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Do colonies of Lytocarpiamyriophyllum, L. 1758 (Cnidaria,Hydrozoa) affect the biochemicalcomposition and the meiofaunaldiversity of surrounding sediments?Carlo Cerranoa, Silvia Bianchellia, Cristina Gioia Di Camilloa,Fabrizio Torsania & Antonio Pusceddua

a Dipartimento di Scienze della Vita e dell'Ambiente, UniversitàPolitecnica delle Marche, Via Brecce Bianche, Ancona 60131, ItalyPublished online: 07 Jan 2015.

To cite this article: Carlo Cerrano, Silvia Bianchelli, Cristina Gioia Di Camillo, Fabrizio Torsani &Antonio Pusceddu (2015) Do colonies of Lytocarpia myriophyllum, L. 1758 (Cnidaria, Hydrozoa)affect the biochemical composition and the meiofaunal diversity of surrounding sediments?,Chemistry and Ecology, 31:1, 1-21, DOI: 10.1080/02757540.2014.966699

To link to this article: http://dx.doi.org/10.1080/02757540.2014.966699

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Chemistry and Ecology, 2015Vol. 31, No. 1, 1–21, http://dx.doi.org/10.1080/02757540.2014.966699

Do colonies of Lytocarpia myriophyllum, L. 1758 (Cnidaria,Hydrozoa) affect the biochemical composition and the

meiofaunal diversity of surrounding sediments?

Carlo Cerrano∗, Silvia Bianchelli, Cristina Gioia Di Camillo, Fabrizio Torsani andAntonio Pusceddu

Dipartimento di Scienze della Vita e dell’Ambiente, Università Politecnica delle Marche,Via Brecce Bianche, Ancona 60131, Italy

(Received 20 January 2014; final version received 3 September 2014)

Lytocarpia myriophyllum, the biggest hydroid of the Mediterranean, lives at soft bottoms. It is severelythreatened by bottom trawling activities. To assess its possible influence on trophodynamics and biodiver-sity of surrounding sediments, we compared the organic matter content and biochemical composition, andmeiofaunal biodiversity in sediments below L. myriophyllum colonies and in surrounding bare sediments.Below L. myriophyllum sediments, the protein to carbohydrate ratio, the lipid contents and meiofaunalabundance showed values higher than in bare sediments, suggesting that L. myriophyllum may influ-ence the availability of resources for benthic consumers. The richness of meiofaunal taxa and nematodespecies generally decreased from below the colonies to bare sediments suggesting that the presence ofL. myriophyllum colonies can have a certain influence also on benthic biodiversity. The possible influenceof L. myriophyllum on trophodynamics and biodiversity of neighbouring soft bottoms claims for effortsto increase our knowledge about the trophodynamics of mesophotic coral forests and to develop adequatemeasures of conservation of these important and threatened habitats.

Keywords: ecosystem engineers; meiofauna; nematodes; hydrozoa; organic matter; Mediterranean Sea

1. Introduction

During the last few years, it is becoming increasingly evident that the loss of biodiversity andhabitats is one of the most important threats to the sustainable use of marine ecosystem goodsand services.[1,2]

Coastal marine environments at depths ranging from 0 to 200 m represent less than 20% ofthe global ocean’s surface, but host the majority of biodiversity of the worldwide oceans.[3] Thisholds particularly true for the Mediterranean Sea, which, although representing less than 1% insurface area and volume as compared to the world’s oceans,[4] is a hot spot of biodiversity.[4–6]

The so-called ‘mesophotic’ or ‘twilight’ zone is located on continental slopes or on the top ofseamounts at depths comprised between 60 and 100–120 m.[7] This zone is defined as the deep-est region of the photic zone in which light-dependent communities can reliably develop.[8,9]

*Corresponding author. Email: c.cerrano@univpm.it

© 2015 Taylor & Francis

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The mesophotic zone, in spite of energy limitation, harbours a significant fraction of coastal bio-diversity and is supposed to function as an important biodiversity reservoir,[10] less prone to thenegative impacts of climate change.[11,12]

Moreover, it hosts communities with putatively key ecological roles that still need to be defi-nitely assessed and species with peculiar physiological adaptations, which might represent alsoan important biotechnological resource.[13] The mesophotic zone hosts large populations of oth-erwise rare and/or threatened species, such as the Mediterranean kelp (Laminaria rodriguezii,Bornet, 1888), the gold (Savalia savaglia, Bertoloni, 1819), black (Antipathes dichotoma; Pallas,1766), and red (Corallium rubrum; Linnaeus, 1758) corals, the purple gorgonian (Paramuriceaclavata; Risso, 1826), and other highly heterogeneous coralligenous assemblages.[14–20]

All the above-mentioned species and assemblages modulate the availability of resources toother species by causing physico-chemical changes in biotic or abiotic materials, modifying,maintaining, and creating habitats, ultimately acting as ecosystem engineers.[21–24]

Ecosystem engineers can cause a multitude of structural transformations of habitats, forinstance through bioconstruction and bioturbation.[25–27] By these activities, ecosystem engi-neers also exert relevant effects on the local biodiversity in a broad range of ecosystems.[28–33]

Coastal ecosystems dominated by habitat formers are included among the economically mostvaluable ecosystems of the world.[34] Nevertheless, they are threatened and declining,[35–38] asa result of a myriad of disrupting anthropogenic factors (e.g. coastal engineering, bottom trawl-ing, introduction of invasive species),[33] many of which acting synergistically.[39] Hence, theknowledge of ecological processes in the mesophotic zone of the Mediterranean Sea is becom-ing increasingly important not only to preserve those habitats and safeguard the communitiesliving there, but also to manage coastal fisheries and other human activities in a more sustainableway.

To date, the few studies conducted in the mesophotic zone of the Mediterranean Seahave dealt almost exclusively with the description of the ecological role of hard-bottomassemblages,[14,16,40] whereas very little attention has been paid to the ecological role of thesoft bottoms’ fauna.[15]

An important goal for area-based management is to protect marine habitats and their associ-ated fauna. Since understanding the impacts of biodiversity loss and ecosystem size reductionappears necessary to estimate the ecosystem goods and services to the mankind,[41,42] we stillneed to know where highly diverse and pristine habitats still exist and what is their importance onthe biodiversity and ecosystem functioning of neighbouring areas. From this point of view, iden-tifying and managing engineering species and responsive ecosystems promise opportunities forrestoration ecology and should be a key priority for marine habitats conservation.[43] The latter,in turn, will necessitate a shift to a process-based understanding functioning of whole systems,which is a large and important step towards ecosystem-based management.[44]

Moreover, as mesophotic zone represents a key transition zone between shallow waters andthe deep sea and because of its relative remoteness to human direct exploitation, this environ-ment may represent today one of the few remnant examples of pristine marine habitat still to beexplored.

To provide new insights on the ecological significance of ecosystem engineers living in softbottoms of the Mediterranean sea mesophotic zone, we focused our attention on a populationof Lytocarpia myriophyllum (Linnaeus, 1758) living on incoherent sediments within the MarineProtected Area of the Portofino Promontory (Ligurian Sea, NW Mediterranean Sea; Figure 1).This species, the biggest hydroid of the Mediterranean sea with colonies of more than 1 m height,colonises soft bottoms down to ca. 1000 m depth.[45] Like other erected sessile species (e.g. gor-gonians and the gold coral S. savaglia, Bertoloni, 1819),[15] since the large size reached by L.myriophyllum, each colony of the hydroid can be considered a miniature forest. This leads us tothink that the entire L. myriophyllum population could have possible effects on the biodiversity

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Figure 1. Large L. myriophyllum colony on the detrital bottom of the Portofino Promontory. Each colony is made byseveral stems and anchored in the sediments by a root-like apparatus of hydrorizae. Colony height is 90 cm (Photocredits:Portofino Divers).

of the fauna living in the neighbouring sediments (Figure 1). To assess whether L. myriophyllumbehaves as an ecosystem engineer in mesophotic soft bottoms, we first characterised the mor-phometry of the colonies adapted to live on the sandy substrates in the study area, and then wetested the null hypothesis that the quantity and biochemical composition of organic matter in thesediment, along with the abundance and taxa richness of meiofauna and nematode biodiversity,does not differ between colonised sediments and neighbouring bare soft bottoms.

2. Methods

2.1. Study area, samplings, and morphometric analysis

The population of the studied species lays at 70 m depth on a detritic bottom (Figure 2)at the basis of a vertical cliff inside the Marine Protected Area of Portofino (44°18′00.54′′

N, 09°13′11.25′′ E; Ligurian Sea, Italy), in an area characterised by strong unidirectionalcurrents.[15] L. myriophyllum, a suspension feeder hydroid, typically forms meadows in soft bot-toms at depths comprised within the mesophotic zone.[46] Samples for the analysis of organicmatter in the sediment and meiofauna were collected by SCUBA (trimix) scientific divers by gen-tly inserting PVC corers (20 cm height and 7 cm diameter) into the sediment. During early May2011, at all stations three replicate samples were collected to account for the small-scale vari-ability of the investigated variables.[47] Samples were collected from five stations, all located at70 m depth: (i) two stations at the basis of two different coral colonies (namely ‘Inner 01’ and‘Inner 02’ stations), (ii) two stations at 1 m far from the two colonies (namely ‘Outer 01’ and‘Outer 02’), and (iii) a bare station (namely ‘bare’ station), without any colony, located downcurrent at about 10 m distance from both colonies. This reduced sampling design was adopted,in spite of a limited representativeness of environmental and faunal variability, to limit as much

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Figure 2. Sampling area located in the Portofino marine protected area, Northern Tyrrhenian Sea (Western Mediter-ranean Sea).

as possible the impacts of an invasive sampling within the marine protected area and around thecoral colonies, uncommon in the area. Unfortunately, the collection of samples from bare sed-iments was limited to only one station due to the intrinsic difficulties in retrieving samples byscuba diving at 70 m water depth.

All samples were immediately frozen at − 20°C until analyses in the laboratory (within twoweeks). In order to figure out the trophic role of L. myriophyllum in the considered habitat, a well-preserved colony has been subjected to the morphometric analysis. Again, since the morphologyof Lytocarpia, and of the aglaopheniids in general, is not much varied among individuals of thesame species, and in order to avoid a disruptive sampling, only one single colony was consideredfor this kind of analysis. To determine the density of hydrocladia (average number of hydrocladiacm−1), five portions of the ramifications were cut and the hydrocladia were counted. Moreover,hydrothecae, hydranths, and cnidocysts were observed at the light microscope and measured bya micrometric lens.[46]

2.2. Biochemical composition of sediment organic matter

Once in the laboratory, the first centimetre of sediment from each core was analysed fororganic matter biochemical composition in terms of total phytopigment, protein, carbohydrate,and lipid contents. Chlorophyll-a and phaeopigments were analysed fluorometrically.[48] Totalphytopigment concentrations were defined as the sum of chlorophyll-a and phaeopigment con-centrations, and utilised as an estimate of the organic material of algal origin, including the living(chlorophyll-a) and senescent/detrital (phaeopigment) fractions.[49] Protein, carbohydrate, and

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lipid analyses were carried out spectrophotometrically.[50] For the analysis of each biochemi-cal class of organic compound, blanks were made with the same sediment samples previouslytreated in a muffle furnace (450°C, 2 h).

Protein, carbohydrate, and lipid concentrations were converted to carbon equivalents usingthe conversion factors 0.49, 0.40, and 0.75 mg C mg−1 respectively, and their sum referred asthe biopolymeric C (BPC).[51] Sediment phytopigment concentrations were converted into car-bon equivalents using a mean value of 40 µg C µg phytopigment−1.[49] The fraction of BPCrepresented by relatively fresh algal material was then assessed as the percentage contribution ofphytopigment C to BPC contents and referred to as the algal fraction of BPC.[49]

We used the percentage contributions of phytopigment and protein C equivalents to BPCconcentrations and the values of the protein to carbohydrate ratio as descriptors of ageing andnutritional quality of organic matter in the sediment, respectively.[52]

2.3. Meiofaunal analyses

Once in the laboratory, sediment samples for meiofaunal analyses were sliced into five sed-iment layers (i.e. 0–1, 1–3, 3–5, 5–10, and 10–15 cm), fixed with 4% buffered formalin andstained with Rose Bengal (0.5 g L−1) until analysis. Sediments were sieved through a 1000-µmmesh, and a 20-µm mesh was used to retain the smallest organisms. The fraction remaining onthe latter sieve was resuspended and centrifuged three times with Ludox HS40 (diluted withwater to a final density of 1.18 g cm−3).[50,53] All animals remaining in the supernatant werepassed again through a 20 µm mesh net, washed with tap water and, after staining with RoseBengal, sorted under a stereomicroscope ( × 40 magnification).[50] The rare taxa were definedas those taxa that represented each < 1% of the total meiofaunal abundance in all investigatedsamples.[54–56]

2.4. Nematode biodiversity

One-hundred nematodes for each of the 3 replicates (or all of the nematodes when the abundancewas lower than 100 specimens per sample) were mounted on slides, following the formalin–ethanol–glycerol technique to prevent dehydration.[57] The nematodes were identified to thespecies level according to the presently used manuals[58–61] and the most recent literature deal-ing with new nematode genera and species. All of the unknown species were indicated as sp1,sp2, sp3, . . . spn.

The nematode α-diversity [62] was estimated using the species richness (SR). The SR ateach site was determined cumulatively as the total number of species retrieved from the threereplicate samples. As SR is strongly affected by sample size, the expected number of species(ES), which provides a standardisation of the values of the SR according to the sample size,was also calculated. The ES for a theoretical sample of 100 specimens (ES100) was used inthis study.

The species diversity was also measured by the Shannon–Wiener information function (H′,using log2), and the evenness was measured by the Pielou’s J index.[63] These indices werecalculated using PRIMER v6.0 + (Plymouth Marine Laboratory, UK).[64]

The β-diversity (i.e. turnover diversity) [62] was measured between sampling stations (i.e.Inner vs. Outer vs. Bare sediments) as the percentage dissimilarity of the nematode assemblagespecies composition, calculated from resemblance matrices based on Bray–Curtis similarityafter presence/absence transformation of the raw data (SIMPER, included in the PRIMERv6.0 + software).

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The γ -diversity (i.e. landscape diversity) [62] was measured as overall nematode SR found inthe investigated area.

The trophic composition of the nematode assemblages was defined based on the assumptionthat nematode stoma morphology is an important determinant of food selection.[65] Nematodeswere divided into four groups: no buccal cavity or a fine tubular one-selective (bacterial) feeder(1A); large but unarmed buccal cavity non-selective deposit feeders (1B); buccal cavity withscraping tooth or teeth, epistrate or epigrowth (diatoms) feeders (2A); and buccal cavity withlarge jaws, predators/omnivores (2B). The index of trophic diversity (ITD) was calculated as1-ITD, where ITD = g1

2 + g22 + g3

2 . . . + gn2, where g is the relative contribution of each

trophic group to the total number of individuals, and n is the number of trophic groups.[66] Forn = 4 (as in the present study), the 1-ITD ranges from 0.00 to 0.75.

To determine the colonisation strategies of the nematodes, the maturity index (MI) was calcu-lated according to the weighted mean of the individual genus scores, as � ν (i) f (i), whereν is the colonisers-persisters (c-p) value of the genus I,[67] and f (i) is the frequency ofthat genus.

2.5. Statistical analyses

The variability in the sedimentary assets below coral colonies having different size and exposedto different near-to-bottom current direction and intensity can be very large. At the expenseof a certain loss of information about the spatial variability across different coral coloniesand the surrounding bare sediments, differences in the organic compounds sedimentary con-tents, total meiofaunal abundance, richness of taxa, and nematode biodiversity indices amongsampling stations were assessed using one-way analysis of variance (ANOVA), separately foreach variable. We used the station as a fixed source of variability (n = 5, Inner 01, Inner02, Outer 01, Outer 02, and Bare). When significant differences were observed between sta-tions, a pairwise test was also applied to ascertain patterns of differences among stations.The same experimental design was used to test variations in the biochemical composition andnutritional quality of sedimentary organic matter, as well as the composition of meiofaunalcommunity (both the whole community and rare taxa) among stations, after presence/absencetransformations using the PERMANOVA tests based on matrixes of Euclidean distance afternormalisation of the data (organic matter) and Bray–Curtis similarity matrices (meiofaunalcommunity).[64]

SIMPER analyses were performed to assess the percentage dissimilarity in the nematodeassemblages among the investigated stations. Analysis of similarities (ANOSIM) was performedto test for the presence of statistical differences in nematode species among the investigated sta-tions. A ranked matrix of Bray–Curtis similarities, after presence/absence transformations, wasused as input for these tests.

To visualise differences among stations in the sedimentary organic matter composition andnutritional quality, meiofaunal community (both the whole community and rare taxa, contrastedwith organic matter composition and nutritional quality) and nematode assemblages compo-sition bi-plots after a Canonical Analysis of Principal Coordinates (CAP) were prepared.[68]CAP analysis was selected as the ordination technique as it allows to find the axis (oraxes) in the principal coordinate space that is best at discriminating among the a priorigroups. Moreover, this analysis allows identifying the environmental variables which guidethe ordination.

All statistical analyses were performed with the software PRIMER 6 + ,[64] except forthe ANOVA tests, which were carried out using the GMAV 5.0 software (Universityof Sydney).

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3. Results

3.1. Characteristics of the L. myriophyllum meadows

Each colony considered to assess the effect of L. myriophyllum on sedimentary biochemical com-position and meiofaunal diversity (see sampling design in the Methods section) hosts up to 10–12main stems with a height of about 90 cm (Figure 1, [46]). As previously reported,[46] the basalportion of the buried hydrocaulae can be 40 cm in diameter and is about 20 cm sunk into the sedi-ments, giving rise to a spongy web of thin anastomosing stolons. L. myriophyllum is characterisedby repeated units called hydrothecae, each containing a feeding polyp (hydranth). Hydrothecaemeasure 480–520 µm in length and 200–265 µm in diameter and contain tiny hydranths (dimen-sions of fixed material: 290–420 µm in length; 110–130 µm in width) bearing small cnidocysts(about 5 × 2.5 µm). Feeding polyps are present on about 80% of the colony. Considering a den-sity of 21.3 ± 1.6 hydrocladia cm−1, on a colony 90 cm heigh and composed of 12 primarybranches, it can be estimated that the total length of branches bearing hydrocladia is ca. 864 cm,overall hosting > 18,000 hydrocladia. Even if hydranths are minute, their number is high ( > 44hydrants per hydrocaldium); hence, the investigated colony hosts, only on primary branches,> 810,000 feeding polyps.

3.2. Content, biochemical composition, and nutritional quality of organic matter in thesediment

The chlorophyll-a, phaeopigment, total phytopigment, protein, carbohydrate, lipid, and BPC con-centrations, as well as the indicators of nutritional quality (i.e. phytopigment and protein to BPCratio and protein to carbohydrate ratio) in the sediment of the investigated stations are reported inTable 1. The results of one-way ANOVAs reveal that the concentrations of all investigated bio-chemical compounds and the descriptors of nutritional quality vary significantly among stations;the pairwise analyses reveal that the values of all investigated variables, except lipid contentsand the values of the protein to carbohydrate ratio, were significantly higher in bare sedimentsthan in L. myriophyllum meadows (Table 2). The PERMANOVA test shows that the biochemicalcomposition of organic matter varies significantly among all sampling stations (Table 3). The bi-plot produced after the CAP analysis shows that bare sediments, being characterised by higherphaeopigment, protein, and carbohydrate contents, display a biochemical composition well apartfrom those in all other stations (Figure 3(a)). Sediments in one of the two Outer stations clus-tered apart from all other stations because of the higher lipid contents (Figure 3(a)). The resultsof the PERMANOVA test on the nutritional quality of organic matter, performed using the con-tributions of phytopigment and protein C equivalents to BPC concentrations and the protein tocarbohydrate ratio as descriptors of nutritional quality of organic matter in the sediment, revealsignificant though less pronounced differences among stations (Table 3), with sediments in Outerstations being characterised by the lowest nutritional values (Figure 3(b)).

3.3. Meiofaunal abundance, richness of taxa, and community structure

The one-way ANOVA reveals that the meiofaunal abundance and the richness of taxa varysignificantly among the sampling stations, with values in station Inner 01 significantly higherthan that in all other stations (Figure 4, Table 2). The meiofaunal community is dominatedat all stations by nematodes (69–86%), followed by copepods (11–25%) and polychaetes (1–2%) (Figure 5(a)). Among the rare taxa, oligochates, isopods, amphipods, acarina, tardigrades,

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Table 1. Contents, nutritional quality, and ageing of organic matter in the sediments of the investigated stations at Portofino’s promontory marine protected area.

Chlorophyll-a Phaeopigment Total phytopigment Protein Carbohydrate Lipid BPC Algal fraction Protein fraction Protein toStation (µg g−1) (µg g−1) (µg g−1) (mg g−1) (mg g−1) (mg g−1) (mgC g−1) of BPC (%) of BPC (%) carbohydrate ratio

Inner 01 0.20 ± 0.01 6.44 ± 0.64 6.64 ± 0.64 0.83 ± 0.04 1.51 ± 0.21 0.40 ± 0.03 1.31 ± 0.08 20.4 ± 2.9 30.9 ± 0.9 0.55 ± 0.06Inner 02 0.28 ± 0.03 5.55 ± 0.64 5.83 ± 0.67 0.74 ± 0.04 1.45 ± 0.09 0.26 ± 0.03 1.14 ± 0.05 20.6 ± 3.3 32.0 ± 2.3 0.51 ± 0.05Outer 01 0.17 ± 0.02 6.10 ± 0.13 6.26 ± 0.11 0.76 ± 0.11 1.80 ± 0.06 0.71 ± 0.16 1.63 ± 0.08 15.4 ± 1.0 23.0 ± 4.5 0.42 ± 0.07Outer 02 0.20 ± 0.02 3.68 ± 0.30 3.88 ± 0.31 0.39 ± 0.04 1.32 ± 0.09 0.19 ± 0.03 0.86 ± 0.04 18.0 ± 1.4 22.4 ± 2.6 0.30 ± 0.04Bare 0.44 ± 0.10 7.59 ± 0.14 8.03 ± 0.24 0.98 ± 0.09 2.04 ± 0.25 0.22 ± 0.01 1.46 ± 0.08 22.1 ± 0.9 33.1 ± 4.5 0.49 ± 0.11

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Table 2. Results of the one-way ANOVA testing for differences among stations in the (a) quantity, (b) nutritionalquality of sedimentary organic matter, and (c) meiofaunal abundance and richness of taxa between stations.

Variable Source df MS F P Pair wise test

(a)Chlorophyll-a Station 4 0.259 60.60 ** Bare, Inner 02 > Inner 01, Outer 01, Outer 02

Residual 10 0.043Phaeopigment Station 4 6.164 32.51 ** Bare > Inner 01, Inner 02, Outer 01 > Outer 02

Residual 10 0.190Total phytopigment Station 4 6.775 32.84 ** Bare > Inner 01, Inner 02, Outer 01 > Outer 02

Residual 10 0.206Protein Station 4 0.138 26.83 ** Bare > Inner 02 > Outer 02

Residual 10 0.005Carbohydrate Station 4 0.255 10.05 ** Bare, Outer 01 > Inner 02, Outer 02

Residual 10 0.026Lipid Station 4 0.139 23.84 ** Outer 01 > Inner 01 > Inner 02, Bare > Outer 02

Residual 10 0.006BPC Station 4 0.259 60.60 ** Bare, Outer 01 > Inner 01 > Inner 02 > Outer 02

Residual 10 0.004

(b)Algal fraction of BPC Station 4 20.248 4.44 * Bare, Inner 01 > Outer 01

Residual 10 4.558Protein fraction of BPC Station 4 79.548 7.39 *** Bare > Outer 01, Outer 02, Inner 01, Inner 02

Residual 10 10.767Protein to carbohydrate ratio Station 4 0.029 5.98 * Inner 01, Inner 02 > Bare > Outer 02

Residual 10 0.005

(c)Abundance Station 4 79.3 18.8 ** Inner 01 > Bare > Inner 02, Outer 01, Outer 02

Residual 10 4.2Richness of Taxa Station 4 3.5 6.4 ** Inner 01 > Inner 02, Outer 01, Outer 02, Bare

Residual 10 0.6

Note: df = degrees of freedom; MS = mean of squares; F = variance ratio; P = significance level.*** = P < 0.001.** = P < 0.01.* = P < 0.05.

ostracods, kinorhynchs, and sipuncula were retrieved, with different relative abundances depend-ing on the investigated station (Figure 5(b)).

The PERMANOVA results show that the composition of the meiofaunal community variessignificantly among stations: the pairwise tests reveal that the community differed significantlyamong all the pairs contrasted, considering separately either the whole or the rare taxa com-munities (Table 4). The bi-plot produced after a CAP analysis (Figure 6(a–d)) reveals that thecomposition of overall meiofaunal and the rare taxa community varied among some but not allstations and that no clear spatial patterns can be associated with the presence or absence of theL. myriophyllum. Moreover, with plotting the taxonomic composition of both whole communityand rare taxa against the sedimentary organic matter quantity, the CAP analysis revealed that thedifferences among stations were mostly driven by pigment concentration (i.e. chlorophyll-a andphaeopigment concentration, Figure 6(a) and 6(c)). With plotting the composition of rare taxaagainst the sedimentary organic matter nutritional quality, the CAP analysis revealed that thedifferences among stations were mostly driven by the contribution of phytopigment and proteinto BPC and by protein to carbohydrate ratio.

3.4. Nematode diversity

The indices of nematode diversity are reported in Table 5(a). The univariate ANOVA reveals thatsome of the biodiversity indices varied significantly among stations (data not shown). ANOSIM

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Table 3. Results of PERMANOVA (a) and pairwise test (b) on biochemical composi-tion and nutritional quality of organic matter in the sediment among stations.

(a) Source df MS F P

Biochemical composition Stations 4 15.448 18.821 ***Residual 10 0.821

Total 14Nutritional quality Stations 4 7.323 **

Residual 10 1.271Total 14

(b) Biochemical composition Nutritional quality

Groups t P t P

Inner01, Inner02 2.068 * 0.418 nsInner01, Outer01 2.398 * 2.747 *Inner01, Outer02 5.565 *** 3.694 *Inner01, Bare01 3.479 ** 0.890 nsInner02, Outer01 3.888 *** 2.628 *Inner02, Outer02 5.134 ** 3.289 *Inner02, Bare01 3.589 * 0.554 nsOuter01, Outer02 6.148 ** 1.825 nsOuter01, Bare01 4.012 ** 2.945 *Outer02, Bare01 6.448 ** 3.314 *

Notes: df = degrees of freedom; MF = mean of squares; F = variance ratio; P = significancelevel; ns = not significant.** = P < 0.01.* = P < 0.05.

and SIMPER analyses reveal that there is an average dissimilarity of 54% (from 49% to > 60%)among all stations, and that the largest differences occur between Bare sediments and all otherstations, between Inner 01 and the Outer stations, between Outer 01 and Outer 02 (Table 5(b)).The bi-plot produced after a CAP analysis (Figure 7) reveals that the composition of nematodeassemblages varied among the investigated stations.

4. Discussion

A peculiar role of ecosystem engineers is structuring habitats characterised by stable edaphicconditions, by creating a sort of buffered zone where oscillation in environmental parameters canbe lower when compared to the surrounding environments both on land and underwater.[69,70]In the presence of ecosystem engineers, indeed, water movement is usually reduced and affectssedimentation and re-suspension processes.[71] Also, ecosystem engineers, altering the sedimenttexture, can influence local biodiversity.[15] L. myriophyllum, with its large colonies and itspeculiar system of anastomosed root-like apparatus allowing the anchorage of the colonies andthe stabilisation of colonised sediments, is a good example of ecosystem engineers.[46]

Hydroids belonging to the family Aglaopheniidae generally show small hydranths bear-ing delicate tentacles extended over the thecal margin in order to collect food. While theliving tissue (coenosarc) and the defensive polyps (nematophores) can be armed with largecnidocysts, feeding polyps (hydranths) in general bear very small capsules. The examined mate-rial of L. myriophyllum reflects these characteristics. These observations lead us to supposethat aglaopheniids, and therefore also L. myriophyllum, feed by intercepting suspended organicparticles.[72] Capture rates of L. myriophyllum are probably lower than those of a hydroidwith larger hydranths, such as Campanularia everta (0.5 mg C polyp−1 day−1 [73]). Consid-ering the minimal capture rate reported in the literature for cnidarians (0.01 mg C polyp−1 day−1

[73]), the entire colony of L. myriophyllum with an estimated total of over 800,000 feeding

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Figure 3. Output of CAP analyses on the (a) biochemical composition and (b) nutritional quality indicators (in termsof contributions of phytopigment and protein to BPC and protein to carbohydrate ratio) of the organic matter in thesediment.

hydranths could collect up to 8 g C day−1. Since the abundance of L. myriophyllum in the con-sidered area is 1.57 ± 0.75 colonies m−2,[46] the hydroid meadow could be estimated as highas ca. 13 g C m−2 day−1, that is, about three orders of magnitude higher than the carnivoroushydroid Eudendrium racemosum (ca. 14 mg C m−2 day−1 [74]). The ingestion rates in hydro-zoans are similar to those estimated for some bivalves and tunicates (active suspension feeders),and despite little information is available on non-carnivorous diet of hydroids, there is evidencethat they are able to feed also on bacteria, particulate, and dissolved organic matter,[73,75,76]

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0

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Figure 4. Meiofaunal abundance (a) and richness of taxa (b) in the sediments of the investigated stations. Displayedare average values ± standard deviation.

which allows ranking this taxon among the most voracious suspension feeding organisms atsea.[77,78]

In this study, the organic loads in sediments colonised by L. myriophyllum are generally lowerthan those in surrounding bare sediments. This finding is in contrast with results previouslyreported for the gold coral S. savaglia living inside a gorgonian forest.[15] In that case, thequantity of organic matter below the anthozoan forest was significantly higher than those inbare sediments and related to the hydrodynamic control of the forest on organic particles andlocal sedimentation rates. As previously reported for other ecosystem engineers like seagrassmeadows and reef-building filter-feeders,[71,79] the accumulation of high quantities of organicmatter in the sediments beneath the S. savaglia colonies and the hosting gorgonians was mostlikely the result of diminished water current speed and re-suspension processes near the coloniesthemselves. Our results let us hypothesise that the discrepancy between this and previous studiescould be related to the different feeding behaviour of the investigated species (i.e. S. savagliavs. L. myriophyllum). We postulate indeed that L. myriophyllum is probably more efficient inconsuming suspended organic matter than S. savaglia, thus limiting the potential accumulationof organic particles at the sea bottom.

Moreover, we report here that the presence of L. myriophyllum may exert some influence onthe nutritional quality of organic matter in the sediment below the meadows, since both univariate

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Chemistry and Ecology 13

0%

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Inner 01 Inner 02 Outer 01 Outer 02 Bare

Oligochaetes Isopods Amphipods AcarinaTardigrades Ostracods kinorhynchs Sipuncula

Figure 5. Whole meiofaunal (a) and rare taxa (b) community structure in the investigated stations.

and multivariate ANOVA revealed differences between sediments below (i.e. Inner stations),outside the colonies (i.e. Outer stations) and Bare sediments. In particular, two of the descriptorsof nutritional quality (algal and protein contribution to BPC) displayed higher values in barethan colonised sediments, suggesting a selective feeding activity by L. myriophyllum on recentlyproduced organic matter. In this regard, studies of energy transfer in Mediterranean rocky coral-ligenous communities estimate that a hydroid species contributing less than 0.5% to the totalbenthic biomass can capture alone ca. 10% of the phytoplankton production, annually.[80]Similarly, our results allow us to hypothesise that the filtering activity of this hydroid doesnot only affect the quantity of organic matter in the sediment, but also its nutritional quality,most probably through the selective interception of sinking particles. However, at this stage ofthe research we cannot exclude that also the root-like system of L. myriophyllum may affect

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Table 4. Results of PERMANOVA and pairwise tests on meiofaunal community: whole community composition(first line) and rare taxa community composition (second line).

Source of variation df MS F P Pairwise tests

Station 4 314.5 5.6 ** [Inner 01 = Inner 02] �= Outer 01 �= Outer 02 �= BareResidual 10 56.4Total 14Station 4 4602.7 9.5 *** Inner 01 �= Inner 02 �= Outer 01 �= Outer 02 �= BareResidual 10 484.9Total 14

Notes: df = degrees of freedom; MS = mean squares; F = variance ratio; P = significance level.*** = P < 0.001.** = P < 0.01.* = P < 0.05.

sediment quality by the absorption of organic matter, so that this hypothesis needs to be testedfurther.

Studies conducted in the mesophotic zone of the Mediterranean Sea revealed that sedimentssurrounding S. savaglia colonies, when compared to the surrounding bare sediments, are charac-terised by significantly higher meiofaunal abundance and diversity.[15] Other studies, revealedthat the presence of coral habitats in the mesophotic zone can have a certain effect on themeiofaunal communities inhabiting the surrounding coral-free sediments, but also that theseeffects are more evident on meiofaunal diversity and community composition rather than ontheir abundance or biomass.[40] In this study, the highest meiofaunal abundance was encoun-tered in organically enriched bare sediments as well as in the high nutritional quality sedimentbelow one of the investigated L. myriophyllum colonies. In this regard, it must be considered thatseveral studies showed that meiofaunal abundance, especially in food-limited environments, likethe mesophotic zone and the deep sea, respond not only to the quantity but also to the quality offood items.[81]

The statistical analysis conducted on single stations apart did not identify clear patterns inthe number of meiofaunal taxa. However, when pooling together the richness of taxa in similarhabitats (e.g. Inner stations vs. Outer stations vs. Bare sediments) the three-dimensional archi-tecture produced by the hydrorhizae of L. myriophyllum apparently determined an increase inthe overall number of meiofaunal taxa, which, from 12 taxa in inner stations, slightly decreasedto 10 in outer stations and 8 in Bare sediments (Figure 8). Moreover, the multivariate analy-sis conducted on the whole meiofaunal community and rare taxa indicated that the samplingstations were characterised by different communities even if no clear spatial patterns can beassociated with the presence or absence of the L. myriophyllum. Recent studies have revealedthat the effects of the presence of coralligenous concretions are more clearly detected if theanalysis is carried out focusing on meiofaunal rare taxa (i.e.. each representing < 1% of thetotal meiofaunal abundance, mostly represented by juveniles of macro-megafaunal species [40]).In addition, nematodes and copepods, representing cumulatively the highest fraction of themeiofaunal assemblages, were responsible for higher dissimilarity between different habitats(i.e. below, outside the colonies and Bare sediments) when considering the entire meiofaunalassemblages.[40,54,55] However, in this study the differences in the taxonomic compositionamong stations are evident also considering the whole meiofaunal communities, despite the largedominance of nematodes and copepods.

The high level of dissimilarity between adjacent distances found in the meiofaunal assem-blages composition indicates that the presence or absence of the L. myriophyllum can any-way contribute to enhance the overall biodiversity of the surrounding soft bottoms seascape.However, the lack clear spatial patterns associated with the presence or absence of the

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–0.4 –0.2 0 0.2 0.4 0.6CAP1

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Inner 01

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Inner 01

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Inner 02

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Figure 6. Output of CAP analyses on the whole meiofaunal community against sedimentary organic matter quantity (a)and nutritional quality (b) and rare taxa meiofaunal community composition against sedimentary organic matter quantity(c) and nutritional quality (d).

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Table 5. Nematode biodiversity in the study area. Reported are (a) diversity indices (SR, Eveness (Pielou’s index, J),expected species number for 100 individuals (ES100), Shannon index (H′), MI and 1-ITD) and (b) results of ANOSIMand SIMPER, testing for differences in nematode assemblages species composition among sampling stations.

(a)Station SR J ES100 H′ log2 MI 1-ITD

Inner 01 35 ± 1 0.91 ± 0.04 35 ± 1 4.67 ± 0.25 3.16 ± 0.02 0.54 ± 0.03Inner 02 36 ± 6 0.89 ± 0.05 36 ± 6 4.58 ± 0.46 2.90 ± 0.18 0.64 ± 0.03Outer 01 38 ± 1 0.93 ± 0.02 38 ± 1 4.86 ± 0.16 3.05 ± 0.21 0.47 ± 0.15Outer 02 29 ± 7 0.84 ± 0.04 29 ± 7 4.06 ± 0.11 2.96 ± 0.01 0.58 ± 0.04Bare 28 ± 6 0.90 ± 0.04 28 ± 6 4.34 ± 0.44 3.11 ± 0.05 0.52 ± 0.14

(b)ANOSIM SIMPER

Groups R statistic Dissimilarity (%)

Inner 01, Inner 02 0.50 58,6Inner 01, Outer 01 1.00 50.0Inner 01, Outer 02 0.88 57.5Inner 01, Bare 0.75 49.3Inner 02, Outer 01 0.00 52.3Inner 02, Outer 02 0.50 55.8Inner 02, Bare 0.75 61.3Outer 01, Outer 02 0.75 54.3Outer 01, Bare 1.00 49.9Outer 02, Bare 1.00 52.7

–0.8 –0.6 –0.4 –0.2 0 0.2 0.4CAP1

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Inner 01Outer 01Inner 02Outer 02Bare

Figure 7. Output of CAP analysis on the nematode species composition in the investigated stations.

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Figure 8. Diversity of meiofaunal taxa and nematode species cumulatively for inner, outer stations, bare sediments,and for the entire study area (i.e. γ -diversity).

L. myriophyllum let us hypothesise that the presence of the colonies under scrutiny was notthe unique source of variability in the composition of meiofaunal assemblages.

Moreover, the results of the multivariate analysis revealed that the taxonomic composition ofmeiofaunal communities are highly sensitive to changes in trophic conditions, since they seem tobe driven by the enrichment in organic matter originated from primary production. Furthermore,the composition of the rare meiofaunal taxa seems to be influenced also by the freshness andnutritional quality of sedimentary organic matter. Overall, our findings let us hypothesise that

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the presence of L. myriophyllum colonies may enhance the habitat heterogeneity in its closeproximity, also modifying the distribution of trophic resources, enhancing the overall meiofaunalbiodiversity of the surrounding soft sediment seascape area, as previously described for areascharacterized by other coralligenous concretions.[40]

This was evident also at the nematode species level. Indeed, despite the ANOVAs did notidentify any significant influence of the hydroid meadows on the α-diversity of nematodes, whenlooking at the their species composition, we report that nematode assemblages differed each otherby at least 50%, indicating a large turnover diversity (i.e. β-diversity) among stations. Again,such a high level of dissimilarity among nematode assemblages suggests that the presence of L.myriophyllum may enhance the local environmental heterogeneity, increase levels of β-diversityand, consequently, the γ -diversity (overall 111 nematode species found) of the whole landscape(Figure 8). Moreover, the high levels of β-diversity were ensured by the high level of nematodeexclusive species found at each habitat: 29 exclusive species in Inner stations sediments, 22 inOuter stations sediments, and only 3 in Bare sediments.

It is worthy of notice that the high level of β-diversity was also encountered between thenematode assemblages below the two L. myriophyllum colonies under scrutiny, that is, betweenthe two Inner stations, suggesting that the presence of several colonies (about 1.6 m−2 in thisstudy) in the same area may enhance the overall biodiversity, also at the landscape level.

The analysis of functional (trophic) diversity and life strategies (1-ITD and MI) did not displayclear differences between sediment below the L. myriophyllum meadows and Bare sediments. Wereport here that the MI always displayed intermediate values of 2.90–3.16, indicating that thenematode assemblages were characterised by a mixture of colonisers and persisters both in thesediment below the L. myriophyllum meadows and in Bare sediments. Similarly, the ITD displaysintermediate values (0.52–0.64), indicating that the nematode assemblages were dominated at allsampling stations by two trophic groups, in particular bacterial and deposit feeders.

5. Conclusions

This study allowed to partially refute the null hypothesis of no influence of the presence of L.myriophyllum meadows on the surrounding environmental characteristics and biodiversity, as wedemonstrated that the presence of L. myriophyllum determines increases in the quality of organicmatter, in meiofaunal abundance and the richness of meiofaunal taxa. Despite no influence onnematode α-diversity was observed, the presence of L. myriophyllum leads to higher levels ofβ-diversity, and apparently promotes high values of γ -diversity.

There is accumulating evidence that species assemblages and communities living in themesophotic zone expand their ecological role to the deeper layers of the ocean,[15] even whendead.[82] Our results, revealing that three-dimensional meadows of ecosystem engineers likethe species investigated here have a pre-eminent influence in controlling β- and γ -diversity ofsurrounding soft bottoms, corroborate previous findings, and claim for an urgent adoption ofmeasures for the study and conservation of mesophotic habitats of the Mediterranean Sea.

Acknowledgements

This study was supported by the PRIN 2008 Italian funds under grant number 2008YBEANX_002; National Geo-graphic under grant number #8876-11; EC Grant Agreement under grant number 287844 (FP7/2007-2013) for the project‘Towards Coast to Coast NETworks of marine protected areas (from the shore to the high and deep sea), coupled withsea-based wind energy potential’ (COCONET), Flagship Project RITMARE – The Italian Research for the Sea – coordi-nated by the Italian National Research Council and funded by the Italian Ministry of Education, University and Researchwithin the National Research Program 2011–2013. Portofino divers provided the technical assistance during samplingactivities.

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