water exchange was again observed and thetide was high.

To assess the variation in the nutrient concentrations over the exper-imental period at the sites where algal individuals were collected, sea-water samples (n = 4) were collected on November 2010, April/May2011 and June 2011 from Sines and Viana do Castelo. Also, water sam-ples were collected from Moledo, where the experiment took place.

Water samples were filtered in situ using portable microfiber fil-ters (Fisherbrand® MF 300) and acid cleaned 50-ml plastic syringes.Immediately after collection, samples were placed on ice, returned tothe laboratory in darkness, and frozen at −20 °C until analysis (lessthan three months after) of nitrates, ammonium and orthophosphate.Water analyses were carried out using a wet chemistry analyser(Sanplus++ System, SKALAR, Breda, The Netherlands), at CIIMAR,Porto.

2.4. Nutrient uptake experiment in the laboratory

2.4.1. Collection of samples and pre-incubationVegetative individuals of S. muticum and C. humiliswere collected in

the rocky intertidal of Viana do Castelo (41°42′25″ N, 8°51′42″ W,Northwest coast of Portugal) and Sines (37°53′12″ N, 8°47′43″ W,Southwest coast of Portugal), respectively, in April 2012. Collectionwas made three days before assaying their uptake kinetics. In the labo-ratory, the material was pre-incubated in 2 L Erlenmeyer flasks filledwith artificial seawater (TropicMarine ZooMix, TropicalMarine Centre,U.K.) enriched with Von Stosch's (VSE) medium (Ott, 1965) but thespecific nutrient (N for ammonium and nitrate or P for the phosphateuptake experiments) to be assayed. Adequate mixing was assured bybubbling filtered air into the culture medium. Flasks were left in awalk-in culture chamber at 15 °C, with constant photon flux density(approx. 150 μmol photons m−2 s−1) and photoperiod (12:12; Light:Dark) until the beginning of the experiment.

2.4.2. Uptake experimentsNutrient uptake kinetics was assessed by measuring the decrease of

nutrient concentration at different time intervals in 250 ml Erlenmeyerflasks filled with 200ml ofmedium set at different initial substrate con-centrations (see Martínez and Rico, 2004). Algal fronds of each species,1 g fresh weight (FW), were incubated in 250 ml Erlenmeyer flasks: 12flasks with increasing initial nutrient concentration (19.01 to132.26 μM, 3.85 to 48.35 μM, and 0.60 to 15.19 μM, for ammonium, ni-trate and phosphate, respectively) plus 3 blanks (no seaweed) as con-trols. In total, 30 flasks were used per nutrient experiment (15 perspecies). Flaskswere randomly arranged into amulti-positionmagneticstirrer (IKA-WERKE). Irradiance was provided with fluorescent lamps(cool white F18W/840) to a final value of 425 μmol photons m−2 s−1

and the temperature was controlled inside the walk-in chamber at15 °C. The nutrient depletion of the medium was determined by nutri-ent analysis ofwater samples.Water samples (10ml)were taken beforethe addition of the algal fronds and then at 15, 30, 60, 120, 180, 240 and300min. After the experiment, algalmaterial was oven-dried at 50 °C toa constant weight (48 h), for determination of dry weight (DW).

Analyses of NH4+, NO3− and HPO4

2− were carried out using a wetchemistry analyser (Sanplus++ System, SKALAR), at CIIMAR, Porto.Blank control flasks registered minor concentration changes and thus

no corrections were made to the algal uptake rates. Mean (±SE) differ-ences between initial andfinal values (0–300min) in controlswere 3.30(±1.57) μM for ammonium, 1.17 (±0.67) μM for nitrate and 0.34(±0.33) μM for phosphate experiment.

2.4.3. Uptake rate calculationNet uptake rates (μmol g DW−1 h−1) for each nutrient source were

calculated following the equation:

V ¼ Si � Volið Þ− Sf � Volfð Þ½ �= DW� tð Þ;

where Si and Sf, voli and volf are the substrate concentration (μM) andthe water volume (l) at the beginning and at the end of the sample in-terval, t is the time of the sampling interval (h), and DW is the algaldry weight (g) (see Pedersen, 1994).

The exhaustion of ammonium in low nutrient flasks was registeredfor the time intervals 30–60 and 60–120 min, for C. humilis andS. muticum, respectively. Therefore, the first 6 uptake measurements,i.e. lower nutrient flasks, were not displayed for both species (n = 6).Moreover, no ammonium uptake measurements were displayed afterthose time intervals, since nutrients in the flasks were exhausted byalgal uptake. Although all glass material used was washed with 10%HCl, anomalous negative uptake rates and outliers were recorded, sug-gesting handling contamination. These corresponded to the 0–15 minintervals for phosphate uptake and 45 wrong uptake measurementsout of 504 in the remainder time intervals. These outliers were notused in the calculation of uptake rates but are shown in the figures ifwithin the graph ranges.

2.5. Statistical analysis

A two-way analysis of variance (ANOVA) was used to test main andinteractive effects of density of macroalgal assemblages (high and low)and neighbour identity (monospecific or mixed assemblages) on thegrowth rate of each species individually. The same two-way crossed de-sign was used to test the variation of algal tissue C and N content usingthe density of macroalgal assemblages and neighbour identity as fixedfactors. Some experimental plates were lost in the field due to roughweather conditions and thus the available number of focal species pertreatment combination varied between 5 and 8. For statistical analyseswe considered an unbalanced design and performed the analysis of var-iance using type III sums of squares (SS) (Underwood, 1997), separatelyfor each species.

Ordinary linear regression analyses were performed to analyse therelationship between nutrient concentrations in the rock-pool over atidal cycle.

Regarding the uptake laboratory experiment, for each time interval,net uptake rateswere plotted against the initial substrate concentrationof the current sampling interval. Although nutrient uptake kinetics usu-ally follows a Michaelis–Menten saturation model (Lobban andHarrison, 1997), other alternative linear responses can also be observed(Martínez et al., 2012; Phillips and Hurd, 2004). Thus, the Michaelis–Menten function was only used when the quadratic fit was significantagainst the corresponding linear fit (p-value b0.05); otherwise a linearfit was applied (see Martínez et al., 2012; Phillips and Hurd, 2004). Thedata was fit using SigmaPlot version 10.0 (Systat Software, Inc., San JoseCalifornia, USA).

Analyses of variance with unequal sample sizes were performedusing Type III SS using the Anova function from the car package and lin-ear models were carried out using the linear model function (lm) in theR-program 2.15.0 (R Development Core Team, 2012). Prior to all analy-ses, the homogeneity of varianceswas examined using Cochran's C-test.Data were transformed when necessary and in those cases in whichtransformation did not remove heterogeneity, the level of significancewas judge at the more conservative probability of 0.01 (Underwood,1997). Post-hoc multiple comparisons were obtained using Fisher's

36 F. Vaz-Pinto et al. / Journal of Experimental Marine Biology and Ecology 454 (2014) 32–41

least significant difference (LSD) test. Data shown represent mean(±SE) throughout.

3. Results

3.1. Nutrient concentrations in the field

Nutrient concentrations varied between algal collection sites and theexperimental site over the experimental period. Overall, our data fromSines presented lower values of nitrate and phosphate, whereas Vianado Castelo showed greater nutrient concentrations of ammonium andphosphate (Fig. 2). Moledo, where the experiment took place, showednitrate values similar to Viana do Castelo and ammonium values closerto Sines (Fig. 2A).

Over a tidal cycle of almost 5 h, nutrient concentrations (nitrate andphosphate) declined drastically in the first 2 h 30 min (R2 = 0.84,p b 0.0001, n = 11 for both nutrient types, Fig. 3). Nitrate concentra-tion varied from1.4 μMto3.8 μM(Fig. 3A) and phosphate concentrationranged from 0.51 μM to 0.38 μM (Fig. 3B). In contrast, ammonium con-centration remained quite constant as a recycling N source (R2 = 0.26,p = 0.112, n = 11) at 3.03 μM (±0.10) (Fig. 3C).

3.2. In situ algal growth rate and tissue C and N

Throughout the field experiment, a greater growth was observed inindividuals of C. humilis compared to S. muticum with a mean value(data of all treatments pooled) of 0.15 cm (±0.08, n = 26) and

Fig. 2.Mean (±SE, n= 4) variation in the nutrient concentration of seawater over the ex-perimental period in Sines and Viana do Castelo, where algae were collected, andMoledo,where the experiment took place. A) shaded bars indicate nitrate concentration; openbarsindicate ammonium concentration; B) phosphate.

Fig. 3.Mean (±SE, n = 3) variation in the nutrient concentration in tidepools through atidal cycle. A) nitrate, B) phosphate and C) ammonium concentration.

0.002 cm (±0.06, n= 23), respectively (Fig. 4). Nonetheless, N contentwas similar between species, 1.843 (±0.068) and 1.836 (±0.060),for C. humilis and S. muticum, respectively (Anova, F1,48 = 0.23,p = 0.634). High variability within replicates of the same treatmentswas, however, observed for both studied species and, in particular, forS. muticum.

The growth rate of C. humilis focal individuals was affected interac-tively by the experimental treatments, i.e., density of macroalgal assem-blages and neighbour identity (Table 1a, Fig. 4A). Specifically, there wasa significant greater growth rate of C. humilis in assemblages with com-petitor compared to assemblages without competitor, but only at highmacroalgal density (Fisher's LSD, p = 0.02). Monospecific stands ofC. humilis at high density showed a slight net biomass loss of 0.14(±0.11) g DW month−1, with only one individual replicate showingsome net tissue increment (0.38 g DW month−1), whereas the pres-ence of S. muticum neighbours leads to an increase in net biomass of0.37 (±0.16) g DW month−1 with 2 out of 7 replicates showing sometissue loss. Moreover, C. humilis at low density assemblages showedan overall increment of 0.17 (±0.10) g DW month−1. The initiallengths of C. humilis focal individuals did not differ significantly at lowand high density (Anova, F1,30 = 0.01, p = 0.934). Additionally, the Cnutrient content of macroalgal individuals also did not vary betweentreatments (overall mean ± SE: 25.87 ± 0.72, Table 2a). N nutrient

Fig. 4. Mean (±SE) variation in the biomass of the focal species between February andJune 2011. Monospecific assemblages (shaded bars, with intra-specific competitors) andmixed assemblages (white bars, with inter-specific competitors) for both studied speciesA) Cystoseira humilis and B) Sargassum muticum. * indicate significant differences atP b 0.05, "ns" indicate non-significant differences.

Table 2Two-way unbalanced analysis of variance (Type III ANOVA) for a) % carbon g−1 DWandb) % nitrogen g−1 DW of the focal species, Sargassum muticum and Cystoseira humilis.Densities of macroalgal assemblages and neighbour identity were fixed factors.

a) Carbon b) Nitrogen

Variables df SS F P SS F P

C. humilisNeighbour 1 19.71 1.496 0.236 0.682 124.53 0.013Density 1 13.94 1.058 0.316 0.348 7.47 0.065Neighbour × density 1 12.09 0.918 0.350 0.263 3.81 0.105Residual 20 263.61 1.826 2.88

S. muticumNeighbour 1 0.40 0.09 0.764 0.0001 0.0001 0.992Density 1 4.10 0.91 0.353 0.108 1.299 0.268Neighbour × density 1 9.50 2.11 0.162 0.001 0.017 0.897Residual 19 85.00 1.585

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content, however, was significantly affected by the neighbour identity(Table 2b). The N nutrient content was greater in mixed assemblagesthan in monospecific assemblages of C. humilis, 1.96 (±0.09) and 1.70(±0.09), respectively.

S. muticum growth rate showed high variability of results betweenmacroalgal assemblages and no significant differences were obtainedbetween treatments. Net biomass loss was themain outcome of the ex-periment in all combination treatments but monospecific stands at lowdensity (Fig. 4B). The initial lengths of S. muticum focal individuals atlow and high density were similar, 15.39 (±0.80) cm and 14.54(±1.16) cm, respectively. C (overall mean ± SE: 29.72 ± 0.48) and N(overall mean± SE: 1.84 ± 0.06) nutrient content also showed no sig-nificant effect of the experimental treatments (Table 2).

3.3. Nutrient uptake

The native C. humilis showed the greatest uptake rates concerningthe N sources (Fig. 5). Nitrate uptake increased linearly with substrate

Table 1Two-way unbalanced analysis of variance (type III ANOVA) for growth rate(g DW month−1) of the focal species, a) Cystoseira humilis and b) Sargassum muticum.Densities of macroalgal assemblages and neighbour identity were fixed factors.

Variables df SS F P

a) C. humilisDensity 1 0.831 6.318 0.020Neighbour 1 0.446 3.387 0.079Density × neighbour 1 0.670 5.091 0.034Residual 22 2.894

b) S. muticumDensity 1 0.002 0.098 0.882Neighbour 1 0.045 0.023 0.426Density × neighbour 1 0.153 0.662 0.152Residual 19 1.302 2.228

concentration, except during the period 30–60 min when a saturationresponse was observed (Fig. 5A; Table 3). S. muticum, on the otherhand, showed mostly saturation kinetics up to 180 min and then linearresponses of low slope were observed (Fig. 5B; Table 3). Nitrate wasexhausted earlier (240 min) by C. humilis and at greater rates (compareaxes in Fig. 5A, B), suggesting greater nitrate uptake potential ofC. humilis than S. muticum.

The ammonium uptake response was linear for both species as itpassively enters the cells (Fig. 5C, D; Table 3). Ammonium was totallyconsumed after 60 min by C. humilis and 60 min later by S. muticum.Moreover, maximal uptake rates were recorded for C. humilis, also sug-gesting greater uptake potential for this nutrient than S. muticum.

Overall, phosphate uptake rates were similar for both species(Fig. 5E, F), although the mechanism varied between species and wefound slightly different affinity. Phosphate uptake response forC. humilis followed a linear uptake response throughout the experimen-tal period, whereas S. muticum alternated between linear and saturatedresponses (Table 3). At 240 min, no phosphate concentration remainedin C. humilis experiment, whereas S. muticum did not show a completephosphate depletion over the experimental period (300 min), thus sug-gesting somewhat higher affinity for P by the former.

4. Discussion

Marine macroalgae compete for space, nutrients and light becausethese resources are often in short supply in coastal ecosystems(reviewed in Carpenter, 1990) and vary at spatial and temporal scales(Bonan, 1988; Connell, 1983; Naeem et al., 1999). The present study re-vealed an effect of the identity of the neighbours in the native C. humilisgrowth and nutritional state. In field conditions, C. humilis grew at afaster rate showing N accumulation if surrounded by S. muticum athigh density assemblages. In addition, the invasive species exhibitedlower uptake potential. Our results suggest a better nutritional strategyof C. humilis to cope with limiting nutrient conditions of intertidal rock-pools, contrary to the expectations.

The population of C. humilis from the Atlantic Portuguese coast de-velops on intertidal rock-pools along semi-exposed and exposed shores(Araújo et al., 2009; Engelen and Santos, 2009). Rock-pools are physical-ly stressful environments, and as observed in the present study, nitrateand phosphate showed a strong decrease in concentration over a tidalcycle and thus short-term nutrient limitation may be attained duringlow tides. Evidence suggest that at low and potentially limiting N avail-ability, slow-growing species, i.e., perennials, should be favoured whencompared to fast-growing species, i.e., opportunistic (Pedersen andBorum, 1997). Fast-growing opportunistic species, however, show agreat nutrient uptake potential, while slow growing perennials havesmaller uptake rates and large nutrient store capacity (Lobban andHarrison, 1997; Martínez et al., 2012). The response obtained in thepresent studywas, however, not the expected for a perennialmacroalga

Fig. 5. Uptake rates of nitrate (A, B), ammonium (C, D) and phosphate (E, F) for Cystoseira humilis and Sargassum muticum. Uptake kinetics are shown as a function of the substrate con-centration at the beginning of 7 different time intervals (as different symbols). X, values not included in the fit.

38 F. Vaz-Pinto et al. / Journal of Experimental Marine Biology and Ecology 454 (2014) 32–41

(e.g. Martínez et al., 2012). C. humilis showed elevated growth and nu-trient uptake rates. In particular, C. humilis showed a greater increasein biomass when in high density mixed assemblages with S. muticumthan in monospecific assemblages, in agreement with its greater nutri-ent uptake rates. The positive effect of S. muticum at high density couldbe related to a balance between the fact that S. muticum is less efficientthan C. humilis in nutrient uptake and also maybe a positive density ef-fect from protection against waves and other physical perturbations.S. muticum has a very plastic response to density (Arenas et al., 2002),thus changes in the effect of S. muticum on C. humilis at high and lowdensity may also be a consequence of that plasticity. Surprisingly,C. humilis not only showed nutrient uptake rates much greater thanthose reported for other perennials, but also presented values compara-ble to the ones reported for ephemeral macroalgae such as Ulvaintestinalis and Ceramium sp. (Martínez et al., 2012; Pedersen andBorum, 1997). This resultmay either be related to the nutrient availabil-ity from the alga recipient region, or from its natural habitat, i.e., pools,which, as demonstrated here, are routinely subjected to decreasing nu-trients over tidal cycles. Field nutrient measurements revealed lowernutrient concentration in Sines compared to Viana do Castelo.

Generally, nutrient-limited algae show greater uptake rates (Lobbanand Harrison, 1997). Thus, our results might be explained by the factthat although C. humilis is a highly efficient species, it may be nutrientlimited in SW Portugal. Observational field results may reinforce thistheory as C. humilis showed a maximum length of 44.8 cm in NWPortugal, while the maximum value recorded from SW Portugal was18.8 cm (unpub. data). Thus, our results are contradictory to the mostfrequent response of perennial slow-growing species, which are sug-gested to be better adapted to pristine temperate rocky shores(Pedersen and Borum, 1997), and exhibit low nutrient uptake rates(Martínez et al., 2012).

The invasive species, S. muticum, is known to develop among com-munities of the genus Cystoseira (e.g. Engelen and Santos, 2009;Otero-Schmitt and Pérez-Cirera, 2002). Despite being dominantthroughout the Portuguese coast, our results do not characterize adultS. muticum as a great competitor or with an opportunistic nutritionalstrategy. The growth of S. muticumwas the same regardless of the pres-ence of C. humilis, contrary to our expectations. Also, the lack of signifi-cant negative effect of densitywas quite surprising as negative effects ofdensity have been previously registered for S. muticum and other

Table 3Estimations (standard error) of the uptake kinetic parameters for Cystoseira humilis and Sargassummuticum using either linear orMichaelis–Menten (MM) functions. Vmax is expressed inμmol g DW−1 h−1 and Ks in μM.

Interval (min) Cystoseira humilis Sargassum muticum

Slope (linear) Vmax Ks n Slope (linear) Vmax Ks n

Nitrate0–15 1.16 (0.06) 10 0.32 (0.08) 1015–30 0.62 (0.08) 10 0.56 (0.07) 1130–60 0.13 (0.02) 9 0.08 (0.02) 1260–120 0.51 (0.02) 9 8.34 (0.89) 7.23 (2.41) 11120–180 15.82 (4.32) 12.06 (8.71) 12 3.68 (0.36) 2.14 (0.90) 10180–240 11.16 (2.43) 9.43 (5.33) 11 4.96 (0.60) 2.20 (1.70) 8240–300 – – – – 0.95 (0.34) 0.06 (0.01) 6

Ammonium0–15 2.66 (0.21) 11 1.03 (0.29) 1015–30 2.60 (0.53) 10 2.04 (0.22) 1130–60 1.67 (0.51) 6 0.58 (0.14) 960–120 – – – – 1.21 (0.08) 6

Phosphate0–15 0.29 (0.04) 10 – – – –

15–30 6.00 (1.57) 7.67 (4.24) 11 10.54 (2.58) 21.51 (8.69) 1130–60 0.43 (0.04) 9 0.27 (0.03) 960–120 0.57 (0.03) 9 2.41 (0.27) 5.10 (1.50) 12120–180 7.40 (4.46) 16.15 (14.20) 10 1.99 (0.23) 4.23 (1.40) 12180–240 0.29 (0.03) 11 3.15 (0.56) 7.90 (3.34) 12240–300 – – – – 0.15 (0.01) 12

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intertidal macroalgae (e.g. Arenas et al., 2002; Viejo and Åberg, 2001).Nonetheless, there was a trend towards the effect of density withgreat biomass losses within this treatment, as expected. In addition,S. muticum showed N-uptake rates comparable to summer-annual spe-cies (Martínez et al., 2012). The fact that S.muticumdid not demonstrateopportunistic characteristics in uptake rates is in agreement with a re-cent study which stated that at a population level, growth ofS. muticum when invading intertidal rock pool habitats follows K-selected traits (Engelen and Santos, 2009). Our results, however, donot support the fact that S. muticum may displace C. humilis, at leastnot due to mechanisms related to density or identity of neighbours.This same competitive outcome has been previously reported betweenS. muticum and the kelp S. latissima (Strong and Dring, 2011). Naturalstands of S. muticum comprise different adult and juvenile stages, i.e.,mixture of sizes and biomass (Baer and Stengel, 2010; Strong andDring, 2011). Thus, our findings may be due to the fact that the presentexperiment was focused on post-recruitment competition and we ex-clusively used fully grown thalli of similar size (≈15 cm). Density de-pendent effects may regulate S. muticum populations from embryosand juveniles individuals, where larger densities can be observed (e.g.Arenas et al., 2002). A similar trend has also been registered forCystoseira spp. settlement and recruitment (Benedetti-Cecchi andCinelli, 1992).

Competition between closely related species, e.g. Family:Sargassaceae, is a deterministic factor in natural selection (Darwin,1875). In this context, our results suggest that algae adapted to sustainmaximum growth rates under conditions of N limitation will have thecompetitive advantage to become dominant inmixed algal assemblagesat rock-pool systems. C. humiliswill then be favoured simply because oftheir greater uptake rates, in accordance with the fact that the thick,slow-growing macroalgae can be very efficient in exploiting limited Nresources to meet their requirements (Pedersen and Borum, 1997).Thus, contrary to the expectations, adult populations of the nativeC. humilis showed a greater competitive potential to exploit transientnutrient pulses than the invasive S. muticum, which may explain itsgreater growth rates.

Overall, at the largest density of C. humilis, monospecific assem-blages intensify intraspecific interactions, which decrease in mixed as-semblages, suggesting that growth in this alga was more sensitive to

its own density. Our results in the mixed assemblages suggest that thepresence of S. muticum entails that: 1) S. muticummay display a positiveinteraction with C. humilis by decreasing the conditions of nutrient lim-itation for C. humilis individuals, or 2) inter-specific competition withS. muticum is lower than intra-specific competition between C. humilisindividuals. Because growth rate in high-density mixed assemblageswas not greater than at monospecific low-density assemblages, resultssuggest that this pattern is due to a greater intra- vs interspecific com-petition in C. humilis. Facilitation by the invader, however, could notbe fully investigated with the present experimental design.

Our results, however, are not consistent with field observationswhere the NIS S. muticum is the dominant macroalga in the intertidalrocky-shore. S. muticum has been previously described as an opportu-nistic, rapidly colonizing available space rather than displacing nativespecies (Farnham, 1974). Evidence suggests that many invasive speciesare, in fact, weak competitors and its establishment and spread are facil-itated by disturbance (e.g. Bando, 2006; Hierro et al., 2006). Nonethe-less, the outcome of competitive interactions varies considerably withthe age or size at which species interact (Olson and Lubchenco, 1990).Previous studies have reported physiological differences in Sargassumspp. at germling and adult stages (Choi et al., 2008; Hales andFletcher, 1989). In this context, our results support the fact that the in-vasive success of S. muticummay be linked to competitive interactionsat the early post-settlement stage (Strong and Dring, 2011).

4.1. Conclusion

The present experiment described the competitive capacity of thenative C. humilis and the invasive S.muticum in adulthood. In conclusion,our study suggested that high density of monospecific assemblagesleads to increased intra-specific competition, as expected (e.g. Viejoand Åberg, 2001). Moreover, both species differed greatly in their nutri-tional physiology as well as growth pattern, although contrary to ourinitial hypotheses. C. humilis presented greater uptake rates of N thanS. muticum, suggesting that adult individuals of S. muticum are weakercompetitors compared to adult individuals of the native C. humilis. Ourresults support the fact that competition between adultmacroalgal indi-viduals may not be the key mechanism linked to the dominance of NISin introduced habitats. In accordance, other factors such as disturbance

40 F. Vaz-Pinto et al. / Journal of Experimental Marine Biology and Ecology 454 (2014) 32–41

have been suggested to enhance the abundance and spread of NIS(Hierro et al., 2006), while invader persistence strongly determinesinvasion patterns (Spence et al., 2011).

Role of the funding source

The funding agencies had no role in the study design, data collection,analysis and interpretation of data or in the writing of this paper.

Author contributions

Planned and designed the experiment: FVP, BM, FA, and CO. Per-formed the experiment: FVP, FA, and CO. Analysed the data: FVP andBM. Wrote the paper: FVP and BM. All authors have approved the finalarticle.

Acknowledgements

This work would not have been possible without the able assis-tance in the field and lab from numerous individuals. In particular,we would like to thank Abel Vaz Pinto, Bernardete Lopes, IgnacioGestoso and Erin Sullivan. Also, we would like to thank Rosa Viejofor the help in biomass estimations. This research was funded bythe AXA-Marine Alien and Climate Change project and the FCTthrough the project CLEF (PTDC/AAC-AMB/102866/2008). FVP wassupported by a PhD grant from the Portuguese Foundation for Scienceand Technology — FCT (SFRH/BD/33393/2008). [RH]

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