continental shelf (60–80 m depth) and the
shelf-edge (110–160 m depth) of the Menorca
Channel (39° 53′ 0.73″ N, 3° 29′ 51.16″ E) in
September 2010 and April 2011. Videos were
recorded with the manned submersible JAGO
(IFM-GEOMAR) equipped with a 1080 horizontal
line color video camera, and two parallel laser
beams that provide a scale for the images (50
cm). In each picture, colonies were perpendicular
to the camera, and lasers were in the same
plane as the colony. Twenty pictures of colonies
of each form (M1 and M2 from the continental
shelf, and M3 from the shelf-edge) were se-
lected for the subsequent analysis. Analyses
were performed with the Macnification 2.0.1
(Orbicule Enhanced Labs) software and laser
beams were used as reference to calibrate
each picture.

From each picture, the maximum height and
width of the gorgonian colony was measured;
mean width was calculated as the mean of
three measurements taken at equidistant po-
sitions and perpendicular to the height (Gori
et al., 2012). Fan surface area was estimated
by measuring the area defined by a continuous
line that linked the tips of all the branches lo-
cated on the external perimeter of the colony
(Weinbauer and Velimorov, 1995). The ramifi-
cation pattern was determined by establishing
a branch ordination pattern following Brazeau
and Lasker (1988). Most distal branches were
defined as first order branches, second order
branches occurred when two first order branch-
es joined. Higher order branches only appear
when two branches of equal lower order join.
Branches were also classified as "tributary"
or "source", where source branches are those
that join same order branches whereas tributary
branches are those that join a higher order
branch (Brazeau and Lasker, 1988; Gori et al.,
2012). Finally, the length of all primary branches
was measured.

For each colony, the following shape features
were calculated: height to width ratio, height to

mean width ratio, ramification density (number
of ramifications/fan surface area), primary branch-
es density (number of primary branches/fan
surface area), order of the colony's base, the
primary branches maximum and mean length,
the bifurcation ratio (R<sub>b</sub> = <sup>1</sup>/<sub>m</sub> Σ<sub>i=1</sub><sup>m</sup> <sup>n<sub>i</sub></sup>/<sub>1 + n<sub>i</sub></sub> where
n is the number of branches of order i, and m
is the total number of orders) and the tributary
to source ratio of primary and secondary order
branches.

Sclerite size and shape

Colonies of *P. macrospina* were collected from
the continental shelf (~75 m depth) and shelf-
edge (~130 m depth) of the Menorca Channel
in April 2011 and July 2012 (Fig. 1). Sampling
was performed by means of the manned sub-
mersible JAGO (IFM-GEOMAR) and the ROV
NEMO (Gavin Newman), both equipped with
a grabber. Fifteen colonies of each of the two
continental-shelf forms (M1 and M2) were
sampled, whereas six colonies were sampled
for the shelf-edge form (M3). A fragment of
each colony was immediately preserved in ab-
solute ethanol for genetic analyses, whereas
the rest of the colonies were fixed in 4% buf-
fered formalin in seawater, and finally preserved
in 70% ethanol.

In order to compare sclerites from polyps
with a similar size, several primary branches
were placed under a binocular stereomicros-
cope (Olympus SZ-60) and photographed in
different angles with a Moticam 2300 camera.
Polyp length was measured with the software
Macnification 2.0.1 (Orbicule Enhanced Labs)
as the distance from the base of the calix to
the tips of the most distal calix sclerites. Me-
asurements were done on fully contracted
polyps placed perpendicular to the camera. A
total of 200 polyps were measured for the
M1 and M2 forms, and 55 polyps could be
measured for the M3 form. Polyps length ran-
ged from 0.3 mm to 1.8 mm. Polyps of 1–1.5
mm were the most abundant, accounting for
62%, 55% and 60% of polyps in M1, M2 and M3 forms, respectively. Consequently, for each sampled colony, a total of 10 polyps 1–1.5 mm in size from the middle section (2 cm below the tip) of primary branches were dissected under a binocular stereomicroscope (Olympus SZ-60). Collected polyps were immersed in a sodium hypochlorite solution until all organic matter was dissolved and sclerites were disaggregated. Sclerites were rinsed several times with distilled water. A minimum of 20 sclerites were haphazardly selected from each colony, placed on a double stick-tape and posteriorly sputter-coated with Au-Pd. Sclerite observations and images were obtained with a Scanning Electron Microscope (SEM) HITACHI S-3500 N at 5.0 KV, images were obtained at X110. Images were pre-processed with Photoshop C5 (Adobe System) creating a black background, while sclerites were kept grey shaded alongside the scale caption. This procedure facilitated the subsequent measurement by means of the software Macnification 2.0.1 (Orbicule Enhanced Labs). The following measures were taken for each sclerite: total length, perimeter length, area, maximum width, and bottom, middle and top widths (Kim et al., 2004; Prada et al., 2008; Gutiérrez-Rodríguez et al., 2009; Gori et al., 2012). The number of ramifications at the base of large sclerites were counted and referred to as root number. Mean width was calculated as the mean among bottom, middle and top width. Sclerite shape was also evaluated by the following ratios: length/maximum width and area/perimeter (Bramanti et al., 2013).

## Genetic markers

Total genomic DNA was extracted from ethanol-preserved samples of the three forms using the E.Z.N.A. DNA kit (OmegaBiotech) following the manufacturer's instructions. The mtMutS (msh1), and Igr1+COI mitochondrial regions were sequenced as an octocoral bar-

code proposed by McFadden et al. (2011) for comparative purposes. The start of the mtMutS (msh1) region was amplified using the primers ND42599F and MUT3458R (France and Hoover, 2002; Sánchez et al., 2003). Igr1+COI region was amplified using the primers COII8068F and COIOCTR (McFadden et al., 2004; France and Hoover, 2002). Each PCR used 1 U of DNA Stream Polymerase (BIORON), 0.2 mM of dNTPs, 0.3 µM of each primer, approximately 25 ng of genomic DNA, and was brought to a final volume of 25 µL with H₂O. MtMutS PCR was carried out using the following cycle profile: initial denaturation at 94°C for 2 min, 35 cycles of denaturation at 94°C for 30 s, annealing at 55–58°C for 30 s, extension at 72°C for 30 s, and a final extension at 72°C for 5 min. The Igr1+COI PCR used the same cycle profile, but 58°C as annealing temperature and 40 s for extension duration on each of the 35 cycles. PCR products were purified using the NucleoSpin® Extract II DNA Purification Kit following the manufacturer's instructions. Purified products were electrophoresed on an ABI PRISM® 3730xl Genetic Analyzer and sequence traces were edited using Sequencher™ 4.0. The obtained sequences were compared with homologous sequences from the EMBL-Bank of Plexauridae species.

The alignments of the sequences were carried out using MUSCLE, implemented in MEGA5 (Tamura et al., 2011). After alignment, the best nucleotide substitution model was selected using Modeltest implemented in MEGA 5, according to Akaike Information Criterion (AIC) and hierarchical likelihood ratio test (hLRT) values.

The mtMutS comparison involved 41 nucleotide sequences, while the COI, mtMutS+COI and mtMutS+Igr1+COI comparisons only involved 11, 11 and 10 nucleotide sequences, respectively. All positions containing missing data were eliminated. Positions containing internal
gaps were maintained but not considered by
the analysis. The aligned mtMutS sequences
had 660 positions, with a total of 121 variable
and 69 parsimony-informative sites. The aligned
COI sequences had 752 positions, with a total
of 63 variable and 32 parsimony-informative
sites. The aligned mtMutS+COI concatenated
sequences had 1412 positions, with a total of
145 variable and 65 parsimony-informative
sites. The aligned mtMutS+Igr1+COI concate-
nated sequences had 1524 positions, with a
total of 139 variable and 69 parsimony-infor-
mative sites. The genus *Swiftia* (*Swiftia pallida*,
HM106337 and FJ264905) was selected as
outgroup, this genus is currently included in

Plexauridae, but molecular analyses often
aligned it close to the Gorgoniidae genera (see
Wirshing and Baker, 2015; Vargas et al., 2014).
The phylogenetic reconstruction was obtained
applying Maximum Likelihood (ML) and
Bayesian inference methods. Maximum likeli-
hood method (ML) was developed in MEGA5,
and based on the T92+G (Tamura, 1992) model
for all set of sequences, using the NNI heuristic
method (Nearest Neighbor Interchange) and
1000 bootstraps replications (Felsenstein, 1985).
The Bayesian Inference was carried out on-
line in Bioportal (Kumar et al., 2009) in MrBayes
3.1.2 program (Huelsenbeck and Ronquist,
2001; Ronquist and Huelsenbeck, 2003), using
the model GTR+G (lset nst=6 rates=gamma),
10⁷ generations and discarding 25% initial
trees.

Statistical analyses

Distance-based permutational multivariate analy-
sis of variance (PERMANOVA) (Anderson, 2001)
was employed to test the null hypothesis of
no significant differences among forms in
colony shape, and sclerite size and shape.
Each term of the analysis was tested using
9,999 permutations. Data were standardized
respect to their mean absolute deviation
(MAD = 1/n Σn j=1 [ xij - x̄i ], where xj is the value
of the i variable observed in the n colonies)
(García Pérez, 2005). Significant relations rele-
vant to the hypothesis were investigated using
a posterior pair-wise post-hoc test. Finally, an
ordination of the analyzed colony shape and
sclerite size and shape was obtained with a
principal component analysis (PCA). PERMA-
NOVA and pair-wise post-hoc tests were per-
formed with the PaST software (Hammer et
al., 2001). PCA was performed with the R-lan-
guage function princomp, which is available in
the Vegan library (Oksanen et al., 2005) of the
R software platform (R Core Team, 2014).

<figure><img src="image_47.png" /><figcaption>**Fig. 3.** Principal component analysis (PCA) biplot showing the ordination of the studied colonies (n = 60) regarding their colony shape, and the role of the analyzed features; H/W = height to width ratio; H/Wm = height to mean width ratio; Rd = ramification density; 1ºd = primary branch density, base = order of the colony base; 1ºmax = maximum length of primary branches; 1ºmean = mean length of primary branches; Rb = bifurcation ratio; T/S1º = tributary to source ratio of primary branches; T/S2º = tributary to source ratio of secondary branches.</figcaption></figure>
# RESULTS

## Colony shape

No significant differences in colony shape (PERMANOVA, Pseudo-F=0.983, p=0.517) were found amongst the three forms. The first two principal components of the PCA explained 55.4% of the data variance, the first component accounted for 21.2% of the variance. A strong correlation occurred between primary branch

density and ramification density, as well as between height to maximum width and mean width ratios (Fig. 3). M1 was characterized by high primary branch and ramification densities (Figs. 3 and 4), whereas M2 by high values of height to maximum width ratio and height to mean width ratio (Figs. 3 and 4). M3 colonies were characterized by long primary branches, high base orders and tributary to source ratio of secondary branches (Figs. 3 and 4).

## Sclerite size and shape

Two different types of sclerites were observed to form the *P. macrospina* polyp calyxes: thorns

<figure><img src="image_48.png" /><figcaption>**Fig. 4.** Colony shape features; box indicates first and third quartiles, line within the box indicates the mean value, and upper and lower lines indicate the range between minimum and maximum values.</figcaption></figure>
<figure><img src="image_49.png" /><figcaption>Fig. 5. Thorns (a–h) and spindles (i–n) from the calyx of *Paramuricea macrospina* M1.</figcaption></figure>
<figure><img src="image_50.png" /><figcaption>Fig. 6. Thorns (a–j) and spindles (k–q) from the calyx of *Paramuricea macrospina* M2.</figcaption></figure>
<figure><img src="image_51.png" /><figcaption>Fig. 7. Thorns (a–i) and spindles (j–q) from the calyx of *Paramuricea macrospina* M3.</figcaption></figure>
and spindles (Figs. 5, 6, and 7). Significant differences amongst the three forms were observed in the thorns (PERMANOVA, Pseudo-F = 6.812, p<0.001) as well as in the spindles (PERMANOVA, Pseudo-F = 6.586, p<0.001) (Table 1). The first two principal components of the PCA explained 84% of the data variance for thorns, and 88% for spindles; first components accounted for 65% and 64% of the variance, respectively. Thorn size and shape changed gradually, being larger in M1, intermediate in M2, and smaller in M3 (Fig. 8). Spindle size and shape presented less variability amongst forms (Fig. 9). M1 colonies were characterized by thorn and spindle with high mean and maximum width, as well as by high number of thorn roots (Figs. 8, 9, 10 and 11). M2 colonies presented intermediate values (Figs. 8, 9, 10 and 11), whereas M3 colonies were mainly characterized by thorn and spindle with high length and high length to width ratios (Figs. 8, 9, 10 and 11).

<table><caption>**Table 1.** Pairwise test for thorns and spindles forms. Significance is indicated with one (p-value <0.05), two (p-value <0.01), or three asterisks (p-value <0.001).</caption><thead><tr><th rowspan="2">Morfotypes</th><th colspan="2">Thorns</th><th colspan="2">Spindles</th></tr><tr><th>p-value</th><th>Significance</th><th>p-value</th><th>Significance</th></tr></thead><tbody><tr><td>M1 vs. M2</td><td>0.0158</td><td>*</td><td>0.0237</td><td>*</td></tr><tr><td>M1 vs. M3</td><td>0.0002</td><td>***</td><td>0.0009</td><td>***</td></tr><tr><td>M2 vs. M3</td><td>0.0423</td><td>*</td><td>0.0366</td><td>*</td></tr></tbody></table>

## Phylogenetic analyses

Uncorrected *p*-distances based on mtMutS pointed out that all *Paramuricea macrospina* genotyped specimens shared the same sequence. The set of sequences obtained in GenBank for *P. biscaya*, *P. multispina* and *P*.

<figure><img src="image_52.png" /><figcaption>**Fig. 8.** Thorn features; box indicates the first and third quartile, line within the box indicates the mean value, and upper and lower lines indicate the range between minimum and maximum values.</figcaption></figure>
placomus show these species to be polyphyletic (Fig. 12). The species used in this comparison of the genera *Paramuricea* and *Placogorgia* are reunited in a well-supported clade, but they are nested in the analysis, showing a possible future generic synonymy when a higher number of species and better diagnostic specific characters will be defined. Internal relationships within the *Paramuricea-Placogorgia* clade are poorly supported, just a set of *P. biscaya* (I, J, and K), the different *Paramuricea macrospina* forms here considered, a group of *P. placomus*, *Placogorgia* spp., and two additional sets of sequences [*P. biscaya* (L)-*Placogorgia* sp. (A), and *P. multispina* (B) - *P. placomus* (A)] are highly supported (see Fig. 12). The two Mediterranean *Paramuricea* species, *P. clavata* and *P. macrospina* are distinctly divergent (p-distance 2.1%). *Paramuricea macrospina* is closely related (p-distance 0.2%) to the set of sequences of *P. biscaya* A to H, *P. multispina*, *P. tenuis*, and *P. terceira*. The "intra-generic" p-

distances considering all species in the *Paramuricea-Placogorgia* clade ranged from 0 to 3.9%, being 0–2.3% when excluding *Placogorgia* sp. (C), which is separated from the rest of *Paramuricea-Placogorgia* species by 2.3–3.9% in p-distance. The analysed species here included in the *Paramuricea-Placogorgia* clade are widely separated from other Plexauridae genera such as *Villogorgia*, *Bebryce*, *Alaskagorgia* and *Swiftia* by distances between 5.6% and 7.30%.

Analyses based on COI, and concatenated mt-MutS+COI and mtMutS+lgr1+COI sets of sequences (Fig. 13), with a reduced number of species represented by the poor knowledge of COI sequences in *Paramuricea-Placogorgia*, have similar results as for mtMutS. *Paramuricea biscaya* is also showed as a polyphyletic taxon, the two Mediterranean *Paramuricea* species are also clearly differentiate by COI, and the *Paramuricea-Placogorgia* clade is well supported,

<figure><img src="image_53.png" /><figcaption>Fig. 9. Spindle features; box indicates the first and third quartile, line within the box indicates the mean value, and upper and lower lines indicate the range between minimum and maximum values.</figcaption></figure>
although internal relationships remain in general poorly resolved. Comparing between the two Mediterranean *Paramuricea* species, and despite the reduced number of bases in the available sequence mtMutS of *Paramuricea clavata*, this species retains 11 mutations respect to its homologue sequence in *P. macrospina*, expressed in 7 amino acid differences. The available COI sequence of *P. clavata* is 205 bases shorter than the homologous in *P. macrospina* (M3), differing in 13 silent mutations that do not produce any amino acid difference.

DISCUSSION

The results of this study show that the three chromatic forms of the gorgonian *Paramuricea macrospina* observed on the continental shelf and slope of the Menorca Channel, significantly differs in terms of sclerite size and shape, but not in terms of molecular markers and colony shape.

Most colonial morphological features barely changed among the three forms (Fig. 4). Therefore, *P. macrospina*'s colonial morphology is a conservative character that experiences little variation across different environments, as similarly observed in other gorgonian species (Mitchell et al., 1993). This invariability contrasts with other studies, where depth-related changes in environmental conditions result in significant morphological variation in coral species (e.g. Helmuth and Sebens, 1993; Prada et al., 2008; Nir et al., 2011; Gori et al., 2012). However, most research has been conducted in shallow littoral environments, where environmental conditions considerably change over a narrow depth range (Garrabou et al., 2002). Conversely, the increased stability of environmental conditions at greater depth (>50 m) (e.g. Puig et al., 2000; Fernández de Puelles et al., 2007), may

explain the lack of variability in the colonial morphology of the three chromatic forms of *P. macrospina*. The shallow forms (M1 and M2) occurring on maërl beds on the continental shelf may only suffer the effects of sporadic intense hydrodynamic events (Puig et al., 2001; Teixidó et al., 2013) that have been shown to cause damage to sessile organism at similar depths (Woodley et al., 1981; Bongaerts et al., 2013). Conversely, the structural stability of shelf-break rocky bottoms sheltered from strong hydrodynamic events may allow deep M3 colonies to grow larger, as previously observed in other gorgonian species (West et al., 1993; Gori et al., 2012). This may explain why M1 and M2 colonies are significantly (PERMANOVA p<0.05) smaller (23 ± 8.7 SD and 22 ± 9.1 S cm, respectively) than deeper M3 ones (34 ±

<figure><img src="image_54.png" /><figcaption>**Fig. 10.** Principal component analysis (PCA) biplot showing the ordination of the studied colonies (n= 36) regarding their thorn size and shape, and the role of the analyzed features; L = thorn length; P = thorn perimeter; A = thorn area; Wmax = thorn maximum width; Wmean = thorn mean width; Rn = number of thorn roots; L/Wmean = thorn length to mean width ratio; A/P thorn area to perimeter ratio.</figcaption></figure>
15 SD cm). Moreover, it has been proposed that in deep environments exposed to unidirectional currents, gorgonians may benefit from developing larger colonies as they maximize surface exposed to perpendicular flow and increase prey capture efficiency (Sebens and Johnson, 1991). However, these differences in the stability of the substrate and possible exposure to intense hydrodynamic events of *P. macrospina* on the continental shelf are evidently not influential enough to cause any significant changes in its colonial morphology.

Unlike colony shape, thorn and spindle size and shape significantly differed among the three forms. Differences in *P. macrospina* sclerite shape mostly derived from width rather than length, differently from what has been

more frequently observed in other gorgonian species (e.g. West, 1997; Gori et al., 2012). Both thorns and spindles had the same length but were much wider and heavily rooted in M1 and M2 than in M3 colonies. Other Mediterranean coastal gorgonian species had also larger sclerites in shallow environments than in deeper ones (Velimirov, 1976; Skoufas, 2006; Gori et al., 2012). It has been suggested that larger sclerites would proportion higher flexion capacity to gorgonian colonies in shallow or exposed habitats (Skoufas, 2006). However, deep colonies of Caribbean gorgonians had longer sclerites than shallow ones (West, 1993; Kim et al., 2004; Prada et al., 2008) as a possible adaptation to reduced water motion, since large sclerites are more susceptible to breakage than smaller ones (West, 1998). This would agree with studies reporting a negative correlation between sclerite size and water motion (West et al., 1993; Kim et al., 2004). In this regard, the less robust morphology of M3 sclerites could be an adaptation to less intense hydrodynamic conditions. In addition to their structural role, sclerites can also act as defense against predators (Van Alstyne et al., 1992; Puglisi et al., 2002), with larger sclerites being more effective than smaller ones in deterring predators (West, 1998). Therefore larger sclerites in M1 and M2 could also be an adaptation to higher predation pressure. This would agree with previous studies that generally reported higher predation pressure in shallow than in deeper areas (West et al., 1993). However predation processes on deep-water gorgonians are still highly unknown.

Overall, it could be supposed that differences in environmental conditions on the continental shelf and shelf-edge are not enough to induce any change in the *P. macrospina* colony shape, but may be influential enough to affect sclerite size and shape. However, this is not supported by the significant differences observed among the two continental shelf forms (M1 and M2), which occurs side by side on the same mäerl

<figure><img src="image_55.png" /><figcaption>**Fig. 11.** Principal component analysis (PCA) biplot showing the ordination of the studied colonies (n=36) regarding their spindle size and shape, and the role of the analyzed features; L = spindle length; P = spindle perimeter; A = spindle area; Wmax = spindle maximum width; Wmean = spindle mean width; L/Wmean = spindle length to mean width ratio; A/P spindle area to perimeter ratio.</figcaption></figure>
beds (Grinyó et al., 2016). In this sense, differ-
ences between M1 and M2 sclerite could in-
deed suggest a genetically controlled processes
rather than an environmental driven one. M2
sclerites present intermediate values between
M1 and M3 forms (Figs. 8 and 9). However,
differences between M2 and M3 sclerites
were less pronounced than between M2 and
M1 (Table. 1). Considering the similar coloration

in M2 and M3 colonies and the less marked
differences in sclerite size and shape, it could
alternatively be suggested that M2 and M3
are a shallow and a deep forms of a single
morphotype occurring over a wide depth range
comprising both the continental shelf and
slope, whereas M1 is a different morphotype
only occurring on the continental shelf.

<figure><img src="image_56.png" /><figcaption>**Fig. 12.** Molecular analysis by ML method. Relationship of Paramuricea-Placogorgia species and related plexauriid genera using *Swiftia pallida* (HM106337) as outgroup; the analysis is based in *mtMutS*. The tree is drawn to scale, with branch lengths measured in the number of substitutions per site.</figcaption></figure>
<figure><img src="image_57.png" /></figure>

<figure><img src="image_58.png" /></figure>

<figure><img src="image_59.png" /></figure>

<figure><img src="image_60.png" /></figure>

**Fig. 13.** Additional molecular analysis. Relationship of Paramuricea-Placogorgia species and related plexauriid genera using *Swiftia pallida* (HM106337 for *mtMutS* and FJ264905 for *Igr1+COI*) as outgroup. The trees are drawn to scale, with branch lengths measured in the number of substitutions per site. Abbreviations: ML, maximum likelihood; BI, Bayesian inference.
However, mtMutS sequences obtained in this study for the three forms of *Paramuricea macrospina* (M1, M2, and M3) were identical. Among the mitochondrial genes, the mtMutS (msh1) is the most rapidly evolving protein-coding region, being genetic distances (uncorrected *p*-distances) usually 2-3 times those for other segments as COI (McFadden et al., 2011). Unfortunately, only a single COI sequence of *P. macrospina* (form M3) could be successfully obtained in this study, being sequences of this marker poorly available in the *Paramuricea-Placogorgia* clade in general. Both mtMutS and COI have proven to be good discriminators sequences among octocoral species (e.g. Quatrini et al., 2013; 2014). However, different octocoral species may also share identical COI or mtMutS sequence (e.g. McFadden et al., 2011; Gori et al., 2012; Doughty et al., 2013). The lack of resolution in mtMutS is not necessary indicative of species identity (see also Moore et al., 2016 for *Primnoisis* species). In this sense, a recent speciation process that separated M1 versus M2+M3 forms of *P. macrospina* cannot be discarded. Indeed, the molecular information currently available neither resolves the relationships between the two Mediterranean endemic *P. macrospina* and *P. clavata*, since basal nodes of the clades including these species are not well supported in the mtMutS nor in the mtMutS+COI analyses (Figs. 12 and 13). The nested structure of the *Paramuricea-Placogorgia* clade is similar to that observed for other pairs of octocoral genera, such *Siphonogrogia-Chronephthya* (see López-González et al., 2015) or *Lepidisis-Keratoisis* (see Dueñas et al., 2014), where apparently clear morphological features distinguishing genera do not found molecular support. The polyphyletic appearance of *P. biscaya*, *P. multispina* and *P. placomus* observed in the mtMutS phylogenetic hypothesis in this study (and at least *P. biscaya* when including COI and lgr1+COI) could be explained as the result of: 1) cryptic speciation, or 2) current instability (or understood variability) of the morphological

diagnostic characters that should be used in the identification of these species. In the first case, cryptic species (morphologically identical or extremely similar, but genetically distinct) have already been detected in octocoral genera such *Cornularia* (McFadden and van Ofwegen, 2012), *Incrustatus* (McFadden and van Ofwegen, 2013), and the soft-coral genera *Sarcophyton-Lobophytum* (McFadden et al., 2006). In the second case, supposed well-understood morphology is diluted when a large sampling effort intra- and interspecies is carried out (e.g. Gori et al., 2012 for Mediterranean *Eunicella* spp.). Additional mistakes in the assignation of species names could also be the result of the identification by direct comparison to sequences in accessible public databases whose reliable identifications had not been fully assessed, or simply by errors in the interpretation of the diagnostic characters proposed. In this sense, the deposit of voucher specimens of the published sequences will surely facilitate further consults, reinterpretation of morphological features, and corrections of the identifications initially proposed, when necessary.

Overall, colonial morphology of the three forms of *P. macrospina* observed in the Menorca Channel did not show any clear relation with possible changes in environmental conditions with depth in the continental shelf versus shelf-edge. However, it would be interesting to extend the morphological comparisons to also include shallow coastal areas, where the species has been recently reported (20-40 m depth, Topçu and Östürk, 2015), and where hydrodynamic conditions are probably much more contrasted. Conversely, the observed differences in sclerite size and shape probably suggest that both environmental and genetic factors are conditioning sclerite size and shape. Transplant experiments between depths would be desirable to further explore the possible role of phenotypic plasticity and genetic divergence on the morphological variability of this very abundant Mediterranean deep gorgonian
species (Grinyó et al., 2016). Gorgonians are
capable to adapt to different environmental
settings that can derive in morphological vari-
ability (Weinbauer and Velimirov, 1995; Gori et
al., 2012). In this regard, *P. macrospina's* broad
bathymetric distribution and occurrence in very
different environments reflects its wide adapt-
ability (Bo et al., 2012; Topçu and Öztürk, 2015;
Grinyó et al., 2016). While it is clear that mor-
phological variability can be driven by environ-
mental features, we hypothesis that morpho-
logical variability could also be reflecting a
more complex speciation process occurring in
the studied species. In this regard, new genetic
markers are needed to elucidate the fine line
between species and morphological variation
among Mediterranean gorgonians.

ACKNOWLEDGEMENTS

The authors thank the crew of the RV *García del Cid*, the JAGO team (J. Schauer and K. Hissmann) from IFM-GEOMAR (Kiel, Germany), the NEMO ROV pilot Gavin Newman, José Manuel Fortuño for his help and advise with the Scanning Electronic Microscope and Guillem Corbera and Stefano Ambroso for their help in some of the laboratory analyses. This work was funded by the European project LIFE+ INDEMARES "Inventario y designación de la red Natura 2000 en áreas marinas del estado español" (LIFE07/NAT/E/000732).

REFERENCES

ANDERSON, M.J., 2001. A new method for non-para-
metric mutlivariate analysis of variance. Austral
Ecology, 26, 32–46.

BO, M., CANESE, S., SPAGGIARI, C., PUSCEDDU, A., BERTOLINO, M., ANGIOLILLO, M., GIUSTI, M., LORETO, M.F., SALVATI, E., GRECO, S., BAVESTRELLO, G., 2012. Deep Coral Oases in the South Tyrrhenian Sea. PLoS One 7. doi:10.1371/journal.pone.0049870

BONGAERTS, P., MUIR, P., ENGLEBERT, N., BRIDGE, T.C.L., HOEGH-GULDBERG, O., 2013. Cyclone damage at mesophotic depths on Myrmidon Reef (GBR). Coral Reefs 32, 935. doi:10.1007/s00338-013-1052-y

BRAMANTI, L., MOVILLA, J., GURON, M., CALVO, E., GORI, A., DOMINGUEZ-CARRIÓN, C., GRINYÓ, J., LOPEZ-SANZ, A., MARTINEZ-QUINTANA, A., PELEJERO, C., ZIVERI, P., ROSSI, S., 2013. Detrimental effects of ocean acidification on the economically important Mediterranean red coral (Corallium rubrum). Global Change Biology 19, 1897–908. doi:10.1111/gcb.12171

BRAZEAU, D.A., LASKER, H.R., 1988. Inter- and in-
traspecific variation in gorgonian colony morphology:
quantifying branching patterns in arborescent ani-
mals. Coral Reefs 7, 139-143.
doi:10.1007/BF00300973

BRUNO, J.F., EDMUNDS, P., 1997. Clonal Variation for Phenotypic Plasticity in the America (NY). 78, 2177-2190.

BUHL-MORTENSEN, L., OLAFSDOTTIR, S., BUHL-MORTENSEN, P., BURGOS, J., RAGNARSSON, S., 2014. Distribution of nine cold-water coral species (Scleractinia and Gorgonacea) in the cold temperate North Atlantic: effects of bathymetry and hydrography. Hydrobiology 759, 39–61. doi:10.1007/s10750-014-2116-x

CARPINE, C., GRASSHOFF, M., 1975. Les gorgonaires de la Méditerranée. Bulletin de l'Institut océanographique Monaco 71, 1-140.

DOUGHTY, C.L., QUATTRINI, A.M., CORDES, E.E., 2013. Insights into the population dynamics of the deep-sea coral genus *Paramuricea* in the Gulf of Mexico. Deep Sea Research Part II: Topical
Studies in Oceanography 99, 71–82.
doi:10.1016/j.dsr2.2013.05.023

DUEÑAS L.F., ALDERSLADE P., SÁNCHEZ J.A., 2014. Molecular systematics of deep-sea bamboo corals (Octocorallia: Isididae: Keratoisidinae) from New Zealand with descriptions two new species of *Keratoisis*. Molecular Phylogenetics and Evolution, 7415–28.

FELSENSTEIN, J., 1985. Confidence limits on phylogenies: An approach using the bootstrap. Evolution 39, 783–791.

FERNÁNDEZ DE PUELLES, M.L., ALEMANY, F., JANSÁ, J., 2007. Zooplankton time-series in the Balearic Sea (Western Mediterranean): Variability during the decade 1994–2003. Progress in Oceanography 74, 329–354. doi:10.1016/j-pocean.2007.04.009

FRANCE, S.C., HOOVER, L.L., 2002. DNA sequences of the mitochondrial COI gene have low levels of divergence among deep-sea octocorals (Cnidaria: Anthozoa). Hydrobiologia 471, 149–155.

GARCÍA PÉREZ, A., 2005. Estadística aplicada: conceptos básicos. UNED Editorial, Madrid.

GARRABOU, J., BALLESTEROS, E., ZABALA, M., 2002. Structure and dynamics of north-western Mediterranean rocky benthic communities along a depth gradient. Estuarine, Coastal and Shelf Science 55, 493–508.

GORI, A., BRAMANTI, L., LÓPEZ-GONZÁLEZ, P., THOMA, J.N., GILI, J.M., GRINYÓ, J., UCEIRA, V., ROSSI, S., 2012. Characterization of the zooxanthellate and azooxanthellate morphotypes of the Mediterranean gorgonian *Eunicella singularis*. Marine Biology 159, 1485–1496. doi:10.1007/s00227-012-1928-3

GRINYÓ, J., GORI, A., AMBROSO, S., PURROY, A., CALATAYUD, C., DOMINGUEZ-CARRIÓ, C., COPPARI, M., LO IACONO, C., LÓPEZ-GONZÁLEZ, P.J., GILI, J.M., 2016. Diversity, distribution and population size structure of deep Mediterranean gorgonian assemblages

(Menorca Channel, Western Mediterranean Sea). Progress in Oceanography 145, 42–56.
doi:10.1016/j-pocean.2016.05.001

GUTIÉRREZ-RODRÍGUEZ, C., BARBEITOS, M.S., SÁNCHEZ, J.A., LASKER, H.R., 2009. Phylogeography and morphological variation of the branching octocoral *Pseudopterogorgia elisabethae*. Molecular Phylogenetics and Evolution 50, 1–15. doi:10.1016/j.ympev.2008.09.019

HAMMER, Ø., HARPER, D.A.T., RYAN, P.D., 2001. Past: paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4, 9.

HELMUTH, B., SEBENS, K., 1993. The influence of colony morphology and orientation to flow on particle capture by the scleractinian coral *Agaricia agaricites* (Linnaeus). Journal of Experimental Marine Biology and Ecology 165, 251–278. doi:10.1016/0022-0981(93)90109-2

HELMUTH, B.S.T., SEBENS, K.P., DANIEL, T.L., 1997. Morphological variation in coral aggregations: branch spacing and mass flux to coral tissues. Journal of Experimental Marine Biology and Ecology 209, 233–259.

HUELSENBECK, J.P., RONQUIST, F., 2001. MRBAYES: Bayesian inference of phylogeny. Bioinformatics 17, 754–755.

HUGES, R.N., 1989. Functional biology of clonal animals. Springer Science and Business Media, London.

JOSEPH, E.O., CARLO, J.M., LASKER, H.R., 2015. Plasticity and conservatism in sclerites of a Caribbean octocoral. Hydrobiologia 759, 109–118. doi:10.1007/s10750-014-2153-5

KIM, E., LASKER, H.R., COFFROTH, M.A., KIM, K., 2004. Morphological and genetic variation across reef habitats in a broadcast-spawning octocoral. Hy-
drobiologia 530, 423-432. doi:10.1007/s10750-004-2646-8

KUMAR, S., SKJÆVELAND, Å., ORR, R., ENGER, P., RUDEN, T., MEVIK, B.H., BURKI, F., BOTNEN, A., SHALCHIAN-TABRIZI, K., 2009. Air: a batch-oriented web program package for construction of supermatrices ready for phylogenomic analyses. BMC Bioinformatics 10, 357.

LÓPEZ-GONZÁLEZ, P.J., GRINYÓ, J., GILI, J.M., 2015. *Chironephthya mediterranea* n. sp. (Octocorallia, Alcyonacea, Nidaliidae), the first species of the genus discovered in the Mediterranean Sea. Marine Biodiversity 45, 667-688. doi:10.1007/s12526-014-0269-5

MCFADDEN, C.S., TULLIS, I.D., HUTCHINSON, M.B., WINNER, K., SOHM, J.A., 2004. Variation in Coding (NADH Dehydrogenase Subunits 2, 3, and 6) and Noncoding Intergenic Spaceer Regions of the Mitochondrial Genome in Octocorallia (Cnidaria: Anthozoa). Marine Biotechnology 6, 516-526.

MCFADDEN, C.S., ALDERSLADE, P., VAN OFWEGEN, L.P., JOHNSEN, H., RUSMEVICHIENTONG, A., 2006. Phylogenetic relationship within the tropical soft coral genera *Sarcophyton* and *Lobophytum* (Anthozoa, Octocorallia). Invertebr Biology 125, 228-305.

MCFADDEN, C.S., BENAYAHU, Y., PANTE, E., THOMA, J.N., NEVAREZ, P.A., FRANCE, S.C., 2011. Limitations of mitochondrial gene barcoding in the cnidarian sub-class Octocorallia. Molecular Ecology Resources 11, 19-31.

MCFADDEN, C.S., VAN OFWEGEN, L.P., 2012. Stoloniferous octocorals (Anthozoa, Octocorallia) from South Africa, with descriptions of a new family of Alcyonacea, a new genus Clavulariidae, and new species of *Cornularia* (Cornulariidae). Invertebrate systematics 26, 331-356.

MCFADDEN, C.S., VAN OFWEGEN, L.P., 2013. A second, cryptic species of the soft coral genus *Incrustatus* (Anthozoa: Octocorallia: Clavulariidae) from Tierra

del Fuego, Argentina, revealed by DNA barcoding. Helgoland Marine Research 67, 137-147.

MITCHELL, N.D., DARDEAU, M.R., SCHROEDER, W.W., 1993. Colony morphology, age structure, and relative growth of two gorgonian corals, *Leptogorgia hebes* (Verrill) and *Leptogorgia virgulata* (Lamarck), from the northern Gulf of Mexico. Coral Reefs 12, 65-70. doi:10.1007/BF00302103

MOORE, K., ALDERSLADE, P., MILLER, K., 2016. A taxonomic revision of the genus *Primnoisis* Studer [&Wrigth], 1887 (Coelenterata: Octocorallia: Isididae) using morphological and molecular data. Zootaxa 4075, 1-141.

MORTENSEN, P.B., BUHL-MORTENSEN, L., 2005. Morphology and growth of the deep-water gorgonians *Primnoa resedaeformis* and *Paragorgia arborea*. Marine Biology 147, 775-788. doi:10.1007/s00227-005-1604-y

NIR, O., GRUBER, D.F., EINBINDER, S., KARK, S., TCHERNOV, D., 2011. Changes in scleractinian coral *Seriatopora hystrix* morphology and its endocellular *Symbiodinium* characteristics along a bathymetric gradient from shallow to mesophotic reef. Coral Reefs 30, 1089-1100. doi:10.1007/s00338-011-0801-z

OKSANEN, J., KINDT, R., LEGENDRE, P., O'HARA, R.B., 2005. Vegan: community ecology package. Version 1.7-81.

PRADA, C., SCHIZAS, N.V., YOSHIOKA, P.M., 2008. Phenotypic plasticity or speciation? A case from a clonal marine organism. BMC Evolution Biology 8, 47 doi:10.1186/1471-2148-8-47

PUGLISI, M.P., PAUL, V.J., BIGGS, J., SLATTERY, M., 2002. Co-occurrence of chemical and structural defenses in the gorgonian corals of Guam. Marine Ecology Progress Series 239, 105-114. doi:10.3354/meps239105

PUIG, P., PALANQUES, A., GUILLÉN, J., 2001. Near-bottom suspended sediment variability caused by
storms and near-inertial internal waves on the
Ebro mid continental shelf (NW Mediterranean).
Marine Geology 178, 81–93. doi:10.1016/S0025-
3227(01)00186-4

PUIG, P., PALANQUES, A., GUILLE, J., GARCÍA-LADONA, E., 2000. Deep slope currents and suspended particle fluxes in and around the Foix submarine canyon (NW Mediterranean). Deep Sea Research Part I: Oceanographic Research Papers 47, 343–366.

PUYANA, M., NARVAEZ, G., PAZ, A., OSORNO, O., DUQUE, C., 2004. Pseudopterosin content variability of the purple sea whip *Pseudopterogorgia elisabethae* at the Island of San Andres and Providencia (SW Caribbean). Journal of chemical ecology 30, 1183–1201.

QUATTRINI, A.M., GEORGIAN, S.E., BYRNES, L., STEVENS, A., FALCO, R., CORDES, E.E., 2013. Niche divergence by deep-sea octocorals in the genus *Callogorgia* across the continental slope of the Gulf of Mexico. Molecular Ecology 22, 4123–4140. doi:10.1111/mec.12370

QUATTRINI, A.M., ETNOYER, P.J., DOUGHTY, C., ENGLISH, L., FALCO, R., REMON, N., RITTINGHOUSE, M., CORDES, E.E., 2014. A phylogenetic approach to octocoral community structure in the deep Gulf of Mexico. Deep-Sea Research Part II: Topical Studies in Oceanography 99, 92–102.

R CORE TEAM, 2014. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org.

RONQUIST, F., HUELSENBECK, J.P., 2003. Mr Bayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19, 1572–1574.

SÁNCHEZ, J.A., MCFADDEN, C.S., FRANCE, S.C., LASKER, H.R., 2003. Molecular phylogenetic analyses of shallow-water Caribbean octocorals. Marine Biology 142, 975–987. doi:10.1007/s00227-003-1018-7

SÁNCHEZ, J.A., AGUILAR, C., DORADO, D., MANRIQUE, N., 2007. Phenotypic plasticity and morphological integration in a marine modular invertebrate. BMC Evolution Biology 7, 122. doi:10.1186/1471-2148-7-122

SEBENS, K.P., 1987. The ecology of indeterminate growth in animals. Annual Review of Ecology and Systematics 18, 371–407.

SEBENS, K.P., JOHNSON, A.S., 1991. Effects of water movement on prey capture and distribution of reef corals. Hydrobiologia 226, 91–101. doi:10.1007/BF00026470

SKOUFAS, G., 2006. Comparative biometry of *Eunicella singularis* (Gorgonian) sclerites at East Mediterranean Sea (North Aegean Sea, Greece). Marine Biology 149, 1365–1370. doi:10.1007/s00227-006-0314-4

STONE, R.P., 2006. Coral habitat in the Aleutian Islands of Alaska: depth distribution, fine-scale species associations, and fisheries interactions. Coral Reefs 25, 229–238. doi:10.1007/s00338-006-0091-z

TAMURA, K., 1992. Estimation of the number of nucleotide substitutions when there are strong transition transversion and G+C-content biases. Molecular Biology and Evolution 9, 678–687.

TAMURA, K., PETERSON, D., PETERSON, N., STECHER, G., NEI, M., KUMAR, S., 2011. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony method. Molecular Biology and Evolution 28, 2731–2739.

TEIXIDÓ, N., CASAS, E., CEBRIÁN, E., LINARES, C., GARRABOU, J., 2013. Impacts on coralligenous out-crop biodiversity of a dramatic coastal storm. PLoS One 8:e53742.

TODD, P.A., 2008. Morphological plasticity in scleractinian corals. Biological Reviews 83, 315–337.
TOPÇU, E.N., ÖZTÜRK, B., 2015. Composition and abundance of octocorals in the Sea of Marmara, where the Mediterranean meets the Black Sea. Scientia Marina 79, 125-135.

TOPÇU, N.E., ÖZTÜRK, B., 2016. Reproduction in the Mediterranean endemic gorgonian *Spinimuricea klavereni* (Anthozoa, Octocorallia, Plexauridae). In-vertebrate Biology 135, 13-19. doi:10.1111/ivb.12112

VAN ALSTYNE, K.L., WYLE, C.R., PAUL, V.J., MEYER, K., 1992. Antipredator Defenses in Tropical Pacific Soft Coral (Coelenterate: Alcyonacea) II. Sclerite as Defense Against Generalist Carnivorous Fishes. The Biological Bulletin 182, 231-240.

VARGAS, S., GUZMAN, H.M., BREEDY, O., WÖRHEIDE, G., 2014. Molecular phylogeny and DNA barcoding of tropical eastern Pacific shallow-water gorgonian octocorals. Marine Biology 161, 1027-1038.

VELIMIROV, B., 1976. Variations in growth form of *Eunicella cavolini* Koch (Octocorallia) related to intensity of water movement. Journal of Experimental Marine Biology and Ecology 21, 109-107.

VERMEIJ, M.J.A., FRADE, P.R., JACINTO, R.I.R., DEBROT, A.O., BAK, R.P.M., 2007. Effects of reproductive mode on habitat-related differences in the population structure of eight Caribbean coral species. Effects of reproductive mode on habitat-related differences in the population structure of eight Caribbean coral species. Marine Ecology Progress Series 351, 91-102. doi:10.3354/meps07085

WEINBAUER, M.G., VELIMIROV, B., 1995. Morphological variations in the Mediterranean Sea fan *Eunicella cavolini* (Coelenteratea: Gorgonacea) in relation to exposure, colony size and colony region. Bulletin of Marine Science 56, 283-295.

WEST, J.M., 1998. The dual role of sclerites in a gorgonian coral: Conflicting functions of suport and defence. Evolutionary Ecology 12, 803-821.

WEST, J.M., 1997. Plasticity in the sclerites of a gorgonian coral: Tests of water motion, light level, and damage cues. The Biological Bulletin 192, 279-289. doi:10.2307/1542721

WEST, J.M., HARVELL, C.D., WALLS, A.M., 1993. Morphological plasticity in a gorgonian coral (*Briareum asbestinum*) over a depth cline. Marine Ecology Progress Series 94, 61-69. doi:10.3354/meps094061

WIRSHING H.H., BAKER A.C., 2015. Molecular and morphological species boundaries in the gorgonian octocoral genus *Pterogorgia* (Octocorallia: Gorgoniidae). PloS One 10:e0133517. doi:10.1371/journal.pone.0133517

WOODLEY, J.D., CHORNESKY, E.A., CLIFFORD, P.A., JACKSON, J.B., KAUFMAN, L.S., KNOWLTON, N., LANG, J.C., PEARSON, M.P., PORTER, J.W., ROONEY, M.C., RYLAARSDAM, K.W., TUNNICLIFFE, V.J., WAHLE, C.M., WULFF, J.L., CURTIS, A.S., DALLMEYER, M.D., JUPP, B.P., KOEHL, M.A., NEIGEL, J., SIDES, E.M., 1981. Hurricane Allen's Impact on Jamaican Coral Reefs. Science 214, 749-755. doi:10.1126/science.214.4522.749
Chapter 4

Reproductive cycle, energy storage and metabolic
requirements of the gorgonian *Paramuricea macrospina*
from the Mediterranean continental shelf
(Menorca Channel, Western Mediterranean Sea)

ABSTRACT

*Paramuricea macrospina* is an endemic Mediterranean gorgonian species found on hard substrates at 40–160 m depth. This study examined the sexual reproductive cycle, energy storage, and metabolic requirements of *P. macrospina* in a continental shelf environment (~75 m depth). *P. macrsopina* resulted to be a gonochoric, internal brooding species with a 1:1 population sex ratio. Oogenesis lasted ~12–14 months, whereas spermatogenesis was significantly shorter only lasting 6 months. Fertilization occurred during late summer (August) and larval release occurred during early autumn (September - October). The organic matter, total lipid content and stable isotopic composition showed very little seasonal variability, reflecting a general stability in gorgonian food sources on the continental shelf. Conversely, the free fatty acid composition varied seasonally reflecting changes in *P. macrospina* energetic demands probably related to the gametogenesis and the larval brooding. *P. macrospina* reproductive ecology and biochemical composition differed from most shallow Mediterranean gorgonian species, most likely reflecting the higher environmental stability of deeper environments.

CO-AUTHORS

N Viladrich, D Díaz, A Muñoz, S Mallol, J Salazar, R Castillo, JM Gili, A Gori

ADDITIONAL RESOURCES

Submitted in Coral Reefs
# 1. INTRODUCTION

In the past few decades there has been a substantial increase in the ecological characterization of coral-dominated communities located at 40–150 m depth. In tropical areas, mesophotic coral reefs represent a direct extension of shallow-water reef ecosystems reaching depths of over 150 m (Hinderstein et al., 2010; Kahng et al., 2010). Mesophotic coral ecosystems have revealed extensive, productive and rich communities, which differ significantly from their shallow-water counterparts (Bongaerts et al., 2010; Kahng et al., 2010; 2014; Loya et al., 2016). High-density gorgonian and black coral assemblages have been also recently reported on rocky bottoms in deep coastal areas, continental shelf and upper slope at 40–200 m depth in the Mediterranean Sea (Bo et al., 2011, Gori et al., 2011; Grinyó et al., 2016). These assemblages are composed of shallow species that extend their distribution to deeper environments (Bo et al., 2011; Gori et al., 2011), as well as by depth specialist species with distribution restricted to the continental shelf and upper slope (Bo et al., 2012; Grinyó et al., 2016). Despite the recent increase in knowledge about species composition and abundance of Mediterranean coral and gorgonian assemblages at intermediate depths (40–200 m depth) (e.g. Cau et al., 2015; Grinyó et al., 2016), very few studies have addressed basic aspects of their biology, such as the reproductive ecology and the dynamic of energy storage and metabolic demands (Gori et al., 2012).

Reproductive success and posterior larval settlement are crucial processes in the maintenance of Mediterranean shallow gorgonian populations (Coma et al., 1995; Ribes et al., 2007) as well as for their recovery after perturbations (Cerrano et al., 2005). In general, oogenesis has a longer

duration than spermatogenesis (e.g. Coelho and Lasker, 2014). In broadcast spawning species, sperms and oocytes are released and fertilized in the water column; whereas in surface brooder species oocytes are retained by mucous material and fertilized on the surface of female colonies, and in internal brooder species oocytes are fertilized inside female polyps, where larvae develop (Kahng et al., 2011). Gorgonian larvae are lecithotrophic, and thus their survival success solely depends on the energetic reserves transferred from the maternal colony during the oogenesis (Thorson, 1950; Pechenik, 1990). However, the quantity of energy transferred by maternal colonies is limited since the energetic reserves are finite and need to be partitioned into respiration, growth, defense, and reproduction (Stearns, 1992). These energetic requirements are primarily supported by lipids (Oku et al., 2002; Grottoli et al., 2004; Brodte et al., 2008), which are also the main structural constituents of cellular membranes (Tchernov et al., 2004; Dalsgaard et al., 2003). Thus, lipid content reflects the nutritional condition of corals and gorgonians, which results from the balance between food inputs and respiration output, tissue replenishment and reproductive investment (Arai et al., 1993; Ward, 1995). When used as energetic source, lipid reserves are oxidized to provide energy in form of free fatty acids (FFA) that produce high ATP per molecule (Gurr et al., 2002). The FFA content can increase under stress situations, such as starvation and thermal anomalies, and thus their content can be used as a measure of metabolic demands (Sargent et al., 1999). On the other hand, FFA composition may reflect the nature of these metabolic demands (i.e., energetic requirements) (Imbs, 2013; Viladrich et al., 2016). For example, Poly Unsaturated Fatty Acids (PUFA) are highly energetic fatty acids (FA), essential for overcoming stress conditions, since they can be converted into many other FA (Müller-Navarra et al., 2000; Wacker and Von Elert, 2001), whereas Mono Unsaturated Fatty Acids (MUFA) and Saturated Fatty Acids (SFA) are mainly used to cover basic
metabolic energy consumption (Sargent et al., 1999; Dalsgaard et al., 2003).

Environmental conditions affecting food avail-
ability, gorgonian metabolism, and the quantity
and quality of the ingested food will determine
lipid storage, as well as their consumption (FFA
content), and thus the nutritional condition of
gorgonian colonies (Ward and Harrison, 2000).
In a temperate sea such as the Mediterranean,
shallow gorgonians exhibit a marked seasonality
of activity and secondary production, as a con-
sequence of the strong seasonal environmental
variability (Coma et al., 2000). Food capture,
growth, and lipid storage are enhanced during
winter-spring, in correspondence with phyto-
and zooplankton blooms (Ribes et al., 1999;
Rossi et al., 2006; Coma and Ribes, 2003).
Conversely, gorgonian activity is significantly
reduced during summer in shallow waters,

when the stratification of the water column re-
sults in severe depletion of food sources (Coma
et al., 2000; Coma and Ribes, 2003), and gor-
gonian mainly rely on their lipid reserves (Rossi
et al., 2006). Environmental variability is damp-
ened with depth in Mediterranean coastal bot-
toms (Garrabou et al., 2002), because temper-
ature and currents are more constant below
the summer thermocline (Riedl, 1971; Balles-
teros, 2006). This major environmental stability
is reflected in the lower but constant lipid
content in gorgonian tissue at 60 m depth, as
well as in their lower reproductive output com-
pared to shallow gorgonians at 20 m depth
(Gori et al., 2012). Continental shelf environments
are more stable than littoral ones, showing
very little variation in seawater temperature
and being sheltered from strong hydrodynamic
forces (Puig et al., 2001; Fernández de Puelles
et al., 2007). Food availability on Mediterranean

<figure><img src="image_61.png" /><figcaption>Fig. 1. Map of the study area, black dots indicate the location of the sampling stations.</figcaption></figure>
continental shelves also follows a seasonal trend with highest inputs during winter and spring although, it is generally more constant during the year than in shallower environments (Danovaro et al., 2000; Rossi et al., 2003). Consequently, gorgonians are exposed to overall more stable environmental conditions on the continental shelf than in coastal areas, which can directly affect their annual reproductive cycle, energetic storage dynamic and metabolic requirements. Recently, *Paramuricea macrospina* has been reported as one of the most frequent and abundant species in Mediterranean gorgonian assemblages at intermediate depths. This species is the dominant gorgonian in maërl

beds on the continental shelf at 65–100 m depth, and can also occur in dense multispecific assemblages on rocky shoals on the continental shelf and shelf edge at 90–160 m depth (Bo et al., 2012; Grinyó et al., 2016).

The aim of this study was to explore, for the first time, the reproductive ecology and the dynamic of energy storage and metabolic demands of a Mediterranean gorgonian on the continental shelf. For this purpose, the annual development of sexual products, lipid content, FFA content and composition, and stable isotopes (δ¹³C and δ¹⁵N) composition were assessed over an annual cycle in the gorgonian *P. macrospina* from 70 m depth, to address the following questions: (1) Are there differences in the reproductive timing and gonadal output compared to shallow species? (2) Are there differences in the annual dynamic of energy storage and metabolic requirements compared to shallow species? (3) How are the gorgonian reproductive cycle, energy storage and metabolic demands related in the Mediterranean continental shelf environment?

# 2 MATERIALS AND METHODS

## 2.1 Sampling procedure

*P. macrospina* colonies were monthly sampled on the continental shelf of the Menorca Channel at 60–75 m depth (Fig. 1), from September 2011 to May 2012 as bycatch from trammel net experimental fisheries (project LANBAL). Since no colonies were caught in the experimental fisheries during the summer 2012, additional colonies were subsequently monthly sampled by SCUBA diving from June 2013 to October 2013. In November 2011, February and April 2012, and September 2013 no sample could be collected due to bad weather condi-

<figure><img src="image_62.png" /><figcaption>**Fig. 2.** Reproductive state of *Paramuricea macrospina* colonies (a) (black = female colonies, gray = male colonies, white = indeterminate colonies), and percentage of female (b) and male (c) fertile polyps (black = fertile polyps, white = empty polyps) (N polyps = 453; N examined colonies = 115).</figcaption></figure>
tions. All sampled colonies were higher than 10 ± 0.5 cm (height distance from the base to the farthest point). Two primary branch fragments (~2 cm) were collected from each colony: one branch was fixed in 10% formalin in order to study the reproduction and population sex ratio; the other one was frozen at -20°C and freeze-dried during 12 h at -110°C and at 100 mbar pressure (Telstar Lyo Alfa 6 lyophilizer) for biochemical analyses.

## 2.2 Gametogenesis

Sex identification was performed under optical microscope and according to the color and appearance of sexual products (Coma et al., 1995; Ribes et al., 2007; Gori et al., 2007).

Spermataries are pale, while oocytes present darker tonalities, harder consistency and are covered by a spotted membrane. Five female and five male colonies were examined for each sampling event, except for September 2011 when only nine colonies were sampled. For each colony, six polyps on the central portion of the branch were haphazardly selected and dissected under a binocular stereomicroscope (Olympus SZ-60). All sexual products were photographed with a Moticam 2300 photo camera, and pictures were analyzed with the image-processing software Macnification (Version 2.0.1 Orbicule Enhanced Labs). This software automatically counts the number of sexual products, and measures area and circularity (the proximity of the shape of an

<figure><img src="image_63.png" /><figcaption>**Fig. 3.** Frequency of oocyte diameter (µm) in *Paramuricea macrospina* colonies.</figcaption></figure>
object to that of a circle) of each one. Since
circularity was always higher than 0.8, all sexual
products were considered as spherical, and
their measured areas (a) were converted to di-
ameters (d = 2 (√a/π)). Diameters (d) were
then transformed to volume (v = (4/3) π (d/2)³)
in order to quantify the produced volume of
sexual products per polyp. When observed in-
side female polyps, larvae were also quantified.
A total of 594 polyps were dissected, and
3631 sexual products measured.

**2.3 Population sex ratio**

Colonies collected in June, July and August
(when polyps are full of sexual products, see
later) were used to quantify the population

sex ratio. Based on Gori et al. (2007) branches
in which no sexual products were found inside
10 polyps were not considered. A total of 90
colonies were examined.

**2.4 Biochemical analyses**

**2.4.1 Organic matter**

Organic matter (OM) in the coenenchyme was
monthly quantified in four colonies. Approxi-
mately 10 mg (± 0.1 mg) of coenenchyme dry
weight from each sample was reduced to ash
during 4 h at 500 °C in a muffle (Relp 2H-M9),
and the OM was calculated as the difference
between the coenenchyme dry weight and
ash weight (Slattery and McClintock, 1995;

MALE COLONIES

<figure><img src="image_64.png" /><figcaption>Fig. 4. Frequency of spermaries diameter (µm) in *Paramuricea macrospina* colonies.</figcaption></figure>
Rossi et al., 2006). Results are expressed in percentage.

### 2.4.2 Lipid content

Total lipid content in the tissue was quantified in five colonies per sampling event. Approximately 10 mg (± 0.1 mg) of coenenchyme dry weight from each sample were homogenized in 3 ml of chloroform:methanol (2:1), and total lipids were quantified colorimetrically (Barnes and Blackstock, 1973) with cholesterol as a standard. Results are expressed in µg of lipid mg⁻¹ of OM.

### 2.4.3 Free Fatty acids and stable isotope composition

Five colonies for each sampling event were used to determine the FFA content and composition, following the methodology previously used by Viladrich et al. (2016). Approximately 11 mg (± 0.1 mg) of coenenchyme dry weight from each sample were dissolved in 3:1 DCM:MeOH (dichloromethane-methanol), and fatty acids were quantified with gas chromatography

technique (Viladrich et al., 2016). Results are expressed in µg FA mg⁻¹ of OM, and in percentage of SFA, MUFA and PUFA mg⁻¹ of OM. The stable isotopes (SI) (δ¹³C and δ¹⁵N) composition of the gorgonian tissue was assessed from monthly samples of three colonies. Approximately 2 mg (± 0.001 mg) of coenenchyme dry weight from each sample was acidified with HCl 1 M during 48 h to eliminate carbonates, and the δ¹³C composition was determined with Thermo Finnigan EA1108 analyzer and a Thermo Finnigan MAT253 spectrometer. Finally, approximately 2 mg (± 0.001 mg) of coenenchyme dry weight from each sample was directly analyzed with the Thermo Flash EA112 analyzer and the Thermo Delta V advantage spectrometer to determine the δ¹⁵N composition.

### 2.5 Statistical analyses

The population sex ratio was tested by means of a chi-square test using the R-language function chisq.test (Patefield, 1981) of the R software platform (R Core Team, 2014).

<figure><img src="image_65.png" /><figcaption>**Fig. 5.** Mean gonadal volume per polyp (mean ± SE) of female (black circles) and male (white circles) *Paramuricea macrospina* colonies (µm³ polyp⁻¹) (N female polyps = 312, N male polyps = 132).</figcaption></figure>
Significant differences amongst seasons in OM, lipid content and SI composition were tested by means of a repeated measure ANOVA with the R-language function aov (Chambers et al., 1992) of the R software platform (R Core Team, 2014). Seasons were defined as follow: Autumn (September 2011, October 2012 and 2013), winter (December 2011 and January 2012), spring (March 2012, May 2012 and June 2013), summer (July and August 2013).

Colonies analyzed for FFA composition (n=50) were ordinated by means of a principal component analysis (PCA) performed on transformed data ($p' = arcsin(\sqrt{p})$) with the R-language function princomp, which is available in the Vegan library (Oksanen et al., 2005) of the R software platform (R Core Team, 2014).

# 3. RESULTS

## 3.1 Population sex ratio

The recorded ratio of male to female colonies was 1.41 (36/51) and did not significantly deviate from 1:1 ($X^2 = 2.586, df = 1, p-value = 0.108$).

## 3.2 Gametogenesis

Colonies with female sexual products were observed during all sampling events (Fig. 2a). During late summer and autumn (August, September and October) fertile polyps were 45-66% of all the dissected polyps, whereas during the rest of the year almost all polyps (>80%) were fertile (Fig. 2b). Colonies with male sexual products were found from early spring to late summer (March to August) (Fig. 2a), with almost 100% of fertile polyps (Fig. 2c). Oocyte development took ~12-14 months to complete, beginning in early summer and

ending in the next late summer (Fig. 3). Oocyte mean diameter progressively increased from early autumn to late summer (Table 1; Fig. 3) and oocyte number increased from early autumn to early summer (Table 1; Fig. 3). Smallest oocytes (<300 mm) were present in all sampling events, reaching its highest abundance during early autumn and winter (~98% and 100% observed oocytes, respectively) (Fig. 3). Large oocytes (>300 mm) were most abundant during late summer (80% of observed oocytes in August), whereas in early autumn their presence was residual (1-2% of observed oocytes in October) and they were completely absent in winter (Fig. 3). Spermaries development was considerably shorter, beginning in early spring (March) and ending in late summer (August) (Fig. 4). Spermaries mean diameter progressively increased from early spring to late summer (Table 1; Fig. 4). Mean number of spermaries per polyp increased from early spring (May) to early summer (June), and decreased during summer (Table 1).

Female gonadal volume per polyp progressively increased from early winter (October) to mid summer (July) when it reached its maximum volume (Fig. 5). From this point onward female gonadal volume decreased reaching its lowest values in mid autumn (October) (Fig. 5). Male gonadal volume per polyp increased from early spring to late summer when it reached its maximum (Fig. 5). A slight decrease in male volume was observed between early and mid summer (July) (Fig. 5).

One to five planulae larvae (1.3 ± 0.13 (mean ± SE)) were found inside 15.5% of female polyps in September and October.

## 3.4 Biochemical analyses

OM represented 27.2 ± 7.1% (mean ± SD) of the coenenchyma dry weight, with seasonal fluctuation ranging from 20.7 ± 2.5% in autumn to 34.1 ± 3.4% in summer (Fig. 6a). Summer
<figure><img src="image_66.png" /><figcaption>**Fig. 6.** Organic matter (a) in the coenenchyme of *Paramuricea macrospina* colonies (N= 35), and lipid content (b) (µm mg⁻¹ OM) in the organic matter of *Paramuricea macrospina* colonies (N= 49).</figcaption></figure>

<figure><img src="image_67.png" /><figcaption>**Fig. 7.** Total free fatty acid (FFA) content (µm mg⁻¹ OM) in the organic matter of *Paramuricea macrospina* colonies (N=46).</figcaption></figure>
OM content was significantly higher than in autumn and winter (ANOVA, F = 11, p<0.001), and spring OM content was significantly higher than in autumn (ANOVA, F = 11.01, p<0.001).

Average total lipid content was 137 ± 53.5 µg lipid mg⁻¹ OM (mean ± SD), with significantly higher values (ANOVA, F = 5.8, p = 0.002) in summer (1778 ± 504 µg lipid mg⁻¹ OM) (Fig. 6b).

The overall FFA concentration progressively increased from early autumn (October) to mid summer (August) (Fig. 7). PUFFAs and SFFAs were the most abundant fractions of the total FFA content, whereas MUFFAs only represented <15% of total FA (Fig. 8). A total of 36 fatty acids were identified (Table 2), with FFA composition showing seasonal changes characterized by SFFA markers during winter, and by PUFFA markers during spring and summer. The first component of the PCA accounted for 57.4%, and the second component accounted for 14.6% of the data variance, for a total 72% of explained variance. The PCA biplot revealed

a seasonal gradient along the first component (Fig. 9), with autumn samples (orange squares) mainly characterized by 18:3 and 24:0, most winter samples (blue squares) characterized by 13:0, 14:0, 15:0 and 17:0 (all SFFA), spring samples (green squares) and most summer samples (red squares) characterized by 22:6, 20:4(n-3) and 18:4(n-3). SI composition presented very little seasonal variation (Fig. 10). The δ¹³C ranged between -21.6 ± 0.3% (mean ± SD) in winter to -21.9 ± 0.1% in summer. The δ¹⁵N ranged between 5.2 ± 0.5% (mean ± SD) in winter to 4.7 ± 0.4% (mean ± SD) in summer. No significant differences amongst seasons were found in the δ¹³C and δ¹⁵N.

## 4 DISCUSSION

The results of this study showed the gorgonian *P. macrospina* from the Mediterranean continental shelf to sexually reproduce annually,

<figure><img src="image_68.png" /><figcaption>**Fig. 8.** Percentage of saturated (SFAs), monounsaturated (MUFAs) and poly-unsaturated (PUFAs) fatty acids in total FFA of *Paramuricea macrospina* colonies (N=46) (SFAs= circles, MUFAs= diamonds, PUFAs = squares).</figcaption></figure>
<table><caption>Table 1. Changes in the diameter and number of *Paramuricea macrospina* sexual products (mean ± SE) (N=3631).</caption><thead><tr><th rowspan="3">Sampling Event</th><th colspan="4">Diameter (µm)</th><th colspan="4">Number</th></tr><tr><th colspan="2">Female</th><th colspan="2">Male</th><th colspan="2">Female</th><th colspan="2">Male</th></tr><tr><th>Mean ± SD</th><th>Max.</th><th>Mean ± SD</th><th>Max.</th><th>Mean ± SD</th><th>Max.</th><th>Mean ± SD</th><th>Max.</th></tr></thead><tbody><tr><td>September 2011</td><td>248 ± 149</td><td>607</td><td></td><td></td><td>0.7 ± 1.1</td><td>4</td><td></td><td></td></tr><tr><td>October 2011</td><td>88 ± 57</td><td>510</td><td></td><td></td><td>2.6 ± 3.4</td><td>11</td><td></td><td></td></tr><tr><td>December 2011</td><td>94 ± 35</td><td>216</td><td></td><td></td><td>9.6 ± 6.2</td><td>21</td><td></td><td></td></tr><tr><td>January 2012</td><td>128 ± 32</td><td>227</td><td></td><td></td><td>7.6 ± 5.9</td><td>28</td><td></td><td></td></tr><tr><td>March 2012</td><td>158 ± 66</td><td>330</td><td>85.8 ± 21</td><td>150</td><td>13.0 ± 5.1</td><td>20</td><td>6.0 ± 6.4</td><td>24</td></tr><tr><td>May 2012</td><td>214 ± 91</td><td>403</td><td>153 ± 45</td><td>291</td><td>6.2 ± 4.7</td><td>15</td><td>14.8 ± 9.6</td><td>35</td></tr><tr><td>June 2013</td><td>236 ± 82</td><td>494</td><td>195 ± 56</td><td>335</td><td>10.5 ± 5.9</td><td>23</td><td>29.1 ± 11.8</td><td>56</td></tr><tr><td>July 2013</td><td>259 ± 99</td><td>509</td><td>195 ± 50</td><td>347</td><td>6.5 ± 3.1</td><td>14</td><td>13.9 ± 8.1</td><td>38</td></tr><tr><td>August 2013</td><td>373 ± 91</td><td>562</td><td>276 ± 81</td><td>491</td><td>1.7 ± 1.8</td><td>6</td><td>7.5 ± 8.2</td><td>30</td></tr><tr><td>October 2013</td><td>168 ± 101</td><td>502</td><td></td><td></td><td>1.1 ± 1.7</td><td>6</td><td></td><td></td></tr></tbody></table>

with larval development taking place inside the female polyps, and larval release occurring in late summer-early fall. Gonochorism of *P. macrospina* colonies, and 1:1 population sex ratio, match the general pattern previously observed in shallow Mediterranean gorgonian species (Coma et al., 1995; Rossi et al., 2003; Ribes et al., 2007), and in the majority of octocorals in general (Kahng et al., 2011). In the same way, oogenesis (12–14 months) (Fig. 3) and spermatogenesis (~6 months) (Fig. 4) duration in *P. macrospina* was within the range observed in other shallow Mediterranean (Coma et al., 1995; Ribes et al., 2007) and temperate gorgonian species (e.g. Excoffon et al., 2004; Seo et al., 2008) with annual sexual reproduction (Kahng et al., 2011; Table 3). This long oogenesis duration results in the presence of a cohort of mature large oocytes during summer, together with a second cohort of immature small oocytes (<200 mm) that will slowly increase in size and number to mature during the following summer (Fig. 3). Conversely, spermataries maturation is much faster, starting in early spring

(March) and ending with its release during late summer (August).

Spawning of male gametes and larval fertilization in *P. macrospina* is delayed 2–3 months respect to shallow Mediterranean gorgonian species, which generally spawn during late spring - early summer coinciding with the spring increase in shallow seawater temperature (Coma et al., 1995; Tsounis et al., 2006; Ribes et al., 2007). Gonadal development has been suggested to be conditioned by seawater temperature (Grigg, 1977; de Putron and Ryland, 2009), since gorgonian colonies occurring or maintained in colder environments showed a delay in gonadal development (Gori et al., 2013) and spawning respect to populations located in warmer environments (Gori et al., 2007; Pakes and Woollacott, 2008). Seawater temperature in the Balearic continental shelf (75 m) slightly increases a few degrees during late summer and early autumn (Fernández de Puelles et al., 2007) coinciding with the *P. macrospina* spawning. This might support that
timing in *P. macrosina* reproductive cycle is conditioned by the late increase in seawater temperature occurring on the Mediterranean continental shelf. It is also interesting that larval release in September and October also coincides with the beginning of the fall phytoplankton bloom in the study area (Grinyó et al., submitted), which could suppose fruitful food availability for the primary polyps resulting from the larvae metamorphosis.

When compared to other internal brooding species, *P. macrospina* showed much smaller oocytes (Table 3), only exceeding those observed in species of the genus *Acabaria* (Ben-Yosef and Benayahu, 1999; Fine et al., 2005). The small size of *P. macrospina* oocytes is however compensated by high fertility compared to other internal brooding species (Table 3), which generally tend to develop few but large

oocytes (e.g. Tsounis et al., 2006; Ribes et al., 2007; Orejas et al., 2007). In this sense, both oocyte size and fertility of *P. macrospina* are within the range observed in the congeneric Mediterranean *Paramuricea clavata* (Table 3; Coma et al., 1995; Gori et al., 2007), which mainly inhabit vertical rocky walls in coastal areas (Linares et al., 2008; Gori et al., 2011; Kipson et al., 2014). However, the two species clearly differ in their reproductive strategy, being *P. macrospina* an internal brooder, and *P. clavata* a surface brooder (Coma et al., 1995). Differences among congeneric species in the reproductive strategy have previously been reported in allopatric species of the genus *Coralium* (Waller and Baco, 2007; Nonaka et al., 2015; Priori et al., 2013) as well as in sympatric species of the genus *Anthillogorgia* (Coelho and Lasker, 2014), although the causes of this variability remain unknown. A possible hypoth-

esis could be that fertilization in surface brooding species is mainly restricted to the few days when oocytes remain attach to the surface of the mother colony (Coma et al., 1995; Fiorillo et al., 2013). Thus, fertilization success is highly conditioned by the currents and proximity of male and female colonies. Conversely, in internal brooding species fertilization may probably occurs over a longer period, and thus colony proximity would possibly be less important for fertilization success. In this sense, surface brooding could be highly effective in a large-sized species with high population density (33 ± 14 colonies m⁻²) such as *P. clavata* (Linares et al., 2008; Kipson et al., 2014). Conversely, internal brooding could be more effective for *P. macrospina*, which has smaller colonies mainly distributed in low to medium densities (3.2 ± 5 colonies m⁻²) over vast ar-

<figure><img src="image_69.png" /><figcaption>**Fig. 9.** Principal component analysis (PCA) biplot showing the ordination of studied colonies of *Paramuricea macrospina* regarding their composition in fatty acids</figcaption></figure>
eas of the continental shelf (Grinyó et al., 2016). Moreover, from an energetic point of view, surface brooding probably requires a higher energetic investment than internal brooding, due to the production and secretion of large quantities of mucus (Viladrich et al., 2016). In this regard, *P. clavata* can probably rely upon high spring food availability in littoral environments (Rossi et al., 2003, 2006; Viladrich et al., 2016) exposed to intense turbulent flows (Riedl, 1971), whereas, food availability might be lower in continental shelf environments, where *P. macrospina* is exposed to mild unidirectional currents (Palanques et al., 2002).

Organic matter and total lipid content in *P. macrospina* showed little seasonal variation, with higher values during summer coinciding with the progressive augment of gonadal volume (Figs. 5 and 7). This fact, together with their lower content after larval release, can suggest a direct transfer of lipid from the parental colonies to the sexual products (Arai et al., 1993; Richmond, 1987). Remarkably, total lipid content was much lower and more constant in *P. macrospina* all year round than previously observed in shallow (25-30 m depth) colonies of *P. clavata* (Rossi et al., 2006; Viladrich et al., 2016). This lower and more constant lipid content in deep than shallow colonies has previously been observed in the Mediterranean gorgonian *Eunicella singularis* (Gori et al., 2012). Thus, these differences between depths may be due to lower but more constant food availability on the Mediterranean continental shelf than in littoral environments (Gori et al., 2012). A general stability in food availability for gorgonians on the Mediterranean continental shelf is also supported by the lack of seasonality in the ∂¹³C and ∂¹⁵N composition of *P. macrospina* tissue (Fig. 10). Both observed ∂¹³C and ∂¹⁵N values are in the range of those from heterotrophic Mediterranean coastal gorgonians (Fig. 11) (Gori et al., 2012; Cocito et al., 2013), and suspension feeders feeding on microzooplankton and particulate organic matter

in general (Carlier et al., 2007). The ∂¹³C signature of *P. macrospina* also resembled that observed in other gorgonian species on the Pacific continental shelf (Sherwood et al., 2005).

<figure><img src="image_70.png" /><figcaption>**Fig. 10.** Stable isotope (δ13C and δ15N) composition of *Paramuricea macrospina* colonies (N=12).</figcaption></figure>

<figure><img src="image_71.png" /><figcaption>**Fig. 11.** Stable isotope (δ13C and δ15N) composition of *Paramuricea macrospina* colonies (N=12) and *Eunicella singularis* from 20 m depth and 60 m depth (From Gori et al., 2012).</figcaption></figure>
<table><caption>Table 2. Fatty acid composition (% of total fatty acids) of *Paramuricea macrospina* colonies (N=46) (mean ± SD).</caption><thead><tr><th>Fatty Acids</th><th>Sep 11<br>(n=5)</th><th>Oct 11<br>(n=5)</th><th>Dec 11<br>(n=4)</th><th>Jan 12<br>(n=4)</th><th>Mar 12<br>(n=5)</th><th>May 12<br>(n=5)</th><th>Jun 13<br>(n=5)</th><th>Jul 13<br>(n=5)</th><th>Aug 13<br>(n=4)</th><th>Oct 13<br>(n=4)</th></tr></thead><tbody><tr><td>C12:0</td><td>0.003 ± 0.002</td><td>0.002 ± 0.001</td><td>0.003 ± 0.002</td><td>0.002 ± 0.000</td><td>0.001 ± 0.000</td><td>0.002 ± 0.001</td><td>0.001 ± 0.001</td><td>0.001 ± 0.001</td><td>0.001 ± 0.001</td><td>0.003 ± 0.001</td></tr><tr><td>C13:0</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.001 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.001 ± 0.000</td><td>0.001 ± 0.001</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td></tr><tr><td>C14:1</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.004 ± 0.009</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td></tr><tr><td>C14:0</td><td>0.014 ± 0.006</td><td>0.02 ± 0.003</td><td>0.023 ± 0.03</td><td>0.030 ± 0.005</td><td>0.016 ± 0.003</td><td>0.019 ± 0.003</td><td>0.026 ± 0.015</td><td>0.031 ± 0.011</td><td>0.016 ± 0.04</td><td>0.017 ± 0.013</td></tr><tr><td>C15:0</td><td>0.007 ± 0.003</td><td>0.006 ± 0.000</td><td>0.010 ± 0.001</td><td>0.012 ± 0.002</td><td>0.017 ± 0.001</td><td>0.009 ± 0.001</td><td>0.010 ± 0.004</td><td>0.013 ± 0.004</td><td>0.006 ± 0.001</td><td>0.007 ± 0.005</td></tr><tr><td>C16:1</td><td>0.014 ± 0.003</td><td>0.012 ± 0.001</td><td>0.019 ± 0.003</td><td>0.023 ± 0.002</td><td>0.018 ± 0.004</td><td>0.022 ± 0.003</td><td>0.031 ± 0.008</td><td>0.027 ± 0.014</td><td>0.022 ± 0.005</td><td>0.017 ± 0.004</td></tr><tr><td>C16:0</td><td>0.0173 ± 0.04</td><td>0.192 ± 0.02</td><td>0.211 ± 0.056</td><td>0.160 ± 0.013</td><td>0.155 ± 0.023</td><td>0.181 ± 0.032</td><td>0.167 ± 0.072</td><td>0.220 ± 0.101</td><td>0.138 ± 0.016</td><td>0.222 ± 0.049</td></tr><tr><td>C17:0</td><td>0.015 ± 0.005</td><td>0.017 ± 0.002</td><td>0.015 ± 0.002</td><td>0.015 ± 0.001</td><td>0.013 ± 0.002</td><td>0.014 ± 0.002</td><td>0.012 ± 0.004</td><td>0.016 ± 0.007</td><td>0.011 ± 0.001</td><td>0.012 ± 0.010</td></tr><tr><td>C18:3(n-6)</td><td>0.01 ± 0.01</td><td>0.000 ± 0.000</td><td>0.001 ± 0.002</td><td>0.005 ± 0.003</td><td>0.006 ± 0.004</td><td>0.006 ± 0.004</td><td>0.002 ± 0.000</td><td>0.002 ± 0.000</td><td>0.002 ± 0.001</td><td>0.007 ± 0.002</td></tr><tr><td>C18:4</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.001</td><td>0.001 ± 0.002</td><td>0.001 ± 0.002</td><td>0.004 ± 0.000</td><td>0.004 ± 0.002</td><td>0.003 ± 0.001</td><td>0.000 ± 0.000</td></tr><tr><td>C18:2(n-6)</td><td>0.02 ± 0.02</td><td>0.000 ± 0.000</td><td>0.013 ± 0.003</td><td>0.023 ± 0.002</td><td>0.026 ± 0.002</td><td>0.030 ± 0.003</td><td>0.026 ± 0.008</td><td>0.026 ± 0.008</td><td>0.021 ± 0.007</td><td>0.012 ± 0.015</td></tr><tr><td>C18:3(n-3)</td><td>0.02 ± 0.03</td><td>0.010 ± 0.001</td><td>0.001 ± 0.001</td><td>0.004 ± 0.001</td><td>0.006 ± 0.001</td><td>0.006 ± 0.000</td><td>0.013 ± 0.003</td><td>0.021 ± 0.021</td><td>0.009 ± 0.005</td><td>0.026 ± 0.039</td></tr><tr><td>C18:1(n-9)</td><td>0.09 ± 0.02</td><td>0.100 ± 0.009</td><td>0.098 ± 0.020</td><td>0.086 ± 0.010</td><td>0.093 ± 0.011</td><td>0.097 ± 0.007</td><td>0.038 ± 0.011</td><td>0.035 ± 0.022</td><td>0.042 ± 0.013</td><td>0.070 ± 0.048</td></tr><tr><td>C18:2(n-4)</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.001 ± 0.003</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td><td>0.003 ± 0.007</td><td>0.011 ± 0.018</td><td>0.000 ± 0.000</td><td>0.000 ± 0.000</td></tr><tr><td>C18:0</td><td>0.09 ± 0.02</td><td>0.116 ± 0.027</td><td>0.106 ± 0.025</td><td>0.084 ± 0.008</td><td>0.080 ± 0.010</td><td>0.088 ± 0.022</td><td>0.062 ± 0.019</td><td>0.096 ± 0.074</td><td>0.062 ± 0.04</td><td>0.102 ± 0.018</td></tr><tr><td>C19:0</td><td>0.002 ± 0.002</td><td>0.003 ± 0.002</td><td>0.004 ± 0.001</td><td>0.003 ± 0.000</td><td>0.003 ± 0.000</td><td>0.008 ± 0.000</td><td>0.003 ± 0.001</td><td>0.001 ± 0.002</td><td>0.002 ± 0.000</td><td>0.002 ± 0.001</td></tr><tr><td>C20:5(n-3)</td><td>0.3 ± 0.2</td><td>0.297 ± 0.158</td><td>0.356 ± 0.862</td><td>0.370 ± 0.021</td><td>0.348 ± 0.033</td><td>0.294 ± 0.021</td><td>0.343 ± 0.052</td><td>0.253 ± 0.110</td><td>0.436 ± 0.047</td><td>0.247 ± 0.151</td></tr><tr><td>C20:4(n-6)</td><td>0.07 ± 0.1</td><td>0.088 ± 0.154</td><td>0.021 ± 0.004</td><td>0.030 ± 0.004</td><td>0.031 ± 0.004</td><td>0.038 ± 0.009</td><td>0.047 ± 0.003</td><td>0.061 ± 0.042</td><td>0.058 ± 0.023</td><td>0.087 ± 0.110</td></tr><tr><td>C20:3(n-6)</td><td>0.01 ± 0.01</td><td>0.014 ± 0.003</td><td>0.02 ± 0.005</td><td>0.006 ± 0.004</td><td>0.010 ± 0.002</td><td>0.013 ± 0.004</td><td>0.011 ± 0.003</td><td>0.012 ± 0.002</td><td>0.009 ± 0.004</td><td>0.019 ± 0.013</td></tr><tr><td>C20:4(n-3)</td><td>0.003 ± 0.007</td><td>0.000 ± 0.001</td><td>0.000 ± 0.001</td><td>0.002 ± 0.002</td><td>0.005 ± 0.003</td><td>0.004 ± 0.005</td><td>0.009 ± 0.004</td><td>0.007 ± 0.006</td><td>0.006 ± 0.007</td><td>0.002 ± 0.003</td></tr><tr><td>C20:2(n-6)</td><td>0.008 ± 0.007</td><td>0.006 ± 0.001</td><td>0.006 ± 0.002</td><td>0.004 ± 0.000</td><td>0.006 ± 0.001</td><td>0.007 ± 0.001</td><td>0.008 ± 0.002</td><td>0.009 ± 0.004</td><td>0.007 ± 0.005</td><td>0.007 ± 0.003</td></tr><tr><td>C20:1(n-9)</td><td>0.007 ± 0.007</td><td>0.007 ± 0.001</td><td>0.007 ± 0.002</td><td>0.006 ± 0.002</td><td>0.007 ± 0.001</td><td>0.008 ± 0.002</td><td>0.011 ± 0.004</td><td>0.009 ± 0.004</td><td>0.006 ± 0.003</td><td>0.007 ± 0.002</td></tr><tr><td>C20:0</td><td>0.002 ± 0.002</td><td>0.002 ± 0.002</td><td>0.002 ± 0.002</td><td>0.002 ± 0.000</td><td>0.003 ± 0.000</td><td>0.003 ± 0.001</td><td>0.001 ± 0.001</td><td>0.002 ± 0.001</td><td>0.001 ± 0.000</td><td>0.004 ± 0.002</td></tr><tr><td>C21:0</td><td>0.002 ± 0.002</td><td>0.000 ± 0.001</td><td>0.001 ± 0.002</td><td>0.004 ± 0.000</td><td>0.005 ± 0.001</td><td>0.004 ± 0.002</td><td>0.005 ± 0.001</td><td>0.001 ± 0.001</td><td>0.004 ± 0.002</td><td>0.001 ± 0.001</td></tr><tr><td>C22:5</td><td>0.03 ± 0.01</td><td>0.026 ± 0.005</td><td>0.036 ± 0.009</td><td>0.006 ± 0.019</td><td>0.076 ± 0.022</td><td>0.078 ± 0.027</td><td>0.086 ± 0.021</td><td>0.076 ± 0.039</td><td>0.078 ± 0.018</td><td>0.044 ± 0.010</td></tr><tr><td>C22:6(n-3)</td><td>0.003 ± 0.002</td><td>0.000 ± 0.001</td><td>0.001 ± 0.001</td><td>0.003 ± 0.000</td><td>0.007 ± 0.002</td><td>0.002 ± 0.001</td><td>0.002 ± 0.001</td><td>0.000 ± 0.000</td><td>0.002 ± 0.001</td><td>0.001 ± 0.002</td></tr><tr><td>C22:4</td><td>0.001 ± 0.002</td><td>0.000 ± 0.000</td><td>0.000 ± 0.001</td><td>0.002 ± 0.001</td><td>0.002 ± 0.001</td><td>0.001 ± 0.001</td><td>0.004 ± 0.002</td><td>0.001 ± 0.000</td><td>0.002 ± 0.002</td><td>0.001 ± 0.001</td></tr><tr><td>C22:5</td><td>0.008 ± 0.006</td><td>0.006 ± 0.007</td><td>0.005 ± 0.004</td><td>0.009 ± 0.002</td><td>0.008 ± 0.004</td><td>0.007 ± 0.003</td><td>0.004 ± 0.001</td><td>0.003 ± 0.002</td><td>0.004 ± 0.001</td><td>0.009 ± 0.003</td></tr><tr><td>C22:2(n-9)</td><td>0.007 ± 0.004</td><td>0.013 ± 0.008</td><td>0.008 ± 0.003</td><td>0.008 ± 0.002</td><td>0.007 ± 0.002</td><td>0.007 ± 0.003</td><td>0.005 ± 0.001</td><td>0.004 ± 0.002</td><td>0.005 ± 0.001</td><td>0.007 ± 0.003</td></tr><tr><td>C22:2(n-6)</td><td>0.003 ± 0.003</td><td>0.03 ± 0.001</td><td>0.003 ± 0.000</td><td>0.002 ± 0.000</td><td>0.003 ± 0.001</td><td>0.003 ± 0.001</td><td>0.002 ± 0.001</td><td>0.002 ± 0.001</td><td>0.001 ± 0.000</td><td>0.008 ± 0.011</td></tr><tr><td>C22:0</td><td>0.014 ± 0.02</td><td>0.021 ± 0.026</td><td>0.003 ± 0.001</td><td>0.002 ± 0.00</td><td>0.002 ± 0.000</td><td>0.003 ± 0.001</td><td>0.001 ± 0.000</td><td>0.001 ± 0.000</td><td>0.001 ± 0.000</td><td>0.018 ± 0.025</td></tr><tr><td>C23:0</td><td>0.03 ± 0.02</td><td>0.012 ± 0.011</td><td>0.019 ± 0.009</td><td>0.017 ± 0.003</td><td>0.024 ± 0.004</td><td>0.019 ± 0.004</td><td>0.028 ± 0.007</td><td>0.021 ± 0.013</td><td>0.019 ± 0.013</td><td>0.015 ± 0.004</td></tr><tr><td>C24:PUFA</td><td>0.012 ± 0.015</td><td>0.004 ± 0.001</td><td>0.004 ± 0.004</td><td>0.011 ± 0.003</td><td>0.013 ± 0.004</td><td>0.013 ± 0.006</td><td>0.020 ± 0.006</td><td>0.017 ± 0.012</td><td>0.011 ± 0.009</td><td>0.005 ± 0.004</td></tr><tr><td>C24:PUFA</td><td>0.009 ± 0.001</td><td>0.007 ± 0.002</td><td>0.008 ± 0.002</td><td>0.006 ± 0.001</td><td>0.014 ± 0.002</td><td>0.014 ± 0.002</td><td>0.012 ± 0.003</td><td>0.009 ± 0.004</td><td>0.009 ± 0.003</td><td>0.008 ± 0.002</td></tr><tr><td>C24:1(n-9)</td><td>0.009 ± 0.002</td><td>0.007 ± 0.001</td><td>0.007 ± 0.003</td><td>0.004 ± 0.001</td><td>0.005 ± 0.001</td><td>0.006 ± 0.002</td><td>0.003 ± 0.001</td><td>0.003 ± 0.001</td><td>0.003 ± 0.001</td><td>0.009 ± 0.005</td></tr></tbody></table>
<table><caption>**Table 3.** Reproductive patterns in different gorgonian species. Med = Mediterranean, Isl. = island, S = sexuality, G = gonochoric, H = hermaphroditic, RS = reproductive strategy, IB = internal brooder, SB = surface brooder, BS = broadcast spawner, Spermat. = spermatogenesis.</caption><thead><tr><th>Environment</th><th>Location</th><th>Species</th><th>S</th><th>RS</th><th>Oogenesis Duration</th><th>Oocyte Diameter (µm)</th><th>Fertility</th><th>Sperm. Duration</th><th>Spermary Diameter (µm)</th><th>Fertility</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="6">Littoral temperate</td><td>Med.</td><td>Paramuricea macropsinæ</td><td>G</td><td>IB</td><td>~12</td><td>87.9±6.5 – 330.04±15.6</td><td>2±0.3 – 10.4±0.2</td><td>5</td><td>85.8±1.5 – 276.1±4.3</td><td>8.36±0.4 – 29.11±0.5</td><td>This study</td></tr><tr><td>Med.</td><td>Paramuricea clavata</td><td>G</td><td>SB</td><td>13 – 18</td><td>72±45 – 425±76</td><td>13±2.2</td><td>6 – 7</td><td>77 ±34 – 326±108</td><td>4.3±0.8 – 35±6.1</td><td>Coma et al., 1995</td></tr><tr><td>Med.</td><td>Eunicella singularis</td><td>G</td><td>IB</td><td>13 – 17</td><td>141±46 – 829±250</td><td>0.69±0.16</td><td>4 – 6</td><td>123 ± 49 – 387±106</td><td>-</td><td>Ribes et al., 2007</td></tr><tr><td>Med.</td><td>Corallium rubrum</td><td>G</td><td>IB</td><td>>12</td><td>170 – 520</td><td>~0.5 – ~2.4</td><td>8</td><td>50 – 480</td><td>~0.5 – ~2.7</td><td>Tsounis et al., 2006</td></tr><tr><td>Med.</td><td>Leptogorgia sarmentosa</td><td>G</td><td>BS</td><td>>12</td><td>500</td><td>3 – 4</td><td>6 – 7</td><td>550</td><td>4 – 5</td><td>Rossi et al., 2009</td></tr><tr><td>Med.</td><td>Spinimuricea klavereni</td><td>G</td><td>BS</td><td>-</td><td>&lt;150 – 538</td><td>43±22 – 87±27</td><td>-</td><td>&lt;150 – 680</td><td>29.7±12.9 – 65±17.5</td><td>Topcu and Öztürk, 2016</td></tr><tr><td>Med.</td><td>Acabaria erythraea</td><td>H</td><td>IB</td><td>-</td><td>100</td><td>10 – 25</td><td>-</td><td>200</td><td>-</td><td>Fine et al., 2005</td></tr><tr><td rowspan="5">Littoral tropical</td><td>S Atlantic</td><td>Tripalea clavaria</td><td>G</td><td>IB</td><td>11 – 12</td><td>40 – 700</td><td>7.2 ±3.7 – 14.3 ±5.2</td><td>6 – 7</td><td>900</td><td>10.1 ±3.8 – 3.8±2.1</td><td>Excoffon et al., 2004</td></tr><tr><td>Jeju Isl.</td><td>Anthoplexaura dimorpha</td><td>G</td><td>BS</td><td>12</td><td>43±8 – 359±62</td><td>-</td><td>6</td><td>56±10 – 315±36</td><td>-</td><td>Seo et al., 2008</td></tr><tr><td>California</td><td>Muricea fruticosa</td><td>G</td><td>IB</td><td>9 – 12</td><td>>750</td><td>-</td><td>-</td><td>~450</td><td>-</td><td>Grigg, 1970</td></tr><tr><td>California</td><td>Muricea californica</td><td>G</td><td>IB</td><td>9 – 12</td><td>~800</td><td>-</td><td>-</td><td>~600</td><td>-</td><td>Grigg, 1970</td></tr><tr><td>Caribbean</td><td>Briareum asbestinum</td><td>G</td><td>SB</td><td>9 – 12</td><td>900</td><td>2.25 – 4.4±2.76</td><td>5</td><td>-</td><td>0.5±1.5 – 4.55±3.87</td><td>Brazeau and Lasker, 1990</td></tr></tbody></table>
<table><caption>**Table 3 (cont.)**. Reproductive patterns in different gorgonian species. Med = Mediterranean, Isl. = island, S = sexuality, G = gonochoric, H = hermaphroditic, RS = reproductive strategy, IB = internal brooder, SB = surface brooder, BS = broadcast spawner, Spermat. = spermatogenesis.</caption><thead><tr><th>Environment</th><th>Location</th><th>Species</th><th>S</th><th>RS</th><th>Oogenesis Duration</th><th>Oocyte Diameter (µm)</th><th>Fertility</th><th>Spermat. Duration</th><th>Spermat. Diameter (µm)</th><th>Fertility</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">Littoral tropical Red Sea</td><td>Briaenum hambrium</td><td>G</td><td>SB</td><td>12</td><td>180 – 750</td><td>14 – 16</td><td>8 – 10</td><td>250 – 550</td><td>-</td><td>Benayahu, 1989</td></tr><tr><td>Plexaura flexuosa</td><td>G</td><td>BS</td><td>-</td><td>597±27</td><td>0.17±0.24 – 1.09±0.73</td><td>-</td><td>~450</td><td>-</td><td>Beiring and Lasker, 2000</td></tr><tr><td>Plexaura sp.</td><td>G</td><td>BS</td><td>~9</td><td>200 – 600</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Brazeau and Lasker, 1989</td></tr><tr><td>Plexaura homomalla</td><td>G</td><td>BS</td><td>18</td><td>>100 – 640</td><td>1.97±0.26</td><td>6 – 8</td><td>-</td><td>-</td><td>Martin et al., 1982</td></tr><tr><td>Antillogorgia hystrix</td><td>G</td><td>IB</td><td>9</td><td>101 – >700</td><td>-0.75 – ~3.5</td><td>4</td><td>101 – >601</td><td>-</td><td>Coelho and Lasker, 2014</td></tr><tr><td>Pseudopterogorgia elisabethae</td><td>G</td><td>SB</td><td>~10</td><td>66±3.1 – 379.6±9.2</td><td>-</td><td>~2</td><td>70±4.5 – 296.6±11.8</td><td>-</td><td>Gutiérrez-Rodriguez and Lasker, 2004</td></tr><tr><td>Acabaria bisertialis</td><td>G</td><td>IB</td><td>10</td><td><40 – 240</td><td>-</td><td>~10</td><td><40 – 160</td><td>-</td><td>Ben-Yosef and Benayahu, 1999</td></tr><tr><td>N Atlantic</td><td>Acanella arbuscula</td><td>G</td><td>-</td><td>20.8±6.6 – 543±71.9</td><td>21.0±17.5</td><td>-</td><td>28.8±14 – 309.7±21</td><td>13.9±13.5</td><td>Beazley and Kenchington, 2012</td></tr><tr><td>S Pacific</td><td>Primnoa notialis</td><td>G</td><td>-</td><td>100 – 690</td><td>18±4.51</td><td>-</td><td>-</td><td>-</td><td>Feehan and Waller, 2015</td></tr><tr><td>N Pacific</td><td>Swiftia beringi</td><td>G</td><td>-</td><td>726.63</td><td>13.6±2.85</td><td>-</td><td>-</td><td>-</td><td>Feehan and Waller, 2015</td></tr><tr><td>N Pacific</td><td>Swiftia kofoidi</td><td>G</td><td>-</td><td>561.81</td><td>3±1.53</td><td>-</td><td>-</td><td>-</td><td>Feehan and Waller, 2015</td></tr><tr><td>N Pacific</td><td>Swiftia pacifica</td><td>G</td><td>-</td><td>150 – 664.81</td><td>4.6±2.06</td><td>-</td><td>-</td><td>-</td><td>Feehan and Waller, 2015</td></tr><tr><td>N Pacific</td><td>Swiftia simplex</td><td>G</td><td>-</td><td>269 – 698.53</td><td>42.53±9.82</td><td>-</td><td>-</td><td>-</td><td>Feehan and Waller, 2015</td></tr><tr><td>N Pacific</td><td>Swiftia torreyi</td><td>G</td><td>-</td><td>241 – 645.07</td><td>8±1.15</td><td>-</td><td>-</td><td>-</td><td>Feehan and Waller, 2015</td></tr></tbody></table>
<table><caption>Table 3 (cont.). Reproductive patterns in different gorgonian species. Med = Mediterranean, Isl. = island, S = sexuality, G = gonochoric, H = hermaphroditic, RS = reproductive strategy, IB = internal brooder, SB = surface brooder, BS = broadcast spawner, Spermat. = spermatogenesis.</caption><thead><tr><th>Environment</th><th>Area</th><th>Species</th><th>S</th><th>RS</th><th>Oogenesis Duration</th><th>Oocyte Diameter (µm)</th><th>Fertility</th><th>Spermat. Duration</th><th>Spermary Diameter (µm)</th><th>Fertility</th><th>Reference</th></tr></thead><tbody><tr><td rowspan="10">Continental shelf and slope</td><td>N Pacific</td><td>Primnoa pacifica</td><td>G</td><td>SB</td><td>~12</td><td>50 – 802</td><td>86±23</td><td>~12</td><td>500 – 1000</td><td>-</td><td>Waller et al., 2014</td></tr><tr><td rowspan="2">N Atlantic</td><td>Primnoa resedaeformis</td><td>G</td><td>-</td><td>-</td><td>&lt;100 – 1000</td><td>84.3±3.1</td><td>-</td><td>-</td><td>-</td><td>Mercier and Hamel, 2011</td></tr><tr><td>Keratoisis ornata</td><td>G</td><td>-</td><td>-</td><td>70 – 700</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Mercier and Hamel, 2011</td></tr><tr><td>N Pacific</td><td>Paracorallium japonicum</td><td>G</td><td>BS</td><td>~9</td><td>102.3 – 227.7</td><td>1 – 3</td><td>-</td><td>162.3 – 261.7</td><td>1 – 6</td><td>Nonaka et al., 2015</td></tr><tr><td>N Pacific</td><td>Corallialum elatius</td><td>G</td><td>BS</td><td>~11</td><td>112.7 – 229.3</td><td>1 – 7</td><td>-</td><td>36.1 – 250.3</td><td>1 – 6</td><td>Nonaka et al., 2015</td></tr><tr><td>N Pacific</td><td>Corallium konjoi</td><td>G</td><td>BS</td><td>-</td><td>76.3 – 168.8</td><td>1 – 7</td><td>-</td><td>50.6 – 287.2</td><td>1 – 8</td><td>Nonaka et al., 2015</td></tr><tr><td>N Pacific</td><td>Corallium lauense</td><td>G</td><td>BS</td><td>-</td><td>~650</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Waller and Baco, 2007</td></tr><tr><td>N Pacific</td><td>Corallium secundum</td><td>G</td><td>BS</td><td>-</td><td>~600</td><td>-</td><td>-</td><td>-</td><td>-</td><td>Waller and Baco, 2007</td></tr><tr><td>Antarctica</td><td>Dasystenella acanthina</td><td>G</td><td>-</td><td>&gt;12</td><td>50 – 1200</td><td>1.2±0.08</td><td>-</td><td>20 – ~790</td><td>2.6±0.19</td><td>Orejas et al., 2007</td></tr><tr><td>Antarctica</td><td>Thouarella sp.</td><td>G</td><td>IB</td><td>&gt;12</td><td>&gt;100 – 550</td><td>1.1±0.1</td><td>-</td><td>&gt;75 – 325</td><td>3.0±0.2</td><td>Orejas et al., 2007</td></tr><tr><td>Antarctica</td><td>Thouarella variabilis</td><td>G</td><td>IB</td><td>&gt;12</td><td>&gt;50 – &gt;800</td><td>-</td><td>-</td><td>&gt;50 – &gt;950</td><td>-</td><td>Brito et al., 1997</td></tr><tr><td>Antarctica</td><td>Fannyella rossii</td><td>G</td><td>IB</td><td>&gt;12</td><td>&gt;100 – &gt;350</td><td>1.5±0.06</td><td>-</td><td>75 – &gt;150</td><td>5.0±0.21</td><td>Orejas et al., 2007</td></tr><tr><td>Antarctica</td><td>Fannyella spinosa</td><td>G</td><td>IB</td><td>&gt;12</td><td>&gt;80 – &gt;300</td><td>1.4±0.8</td><td>-</td><td>&gt;80 – &gt;300</td><td>2.6±0.21</td><td>Orejas et al., 2007</td></tr></tbody></table>
Seasonality in the energetic requirements of *P. macrospina* is highlighted by the seasonal changes observed in the FFA content and composition. Indeed, the slight increase of FFA content from early autumn to mid summer (Figs. 7 and 8), in coincidence with progressive augment of gonadal volume (Fig. 5), suggests that gonadal development supposes a high energetic demand that *P. macrospina* meets by mobilizing FFA as a source of immediate energy (Sargent et al., 1988; Viladrich et al., 2016). On the contrary, larval development inside the maternal polyp does not seem to require any high metabolic investment, as FFA content was minimum in September and October when larvae were found inside the polyps. This seasonality was also reflected in the FFA composition of *P. macrospina* tissue, which is mainly characterized by SFFA markers during winter, and by PUFFA markers during spring and summer (i.e., when gonadal volume progressively increases) (Fig. 9). Besides, the predominance of 18:4(n-3), 20:4(n-3) and 22:6(n-3) during gonadal development might have been directly related to increased fecundity, fertility and egg quality (Pernet et al., 2002; Viladrich et al., 2016). On the other hand, 18:3(n-3) and 24:0 predominate during late summer and early fall, when larvae are present inside the female polyps. The 18:3(n-3) is an essential FA that can be converted into the high energy and biologically active FFA 20:5(n-3) and 22:6(n-3) (Kelly and Scheibling, 2012). In this sense, larvae could be directly using 18:3(n-3) to fulfil their metabolic demands (Sargent et al., 1999).

Overall, dampening of environmental variability with depth (Puig et al., 2001; Garrabou et al., 2002; Fernández-Puelles et al., 2007) is reflected in the constant lipid content and SI composition of *P. macrospina* from the Mediterranean continental shelf, contrasting with the strong seasonality observed in shallow species (Coma et al., 2000; Rossi et al. 2006; Gori et al., 2012). The slight temperature increase occurring on the continental shelf in late summer (Fer-

nández de Puelles et al., 2007) is probably driving the reproductive cycle of *P. macrospina*, with gametogenesis likely affecting its metabolic requirements (with a mobilization of high-energy PUFFA in spring and early summer). Internal brooding in *P. macrospina* contrasts with surface brooding of the congeneric *P. clavata*. The differences in the habitats where the two species occur, arises the question about the possible adaptive advantage of their reproductive strategies. Nevertheless, the observed differences in the reproductive features of *P. macrospina* compared to the shallower Mediterranean species, contributes to highlight the ecological diversity of gorgonians as a possible explanation of their wide spatial and bathymetric distribution in the Mediterranean Sea (Carpine and Grasshoff, 1975; Bo et al., 2012; Grinyó et al., 2016).

## ACKNOWLEDGMENTS

The authors thank the fishermen from the artisanal fishing boat GOGA (Cala Ratjada, Mallorca) for their help in the collection of the samples, and Núria Moraleda, Stefano Ambroso, Maria Montseny, Carlos Dominguez-Carrió and Ivana Simunovic for their help in laboratory analysis and data treatment. This work was funded by the European project LIFE+INDEMARES "Inventario y designación de la red Natura 2000 en áreas marinas del estado español" (LIFE07/NAT/E/000732).

## REFERENCES

* ARAI, T., KATO, M., HEYARD, A.; IKEDA, Y., IIZUKA, T., MARUYAMA, T., 1993. Lipid composition of positively
buoyant eggs of reef building corals. Coral Reefs 12, 71–75.

BALLESTEROS, E., 2006. Mediterranean coralligenous assemblages: a synthesis of present knowledge. *Oceanography and Marine Biology: An Annual Review* 44, 123–195.

BARNES, H., BLACKSTOCK, J., 1973. Estimation of lipis in marine animals and tissues: detailed investigation of the sulphosphovanillin method for "total" lipids. *Journal of Experimental Marine Biology and Ecology* 12, 103–118.

BEAZLEY, L.I., KENCHINGTON, E.L., 2012. Reproductive biology of the deep-water coral *Acanella arbuscula* (Phylum Cnidaria: Class Anthozoa: Order Alcyonacea), northwest Atlantic. Deep Sea Research Part I: Oceanographic Research Papers 68, 92–104. doi:10.1016/j.dsr.2012.05.013

BEIRING, E.A, LASKER, H.R., 2000. Egg production by colonies of a gorgonian coral. Marine Ecology Progress Series 196, 169–177.

BEN-YOSEF, D.Z., BENAYAHU, Y., 1999. The gorgonian coral *Acabaria bisensis*: life history of a successful colonizer of artificial substrata. Marine Biology 135, 473–481. doi:10.1007/s002270050647

BENAYAHU, Y., 1989. Reproductive cycle and developmental processes during embryogenesis of *Clavularia hamra* (Cnidaria, Octocorallia). Acta Zoologica 70, 29–36.

BO, M., BERTOLINO, M., BORGHINI, M., CASTELLANO, M., HARRIAGUE, A.C., DI CAMILLO, C.G., GASPARINI, G., MISIC, C., POVERO, P., PUSCEDDU, A., SCHROEDER, K., BAVESTRELLO, G., 2011. Characteristics of the mesophotic megabenthic assemblages of the Vercelli seamount (North Tyrrhenian Sea). PLoS One 6. doi:10.1371/journal.pone.0016357

BO, M., CANESE, S., SPAGGIARI, C., PUSCEDDU, A., BERTOLINO, M., ANGIOLILLO, M., GIUSTI, M., LORETO, M.F., SALVATI, E., GRECO, S., BAVESTRELLO, G., 2012.

Deep Coral Oases in the South Tyrrhenian Sea. PLoS One 7. doi:10.1371/journal.pone.0049870

BONGAERTS, P., RIDGWAY, T., SAMPAYO, E.M., HOEGH-GULDBERG, O., 2010. Assessing the "deep reef refugia" hypothesis: Focus on Caribbean reefs. Coral Reefs 29, 1–19. doi:10.1007/s00338-009-0581-x

BRITO, T.A.S., TYLER, P.A., CLARKE, A. 1997. Reproductive biology of the Antarctic octocoral *Thouarella variabilis* Wright and Studer 1889. Proceedings 6th International Conference of Coelenterate Biology, Natural History Museum of Leiden, The Netherlands, p 63–69.

BRAZEAU, D.A., LASKER, H., 1989. The Reproductive Cycle and Spawning in a Caribbean Gorgonian. The Biological Bulletin 176, 1–7.

BRAZEAU, D.A., LASKER, H.R., 1990. Sexual reproduction and external brooding by the Caribbean gorgonian *Briareum asbestinum*. Marine Biology 104, 456–474.

BRODTE, E., GRAEVE, M., JACOB, U., KNUST, R., PÖRTNER, H.O., 2008. Temperature-dependent lipid levels and components in polar and temperate eelpout (Zoarcidae). Fish Physiology and Biochemistry 34, 261–274. doi:10.1007/s10695-007-9185-y

CARLIER, A., RIERA, P., AMOUROUX, J.M., BODIOU, J.Y., GRÉMARE, A., 2007. Benthic trophic network in the Bay of Banyuls-sur-Mer (northwest Mediterranean, France): An assessment based on stable carbon and nitrogen isotopes analysis. Estuarine and Coastal Shelf Science 72, 1–15. doi:10.1016/j.ecss.2006.10.001

CARPINE, C., GRASSHOFF, M., 1975. Les gorgonaires de la Méditerranée. Bulletin de l'Institut océanographique Monaco 71, 1–140.

CAU, A., FOLLESA, M.C., MOCCIA, D., ALVITO, A., BO, M., ANGIOLILLO, M., CANESE, S., PALIAGA, E.M., ORRÜ, P.E., SACCO, F., CANNAS, R., 2015. Deepwater
corals biodiversity along roche du large ecosystems with different habitat complexity along the south Sardinia continental margin (CW Mediterranean Sea). Marine Biology 162, 1865–1878. doi:10.1007/s00227-015-2718-5

CHAMBERS, J.M., FREENY, A., HEIBERGER, R.M., 1992. *Analysis of variance; designed experiments*. Chapter 5 of *Statistical Models in S* eds J. M. Chambers and T. J. Hastie, Wadsworth and Brooks/Cole. Pacific Grove, California.

FEEHAN, K.A., WALLER, R.G., 2015. Notes on reproduction of eight species of Eastern Pacific cold-water octocorals. Journal of the Marine Biological Association of the United Kingdom 95, 691–696. doi:10.1017/S0025315415000053

CERRANO, C., ARILLO, A., AZZINI, F., CALCINAI, B., CASTELLANO, L., MUTI, C., VALISANO, L., ZEGA, G., BAVESTRELLO, G., 2005. Gorgonian population recovery after a mass mortality event. Aquatic Conservation: Marine and Freshwater Ecosystems 15, 147–157. doi:10.1002/aqc.661

CHANG, Tc., 2007. Sexual reproduction of four gorgonian corals in southern Taiwan. Masters thesis, National Sun Yat-sen University p 46.

COCITO, S., FERRIER-PAGÈS, C., CUPIDO, R., ROTTIER, C., MEIER-AUGENSTEIN, W., KEMP, H., REYNAUD, S., PEIRANO, A., 2013. Nutrient acquisition in four Mediterranean gorgonian species. Marine Ecology Progress Series 473, 179–188. doi:10.3354/meps10037

COELHO, M.A.G., LASKER, H.R., 2014. Reproductive biology of the Caribbean brooding octocoral *Antilogorgia hystrix*. Invertebrate Biology 133, 299–313. doi:10.1111/ivb.12070

COMA, R., RIBES, M., 2003. Seasonal energetic constraints in Mediterranean benthic suspension feeders: effects at different levels of ecological organization. Oikos 101, 205–215. doi:10.1034/j.1600-0706.2003.12028.x

COMA, R., RIBES, M., GILI, J.M., 2000. Seasonality in coastal benthic ecosystems. Trends in Ecology and Evolution 15, 448–453.

COMA, R., RIBES, M., ZABALA, M., GITI, J., 1995. Reproduction and cycle of gonadal development in the Mediterranean gorgonian *Paramuricea clavata*. Marine Ecology Progress Series 117, 173–183. doi:10.3354/meps117173

DALSGAARD, J., ST JOHN, M., KATTNER, G., MÜLLER-NAVARRA, D., HAGEN, W., 2003. Fatty Acid Trophic Markers in the Pelagic Marine Environment. Advances in Marine Biology 46, 225–340.

DANOVARO, R., DELLA CROCE, N., DELL'ANNO, A., FABIANO, M., MARRALE, D., MARTORANO, D., 2000. Seasonal changes and biochemical composition of the labile organic matter flux in the Cretan Sea. Progress in Oceanography 46, 259–278. doi:10.1016/S0079-6611(00)00022-7

DE PUTRON, S.J., RYLAND, J.S., 2009. Effect of seawater temperature on reproductive seasonality and fecundity of *Pseudoplexaura porosa* (Cnidaria: Octocorallia): latitudinal variation in Caribbean gorgonian reproduction. Invertebrate Biology 128, 213–222. doi:10.1111/j.1744-7410.2009.00170.x

EXCOFFON, A.C., ACUÑA, F.H., ZAMPONI, M.O., GENZANO, G.N., 2004. Reproduction of the temperate octocoral *Tripalea clavaria* (Octocorallia: Anthothelidae) from sublittoral outcrops off Mar del Plata, Argentina. Journal of the Marine Biology Association of the United Kingdom 84, 695–699. doi:10.1017/S0025315404009774h

FERNÁNDEZ DE PUELLES, M.L., ALEMANY, F., JANSÁ, J., 2007. Zooplankton time-series in the Balearic Sea (Western Mediterranean): Variability during the decade 1994–2003. Progress in Oceanography 74, 329–354. doi:10.1016/j-pocean.2007.04.009

FINE, M., ALUMA, Y., MEROZ-FINE, E., ABELSON, A., LOYA, Y., 2005. *Acabaria erythraea* (Octocorallia: Gorgonacea) a successful invader to the Mediter-
ranean Sea? Coral Reefs 24, 161–164.
doi:10.1007/s00338-004-0462-2

FIORILLO, I., ROSSI, S., ALVA, V., GILI, J.M., LÓPEZ-GONZÁLEZ, P.J., 2013. Seasonal cycle of sexual re-production of the Mediterranean soft coral *Alcyonium acaule* (Anthozoa, Octocorallia). Marine Biology 160, 719–728. doi:10.1007/s00227-012-2126-z

GARRABOU, J., BALLESTEROS, E., ZABALA, M., 2002. Structure and Dynamics of North-western Mediterranean Rocky Benthic Communities along a Depth Gradient. Estuarine Coastal and Shelf Science 55, 493–508. doi:10.1006/ecss.2001.0920

Gori, A., Linares, C., Rossi, S., Coma, R., Gili, J.M., 2007. Spatial variability in reproductive cycle of the gorgonians *Paramuricea clavata* and *Eunicella singularis* (Anthozoa, Octocorallia) in the Western Mediterranean Sea. Marine Biology 151, 1571–1584. doi:10.1007/s00227-006-0595-7

GORI, A., VILADRICH, N., GILI, J.-M., KOTTA, M., CUCIO, C., MAGNI, L., BRAMANTI, L., ROSSI, S., 2012. Reproductive cycle and trophic ecology in deep versus shallow populations of the Mediterranean gorgonian *Eunicella singularis* (Cap de Creus, northwestern Mediterranean Sea). Coral Reefs 31, 823–837. doi:10.1007/s00338-012-0904-1

GORI, A., ROSSI, S., LINARES, C., BERGANZO, E., OREJAS, C., DALE, M.R., GILI, J.M., 2011. Size and spatial structure in deep versus shallow populations of the Mediterranean gorgonian *Eunicella singularis* (Cap de Creus, northwestern Mediterranean Sea). Marine Biology 158, 1721–1732. doi:10.1007/s00227-011-1686-7

GORI, A., LINARES, C., VILADRICH, N., CLAVERO, A., OREJAS, C., FIORILLO, I., AMBROSO, S., GILI, J.M., ROSSI, S., 2013. Effects of food availability on the sexual reproduction and biochemical composition of the Mediterranean gorgonian *Paramuricea clavata*. Journal of Experimental Marine Biology and Ecology 444, 38–45. doi:10.1016/j.jembe.2013.03.009

GRIGG, R.W., 1970. Ecology and population dynamics of the gorgonians *Muricea californica* and *Muricea fruticosa*. Ph D. Thesis, University of California at San Diego.

GRIGG, R.W., 1977. Population Dynamics of Two Gorgonian Corals. Ecology 58, 278–290.

GRINYÓ, J., GORI, A., AMBROSO, S., PURROY, A., CALATAYUD, C., DOMINGUEZ-CARRIÓ, C., COPPARI, M., LO IACONO, C., LÓPEZ-GONZÁLEZ, P.J., GILI, J.M., 2016. Diversity, distribution and population size structure of deep Mediterranean gorgonian assemblages (Menorca Channel, Western Mediterranean Sea). Progress in Oceanography 145, 42–56. doi:10.1016/j.pcean.2016.05.001

GRINYÓ, J., ISLA, E., PERAL, L., GILI, J.M. (submitted) Composition and temporal variability of particle fluxes in an insular canyon of the northwestern Mediterranean Sea. Progress in Oceanography.

GROTTOLI, A.G., RODRIGUES, L.J., JUAREZ, C., 2004. Lipids and stable carbon isotopes in two species of Hawaiian corals, *Porites compressa* and *Montipora verrucosa*, following a bleaching event. Marine Biology 145, 621–631. doi:10.1007/s00227-004-1337-3

GURR, M.I., HARWOOD, J.L., FRAYN, K.N., 2002. Lipid biochemistry. Blackwell Science, Oxford, UK.

GUTIÉRREZ-RODRÍGUEZ, C., LASKER, H.R., 2004. Reproductive biology, development, and planula behavior in the Caribbean gorgonian. Invertebrate Biology 123, 54–67.

HINDERSTEIN, L.M., MARR, J.C.A., MARTINEZ, F.A., DOWGIALLO, M.J., PUGLISE, K.A., PYLE, R.L., ZAWADA, D.G., APPELDOORN, R., 2010. Theme section on "Mesophotic Coral Ecosystems: Characterization, Ecology, and Management." Coral Reefs 29, 247–251. doi:10.1007/s00338-010-0614-5

IMBS, A.B., 2013. Fatty acids and other lipids of corals: Composition, distribution, and biosynthesis.
Russian Journal of Marine Biology 39, 153–168.
doi:10.1134/S1063074013030061

KAHNG, S.E., COPUS, J., WAGNER, D., 2014. Recent advances in the ecology of mesophotic coral ecosystems (MCEs). Current Opinion in Environmental Sustainability 7, 72–81. doi:10.1016/j.co-sust.2013.11.019

KAHNG, S.E., BENAYAHU, Y., LASKER, H.R., 2011. Sexual reproduction in octocorals. Marine Ecology Progress Series 443, 265–283. doi:10.3354/meps09414

KAHNG, S.E., GARCIA-SAIS, J.R., SPALDING, H.L., BROKOVICH, E., WAGNER, D., WEIL, E., HINDERSTEIN, L., TOONEN, R.J., 2010. Community ecology of mesophotic coral reef ecosystems. Coral Reefs 29, 255–275. doi:10.1007/s00338-010-0593-6

KELLY, J.R., SCHEIBLING, R.E., 2012. Fatty acids as di-
etary tracers in benthic food webs. Marine Ecology
Progress Series 446, 1–22. doi:10.3354/meps09559

KIPSON, S., LINARES, C., CIŽMEK, H., CEBRIÁN, E., BALLESTEROS, E., BAKRAN-PETRICIOLI, T., GARRABOU, J., 2014. Population structure and conservation status of the red gorgonian *Paramuricea clavata* (Risso, 1826) in the Eastern Adriatic Sea. Marine Ecology 34, 982–993. doi:10.1111/maec.12195

LINARES, C., COMA, R., GARRABOU, J., DÍAZ, D., ZABALA, M., 2008. Size distribution, density and disturbance in two Mediterranean gorgonians: *Paramuricea clavata* and *Eunicella singularis*. Journal of Applied Ecology 45, 688–699. doi:10.1111/j.1365-2664.20070

LOYA, Y., EYAL, G., TREIBITZ, T., LESSER, M.P., 2016. Theme section on mesophotic coral ecosystems: advances in knowledge and future perspectives. Coral Reefs 35, 1–9. doi:10.1007/s00338-016-1410-7

MARTIN, E., FITZSIMMONS-SOSA, K., HALLOCK, P., WHEATON, J., HACKETT, K.E., CALLAHAN, M.K., 1982. Ciclo reproductivo, proporcion sexual y fecundidad del coral blando *Plexaura homomalla* (Esper.) en el mar Caribe Mexicano (Octocoralla: Plexauridae).

An. Inst. Cienc. del Mar y Limnol. Univ. nal. Auton.
México 1, 359–380.

MERCIER, A., HAMEL, J.-F., 2011. Contrasting reproductive strategies in three deep-sea octocorals from eastern Canada: *Primnoa resedaeformis*, *Keratoisis ornata*, and *Anthomastus grandiflorus*. Coral Reefs 30, 337–350. doi:10.1007/s00338-011-0724-8

MÜLLER-NAVARRA, D.C., BRETT, M.T., LISTON, A.M., GOLDMAN, C.R., 2000. A highly unsaturated fatty acid predicts carbon transfer between primary producers and consumers. Nature 403, 74–77. doi:10.1038/47469

NONAKA, M., NAKAMURA, M., MUZIK, K., 2015. Sexual Reproduction in Precious Corals (Coralliidae) Collected in the Ryukyu Archipelago. Pacific Science 69, 15–46. doi:10.2984/69.1.2

OKSANEN, J., KINDT, R., LEGENDRE, P., O'HARA, R.B., 2005.
Vegan: community ecology package. Version 1.7–81.

OKU, H., YAMASHIRO, H., ONAGA, K., IWASAKI, H., TAKARA, K., 2002. Lipid distribution in branching coral *Montipora digitata*. Fisheries Science 68, 517–522. doi:10.1046/j.1444-2906.2002.00456.x

OREJAS, C., GILI, J.M., LÓPEZ-GONZÁLEZ, P.J., HASEMANN, C., ARNTZ, W.E., 2007. Reproduction patterns of four Antarctic octocorals in the Weddell Sea: An inter-specific, shape, and latitudinal comparison. Marine Biology 150, 551–563. doi:10.1007/s00227-006-0370-9

PAKES, M.J., WOOLLACOTT, R.M., 2008. Reproduction of the gorgonian *Plexaura flexuosa* in Bermuda. Journal of Experimental Marine Biology and Ecology 357, 121–127. doi:10.1016/j.jembe.2008.01.003

PALANQUES, A., PUIG, P., GUILLÉN, J., JIMÉNEZ, J., GRACIA, V., SÁNCHEZ-ARCILLA, A., MADSEN, O., 2002. Near-bottom suspended sediment fluxes on the microtidal low-energy Ebro continental shelf (NW Mediterranean). Continental Shelf Research 22, 285–303. doi:10.1016/S0278-4343(01)00058-9
PATEFIELD, W.M., 1981. Algorithm AS159. An efficient method of generating r x c tables with given row and column totals. Applied Statistics 30, 91–97.

PECHENIK, J.A., 1990. Delayed metamorphosis by larvae of benthic marine invertebrates: Does it occur? Is there a price to pay? Ophelia 32, 63–94. doi:10.1080/00785236.1990.10422025

PERNET, V., GAVINO, V., GAVINO, G., ANCTIL, M., 2002. Variations of lipid and fatty acid contents during the reproductive cycle of the anthozoan *Renilla koellikeri*. Journal of Comparative Physiology 172, 455–465. doi:10.1007/s00360-002-0268-x

PRIORI, C., MASTASCUSA, V., ERRA, F., ANGIOLILLO, M., CANESE, S., SANTANGELO, G., 2013. Demography of deep-dwelling red coral populations: Age and reproductive structure of a highly valued marine species. Estuarine and Coastal Shelf Science 118, 43–49. doi:10.1016/j.ecss.2012.12.011

PUIG, P., PALANQUES, A., GUILLÉN, J., 2001. Near-bottom suspended sediment variability caused by storms and near-inertial internal waves on the Ebro mid continental shelf (NW Mediterranean). Marine Geology 178, 81–93. doi:10.1016/S0025-3227(01)00186-4

R CORE TEAM, 2014. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL http://www.R-project.org.

RIBES, M., COMA, R., GILI, J.M., 1999. Heterogeneous feeding in benthic suspension feeders: The natural diet and grazing rate of the temperate gorgonian *Paramuricea clavata* (Cnidaria: Octocorallia) over a year cycle. Marine Ecology Progress Series 183, 125–137. doi:10.3354/meps183125

RIBES, M., COMA, R., ROSSI, S., MICHELI, M., 2007. Cycle of gonadal development in *Eunicella singularis* (Cnidaria: Octocorallia): trends in sexual reproduction in gorgonians. Invertebrate Biology 126, 307–317. doi:10.1111/j.1744-7410.2007.00101.x

RICHMOND, R.H., 1987. Energetic relationships and biogeographical differences among fecundity, growth and reproduction in the reef coral *Pocillopora damicornis*. Bulletin of Marine Science 41, 594–604.

RIEDL, R., 1971. Water movement: animals. Kinne, O. ed Marine ecology vol 1, part 2., Wiley inter-science, London, pp 1123–1149.

ROSSI, S., GRÉMARE, A., GILI, J.M., AMOUROUX, J.M., JORDANA, E., VÉTION, G., 2003. Biochemical characteristics of settling particulate organic matter at two north-western Mediterranean sites: A seasonal comparison. Estuarine Coastal Shelf Science 58, 423–434. doi:10.1016/S0272-7714(03)00108-2

ROSSI, S., SNYDER, M.J., GILI, J.M., 2006. Protein, carbohydrate, lipid concentrations and HSP 70-HSP 90 (stress protein) expression over an annual cycle: useful tools to detect feeding constraints in a benthic suspension feeder. Helgoland Marine Research 60, 7–17. doi:10.1007/s10152-005-0009-0

ROSSI, S., GILI, J.M., 2009. Reproductive features and gonad development cycle of the soft bottom-gravel gorgonian *Leptogorgia sarmentosa* (Esper, 1791) in the NW Mediterranean Sea. Invertebrate Reproduction and Development 53, 175–190. doi:10.1080/07924259.2009.9652304

SARGENT, J.R., PARKS, R.J., MUELLER-HARVEY, I., HENDERSON, R.J., 1988. Lipid biomarkers in marine ecology. In Sliegh, MA (Eds.) Microbes in the sea. Ellis Horwood Ltd, Chichester, pp 119–138.

SARGENT, J.R., MCEVOY, L., ESTÉVEZ, A., BELL, J.G., BELL, M., HENDERSON, J., TOCHER, D.R., 1999. Lipid nutrition of marine fish during early development: current status and future directions. Aquaculture 179, 217–230.

SEO, S.Y., HWANG, S.J., SONG, J.I., 2008. Sexual reproduction of *Anthoplexaura dimorpha* (Gorgonacea: Octocorallia) from Munseom, Jejudo islands, Korea. Animal Cells and Systems 12, 231–240. doi:10.1080/19768354.2008.9647177
SHERWOOD, O.A., HEIKOOP, J.M., SCOTT, D.B., RISK, M.J., GUILDERSON, T.P., MCKINNEY, R.A., 2005. Stable isotopic composition of deep-sea gorgonian corals. Marine Ecology Progress Series 301, 135–148. doi:10.3354/meps301135

SLATTERY, M., MCCLINTOCK, J.B., 1995. Population structure and feeding deterrence in three shallow-water antartic soft corals. Marine Biology 122, 461–470.

STEARNS, S.C., 1992. The evolution of life histories. Oxford University Press, Oxford, UK.

TCHERNOV, D., GORBUNOV, M.Y., DE VARGAS, C., NARAYAN YADAV, S., MILLIGAN, A.J., HÄGBLOM, M., FALKOWSKI, P.G., 2004. Membrane lipids of symbiotic algae are diagnostic of sensitivity to thermal bleaching in corals. Proceedings of the National Academy of Sciences of the United States of America 101, 13531–13535. doi:10.1073/pnas.0402907101

THORSON, G., 1950. Reproductive and Larval Ecology of Marine Bottom Invertebrates. Biological Review of the Cambridge Philosical Society 25, 1–45.

TOPÇU, N.E., ÖZTÜRK, B., 2016. Reproduction in the Mediterranean endemic gorgonian *Spinimuricea klavereni* (Anthozoa, Octocorallia, Plexauridae). In-vertebrate Biology. doi:10.1111/ivb.12112

TSOUNIS, G., ROSSI, S., ARANGUREN, M., GILI, J.M., ARNTZ, W., 2006. Effects of spatial variability and colony size on the reproductive output and gonadal development cycle of the Mediterranean red coral (*Corallium rubrum* L.). Marine Biology 148, 513–527. doi:10.1007/s00227-005-0100-8

VILADRICH, N., BRAMANTI, L., TSOUNIS, G., CHOCARRO, B., MARTÍNEZ-QUINTANA, A., AMBROSO, S., MADURELL, T., ROSSI, S., 2016. Energetic resource allocation in reproduction for two temperate octocorals with contrasting reproductive strategies: surface versus internal brooder. Coral Reefs. doi:10.1007/s00338-016-1440-1

WACKER, A., VON ELERT, E., 2001. Polyunsaturated fatty acids: Evidence for non-substitutable bio-chemical resources in *Daphnia galeata*. Ecology 82, 2507–2520. doi:10.1890/0012-9658(2001)082

WALLER, R.G., BACO, A.R., 2007. Reproductive Morphology of Three Species of Deep-Water Precious Corals from the Hawaiian Archipelago: Bullentine of Marine Science 81, 533–542.

WALLER, R.G., STONE, R.P., JOHNSTONE, J., MONDRAGON, J., 2014. Sexual reproduction and seasonality of the Alaskan red tree coral, *Primnoa pacifica*. PLoS One 9, e90893. doi:10.1371/journal.pone.0090893

WARD, S., 1995. Two patterns of energy allocation for growth, reproduction and lipid storage in the scleractinian coral *Pocillopora damicornis*. Coral Reefs 14, 87–90. doi:10.1007/BF00303428

WARD, S., HARRISON, P., 2000. Changes in gametogenesis and fecundity of acroporid corals that were exposed to elevated nitrogen and phosphorus during the ENCORE experiment. Journal of Experimental Marine Biology and Ecology 246, 179–221. doi:10.1016/S0022-0981(99)00182-3
Chapter 5

Composition and temporal variability of
particle fluxes in an insular canyon
of the northwestern Mediterranean Sea

ABSTRACT

Particle fluxes have widely been studied in canyons located in continental margins conversely particle fluxes in canyons incising small island margins have received very little attention and remain poorly understood. The Menorca Canyon is the only canyons system in the Balearic Archipelago. Moreover, the Menorca Canyon and surrounding areas host highly diverse communities dominated by benthic suspension feeders. Understanding the magnitude and variability of environmental factors influencing these communities thus remains crucial. In order to characterize the temporal variability of particle fluxes, analyze its geochemical and macroscopic composition and identify the main processes that modulate particle fluxes in the Menorca Canyon one instrumented line was deployed at 430 m from September 2010 to October 2012. Particle fluxes ranged between 0.2 – 2.3 g m² d⁻¹ being one of the lowest ever registered in a Mediterranean submarine canyon's head. Asides opal, geochemical constituents were temporally highly correlated with particle fluxes. CaCO₃ fraction was the major constituent contrasting with the general trend observed in other Mediterranean canyons. Macroscopic constituents (fecal pellets, *Posidonia oceanica* detritus and pelagic and benthic foraminifera) constituents presented a wide variability throughout the sampling period and were poorly correlated with the total mass flux. The low magnitude of the registered fluxes and the lack of correlation with the observed environmental variables (e.g., currents, winds, wave height, chlorophyll-a biomass) suggest that there is no evident controlling mechanism. However, we could infer that resuspension processes and the presence of different hydrodynamic features (e.g. water masses) condition the magnitude and composition of particle fluxes.

CO-AUTHORS

E Isla, L Peral, JM Gili

ADDITIONAL RESOURCES

Submitted in Progress in Oceanography
# 1. INTRODUCTION

Continental and island margins receive particulate matter derived from fluvial discharges (Syvitski and Morehead, 1999; Kineke et al., 2000), which stimulate primary production (González et al., 2010) that can result in higher sediment accumulation (Sanchez-Cabeza et al., 1999). Continental and insular margins are irrupted by submarine canyons (Harris and Whiteway, 2011) that act as conduits of sediments and organic matter from the continental shelf to deeper environments (Mullenbach et al., 2004; Lopez-Fernandez et al., 2013a). Particle fluxes within submarine canyons are characterized by strong seasonal fluctuations (e.g. Fabres et al., 2008) and can be orders of magnitude larger than those over the adjacent slopes (Martín et al., 2006; Zúñiga et al., 2009; Pasqual et al., 2013). The interaction of along slope currents and canyon topography may result in alterations of local currents that can promote cross shelf edge water exchange (Ahumada-Sempoal et al., 2015) leading to downwelling (Jordi et al., 2005) and upwelling processes within the canyon (Sobarzo and Djurfledt, 2004). Moreover, several hydrodynamic and atmospheric driven mechanisms can punctually increase particle fluxes in submarine canyons through resuspension and gravity-driven processes over short periods of time (Puig et al., 2014). Storms can cause sediment resuspension and gravity flows enhancing particle fluxes (Puig et al., 2004; Ross et al., 2009). Dense shelf water cascading can also cause dramatic increases in downward particle fluxes over few days span (Heussner et al., 2006) and can represent a major source of organic matter for deep environments (Tesi et al., 2010).

Downward particle fluxes and the main mechanisms controlling them have been widely studied in submarine canyons associated to continental landmasses (e.g. Shepard et al., 1974; Xu et al., 2002; Khripounoff et al., 2003) and large islands with high mountain ranges (≥ 2000 m) and permanent rivers systems (e.g. Kineke et al., 2000; Liu et al., 2002). However, downward particle fluxes in canyons incising small island margins remain widely unknown.

In the Mediterranean, this situation is rather surprising due to the elevated number of islands found in this basin (~200), where many of them are incised by submarine canyons (e.g. Lo Iacono et al., 2011). Despite the relative abundance of Mediterranean insular canyons (Harris and Whiteway, 2011), most studies concerning particles have been conducted in less than 25 canyons located in the European margin. Moreover, many of them are located in river-dominated shelves (e.g. Bonnin et al., 2008) with high productivity relative to the Mediterranean average (Bosc et al., 2004). The Baleric Archipelago is one of the most oligotrophic environments in the Western Mediterranean Sea (Bosc et al., 2004). In this archipelago there is only one large canyon system, the Menorca Canyon, located in the southern slope of Menorca Island (Fig. 1). Benthic communities associated with the head of the submarine canyons and surrounding shelves are characterized by high biodiversity and biomass (De Leo et al., 2010; Fabri et al., 2014). Despite the high oligotrophy of the Balearic Sea, benthic communities in the proximities of the Menorca Canyon are highly diverse (Barberá et al., 2012), giving an interesting opportunity to assess the environmental characteristics, including the flux of organic matter, where these communities thrive.

In this sense the aims of this study are to characterize (1) the temporal variability of downward particle fluxes analysing its geochemical
and (2) macroscopic composition and (3) identify the main processes that modulate particle fluxes in the Menorca Canyon, and (4) set the present results within previous research mainly developed in the Mediterranean.

## 1.2. Regional setting

The Menorca Canyon is located in the southwestern slope of Menorca Island (Fig. 1) and is the largest submarine canyon system in the Balearic Archipelago (Acosta et al., 2002). The canyon's head is at approximately 80 m water depth and is less than 5 km off Menorca's coastline (Acosta et al., 2002). The axis of the canyon has a NNE-SSW orientation and reaches approximately 1000 m water depth (Acosta et al., 2002). The flanks are steep and characterized by vertical walls and escarpments up to 20 m height (Lo Iacono et al., 2014). The continental shelf surrounding the canyon is relatively narrow and it only extends few kilometres (3 to 6 km) (Alonso et al., 1988). Biogenic sands with > 65% of carbonate content constitute the sediments in the canyon and surrounding shelves (Alonso et al., 1988).

The Balearic archipelago separates the Balearic-subbasin in the north from the Algerian-subbasin in the south (Amores and Monserrat, 2014) in such a way that different hydrodynamic processes and water masses influence the northern and southern slopes of the archipelago (Balbín et al., 2014). The northern slope is influenced by the Balearic current (Balbín et al., 2012; Amores et al., 2014) and is mostly characterized by the presence of resident Atlantic water (AW) (Salinity >37.5 psu) (Balbín et al., 2014). Conversely, the southern slope is influenced by the sporadic arrivals of mesoscale structures detaching from the Algerian current and from the instability of the Almería-Oran front (Millot, 1987), which is characterized by the presence of recent AW (Salinity <37.5 psu) (Balbín et al., 2014). During spring and summer a density front that separates the

resident AW from the recent AW develops south of the archipelago when western intermediate water (WIW) is present in the Ibiza and Mallorca channels (Balbín et al., 2014). Under these conditions anticyclonic gyres

<figure><img src="image_72.png" /><figcaption>**Fig. 1.** (a) Three-dimensional bathymetry of the northern Balearic Archipelago and its position in the western Mediterranean Sea. (b) Detail of the Menorca canyon. The white dot indicates the position of the sediment trap.</figcaption></figure>
have been detected in the southern slope of the Menorca Island (García et al., 2005; Balbín et al., 2014). Waters surrounding the archipelago are considered oligotrophic (Fernández de Puelles et al., 2007) as they receive little quantities of nutrients from land runoff due to low precipitation and the absence of rivers (Estrada et al., 1996). In some areas of the archipelago these conditions are enhanced by the intrusion of nutrient-poor recent AW including the southern slope of Menorca Island (García et al., 2005).

## 2. MATERIAL AND METHODS

### 2.1 Field work and instrumentation

One mooring array was maintained for two consecutive years in the Menorca Canyon (39°50.6601′N, 004°01.2600′E) on a site with approximately 430 m water depth. The mooring array was equipped with one cylindrical-sediment trap Technicap PPS3 and a Aanderaa current meter RCM9, tethered 30 m above the seabed and 25 m above the seabed, respectively. The sediment trap was set with 24 sequential collecting cups filled with a buffered 5% formaldehyde solution. The first study period (T1) continuously operated for 412 days (09/15/2010 – 10/11/2011). Sediment trap cups sampled during sequential intervals of 17-day except the last cup that sampled for 21 days. The second study period (T2) continuously operated for 365 days (11/03/2011 – 11/02/2012) and the sediment trap cups sampled for 15-day intervals except for the last five cups that sampled for 16-days intervals. The current meter was equipped with oxygen, turbidity, temperature and conductivity (salinity) sensors and it acquired measurements every 10 min.

### 2.2. Processing of sediment trap samples

Refrigerated (4 °C) sediment trap samples were processed in the laboratory according to the methodology described in Heussner et al. (1990) to produce aliquot sub-samples to carry out different analyses. Sub-samples were sieved with 0.4 µm-filtered seawater through a 1 mm nylon mesh. All "swimmers" (organisms that swam actively into the trap and died) were removed with forceps from the mesh. Sieved material was poured into a 2000-ml flask and filled up with 0.4 µm-filtered seawater. The flask was placed in a shaking table to generate homogenized aliquots separated with a robotized peristaltic pump. Aliquots were filtered onto pre-weighted 0.45 µm mesh nitro-cellulose white HAWP Millipore filters and onto pre-weighted Whatman GF/F filters both kinds, 47 mm diameter. Total mass was calculated as the dry mass weight of the filtered subsamples multiplied by the fraction of the aliquot. The total mass flux (TMF) expressed as, $mg m^{-2} d^{-1}$, was calculated from the total mass weight divided by the trap collecting area ($0.5 m^2$) and the sampling period in days.

### 2.3. Geochemical analyses

Total and organic carbon and total nitrogen were measured with a True Spec Carbon Nitrogen analyser LECO. Organic carbon (OC) was measured in samples pre-treated in a 1 M HCl vapour-bath for 24 h. Inorganic carbon was calculated as the difference between total and organic carbon. The inorganic carbon value was multiplied by 8.3331 to determine the calcium carbonate ($CaCO_3$) concentration. Carbon related analyses were performed with samples filtered onto Whatman GF/F filters. Biogenic opal content was obtained by alkaline extraction following the Mortlock and Froelich (1989) method modified by DeMaster (1981). Biogenic opal analyses were performed on samples filtered onto nitro-cellulose white
HAWP Millipore filters. Lithogenic content equals the difference between the total mass and the sum of the main biogenic components: biogenic opal, CaCO₃ and OM content (2x the OC content). Total concentrations of each geochemical component was calculated as the concentration of each subsamples multiplied by the fraction of the aliquot.

## 2.4. Macroscopic components

Fecal pellet, *Posidonia oceanica* detritus and foraminifera abundance were counted in aliquots using a Wild, Heerbrugg, (Switzerland) stereomicroscope (10x). Fecal pellets and *P. oceanica* detritus were measured with an eyepiece micrometer (± 10 µm). Fecal pellet volume was calculated with the formulas for cylindrical, ellipsoidal and spherical bodies, $V = \frac{4}{3} \pi r^3$, $V = \pi r^2 h$ and $V = \frac{4}{3} \pi r_a r_b r_c$ (where V is volume, r is radius and h is height, assuming that $r_a$ and $r_b$ had the same length in the case of ellipsoidal shapes), respectively. The volume of *P. oceanica* detritus was calculated with the formula of cylindrical bodies. Foraminifera tests were counted as single units regardless of their individual size. Pelagic and benthic species were differentiated based on the existing taxonomic works on Mediterranean foraminifera (Colom, 1974).

Additionally, images of selected samples were obtained with a Scanning Electron Microscope (SEM) HITACHI S-3500 N at 5.0 KV.

## 2.5 Wind and precipitation

The Spanish Meteorological Agency (AEMET) provided hourly data on precipitation, wind velocity and heading, obtained from the Maó Airport meteorological station (39°51′50.04″ N, 004°13′26.04 E, 91 m above the sea level) located ~17 km from the Menorca canyon's head. For each sampling cup interval we calculated: total precipitation, heading percentages (based on individual observations over the

total) and maximum wind velocity values. Additionally, for each sampling interval, hourly wind velocity percentages were grouped from 0 to 60 km h⁻¹ into 10 km h⁻¹ intervals.

## 2.5 Wave height

Large wave height events (Hs) were obtained from an oceanographic buoy of the "Puertos del Estado" (Spanish Ports Authority) located at 50 km from the canyons head (39°39′00′′ N, 003°29′2″ E). Although there is another buoy closer to the canyon's head (Maó), it was discarded due to multiple absences of registers during the winter and spring months.

## 2.7 Chlorophyll-a concentration and sea surface salinity:

Daily superficial chlorophyll a (Chl-a) concentration of waters surrounding Menorca Island and monthly average Chl-a concentration maps of the southern part of the Balearic subbasin and most of the Algerian subbasin were obtained from the satellite database of the Ocean Color Climate Change Initiative project (ocean-color.gsfc.nasa.gov/cms/). Daily satellite-derived Sea Surface Salinity (SSS) maps from the Marine Copernicus Environmental Monitoring Services (http://marine.copernicus.eu/web/69-interactive-catalogue.php) were revised in order to identify fronts or eddies in the study area.

## 2.8 Statistical analyses

For statistical comparisons two sampling intervals that lasted one-year approximately, and covered the same seasonal range were defined. The first interval, from hereby referred as 2011, ranged from 9/15/10 – 9/23/11. The second interval, from hereby referred as 2012, ranged from 9/24/11 – 9/12/12. Due to the mismatch between sampling cup intervals in T1 and T2 both sampling intervals do not comprise the same duration, being 2012 18 days shorter
than 2011. Distance-based permutational multivariate analysis of variance (PERMANOVA) (Anderson, 2001) was used to test the null hypothesis of no significant differences between 2011 and 2012 fluxes.

Each term of the analysis was tested using 9.999 permutations. Previous to the analysis data were standardized respect to their mean absolute deviation (MAD = $\frac{1}{n} \sum_{j=1}^{n} [x_{ij} - \bar{x}]$, where $x_{ij}$ is the value of the *i* variable observed in the *n* colonies) (García Pérez, 2005). The PERMANOVA was performed with the PaST software (Hammer et al., 2001). In order to explore the relationship among the TMF magnitude and the fluxes of its geochemical (CaCO$_3$, the lithogenic fraction, OC and biogenic opal) and macroscopic components (fecal pellet, *P. oceanica* detritus and foraminifera) simple linear regression analyses were performed using the lm function (Chambers, 1992) of the R software platform (R Development CoreTeam. 2011) for both 2011 and 2012 intervals separately.

## 3. RESULTS

### 3.1 Total mass flux and geochemical components

Forty-seven samples were obtained from mid-September 2010 (9/15/2010) to mid October 2012 (10/17/12). The 24th sampling cup of T1 (October 2011) was lost during the recovery process. During T1 three peaks in TMF were observed, a first one during mid autumn 2010, a second one during late spring and early summer 2011 and the third one during late summer 2011 (Fig. 2). The largest peak was the one registered during late spring and early summer 2011 (Fig. 2). During T2 four peaks in TMF were observed (Fig. 2). The first two peaks occurred during spring 2012, the third one during

late summer 2012 and the fourth and largest during mid autumn 2012 (Fig. 2).

During both T1 and T2 CaCO$_3$ fluxes were the largest fraction contributing 44% to 70% of the TMF, and the lithogenic fraction was the second most abundant component of the TMF ranging between 22% to 45%. OC contributed between 2% and 3% of the TMF. Biogenic opal fluxes contributed between 0.7% and 4% to the TMF.

Fluxes of CaCO$_3$ and the lithogenic fraction were significantly correlated with TMF (p<0.01) in both T1 and T2 (Fig. 3). OC fluxes were also significantly correlated with TMF. However, correlations were weaker during T1 (R$^2$ = 0.678) than during T2 (R$^2$ = 0.922) (Fig. 3).

During T1 biogenic opal fluxes presented two peaks, one in autumn and a second one in late spring that coincided with TMF peaks. During T2, this coincidence did not occur and biogenic opal fluxes progressively increased from early spring to early summer (Fig. 2). Biogenic opal fluxes were significantly correlated with TMF in both T1 and T2 (p<0.01 and p<0.05, respectively) but correlations were weaker than other geochemical components (Fig.3). However, during T1 and T2, the highest proportions of biogenic opal took place during the transition between autumn and winter (Fig. 2).

No significant differences were found, between the two annually defined periods of 2011 and 2012, when comparing the magnitude of the TMF and the fluxes of the geochemical components (Table 1).

### 3.2 Macroscopic characterization

During the T1 fecal pellets volumes (Fig. 4g) presented four peaks (Fig. 5). The first and second peaks occurred during mid autumn 2010 and late winter 2011, respectively (Fig. 5). The
third and fourth peak occurred during late spring 2011 and mid summer 2011 and presented higher magnitudes than the earlier two (Fig. 5).

During T2 fecal pellet volumes were one order of magnitude larger than in T1. The highest fecal pellet volumes during T2 were registered during the autumn 2012 (Fig. 5).

<figure><img src="image_73.png" /><figcaption>**Fig. 2.** Time series of surface Chl-a concentration, total mass, and the major constituent fluxes. Gray bands indicate the period with highest surface Chl-a concentration. Connected crosses represent each constituent percentage and the black crosses represent the OC/N (mol) values. The different bar colours indicate the sampling season (fall = orange, blue = winter, green = spring, summer = red).</figcaption></figure>
<figure><img src="image_74.png" /><figcaption>Fig. 3. Linear correlations between fluxes of CaCO₃, the lithogenic fraction (Litho), OC, biogenic opal and total mass flux.</figcaption></figure>
<table><caption>**Table 1.** Total mass and geochemical component mean fluxes for the annually defined periods of 2011 and 2012 and PERMANOVA analysis. SD = Standard deviation, Litho. = lithogenic.</caption><thead><tr><th rowspan="2">Fluxes</th><th colspan="2">2011</th><th colspan="2">2012</th><th colspan="2">PERMANOVA</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Pseudo-F</th><th>p value</th></tr></thead><tbody><tr><td>Total Mass (mg m<sup>-2</sup> d<sup>-1</sup>)</td><td>1023.3</td><td>388</td><td>1018.4</td><td>453</td><td>0.001</td><td>0.971</td></tr><tr><td>CaCO<sub>3</sub> (mg m<sup>-2</sup> d<sup>-1</sup>)</td><td>585.6</td><td>216.6</td><td>596.2</td><td>251.6</td><td>0.022</td><td>0.833</td></tr><tr><td>Litho. (mg m<sup>-2</sup> d<sup>-1</sup>)</td><td>362</td><td>166</td><td>349.8</td><td>174</td><td>0.069</td><td>0.792</td></tr><tr><td>OC (mg m<sup>-2</sup> d<sup>-1</sup>)</td><td>579</td><td>225</td><td>665</td><td>316</td><td>0.17</td><td>0.687</td></tr><tr><td>Biogenic opal (mg m<sup>-2</sup> d<sup>-1</sup>)</td><td>404</td><td>144</td><td>513</td><td>270</td><td>0.022</td><td>0.883</td></tr></tbody></table>

During T1 *P. oceanica* detritus (Figs. 4e and f) volumes were comparatively low with no remarkable peak. During T2 several peaks were observed during winter, one occurring in December 2011 and two occurring in February 2012 (Fig. 5).

During the T1 both pelagic (Figs. 4a and 4b) and benthic foraminifera (Figs. 4c and 4d) fluxes were low from early autumn until late winter (Fig. 5). In early spring their values increased and remained more or less stable until early autumn (Fig. 5). During T2 both pelagic and benthic foraminifera fluxes remained low except for two isolated peaks during winter (Fig. 5).

Fecal pellets and *P. oceanica* detritus volumes were significantly larger during the annual period of 2012 (Table 2). Conversely, the fluxes of pelagic foraminifera were significantly larger during the annual period of 2011 (Table 2). No significant differences were found among benthic foraminifera fluxes.

None of the macroscopic components fluxes were significantly correlated with TMF (Fig. 6). Microplastic fibres were found in all sampling

cups for both T1 and T2 (Fig. 4h); however, due to their irregular shape and small dimensions they were not quantified.

### 3.3 Environmental factors

#### 3.3.1 Water currents

During both sampling periods current mainly followed a north-south direction (Fig. 7). Average current velocity during T1 was 3.5 cm s⁻¹ and 4.4 cm s⁻¹ during T2, and current velocity rarely exceeded 20 cm s⁻¹. During T1, turbidity values were very low (<0.27 NTU); however, after mid spring the values substantially increased and remained above 0.28 NTU for most of the remaining sampling period. Simultaneously to this turbidity increment a substantial reduction in dissolved oxygen was observed. In T2 turbidity was comparatively very low for the whole sampling period with two peaks, one in mid-summer and a second one in early winter (Fig. 7).

#### 3.3.2 Wind and precipitation

During T1 the highest precipitation rates were registered during mid-September 2010 to mid-November 2010 ranging from 818 ml/m³ to
<figure><img src="image_75.png" /><figcaption>**Fig. 4.** Scanning electron microscope images of pelagic foraminifera of the genus a), b) *Globigerina*; benthic foraminifera of the genus c) *Cibicides*, and d) *Bulimina*, e), f) *P. oceanica* detritus, g) fecal pellet and h) microplastic fiber.</figcaption></figure>
<table><caption>**Table 2.** Mean fluxes of the macroscopic components for the annually defined periods of 2011 and 2012 and PERMANOVA analysis. SD = Standard deviation, detr. = detritus.</caption><thead><tr><th rowspan="2">Fluxes</th><th colspan="2">2011</th><th colspan="2">2012</th><th colspan="2">PERMANOVA</th></tr><tr><th>Mean</th><th>SD</th><th>Mean</th><th>SD</th><th>Pseudo-F</th><th>p value</th></tr></thead><tbody><tr><td>Fecal Pellets (µm³ m⁻² d⁻¹)</td><td>10.1 10⁵</td><td>486 10⁶</td><td>57.4 10⁶</td><td>8.4 10⁶</td><td>6.85</td><td>0.002</td></tr><tr><td>P. oceanica detr. (µm³ m⁻² d⁻¹)</td><td>10.7 10⁵</td><td>6.5 10⁵</td><td>27.4 10⁵</td><td>27.7 10⁵</td><td>7.54</td><td>0.002</td></tr><tr><td>Pel. Foram. (ind. m⁻² d⁻¹)</td><td>1615</td><td>1620</td><td>697.7</td><td>148.3</td><td>5.95</td><td>0.016</td></tr><tr><td>Bent Foram. (ind. m⁻² d⁻¹)</td><td>729</td><td>634</td><td>938</td><td>1169</td><td>0.543</td><td>0.524</td></tr></tbody></table>

2430 ml/m³. During T2 the highest precipitation rates were registered during November 2011 and 2012 ranging between 912 ml/m³ to 3112 ml/m³ and in February 2012 reaching precipitation values of 1090 ml/m³. Northerly winds were dominant during both sampling periods representing 47.9% and 45.9% of total hourly wind registers for T1 and T2, respectively. Windiest episodes were registered during January and February for both T1 and T2. During this period the percentage of the hourly winds exceeding 30 km/h ranged between 10% and 14% in T1 and between 7% and 46% in T2 and presented a northerly component.

### 3.3.3 Wave height

Wave measuring between 1.0 – 1.5 m were the most abundant size class and represented 36% of total wave registers for T1 and T2. Several wave height events ranging between 3.5 – 5 m were registered. Most events occurred during winter months and were associated to high wind speeds (> 40 km h⁻¹) (Table 3), few coincided with heavy precipitation (Table 3). Most registered waves presented a NNE orientation.

### 3.3.4 Chlorophyll-a concentration and sea surface salinity

Chl-a superficial concentration progressively increased from mid-October to early February when it reached its maximum values of ~0.7 mg m⁻³ and ~0.4 mg m⁻³ for T1 and T2, respectively (Figs. 2 and 4). For both T1 and T2, the lowest Chl-a concentrations ranging between ~0.2 mg m⁻³ to ~0.1 mg m⁻³, were registered from early April to early October (Figs. 2, 5, 8 and 9).

Sea surface salinity maps revealed that from September 2010 until February 2011 resident AW occupied the southern slope of Balearic archipelago (Fig. 10). However, from March 2011 until August 2011 fresher recent AW progressively moved northward from the Al-gerian sub-basin and reached the southern slope of the archipelago (Fig. 10). Moreover, an eddy was present in the southern slope of Menorca from June to August 2011 remaining in this area for approximately 75 days (Fig. 10).

During the T2, sea surface salinity was comparatively stable and no major intrusions of
fresher recent AW were observed in the south-
ern slope of the archipelago (Fig. 11).

4. DISCUSSION

4.1 Particle flux into the Mediterranean
context

TMF in the Menorca canyon were smaller than
those registered elsewhere in western Mediter-
ranean submarine canyons (Table 4). The ex-

ceptions were the TMF registered in canyons
and slopes in the Cretan margin (Table 4) and
some open sea environments (Zúñiga et al.,
2007; Khripounoff et al., 2012). The TMF range
in the Menorca Canyon was comparable to
those observed in the deep continental margin
of the Alboran Sea (Sanchez-Vidal et al., 2005)
and the open slope of the southern Adriatic
Sea (Miserocchi et al., 1999). However, TMF in
the Menorca canyon exceeded those collected
in the southern and northern Balearic slope
(Danovaro et al., 1999; Pasqual et al., 2015),
which suggest that the canyon acts as a conduit
of sediment from the continental shelf to
deeper environments agreeing with the general

<figure><img src="image_76.png" /><figcaption>**Fig. 5.** Time series of fecal pellet, *P. oceanica* detritus and pelagic and benthic foraminifera fluxes. Gray bands indicate the period with highest surface Chl-a concentration. *detr* = detritus, *Pel. Foram.* = pelagic foraminifera, *Ben. Foram.* = benthic foraminifera. The different bar colours indicate the sampling season (fall = orange, blue = winter, green = spring, summer = red).</figcaption></figure>
<figure><img src="image_77.png" /><figcaption>**Fig. 6.** Linear correlations between fluxes of fecal pellets, *P. oceanica* detritus, pelagic and benthic foraminifera and total mass flux. *detritus* = detritus, *Pel. Foram.* = pelagic foraminifera, *Ben. Foram.* = benthic</figcaption></figure>
<table><caption>**Table 3.** Large wave height events during both sampling intervals. Max WH = maximum wave height. Gray rows indicate those events that coincided with particle flux peaks. Max WS = maximum wind speed.</caption><thead><tr><th>Date</th><th>Max WH (m)</th><th>Max WS (km/h)</th><th>Precipitation (ml/m³)</th></tr></thead><tbody><tr><td>10/18/10</td><td>4.07</td><td>44</td><td>0</td></tr><tr><td>10/25/10</td><td>4.25</td><td>50</td><td>41</td></tr><tr><td>11/02/10</td><td>4.08</td><td>46</td><td>46</td></tr><tr><td>12/15/10</td><td>5</td><td>53</td><td>0</td></tr><tr><td>12/26/10</td><td>3.8</td><td>53</td><td>18</td></tr><tr><td>01/21/11</td><td>3.48</td><td>36</td><td>0</td></tr><tr><td>02/02/11</td><td>3.78</td><td>51</td><td>0</td></tr><tr><td>03/01/11</td><td>4.02</td><td>47</td><td>194</td></tr><tr><td>08/03/11</td><td>3.98</td><td>36</td><td>0</td></tr><tr><td>08/10/11</td><td>4.57</td><td>57</td><td>0</td></tr><tr><td>12/19/11</td><td>4.13</td><td>28</td><td>0</td></tr><tr><td>12/24/11</td><td>4.17</td><td>51</td><td>0</td></tr><tr><td>12/30/11</td><td>4.23</td><td>41</td><td>0</td></tr><tr><td>01/06/12</td><td>4.69</td><td>60</td><td>0</td></tr><tr><td>02/03/12</td><td>4.88</td><td>58</td><td>62</td></tr><tr><td>02/07/12</td><td>4.86</td><td>49</td><td>0</td></tr><tr><td>03/05/12</td><td>3.57</td><td>61</td><td>2</td></tr><tr><td>04/16/12</td><td>4.31</td><td>40</td><td>314</td></tr><tr><td>09/14/12</td><td>3.74</td><td>55</td><td>0</td></tr><tr><td>10/27/10</td><td>4.70</td><td>59</td><td>611</td></tr></tbody></table>

pattern observed in Mediterranean canyons (e.g. Monaco et al., 1990; Martín et al., 2006).

The temporal evolution of the TMF within the Menorca Canyon showed a marked seasonal variability. During both sampling periods there were particle flux peaks during spring, summer and fall (Fig. 2) and their individual contribution to the TMF in T1 and T2 ranged between 4% and 6%. Winter fluxes were comparatively low (Fig. 2). The seasonal pattern and low contributions of the winter peaks contrast with the seasonal trends reported in submarine canyons related to fluvial systems and affected by intense across slope current events (Table 4). In these kind of canyons, enhanced particle

fluxes mostly occur during fall and winter (e.g. Martín et al., 2006; Lopez-Fernandez et al., 2013b), can account for very large proportions (40% – 60%) of the TMF input (Durrieu de Madron et al., 1999; Turchetto et al., 2007) and mainly derive from brief and intense forcing mechanisms (e.g., river floods, cascading, storms) that trigger sediment resuspension and down canyon particle mobilization (Pasqual et al., 2010; Khripounoff et al., 2012). Overall, these differences suggest that the seasonal meteorological attributes of the Mediterranean basin (e.g., winter precipitation and storms) have little effect in the TMF collected at the southern slope of Menorca Island, accentuated by the absence of large fluvial systems.
## 4.2 Particle flux triggering factors

In the Menorca Canyon only few particle flux peaks coincided with atmospheric or hydrodynamic events that could act as forcing mechanisms to resuspend and trigger the transport

particulate material. The peaks reported during mid autumn 2010 (October 2010) and mid spring 2012 (April 2012) (Fig. 2) coincided with large wave high events ($Hs$ = > 4 m) (Table 3) that could potentially cause inner shelf bottom sediment resuspension (Guillén et al., 2006; Ulses et al., 2008). These peaks were charac-

<figure><img src="image_78.png" /></figure>

**Fig. 7.** Times series plots of turbidity, dissolved oxygen and current direction and velocity during T1 and T2 sampling intervals.
terized by a decrease in the CaCO₃ fraction and a slight increment in turbidity, in OC/N ratio (6.5 – 8) and the lithogenic fraction (Figs. 2 and 7), which provides further support to this hypothesis (Martín et al., 2006; Zúñiga et al., 2009).

From mid June to mid August 2011 an anticyclonic eddy was detected over the southern slope of Menorca (Fig. 9). Although it is unclear

whether the effect of the eddy reached the mooring site (it was not clearly detected in the currentmeter), and during this period a peak in TMF was collected (Fig. 2). This peak coincided with a slight increment in turbidity (Fig. 7), the lithogenic fraction and a punctual decrease in the CaCO₃ (Fig. 2). During this peak OC/N ratio presented intermediate values (7.5). This association would agree with previous observations in the Balearic Archipelago, that reported similar

<figure><img src="image_79.png" /></figure>

**Fig. 8.** Horizontal distribution of monthly averaged Chl-a surface concentration during T1.
TMF patterns in relation to anticyclonic eddies (Amores et al., 2013; Pasqual et al., 2014).

TMF peaks occurring during late summer 2011 (September 2011), and late summer and mid autumn 2012 (Fig. 2, August and October 2012) were associated with slight increments in the lithogenic fraction and turbidity, indicating a possible association with resuspension processes (Martín et al., 2006). However, the forcing mechanism that triggered these processes remains unclear given that no strong meteorological or hydrodynamic event was observed (Figs. 7, 10, 11 and Table 3).

In other areas of the Mediterranean, TMF peaks that did not present any evident relationship with natural forcing mechanisms were found to derive from trawling activities (Martín et al., 2006; Palanques et al., 2006). Indeed, trawling has been identified as a forcing mechanism that can cause abrupt increments in turbidity and particle fluxes (Martín et al., 2006; Palanques et al., 2006). However, it is unlikely that the observed particle flux peaks derive from this fishing activity given that in the study area, trawling is mostly conducted below 500 m (Moranta et al., 2014).

<figure><img src="image_80.png" /><figcaption>Fig. 9. Horizontal distribution of monthly averaged Chl-a surface concentration during T2.</figcaption></figure>
<figure><img src="image_81.png" /></figure>

Fig. 10. Sea surface salinity maps of the 15th day of every month during T1. Salinity values were extracted at 1.47 m depth.
Spring and summer TMF peaks have been as-
sociated to primary production blooms (Sanchez-
Vidal et al., 2005; Stavrakakis et al., 2013).
These TMF peaks are characterized by material
with relative high organic carbon and opal con-
centrations (Sanchez-Vidal et al., 2005) derived

from exports from the euphotic zone (Rigual-
Hernandez et al., 2013). In our study only the
peak in March 2012 coincided with the winter-
spring bloom and presented high organic carbon
but low opal percentages (Fig. 2), suggesting
that non-siliceous phytoplankton dominated

<figure><img src="image_82.png" /><figcaption>Fig. 11. Sea surface salinity maps of the 15th day of every month during T2. Salinity values were extracted at 1.47 m depth.</figcaption></figure>
the bloom. The high OC/N ratio (9.5 molar OC/N) registered during this peak (Fig. 2) suggests that the presence of resuspended sediments, with remineralized organic matter diluted the bloom signal (Martín et al., 2011). This peak appears to be related to both, resuspension processes and primary production and suggests that the fraction of resuspended material in the TMF is larger than the input of fresh organic matter from the euphotic zone as probably happened in the earlier spring and summer (2011) samples.

## 4.3 Geochemical components

The high CaCO₃ fraction (58±4% and 59±2% for T1 and T2, respectively) observed in the Menorca canyon's TMF (Fig. 2) contrasts with most studies performed in Mediterranean canyons, slopes and open sea environments, where the lithogenic material was the highest fraction comprising more than 50% of the total (Table 4, Stavrakakis et al., 2013; Fabrés et al., 2002). Conversely, in the Menorca canyon the lithogenic material barely represents 35% of all collected material. These unusual patterns in the CaCO₃ and lithogenic fractions may derive from the synergy of low continental inputs (Estrada et al., 1996) and the high benthic carbonate productivity occurring in the adjacent continental shelf, derived from thriving communities of calcareous algae (Canals and Ballesteros, 1997). The relatively high CaCO₃ fraction values resembled the surface sediment composition of the adjacent slope (Alonso et al., 1988) suggesting that most of the CaCO₃ was resuspended and transported along the canyon, supporting previous hypothesis on particle transport in the area (Canals and Ballesteros, 1997). This observation implies that resuspended material might be the most important source of lithogenic matter in this area, as expected for a setting without substantial river inputs.

Moreover, during mid January 2011 there was a sharp increment in the CaCO₃ concentration

(70%) coinciding with the Chl-a concentration maximum (Fig. 2). It has been observed that coccolithophore blooms may trigger rapid increments in CaCO₃ and OC (Malinverno et al., 2009). Although coccolithophores have been identified as one of the most representative groups during early stages of the phytoplankton bloom in the Balearic Sea (Valencia-Vila et al., 2015), the observed CaCO₃ peak was not associated with an increment in the OC fraction and the OC/N ratio (8) (Fig. 2), and was rather high to be associated with a phytoplankton bloom (Middelburg and Nieuwenhuize, 1998). Thus, with the present information it cannot be distinguished the potential contribution of lithogenic CaCO₃ from that of phytoplanktonic origin.

The OC and opal were the two constituents with the highest seasonal fluctuation (Fig. 2). Both components presented similar percentages to those observed in other Mediterranean submarine canyons (Table 4; Bonnin et al., 2008; Pasqual et al., 2013) but were lower than values from oligotrophic continental slope environments where pelagic inputs are the main source of particulate matter (Pasqual et al., 2014). The Menorca canyon is also located in an oligotrophic environment. However, the pelagic inputs are probably diluted by continental shelf and slope inputs with lower OC and opal contents due to the funnel effect of the canyon's shape in concert with the general trend in Mediterranean canyons (Table 4).

Highest organic carbon percentages were registered from early autumn to early winter during T1 and from early autumn to early spring during T2. During these periods the CaCO₃ and lithogenic fractions remained constant except for an abrupt increment in the CaCO₃ fraction and a simultaneous decrease in the lithogenic fraction during January 2011 (Fig. 2). The OC/N ratio widely fluctuated, ranging from 5 to 9.5 (Fig. 2). The highest OC/N ratios (8 – 9.5) were registered in periods were large wave height ($Hs$ = > 4 m) events associated to strong
<table><caption>**Table 4.** Register of TMF and geochemical constituents in Mediterranean submarine canyons and slopes. Med. = Mediterranean, S Adr. = South Adriatic, Lig. = Ligurian, GL = Gulf of Lions, Cat. Mar. = Catalan Margin, Bal. Arc. = Balearic Archipelago, Alb. = Alboran Sea, Forc. Mech. = forcing mechanism, am = annual mean, twf = time weighted flux, CCC= Cap de Creus canyon, LDC = Lacaze-Duthiers canyon, PP = primary production, R = resuspension, NADW = North Adriatic Dense Sea Water, RF = river flood, S = storm, SD = Saharan dust, HS = hydrodynamic setting, ~ = approximate values.</caption><thead><tr><th rowspan="2">Med. Area</th><th rowspan="2">Sub-Area</th><th colspan="3">Depth (m)</th><th rowspan="2">Duration (days)</th><th colspan="3">TMF (mg m<sup>-2</sup> d<sup>-1</sup>)</th><th colspan="4">Maximum</th><th rowspan="2">Forc. Mech.</th><th rowspan="2">Reference</th></tr><tr><th>Sea bottom</th><th>Current meter</th><th>Trap</th><th>Mean</th><th>Max.</th><th>Max. Season</th><th>Lit.%</th><th>CaCO<sub>3</sub>%</th><th>OC%</th><th>Opal%</th></tr></thead><tbody><tr><td>Crete</td><td>Samaria Canyon</td><td>1246</td><td>-</td><td>1231</td><td>350</td><td>0.332</td><td>~1.2</td><td>March</td><td>-</td><td>-</td><td>-</td><td>-</td><td>PP</td><td>Malinverno et al., 2009</td></tr><tr><td>Crete</td><td>Samaria Canyon</td><td>1316</td><td>-</td><td>1301</td><td>350</td><td>0.056</td><td>~0.1</td><td>September</td><td>-</td><td>-</td><td>-</td><td>-</td><td>PP</td><td>Malinverno et al., 2009</td></tr><tr><td>Crete</td><td>Samaria Canyon</td><td>1982</td><td>-</td><td>1967</td><td>350</td><td>0.077</td><td>0.225</td><td>July</td><td>-</td><td>-</td><td>-</td><td>-</td><td>PP</td><td>Malinverno et al., 2009</td></tr><tr><td>Crete</td><td>Samaria Canyon</td><td>3568</td><td>-</td><td>3553</td><td>350</td><td>0.060</td><td>0.11</td><td>March</td><td>-</td><td>-</td><td>-</td><td>-</td><td>PP</td><td>Malinverno et al., 2009</td></tr><tr><td>Crete</td><td>N Cretan Margin</td><td>1150</td><td>1135</td><td>1515</td><td>363</td><td>0.209</td><td>0.459</td><td>March</td><td>68.3</td><td>52.7</td><td>3.57</td><td>16.1</td><td>R / PP</td><td>Stavrakakis et al., 2000</td></tr><tr><td>S Adr.</td><td>Bari Slope</td><td>642</td><td>570</td><td>607</td><td>~365</td><td>1.7 am</td><td>6.8</td><td>March – April – May</td><td>-</td><td>-</td><td>~2</td><td>-</td><td>NADW</td><td>Turchetto et al., 2007</td></tr><tr><td>S Adr.</td><td>N Bari Canyon</td><td>600</td><td>570</td><td>565</td><td>~365</td><td>3.1</td><td>15.6</td><td>March – April – May</td><td>-</td><td>-</td><td>~2</td><td>-</td><td>NADW</td><td>Turchetto et al., 2007</td></tr><tr><td>S Adr.</td><td>S Bari Canyon</td><td>595</td><td>570</td><td>560</td><td>&lt;150</td><td>8.1</td><td>24.6</td><td>March – April – May</td><td>-</td><td>-</td><td>~1.4</td><td>-</td><td>NADW</td><td>Turchetto et al., 2007</td></tr><tr><td>Lig. Sea</td><td>Var Canyon</td><td>1280</td><td>1245</td><td>1235</td><td>121</td><td>-</td><td>120</td><td>December</td><td>-</td><td>-</td><td>2</td><td>-</td><td>RF</td><td>Khripounoff et al., 2012</td></tr><tr><td>Lig. Sea</td><td>Var Canyon</td><td>1575</td><td>1545</td><td>1555</td><td>121</td><td>-</td><td>218</td><td>February</td><td>-</td><td>-</td><td>1.5</td><td>-</td><td>RF</td><td>Khripounoff et al., 2012</td></tr></tbody></table>
<table><caption>**Table 4 (cont.)**. Register of TMF and geochemical constituents in Mediterranean submarine canyons and slopes. Med. = Mediterranean, S Adr. = South Adriatic, Lig. = Ligurian, GL = Gulf of Lions, Cat. Mar. = Catalan Margin, Bal. Arc. = Balearic Archipelago, Alb. = Alboran Sea, Forc. Mech. = forcing mechanism, am = annual mean, twf = time-weighted flux, CCC= Cap de Creus canyon, LDC = Lacaze-Duthiers canyon, PP = primary production, R = resuspension, NADW = North Adriatic Dense Sea Water, RF = river flood, S = storm, SD = Saharan dust, HS = hydrodynamic setting, ~ = approximate values.</caption><thead><tr><th rowspan="2">Med. Area</th><th rowspan="2">Sub-Area</th><th colspan="3">Depth (m)</th><th rowspan="2">Duration (days)</th><th colspan="3">TMF (mg m<sup>-2</sup> d<sup>-1</sup>)</th><th colspan="4">Maximum</th><th rowspan="2">Forc. Mech.</th><th rowspan="2">Refrence</th></tr><tr><th>Sea bottom</th><th>Current meter</th><th>Trap</th><th>Mean</th><th>Max.</th><th>Max. Season</th><th>Lit.%</th><th>CaCO<sub>3</sub>%</th><th>OC%</th><th>Opal%</th></tr></thead><tbody><tr><td>GL</td><td>CCC</td><td>302</td><td>297</td><td>84</td><td>84</td><td>7.5</td><td>~30</td><td>Spring</td><td>64.8</td><td>29.4</td><td>1.8</td><td>2.2</td><td>S-DSWC</td><td>Bonnin et al., 2008</td></tr><tr><td>GL</td><td>LCD</td><td>309</td><td>304</td><td>140</td><td>140</td><td>11.7</td><td>~110</td><td>Autumn</td><td>66.1</td><td>31.0</td><td>1.9</td><td>3.2</td><td>S-DSWC</td><td>Bonnin et al., 2008</td></tr><tr><td>GL</td><td>Aude Canyon</td><td>309</td><td>304</td><td>84</td><td>84</td><td>7.8</td><td>~25</td><td>Autumn</td><td>65.3</td><td>31.4</td><td>1.3</td><td>1.3</td><td>S-DSWC</td><td>Bonnin et al., 2008</td></tr><tr><td>GL</td><td>Hérault Canyon</td><td>302</td><td>297</td><td>168</td><td>168</td><td>9.6</td><td>~75</td><td>Autumn</td><td>64.6</td><td>30.4</td><td>1.6</td><td>0.3</td><td>S-DSWC</td><td>Bonnin et al., 2008</td></tr><tr><td>GL</td><td>Petit Rhône Canyon</td><td>302</td><td>297</td><td>168</td><td>168</td><td>8.6</td><td>~30</td><td>Autumn</td><td>63.8</td><td>33.1</td><td>1.9</td><td>2.8</td><td>S-DSWC</td><td>Bonnin et al., 2008</td></tr><tr><td>GL</td><td>Planiere Canyon</td><td>307</td><td>302</td><td>84</td><td>84</td><td>3.1</td><td>3.1</td><td>Autumn</td><td>45.1</td><td>35.1</td><td>2.5</td><td>0.9</td><td>S-DSWC</td><td>Bonnin et al., 2008</td></tr><tr><td>GL</td><td>LDC</td><td>645</td><td>620</td><td>600</td><td>304</td><td>6.02</td><td>20</td><td>February</td><td>68</td><td>30</td><td>7</td><td>15</td><td>S</td><td>Courp and Monaco, 1990</td></tr><tr><td>GL</td><td>LDC</td><td>300</td><td>295</td><td>270</td><td>360</td><td>6.7</td><td>18.1</td><td>November</td><td>71.6</td><td>36.6</td><td>4.1</td><td>3.7</td><td>DSWC</td><td>Pasqual et al., 2010</td></tr><tr><td>GL</td><td>LDC</td><td>1000</td><td>995</td><td>970</td><td>300</td><td>6.7</td><td>11.2</td><td>January</td><td>68.6</td><td>42.9</td><td>5.5</td><td>5.2</td><td>DSWC</td><td>Pasqual et al., 2010</td></tr><tr><td>GL</td><td>LDC</td><td>1500</td><td>1495</td><td>1470</td><td>345</td><td>1.4</td><td>11.9</td><td>March</td><td>65.8</td><td>34.9</td><td>5.4</td><td>10.8</td><td>DSWC</td><td>Pasqual et al., 2010</td></tr><tr><td>GL</td><td>CCC</td><td>300</td><td>295</td><td>270</td><td>150</td><td>7.0</td><td>20</td><td>October</td><td>67.6</td><td>29</td><td>3.2</td><td>3.5</td><td>DSWC</td><td>Pasqual et al., 2010</td></tr><tr><td>GL</td><td>CCC</td><td>1000</td><td>995</td><td>970</td><td>360</td><td>11.7</td><td>90.1</td><td>January</td><td>73.1</td><td>29.9</td><td>3.4</td><td>9</td><td>DSWC</td><td>Pasqual et al., 2010</td></tr><tr><td>GL</td><td>CCC</td><td>1500</td><td>1495</td><td>1470</td><td>345</td><td>1.3</td><td>6.3</td><td>January</td><td>68.6</td><td>36.3</td><td>9.5</td><td>13.1</td><td>DSWC</td><td>Pasqual et al., 2010</td></tr></tbody></table>
<table><caption>Table 4 (cont.). Register of TMF and geochemical constituents in Mediterranean submarine canyons and slopes. Med. = Mediterranean, S Adr. = South Adriatic, Lig. = Ligurian, GL = Gulf of Lions, Cat. Mar. = Catalan Margin, Bal. Arc. = Balearic Archipelago, Alb. = Alboran Sea, Forc. Mech. = forcing mechanism, am = annual mean, twf = time-weighted flux, CCC= Cap de Creus canyon, LDC = Lacaze-Duthiers canyon, PP = primary production, R = resuspension, NADW = North Adriatic Dense Sea Water, RF = river flood, S = storm, SD = Saharan dust, HS = hydrodynamic setting, ~ = approximate values.</caption><thead><tr><th rowspan="2">Med. Area</th><th rowspan="2">Sub-Area</th><th colspan="3">Depth (m)</th><th rowspan="2">Duration (days)</th><th colspan="3">TMF (mg m<sup>-2</sup> d<sup>-1</sup>)</th><th colspan="4">Maximum</th><th rowspan="2">Forc. Mech.</th><th rowspan="2">Reference</th></tr><tr><th>Sea bottom</th><th>Current meter</th><th>Trap</th><th>Mean</th><th>Max.</th><th>Max. Season</th><th>Lit.%</th><th>CaCO<sub>3</sub> %</th><th>OC %</th><th>Opal %</th></tr></thead><tbody><tr><td>Cat. Mar.</td><td>Blanes Canyon</td><td>300</td><td>275</td><td>275</td><td>181</td><td>12.06</td><td>82.67</td><td>December</td><td>78.27</td><td>43</td><td>7.68</td><td>4.19</td><td>S-RF</td><td>Lopez-Fernandez et al., 2013b</td></tr><tr><td>Cat. Mar.</td><td>Blanes Canyon</td><td>900</td><td>875</td><td>875</td><td>359</td><td>14.75</td><td>35.29</td><td>December</td><td>75.63</td><td>30.1</td><td>4.44</td><td>2.17</td><td>S-RF</td><td>Lopez-Fernandez et al., 2013b</td></tr><tr><td>Cat. Mar.</td><td>Slope</td><td>900</td><td>875</td><td>875</td><td>359</td><td>3.43</td><td>7.81</td><td>April</td><td>74.01</td><td>26.2</td><td>10.3</td><td>3.39</td><td>SD</td><td>Lopez-Fernandez et al., 2013b</td></tr><tr><td>Cat. Mar.</td><td>Blanes Canyon</td><td>600</td><td>568</td><td>570</td><td>319</td><td>14 twf</td><td>40.78</td><td>December</td><td>-</td><td>24</td><td>5.5</td><td>-</td><td>RF</td><td>Zuñiga et al., 2009</td></tr><tr><td>Cat. Mar.</td><td>Palamós Canyon</td><td>470</td><td>~446</td><td>448</td><td>~274</td><td>28.75</td><td>94</td><td>November</td><td>77</td><td>33</td><td>3.5</td><td>12.5</td><td>S</td><td>Martin et al., 2006</td></tr><tr><td>Cat. Mar.</td><td>Foix Canyon</td><td>600</td><td>-</td><td>570</td><td>~91</td><td>3.8 am</td><td>19</td><td>July</td><td>-</td><td>27.5</td><td>2</td><td>4</td><td>S-RF</td><td>Puig and Palanques, 1998</td></tr><tr><td>Cat. Mar.</td><td>Foix Canyon</td><td>1180</td><td>-</td><td>1150</td><td>~516</td><td>12 am</td><td>7.5</td><td>October</td><td>-</td><td>28</td><td>-2.5</td><td>5</td><td>S-RF</td><td>Puig and Palanques, 1998</td></tr><tr><td>Cat. Mar.</td><td>Slope</td><td>980</td><td>-</td><td>950</td><td>~516</td><td>0.8 am</td><td>1.8</td><td>April</td><td>-</td><td>30</td><td>5.9</td><td>8.75</td><td>S-RF</td><td>Puig and Palanques, 1998</td></tr><tr><td>Bal. Arc.</td><td>Menorca Canyon T1</td><td>430</td><td>405</td><td>400</td><td>412</td><td>1</td><td>2.06</td><td>July</td><td>45.7</td><td>70</td><td>3.5</td><td>3</td><td>R-HS</td><td>Present Study</td></tr><tr><td>Bal. Arc.</td><td>Menorca Canyon T2</td><td>430</td><td>405</td><td>400</td><td>365</td><td>1.09</td><td>2.30</td><td>October</td><td>40.7</td><td>63</td><td>3.2</td><td>4.3</td><td>R-HS</td><td>Present Study</td></tr><tr><td>Bal. Arc.</td><td>N slope Mallorca</td><td>900</td><td>870</td><td>870</td><td>344</td><td>0.31</td><td>0.61</td><td>March</td><td>65.2</td><td>43</td><td>5.8</td><td>17.1</td><td>S-RF</td><td>Pasqual et al., 2015</td></tr><tr><td>Bal. Arc.</td><td>S slope Mallorca</td><td>900</td><td>870</td><td>870</td><td>225</td><td>0.16</td><td>0.337</td><td>February</td><td>66.4</td><td>47.3</td><td>7.1</td><td>9.1</td><td>PP</td><td>Pasqual et al., 2015</td></tr><tr><td>Alb.</td><td>Guadiaro Canyon</td><td>592</td><td>567</td><td>567</td><td>155</td><td>6.1</td><td>27.4</td><td>May</td><td>~80</td><td>~14</td><td>~2.3</td><td>~8</td><td>RF</td><td>Palanques et al., 2005</td></tr><tr><td>Alb.</td><td>Channel</td><td>717</td><td>692</td><td>692</td><td>346</td><td>1.87</td><td>5.7</td><td>December - January</td><td>~82</td><td>~15</td><td>>5</td><td>~12</td><td>RF</td><td>Palanques et al., 2005</td></tr><tr><td>Alb.</td><td>Oppen Slope</td><td>720</td><td>695</td><td>695</td><td>191</td><td>3.03</td><td>6</td><td>December</td><td>82</td><td>~15</td><td>3</td><td>~8</td><td>RF</td><td>Palanques et al., 2005</td></tr></tbody></table>
winds (> 40 km h⁻¹) occurred, suggesting a resuspended (remineralized) origin of the collected OC (Fabres et al., 2008) (Fig. 2 and Table 3). Conversely low OC/N ratios (5 – 7) may have resulted from biogenic exports from the photic zone (Middelburg and Nieuwenhuize, 1998). In this regard, strong winds and large wave, registered during these periods, can induce turbulent mixing, increasing nutrient supply in shallow environments that can result in a posterior increment of phytoplankton production and biogenic exports (Marty et al., 2008; Isla et al., 2009). Thus we suggest that the wide fluctuation of the OC/N ratio, registered during these periods, may have resulted from the alternated supply of resuspended and pelagic OC.

During spring and summer of 2011 and 2012, we observed a decline in OC fraction (Fig. 2) that could be related to water stratification. Under these conditions nutrients undergo a rapid depletion in superficial layers limiting primary production (Marty et al., 2002) and reducing the pelagic particulate matter supply (Coma et al., 2000).

However, most spring and summer samples presented low OC/N ratios (5 – 6.5) (Fig. 2) indicating pelagic origin of OC and a low contribution of resuspended matter. This would agree with wind (14.6 ± 9.2 SD km h⁻¹ and 14.5 ± 9.2 SD km h⁻¹ T1 and T2 respectively) and wave height (Table 3) that were relatively low during these periods.

Highest opal percentages did not coincide with surface Chl-a maximum and were detected during spring and early summer (Fig. 2). This temporal pattern contrasts with previous studies in Mediterranean submarine canyons (Martín et al., 2006; Pasqual et al., 2010) and slopes (Sanchez-Vidal et al., 2005; Stavrakakais et al., 2013) that detected a simultaneous increment in OC and opal associated with the winter-spring phytoplankton bloom. In this sense, the low opal concentrations registered in the

present study during the winter surface Chl-a maximum (Fig. 2) may imply that siliceous phytoplankton was overshadowed by other groups. This would agree with Estrada et al. (1999) who observed that during the winter spring bloom phytoplankton biomass was not associated with diatom dominance in the Balearic Sea. The weak relationship between surface Chl-a concentrations and spring and early summer opal maximums may be explained by the development of a deep chlorophyll maximum (~60 m) closely related with the nutricline (Crombet et al., 2011). In other oligotrophic areas of the Mediterranean Sea it has been reported that under high stratified conditions, diatoms can dominate the deep chlorophyll maximum (Crombet et al., 2011). Nonetheless, the low values of OC during opal maximums (Fig. 2) could indicate that opal came from resuspended biological remains as it has been suggested in other areas of the Mediterranean (Lopez-Fernandez et al., 2013b). However, the low OC/N ratio (5 – 6.5), the absence of other sediment resuspension indicators (enhanced current velocity, enhanced turbidity, and increments in the lithogenic fraction), (Figs. 2 and 7) and the poor correlation of opal with TMF (Fig. 3) do not support this environmental approach. Consequently, the mechanisms involved in both opal maximums remain unclear.

Moreover, T1 maximum opal percentages, registered during early May to late June 2011, were 47% lower than T2 maximum opal percentages, registered during early May to late June 2012 (Fig. 2). This could be related with the characteristics of the water masses influencing the study area during both sampling periods. During spring and early summer 2011 the study area was influenced by recent AW (Fig. 10) considered highly oligotrophic (García et al., 2005). Conversely during 2012 spring and summer the study area was influenced by resident AW (Fig. 11). Resident AW has higher H₄SiO₄ than recent AW (Crombet et al., 2011). In this regard, we hypothesized that higher
opal fluxes observed during T2 could result
from higher H₄SiO₄ availability, which could
trigger the deep Chl-a peaks and sustain larger
siliceous phytoplankton populations.

4.4 Macroscopic components

Throughout both sampling intervals fecal pellet
fluxes (Fig. 4g) presented several peaks with
only one peak coinciding with the 2011 winter
spring bloom (Fig. 5). This temporal pattern
contrasts with zooplankton dynamics in the
Balearic archipelago (Cartes et al., 2008) and
long-term fecal pellet monitoring in other areas
of the western Mediterranean that showed a
clear relationship between the winter-spring
phytoplankton bloom and the fecal pellet max-
imums (Fowler et al., 1991). During T2, fecal
pellet fluxes were one order of magnitude
larger than during T1. This is surprising as
surface chlorophyll satellite observations re-
vealed that during T2 winter-spring bloom was
less intense and shorter than the one observed
during T1 (Figs. 8 and 9). However, it has been
observed that zooplankton abundances may
experience a wide variability under different
hydrographic settings (Guerrero et al., 2016).
In the Balearic Archipelago higher zooplankton
abundances have been observed under the
prevalence of resident AW (Fernández de
Puelles et al., 2007). This would agree with
our results as during 2012 resident AW strongly
influenced the southern slope of the archipelago
(Fig. 11). Moreover, fecal pellets may induce
rapid vertical transports enhancing biogenic
components fluxes (Monaco et al., 1990; Stone
and Steinberg, 2016). In the Menorca canyon
fecal pellet fluxes were not significantly corre-
lated with CaCO₃ (p=0.51, p=0.34 during 2011
and 2012 respectively), OC (p = 0.32, p = 0.34
during 2011 and 2012 respectively) and opal
fluxes (p = 0.06, p = 0.07 during 2011 and
2012, respectively) suggesting a low contribution
to biogenic fluxes. This could also reflect the
strong diluting influence of resuspended material
into the sediment trap.

The fluxes of *P. oceanica* fragments (Figs. 4e and 4f) presented high variability throughout both sampling periods and were poorly correlated with TMF (Figs. 5 and 6). During the T1 fluxes of *P. oceanica* detritus remained quite stable and could not be related to any environmental pattern. Conversely, during the T2, fluxes were more variable and there seemed to be a relationship between relatively high *P. oceanica* detritus fluxes and large wave high events (Fig. 5 and Table 3). Indeed, shelf resuspension processes have been identified, as a major source of macrophyt and sea grass detritus to deeper environments (Britton-Simmons et al., 2012). In North Pacific canyons macrophyt and seagrass detritus have shown to be a major carbon source, accounting for 20–83% of the particulate organic carbon (Harrold et al., 1998). *P. oceanica* detritus does not seem to widely contribute to the bulk OC flux as both variables were not significantly correlated (p = 0.33 and p = 0.35 during 2011 and 2012 respectively). Despite this lack of correlation, *P. oceanica* detritus cannot be ruled out as major carbon sink in deep Mediterranean environments due to its refractory nature (Danovaro et al., 1994) and high carbon contents (Romero et al., 1992). In this regard, peaks in *P. oceanica* detritus registered during mid December 2011 and early February 2012 coincided with high OC/N ratio (9 – 9.5) (Figs 5 and 2). However, it should be mentioned that very small, reworked fragments represented the *P. oceanica* detritus fraction and this material should behave as a particle lighter than CaCO₃ or lithogenic sediment of the same size. That implies a more complicated transport mechanisms related to the coincidence of *P. oceanica* fragments availability at the beach and shallow coastal areas and resuspension and downslope transport mechanisms. With the available information, explaining the presence of this fraction represents the strongest challenge in this study and it cannot be clarified.
Benthic and pelagic foraminifera presented similar trends during both sampling periods, with poor correlation with the TMF pattern (Figs. 5 and 6). From early spring to late summer 2011 there was a substantial increment in the fluxes of both foraminifera groups (including resuspended dead pelagic foraminifera). Coinciding with this increment, we observed a turbidity enhancement, suggesting that these peaks in foraminifera fluxes derive from resuspension processes (Figs. 5 and 7). Moreover, in the water column pelagic foraminifera reached its maximum concentrations during winter months (Pujol and Grazzini, 1995), which points to resuspension as the main responsible process to explain the spring and summer 2011 pelagic foraminifera peaks (Fig. 5). During the T2 foraminifera fluxes presented two peaks during mid December 2011 and early February 2012 coinciding with *P. oceanica* detritus fluxes. Pelagic foraminifera may produce high densities during winter months (Pujol and Grazzini, 1995) and probably the signal of these developments was recorded in the trap collection. Coinciding with both peaks several large wave height events (Table 3) were registered, which may indicate that the observed peaks during the second sampling periods could also derive from resuspension events, especially that of benthic foraminifera. Nonetheless these peaks were not associated with increments in turbidity nor in the lithogenic fraction (Figs. 2 and 7). Moreover, benthic (p = 0.11 and p = 0.17 during 2011 and 2012, respectively) and pelagic (p = 0.17 and p = 0.07 during 2011 and 2012, respectively) foraminifera fluxes were poorly correlated with $CaCO_3$ suggesting that the contribution of foraminifera to the $CaCO_3$ is diluted by other $CaCO_3$ sources. During both sampling periods microplastic fibres (Fig. 4h) were found in all samples. Future research should quantify the amount of carbon that these artificial polymers introduce into the carbon flux and the possible bias to the budget of naturally produced OC carbon.

## 4.5 TMF: general approach

The southern area of the Balearic archipelago has been described as a sediment-starved margin where no major sources of continental sediments are present (Lo Iacono et al., 2014). Consequently, lithogenic fluxes within the insular system of the Menorca canyon were rather poor within the Mediterranean context and were far exceeded by fluxes registered in canyons and slopes that receive sediment inputs from fluvial systems and where intense across slope current occur (Table 4). To our knowledge, the registered fluxes were the lowest ever registered in a western Mediterranean canyon head. These observations suggest that Mediterranean submarine canyons related to insular environments, without important fluvial networks, receive particle fluxes similar to those collected in deep-sea environments (Sanchez-Vidal et al., 2005). No clear relationship was found between environmental parameters and TMF temporal variation. Unlike other studies in the Mediterranean, where the relation between hydrodynamic, meteorological and biological processes and TMF peaks was more evident, we advocate that in the present study, the low magnitude of the TMF difficulties the identification of a main mechanism that explained its magnitude and temporal variation (Table 4). The fraction represented by biogenic material was smaller than the resuspended, degraded material. Nonetheless, our results suggest that physical mechanisms, (e.g., resuspension) were more important than biological processes in the control of the temporal variation and chemical composition of the TMF. TMF were poorly related with phytoplankton blooms in contrast to the patterns observed in other Mediterranean oligotrophic environments with similar surface Chl-a concentrations (Malinverno et al., 2009). Although TMF and geochemical components varied between both sampling periods (Fig. 2), the absence of significant differences between both periods (Table 1) indicates that overall,
the magnitude and composition of the TMF showed little interannual variation between 2011 and 2012 indicating that overall sediment sources remain constant throughout the year. Except for benthic foraminifera, macroscopic components significantly differed between 2011 and 2012 (Table 2) suggesting that the fluxes of the macroscopic particles may develop according to a different combination of sources and physical-biological mechanisms than the TMF and its main components.

The amount of OC collected in the Menorca Canyon (~3%) is similar to that of areas with higher TMF that receive continental inputs such as the Gulf of Lyons or the Catalan Margin (Table 4). This suggests that the dilution effect of the lithogenic fraction, in the Menorca Canyon, is less intense than in areas adjacent to continental landmasses. Submarine canyons have been identified as macrobenthic community hotspots of diversity and biomass (e.g. Fabri et al., 2014; De Leo et al., 2010). However, high sedimentation rates may generate instability (erosion, smothering) restricting the development of macrobenthic communities to areas with low sedimentation rates (Sousa, 2001). Thus, the synergy of the magnitude of the OC fluxes and low TMF measured in the Menorca Canyon presumably are sufficient to maintain the high biodiversity of the benthic communities in the Menorca Canyon and surrounding areas (Barberá et al., 2012, Grinyó et al., 2016). The results of the present work stimulate further research on the interesting relationship between benthos and particle fluxes in oligotrophic environments.

## ACKNOWLEDGEMENTS

The authors thank the crew of the R/V *García del Cid*, Javier Romero and Aurora M. Ricart

for their help in the identification of *P. oceanica* detritus, Claudio Lo Iacono for providing the study area figure and Silvia De Diago and Neus Maestro for their technical support. This work was funded by the European project LIFE+INDEMARES "Inventario y dsignación de la red Natura 2000 en áreas marinas del estado español" (LIFE07/NAT/E/000732).

## REFERENCES

* ACOSTA, J., CANALS, M., LÓPEZ-MARTÍNEZ, J., MUÑOZ, A., HERRANZ, P., URGELES, R., PALOMO, C., CASAMOR, J.L., 2002. The Balearic Promontory geomorphology (western Mediterranean): Morphostructure and active processes. Geomorphology 49, 177-204. doi:10.1016/S0169-555X(02)00168-X.

* AHUMADA-SEMPOAL, M.A., FLEXAS, M.M., BERNARDELLO, R., BAHAMON, N., CRUZADO, A., REYES-HERNÁNDEZ, C., 2015. Shelf-slope exchanges and particle dispersion in Blanes submarine canyon (NW Mediterranean Sea): A numerical study. Continental Shelf Research 109, 35-45. doi:10.1016/j.csr.2015.09.012.

* ALONSO, B., GUILLÉN, J., CANALS, M., SERRA, J., ACOSTA, J., HERRANZA, P., SANZ, J.L., CALAFAT, A., CATAFAU, E., 1988. Los sedimentos de la plataforma continental balear. Acta Geológica Hispánica 23, 185-196.

* AMORES, A., MONSERRAT, S., 2014. Hydrodynamic comparison between the north and south of Mallorca Island. Journal of Marine Systems 138, 40-50. doi:10.1016/j.jmarsys.2014.01.005.

* AMORES, A., MONSERRAT, S., MARCOS, M., 2013. Vertical structure and temporal evolution of an anticyclonic eddy in the Balearic Sea (western Mediterranean). Journal of Geophysical Research: Oceans 118, 2097-2106. doi:10.1002/jgrc.20150.
AMORES, A., RUEDA, L., MONSERRAT, S., GUIJARRO, B., PASQUAL, C., MASSUTÍ, E., 2014. Influence of the hydrodynamic conditions on the accessibility of *Aristeus antennatus* and other demersal species to the deep water trawl fishery off the Balearic Islands (western Mediterranean). Journal of Marine Systems 138, 203-210.
doi:10.1016/j.jmarsys.2013.11.014.

ANDERSON, M.J., 2001. A new method for non-parametric multivariate analysis of variance. Austral Ecology 26, 32-46.

BALBÍN, R., FLEXAS, M.M., LÓPEZ-JURADO, J.L., PEÑA, M., AMORES, A., ALEMANY, F., 2012. Vertical velocities and biological consequences at a front detected at the balearic sea. Continental Shelf Research 47, 28-41. doi:10.1016/j.csr.2012.06.008.

BALBÍN, R., LÓPEZ-JURADO, J.L., FLEXAS, M.M., REGLERO, P., VÉLEZ-VELCHÍ, P., GONZÁLEZ-POLA, C., RODRÍGUEZ, J.M., GARCÍA, A., ALEMANY, F., 2014. Interannual variability of the early summer circulation around the Balearic Islands: Driving factors and potential effects on the marine ecosystem. Journal of Marine Systems 138, 70-81.
doi:10.1016/j.jmarsys.2013.07.004.

BARBERÀ, C., MORANTA, J., ORDINES, F., RAMÓN, M., DE MESA, A., DIAZ-VALDÉS, M., GRAU, A.M., MASSUTÍ, E., 2012. Biodiversity and habitat mapping of Menorca Channel (western Mediterranean): Implications for conservation. Biodiversity and Conservation 21, 701-728. doi:10.1007/s10531-011-0210-1.

BONNIN, J., HEUSSNER, S., CALAFAT, A., FABRES, J., PALANQUES, A., DURRIEU DE MADRON, X., CANALS, M., PUIG, P., AVRIL, J., DELSAUT, N., 2008. Comparison of horizontal and downward particle fluxes across canyons of the Gulf of Lions (NW Mediterranean): Meteorological and hydrodynamical forcing. Continental Shelf Research 28, 1957-1970.
doi:10.1016/j.csr.2008.06.004.

BOSC, E., BRICAUD, A., ANTOINE, D., 2004. Seasonal and interannual variability in algal biomass and primary production in the Mediterranean Sea, as derived from 4 years of SeaWiFS observations. Global Biogeochemical Cycles 18, 1-17.
doi:10.1029/2003GB002034.

BRITTON-SIMMONS, K.H., RHOADES, A.L., PACUNSKI, R.E., GALLOWAY, A.W.E., LOWE, A.T., SOSIK, A.E., DETHIER, M.N., DUGGINS, D.O., 2012. Habitat and bathymetry influence the landscape-scale distribution and abundance of drift macrophytes and associated invertebrates. Limnology and Oceanography 57, 176-184. doi:10.4319/lo.2012.57.1.0176.

CANALS, M., BALLESTEROS, E., 1997. Production of carbonate particles by phytobenthic communities on the Mallorca-Menorca shelf, northwestern Mediterranean Sea. Deep Sea Research: Part II Topical Studies in Oceanography 44, 611-629.
doi:10.1016/S0967-0645(96)00095-1.

CARTES, J.E., MADURELL, T., FANELLI, E., LÓPEZ-JURADO, J.L., 2008. Dynamics of suprabenthos-zooplankton communities around the Balearic Islands (western Mediterranean): Influence of environmental variables and effects on the biological cycle of Aristeus antennatus. Journal of Marine Systems 71, 316-335. doi:10.1016/j.jmarsys.2006.11.013.

CHAMBERS, J.M., 1992. Linear models. Chapter 4. In: Chambers JM, Hastie TJ (eds) Statistical models. S. Wadsworth & Brooks, Cole, Pacific Grove, California.

COLOM, G., 1974. Foraminíferos ibéricos. Introducción al estudio de las espécies bentónicas recientes. Investigación Pesquera, Consejo Superior de Investigaciones Científicas, vol. 38. Patrono Juán de la Cierva, Barcelona.

COMA, R., RIBES, M., GILI, J.M., ZABALA, M., 2000. Seasonality in coastal benthic ecosystems. Trends in Ecology and Evolution 15, 448-453.
COURP, T., MONACO, A., 1990. Sediment and acculation on the continental margin of the Gulf of Lions: sedimentary budget. Continental Shelf Research 10, 1063-1087.

CROMBET, Y., LEBLANC, K., QUÉGUINER, B., MOUTIN, T., RIMMELIN, P., RAS, J., CLAUSTRE, H., LEBLOND, N., ORIOL, L., PUJO-PAY, M., 2011. Deep silicon maxima in the stratified oligotrophic Mediterranean Sea. Biogeosciences 8, 459-475. doi:10.5194/bg-8-459-2011.

DANOVARO, R., FABIANO, M., BOYER, M., 1994. Seasonal changes of benthic bacteria in a seagrass bed (*Posidonia oceanica*) of the Ligurian Sea in relation to origin, composition and fate of the sediment organic matter. Marine Biology 119, 489-500. doi:10.1007/BF00354310.

DANOVARO, R., DINET, A., DUINEVELD, G., TSELEPIDES, A., 1999. Benthic response to particulate fluxes in different trophic environments: a comparison between the Gulf of Lions-Catalan Sea (western-Mediterranean) and the Cretan Sea (eastern-Mediterranean). Progress in Oceanography 44, 287-312. doi:10.1016/S0079-6611(99)00030-0.

DANOVARO, R., DELLA CROCE, N., DELL'ANNO, A., FABIANO, M., MARRALE, D., MARTORANO, D., 2000. Seasonal changes and biochemical composition of the labile organic matter flux in the Cretan Sea. Progress in Oceanography 46, 259-278. doi:10.1016/S0079-6611(00)00022-7.

DE LEO, F.C., SMITH, C.R., ROWDEN, A.A., BOWDEN, D.A., CLARK, M.R., 2010. Submarine canyons: hotspots of benthic biomass and productivity in the deep sea. Proceedings of the Royal Society B 277, 2783-2792. doi:10.1098/rspb.2010.0462.

DEMASTER, D., 1981. The supply and accumulation of silica in the marine environment. Geochimica et Cosmochimica Acta 45, 1715-1732.

DURRIEU DE MADRON, X., RADAKOVITCH, O., HEUSSNER, S., LOYE-PILOT, M.D., MONACO, A., 1999. Role of

the climatological and current variability on shelf-slope exchanges of particulate matter: Evidence from the Rhone continental margin (NW Mediterranean). Deep Sea Research Part I: Oceanographic Research Papers 46, 1513-1538. doi:10.1016/S0967-0637(99)00015-1.

ESTRADA, M., 1996. Primary production in the north-western Mediterranean. Scientia Marina 60, 55-64.

ESTRADA, M., VARELA, R.A., SALAT, J., CRUZADO, A., 1999. Spatio-temporal variability of the winter phytoplankton distribution across the Catalan and North Balearic fronts (NW Mediterranean). Journal of Plankton Research 21, 1-20.

FABRÉS, J., CALAFAT, A., SANCHEZ-VIDAL, A., CANALS, M., HEUSSNER, S., 2002. Composition and spatial variability of particle fluxes in the Western Alborán Gyre, Mediterranean Sea. Journal of Marine Systems 33-34, 431-456.

FABRES, J., TESI, T., VELEZ, J., BATISTA, F., LEE, C., CALAFAT, A., HEUSSNER, S., PALANQUES, A., MISERCCHI, S., 2008. Seasonal and event-controlled export of organic matter from the shelf towards the Gulf of Lions continental slope. Continental Shelf Research 28, 1971-1983. doi:10.1016/j.csr.2008.04.010.

FABRI, M., PEDEL, L., BEUCK, L., GALGANI, F., HEBBELN, D., FREIWALD, A., 2014. Deep-Sea Research II Megafauna of vulnerable marine ecosystems in French Mediterranean submarine canyons: Spatial distribution and anthropogenic impacts. Deep Sea Research Part II: Topical Studies in Oceanography 104, 184-207. doi:10.1016/j.dsr2.2013.06.016.

FERNÁNDEZ DE PUELLES, M.L., ALEMANY, F., JANSÁ, J., 2007. Zooplankton time-series in the Balearic Sea (Western Mediterranean): Variability during the decade 1994-2003. Progress in Oceanography 74, 329-354. doi:10.1016/j-pocean.2007.04.009.

FOWLER, S.W., SMALL, L.F., LAROSA, J., 1991. Seasonal Particulate Carbon Flux in the Coastal Northwestern
Mediterranean-Sea, and the Role of Zooplankton
Fecal Matter. Oceanologica Acta 14, 77–85.

GARCÍA, A., ALEMANY, F., VELEZ-BELCHÍ, P., LÓPEZ JURADO, J.L., CORTÉS, D., DE LA SERNA, J.M., GONZÁLEZ POLA, C., RODRÍGUEZ, J.M., JNASÁ, J., RAMÍREZ, T., 2005. Characterization of the bluefin tuna spawning habitat off the Balearic Archipelago in relation to key hydrographic features and associated environmental. ICCAT Collective Volume of Sicentific Papers 58, 535–549.

GARCÍA PÉREZ A., 2005. Estadística aplicada: conceptos básicos. UNED Editorial, Madrid.

GONZÁLEZ, H.E., CALDERÓN, M.J., CASTRO, L., CLEMENT, A., CUEVAS, L.A., DANERI, G., IRIARTE, J.L., LIZÁRRAGA, L., MARTÍNEZ, R., MENSCHEL, E., SILVA, N., CARRASCO, C., VALENZUELA, C., VARGAS, C.A., MOLINET, C., 2010. Primary production and plankton dynamics in the Reloncaví Fjord and the Interior Sea of Chiloé, northern Patagonia, Chile. Marine Ecology Progress Series 402, 13–30. doi:10.3354/meps08360.

GRINYÓ, J., GORI, A., AMBROSO, S., PURROY, A., CALATAYUD, C., DOMINGUEZ-CARRIÓ, C., COPPARI, M., LO IACONO, C., LÓPEZ-GONZÁLEZ, P.J., GILI, J.-M., 2016. Diversity, distribution and population size structure of deep Mediterranean gorgonian assemblages (Menorca Channel, Western Mediterranean Sea). Progress in Oceanography 145, 42–56. doi:10.1016/j.pocean.2016.05.001

GUERRERO, E., MARRODÁN, A., SABATÉS, A., OREJAS, C., GILI, J.M., 2016. High spatial heterogeneity of two planktonic cnidarian species related to the variability of a shelf-slope front at short time scales. Scientia Marina. In press.

GUILLÉN, J., BOURRIN, F., PALANQUES, A., DURRIEU DE MADRON, X., PUIG, P., BUSCAIL, R., 2006. Sediment dynamics during wet and dry storm events on the Têt inner shelf (SW Gulf of Lions). Marine Geology 234, 129–142. doi:10.1016/j.margeo.2006.09.018.

HAMMER, T., HARPER, D.A.T., RYAN, PD., 2001, PAST: Paleontological statistics software package for education and data analysis: Palaeontologia Electronica 4, 1–9.

HARRIS, P.T., WHITEWAY, T., 2011. Global distribution of large submarine canyons: Geomorphic differences between active and passive continental margins. Marine Geology 285, 69–86. doi:10.1016/j.margeo.2011.05.008.

HARROLD, C., LIGHT, K., LISIN, S., 1998. Organic enrichment of submarine-canyon and continental-shelf benthic communities by macroalgal drift imported from nearshore kelp forests. Limnology and Oceanography 43, 669–678.

HEUSSNER, S., DURRIEU DE MADRON, X., CALAFAT, A., CANALS, M., CARBONNE, J., DELSAUT, N., SARAGONI, G., 2006. Spatial and temporal variability of downward particle fluxes on a continental slope: Lessons from an 8-yr experiment in the Gulf of Lions (NW Mediterranean). Marine Geology 234, 63–92. doi:10.1016/j.margeo.2006.09.003.

Heussner, S., Ratti, C., Carbonne, J., 1990. The PPS 3 time series sediment traps and the trap sample processing techniques used during the ECOMARGE experiment. Continental Shelf Research 10, 943–958.

Isla, E., Gerdes, D., Palanques, A., Gili, J.M., Arntz, W.E., König-Langlo, G., 2009. Downward particle fluxes, wind and a phytoplankton bloom over a polar continental shelf: A stormy impulse for the biological pump. Marine Geology 259, 59–72. doi:10.1016/j.margeo.2008.12.011.

Jordi, A., Orfila, A., Basterretxea, G., Tintoré, J., 2005. Shelf-slope exchanges by frontal variability in a steep submarine canyon. Progress in Oceanography 66, 120–141. doi:10.1016/j.pocean.2004.07.009.

KHRIPOUNOFF, A., VANGRIESHEIM, A., BABONNEAU, N., CRASSOUS, P., DENNIELOU, B., SAVOYE, B., 2003. Direct observation of intense turbidity current
activity in the Zaire submarine valley at 4000 m water depth. Marine Geology 194, 151–158. doi:10.1016/S0025-3227(02)00677-1.

KHRIPOUNOFF, A., CRASSOUS, P., LO BUE, N., DENNIELOU, B., JACINTO, R.S., 2012. Different types of sediment gravity flows detected in the Var submarine canyon (northwestern Mediterranean Sea). Progress in Oceanography 106, 138–153. doi:10.1016/j-pocean.2012.09.001.

KINEKE, G.C., WOOLFE, K.J., KUEHL, S.A., MILLIMAN, J.D., DELLAPENNA, T.M., PURDON, R.G., 2000. Sediment export from the Sepik River, Papua New Guinea: Evidence for a divergent sediment plume. Continental Shelf Research 20, 2239–2266. doi:10.1016/S0278-4343(00)00069-8.

KNOWLTON, N., JACKSON, J.B.C., 2001. THE ECOLOGY OF CORAL REEFS. IN: BERTNESS, M.D., GAINES, S.D., HAY, M.E. (Eds.), Marine Community Ecology. Sinauer Associates, Sunderland, Massachusetts, USA.

LIU, J.T., LIU, K.J., HUANG, J.C., 2002. The effect of a submarine canyon on the river sediment dispersal and inner shelf sediment movements in southern Taiwan. Marine Geology 181, 357–386. doi:10.1016/S0025-3227(01)00219-5.

LO IACONO, C. LO, SULLI, A., AGATE, M., LO PRESTI, V., PEPE, F., CATALANO, R., 2011. Submarine canyon morphologies in the Gulf of Palermo (Southern Tyrrhenian Sea) and possible implications for geo-hazard. Marine Geophysical Research 32, 127–138. doi:10.1007/s11001-011-9118-0.

LO IACONO, C., URGELES, R., POLIZZI, S., GRINYÖ, J., DRUET, M., AGATE, M., GILI, J.M., ACOSTA, J., 2014. Submarine Mass Movements and Their Consequences, in: Submarine Mass Movements Along a Sediment Starved Margin: The Menorca Channel (Balearic Islands-Western Mediterranean). pp. 329–338. doi:10.1007/978-1-4020-6512-5.

LOPEZ-FERNANDEZ, P., BIANCHELLI, S., PUSCEDDU, A., CALAFAT, A., DANOVARO, R., CANALS, M., 2013a.

Bioavailable compounds in sinking particulate organic matter, Blanes Canyon, NW Mediterranean Sea: Effects of a large storm and sea surface biological processes. Progress in Oceanography 118, 108–121. doi:10.1016/j-pocean.2013.07.022.

LOPEZ-FERNANDEZ, P., CALAFAT, A., SANCHEZ-VIDAL, A., CANALS, M., FLEXAS, M.M., CATEURA, J., COMPANY, J.B., 2013b. Multiple drivers of particle fluxes in the Blanes submarine canyon and southern open slope: Results of a year round experiment. Progress in Oceanography 118, 95–107. doi:10.1016/j-pocean.2013.07.029.

MACKIE, A.S., OLIVER, P.G., DARBYSHIRE, T., MORTIMER, K., 2005. Shallow marine benthic invertebrats of the Seychelles Plateau: high diversity in a tropical oligotrophic environment. Philosophical Transactions of the Royal Society of London A: Mathematical, Physical and Engineering Sciences 363, 203–228.

MALINVERNO, E., TRIANTAPHYLLOU, M. V., STAVRAKAKIS, S., ZIVERI, P., LYKOUSIS, V., 2009. Seasonal and spatial variability of coccolithophore export production at the South-Western margin of Crete (Eastern Mediterranean). Marine Micropaleontology 71, 131–147. doi:10.1016/j.marmicro.2009.02.002.

MARTÍN, J., PALANQUES, A., PUIG, P., 2006. Composition and variability of downward particulate matter fluxes in the Palamós submarine canyon (NW Mediterranean). Journal of Marine Systems 60, 75–97. doi:10.1016/j.jmarsys.2005.09.010.

MARTÍN, J., PALANQUES, A., VITORINO, J., OLIVEIRA A., DE STIGTER, H.C., 2011. Near-bottom particulate matter dynamics in the Nazaré submarine canyon under calm and stormy conditions. Deep Sea Research II: Topical Sutdies in Oceanography 58, 2388–2400. doi:10.1016/j.dsr2.2011.04.004.

MARTY, J.-C., GARCIA, N., RAIMBAULT, P., 2008. Phytoplankton dynamics and primary production under late summer conditions in the NW Mediterranean Sea. Deep Sea Research Part I: Oceanography