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# Ecological study of benthic communities in the continental shelf and upper slope in the Menorca Channel (North Western Mediterranean Sea)

Jordi Grinyo

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Jordi Grinyo. Ecological study of benthic communities in the continental shelf and upper slope in the Menorca Channel (North Western Mediterranean Sea). Life Sciences [q-bio]. Programa doctoral de Ciències del Mar, 2016. English. NNT: tel-04429128

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Ecological study of benthic communities
in the continental shelf and upper slope
in the Menorca Channel
(North Western Mediterranean)

JORDI GRINYÓ

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Barcelona 2016

PhD Thesis
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Ecological study of benthic communities
in the continental shelf and upper slope
in the Menorca Channel
(North Western Mediterranean Sea)

JORDI GRINYÓ

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UNIVERSITAT POLITÈCNICA
DE CATALUNYA
BARCELONATECH

ICM

Institute
of Marine
Sciences

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CSIC

CONSEJO SUPERIOR DE INVESTIGACIONES CIENTÍFICAS

Barcelona 2016

PhD Thesis
# Ecological study of benthic communities in the continental shelf and upper slope in the Menorca Channel (North Western Mediterranean Sea)

## Estudi ecològic de les comunitats bentòniques a la plataforma i talús continental somer al Canal de Menorca (Mediterrani Nord Occidental)

Jordi Grinyó Andreu

Tesi presentada per l'obtenció del títol de Doctor
per la Universitat Politècnica
de Catalunya

Programa de Doctorat de Ciències del Mar 2016

Director:
Prof. **Josep-Maria Gili Sardà**
Dept. Biologia Marina i Oceanografia
Institut de Ciències del Mar (ICM-CSIC)

Co-Director:
Dr. **Andrea Gori**
Dept. de Biologia Evolutiva, Ecologia
i Ciències Ambientals
Universitat de Barcelona
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"Ecological study of benthic communities in the continental shelf and upper slope in the Menorca Channel (North Western Mediterranean)"

This work has been developed within the LIFE+ INDEMARES project "Inventario y designación de la Red Natura 2000 en áreas marinas del estado español" (LIFE07/NAT/E/000732)

External referees: Dr. Lorenzo Bramanti from the French National Centre for Scientific Research and Dr. Sergio Rossi Heras from the Autonomous University of Barcelona.
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"The sea, once it casts its spell, holds one in its net of wonder forever."

Jacques Yves Cousteau
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**Chapter 3** Morphological and genetic features of the gorgonian *Paramuricea macrospina* on the Mediterranean continental shelf (Menorca Channel, Western Mediterranean Sea)

**77**

<table><tr><td>Abstract</td><td>77</td></tr><tr><td>Introduction</td><td>78</td></tr><tr><td>Material and methods</td><td>79</td></tr><tr><td>Results</td><td>83</td></tr><tr><td>Discussion</td><td>89</td></tr><tr><td>References</td><td>94</td></tr></table>

**Chapter 4** Reproductive cycle, energy storage and metabolic requirements of the gorgonian *Paramuricea macrospina* from the Mediterranean continental shelf (Menorca Channel, Western Mediterranean Sea)

**99**

<table><tr><td>Abstract</td><td>99</td></tr><tr><td>Introduction</td><td>100</td></tr><tr><td>Material and methods</td><td>102</td></tr><tr><td>Results</td><td>106</td></tr><tr><td>Discussion</td><td>108</td></tr><tr><td>References</td><td>117</td></tr></table>

**Chapter 5** Composition and temporal variability of particle fluxes in an insular canyon of the northwestern Mediterranean Sea

**123**

<table><tr><td>Abstract</td><td>123</td></tr><tr><td>Introduction</td><td>124</td></tr><tr><td>Material and methods</td><td>126</td></tr><tr><td>Results</td><td>128</td></tr><tr><td>Discussion</td><td>134</td></tr><tr><td>References</td><td>149</td></tr></table>

**Conclusions and future perspectives**

**157**

**Annex I**

**161**

**Annex II**

**175**

**Acknowledgements**

**199**
Summary

GRINYÓ J (2016) PhD Thesis pp 11-12

Technological development and increased availability of remotely operated vehicles (ROVs) and manned submersibles have significantly increased accessibility to deep environments revealing the presence of rich and diverse macrobenthic assemblages dominated by suspension feeders. These assemblages have been largely exposed to the impacts of fishing activities (especially bottom trawling) in the Mediterranean Sea. Consequently, it is currently urgent to increase knowledge on their distribution, as well as on the ecology and biology of the main species that constitute these macrobenthic assemblages in order to implement effective management and conservation measures. This thesis addressed the characterization of macrobenthic assemblages in the continental shelf and upper slope (40–360 m depth) of the Menorca Channel through a multidisciplinary approach at different ecological levels.

Six macrobenthic assemblages mainly segregated by substrate and depth. Hard substrates hosted sponge grounds and coral gardens, whereas crinoid and brachiopod beds occurred on soft sediments. Highest diversity values were found in the shelf edge, probably as a consequence of the bottom heterogeneity and the constant hydrodynamic conditions. Gorgonians were one of the most important and diverse bioengineering organisms in the Menorca Channel, forming dense assemblages that extended over vast areas. Gorgonian assemblages on the continental shelf and upper slope were mostly monospecific, whereas shelf edge assemblages were highly multispecific. Small colonies were dominant throughout the studied bathymetric range, but in deeper environments intermediate and large colonies were more abundant. The good preservation state of most of the observed benthic assemblages and gorgonian populations is probably related to the low pressure of bottom trawling, which is mostly concentrated in deeper areas of the continental and the exceptional fishing practices exerted by the local artisanal fisherman. *Paramuricea macrospina* is the most abundant gorgonian species in the Menorca Channel. Throughout its distribution, three different chromatic forms were observed. Two forms occurred on the continental shelf, and a third occurred on the shelf edge. Colony shape, sclerite size and shape, and the genetic variability of mitochondrial markers were compared to elucidate if these chromatic forms represented different taxonomic units. Colony morphology barely changed
among the three forms resulting as a high conservative character. Conversely, sclerite size and
shape significantly differed amongst the three forms, possibly conditioned by genetic and envi-
ronmental factors. However, no significant differences were observed in the studied mitochondrial
markers. The reproductive cycle, energy storage and metabolic requirements of *P. macrospina*
showed significant differences compared to Mediterranean coastal gorgonians. Its reproductive
cycle was delayed 2-3 months respect to shallow species, possibly following the late summer
increase in seawater temperature occurring on the Mediterranean continental shelf. Moreover,
internal brooding in *P. macrospina* contrasts with surface brooding in the congeneric *Paramuricea
clavata*. Lipid content was lower and more constant in *P. macrospina* than in shallow species, and
$\partial^{13}$C and $\partial^{15}$N composition showed almost no seasonal variation, suggesting that food availability
in the continental shelf is lower but more constant than in shallower environments. The high olig-
otrophic conditions of the Balearic Sea apparently contrasts with the high abundance and diversity
of active and passive suspension feeders observed in the Menorca Channel. Indeed, the down-
ward particle fluxes quantified during two consecutive years in the Menorca Canyon were com-
paratively low within the Mediterranean context, reflecting the oligotrophic nature of the study
area and the lack of continental inputs of particulate matter. Hydrodynamic settings and physical
processes that cause sediment resuspension appeared to be more important than surface pri-
mary production in the control of the magnitude and composition of the total mass flux in the
study area, likely playing a major role in determining the distribution of the macrobenthic assem-
blages.
Les millores tecnològiques i la major disponibilitat de vehicles operats remotament (ROVs) i sub-
marins tripulats han incrementat l'accessibilitat a entorns profunds, revelant la presència asso-
ciacions d'organismes bentòniques riques i diverses, dominades per suspensívors. Aquestes
agregacions han estat altament impactades per la pesca (en especial el ròssec) al Mediterrani.
És urgent incrementar el coneixement, sobre la distribució d'aquestes associacions i aspectes
claus de la biologia i ecologia de les principals espècies que les constitueixen per tal d'establir
mesures de conservació efectives. En aquesta tesi es caracteritzen les associacions bentòniques
de la plataforma i talús continentals del Canal de Menorca a través d'una aproximació multidisci-
plinària a diferents nivells ecològics.

S'han identificat sis associacions bentòniques diferents, que es diferencien principalment pel tipus de substrat i el rang batimètric. En els substrats durs s'hi ha trobat fons d'esponges i coralls, en els fons tous s'hi han trobat camps de crinoïdeus i braquiòpodes. Al marge continental s'hi concentren la diversitat més elevada, probablement resultant de l'heterogeneïtat ambiental i l'hi-drodinamisme d'aquest entorn. Al Canal de Menorca les gorgònies són un dels organismes bio-enginyers més importants i diversos, formant denses agregacions sobre amplies zones. Les agregacions de gorgònies a la plataforma i part superior del talús eren principalment monoespe-cífiques, mentre que les del marge continental eren multiespecífiques. Les colònies petites eren les més abundants al llarg de tot el rang batimètric. Tot i així, en entorns profunds les colònies mitjanes i grans incrementaven la seva abundància. El bon estat de les associacions bentòniques i les poblacions de gorgònies probablement resulta de la baixa pressió del ròssec, que es con-centra a major fondària, i a les pràctiques excepcionals dels pescadors artesanals. *Paramuricea macrospina* és la gorgònia més abundant del Canal de Menorca. Al llarg de la seva distribució ba-timètrica es varen observar tres variants cromàtiques diferents. Dues es varen trobar a la pla-ta-forma i una tercera es trobà al marge continental. Per tal d'esbrinar si les diferents variants eren unitats taxonòmiques diferents s'han estudiat la forma colonial, la variabilitat de tamany i formes dels esclerits així com la variabilitat en marcadors mitocondrials. Es va detectar molt poca varia-bilitat en la morfologia colonial de les tres variants, suggerint que és un caràcter conservatiu. El
tamany i la morfologia dels esclerits diferia significativament entre les tres variants, suggerint que estan influenciats per factors ambientals i genètics. No es varen trobar diferències entre els tres marcadors mitocondrials. S'ha avaluat el cicle reproductor, l'acúmul energètic i les necessitats metabòlics de *P. macrospina*. El cicle reproductiu d'aquesta espècia està retardat 2-3 mesos respecte les espècies de gorgònia mediterrànies somes. Aquesta situació podria sorgir de l'augment tardà de la temperatura de l'aigua a la plataforma continental mediterrània. El fet que *P. macrospina* sigui una internal brooder contrasta amb que *Paramuricea clavata* sigui una surface brooder. El contingut lipídic de *P. macrospina* és més baix però més constant que el d'espècies somes mediterrànies. D'altra banda la composició de la ¹³C i la ¹⁵N gariebé no presentaven fluctuacions estacionals. Suggerint que la disponibilitat d'aliment a la plataforma és més baixa però més constant que en entorns soms. Al marge sud del Canal de Menorca hi trobem el Canyó de Menorca. Tot i l'elevada oligotròfia del mar Balear, les associacions d'organismes bentònics a la capçalera del canyó i proximitats són d'una elevada riquesa. Per aquest motiu s'ha avaluat el flux de partícules durant dos anys consecutius al canyó. Els fluxos recollits eren comparativament inferiors que els registrats en d'altres canyons mediterranis, reflectint la manca d'aportacions continentals i l'oligotròfia de l'àrea d'estudi. Els processos físics que causaven resuspensió i diferents paràmetres hidrodinàmics, semblen ser més importants en el control, la magnitud i la composició del flux de partícules que no pas la producció primària superficial. Aquest resultats suggereixen que els fluxos de partícules en canyons insulars Mediterranis, sense sistemes fluvials importants, són semblants als observats en el mar profund, però segurament juguen un paper important en la distribució d'associacions d'organismes macrobentònics.
Introduction

GRINYÓ J (2016) PhD Thesis pp 15-23

Benthic assemblages in shallow environments have received plenty of attention due to the wide diffusion of SCUBA diving. The recent technological development and increased availability of video-equipped towed gears, remotely operated vehicles (ROVs), manned submersibles and autonomous under water vehicles (AUVs) have remarkably increased accessibility to deep environments, allowing the direct observation and quantitative study of benthic assemblages (e.g. Miller et al., 2012; White et al., 2012; Pineda et al., 2016). Several surveys using ROVs and manned submersible vehicles have been carried out in the deep sea, below a depth of 150 m (Sink et al., 2006). Conversely, continental shelf and upper slopes have received relatively less attention (Cau et al., 2015).

Highly diverse benthic assemblages dominated by passive (e.g. gorgonians, corals, black corals) and active (e.g. sponges, brachiopods) benthic suspension feeders have been recently found in continental shelf, slopes and seamounts worldwide (e.g. Clark et al., 2010; Kenchington et al., 2013; Buhl-Mortensen et al., 2016). In these deep environments, hydrodynamic processes provide high and stable food supply allowing the development of dense aggregations

of suspension feeders, such as crinoid beds, sponge grounds, coral gardens and cold-water coral reefs (Lavaleye et al., 2002; Rice, 1990; Thiem et al., 2006). Benthic suspension feeders induce a significant flow of matter and energy from the pelagic to the benthic system by capturing plankton and particulate organic matter, playing a remarkable role in the benthopelagic coupling and biogeochemical cycles (e.g. Bell, 2008; Cathalot et al., 2015). From a structural point of view, they play an important ecological role by forming complex three-dimensional structures that may provide shelter, enhance food availability and act as nursery grounds for numerous associated species (Bo et al., 2015; Buhl-Mortensen and Mortensen, 2005; Henry et al., 2013). Consequently, high diversity and biomass of associated fauna is promoted in these benthic assemblages (Beazley et al., 2013). Amongst the associated fauna there are numerous decapod and fish species of commercial interest, which are targeted by the fishing industry (Clark and O'Driscoll, 2003; Colloca et al., 2004; Shester and Ayers, 2005). Consequently, many sponge grounds, coral gardens and cold-water coral reefs have been widely degraded due to the repeated exposure to destructive fishing activities, such as bottom
trawling and long line fishing (Althaus et al., 2009; Taylor et al., 2013). Due to the slow growth and high longevity of most of the macrobenthic species that constitute these assemblages, their recovery may last centuries to millennia (Clark et al., 2016).

In the Mediterranean Sea, the first studies concerning benthic assemblages on the continental shelf and upper slope date back to the 1950s, when the Swiss engineer August Piccard performed several dives in areas of the Western Mediterranean Sea (Martin, 1960). However, it was not until the 1960s and 1970s when the first bionomical studies with detailed characterizations of deep Mediterranean benthic assemblages were performed (e.g. Pérès and Picard, 1964; Pérès, 1967; Vacelet, 1969; Fredj,

1972). These studies were based on the observations made on board of the manned submersible Scoupe Plongeante, developed by the French marine pioneer Jacks Costeau (Laban et al., 1963). After 1970s, research concerning deep Mediterranean benthic environments abruptly decreased, mainly due to the technical difficulties and high economic costs that imply working in deep-waters. During this period, most research derived from by-cached or withdrawn by blind destructive techniques (Relini et al., 1986; Gili et al., 1987). It was not until the 2000s when the increased affordability of ROVs and manned submersibles, and the increasing interest for cold-water corals, reinforced research in deep Mediterranean benthic environments (Taviani et al., 2005; Rossi et al., 2008; Orejas et al., 2009). In recent years, ROVs and manned submersibles have been used to characterize benthic assemblages on the continental shelf and slopes, reporting the presence of rich and dense assemblages such as coral gardens, sponge grounds or bryozoan beds (e.g. Bo et al., 2013, 2011; Fabri et al., 2014; Michez et al., 2014; Cau et al., 2015). However, in many cases these assemblages also presented clear signs of fishing derived damages (Orejas et al., 2009; Bo et al., 2014; Fabri et al., 2014; Angiolillo et al., 2015) (Fig. 2).

The increasing demand for fishing resources in Mediterranean countries, has lead to an intensification of fishing activities, resulting in overexploitation of fish stocks and the severe damage of benthic habitats, turning the Mediterranean Sea in one of the most impacted seas in the world (Lotze et al., 2011; Coll et al., 2012). Bottom trawling fishing has gradually increased in intensity since the first half of the 20th century, progressively expanding to greater depth (Sacchi, 2008). Consequently, decades of chronic trawling have widely impacted large areas of the Mediterranean continental shelf and slope (e.g. Fabri et al., 2014), probably constraining the presence of macrobenthic assemblages to remote (Díaz et al., 2015) or in-

<figure><img src="image_9.png" /><figcaption>**Fig. 1.** a) the remotely operated vehicle NEMO (G. Newman) b) the manned submersible JAGO (IFM-GE-OMAR) used during the field work of this thesis.</figcaption></figure>
<figure><img src="image_10.png" /><figcaption>**Fig. 2.** a) Colony of the cold-water coral *Dendrophylia cornigera* entangled in a long line b) A large sponge *Haliclona magna* cut by a long line c) Long lines in a coral garden d) Trammel net entangled in the gorgonian *Paramuricea clavata*. Scale bar = 10 cm.</figcaption></figure>

accessible areas (Bo et al., 2015). The vulnerability and low resilience of these assemblages (Althaus et al., 2009) have motivated the implementation of management measures, such as the restriction in the use of bottom trawling and other bottom contact gears (Pipitone et al., 2000) and the establishment of large marine protected areas on continental shelf and slope (Barberá et al., 2012). The European Union Natura 2000 initiative has developed a strong policy framework to deal with the multiple challenges that shallow and deep marine environments are facing, and ensure a sustainable-based approach for their marine resources (http://ec.europa.eu/environment/nature/natura2000/marine/index_en.htm9). From 2009 to 2014, the LIFE+ INDEMARES project has been studying deep benthic environments on Spain's territorial waters in order to contribute to the protection and sustainable use of 10 different sites of community interest (Fig. 3)

that eventually will become special areas of conservation. These areas represent an increment of more than 7 million hectares to the Spanish Natura 2000 Network, covering more than 8% of Spain territorial waters (http://www.indemares.es/en/project/description). Ultimately, the LIFE+ INDEMARES project laid the foundation for future management plans for Natura 2000 marine areas. Moreover the project also contributes to fulfill the objective established by the Convention on Biological Diversity from the United Nations, to reach the goal of protecting 10% of coastal and marine areas by 2020 (https://www.cbd.int/2011-2020/goals). Amongst the 10 areas studied in the LIFE+ INDEMARES project, the Menorca Channel covers a vast extension of the continental shelf and upper slope between the Mallorca and Menorca Islands (Balearic Archipelago) from 40 to 360 m depth.
<figure><img src="image_11.png" /><figcaption>Fig. 3. Project LIFE+ INDEMARES study areas (http://www.indemares.es/areas-marinas).</figcaption></figure>

GENERAL OBJECTIVES

The present thesis addressed the characteri-
zation of the benthic assemblages in the con-
tinental shelf and upper slope of the Menorca
Channel by means of five different studies:

**Chapter 1** - Macrobenthic assemblages on the Mediterranean continental shelf edge and upper slope (Menorca Channel, Western Mediterranean Sea).

This study focuses on the exploration of the
deep continental shelf and upper slope of the

Menorca Channel in order (1) to characterize
the composition of the macrobenthic assem-
blages, (2) to asses their geographical and ba-
thymetric distribution, (3) to quantify their bio-
diversity variation with depth, and finally (4) to
discuss the possible relationship between mac-
robenthic assemblages distribution and the
pressure of bottom trawling fishing in the area.

**Chapter 2** - Diversity, distribution and population size structure of deep Mediterranean gorgonian assemblages (Menorca Channel, Western Mediterranean Sea).

Gorgonians resulted to be the most relevant
macrobenthic species forming highly diverse
and dense assemblages in the Menorca Chan-
nel. This study aimed at (1) characterize the di-
versity and quantify the abundance of gorgonians species on a large area of the continental shelf and upper slope at 40–360 m depth, (2) assess their geographical and vertical distribution, (3) explore their population size structure, and (4) to gain insight into the possible factors affecting their abundance and distribution.

**Chapter 3** - Morphological and molecular variability of the gorgonian *Paramuricea macrospina* on the Mediterranean continental shelf (Menorca Channel, Western Mediterranean Sea).

*Paramuricea macrospina* resulted to be the most abundant gorgonian species in the Menorca Channel. Throughout its range of distribution, three different chromatic forms were observed. Two forms occurred on the continental shelf, and a third one occurred deeper on the shelf edge. This study aims to elucidate if the different *P. macrospina* chromatic forms represent distinct taxonomic units, by exploring differences in their (1) colony shape, (2) sclerite size and shape, and (3) genetic variability in mitochondrial markers (msh1, Igr1 and COI).

**Chapter 4** - Reproductive cycle, energy storage and metabolic requirements of the gorgonian *Paramuricea macrospina* from the Mediterranean continental shelf (Menorca Channel, Western Mediterranean Sea).

The aim of this chapter 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, free fatty acid content and composition, and stable isotope (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?

**Chapter 5** - Composition and temporal variability of particle fluxes in an insular canyon of the northwestern Mediterranean Sea.

The Baleric Archipelago is one of the most oligotrophic environments in the Western Mediterranean Sea. The only submarine canyon of the Archipelago is the Menorca Canyon. Despite the high oligotrophy of the Balearic Sea, benthic assemblages around the Menorca Canyon are highly diverse, with dense gorgonian and black coral assemblages. This study explored the environmental characteristics and flux of organic matter in which these communities thrive by characterizing (1) the temporal variability of downward particle fluxes, (2) assessing their geochemical and macroscopic composition, (3) identifying the main processes that modulate particle fluxes in the Menorca Canyon, and (4) compare the studied canyon with previous research developed in the Mediterranean Sea.

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Chapter 1

# Macrobenthic assemblages on the
Mediterranean continental shelf edge and upper slope
(Menorca Channel, Western Mediterranean Sea)

ABSTRACT

Highly diverse macrobenthic assemblages dominated by passive and active suspension feeders have been recently reported in shelf edge environments of the Mediterranean Sea. Due to the frequent association with species of commercial interest, these assemblages have been heavily impacted by fishing practices. The vulnerability and low resilience of these assemblages, composed mainly by long-living and slow-growing species, have motivated the implementation of management measures such as the restriction of bottom trawling fishing, and the establishment of large protected areas including deep environments. The Menorca Channel is one of these large areas recently included in the protection frame of the European Union Natura 2000 network. Quantitative analysis of video transects recorded at 95-360 m depth by manned submersible and remotely operated vehicles were used to characterize macrobenthic assemblages, and assess their geographical and bathymetric distribution. Six different assemblages were identified, mainly segregated by substrate and depth. Hard substrates hosted coral gardens and sponge grounds, whereas soft sediments were mainly characterized by vast extensions of the crinoid *Leptometra phalangium* and the brachiopod *Gryphus vitreus*. The good preservation of most of the observed assemblages is probably related to the low pressure of bottom trawling fishing in this area, mainly concentrated deeper, on the continental slope. Because of their biological and ecological value, management and conservation measures need to be established to preserve these benthic assemblages.

CO-AUTHORS

A Gori, M Greenacre, S Requena, A Canepa, C Lo Iacono, S Ambroso, A Purroy, JM Gili

ADDITIONAL RESOURCES

Submitted in Progress in Oceanography
# 1. INTRODUCTION

Knowledge about macrobenthic assemblages in coastal areas has greatly increased during the past decades thanks to the wide diffusion of SCUBA diving; conversely, continental shelf and slope have received relative less attention due to the difficulties related to working in deep environments (Cau et al., 2015). However, the most recent technological developments and increased availability of remotely operated vehicles (ROVs), manned submersibles and video-equipped towed gears have significantly increased accessibility to these deep environments, allowing the direct observation and quantitative study of macrobenthic assemblages (e.g. Etiope et al., 2010; Buhl-Mortensen et al., 2016). Rich and high diverse macrobenthic assemblages mostly dominated by passive (e.g. gorgonians, corals and black corals) and active (e.g. sponges) suspension feeders (e.g. Emig, 1997; Bo et al., 2013; Bertolino et al., 2013), have been recently reported on the continental shelf, slope and seamounts in several locations of the Mediterranean Sea (Bo et al., 2009; 2012; 2015; Deidun et al., 2014). These assemblages have shown to provide habitat (Mastrototaro et al., 2010; Porteiro et al., 2013) and act as nurseries (Colloca et al., 2004; Bo et al., 2015) for a wide variety of associated species, many of which are of commercial interest (Abella et al., 2005; Maynou and Cartes, 2012).

Due to their distribution deeper than 50 m, these assemblages have been largely exposed to the impacts of bottom trawling (Maynou and Cartes, 2012; Fabri et al., 2014) and, to a lesser extent, long-line and trammel net fishing (Orejas et al., 2009; Sampaio et al., 2012; Mytilineou et al., 2014). These fishing practices have dramatic effects on macrobenthic species, which are often

removed or severely damaged (Fosså et al., 2002; Mytilineou et al., 2014), resulting in a decline in the biodiversity and abundance of the associated fauna (Althaus et al., 2009; Clark et al., 2016). In the Mediterranean Sea, bottom trawling has progressively increased in intensity since the first half of the 20th century, progressively expanding to greater depth (Sacchi, 2008). Consequently, decades of chronic trawling have widely impacted large areas of the Mediterranean continental shelf and slope (e.g. Fabri et al., 2014), reducing the presence of relatively well preserved macrobenthic assemblages to remote (Díaz et al., 2015) or inaccessible areas (Bo et al., 2015). The ecological effects of bottom trawling are extremely long lasting, as no signs of recovery of macrobenthic assemblages have been observed in areas closed to trawling after more than ten years (Althaus et al., 2009; Williams et al., 2010). Due to the slow-growth and high longevity of most of the macrobenthic species that constitute these assemblages, it has been suggested that their possible recovery may take centuries to millennia (Clark et al., 2016).

The vulnerability and low resilience of these communities (Althaus et al., 2009) have motivated the recent implementation of management measures, such as the restriction in the use of bottom trawling and other bottom contact gears (Pipitone et al., 2000; Armstrong et al., 2014), and the establishment of managed and protected areas on continental shelf, continental slope (Spalding et al., 2013; Bennecke and Metaxas, 2016) and seamounts (Sheppard et al., 2012; Huvene et al., 2016) worldwide. Deep-water protected areas are often much more extensive than coastal ones (De Santo, 2013; Spalding et al., 2013; Bennecke and Metaxas, 2016). Large marine protected areas extend over wide bathymetrical and geographical range, covering several habitat types (Fernandes et al., 2005; Sheppard et al., 2012) and offering refuge to a wide variety of mobile and sessile species with high larval dispersal (Roberts et al., 2003). In the Mediterranean Sea, marine protected areas are often
eas are mostly restricted to littoral environments (e.g. Francour et al., 2001). However, the establishment of large marine protected areas (>100 km²), covering large areas of continental shelf and slope, has been recently proposed as a tool for the ecosystem based management of marine resources and environments (European Union, Natura 2000 network, http://www.eea.europa.eu/data-and-maps/data/natura-2). The Menorca Channel (Fig. 1) is one of these large area recently included in the protection frame of the European Union Natura 2000 network. Previous studies have investigated and characterized benthic assemblages in the coastal (e.g. Coma et al., 2006) and shallow continental shelf (<100 m) (e.g. Joger et al., 2012; Barberá et al., 2012) of the Menorca Channel. Conversely, composition and distribution of benthic assemblages on the deep continental shelf and upper slope still remain widely unknown. This information is basic for the establishment of effective management and conservation measures, as well as to monitor their effectiveness.

To this purpose, the aims of this study were:
(1) to characterize the composition of macrobenthic assemblages on the deep continental shelf and upper slope of the Menorca Channel,
(2) to assess their geographical and bathymetric distribution,
(3) to quantify their biodiversity variation with depth, and finally (4) to identify a potential relationship between macrobenthic assemblages distribution and the pressure of bottom trawling fishing in the area.

## 2. MATERIALS AND METHODS

### 2.1 Study area

The Menorca Channel is located in the Western Mediterranean Sea between Mallorca and

Menorca Islands (39° 53' 0.73" N, 3° 29' 51.16" E) (Fig. 1a), as part of the Balearic Promontory (Acosta et al., 2002). The study area covered the deep continental shelf (90–110 m), the shelf edge (110–180 m) and the upper slope (180–350 m) of the channel. The continental shelf is characterized by smooth reliefs covered by maërl beds alternated with coralligenous outcropping rocks and detritic coarse sediments (Barberá et al., 2012). Fine sands cover vast areas of the northern continental shelf at 100–110 m depth. Smooth reliefs and large extensions of detritic sediments with few isolated patches of outcropping rocks characterize most of the shelf edge and continental slope. Only near Cap Formentor (Fig. 1b) and in the Menorca Canyon (Fig. 1c) sharply edged rock outcroppings and rocky vertical walls are the dominant substrates.

The northern shelf edge and continental slope of the study area is mainly influenced by the Balearic Current (Balbín et al., 2012) and its associated front (Ruiz et al., 2009), which originates from a branch of the Liguro-Provençal-Catalan current flowing northward over the continental slope of the Balearic archipelago at ~200 m depth (Ruiz et al., 2009). Secondary currents flow northward from the Algerian sub-basin entering the Balearic sub-basin and feed the Balearic current (Pinot et al., 2002; Amores et al., 2013). Conversely, the southern shelf edge and upper slope are not influenced by a steady current (Amores and Montserrat, 2014), but by the sporadic arrival of mesoscale structures detached from the Algerian Current and the Almería-Oran front (Millot, 1987; García et al., 2005).

### 2.2 Sampling procedure

A total of 45 video transects (Fig. 1) were recorded during four surveys conducted on board of the R/V "García del Cid" (September 2010, April 2011, October 2011, June 2012). During the first two surveys, 17 video transects
were recorded with the manned submersible JAGO (IFM-GEOMAR), whereas 28 video transects were recorded with the ROV NEMO (Gavin Newman) during the last two surveys. Both instruments were equipped with a 1080 horizontal line resolution camera, a grabber and two parallel laser beams which provided a scale to define a fixed width within the transects (0.5 m) during the subsequent video analysis. Transects were recorded in a close-

zoom (~0.5–1.5 m width) and in a digital format. Positioning of JAGO and NEMO was achieved with underwater acoustic positioning system (LinkQuest TrackLink 1500 HA). Both JAGO and NEMO moved at a constant speed of ~0.3 knots, and transect lengths ranged between 309 and 2375 m, over depths ranging from 90 to 347 m. Transects were haphazardly located in order to cover the entire study area, but areas showing morphological features possibly related to the presence of rocky bottoms were explored more intensively (Fig. 1). Overall, a total of 37.7 km were video recorded along the seabed.

In order to confirm the taxonomic identification of the macrobenthic organisms observed in the video transects, voucher organisms were collected by means of the JAGO and NEMO grabbers. Sampled organisms were fixed and preserved in 10% formalin as well as in absolute ethanol for posterior taxonomical and genetic analyses. Organisms were identified to the lowest possible taxon, which corresponded to species or genus.

## 2.3 Video analysis

Quantitative video analysis was performed according to the methodology described in Gori et al. (2011) using Apple's Final Cut Pro software. In order to correctly estimate the length of each transect, all the pauses in the movement of the JAGO or NEMO were removed from the footage. Sequences with poor image quality or too far away from the seafloor were considered unsuitable for analysis. The remaining useful sequences comprised 93.3% of the total recorded material and corresponded to a total distance of 35 km. All macrobenthic organisms observed within a width of 0.5 m (based on the laser beams) along each video transect were identified and assigned a time reference derived from the time elapsed since the beginning of the video transect to the crossing of the laser beams with the organism (Gori

<figure><img src="image_12.png" /><figcaption>**Fig. 1.** The study area. (a) Three-dimensional bathymetry of the Menorca Channel: the map shows the location of the video transects (1 to 45) and the location of the study area in the western Mediterranean. Detail of the (b) northern and (c) southern subareas.</figcaption></figure>
et al., 2011). This time reference was posteriorly converted into position along the transect according to the known velocity from the positioning system P = t · v, where t is the time reference expressed in seconds, and v is the velocity expressed in meters per second). A similar procedure was used to characterize seabed substrate types and slope along every transect, within the same width of 0.5 m. Seabed substrate type was classified (based on the Wentworth scale) into five categories: fine sands, medium sands to gravels, cobbles and pebbles, maërl, and rock. Seabed slope was classified into three categories: horizontal (0°-30°), sloping (30°-80°) and vertical (80°-90°); slope was estimated from the video by looking at the two parallel laser beams and the depth sensor (Gori et al., 2011; Ambroso et al., 2013).

## 2.4 Data treatment

### 2.4.1 Sampling unit characterization

Each transect was divided into a string of sampling units. Different size of sampling units (2, 5, 10, 15, 20, 30, 40 and 50 m²) were used in the canonical correspondence analysis (see below) in order to explore how the size of the sampling units may affect the identification of the benthic assemblages, and their associated main environmental features. Each sampling unit was characterized by the number of macrobenthic organisms of each identified species, as well as by the average depth and coverage percentage for each substrate and slope category.

### 2.4.2 Canonical correspondence analysis (CCA)

Relationship between benthic macrofauna abundance and depth, substrate type and slope were explored by means of canonical correspondence analysis (CCA). CCA is a multivariate constrained ordination technique used to elucidate the relationships between species abundances (response variables) and environ-

mental variables (explanatory variables) (Greenacre and Primiceiro, 2013). Taxa that appeared with less than three individuals were discarded from the analysis in order to avoid distortions. Depth of sampling units was coded into three fuzzy categories that conserve all the information in the variables while reducing it to a categorical scale (Aschan et al., 2013). This fuzzy-coding allows taking into account possible nonlinear relationships between fauna abundance and depth (Greenacre and Primiceiro, 2013). Environmental variables were standardized by standard deviation, and CCA were performed with the R-language function cca in the ca library (Nenadic and Greenacre, 2007) of the R software platform (R Core Team 2014). Additionally, environmental factors were tested to determine which was the best predictor by means of the R-language function anova (Chambers and Hastie, 1992). CCA was performed with several sampling unit sizes (2, 5, 10, 15, 20, 30, 40 and 50 m²) in order to explore how the identification of the benthic assemblages, and their associated main environmental features, change with the size of the sampling units.

### 2.4.3 Geographic distribution of macrobenthic assemblages

Geographical distribution of each identified macrobenthic assemblage in the study area was reported by mapping its occurrence in the video transects on a geographically referenced map using GIS (ESRI ArcGIS ArcInfo v10).

### 2.4.4 Variation of macrobenthic diversity with depth

Sampling units were grouped in 5 m depth intervals, and the exponential of Shannon's diversity index was calculated based on the observed macrobenthic abundance. This diversity index was chosen as it weights all species by their frequency without favoring rare or most common ones (Jost, 2006). Analysis was per-
<table><caption>Table 1. Results of the CCA analysis performed at different sampling unit size. env. fact. = environmental factors.</caption><thead><tr><th>Sampling Unit Size</th><th>Species N°</th><th>Sampling Units N°</th><th>Total Inertia</th><th>Restricted Inertia</th><th>Unrestricted Inertia</th><th>% of Inertia explained by env. fact.</th><th>Macrobenthic assemblage N°</th></tr></thead><tbody><tr><td>2</td><td>69</td><td>8639</td><td>28.36</td><td>3.03</td><td>25.32</td><td>10.69</td><td>6</td></tr><tr><td>5</td><td>69</td><td>3372</td><td>20.96</td><td>3.28</td><td>17.68</td><td>15.6</td><td>6</td></tr><tr><td>10</td><td>69</td><td>1537</td><td>18.5</td><td>3.39</td><td>15.11</td><td>18.3</td><td>5</td></tr><tr><td>15</td><td>69</td><td>985</td><td>16.15</td><td>2.46</td><td>13.69</td><td>15.2</td><td>5</td></tr><tr><td>20</td><td>69</td><td>713</td><td>15.17</td><td>3.44</td><td>11.73</td><td>22.6</td><td>5</td></tr><tr><td>30</td><td>69</td><td>443</td><td>13.8</td><td>3.45</td><td>10.37</td><td>24.97</td><td>5</td></tr><tr><td>40</td><td>67</td><td>316</td><td>13.25</td><td>3.57</td><td>9.67</td><td>13.25</td><td>5</td></tr><tr><td>50</td><td>67</td><td>248</td><td>12.59</td><td>3.61</td><td>8.98</td><td>28.71</td><td>4</td></tr></tbody></table>

formed with the diversity function of the vegan library (Oksanen et al., 2015) of the R software platform.

### 2.4.5 Fishing activity in the study area

Fishing activity and location of fishing grounds (i.e. areas consistently fished through time) in the study area were assessed by means of the Vessel Monitoring System (VMS) through the analysis of the number of fishing events per surface units. VMS data were obtained in the framework of the LIFE+ INDEMARES project from the General Directorate of Fisheries Management of the Spanish Ministry of Agriculture, Food and Environment (MAGRAMA). Available VMS records for the period January 2007 to July 2012 were included after examination to exclude erroneous vessel identity, position or speed. To select the records referring to fishing activity, a set of common criteria (Lee et al., 2010) was followed: duplicated records and records close to ports were removed, and the interval between records was calculated in order to only retain vessels moving at 2–5 knots. Distribution of fishing activity in the study area was assessed based on

counts of fishing events per cell unit based on a point summation method (Hintzen et al., 2010). A 250 m² cell size was selected as the most suitable considering geographical factors and dataset (for further details, see Piet and Quirijns, 2009). Only one record per vessel, day and cell was retained to avoid overestimation of fishing activity (Hintzen et al., 2010). The centroids of each cell were extracted and weighted by the sum of fishing events for this cell. These weighted centroids were used to identify statistically significant hotspots of fishing activity in the study area using the Gi* statistic (Getis and Ord, 1996).

## 3. RESULTS

### 3.1 Canonical Correspondence Analysis (CCA)

CCA analysis performed with different sampling unit sizes revealed a general increase of the amount of inertia explained by environmental factors as sampling unit size increased
<table><caption>**Table 2.** Macrobenthic taxa occupancy and abundance in the study area. Occupancy (frequency of occurrence in the set of sampling units); abundance (number of organisms). (subc. = subclass).</caption><thead><tr><th rowspan="2">Phylum</th><th rowspan="2">Class</th><th rowspan="2">Nº Species</th><th colspan="2">Occupancy</th><th colspan="2">Abundance</th></tr><tr><th>Number</th><th>(%)</th><th>Number</th><th>(%)</th></tr></thead><tbody><tr><td>Porifera</td><td>Desmospongia</td><td>21</td><td>936</td><td>27.76</td><td>11805</td><td>30.96</td></tr><tr><td rowspan="3">Cnidaria</td><td>Octocorallia (subc.)</td><td>19</td><td>858</td><td>25.44</td><td>6861</td><td>17.99</td></tr><tr><td>Hexacorallia (subc.)</td><td>8</td><td>248</td><td>7.35</td><td>1170</td><td>3.07</td></tr><tr><td>Hydrozoa</td><td>3</td><td>14</td><td>0.42</td><td>16</td><td>0.04</td></tr><tr><td>Briozoa</td><td>Gymnolaemata</td><td>1</td><td>31</td><td>0.92</td><td>47</td><td>0.12</td></tr><tr><td>Brachiopoda</td><td>Rhynchonellata</td><td>1</td><td>467</td><td>13.85</td><td>3219</td><td>8.20</td></tr><tr><td rowspan="2">Annellida</td><td>Polychaeta</td><td>4</td><td>491</td><td>14.56</td><td>2334</td><td>6.12</td></tr><tr><td>Echiura</td><td>1</td><td>49</td><td>1.45</td><td>55</td><td>0.14</td></tr><tr><td rowspan="5">Echinodermata</td><td>Crinoidea</td><td>1</td><td>549</td><td>16.28</td><td>12126</td><td>31.8</td></tr><tr><td>Ophiuroidea</td><td>1</td><td>64</td><td>1.90</td><td>416</td><td>1.09</td></tr><tr><td>Echinoidea</td><td>4</td><td>127</td><td>3.77</td><td>144</td><td>0.38</td></tr><tr><td>Holothuridae</td><td>2</td><td>15</td><td>0.44</td><td>15</td><td>0.04</td></tr><tr><td>Astroidea</td><td>1</td><td>11</td><td>0.33</td><td>12</td><td>0.03</td></tr><tr><td>Chordata</td><td>Ascidiacea</td><td>2</td><td>6</td><td>0.18</td><td>6</td><td>0.02</td></tr></tbody></table>

(Table 1). However, as sampling unit size increased, the resolution of the CCA output decreased (Table 1). This was due to (1) a reduction in the total number of sampling units, (2) an increase in the number of sampling units discarded from the analysis affected by unsuitable sequences, and (3) an increase in the variability of each sampling unit composition (due to the presence of several bottom types in large sampling units) (Table 1). A 5 m² sampling unit size was chosen as the best balance between the inertia explained by environmental factors, and the number of macrobenthic species assemblages identified (Table 1).

Using 5 m² sampling units, a total of 38,136 organisms belonging to 69 macrobenthic species were considered in the analysis. Crinoidea, only

represented by *Leptometra phalangium*, was the most abundant and the third most frequent taxon accounting for 31.8% of all observed organisms, occurring in 16.3% of all sampling units (Table 2). Desmospongia, represented by 21 species, was the second most abundant and the first most frequent taxon, accounting for 30.9% of all observed organisms, in 27.7% of all sampling units (Table 2). The subclass octocorallia, represented by 19 species, was the third most abundant and the second most frequent taxon, accounting for 17.9% of observed organisms, in 25.4% of all sampling units (Table 2). The remaining taxa were represented by 1–4 species, accounting for 0.02–8.2% of observed organisms, in 0.2–14.5% of all sampling units (Table 2).
In the CCA, sampling units and species were displayed constrained according to environmental factors. Environmental factors explained 15.6% of the variation in species abundance. The first axis (CCA1) explained 26.8% of the variance, and the second axis (CCA2) explained 22.7% of the variance. Substrate was the best predictor (inertia = 2.1935) followed by depth (inertia = 1.3017), and slope (inertia = 0.6974). Six different macrobenthic assemblages (Fig. 2a), characterized by environmental factors (Fig. 2b) and most contributing species (Fig. 2c) were identified in the CCA analysis:

Assemblage A: occurring on shallow maërl beds (Fig. 2b), is mostly characterized by the sponges *Haliclona cf. elegans* and *Aplysina cavernicola* (Figs. 2c, 3a and 3b).

**Fig. 2.** Canonical correspondence analysis (CCA) ordination biplots of macrobenthic species. In figure 2a the biplot shows the distribution of the sampling units (n = 3372), sampling units are colored according to the dominant substrate: fine sands (yellow), medium sands to gravels (green), cobbles and pebbles (orange), maërl (blue), outcropping rock (red). In figure 2b the biplot shows the substrate type (FS = fine sands, MG = medium sands to gravels, PC = cobbles and pebbles, M = maërl, OR = outcropping rock), seabed slope (Hor = horizontal, Slo = slopping, Ver = vertical) and depth, being dep1 the shallowest and dep4 the deepest. In figure 2c the biplot shows the contribution of the different species to the macrobenthic assemblages, the scale of grays in the vectors indicate the degree of correlation, being in black the highest correlation degree. Hal_eleg_ = *Haliclona cf. elegans*, Ap_cav_ = *Aplysina cavernicola*, Ham_sp2_ = *Hamacantha* sp., Hal_sp20_ = *Haliclona* sp., Sal_dys_ = *Salmacina* dysteri, Lep_pha_ = *Leptometra phalangium*, Gry_vit_ = *Gryphus vitreus*, Tha_gas_ = *Thalamophyllia gasti*, Ham_fal_ = *Hamacantha falcula*, Vim_fla_ = *Viminella flagellum*, Eun_cav_ = *Eunicella cavolinii*, Nid_stud_ = *Nidalia studeri*, Rhab_sp2_ = *Rhabderemia* sp.

<figure><img src="image_13.png" /></figure>
Assemblage B: occurring on horizontal grounds covered by fine and coarse sands throughout most of the explored depth range (Fig. 2b), is mostly characterized by the presence of the crinoid *Leptometra phalangium* (Figs. 2c and 3c).

Assemblage C: occurring on horizontal grounds covered by medium sands to gravels in deep environments (Fig. 2b), is characterized by the presence of the brachiopod *Gryphus vitreus* (Figs. 2c and 3d).

Assemblage D: mostly found at intermediate depth on mixed cobbles and pebbles bottoms (Fig. 2b), is characterized by the polychaete *Salmacina dysteri* and two unidentified sponge species belonging to the genus *Hamacantha* and *Haliclona* (Figs. 2c, 3e and 3f).

Assemblage E: occurring in slopping rocky out-crops at shallow and intermediate depths (Fig. 2b), is characterized by the presence of the gor-gonians *Eunicella cavolinii*, *Viminella flagellum*, the soft coral *Nidalia studeri*, the solitary coral *Thalamophyllia gasti* and the incrusting sponge *Hamacantha falcula* (Figs. 2c, 3g, 3h, 3i, 3j and 3k).

Assemblage F: occurring on vertical rock out-cropping mostly at intermediate and deep en-vironments (Fig. 2c), is characterized by an in-crusting sponge of the genus *Rhabderemia* (Figs. 2c and 3l).

## 3.2 Geographic distribution of macrobenthic assemblages

Assemblage A was only observed in one single transect in the northeastern side of the Menorca Channel (Fig. 4). Assemblages B and C were widely distributed in both the northern and southern areas of the channel (Fig. 4). Both assemblages presented a certain degree of overlapping, but assemblage C was restricted to deeper environments. Assemblage D was ob-

served in only four locations in the channel, three in the northern slope and one in the head of the Menorca Canyon (Fig. 4), and partially coincided with assemblages A, E and F. Finally, assemblages E and F mostly co-occurred in the transects located in the proximity of Cap For-mentor and the Menorca Canyon's head (Fig. 4).

## 3.3 Variation of macrobenthic diversity with depth

Highest sampling effort was conducted at 90-240 m depth, with sampling effort progres-sively decreasing below this depth. The expo-nential of Shannon's index presented the highest values (3.5-4.0) in the shelf edge depth range (110-160 m depth) (Fig. 5). Intermediate values of the Shannon's index (2.2-3.0) oc-curred at 160-250 m depth, whereas values decreased (<2.5) below 250 m depth, reaching minimum values at 330-350 m depth (Fig. 5).

## 3.4 Fishing activity in the study area

A total of 34 fishing vessels, from eleven ports were observed to regularly trawl in the Menorca Channel. The vessels from the ports of Cala Ratjada, Alcúdia and Ciutadella (Fig. 6) accounted for 77.5% of the localizations. One large (E) and two small (D and C) hot spot areas of bottom trawling were identified on the continental shelf of the channel at 50-75 m depth (Fig. 6). Four additional large hotspots were identified deeper on the continental slope in the north (A) and south area (F, G and H) of the channel at 500-600 m depth (Fig. 6). Fi-nally, a small hot spot area was also observed near Cap Formentor (B) at ~50 m depth (Fig. 6). Three hotspots (A, E and G) concentrated 78.8% of the total bottom trawling fishing ef-fort in the area.
<figure><img src="image_14.png" /></figure>

a

<figure><img src="image_15.png" /></figure>

b

<figure><img src="image_16.png" /></figure>

c

<figure><img src="image_17.png" /></figure>

d

<figure><img src="image_18.png" /></figure>

e

<figure><img src="image_19.png" /></figure>

f

<figure><img src="image_20.png" /></figure>

g

<figure><img src="image_21.png" /></figure>

h

<figure><img src="image_22.png" /></figure>

i

<figure><img src="image_23.png" /></figure>

j

<figure><img src="image_24.png" /></figure>

k

<figure><img src="image_25.png" /></figure>

l
## 4. DISCUSSION

Six different macrobenthic assemblages distributed throughout the continental shelf and upper slope were identified in the Menorca Channel. These assemblages were mainly separated by the substrate type and depth range.

Sponge grounds composed by large desmosponge species (>15 cm in height), mostly characterized by the branched *Haliclona* cf. *elegans* and the tubular sponge *Aplysina cavernicola* (Assemblage A), occurred on a shoal located near the shelf edge at 95-110 m depth (Fig. 4). This shoal presented a highly irregular topography that alternated vertical walls with flat areas covered by maërl and flat coralligenous outcrops. The close proximity of the shoal to the shelf edge (Fig. 4) and its highly irregular topography, agrees with sponge ground distribution in other areas of the world (e.g. Klitgaard and Tendal, 2004). It has been suggested that the irregular topography of these environments may cause the acceleration of local currents (Rice et al., 1990) potentially favoring the presence of sponges (Beazley et al., 2015). A. *cavernicola* and *H.* cf. *elegans* accounted for 73% of all observed organisms, followed by other desmosponge species such us *Poecillastra compressa*, *Aaptos aaptos*, *Haliclona poecilastroides* and *Syphoncahlina* sp., representing 21% of the observed sponges. Species composition resembled that of other sponge grounds from rocky outcrops of the Tyrrhenian Sea continental shelf (Bertolino et al., 2013). Overall, sponge densities (9.2 ± 8.4 individuals

$m^2$, (mean ± SD), max = 43 individuals $m^{-2}$) were similar to those registered in other other Mediterranean sponge grounds (Bo et al., 2011a), and exceeded those observed in deep multispecific sponge grounds in the North Atlantic (Kutti et al., 2013; Howell et al., 2016). The lack of gorgonians (only represented by a few colonies of *Paramuricea macrospina*) and antipatharians basically distinguished this assemblage from Assemblage E, in which almost all the same sponge species (except *A. cavernicola*) co-occurred together with large gorgonians and antipatharians (see below). It has been suggested that under high oligotrophic conditions, such as those observed in the Balearic Archipelago, sponge and other active suspension feeders may become the dominant group in coralligenous community replacing gorgonians (Ballesteros, 2006). However, since gorgonians dominate vast areas of the continental shelf and upper slope of the Menorca Channel (Grinyó et al., 2016), the existence of the observed sponge ground might have been driven by other environmental or historical settings. Sponge grounds increase the structural complexity of habitats and the biodiversity of the associated macrofaunal community (Klitgaard, 1995; Beazley et al., 2013). Moreover, the high water processing capacity of sponges (Morganti, 2016) indicates that sponge grounds play a major role in the benthic-pelagic coupling (Ribes et al., 2005) through their influence on the microbial loop (Yahel et al., 2007) and the cycling of carbon and other elements (Kutti et al., 2013; Cathalot et al., 2015; Maldonado et al., 2015).

*Leptometra phalangium* (Fig. 3c) was widely distributed in both coarse and fine sands (Assemblage B), reaching high densities of more

<figure><img src="image_26.png" /></figure>

**Fig. 3.** Most contributing species a) *Haliclona* cf. *elegans*, b) *Aplysina* *cavernicola*, c) *Leptometra* *phalangium*, d) *Gryphus* *vitreus*, e) *Salmacina* *dysteri*, f) *Hamacantha* sp. 2 (white circle) and *Haliclona* sp. 1 (black circle), g) *Eunice* *cavolinii*, h) *Viminella* *flagellum* i) *Nidalia* *studeri*, j) *Thalamophyllia* *gasti*, k) *Hamacantha* *falcula*, l) *Rhabderemia* sp. Scale Bar: 10 cm.
than 30 individuals m⁻² (5.3 ± 3.2 individuals m⁻² (mean ± SD)), as previously observed on the base of seamounts in the Tyrrhenian Sea (Bo et al., 2010). In the Menorca Channel, this almost monospecific assemblage (*L. phalangium* represented 95% of all observed organisms) mostly occurred on the shelf edge (96% of occupied sampling units) (Fig. 4), agreeing with studies that characterized it as a typical shelf edge assemblage (e.g. Fredj, 1964; Kallianiotis et al., 2000; Mangano et al., 2010). It has been suggested that *Leptometra* beds are mainly constrained to shelf edge environments as they depend upon the exposition to bottom currents that regularly carry high concentrations of suspended organic particles (Lavaleye et al., 2002; Colloca et al., 2003). Absence of trawling activity is fundamental for the persistence of high-density *L. phalangium* populations, since the fragility of this organism makes it extremely vulnerable to mechanical damage (Smith et al., 2000). *L. phalangium* beds have been associated with elevated densities of benthopelagic fish, with high juvenile

abundances, suggesting that this assemblage may play a crucial role in the life cycle of numerous fish species, some of which are of high commercial interest (e.g., *Merlucius merlucius* and *Mullus barbatus*) (Colloca et al., 2004). The sea pen *Funiculina quadrangularis*, the polychaete *Lanice conchilega* and the anemones *Cerianthus membranaceus* and *Arachnanthus oligopodus* accompanied *L. phalangium* on coarse sands, together with the sea pen *Virgularia mirabilis*, the soft coral *Alcyonium palmatum*, and the sponge *Thenea muricata* on fine sands. The presence of *T. muricata* on the shelf edge (~130 m depth) was rather surprising, as it has mostly been reported in deeper areas of the continental slope (e.g. Pérès, 1967; Michez et al., 2014).

The brachiopod *Gryphus vitreus* (Fig. 3d) constituted 81% of all observed organisms in the Assemblage C. As previously described in other areas of the Mediterranean, this species sparsely covered (2 ± 3 individuals m⁻² (mean ± SD)) sandy grounds on and beyond the shelf

<figure><img src="image_27.png" /></figure>

**Fig. 4.** Macrobenthic assemblages distribution. Assemblages were represented on the study area based on the transects where they occur, and following the colors as in figure 2: fine sands (yellow), medium sands to gravels (green), cobbles and pebbles (orange), maërl (blue), outcropping rock (red).
edge, at 110–330 m depth (e.g. Emig, 1987; 1989). Assemblage C presented similar distribution of Assemblage B, occasionally co-occurring for tens of meters. However, while assemblage B was observed on both fine and coarse sands, Assemblage C was basically restricted to coarse sands (Figs. 2 and 4). This substrate segregation responds to the necessity of *G. vitreus* to fix to small pebbles or shells on the surface of the sediment (Emig, 1987). In the upper slope, at 180–200 m depth, *G. vitreus* formed dens aggregations with more than 25 individuals m⁻². However, these densities observed in the study area are rather low

if compared to those reported from continental slopes exposed to intense bottom currents (600 individuals m⁻²) (Emig, 1987). In this regard, *G. vitreus* and other brachiopod occurrence and abundance appear to be directly related to hydrodynamic conditions (Eshleman and Wilkens, 1979; Emig, 1987). The composition of accompanying species in coarse sands of assemblage B and C was very similar, with the sea pen *F. quadrangularis*, the polychaete *L. conchilega* and the anemones *C. membranaceus* and *A. oligopodus* amongst the most abundant. *A. oligopodus* and *L. conchilega*, have commonly been reported in soft sediments of the shelf edge and continental slope of the Mediterranean Sea (e.g. Pérès and Picard, 1964; Emig, 1997; Michez et al., 2014), occasionally formed high density (9 individuals m⁻²) aggregation in the Menorca Channel.

The polychaete *Salmacina distery* (Fig. 3e) and two unidentified sponges belonging to the genus *Hamacantha* and *Haliclona* (Fig. 3f) characterized the Assemblage D, occurring on coarse sand scattered with cobbles and pebbles, at 95–140 m depth. These three species represented 63% of all observed organisms, and rarely exceeded densities of 1.5 individuals m⁻². Despite the mixed substrate where this assemblage was observed, the vast majority of the organisms were associated to hard substrates (e.g. the sponges *P. compressa*, *A. aaptos* and *Pachastrella monilifera* (26% of observed organisms), and the gorgonians *E. cavolinii* and *Swiftia pallida* (10% of observed organisms). Conversely, coarse sand patches were mainly occupied by echinoids, holothurians and polychaetes (1% of observed organisms). Isolated *L. phalangium* were also occasionally observed. The occurrence of Assemblage D in the same transects than Assemblages A and E (Fig. 4) (mainly in the immediate proximities of outcropping rocks and vertical walls on the shelf edge, and in areas of dead maërl rodolyths in the proximities of

<figure><img src="image_28.png" /><figcaption>**Fig. 5.** Vertical distribution of the Shannon's exponential entropy index along the studied bathymetric range. Depth was subdivided at 5 m intervals. Gray-scale histograms represent the total number of sampling units for each substrate type (see legend).</figcaption></figure>
shelf shoals) suggests that Assemblage D is a transition between assemblages occurring on hard substrates and those located in coarse sand ones.

The gorgonians *Eunicella cavolinii* and *Viminella flagellum*, the soft coral *Nidalia studeri*, the small solitary coral *Thalamophyllia gasti* and the incrusting sponge *Hamacantha falcula* (Figs. 3g, 3h, 3i, 3j and 3k) characterized (43% of all observed organisms) the sloping rocky substrates (Assemblage E) from the continental shelf to the upper slope (95–340 m depth). Despite this wide bathymetric distribution, this coral garden assemblage (sensu Stone, 2006; Buhl-Mortensen and Buhl-Mortensen, 2013) mostly occurred on the shelf edge at 110–190 m depth (72% of observed sampling units), on rocky outcrops and vertical walls near Cap

Formentor and the Menorca canyon head (Fig. 4). These environments are most likely exposed to regular currents that may enhance food availability for the large sized passive suspension feeders that characterize this assemblage (Shepard et al., 1974; Balbín et al., 2012). Both the gorgonians *E. cavolinii* and *V. flagellum* had previously been reported as dominant species of rocky assemblages on the Mediterranean shelf edge (Bo et al., 2011b; Angiolillo et al., 2014). In the Menorca Channel, both species mostly formed mixed assemblages along with other species (Grinyó et al., 2016), but they can punctually form dense monospecific patches of up to 15 colonies m⁻², as also observed in other areas of the Mediterranean (Pedel and Fabri, 2011; Angiolillo et al., 2014). *N. studeri* and *T. gasti* formed dense monospecific assemblages (30 colonies m⁻²). *N. studeri* and *T. gasti* formed

<figure><img src="image_29.png" /><figcaption>Fig. 6. Bottom trawling fishing effort in the Menorca Channel.</figcaption></figure>
eri mostly occurred on gently sloping rocky outcrops, whereas *T. gasti* on steeply sloping rocky outcrops. The incrusting sponge *H. falcula* showed low maximum densities of 5 individuals m⁻², but large dimensions (>20 cm diameter), covering large areas of the rocky bottom, and perhaps excluding other species (Fig. 3k). Assemblage E was the most diverse of the six macrobenthic assemblages identified in the study area. Eighteen desmosponges were observed as accompanying species (25% of all observed organisms) mostly as scattered individuals spread throughout the transects (2.4 ± 4 individuals m⁻² (mean ± SD)), and only punctually reaching high densities (15 individuals m⁻²). Some of these sponges reached large dimensions (>20 cm height and width) such as the massive *Haliclona magna* or the fan shaped *Phakellia robusta*, *P. monilifera* and *P. compressa*. Anthozoans accounted for 57% of all observed organisms, with 22 different species. Besides *E. singularis* and *V. flagellum*, eight gorgonian species were also observed in this assemblage: *Acanthogorgia hirsuta*, *Bebryce mollis*, *Callogorgia verticillata*, *Corallium rubrum*, *Muriceides lepida*, *Paramuricea clavata*, *P. macrospina* and *S. pallida*. Gorgonians formed multispecific high-density patches (~20 colonies m⁻²) (Grinyó et al., 2016), which contrast with the lower gorgonian diversity in coastal and bathyal Mediterranean zones (e.g. Gori et al., 2011; Cartes et al., 2013). The antipatharians *Antipathella subpinnata*, *Antipathes dichotoma*, and *Leiopathes glaberrima* occurred at low densities (1–4 colonies m⁻²) in mixed assemblages with gorgonians, as also observed at similar depth in other areas of the Mediterranean Sea (e.g. Bo et al., 2009, 2012; Deidun et al., 2014). Finally, the recently described soft coral *Chronephthya mediterranea* (López-González et al., 2015), and *Paralcyonium spinulosum* were also largely represented in these rocky bottoms. Coral gardens are known to provide habitat to numerous organisms (Auster et al., 2013; De Clippele et al., 2015) and act as nursery grounds for several fish

species (Bo et al., 2015; Etnoyer and Warrenchuk, 2007). Due to the complex three-dimensional structure they form, and the slow growth of gorgonian and antipatharian species (Sherwood and Edinger, 2009), coral gardens are extremely vulnerable to fishing damages (Angiolillo et al., 2015) and pollution (Silva et al., 2015), showing extremely slow recovery from anthropogenic impacts (Althaus et al., 2009).

Finally, vertical rocky substrates at 95–210 m depth were dominated by incrusting sponges (Assemblage F), with an unidentified incrusting sponge of the genus *Rhabderemia* (Fig. 3l) accounting for 64% of all observed organisms (max = 23 individuals m⁻²), and other incrusting sponges representing 10% of all observed organisms. Incrusting sponges seem to be the only organisms adapted to colonize this environment, alternating with the conversely highly diverse coral gardens (Assemblage E), depending on the substrate inclination.

Unlike areas of the Gulf of Lions (Pérès and Picard, 1964; Orejas et al., 2009; Gori et al., 2013; Fabri et al., 2014) and the Catalan margin (Lastras et al., 2016), only a few colonies of the cold-water coral *Madrepora oculata* were observed on vertical rocky walls at 320 m depth in the Menorca Channel. This could suggest that this species might be restricted to deeper environments in the study area (> 350 m depth), as observed in other areas of the Mediterranean (e.g. Etiope et al., 2010; Taviani et al., 2015).

Macrofauna biodiversity was higher on the shelf edge, and progressively decline with depth (Fig. 5). This diversity pattern probably results from the synergy caused by habitat heterogeneity and hydrodynamic conditions in the shelf edge. Indeed, rocky outcrops and vertical walls alternate with different grain sized soft sediments grounds in the shelf edge. Moreover, both passive and active suspension
feeders may benefit from the stable environ-
mental conditions and hydrodynamic
processes that enhance particle suspension in
the near-bottom water layers on the shelf edge
(Thiem et al., 2006). It has also been suggested
that the observed increment in macrofauna di-
versity at intermediate depth may result from
the merging of species with shallower and
deeper distributions, causing a mid domain ef-
fect (Colwell and Less, 2000) as previously ob-
served for deep coral communities in the north
Pacific (Stone, 2006; Matsumoto et al., 2007).
Benthic macrofauna in general, erected and
massive sponges (Klitgaard and Tendal, 2004),
gorgonians (Maynou and Cartes, 2012), an-
tipatharians (Koslow et al., 2001) and crinoids
(Smith et al., 2000) in particular, are extremely
sensitive to bottom trawling fishing. Conse-
quently, the low trawling pressure occurring
at 100–500 m depth in the study area (Fig. 6)
may be one of the main reasons for the preser-
vation of the observed high-density and high-
diverse macrofauna assemblages on the con-
tinental shelf edge. Decades of chronic bottom
trawling on the continental shelf and edge
(Watling and Norse, 1998) have probably heav-
ily impacted and fragmented macrobenthic as-
semblages (Hall-Spencer et al., 2002), confin-
ing them to remote an inaccessible
environments (Díaz et al., 2015; Bo et al., 2015).
In this sense, the observed complexity and
high-diversity of benthic macrofauna, could
suggest how benthic community had been in
several areas of the Mediterranean continental
shelf and shelf edge, before the cumulative
impact of decades of bottom trawling fishing.
Management and conservation measures
based on the knowledge of deep benthic com-
munities composition and distribution should
aim at limiting towed bottom fishing gears over
the most sensitive areas, and establish large
protection zones to protect and ensure con-
servation of these increasingly rare macroben-
thic assemblages. Although the establishment
of these large protection zones may pose po-
tentially challenging situations amongst stake-

holders, large protection zones may have eco-
nomic benefits including the recovery of de-
graded fisheries (Pipitone et al., 2000) and the
maintenance of ecosystem services (Balmford
et al., 2002).

ACKNOWLEDGEMENTS

The authors thank the crew of the R/V *García del Cid*, the JAGO team (J. Schauer and K. Hissmann) from IFM-GEOMAR (Kiel, Germany), the NEMO ROV pilot Gavin Newman. David Díaz, Andreu Santín, Maria Montseny, Anthi Oikonomou, Artemis Atsalaki, Pablo J. López-González, Maria Jesus Uriz and Mikel Zabala for their advice and help during the oceanographic surveys and the video analysis. 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).

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Chapter 2

# Diversity, distribution and population size structure of deep Mediterranean gorgonian assemblages (Menorca Channel, Western Mediterranean Sea)

## ABSTRACT

Gorgonians are a key group of organisms in benthic marine communities with a wide bathymetric and geographical distribution. Although their presence on continental shelves and slopes has been known for more than 100 years, knowledge concerning the ecology of deep gorgonian species is still in a very preliminary stage. To overcome this situation, gorgonian assemblages located at 40–360 m depth were studied over a large geographical area on the continental shelf and upper slope of the Menorca Channel (Western Mediterranean Sea). A quantitative analysis of video transects recorded by a manned submersible and a remotely operated vehicle, were used to examine the diversity, distribution and demography of gorgonian species. Results showed high gorgonian diversity within this depth range (a total of nine species were observed) compared to Mediterranean coastal areas. Gorgonian assemblages on the continental shelf and upper slope were mostly monospecific (respectively 73% and 76% of occupied sampling units contained one single species), whereas shelf edge assemblages were highly multispecific (92% of occupied sampling units contained several species). This contrasts with the monospecificity of Mediterranean coastal gorgonian assemblages. Gorgonian populations on the continental shelf were mostly dominated by small colonies (88% of measured colonies) with few intermediate and large colonies (12% of measured colonies). In deeper areas small colonies were still dominant (60% of measured colonies), but intermediate and large colonies were much more abundant (40% of measured colonies). This suggests high recruitment rates on the continental shelf, but perturbations (trammel nets, long lines and strong storms) may limit the presence of intermediate and large colonies. Conversely, on the shelf edge and upper slope a more stable environment may allow colonies to reach larger dimensions. The identification and ecological characterization of these deep assemblages further extends the current knowledge about Mediterranean gorgonians, and is fundamental in improving the management and conservation of deep benthic ecosystems.

## CO-AUTHORS

A Gori, S Ambroso, A Purroy, C Calatayud, C Domínguez-Carrió, M Coppari, C L Iacono, P J López-González, J M Gili

## ADDITIONAL RESOURCES

Progress in Oceanography (2016) 145, 42–56,
# 1. INTRODUCTION

Gorgonian assemblages play an important structural and functional role in several marine benthic ecosystems all over the world, promoting a high diversity and biomass of associated fauna (Gili and Coma, 1998). From a structural point of view, gorgonians act as ecosystem engineers (sensu Jones et al., 1994) and foundation species (sensu Dayton, 1972) forming complex three-dimensional structures that provide a suitable habitat and refuge for numerous species (Buhl-Mortensen and Mortensen, 2005; Roberts et al., 2009; De Clippele et al., 2015). Current flow, food availability, and sediment re-suspension, vary widely within the complex structures formed by the gorgonian colonies, and this heterogeneity increases the abundance and functional diversity of the associated fauna (Witherell and Coon, 2001; Stone, 2006; Cerrano et al., 2010). From a functional point of view, gorgonians promote a significant flow of matter and energy from the pelagic to the benthic system by capturing plankton and suspended particulate organic matter (Lewis, 1982; Ribes et al., 1999; Sherwood et al., 2008), playing a paramount role in benthic-pelagic coupling processes and biogeochemical cycles (Gili and Coma, 1998; Coma et al., 2001; Hill et al., 2014).

Knowledge about diversity, distribution, ecology and state of conservation of gorgonian assemblages in coastal areas has significantly increased in recent decades based on the research carried out within the depth range of traditional SCUBA diving (~40 m depth) (e.g. Grigg, 1977; Yoshioka and Yoshioka, 1989; Linares et al., 2008). Far less is known about deep gorgonian assemblages located on the continental shelf and slope (e.g. Mortensen

and Buhl-Mortensen, 2004; Matsumoto et al., 2007; Salomidi et al., 2009; Stone et al., 2014), where gorgonian presence was mostly known due to specimens by-cached by fishermen or withdrawn by blind destructive techniques (Relini et al., 1986) for taxonomical studies (Carpine and Grasshoff, 1975; Grasshoff, 1992). The most recent technological development and increased availability of remotely operated vehicles (ROVs), manned submersibles, and video-equipped towed gears have significantly increased accessibility to deeper areas, allowing for controlled sampling and quantitative study of deep rocky bottoms communities (e.g. Reed et al., 2006; Mortensen et al., 2008; Orejas et al., 2009). Abiotic features change considerably with depth (Bell and Barnes, 2000; Garrabou et al., 2002; Bak et al., 2005), and the overall variability in environmental conditions is dampened at greater depths (below ~100 m depth) because temperature, currents, and other water column features are more constant than in shallower areas (e.g. Puig et al., 2000; De Mole et al., 2002; Fernández de Puelles et al., 2007). Such stability in the main environmental conditions could allow deep gorgonians to grow larger and develop populations dominated by medium and large-sized colonies (Grigg, 1975; Watanabe et al., 2009; Gori et al., 2011b). Toppling and detachment by strong currents or wave action is considered one of the main causes of mortality of large gorgonian colonies (Grigg, 1977; Weinbauer and Velimirov, 1996; Weinberg and Weinberg, 1979). Indeed, both the environmental suitability and stability of a habitat may be reflected in the size structure of gorgonian and coral populations, because the size structure reflects the factors affecting recruitment, growth, and mortality rates in a particular habitat for a period of time matching the longevity of the population (Grigg, 1975; Gilmour, 2004; Alvarado-Chacón and Acosta, 2009).

Earlier ecological studies on deep gorgonian assemblages in the Mediterranean Sea re-
vealed that they can reach density values as high as those observed in littoral environments (e.g. Bo et al., 2009; Angiolillo et al., 2014). However, both the continental shelf and upper slope are areas where fishing is more intense (Watling and Norse, 1998; Hall-Spencer, 2002). Gorgonians are among the most common species of the by-catch of bottom trawling, trammel nets, and long line fishing (Van Dolah et al., 1987; Krieger, 2001; Mytilineou et al., 2014). Since gorgonians are usually long-lived and slow growing (Linares et al., 2007; Risk et al., 2002; Sherwood and Edinger, 2009), fishing impacts can have far-reaching and long-lasting effects (Althaus et al., 2009). Consequently, deep gorgonian assemblages located on the continental shelf and upper slope are nowadays highly threatened by fishing activity (Hall-Spencer et al., 2002), and knowledge about their diversity, distribution and state of conservation is urgently needed to implement effective management and conservation measures of deep benthic ecosystems.

To this purpose, the aims of this study were: (1) to characterize the diversity and quantify the abundance of gorgonians forming Mediterranean gorgonian assemblages on a large area of the continental shelf and upper slope at 40–360 m depth (2) to assess the geographical and vertical distribution patterns of the observed species; (3) to appraise their population size structure, and (4) to gain insight into the possible factors affecting their abundance and distribution.

## 2. MATERIAL AND METHODS

### 2.1 Study area

The Menorca Channel is located in the Western Mediterranean Sea between Mallorca and

Menorca Islands (39° 53' 0.73" N, 3° 29' 51.16" E) (Fig. 1a), as part of the Balearic Promontory (Acosta et al., 2002). The continental shelf in this area extends between 40 and ~110 m depth, and is largely covered by maërl alternating with patches of coastal detritic sediments and coralligenous outcrops (Barberá et al., 2012). Conversely, smooth reliefs and large extensions of detritic sediments with few isolated patches of outcropping rocks characterize most of the shelf edge and continental slope. Only near Cap Formentor (Fig. 1b) and in the Menorca Canyon (Fig. 1c) sharply edged rock outcroppings and rocky vertical walls are the dominant substrates.

The northern shelf edge and continental slope of the study area is mainly influenced by the Balearic Current (Balbín et al., 2012) and its associated front (Ruiz et al., 2009), which originates from a branch of the Liguro-Provençal-Catalan current, flowing northward over the continental slope of the Balearic archipelago at ~200 m depth (López García et al., 1994; Ruiz et al., 2009). Secondary currents flow northward from the Algerian subbasin entering the Balearic subbasin and feed the Balearic current (Alemany et al., 2006; Amores et al., 2013). Conversely, the southern shelf edge and upper slope is not influenced by a steady current (Amores and Montserrat, 2014), but by the sporadic arrival of mesoscale structures detached from the Algerian Current and the Almería-Oran front (Millot, 1987; García et al., 2005).

### 2.2 Sampling procedure

A total of 73 video transects (Fig. 1a) were recorded during five surveys conducted on board of the R/V "García del Cid" (September 2010, April 2011, October 2011, June 2012) and the R/V "Miguel Oliver" (August 2011). During the first two surveys, 20 video transects were recorded with the manned submersible JAGO (IFM-GEOMAR), whereas 53 video transects were recorded with the ROV NEMO (Gavin
Newman) during the last three campaigns. Both instruments were equipped with a 1080 horizontal line resolution camera, a grabber and two parallel laser beams which provided a scale to define a fixed width of the transects (0.3 m) during the subsequent video analysis. Transects were recorded in a close-zoom (~0.5-1.5 m width of view) and in a digital format. Positioning of JAGO and NEMO was achieved with underwater acoustic positioning

system (LinkQuest TrackLink 1500 HA). Both JAGO and NEMO moved at a constant speed of ~0.3 knots, and transect lengths ranged between 309 and 2375 m, over depths ranging from 52 to 347 m. Transects were haphazardly located in order to cover the whole study area, but areas showing morphological features possibly related to the presence of rocky bottoms were explored more intensively (Fig. 1). Overall, a total of 57.8 km was video recorded along the seabed.

In order to confirm the taxonomic identification of the species observed in the video-transects, voucher colonies of gorgonian species observed along the transects were also collected. Colonies were fixed and preserved in 10% formalin until analyzed in the laboratory.

## 2.3 Video analysis

Quantitative video analysis was performed according to the methodology described in Gori et al. (2011a) using Apple's Final Cut Pro software. All the pauses in the movement of the JAGO or NEMO were removed from the footage, in order to correctly estimate the length in each transect, and those sequences with poor image quality or too far away from the seafloor were considered unsuitable for analysis. The remaining useful sequences comprised 94.6% of the total recorded material and corresponded to a total distance of 54.7 km. Every gorgonian observed within a width of 0.3 m (based on the laser beams) along each video transect was identified with a time reference derived from the time elapsed since the beginning of the video transect to the crossing of the laser beams with the base of the colony (Gori et al., 2011a). This time reference was posteriorly converted into position along the transect according to known velocity $P = t \cdot v$, where t is the time reference expressed in seconds, and v is the velocity expressed in meters per second). A similar procedure was used to characterize seabed

<figure><img src="image_30.png" /><figcaption>**Fig. 1.** The study area. (a) Three-dimensional bathymetry of the Menorca Channel: the map shows the location of the video transects (1 to 73) and the location of the study area in the western Mediterranean. Detail of the (b) northern and (c) southern subareas.</figcaption></figure>
substrate types and slope along every transect, within the same width of 0.3 m. Seabed substrate type was classified (based on the Wentworth scale) into four categories: sand to gravel, cobbles and pebbles, maërl, and rock. Seabed slope was classified into three categories: horizontal (0°–30°), sloping (30°–80°) and vertical (80°–90°); slope was estimated from the video by looking at the two parallel laser beams and the depth sensor (Gori et al., 2011a; Ambroso et al., 2013).

To study population size structure, the maximum height of each observed gorgonian colony was measured using the Macnification 2.0.1 software (Schols and Lorson, 2008) on still images extracted from recorded footage. The distance from the two lasers beams was used to calibrate the images. Measurements were done on still images in which the colony base was in the same plane as the laser beams, while the colony lied perpendicular to the video (Gori et al., 2011b). This methodological constraint entails that only a subsample of observed gorgonians could be measured for the study of population size structure (81% of the total observed colonies, 63%–100% of the colonies in each transect).

## 2.4 Species identification

Identification of the observed species was based on the existing taxonomic works on Atlanto-Mediterranean gorgonians (Carpine and Grasshoff, 1975; Grasshoff, 1992), and confirmed by the examination of the voucher specimen collected (see section 2.2), looking at the colonial branching pattern, the distribution and shape of calyces and polyps along branches, as well as the shape and size of sclerites. For this purpose, small fractions of the sampled colonies were placed in a sodium hypochlorite solution until organic matter was dissolved and sclerites disaggregated. Sclerites were rinsed with distilled water, and observed under a stereoscope (Olympus SZ-60). Three Para-

*muricea macropsina* (Koch, 1882) chromatic morphotypes were differentiated in the video, two chromatic morphotypes on the continental shelf (M1, M2) and a third one on the shelf edge (M3) (more details in Grinyó et al. (submitted) (Figs. 2c, d and e)).

## 2.5 Data treatment

### 2.5.1 Gorgonian occupancy and abundance

To quantify gorgonian occupancy (frequency of occurrence in the set of sampling units) and abundance (number of colonies per sampling unit), and examine the species composition of gorgonian assemblages within the study area, each transect track was divided into a string of 2 m² (0.3 m width and 6.66 m long) sampling units, using ArcMap 10.1 software (ESRI ArcGIS ArcInfo v10). Such sampling unit size was chosen as representative of Mediterranean oc-to-corals on rocky substrate (based on Weinberg, 1978), as well as to allow a comparison with previous studies on shallow gorgonian and coral species (Gori et al., 2011a; Ambroso et al., 2013). A total of 8221 sampling units were obtained from the 73 transects, corre-sponding to a total area of 0.016 km². Each sampling unit was characterized by the number of colonies of each gorgonian species, as well as by its depth and coverage percentage for each substrate and slope type. For each gor-gonian species, occupancy and abundance were quantified. Gorgonian assemblages were assessed based on species composition using a non-metric multidimensional scaling ordina-tion (nMDS) of the sampling units, with gor-gonian abundances square root transformed, and ordination by a Bray-Curtis similarity ma-trix. Adonis permutation multivariate analysis of variance and subsequent pairwise tests were used to test for significance of differ-ences in gorgonian assemblages based on depth: continental shelf (40–100 m depth), shelf edge (100–180 m depth), upper slope (180–360 m depth), as well as substrate type
(sand to gravel, cobbles and pebbles, maërl and rock), and slope (horizontal, sloping and vertical). Dominant substrate and slope type was assigned to each sampling unit for this analysis. Ordination and permutation multivariate analysis of variance and subsequent pairwise tests were performed using the r-language functions metaMDS and adonis, respectively, which are available in the vegan library (Oksanen et al., 2015) of the R software platform (R Core Team 2014). The adonis test was performed considering depth, substrate and slope simultaneously. Additionally, a similarity percentage procedure analysis (SIMPER, Clarke and Warwick, 1994) was performed to identify which gorgonian species contributed the most to the different assemblages considering the previously mentioned environmental features using the software PRIMER v6 (Clarke and Warwick, 2001).

## 2.5.2 Geographical and vertical distribution

Geographical distribution of each species in the study area was studied by mapping the observed density on a geographically referenced map using GIS (ESRI ArcGIS ArcInfo v10). Vertical distribution of each species was studied grouping sampling units in 20 m depth intervals (based on their depth), and estimating the median (first and third quartile, and the range between minimum and maximum values) of gorgonian density in each depth interval.

## 2.5.3 Population size structure

Size structure of gorgonian populations was analyzed in terms of descriptive statistics using distribution parameters such as skewness and kurtosis. Skewness is a measure of the symmetry of a distribution using its mean, reflecting the proportion of small versus large colonies in a gorgonian population. If skewness is significant (p<0.05) population size structure is asymmetrical. Positive skewness denotes the prevalence of small size colonies,

while negative skewness denotes the dominance of large size colonies in the population. Kurtosis is a measure of the peakedness of a distribution near its central mode. A significant kurtosis value (p<0.05) indicates longer tails than would be expected for a normal distribution, and therefore a particular colony size prevails in the population. Skewness and kurtosis were calculated by means of the r-language functions agostino.test (Komsta and Novomestky, 2012) and anscombe.test (Anscombe and Glynn, 1983), which are available in the moments library of the R software platform. Only populations with more than 40 colonies were studied for their population size structure in order to perform meaningful skewness and kurtosis estimates.

## 2.5.4 Relationship with environmental features

Relationships between gorgonian abundances and depth, substrate type and slope were explored by means of canonical correspondence analysis (CCA), using the ordination software Canoco 4.5 (ter Braak and Smilauer, 2002). For this analysis, each sampling unit was characterized by its depth and the percentage coverage of each substrate and slope type. Gorgonian's abundance was log transformed, and the statistical significance of the species-environment relationship was evaluated using Monte Carlo permutation test using 999 unrestricted permutations.

## 2.5.5 Spatial structure

Finally, spatial structure in the distribution of one gorgonian species respect to the others was analyzed along 3 of the video transects, chosen in order to be representative of the assemblages identified in the study area. Abundance of each gorgonian species along the transects was displayed in density plots, obtained by transforming each transect into a string of contiguous quadrats (0.2 x 0.3 m) and
counting the number of colonies of each
species inside each quadrat. Spatial covariance
between pairs of species was analyzed with
the Three-Term Local Quadrat Covariance
(3TLQC) function, using the Passage 2.0 soft-
ware (Rosenberg, 2008). In the 3TLQC, the
variance as a function of block size for species
A (Var A), specie B (Var B), and the combined
number (Var A+B) (Cov=Var A+B-Var A-Var B)

is examined (Dale, 1999). To test the null hy-
pothesis of an independent distribution of one
species with respect to the other along each
transect, randomized transects were generated
for comparison by randomly permuting the "la-
bels" (species A or B) of all the observed
colonies along each transect. For statistical sig-
nificance (95% confidence interval) the values
at the limit of 2.5% tails of 999 of these ran-

<figure><img src="image_31.png" /><figcaption>**Fig. 2.** Studied species images. (a) *Eunicella singularis*, (b) *Paramuricea clavata*, (c) *Paramuricea macrospina* morphotypes (M1), (d) M2, (e) M3, (f) *Eunicella cavolinii*, (g) *Viminella flagellum*, (h) *Acanthogorgia hirsuta*, (i) *Callolgorgia verticillata*, (j) *Swiftia pallida*, (k) *Bebryce mollis*. Scale Bar: 10 cm.</figcaption></figure>
domizations was used. In the produced plots of covariance as a function of block size, position of a significantly positive deviated sample statistic from the bounds of the confidence interval (peaks) indicates the scale of any positive association (attraction) between species, whereas significant negative deviations (valleys) indicate the scale of any negative association (repulsion) (Dale, 1999).

## 3. RESULTS

### 3.1 Gorgonian occupancy and abundance

A total of 7802 colonies of nine gorgonian species (Fig. 2) were observed along all transects (Table 1), occurring in 18.9% of the 8220 sampling units. Overall, *P. macrospina* was the most abundant and frequent species, with its three morphotypes representing 44% of observed colonies present in 6.8% of the sampling units. *Eunicella cavolinii* (Koch, 1887) was the second most abundant and the third most frequent species, whereas *Eunicella singularis* (Esper, 1791) was the third most abundant and the second most frequent species. *Swiftia pallida* Madsen, 1970 and *Viminella flagellum* (Johnson, 1863) were the fourth and fifth most abundant and frequent species. The other species accounted for less than 2% of the observed colonies, occurring in less than 1% of the sampling units.

**Fig. 3.** Non-metric multidimensional scaling (nMDS) ordination plot. Gorgonian abundance was square root transformed and analysis performed on Bray-Curtis dissimilarity matrix. Sampling units (n= 1562) containing gorgonians are represented considering (a) depth range, (b) bottom type and (c) seabed slope. A stress estimate of 0.025 was obtained.

<figure><img src="image_32.png" /></figure>

<figure><img src="image_33.png" /></figure>

<figure><img src="image_34.png" /></figure>
<table><caption>**Table 1.** Gorgonian occupancy and abundance in the study area. Occupancy (frequency of occurrence in the set of sampling units) is given for each bathymetric range and species; abundance (number of colonies); mean and maximum density of each species is given per each bathymetric range.</caption><thead><tr><th rowspan="2">Depth-range</th><th colspan="3">Sampling Units</th><th rowspan="2">Species</th><th colspan="2">Occupancy</th><th colspan="2">Abundance</th><th rowspan="2">Mean density ± SD<br>(colonies · m<sup>-2</sup>)</th><th rowspan="2">Max density<br>(colonies · m<sup>-2</sup>)</th></tr><tr><th>N°</th><th>with gorgonians</th><th>(%)</th><th>N°</th><th>(%)</th><th>N°</th><th>(%)</th></tr></thead><tbody><tr><td rowspan="4">Continental shelf<br>(40 - 100 m)</td><td rowspan="4">2723</td><td rowspan="4">735</td><td rowspan="4">(27)</td><td>E. singularis</td><td>357</td><td>(48.5)</td><td>966</td><td>(23.0)</td><td>1.2 ± 1</td><td>7.5</td></tr><tr><td>P. clavata</td><td>22</td><td>(3.1)</td><td>48</td><td>(1.1)</td><td>1.1 ± 0.8</td><td>3</td></tr><tr><td>P. macrospina M1</td><td>292</td><td>(39.7)</td><td>1899</td><td>(44.0)</td><td>3.2 ± 5</td><td>33</td></tr><tr><td>P. macrospina M2</td><td>208</td><td>(29.0)</td><td>1370</td><td>(32.0)</td><td>3.6 ± 3.9</td><td>18.5</td></tr><tr><td rowspan="10">Shelf edge<br>(100 - 180 m)</td><td rowspan="10">3338</td><td rowspan="10">751</td><td rowspan="10">(22.5)</td><td>P. clavata</td><td>10</td><td>(1.3)</td><td>16</td><td>(0.5)</td><td>1.2 ± 1</td><td>3.0</td></tr><tr><td>P. macrospina M1</td><td>3</td><td>(0.4)</td><td>4</td><td>(0.1)</td><td>0.7 ± 0.3</td><td>1.0</td></tr><tr><td>P. macrospina M2</td><td>7</td><td>(0.9)</td><td>11</td><td>(0.3)</td><td>0.8 ± 0.7</td><td>2.5</td></tr><tr><td>P. macrospina M3</td><td>51</td><td>(6.8)</td><td>149</td><td>(4.6)</td><td>1.5 ± 1.7</td><td>9.0</td></tr><tr><td>E. cavolinii</td><td>308</td><td>(41.0)</td><td>1768</td><td>(55.1)</td><td>2.9 ± 2.7</td><td>24</td></tr><tr><td>V. flagellum</td><td>65</td><td>(8.7)</td><td>425</td><td>(13.2)</td><td>3.2 ± 5.4</td><td>27.5</td></tr><tr><td>A. hirsuta</td><td>17</td><td>(2.3)</td><td>34</td><td>(1.1)</td><td>1.0 ± 1.0</td><td>4.5</td></tr><tr><td>C. verticillata</td><td>102</td><td>(13.6)</td><td>102</td><td>(3.2)</td><td>1.0 ± 0.9</td><td>5</td></tr><tr><td>S. pallida</td><td>141</td><td>(18.8)</td><td>606</td><td>(18.9)</td><td>2.1 ± 2.6</td><td>13</td></tr><tr><td>B. mollis</td><td>47</td><td>(6.3)</td><td>93</td><td>(2.9)</td><td>1.0 ± 1.0</td><td>5.5</td></tr><tr><td rowspan="5">Upper slope<br>(180 - 360 m)</td><td rowspan="5">2159</td><td rowspan="5">75</td><td rowspan="5">(3.5)</td><td>E. cavolinii</td><td>8</td><td>(10.6)</td><td>16</td><td>(5.1)</td><td>1.0 ± 0.9</td><td>3</td></tr><tr><td>V. flagellum</td><td>19</td><td>(25.3)</td><td>118</td><td>(37.9)</td><td>3.1 ± 4.5</td><td>17</td></tr><tr><td>C. verticillata</td><td>23</td><td>(30.7)</td><td>32</td><td>(10.3)</td><td>0.7 ± 0.3</td><td>1.5</td></tr><tr><td>S. pallida</td><td>23</td><td>(30.7)</td><td>79</td><td>(25.4)</td><td>1.6 ± 2.8</td><td>14</td></tr><tr><td>B. mollis</td><td>26</td><td>(34.2)</td><td>66</td><td>(21.2)</td><td>1.2 ± 0.8</td><td>3</td></tr></tbody></table>

Two major groups could be identified in the nMDS, one represented by sampling units from the continental shelf covered by rock and maërl and presenting an horizontal inclination, and a second one formed by sampling units on the shelf edge and upper slope mostly covered by rock and presenting a flat or sloping inclination (Fig. 3).

Permutation multivariate analysis of variance and subsequent pairwise test revealed that for

all environmental categorizations gorgonian assemblages were significantly different (p<0.001) from one another. The SIMPER analysis showed an average similarity in species composition that ranged from 17.89% to 31.68% (Table 2). The number of species contributing up to 90% of the similarity varied between two to four (Table 2). *E. singularis* dominated assemblages located on the continental shelf as well as those located on rocky and horizontal sampling units (Table 2). *E.*
*cavolinii* was especially relevant on assemblages located on the shelf edge as well as on sloping and vertical rocky outcrops (Table 2). *Bebryce mollis* Phillipi, 1842 dominated assemblages located in the upper slope, whereas *P. macrospina* M1 on those located on maërl beds, and *V. flagellum* dominate in vertical rocky bottoms (Table 2).

## 3.2 Geographic and vertical distribution

Only three species were observed on the continental shelf of the study area, *P. macrospina* (M1, M2), *E. singularis* and *Paramuricea clavata* (Risso, 1826) (Fig. 4, Table 1). *P. macrospina* (M1, M2) and *E. singulairs* were the most frequent species scattered over the continental shelf (Fig. 4) at 65–100 m and 52–88 m depth, respectively (Fig. 5). Both morphotypes of *P. macrospina* were much more abundant in terms of colony number than *E. singularis* (Table 1). Conversely, *P. clavata* was only found on the continental shelf in two low-density patches on coralligenous banks at 67–92 m depth, and few colonies on the shelf edge at 109–120 m depth (Figs. 4 and 5). The shelf edge and upper slope were much more diverse than the continental shelf in terms of species richness. In this depth range, gorgonian assemblages were highly localized in the areas dominated by rocky outcrops and vertical walls near Cap Formentor and in the Menorca Canyon (Fig. 4). *E. cavolinii* was the most frequent and abundant species of the shelf edge, with the highest abundances concentrated at 100–160 m depth, together with *P. macrospina* M3 (Figs. 4 and 5). *V. flagellum*, *S. pallida*, *C. verticillata* were also abundant on the shelf edge, but extended their distribution beyond 240 m depth, with one colony of *S. pallida* observed at 324 m (Figs. 4 and 5). *Acanthogorgia hirsuta* Gray, 1857 was the species showing the most restricted distribution, occurring in few locations of the shelf edge at 149–176 m depth (Fig. 5). *B. mollis* extended its distribution from the shelf edge to the upper slope,

where it was the most frequent species, followed by *C. verticillata* and *V. flagellum* (Table 1). *V. flagellum* was the most abundant species in the upper slope, followed by *S. pallida*, *B. mollis* and *C. verticillata* (Table 1). Finally, some colonies (~20) of the precious coral, *Corallium ruburm*, were observed at 80–120 m depth, whereas only few isolated colonies were seen below 150 m depth.

## 3.3 Population size structure

Overall, the maximum height of 81% of all the observed colonies was measured (63–100% of the colonies in each transect). All the analyzed populations, indistinctively of the species, were unimodal (Figs. 6a and b). Most populations of *P. macrospina* M1, *V. flagellum* and the only analyzed population of *C. verticillata* were positively skewed, indicating the dominance of small colonies (Figs. 6a, b and Table 3). Conversely, most population of *P. macrospina* M2, *E. cavolinii*, *S. pallida* and all the *E. singularis* populations were not skewed, being dominated by medium sized colonies (Figs. 6a, b and Table 3). One of the two-studied populations of *P. macrospina* M3 was positively skewed (Fig. 6b, Table 3). All the *P. macrospina* M1 populations showed significant kurtosis (Table 3), indicating that they were slightly more peaked or over-centralized than normal distributions. Conversely, only a few populations showed significant kurtosis in the other species (Table 3). The shrub-like morphology, and the densely intertwined branches of *B. mollis* did not allow the study of population size structure in this species.

## 3.4 Relationship with environmental features

Depth, substrate and slope explained ~20.8% of the total inertia (i.e. explained variation of the data) in the CCA, with the first two axis accumulating 17.1% of the species variances, and 82.3% of the species-environment relation variance (Fig. 7). According to the Monte Carlo
<table><caption>**Table 2.** SIMPER analysis. Species that belong to the similarity group of 90% of cumulative contribution are present. Percentages in bold letters are the average similarity values for each category.</caption><thead><tr><th>Depth range</th><th>Species</th><th>Sim/SD</th><th>Contribution%</th><th>Cum. Contribution%</th></tr></thead><tbody><tr><td rowspan="3">Continental shelf: 22.43%</td><td>E. singularis</td><td>0.48</td><td>60.33</td><td>60.33</td></tr><tr><td>P. macrospina M1</td><td>0.34</td><td>24.59</td><td>84.92</td></tr><tr><td>P. macrospina M2</td><td>0.25</td><td>14.86</td><td>99.79</td></tr><tr><td rowspan="2">Shelf edge: 21.45%</td><td>E. cavolinii</td><td>0.65</td><td>78.43</td><td>78.43</td></tr><tr><td>S. pallida</td><td>0.25</td><td>14.70</td><td>93.14</td></tr><tr><td rowspan="4">Upper slope: 17.89%</td><td>B. mollis</td><td>0.33</td><td>38.55</td><td>38.55</td></tr><tr><td>C. verticillata</td><td>0.26</td><td>24.83</td><td>63.38</td></tr><tr><td>S. pallida</td><td>0.27</td><td>23.30</td><td>86.68</td></tr><tr><td>V. flagellum</td><td>0.21</td><td>12.13</td><td>98.81</td></tr><tr><th>Bottom type</th><th>Species</th><th>Sim/SD</th><th>Contribution%</th><th>Cum. Contribution%</th></tr><tr><td rowspan="3">Rock: 18.63%</td><td>E. singularis</td><td>0.37</td><td>54.89</td><td>54.89</td></tr><tr><td>E. cavolinii</td><td>0.33</td><td>32.61</td><td>87.50</td></tr><tr><td>S. pallida</td><td>0.12</td><td>3.68</td><td>91.19</td></tr><tr><td rowspan="4">Cobbles and pebbles: 17.67%</td><td>S. pallida</td><td>0.42</td><td>60.77</td><td>60.77</td></tr><tr><td>E. cavolinii</td><td>0.21</td><td>14.08</td><td>74.85</td></tr><tr><td>P. macrospina M1</td><td>0.16</td><td>10.86</td><td>85.70</td></tr><tr><td>B. mollis</td><td>0.15</td><td>9.93</td><td>95.64</td></tr><tr><td rowspan="2">Maërl: 28.68%</td><td>P. macrospina M1</td><td>0.62</td><td>60.20</td><td>60.20</td></tr><tr><td>P. macrospina M2</td><td>0.42</td><td>33.55</td><td>93.75</td></tr><tr><th>Seabed Slope</th><th>Species</th><th>Sim/SD</th><th>Contribution%</th><th>Cum. Contribution%</th></tr><tr><td rowspan="4">Horizontal: 18.11%</td><td>E. singularis</td><td>0.35</td><td>49.09</td><td>49.09</td></tr><tr><td>P. macrospina M1</td><td>0.28</td><td>25.11</td><td>74.19</td></tr><tr><td>P. macrospina M2</td><td>0.29</td><td>14.93</td><td>89.13</td></tr><tr><td>E. cavolinii</td><td>0.15</td><td>7.12</td><td>96.25</td></tr><tr><td rowspan="4">Slopping: 19.66%</td><td>E. cavolinii</td><td>0.50</td><td>66.28</td><td>66.28</td></tr><tr><td>B. mollis</td><td>0.17</td><td>9.94</td><td>76.22</td></tr><tr><td>S. pallida</td><td>0.17</td><td>9.26</td><td>85.48</td></tr><tr><td>V. flagellum</td><td>0.17</td><td>7.03</td><td>92.51</td></tr><tr><td rowspan="2">Vertical: 31.68%</td><td>E. cavolinii</td><td>0.56</td><td>53.09</td><td>53.09</td></tr><tr><td>V. flagellum</td><td>0.51</td><td>44.75</td><td>97.84</td></tr></tbody></table>
<figure><img src="image_35.png" /><figcaption>Fig. 4. Geographical distribution. *E. singularis*, *P. clavata*, *P. macrospina* (M1, M2, M3), *E. cavolini*, *V. flagellum*, *A. hirsuta*, *C. verticillata*, *S. pallida*, and *B. mollis* distribution is represented on the study area based on sampling unit density. Species have been displayed from shallowest to deepest distribution.</figcaption></figure>
<figure><img src="image_36.png" /></figure>

Fig. 5. Vertical distribution. *E. singularis*, *P. clavata*, *P. macrospina* (M1, M2, M3), *E. cavolini*, *V. flagellum*, *A. hirsuta*, *C. verticillata*, *S. pallida*, and *B. mollis* distribution is represented along the studied bathymetric range based on sampling unit density. Black square indicates the median value; the box indicates the first and third quartiles; and the line indicates the range between minimum and maximum values. Gray scale histograms represent the total number of sampling units for each substrate type (see legend) over the studied bathymetric range. The numbers on the right indicate the percentage of sampling units with a certain species presence (n = number of colonies).
<table><caption>**Table 3.** Size structure characteristics of the studied morphotypes and species: colony height, skewness and kurtosis. Significant skewness or kurtosis are 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">Species</th><th rowspan="2">Transect</th><th rowspan="2">Colonies</th><th colspan="3">Height (cm)</th><th rowspan="2">Max.</th><th rowspan="2">Skew.</th><th colspan="2">Skewness</th><th colspan="2">Kurtosis</th></tr><tr><th>Mean</th><th>SD</th><th>p-value</th><th>Sig.</th><th>Kurt.</th><th>p-value</th><th>Sig.</th></tr></thead><tbody><tr><td rowspan="2">E. singularis</td><td>37</td><td>169</td><td>25.30</td><td>10.35</td><td>55.1</td><td>0.541</td><td>0.062</td><td>***</td><td>2.708</td><td>0.497</td><td></td></tr><tr><td>47</td><td>45</td><td>8.86</td><td>4.58</td><td>23</td><td>0.066</td><td>0.209</td><td>***</td><td>3.216</td><td>0.449</td><td></td></tr><tr><td rowspan="10">P. macrospina<br>(M1)</td><td>28</td><td>98</td><td>6.26</td><td>5.83</td><td>29.4</td><td>1.775</td><td>&lt;0.001</td><td>***</td><td>5.870</td><td>&lt;0.001</td><td>***</td></tr><tr><td>29</td><td>81</td><td>5.90</td><td>3.36</td><td>18.4</td><td>1.377</td><td>0.004</td><td>**</td><td>4.939</td><td>0.008</td><td>**</td></tr><tr><td>30</td><td>1099</td><td>8.72</td><td>6.9</td><td>33.1</td><td>1.056</td><td>&lt;0.001</td><td>***</td><td>3.335</td><td>0.037</td><td>*</td></tr><tr><td>37</td><td>65</td><td>10.82</td><td>6.10</td><td>33.8</td><td>0.829</td><td>0.075</td><td>***</td><td>4.550</td><td>0.027</td><td>*</td></tr><tr><td>38</td><td>276</td><td>8.24</td><td>6.17</td><td>41.9</td><td>2.013</td><td>&lt;0.001</td><td>***</td><td>7.596</td><td>&lt;0.001</td><td>***</td></tr><tr><td>43</td><td>251</td><td>12.10</td><td>6.24</td><td>29.4</td><td>0.185</td><td>0.421</td><td>***</td><td>2.247</td><td>&lt;0.001</td><td>***</td></tr><tr><td>P. macrospina<br>(M2)</td><td>28</td><td>461</td><td>6.82</td><td>5.13</td><td>30.1</td><td>1.302</td><td>&lt;0.001</td><td>***</td><td>4.415</td><td>&lt;0.001</td><td>***</td></tr><tr><td>42</td><td>166</td><td>15.05</td><td>5.75</td><td>31.35</td><td>0.346</td><td>0.222</td><td>***</td><td>2.570</td><td>0.222</td><td></td></tr><tr><td>43</td><td>57</td><td>14.52</td><td>5.61</td><td>25.1</td><td>-0.242</td><td>0.590</td><td>***</td><td>2.366</td><td>0.286</td><td></td></tr><tr><td rowspan="2">P. macrospina<br>(M3)</td><td>2</td><td>70</td><td>17.22</td><td>9.07</td><td>41.31</td><td>0.784</td><td>0.086</td><td>*</td><td>2.831</td><td>0.967</td><td></td></tr><tr><td>3</td><td>52</td><td>17.95</td><td>11.43</td><td>55.65</td><td>13.566</td><td>0.018</td><td>***</td><td>4.037</td><td>0.272</td><td></td></tr><tr><td rowspan="10">E. cavolinii</td><td>2</td><td>206</td><td>10.55</td><td>7.43</td><td>35.63</td><td>0.995</td><td>&lt;0.001</td><td>***</td><td>3.205</td><td>0.417</td><td></td></tr><tr><td>3</td><td>384</td><td>15.16</td><td>10.06</td><td>50.45</td><td>0.739</td><td>&lt;0.001</td><td>***</td><td>2.912</td><td>0.843</td><td></td></tr><tr><td>4</td><td>78</td><td>16.03</td><td>7.98</td><td>43.51</td><td>0.081</td><td>0.059</td><td>***</td><td>3.621</td><td>0.177</td><td></td></tr><tr><td>6</td><td>156</td><td>14.59</td><td>8.77</td><td>47.15</td><td>0.579</td><td>0.056</td><td>***</td><td>2.909</td><td>0.990</td><td></td></tr><tr><td>7</td><td>82</td><td>13.03</td><td>6.65</td><td>31.94</td><td>0.694</td><td>0.092</td><td>***</td><td>3.136</td><td>0.542</td><td></td></tr><tr><td>10</td><td>63</td><td>19.07</td><td>9.83</td><td>47.65</td><td>0.276</td><td>0.524</td><td>***</td><td>2.775</td><td>0.950</td><td></td></tr><tr><td>11</td><td>207</td><td>9.08</td><td>7.30</td><td>38.55</td><td>1.492</td><td>&lt;0.001</td><td>***</td><td>4.940</td><td>&lt;0.001</td><td>***</td></tr><tr><td>13</td><td>110</td><td>11.93</td><td>6.93</td><td>29.75</td><td>0.442</td><td>0.202</td><td>***</td><td>2.468</td><td>0.198</td><td></td></tr><tr><td>22</td><td>71</td><td>9.81</td><td>4.92</td><td>22.42</td><td>0.686</td><td>0.116</td><td>***</td><td>2.703</td><td>0.785</td><td></td></tr><tr><td rowspan="3">V. flagellum</td><td>2</td><td>56</td><td>42.35</td><td>22.20</td><td>96.41</td><td>0.308</td><td>0.500</td><td>***</td><td>2.558</td><td>0.606</td><td></td></tr><tr><td>4</td><td>230</td><td>38.19</td><td>25.14</td><td>148.3</td><td>1.045</td><td>&lt;0.001</td><td>***</td><td>4.721</td><td>&lt;0.001</td><td>***</td></tr><tr><td>7</td><td>130</td><td>46.00</td><td>30.86</td><td>136.6</td><td>0.740</td><td>0.0300</td><td>*</td><td>3.045</td><td>0.689</td><td></td></tr><tr><td rowspan="2">C. verticillata</td><td>73</td><td>54</td><td>26.43</td><td>21.52</td><td>115.31</td><td>2.513</td><td>&lt;0.001</td><td>***</td><td>9.467</td><td>&lt;0.001</td><td>***</td></tr><tr><td>3</td><td>186</td><td>5.35</td><td>1.98</td><td>13.54</td><td>0.932</td><td>&lt;0.001</td><td>***</td><td>4.452</td><td>&lt;0.001</td><td>***</td></tr><tr><td rowspan="5">S. pallida</td><td>4</td><td>88</td><td>7.12</td><td>2.12</td><td>12.17</td><td>0.224</td><td>0.548</td><td>***</td><td>2.655</td><td>0.615</td><td></td></tr><tr><td>7</td><td>83</td><td>6.83</td><td>2.71</td><td>13.96</td><td>0.753</td><td>0.069</td><td>***</td><td>3.190</td><td>0.480</td><td></td></tr><tr><td>9</td><td>90</td><td>6.66</td><td>2.81</td><td>17.46</td><td>1.061</td><td>0.013</td><td>*</td><td>5.329</td><td>0.002</td><td>**</td></tr><tr><td>13</td><td>91</td><td>4.62</td><td>1.75</td><td>10.47</td><td>0.552</td><td>0.151</td><td>***</td><td>3.641</td><td>0.156</td><td></td></tr><tr><td>69</td><td>40</td><td>5.32</td><td>1.99</td><td>10.39</td><td>0.439</td><td>0.408</td><td>***</td><td>2.571</td><td>0.759</td><td></td></tr></tbody></table>
permutation test, the three factors contributed significantly (p<0.001) to the ordination. Depth clearly segregates gorgonians found on the continental shelf (P. macrospina M1 and M2, E. singularis, P. clavata) from those from the shelf edge and continental slope (P. macrospina M3, V. flagellum, E. cavolinii, S. pallida, B. mollis, C. verticillata, and A. hirsuta). On the continental shelf, P. macrospina M1 and M2 were mainly associated with horizontal maërl beds, whereas E. singularis and P. clavata with rocky bottoms (Fig. 7). On the shelf edge and continental slope, E. cavolinii and P. macrospina M3 were mainly associated with sloping rocky bottoms. At increasing depth V. flagellum, S. pallida, C. verticillata, and A. hirsuta were also mainly associated with sloping bottoms (Fig. 7). Finally, B. mollis was the species most strongly associated with highest depths (Fig. 7).

## 3.5 Spatial structure

Spatial structure of gorgonian distribution was analyzed along three of the video transects (Fig. 8), representative of the continental shelf (transect 30) and slope (transects 4 and 6). On the continental shelf, *E. singularis* was positively associated with *P. macrospina* M1 at large spatial scales (more than 30 m), whereas they were independently distributed at smaller scales (Fig. 8, Table 4). *E. singularis* was negatively associated to *P. clavata* at small scales (less than 5 m) and tended to be independently distributed at large and intermediate scale. *P. clavata* was independently distributed with respect to *P. macrospina* at all scales (Fig. 8 and Table 4). On the continental slope, in transect 4, all species were independently distributed at small and medium scales (few to tens of meters) (Fig. 8 and Table 4). At larger spatial scales (more than 30 m), *C. verticillata*, *S. pallida* and *E. cavolinii* were positively associated, whereas *V. flagellum* occurred in exclusion to the other species (Fig. 8, Table 4). In transect 6, *E. cavolinii*, *P. macrospina* M3, *V. flagellum*

and *B. mollis* were positively associated at large spatial scales (more than 30 m) (Fig. 8 and Table 4). *A. hirsuta* tended to be independently distributed at small (less than 30 m) and large spatial scale (more than 30 m) (Fig. 8 and Table 4). *E. cavolinii* presented no association with *B. mollis* and *V. flagellum*, and the same situation was found for *P. macrospina* M3 when compared to *A. hirsuta*.

## 4. DISCUSSION

The diversity of the studied deep gorgonian assemblages was much higher than in shallow Mediterranean coastal areas (Weinberg, 1978; Linares et al., 2008; Gori et al., 2011a). Nine species were observed to dwell between 40 and 360 m depth within the study area, while only 4–5 species are commonly present in littoral areas (Velimirov, 1973; Gori et al., 2011a; Garrabou et al., 2001). Gorgonian diversity resembled that reported of Atlantic coastal areas (Cúrdia et al., 2013), subtropical areas (Opresko, 1973) and similar depth ranges in the Aleutian Islands (Stone, 2006). Conversely, gorgonian density observed in these deep multispecific assemblages reached high values (~20 colonies m⁻²) comparable to those reported for Mediterranean coastal species (Weinbauer and Velimirov, 1996; Linares et al., 2008; Gori et al., 2011a). These high density values are similar to those found in temperate (Grigg, 1975; 1977; Cúrdia et al., 2013) and tropical (Lasker and Coffroth, 1983; Yoshioka and Yoshioka, 1989) coastal gorgonian assemblages, and clearly exceed those found at similar depths (180–500 m) in the North Atlantic (Mortensen and Buhl-Mortensen, 2004; Buhl-Mortensen et al., 2014), and polar continental shelves and slopes (Orejas et al., 2002; Miller et al., 2012).
## *Paramuricea macrospina*

<figure><img src="image_37.png" /></figure>

## *Eunicella cavolinii*

<figure><img src="image_38.png" /></figure>

## *Eunicella singularis*

<figure><img src="image_39.png" /></figure>

Fig. 6a. *P. macrospina* (M1, M2, M3), *E. cavolinii* and *E. singularis* population size frequency distribution (transect number as in Fig. 1 and Table 3, depth range, n = number of colonies).
However, the observed high gorgonian diversity was mainly concentrated on the shelf edge (100–180 m depth). A clear vertical zonation can be established regarding species distribution: (1) coastal species extending their distribution to deep coralligenous banks (E. sin-

gularis and *P. clavata*); (2) dominance of one single species on the continental shelf (*P. macrospina*); (3) concentration of several gorgonian species on the shelf edge (*E. cavolinii*, *P. macrospina*, *S. pallida*, *A. hirsuta*, *V. flagellum*, *B. mollis* and *C. verticillata*); and (4)

## *Viminella flagellum*

<figure><img src="image_40.png" /></figure>

## *Swiftia pallida*

<figure><img src="image_41.png" /></figure>

## *Callogorgia verticillata*

<figure><img src="image_42.png" /></figure>

**Fig. 6b.** *V. flagellum*, *S. pallida* and *C. verticillata* population size frequency distribution (transect number as in Fig. 1 and Table 3, depth range, n = number of colonies).
<table><caption>**Table 4.** Summary of the three-term local quadrat covariance (3TLQC). Significant spatial covariance between pairs of species along the three analyzed transects.</caption><thead><tr><th>Transect Number</th><th>Compaired species</th><th>Negative</th><th>Random</th><th>Positive</th></tr></thead><tbody><tr><td rowspan="3">30</td><td>P. macrospina M1 vs. E. singularis</td><td></td><td>0-30 // 55-75</td><td>30-55 // &gt;75</td></tr><tr><td>P. macrospina M1 vs. P. clavata</td><td></td><td></td><td>All transect</td></tr><tr><td>P. clavata vs. E. singularis</td><td>2-5</td><td>&gt;5</td><td></td></tr><tr><td rowspan="6">4</td><td>C. verticillata vs. E. cavolinii</td><td></td><td>0-50</td><td>&gt;50</td></tr><tr><td>C. verticillata vs. S. pallida</td><td></td><td>0-5</td><td>&gt;5</td></tr><tr><td>C. verticillata vs. V. flagellum</td><td>&gt;55</td><td>0-55</td><td></td></tr><tr><td>E. cavolinii vs. S. pallida</td><td></td><td>0-50</td><td>&gt;50</td></tr><tr><td>E. cavolinii vs. V. flagellum</td><td>&gt;34</td><td>0-34</td><td></td></tr><tr><td>S. pallida vs. V. flagellum</td><td>&gt;48</td><td>0-48</td><td></td></tr><tr><td rowspan="10">6</td><td>A. hirsuta vs. B. mollis</td><td>30-60</td><td>0-30 // &gt;60</td><td></td></tr><tr><td>A. hirsuta vs. E. cavolinii</td><td>11-20</td><td>0-11 // 20-40</td><td>&gt;40</td></tr><tr><td>A. hirsuta vs. P. macrospina M3</td><td></td><td>All transect</td><td></td></tr><tr><td>A. hirsuta vs. V. flagellum</td><td>30-65</td><td>0-30 // &gt;65</td><td></td></tr><tr><td>B. mollis vs. E. cavolinii</td><td></td><td>All transect</td><td></td></tr><tr><td>B. mollis vs. P. macrospina M3</td><td></td><td>20-30</td><td>0-20 // &gt;30</td></tr><tr><td>B. mollis vs. V. flagellum</td><td>11-20</td><td>0-11 // 20-32</td><td>&gt;32</td></tr><tr><td>E. cavolinii vs. P. macrospina M3</td><td></td><td>0-35</td><td>&gt;35</td></tr><tr><td>E. cavolinii vs. V. flagellum</td><td></td><td>All transect</td><td></td></tr><tr><td>P. macrospina M3 vs. V. flagellum</td><td>12-22</td><td>0-12 // 22-60</td><td>&gt; 60</td></tr></tbody></table>

species extending their distribution deeper into the upper slope (*B. mollis*, *C. verticillata*, *V. flagellum* and *S. pallida*).

The common Mediterranean coastal species *E. singularis* and *P. clavata* were found on deep coralligenous banks on the continental shelf of the studied area (45–100 m depth). The high water transparency allows a very deep distribution of coralligenous banks (Ballesteros and Zabala, 1993), which in turns could explain the deep distribution of *E. singularis* within the study area (down to 100 m depth). In coastal environments characterized by high water turbidity, this species is limited to 70 m depth (Gori et al., 2011a). In the study area continental shelf both species' density was much lower

than that reported in shallow coastal environments (Linares et al., 2008; Gori et al., 2011a). This could indicate that these species' deep bathymetric limit lies around this depth, according to our data. Interestingly, the observed colonies of *E. singularis* presented the same morphology as the deep asymbiotic morphotype reported in coastal environments below 40 m depth (Théodor, 1969; Gori et al., 2012). These populations were dominated by medium sized colonies, corresponding to what has been observed in deep coastal environments (60 m depth) (Gori et al., 2011b). Conversely, in shallow coastal environments (20 m depth) these species populations are mainly composed of small colonies (Linares et al., 2008). Populations of *P. clavata* extending beyond 40 m have
also been reported in several coastal areas (Gori et al., 2011a; Angiolillo et al., 2015), as well as on top of seamounts (Bo et al., 2011) and on rocky bottoms of the continental shelf and slope (Bo et al., 2012, 2014). In these deep environments, *P. clavata* colonies generally achieve larger sizes (50 ± 15 cm (mean ± SD), min = 25 cm, max = 90 cm) than in shallower environments (Linares et al., 2008), as a possible consequence of the higher environmental stability of deeper areas (Grigg, 1975). Deep populations of *E. singularis* and *P. clavata* are probably protected from high hydrodynamic processes (Teixidó et al., 2013) as well as from unusually high-temperature conditions associated to mass mortality events in shallow benthic communities (Garrabou et al., 2009).

Maërl beds on the continental shelf of the studied area are covered by very high abundances of *P. macrospina* (M1 and M2). In this environment this species presents high frequency of occurrence over large areas (Figs. 4 and 8) and can reach densities of up to 33 colonies m⁻², which is at odds with previous data on this species, having been only reported on rocky substrates and never as a dominant species (Bo et al., 2011; Topçu and Öztürk, 2015). Preservation of these *P. macrospina* populations may be a consequence of low trawling pressure over large areas of the continental shelf (Moranta et al., 2014), as well as the habit of local artisanal fishermen to clean their nets *in situ* (Díaz et al., 2015). This habit consists of releasing by-catch over the same fishing grounds. This practice might result in partial damage of entangled colonies of *P. macrospina*, but as long as their holdfast remains attached to the maërl rodholite, their survival might be favored (Díaz et al., 2015). Population size structures of *P. macrospina* were mostly asymmetrical and positively skewed (Table 4), suggesting high recruitment rates (Linares et al., 2008) and implying that maërl beds are particularly suitable habitats for this species. However, unlike *E*.

*P. singularis* and *P. clavata* that occur on corallige nous banks, the structural instability of maërl may limit the presence of large colonies of *P. macrospina* in the continental shelf (Tunnicliffe and James, 1983). Furthermore, fishing activi ties might be particularly detrimental to large colonies, which are probably more susceptible than smaller colonies to get entangled and bro ken by nets used by artisanal fishermen in the area. Indeed, populations dominated by small colonies have been reported in intensely fished areas (Althaus et al., 2009).

Highest gorgonian diversity was concentrated on the rocky bottoms of the shelf edge (at 100–180 m depth). In this environment, gorgonian assemblages were mostly multispecific (92% of occupied sampling units contained several species) and included seven of the nine observed species (Figs. 4 and 5). This diversity is probably influenced by the joint effect of habitat heterogeneity and hydrodynamic sta-

<figure><img src="image_43.png" /><figcaption>**Fig. 7.** Canonical correspondence analysis (CCA): biplot showing the ordination of gorgonian species and the roles of the significant environmental variables. M: maërl, CSP: cobbles and pebbles and R: rocky substrates.</figcaption></figure>
bility. In the study area's shelf edge, boulders, slaps and vertical walls alternate over short distances. Shelf edge environments are mainly influenced by steady flow currents and stable hydrodynamic conditions (e.g. Puig et al., 2000), such as the Balearic current associated with the Balearic front, which flows from south to north along the western shelf edge of the Balearic Promontory (Ruiz et al., 2009). Stable hydrodynamic conditions, together with the hydrodynamic processes that increase particle suspension in the near-bottom water layers, may also suppose enhanced food availability for gorgonians on the shelf edge (Thiem et al., 2006). Finally, the observed increase in gorgonian diversity on the shelf edge could also result from the merging of species with shallower and deeper distributions, causing a mid-domain effect (Colwell and Lees, 2000), as previously suggested for deep coral diversity in other locations (Stone, 2006; Matsumoto et al., 2007). Most gorgonian populations located on the shelf edge had a bell-shaped, unimodal size structure dominated by medium sized colonies (Figs. 6a and b). *P. macrospina* (M3) populations on the shelf edge were bell-shaped, in contrast to its populations on the continental shelf (M1 and M2) that were mostly dominated by small size colonies. When compared to maërl, rocky substrates provide higher structural stability allowing

<figure><img src="image_44.png" /><figcaption>**Fig. 8.** Density plots. Species densities are plotted along representative transects from the continental shelf (transect 30) continental shelf edge and slope (transect 4 and 6, respectively). Bottom type and seabed slope are indicated (see legend). Vertical gray rectangles indicate invalid sequences for the analysis (Tr = transect, n = number of colonies).</figcaption></figure>

BOTTOM TYPES LEGEND

Sand to Gravel

Cobbles & Pebbles

Maërl

Rock

SLOPE TYPES LEGEND

Vertical

Slopping

Horizontal
colonies to achieve larger sizes. In these multispecific gorgonian assemblages of the shelf edge, the fine scale spatial covariance of species appears to be related to the combined effect of density, occupancy, and colony size. When a large species such as *V. flagellum* achieves high-densities over extended areas, it tends to exclude any other species (Fig. 8). Conversely, coexistence is observed at low and intermediate densities among large (*C. verticillata*), medium (*E. cavolinii*) and small sized species (*S. pallida*). This could indicate that differences in size may reduce interspecific competition for space and food (Schoener, 1974). Species with similar size probably feed on the food particles that are transported by the same water layer, with a potential competition for food (Kim and Lasker, 1997). Conversely, species with different size are probably feeding on different water layer, thus avoiding potential shading processes. Coexistence at medium spatial scales (~30 m) may also occur at low and intermediate densities among species of similar size and morphology (*E. cavolinii* and *A. hirsuta*). In this case, however, spatial exclusion occurred at smaller scales (~10 m), suggesting that these species share the same habitat but partially compete for space.

Overall, the species dwelling on the shelf edge within the study area, have been recently found with increasing frequency on rocky substrates at similar depths in other areas of the Mediterranean Sea (Bo et al., 2009; 2011; 2012; 2014), suggesting the general validity of the observed pattern of high gorgonian diversity.

Below the shelf edge, gorgonian abundance strongly decreases, and only *B. mollis*, *S. pallida*, *V. flagellum* and *C. verticillata* were observed to extend their distribution under 200 m depth (Fig. 4). Gorgonian assemblages are here mainly monospecific, (76% of occupied sampling units contained one single species)

and with low densities (~0.75 ± 0.3 colonies m² (mean ± SD)). These species are typically found along the continental slope in other Mediterranean locations (Mytilineou et al., 2014; Deidun et al., 2014; Bo et al., 2015) where they mostly concentrate along the edges, maximizing their exposure to currents (Genin et al., 1986). On the scarce rocky substrates occurring in these deeper environments, gorgonian abundance tends to decrease with increasing depth, and antipatharians become the dominant arborescent species (Deidun et al., 2014; Bo et al., 2015). Finally, the soft sediments of these deeper environments can be colonized by the bamboo coral *Isidella elongata* (Maynou and Cartes, 2012; Bo et al., 2015), which can form extended assemblages in bathyal muds (Pérès, 1967).

Due to their arborescent morphology, gorgonians are especially vulnerable to fishing activities (Mytilineou et al., 2014; Bo et al., 2014), and their slow growth rate (Coma et al., 1998; Sherwood and Edinger, 2009) makes their recovery from related fishing damages very slow (Althaus et al., 2009). The high diversity and abundance of deep gorgonian assemblages in the rocky areas of the shelf edge, and the vast area covered by high densities of *P. macrospina* on the continental shelf are probably related to the low trawling pressure and the exceptional fishing practices exerted by local artisanal fishermen. This represents an example of the possibility of conserving deep gorgonian assemblages and their associated high-diverse fauna (e.g. Buhl-Mortensen and Mortensen, 2005) through better management of fishing activities. These high-density deep gorgonian assemblages may provide an approximate idea of how Mediterranean continental shelves and upper slopes stood before decades of bottom trawling.
## ACKNOWLEDGEMENTS

The authors thank the crew of the R/V *García del Cid* and *Miguel Oliver*, the JAGO team (J. Schauer and K. Hissmann) from IFM-GEOMAR (Kiel, Germany), the NEMO ROV pilot Gavin Newman and Plàcid Griño, David Díaz, Joan Llúis Riera and Guillem Salazar for their advice. 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).

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Chapter 3

# Morphological and genetic features of the gorgonian *Paramuricea macrospina* on the Mediterranean continental shelf (Menorca Channel, Western Mediterranean Sea)

## ABSTRACT

Morphological variability in gorgonians is frequent and commonly associated to habitat variability, often resulting in segregated morphotypes. *Paramuricea macrospina* is an endemic Mediterranean gorgonian species found on rocky bottoms between 40–100 m depth. It has recently been reported as one of the most abundant species in continental shelves and shelf edges. Three different chromatic forms of *P. macrospina* were observed in the Menorca Channel: a yellow and a light purple forms occurring on maërl beds of the continental shelf, and a dark purple form occurring on rocky substrates of the shelf edge. The objective of the present work is to verify if these *P. macrospina* forms may represent distinct taxonomic units by analyzing colony shape, sclerite size and shape, and mitochondrial markers of the three chromatic forms of *P. macrospina*. No significant differences were found in colony shape, suggesting that environmental variability between the continental shelf and the shelf edge is not influential enough to significantly alter colony morphology. Significant differences in sclerite size and shape were found amongst all forms, suggesting that sclerites may be more likely influenced by environmental conditions. However, the coexistence of yellow and light purple forms on the continental shelf suggests that both environmental factors and genetic differentiation are conditioning sclerite size and shape. Molecular analysis revealed no differences amongst the three forms but proportioned new insight on the phylogenetic relationship of this gorgonian genus.

## CO-AUTHORS

A Gori, PJ López-González, A Santín, P Baena, JM Gili

## ADDITIONAL RESOURCES

Submitted in Marine Biodiversity
# 1. INTRODUCTION

Modularity is a successful strategy widely spread amongst sessile organisms as it facilitates adaptation to different environmental conditions (Huges, 1989). In marine environments, morphological variability of sessile modular organisms is very common, especially among coral and gorgonian species (Bruno and Edmunds, 1997; Sánchez et al., 2007; Vermeij et al., 2007). Indeed, these organisms can present a wide plasticity in their growth under different environmental conditions (Todd, 2008). Variability can affect colony color and morphology (Sánchez et al., 2007; Prada et al., 2008), as well as sclerite size and shape (Gutiérrez-Rodríguez et al., 2009) and the

presence of specific secondary compounds (Puyana et al., 2004). This can suppose an additional challenge for taxonomical, evolutionary and ecological studies (Vermeij et al., 2007).

Coral and gorgonian morphological variability has commonly been related to habitat differences (Bruno and Edmunds, 1997; Helmuth et al., 1997), often resulting in segregated morphotypes adapted to different hydrodynamic or light conditions (Sebens, 1987; Sánchez et al., 2007; Gori et al., 2012). Colony transplant experiments have shown that the same genotype can generate different morphological phenotypes confirming the importance of phenotypic plasticity amongst gorgonians (West et al., 1993; Joseph et al., 2015).

In the last 20 years, several studies have addressed morphological variability in shallow gorgonian species (<40 m depth) mainly in relation with the

<figure><img src="image_45.png" /><figcaption>**Fig. 1.** Map of the Menorca Channel. Black dots indicate the sampling locations of each chromatic form.</figcaption></figure>
dominant hydrodynamic conditions (e.g. Weinbauer and Velimirov, 1995; Skoufas, 2006; Sánchez et al., 2007). On the contrary, studies concerning deep gorgoninan morphological variability are still very scarce (e.g. Mortensen and Buhl-Mortensen, 2005; Quattrini et al., 2013; Doughty et al., 2013). However the increased accessibility to deep environments proportioned by remotely operated vehicles (ROVs) and manned submersibles has revealed that deep gorgonians species may occur over a wide bathymetric range and through different environmental settings (Stone, 2006; Buhl-Mortensen et al., 2014). The increasing observation of deep gorgonian assemblages is showing that, as their shallow counterpart, deep gorgonian species may also present a certain degree of morphological variability (Mortensen and Buhl-Mortensen, 2005). *Paramuricea macrospina* (Koch, 1882) is an endemic Mediterranean gorgonian mostly found on rocky bottoms at 40–100 m depth (Carpine and Grasshoff, 1975; Bo et al., 2012). This species has been recently found to be amongst the most abundant gorgonian on the Mediterranean continental shelf (Bo et al., 2012; Grinyó et al., 2016). In the Menorca Channel (Western Mediterranean Sea) (Fig. 1), *P. macrospina* is the dominant gorgonian species at 65–90 m depth on the continental shelf, but is also commonly found on the shelf-edge associated to other gorgonians at 110–160 m depth (Grinyó et al., 2016).

Two different chromatic forms of *P. macrospina* were observed on the continental shelf, a yellow one (M1) and a light purple one (M2) (Figs. 2a and 2b, respectively). Both forms occur on maërl beds, covering vast areas and punctually reaching very high densities (Grinyó et al., 2016). On the shelf edge, a third dark purple form (M3) was observed on rocky substrates (Fig. 2c).

In the last few years several studies started to explore the biology and ecology of deep Mediterranean gorgonian species (e.g. Bo et al., 2012; Topçu and Öztürk, 2016). In this context, this study aims to elucidate if the different *P. macrospina* forms observed in the Menorca Channel represent distinct taxonomic units, by exploring differences in their (1) colony shape, (2) sclerite size and shape, and (3) genetic variability in mitochondrial markers (msh1, lgr1 and COI).

## MATERIAL AND METHODS

### Colony shape

*P. macrospina*'s colony shape was analyzed on pictures obtained from videos recorded on the

<figure><img src="image_46.png" /><figcaption>**Fig. 2.** Images of colonies of different *Paramuricea macrospina*'s chromatic forms (a) M1, (b) M2 and (c) M3. Scale Bar: 10 cm.</figcaption></figure>