Disentangling the effects of solar radiation, wrack macroalgae and beach macrofauna on associated bacterial assemblages

Marine Environmental Research 112 (2015) 104e112

Contents lists available at ScienceDirect

Marine Environmental Research journal homepage: www.elsevier.com/locate/marenvrev

Disentangling the effects of solar radiation, wrack macroalgae and beach macrofauna on associated bacterial assemblages
n F. Rodil*, Joana P. Fernandes, Ana P. Mucha Iva Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, 4050-123 Porto, Portugal

a r t i c l e i n f o

a b s t r a c t

Article history: Received 28 July 2015 Received in revised form 2 October 2015 Accepted 6 October 2015 Available online 21 October 2015

Wrack detritus plays a significant role in shaping community dynamics and food-webs on sandy beaches. Macroalgae is the most abundant beach wrack, and it is broken down by the combination of environmental processes, macrofauna grazing, and microbial degradation before returning to the sea as nutrients. The role of solar radiation, algal species and beach macrofauna as ecological drivers for bacterial assemblages associated to wrack was investigated by experimental manipulation of Laminaria ochroleuca and Sargassum muticum. We examined the effects of changes in solar radiation on wrack-associated bacterial assemblages by using cut-off filters: PAR þ UVA þ UVB (280e700 nm; PAB), PAR þ UVA (320 e700 nm; PA), PAR (400e700 nm; P), and a control with no filter (C). Results showed that moderate changes in UVR are capable to promote substantial differences on bacterial assemblages so that wrack patches exposed to full sunlight treatments (C and PAB) showed more similar assemblages among them than compared to patches exposed to treatments that blocked part of the solar radiation (P and PA). Our findings also suggested that specific algal nutrient quality-related variables (i.e. nitrogen, C:N ratio and phlorotannins) are main determinants of bacterial dynamics on wrack deposits. We showed a positive relationship between beach macrofauna, especially the most abundant and active wrack-users, the amphipod Talitrus saltator and the coleopteran Phaleria cadaverina, and both bacterial abundance and richness. Moderate variations in natural solar radiation and shifts in the algal species entering beach ecosystems can modify the role of wrack in the energy-flow of nearshore environments with unknown ecological implications for coastal ecosystems. © 2015 Elsevier Ltd. All rights reserved.

Keywords: Bacterial assemblages Cut-off filters Macroinvertebrates Sandy beaches Solar radiation Wrack Laminaria ochroleuca Sargassum muticum

1. Introduction Sandy beaches are ubiquitous ecosystems mostly appreciated for providing valuable recreational and economical services to society. Sandy beaches also comprise an important part of any coastal region and harbour a range of often under-appreciated biodiversity commonly subjected to strong variations in the rates of waves, tides, sun exposure and nutrients (McLachlan and Brown, 2006). Simultaneously, allochthonous subsidies of organic material, also known as wrack, play a critical role in shaping community dynamics on beaches with profound implications on coastal food-web dynamics and nearshore ecosystem functioning (e.g. Dugan et al., 2003, 2011; Marczak et al., 2007; Lastra et al., 2008; Spiller et al., 2010).

€rminne Zoological Station, Univer* Corresponding author. Present address: Tva sity of Helsinki, Finland/Baltic Sea Centre, Stockholm University, Sweden. E-mail addresses: [email protected], ivan.rodil@helsinki.fi (I.F. Rodil). http://dx.doi.org/10.1016/j.marenvres.2015.10.002 0141-1136/© 2015 Elsevier Ltd. All rights reserved.

The lack of in situ beach primary production means that beach communities are almost entirely dependent on the accumulation of allochthonous organic debris; i.e., macroalgal wrack, dead animals, and/or dissolved and particulate organics flushed into the sand by waves and tides (Colombini and Chelazzi, 2003). These communities play a key role in the decomposition and transformation of the organic matter accumulated in sandy beaches (Orr et al., 2005; Lastra et al., 2008; Dugan et al., 2011). Once wrack is cast ashore it undergoes physical processes that allow algal loss, including breakdown via dryingerewetting cycles through solar radiation or morning dew, sedimentation, leaching, and eventually fragmentation, decomposition and remineralisation by bacteria, meiofauna and grazers (see Orr et al., 2005). Bacteria colonize wrack deposits releasing dissolved and particulate organic matter into the sediments. Also, a diverse assemblage of meio- and macrofauna rapidly colonizes wrack and strongly influences the microbial community (e.g. Koop and Griffiths, 1982; Griffiths et al., 1983; Inglis et al., 1989). Thus, scavengers, such as amphipods, isopods and insects feed directly upon the macrophytes or other wrack-dependent

I.F. Rodil et al. / Marine Environmental Research 112 (2015) 104e112

organisms, and their activities release particulates, leachates, and faecal pellets, which stimulate the growth of bacteria and other invertebrates (Koop and Griffiths, 1982). Detrital processing by beach organisms plays a key role in coastal nutrient cycling (Colombini and Chelazzi, 2003; McLahlan and Brown, 2006). Previous studies on the biodegradation of beach wrack showed that the role of invertebrates is largely variable, with measured consumptions ranging from 5 to 75% of the biomass of detrital wrack in different beach systems (e.g. Koop et al., 1982; Griffiths et al., 1983; Lastra et al., 2008). Similar studies showed that microorganisms and leaching play a more significant role than macrofauna in the breakdown of wrack and in the ecological functioning of this ecosystem, accounting for as much as 87% of the annual beach production (e.g. Koop and Griffiths, 1982; Koop et al., 1982; Inglis, 1989). In this process, fragments and mineralized wrack components are transported to the nearshore environment, the atmosphere or stored within the beach (Koop and Griffiths, 1982; Koop et al., 1982; McLachlan, 1985; Dugan et al., 2011). Therefore, microorganisms serve as a natural biofilter that mineralizes organic material, providing essential nutrient recycling in nearshore environments, maintaining coastal ecosystem health. The number of microorganisms on a beach depends on many environmental factors, although the most important one is the supply and content of organic matter (Nair and Bharathi, 1980; Novitsky and MacSween, 1989). Large standing stocks of sedimentary bacteria are expected under wrack deposits even if the initial agents of biodegradation are macroinvertebrates. Bacteria can show preferences for different algal species with specific physical and biochemical traits in a similar way as beach macrofauna does (Duarte et al., 2011; Rodil et al., 2015a,b). However, although microorganisms play a critical ecological role on the biodegradation of wrack within beach ecosystems, there is limited knowledge of the microbial function linked to wrack (e.g. Koop and Griffiths, 1982; Inglis, 1989; Colombini and Chelazzi, 2003). Over the next decades, climate change is expected to increase the exposure of marine organisms to damaging UV wavelengths (Andrady et al., 2010). In temperate latitudes, the amount of UVR reaching Earth's ecosystems has not been stable over the past years due to anthropogenic-related changes in cloudiness and aerosol concentrations, causing what is known as the “global dimming and brightening effect” (sensu Wild, 2009). This effect is hold responsible for promoting detrimental variations in the UV levels in midlatitude regions with implications for climate change and farreaching ecological consequences (Wild, 2009; Mateos et al., 2013) that can affect beach communities (Rodil et al., 2015a,b). Several studies focused on the role of solar radiation (UVR) on marine bacteria have provided evidence that UVR-alteration, particularly UVB (280e320 nm), has significant negative effects
rez on productivity, activity and abundance of bacterial cells (e.g. Pe and Sommaruga, 2007; Manrique et al., 2012). It has also been demonstrated that shifts in the natural levels of UVR have direct effects on macrophytes with consequences on consumers (e.g. Swanson and Fox, 2007; Rodil et al., 2015a,b). Microbial communities in sandy beach sediments are organized in response to their requirements for chemical and light needs (Bühring et al., 2014). Therefore, UVR-induced changes in wrack biochemical traits (i.e. nutrients, pigments or phlorotannins) and effects on associated macrofauna might influence microbial responses with consequences on wrack degradation process and fate. To our knowledge this is the first beach study regarding the effects of UVR on wrackassociated bacterial assemblages. We performed a wrack experimental manipulation by using two different species of brown macroalgae: the native Laminaria ochroleuca Bachelot de la Pylaie, 1824, and the non-indigenous Sargassum

105

muticum (Yendo) Fensholt, 1955. We manipulated the ambient solar radiation using cut-off filters to simulate a moderate change in UVB, and we tested the responses of the associated bacterial assemblages. This work was nested within a larger experiment that tested the joint effects of wrack identity and solar radiation on macrofauna assemblages (Rodil et al., 2015b). The aim is to determine whether two different algal species with inherent biochemical composition and typical beach macrofauna can promote different microbial responses when combined with different UVR treatments. Recently, it was suggested that beach grazers, such as talitridae sand-hoppers, might obtain their food source indirectly from wrack via bacterial communities that are specific to different types of imported material (Porri et al., 2011). Therefore, we take the opportunity to evaluate the top-down impact of macroinvertebrates on bacterial assemblages colonizing wrack in a sandy beach. 2. Methods 2.1. Study site and experimental design This study was based on a subset of treatments that were part of a larger experiment that examined the role of wrack as beach macrofauna shelter, and aimed at determining how different UVR treatments affected the algal biochemical composition of two macroalgal species, and the consequent effects on associated macrofauna (Rodil et al., 2015b). Brown canopy-forming macroalgae L. ochroleuca and S. muticum are abundant species frequently found stranded on beaches from the Atlantic coasts of the Iberian Peninsula. We performed the experiment in Ladeira beach (Corrubedo Natural Park), an exposed sandy beach from NW Spain (42 340 3600 N; 9 30 2000 W, NW Spain), where wrack is abundant and diverse (see Rodil et al., 2015b). The day before starting the experiment, we collected by hand fresh amounts of L. ochroleuca and S. muticum from nearby rocky areas, taken to the laboratory, washed, and separated in wrack patches of similar weight (~1.0 ± 0.2 kg wet weight, 16 patches per species). At the field (20 September 2013), 4-replicated squaredpatches (20  20 cm) per algal species and UVR-treatment (i.e., a total of 32 experimental wrack patches) were placed at the northern part of the beach between the highest mark of the drift line and the toe of the dunes parallel to the shoreline. Each patch was placed ~2 m apart, and its treatment determined previously by a random distribution. All patches were covered by a bird-net (1 cm mesh size) attached to the sand by aluminium pegs to prevent aeolian dispersion, and left in place for five days. This time gap was chosen because wrack in natural conditions loses exponentially most of the biomass in a few days, and most of the associated invertebrates are early colonizers (see Rodil et al., 2015b). Specific information on the experimental UVR-treatments is available somewhere else (Rodil et al., 2015b). Briefly, four ambient solar radiation treatments were established by suspending cut-off filters immediately above the patches (10 cm), supported over 4legged polyethylene squared structures (buried 5 cm into the sand) to modify the quality of the solar radiation: (a) The photosynthetically active radiation treatment (400e700 nm, Lee 226), which blocks radiation < 400 nm (i.e., UVA and UVB), but allows for full transmission of PAR (hereafter, P). (b) The PAR þ UVA treatment (320e700 nm, Lee 130), which blocks UVB radiation (hereafter, PA). (c) The full sunlight treatment (PAR þ UVA þ UVB; 280e700 nm) using a commercial polyethylene film as a procedural control (hereafter, PAB), which allowed penetration of full spectrum light (>90% for PAR, UVA, and UVB). (d) The full sunlight with no filter as a control for filter artefacts (hereafter, C). Information on the solar radiation recorded through the filters is available somewhere else (Rodil et al., 2015b).

106

I.F. Rodil et al. / Marine Environmental Research 112 (2015) 104e112

2.2. Biochemical composition of the macroalgae and associated macrofauna After 5 days, 4 random algal subsamples (±5 g) of each replicate wrack patch were collected for biochemical analysis; i.e. total organic matter (OM, %), chlorophyll a (chl a, mg g1), total carbon (TC) and nitrogen (TN) content (%), C/N ratio, and protective phlorotannins (see supplementary material, Table S1 for a summary). Macrofauna data (i.e. total abundance, abundance of specific taxa and species richness) from each wrack patch was also obtained (see Table S1). Sampling methods and specific information on wrack biochemical composition and associated macrofauna are available somewhere else (see Rodil et al., 2015b), though it is not discussed here. 2.3. Wrack-associated microbial abundance After 5 days, 4 random algal subsamples of each replicate wrack patch were collected to estimate microbial abundance in the wrack, measured as total cell counts (TCC). TCC was obtained by the DAPI (40 , 60 -diamidino-2-phenylindole) direct count method (Porter and Feig, 1980; Kepner and Pratt, 1994). For that, 2.5 mL of formaldehyde (4% (v/v)) were added to 0.5 g of homogenized wrack subsamples. Then, 2 drops of Tween (0.2 mm-filtered, 12.5% (v/v)) were added and samples were stirred for 15 min, resting for 15 min. These samples were then sonicated for 10 min, stirred again for 1 min and maintained overnight at 4  C. After this, 100 mL of the solution was added to 2.5 mL of saline solution (0.2 mm-filtered, 9 g/L NaCl) and 2 drops of Tween were added. Samples were then stained with DAPI and incubated in the dark for 15 min. Solutions were filtered onto black Nuclepore polycarbonatefilters (0.2 mm pore size, 25 mm diameter, Whatman, UK) under gentle vacuum and washed with 5 mL of autoclaved 0.2 mm-filtered distilled water. Membranes were set up in glass slides and cells counted in an epifluorescence microscope (Leica DM6000B). 2.4. Bacterial assemblages in the sediment under wrack patches Bacterial assemblages under wrack patches were evaluated by ARISA (Automated rRNA Intergenic Spacer Analysis), one of the widely used methods for fingerprinting bacterial assemblages (e.g., Ranjard et al., 2001; Cardinale et al., 2004). This technique exploits the variability in the length of the intergenic spacer (IGS) between the small (16S) and large (23S) subunit rRNA genes in the rrn operon (Ranjard et al., 2001). The IGS, depending on the bacterial species, displays significant heterogeneity in both length and nucleotide sequence (Fisher and Triplett, 1999). Recent studies considered that ARISA is still a powerful tool for analysing bacterial communities in environmental samples, especially for controlled experiments (e.g. Gobet et al., 2014; Purahong et al., 2015). Total DNA was extracted from 0.5 g wet weight of homogenized sediment subsamples using the Ultra Clean Soil DNA Isolation Kit (MoBio, Carlsbad, CA, USA). DNA was amplified using ITSF (50 GTCGTAACAAGGTAGCCGTA-30 ) and ITSReub (50 -GCCAAGGCATCCACC-30 ) primers sets (Cardinale et al., 2004), which amplifies the ITS1 region in the rRNA operon plus ca. 282 bases of the 16S and 23S rRNA (Hewson and Fuhrman , 2004). ITSReub was labelled with the phosphoramidite dye 6-FAM (6-carboxyfluorescein). PCRs were performed in duplicate 25 m L volumes containing between 10 and 50 ng of DNA, 400 nM of both primers, 200 mM dNTPs, 3  Taq PCR buffer, 2.5 U Taq DNA polymerase, 2.5 mM MgCl and 1 mg bovine serum albumin. The PCR mixture was held at 94  C for 2 min, followed by 30 cycles of 94  C for 45 s, 55  C for 30 s, 72  C for 2 min, and a final extension at 72  C for 7 min. Duplicate PCR products were combined, examined on 1.5% agarose gel, purified using a GFX

PCR DNA kit (GE-Healthcare) and eluted in 30 mL. Purified product was quantified using the Quant-it dsDNA assay kit and the Qubit fluorometer (Invitrogen). A standardised amount of the purified PCR product was diluted 1:2 and mixed with 0.5 mL of ROX-labelled genotyping internal size standard (ROX 1000, Applied Biosystems). The sample fragments were run on a ABI3730 XL genetic analyser at STABVIDA Sequencing Facilities (Lisbon, Portugal). ARISA fragment lengths were analysed by Peak Scanner Software (Applied Biosystems). Fragments that differed by less or equal to 2 bp (base pair) were considered identical and fragments with Fluorescence Units below 50 were considered “background noise”. Fragments of less than 200 bp were removed since were considered too short ITS for bacteria. The bacterial richness was estimated as the total number of unique Operational Technical Units (OTUs) identified within each ARISA electropherogram profile (see Supplementary material, Fig. S1), assuming that the number of peaks represented the species number (phylotype/genotype richness), and that the peak height (fluorescence units) represented the abundance of each bacterial species (see Supplementary material, Fig. S1 and Table S1). 2.5. Statistical analyses Changes in microbial abundance (log-transformed TCC), and in bacterial abundance (log-transformed) and richness associated to wrack patches were analysed using a two-way analysis of variance (ANOVA). Algae (A: L. ochroleuca and S. muticum) and radiation (R: C, PAB, PA, P) were considered orthogonal fixed factors (wrack subsamples were pooled). The homogeneity of variances was evaluated with Cochran's test. A posteriori comparisons were done using StudenteNewmaneKeul's (SNK) tests (p < 0.05). For bacterial assemblages, a matrix of ARISA aligned fragments (OTUs) and fluorescence values (bacterial abundance) was created as previously described (Mucha et al., 2013), and then imported to the PRIMER 6þ PERMANOVA® software (Anderson et al., 2008). Data were normalized by presence/absence, and analysed through a BrayeCurtis resemblance matrix (4999 permutations). Twofactor non-parametric multivariate analyses of variance (PERMANOVA) were run to test the hypothesis about differences among wrack bacterial assemblages (same factors as described above for ANOVA). Only significant effects (p < 0.05) were further investigated through pair-wise comparisons using the appropriate terms in the model. PERMDISP was used to check if data showed homogeneity in multivariate dispersion. Non-metric multidimensional scaling (nMDS) was used to visualize multivariate patterns in bacterial assemblages. We used generalized linear models (GzLM) with continuous predictor variables to better understand the interplay of factors that determine levels of bacterial responses (i.e., bacterial abundance and richness) to wrack accumulations. Patterns in the data were first assessed with Cleveland dotplots and pair plots to identify outliers and the potential for collinearity among explanatory variables. We did not categorize the data by algae or UVR treatment, but let the natural and experimentally induced variability in wrack biochemical (i.e. chl a, organic matter, phlorotannins, nutrients) and macrofaunal predictors (i.e. total abundance, abundance of specific taxa and number of species) explain the data. We applied a negative binomial model distribution with a log-link structure for the raw data of the response variables (i.e., TCC, bacterial abundance and richness) to avoid overdispersion (Zuur et al., 2009). The initial models included all the predictor variables listed previously. A variable was retained in the model only if it caused a significant increase in deviance when it was removed from the current model. Model fit was evaluated through Akaike's Information Criterion (AIC). Model assumptions were checked: (i) plots of residuals

I.F. Rodil et al. / Marine Environmental Research 112 (2015) 104e112

107

versus fitted values to verify homogeneity, (ii) quantileequantile plots or histograms of the residuals for the assessment of normality, and (iii) residuals versus each explanatory variable to check for data independence. Analyses were fitted in R (R Development Core Team, 2014). 3. Results 3.1. Solar radiation and filter treatments The irradiances for PAR, UVA and UVB were successfully manipulated by the treatments. The maximum PAR irradiances reached mean (±SD) values of 578 ± 2.3, 541 ± 2.9, 527 ± 2.1, and 526 ± 5.9 W m2 for C, PAB, PA, and P treatments, respectively. The maximum UVA irradiances reached mean values of 5.9 ± 0.03 (C), 5.5 ± 0.01 (PAB), 5.3 ± 0.01 (PA), and 0.33 ± 0.01 (P) W m2. Finally, the maximum UVB irradiances reached mean values of 0.46þ ± 0.002 (C), 0.43 ± 0.001 (PAB), 0.18 ± 0.001 (PA), and 0.04 ± 0.000 (P) W m2 (see Rodil et al., 2015b for further details). 3.2. Changes in wrack-associated microbial abundance Microbial abundance, measured as total cell counts (TCC), ranged from 106 to 107 cells g1wet tissue (Table S1). TCC did not show significant differences between algal species, but this pattern was not consistent among UVR treatments (i.e. significant A:R interaction, Table 1). Thus, TCC was significantly larger in S. muticum than in L. ochroleuca only in wrack controls (C) with no radiation treatment (SNK test, p < 0.05; Fig. 2). TCC in wrack patches made of L. ochroleuca was significantly larger in C than P treatments (SNK test, p < 0.05; Fig. 1). 3.3. Analysis of bacterial descriptors and bacterial assemblages under wrack Bacterial abundance and richness ranged from 26,685 to 180,772 fluorescence units (peak heights), and from 52 to 189 OTUs, respectively (Table S1 and Fig. S1). No significant differences were found between wrack algal species and radiation treatments for both, bacterial abundance and richness (Table 1). Wrack-associated bacterial assemblages varied significantly between algal species, but these differences were not consistent over radiation treatments (i.e. significant A:R interaction; Table 1). The interaction was caused by variations in the assemblage similarities between wrack patches through different radiation treatments (Fig. 2). Thus, the greatest dissimilarity (60.1%) in bacterial assemblages between wrack species was recorded in patches treated with PA (i.e. no UVB) (Table 2). For L. ochroleuca alone

Fig. 1. Microbial abundance from wrack patches (Laminaria ochroleuca and Sargassum muticum) measured as total cell counts (TCC: cell g1, log-transformed) and subjected to different UVR-treatments: PAR þ UVA þ UVB (280e700 nm; PAB), PAR þ UVA (320e700 nm; PA), PAR (400e700 nm; P), and a control with no filter (C). Different letters represent significant differences.

(Fig. 2), the greatest dissimilarities were recorded between C vs P (54.6%), and PA vs P (59.9%) wrack-treated patches (Table 2). For S. muticum alone (Fig. 2), the greatest dissimilarities were recorded between C vs PA (58.6%), and P vs PA (56.3%) wrack-treated patches (Table 2). 3.4. Influence of algal biochemical composition and wrackassociated macrofauna on bacterial assemblage descriptors A summary of the main algal biochemical content and wrackassociated macrofauna can be found in Table 1 and somewhere else (see Rodil et al., 2015b). Total nitrogen and the C:N ratio were significant predictors of the bacterial abundance associated to wrack (Table 3). The relationships between bacterial abundance and TN was significant and positive (Fig. 3a), and between bacterial abundance and C:N ratio was significant and negative (Fig. 3b). The abundance of talitridae (specifically two talitrid species: Talitrus saltator (Montagu, 1808) and Deshayesorchestia deshayesii (Audouin, 1826)), the abundance of coleopteran Phaleria cadaverina (Fabricius, 1792), and the number of macrofaunal species recorded in wrack patches (Table S1) were all significant and positive predictors of the bacterial abundance (Table 3 and Fig. 3cee). Both, TN and phlorotannins were significant predictors of the bacterial richness (Table 3). Thus, the relationship between bacterial richness and TN was positive (Fig. 4a); meanwhile, the relationship between bacterial richness and phlorotannins was negative (Fig. 4b) (Table 3). The relationship between bacterial richness and C:N ratio was also significant (Table 3) and negative (Fig. 4c). Finally, the abundance of talitridae, the abundance of T. saltator alone, and the number of macrofaunal species were all significant and positive

Table 1 Summary of ANOVA for the total cell counts (TCC: cell g1), bacterial abundance (peak height, fluorescence units) and bacterial richness (operational technical units, OTUs), and PERMANOVA for the entire bacterial assemblage (n ¼ 4). TCC estimated the microbial abundance in wrack patches (DAPI, see methods). Bacterial abundance and richness in sediment under wrack calculated from the ARISA profiles (see methods and Fig. S1). C: Cochran's homegeneity test. PERMDISP: test for homogeneity in multivariate dispersion. Source

Algae (A) Treatment (R) A: R Residuals Total

TCCa

DF

1 3 3 24 31

Bacterial abundancea

Bacterial richness

Entire assemblageb

MS

F

MS

F

MS

F

MS

pseudo-F

0.1708 0.3501 0.4635 0.1325

1.29 2.64+ 3.5*

0 0.059 0.048 0.037

0 1.61 1.3

1596 1903 1551 1093

1.46 1.74 1.42

4269 3549 3410 731

5.84*** 4.85*** 4.66***

C: 0.355 (p > 0.05) Significant results in italics (+ 0.1 < p < 0.05; *p < 0.05; **p < 0.01; a Log-tranformed data. b Presence/absence data, Bray-Curtis similarity matrix.

C: 0.250 (p > 0.05) ***

p < 0.001).

C: 0.233 (p > 0.05)

PERMDISP: 2.621 (p > 0.05)

108

I.F. Rodil et al. / Marine Environmental Research 112 (2015) 104e112

Colombini and Chelazzi, 2003 for a review). Recent experiments showed that modifications in the natural solar radiation affected wrack biochemical traits as well as macrofauna activity (Rodil et al., 2015a,b). To our knowledge, this is the first attempt to investigate the responses of bacterial assemblages to wrack deposits subjected to UVR manipulations. We found that changes in the algae subsidising beaches, and moderate manipulation of the incident natural solar radiation affected the microbial abundance and bacterial assemblages associated to wrack patches. 4.1. Changes in wrack-associated microbial abundance

Fig. 2. Multidimensional scaling (MDS) ordination based on BrayeCurtis similarities (presence/absence, BrayeCurtis similarity matrix) obtained from ARISA fingerprints (Fig. S1) of bacterial assemblages from sediments underneath wrack patches (Laminaria ochroleuca and Sargassum muticum) and subjected to UVR-treatments: PAR þ UVA þ UVB (280e700 nm; PAB), PAR þ UVA (320e700 nm; PA), PAR (400e700 nm; P), and a control with no filter (C).

Table 2 Mean BrayeCurtis dissimilarities (%) of bacterial assemblages between wrack patches (L. ochroleuca vs. S. muticum) and among UVR treatments: PAR+UVA+UVB (280e700 nm; PAB), PAR + UVA (320e700 nm; PA), PAR (400e700 nm; P), and a control with no filter (C). Treatments

Wrack algae L. ochroleuca vs. S. muticum

C PAB PA P

C vs PAB C vs PA C vs P PAB vs P PAB vs PA P vs PA

Our experiment showed interactive effects of UVR and algae on wrack microbial abundance in terms of total cell counts (i.e. TCC). We found that in natural UVR conditions, TCC was larger in S. muticum than in L. ochroleuca patches. In a previous study (Rodil et al., 2015b), macrofauna abundance (mainly communities of larval flies) was also found larger in S. muticum than in L. ochroleuca. This could be linked to the fact that macrofauna, including larvae, plays a vital role in optimising conditions for microbial growth and bacteria mineralization (Koop and Griffiths, 1982; Inglis, 1989). Previous studies on the role of UVR on marine bacteria have provided evidence that the most energetic waveband, mainly UVB (280e320 nm), have detrimental effects on microbial communities
rez and Sommaruga, 2007; Manrique et al., 2012). We found a (Pe subtle, but significant TCC increase in L. ochroleuca when subjected to P treatments (i.e. neither UVA nor UVB). TCC associated to S. muticum was not affected by any UVR treatment. This could be related to the hypothesis that wrack species composed of more structurally complex materials and with slower decomposition rates, such as S. muticum, offer a more long-lasting habitat to support higher community diversity than less complex habitats, such as L. ochroleuca, by increasing habitat heterogeneity (Inglis, 1989; Colombini and Chelazzi, 2003; Rodil et al., 2008). This implies an increase in the area available, not only for microbial colonisation but also for UVR protection. Although the habitat complexity hypothesis is generally linked to macrofaunal communities, the diversity of microorganisms is also strongly related to the heterogeneity of the environment (Kuzyakov and Blagodatskaya, 2015). The architecture of different wrack species might be playing a protective role in the response of beach microbial communities to the environment that deserves further research.

54.4 47.0 60.1 52.5 L. ochroleuca

S. muticum

47.4 50.6 54.6 54.5 53.9 59.9

51.0 58.6 50.3 45.2 51.4 56.3

predictors of the bacterial richness associated to wrack patches (Table 3 and Fig. 4def). 4. Discussion Despite the importance of bacterial communities as direct contributors to wrack dynamics and decomposition, few studies on the microbial ecology of beach wrack have been performed (see

4.2. Response of bacterial assemblages to wrack and UVR treatments We found some evidence to support the hypothesis that bacterial assemblages changed in response to the type of wrack, although patterns varied depending on the UVR treatment. Light environment, together with nutrients, is one of the main regulatory factors of natural microbial communities in beach sediments (Bühring et al., 2014). Multivariate analyses and assemblage fingerprinting (ARISA profiles) showed considerable bacterial assemblage heterogeneity between L. ochroleuca and S. muticum. However, the dissimilarity in bacterial assemblages between these two algal species was larger when patches were subjected to PA treatments (i.e. no UVB). Furthermore, we found significant differences in the bacterial assemblages so that wrack exposed to full sunlight treatments (i.e. C and PAB) presented more similar assemblages among them than compared to the UVR-treatments (i.e. P and PAB). Our findings are in agreement with other studies from different ecosystems that have reported that the exposure to different radiation conditions, either UVB alone or UVR, can induce changes in the bacterial community (Langenheder et al., 2006;
rez and Sommaruga, 2007; Manrique et al., 2012). Changes in Pe

I.F. Rodil et al. / Marine Environmental Research 112 (2015) 104e112

109

Table 3 Final generalized linear models (GzLM model fit was evaluated using backward selection and Akaike's information criterium; AIC) indicating the significance of wrack biochemical and macrofauna predictor variables on bacterial assemblage descriptors (i.e. bacterial abundance and bacterial richness; see methods and Fig. S1). Macrofaunal predictors are counts in each wrack patch. (*p < 0.05; **p < 0.01). Best model fit

Biochemical predictors

Response variables (Rv) Bacterial abundance

Rv ~ TN Rv ~ C:N Rv ~ TN + phlorotannins

TN C:N TN phlorotannins

Bacterial richness

Estimate

SE

z value

AIC

Estimate

SE

z value

AIC

0.684 0.059 e e

0.228 0.031 e e

3.01** 1.9* e e

763.0 767.3 e e

e 0.046 0.447 0.217

e 0.022 0.153 0.115

e 2.08* 2.92** 1.89*

e 317.9 310.9

0.029 e 0.037 0.124

0.013 e 0.018 0.039

2.26* e 2.04* 3.14**

766.1 e 766.9 761.6

0.027 0.025 e 0.084

0.008 0.011 e 0.028

3.28** 2.3* e 2.99**

312.6 317.0 e 313.9

Macrofaunal predictors Rv ~ Talitridae Rv ~ T. saltator Rv ~ P. cadaverina Rv ~SR

Talitridae Talitrus saltator Phaleria cadaverina Species Richness

Fig. 3. Response of bacterial abundance (fluorescence units from ARISA, Fig. S1) to a) total nitrogen content (%), b) C:N ratio, c) abundance of talitridae amphipods (i.e., Talitrus saltator and Deshayesorchestia deshayesii), d) abundance of Phaleria cadaverina, and e) the number of macroinvertebrate species. Macrofauna abundance measured as counts.

110

I.F. Rodil et al. / Marine Environmental Research 112 (2015) 104e112

Fig. 4. Response of bacterial richness (OTUs from ARISA, Fig. S1) to a) total nitrogen content (%), b) C:N ratio, c) phlorotannins (mg g1), d) abundance of talitridae amphipods (i.e., T. saltator and D. deshayesii), e) abundance of T. saltator, and f) the number of macroinvertebrate species. Macrofauna abundance measured as counts.

bacterial assemblages might occur due to a direct effect of UVR on bacterial activity and survival, and consequently differential sensitivity among various bacterial strains may result in altered assemblages. Therefore, bacterial groups with specific photosynthetic capabilities can possess an advantage under different solar conditions (Langenheder et al., 2006). 4.3. Influence of wrack biochemical composition and associated macrofauna on bacterial assemblage descriptors Changes in the bacterial assemblages associated to UVR-altered wrack patches can be partly explained by the underlying biochemical composition of the algae. Previous studies established that most of the nitrogen from algal debris is incorporated to bacteria (Koop et al., 1982; Sosik and Simenstad, 2013), and also that microbial colonization increases the percentage of TN in algal tissues leading to a gradual decrease in the C:N ratio (Duggins and Eckman, 1997; Norderhaug et al., 2003). In our study, bacterial abundance and richness increased significantly with increasing TN, and decreased significantly with increasing C:N. TN is considered to have a high nutritional value, and C:N is a common measure of food quality and decomposition rate for different types of plant detritus (Duggins and Eckman, 1997; Norderhaug et al., 2003). Therefore, algal detritus characterized with a high TN and low C:N reflect highly degraded food ready, not only for macrofauna foraging (Pennings et al., 2000; Rodil et al., 2015a), but also for microbial colonization (Norderhaug et al., 2003). We found that bacterial richness decreased with enhanced phlorotannins, while bacterial abundance was not significantly affected. Phlorotannins are a wellknown class of plant secondary metabolite that not only function as

herbivore deterrents, but also have antibacterial activity (Nagayama, 2002; Sosik and Simenstad, 2013). Phlorotannins leach and breakdown once the wrack begin to desiccate, suggesting that they are rapidly lost during decomposition, allowing numbers of bacteria to quickly colonize well-degraded algal deposits (Duggins and Eckman, 1997; Norderhaug et al., 2003). Data on bacterial abundance was collected after almost a week-long wrack decomposition period. At that point, most of the phlorotannin concentration was probably gone and ineffective to bacteria. Changes in bacterial richness due to phlorotannins might be caused by differential effects of the secondary metabolites to specific bacterial species. Previous studies showed that bacterial degradation and weathering are probably the major causes of wrack weight loss (Koop et al., 1982; Inglis, 1989), highlighting the importance of bacteria and environment in the breakdown of organic material and in making primary production available for beach consumers. Meanwhile, the importance of macrofauna in the breakdown of wrack, although significant, is considered highly variable (Inglis, 1989). However, the presence of associated macrofaunal species with differential activities also drives significant shifts on bacterial assemblages. For instance, it is expected that bacteria and algal nutritional quality increases throughout wrack degradation making algae more suitable as invertebrate food (Koop et al., 1982; Duggins and Eckman, 1997; Norderhaug et al., 2003). Therefore, beach macrofauna is inevitably attracted to the beds of wrack within a few hours of its deposition, consuming the wrack and accelerating its decay (Griffiths et al., 1983; Colombini and Chelazzi, 2003; Lastra et al., 2008). Recently, it was suggested that beach grazers, such as talitrid sand-hoppers, might obtain their food source indirectly

I.F. Rodil et al. / Marine Environmental Research 112 (2015) 104e112

from wrack via bacterial communities that are specific to different types of imported material (Porri et al., 2011). Our results indicated a positive and significant relationship between wrack-associated macrofauna and bacterial assemblage descriptors. For instance, the presence of the amphipod T. saltator and the coleopteran P. cadaverina were related to some of the changes in the responses of wrack bacterial assemblages. Both species are early wrack colonizers and voracious consumers commonly found in the supratidal beach area, where most of the wrack deposits occur (Rodil et al., 2008). Our results support the fact that macrofauna plays a vital role in optimising conditions for bacteria mineralization and microbial activity (Koop and Griffiths, 1982; Inglis, 1989). It is also possible that the initial breakdown of wrack is driven by bacteria and that these in turn constitute an early food source of the macrofauna using wrack-colonizing microbiota as a source of easily accessible nitrogen, while an important carbon source is obtained from wrack material (Mews et al., 2006; Porri et al., 2011). However, the dependence of macrofauna on bacteria as a food source is probably related to the availability of organic matter in the sedimentary beach (Papageorgiou et al., 2007). The complex associations among wrack, invertebrates and bacteria would benefit from further studies using a larger number of wrack algal species, and stable isotope analysis and biomarkers (when isotopic signatures do not differ significantly as is the case for brown macroalgae [e.g. Crawley et al., 2009]) to clearly delineate beach trophic relationships. 5. Conclusions Several authors stressed that the microbial community occupies a central role in the rapid regeneration of detritus material, and subsequently all this material is returned to the sea supporting nearshore food-webs (Koop et al., 1982; Koop and Griffiths, 1982; McLachlan, 1985; Inglis, 1989). The present study is a first attempt to unravel the effects of wrack algae and beach macrofauna on bacterial assemblages associated to wrack, meanwhile subjected to changes in solar radiation. We suggest that moderate changes in ambient UVR are capable to promote shifts on bacterial assemblages modifying the status and ecological significance of beach wrack. Our findings showed that algal species and their underlying biochemistry are determinants of bacterial descriptors on decomposing wrack. Also, our study showed positive and significant relationships between wrack-associated invertebrate species and bacterial descriptors. Changes in some of these factors can modify the microbial regeneration of nutrients from stranded wrack macroalgae with immediate effects on the natural energy-flow of sandy beaches and unpredictable ecological consequences in coastal ecosystems. Acknowledgements We acknowledge the authorities of the Corrubedo Nature Park for permission and assistance. We would like to thank F. Arenas, M.
ndez, P. Rodrigues, S. Rodríguez, E. Sampaio, and F. Lastra, A. Ferna Vaz-Pinto for field assistance. We thank P. Lucena-Moya for critically reading a version of this manuscript. The constructive criticisms of two anonymous reviewers greatly improved the final version of the manuscript. Founding was partially supported by the European Regional Development Fund (ERDF) through the COMPETE - Operational Competitiveness Program, and national funds through FCT- Foundation for Science and Technology - under “PEstC/MAR/LA0015/2013 CLEF (PTDC-AAC-AMB-102866-2008). This research was also partially supported by the Strategic Funding UID/ Multi/04423/2013 in the framework of the programme PT2020. IFR was supported by a postdoctoral fellowship from the Portuguese -

111

FCT (SFRH/BPD/87042/2012). Appendix A. Supplementary material Supplementary material related to this article can be found at http://dx.doi.org/10.1016/j.marenvres.2015.10.002. References Anderson, M.J., Gorley, R.N., Clarke, K.R., 2008. PERMANOVAþ for FRIMER: Guide to Software and Statistical Methods. PRIMER-E, Plymouth, UK.
, C.L., et al., 2010. Environmental effects of Andrady, A., Aucamp, P.J., Bais, A.F., Ballare ozone depletion and its interactions with climate change: progress report, 2009. Photochem. Photobiol. Sci. 9, 275e294. €rmer, L., Ho, S., Hinrichs, K.-U., 2014. Functional structure Bühring, S.I., Kamp, A., Wo of laminated microbial sediments from a supratidal sandy beach of the German Wadden Sea (St. Peter-Ording). J. Sea Res. 85, 463e473. Cardinale, M., Brusetti, L., Quatrini, P., Borin, S., Puglia, A.M., Rizzi, A., Zanardini, E., Sorlini, C., Corselli, C., Daffonchio, D., 2004. Comparison of different primer sets for use in automated ribosomal intergenic spacer analysis of complex bacterial communities. Appl. Environ. Microbiol. 70, 6147e6156. Colombini, I., Chelazzi, L., 2003. Influence of marine allochthonous input on sandy beach communities. Oceanogr. Mar. Biol. Annu. Rev. 41, 115e159. Crawley, K.R., Hyndes, G.A., Vanderklift, M.A., Revill, A.T., Nichols, P.D., 2009. Allochthonous brown algae are the primary food source for consumers in a temperate, coastal environment. Mar. Ecol. Prog. Ser. 376, 33e44. ~ a, K., Navarro, J.M., Go
mez, I., 2011. Intra-plant differences in Duarte, C., Acun seaweed nutritional quality and chemical defenses: importance for the feeding behavior of the intertidal amphipod Orchestoidea tuberculata. J. Sea. Res. 66, 215e221. Dugan, J.E., Hubbard, D.M., McCrary, M.D., Pierson, M.O., 2003. The response of macrofauna communities and shorebirds to macrophyte wrack subsidies on exposed sandy beaches of southern California. Est. Coast. Shelf Sci. 58, 25e40. Dugan, J.E., Hubbard, D.,M., Page, H.,M., Schimel, J.,P., 2011. Marine macrophyte wrack inputs and dissolved nutrients in beach sands. Estuaries Coasts 34, 839e850. Duggins, E.O., Eckman, J.E., 1997. Is kelp detritus a good food for suspension feeders? Effects of kelp species, age and secondary metabolites. Mar. Biol. 128, 489e495. Fisher, M.M., Triplett, E.W., 1999. Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities. Appl. Environ. Microbiol. 65, 4630e4636. Gobet, A., Boetius, A., Ramette, A., 2014. Ecological coherence of diversity patterns derived from classical fingerprinting and next generation sequencing techniques. Environ. Microbiol. 16, 2672e2681. Griffiths, C.L., Stenton-Dozey, J., Koop, K., 1983. Kelp wrack and the flow of energy through a sandy beach ecosystem. In: McLachlan, A., Erasmus, T. (Eds.), Sandy Beaches as Ecosystems. W. Junk Publishers, The Hague, pp. 547e556. Hewson, I., Fuhrman, J.A., 2004. Richness and diversity of bacterioplankton species along an estuarine gradient in Moreton Bay, Australia. Appl. Environ. Microbiol. 70, 3425e3433. Inglis, G., 1989. The colonisation and degradation of stranded Macrocystis pyrifera (L.) C. Ag. by the macrofauna of a New Zealand sandy beach. J. Exp. Mar. Biol. Ecol. 125, 203e217. Kepner, R.L., Pratt, J.R., 1994. Use of fluorochromes for direct enumeration of total bacteria in environmental samples: past and present. Microbiol. Rev. 58, 603e615. Koop, K., Griffiths, C.L., 1982. The relative significance of bacteria, meio- and macrofauna on an exposed sandy beach. Mar. Biol. 66, 295e300. Koop, K., Newell, R.C., Lucas, M.I., 1982. Microbial regeneration of nutrients from the decomposition of macrophyte debris on the shore. Mar. Ecol. Prog. Ser. 9, 91e96. Kuzyakov, Y., Blagodatskaya, E., 2015. Microbial hotspots and hot moments in soil: concept & review. Soil Biol. Biochem. 83, 184e199. Langenheder, S., Sobek, S., Tranvik, L.J., 2006. Changes in bacterial community composition along a solar radiation gradient in humic waters. Aquat. Sci. 68, 415e424. Lastra, M., Page, H.M., Dugan, J.E., Hubbard, D.M., Rodil, I.F., 2008. Processing of allochthonous macrophyte subsidies by sandy beach consumers: estimates of feeding rates and impacts on food resources. Mar. Biol. 154, 163e174. ~ e, V.E., Jones, L.R., Helbling, E.W., Manrique, J.M., Calvo, A.Y., Halac, S.R., Villafan 2012. Effects of UV radiation on the taxonomic composition of natural bacter~ o (Patagonia, Argentina). ioplankton communities from Bahía Engan J. Photochem. Photobiol. B Biol. 117, 171e178. Marczak, L.B., Thompson, R.M., Richardson, J.S., 2007. Meta-analysis: trophic level, habitat, and productivity shape the food web effects of resource subsidies. Ecology 88 (1), 140e148.
n, M., Sa
nchez-Lorenzo, A., Calbo
, J., Wild, M., 2013. Long-term Mateos, D., Anto changes in the radiative effects of aerosols and clouds in a mid-latitude region (1985e2010). Glob. Planet Change 111, 288e295. McLachlan, A., 1985. The biomass of macro- and interstitial fauna on clean and wrack-covered beaches in western Australia. Estuar. Coast. Shelf Sci. 21,

112

I.F. Rodil et al. / Marine Environmental Research 112 (2015) 104e112

587e599. McLachlan, A., Brown, A.C., 2006. The Ecology of Sandy Shores. Academic Press, Burlington, MA, USA, p. 373. Mews, M., Zimmer, M., Jelinski, D.E., 2006. Species-specific decomposition rates of beach-cast wrack in Barkley Sound, British Columbia, Canada. Mar. Ecol. Prog. Ser. 328, 155e160. Mucha, A.P., Teixeira, C., Reis, I., Magalh~ aes, C., Bordalo, A.A., Almeida, C.M.R., 2013. Response of a salt marsh microbial community to metal contamination. Estuar. Coast Shelf. Sci. 130, 81e88. Nagayama, K., 2002. Bactericidal activity of phlorotannins from the brown alga Ecklonia kurome. J. Antimicrob. Chemother. 50, 889e893. Nair, S., Bharathi, L., 1980. Heterotrophic bacterial population in tropical sandy beaches. Mahas. Bull. Nat. Inst. Oceanogr. 13, 261e267. Novitsky, J.A., MacSween, M.C., 1989. Microbiology of a high energy beach sediment: evidence for an active and growing community. Mar. Ecol. Prog. Ser. 52, 71e75. Norderhaug, K.M., Fredriksen, S., Nygaard, K., 2003. Trophic importance of Laminaria hyperborea to kelp forest consumers and the importance of bacterial degradation to food quality. Mar. Ecol. Prog. Ser. 255, 135e144. Orr, M., Zimmer, M., Jelinski, D.E., Mews, M., 2005. Wrack deposition on different beach types: spatial and temporal variation in the pattern of subsidy. Ecology 86, 1496e1507. Papageorgiou, N., Moreno, M., Marin, V., Baiardo, S., Arvanitidis, C., Fabiano, M., Eleftheriou, A., 2007. Interrelationships of bacteria, meiofauna and macrofauna in a Mediterranean sedimentary beach (Maremma Park, NW Italy). Helgol. Mar. Res. 61 (1), 31e42. Pennings, S., Carefoot, T., Zimmer, M., Danko, J.P., Ziegler, A., 2000. Feeding preferences of supralittoral isopods and amphipods. Can. J. Zool. 78, 1918e1929.
rez, M.T., Sommaruga, R., 2007. Interactive effects of solar radiation and dissolved Pe organic matter on bacterial activity and community structure. Environ. Microbiol. 9 (9), 2200e2210. Porri, F., Hill, J.M., McQuaid, C.D., 2011. Associations in ephemeral systems: the lack of trophic relationships between sandhoppers and beach wrack. Mar. Ecol. Prog. Ser. 426, 253e262.

Porter, K.G., Feig, Y.S., 1980. The use of DAPI for identifying and counting aquatic microflora. Limnol. Oceanogr. 25, 943e948. Purahong, W., Stempfhuber, B., Lentendu, G., Francioli, D., Reitz, T., Buscot, F., Schloter, M., Krüger, D., 2015. Influence of commonly used primer systems on automated ribosomal intergenic spacer analysis of bacterial communities in environmental samples. PLoS One 10, e0118967. R Development Core Team, 2014. R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria, ISBN 3900051-07-0. Available: http://www.R-project.org/. Ranjard, L., Poly, F., Lata, J.-C., Mougel, C., Thioulouse, J., Nazaret, S., 2001. Characterization of bacterial and fungal soil communities by automated ribosomal intergenic spacer analysis fingerprints: biological and methodological variability. Appl. Environ. Microbiol. 67, 4479e4487.
pez, J., 2008. Differential effects of native and Rodil, I.F., Olabarria, C., Lastra, M., Lo invasive algal wrack on macrofaunal assemblages inhabiting exposed sandy beaches. J. Exp. Mar. Biol. Ecol. 358, 1e13. Rodil, I.F., Lucena-Moya, P., Olabarria, C., Arenas, F., 2015a. Alteration of macroalgal subsidies by climate-associated stressors affects behaviour of wrack-reliant beach consumers. Ecosystems 18, 428e440. Rodil, I.F., Olabarria, C., Lastra, M., Arenas, F., 2015b. Combined effects of wrack identity and solar radiation on associated beach macrofaunal assemblages. Mar. Ecol. Prog. Ser. 531, 167e178. Sosik, E.A., Simenstad, C.A., 2013. Isotopic evidence and consequences of the role of microbes in macroalgae detritus-based food webs. Mar. Ecol. Prog. Ser. 494, 107e119. Spiller, D.A., Piovia-Scott, J., Wright, A.N., Yang, L.H., Takimoto, G., Schoener, T.W., Iwata, T., 2010. Marine subsidies have multiple effects on coastal food webs. Ecology 91, 1424e1434. Swanson, A.K., Fox, C.H., 2007. Altered kelp (Laminariales) phlorotannins and growth under elevated carbon dioxide and ultraviolet-B treatments can influence associated intertidal food webs. Glob. Change Biol. 13, 1696e1709. Wild, M., 2009. Global dimming and brightening: a review. J. Geophys. Res. 114. Zuur, A.F., Ieno, E.N., Walker, N.J., Savelievev, A.A., Smith, G.M., 2009. Mixed Effects Models and Extensions in Ecology with R. Springer-Verlag, New York, p. 574.