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# Conclusions

GRINYÓ J (2016) PhD Thesis pp 157-160

## ● Chapter 1

Six different assemblages of macrobenthic species were identified in the Menorca Channel, mainly segregated by substrate and depth. Hard substrates hosted coral gardens and sponge grounds. Both assemblages presented a very similar sponge species composition, being basically differentiated by the absence of gorgonians and antipatharians in sponge grounds. Soft sediments were characterized by vast extensions of the crinoid *Leptometra phalangium* and the brachiopod *Gryphus vitreus*. *L. phalangium* mostly occurred on the shelf edge, whereas *G. vitreus* was mostly found in deeper areas. The highest diversity of benthic macrofauna was concentrated in the shelf edge. This high diversity most likely results from the synergy of habitat heterogeneity and hydrodynamic conditions in the shelf edge. The good preservation of most of the observed assemblages is probably related to the low pressure of bottom trawling fishing, which is mainly concentrated above 100 m on the continental shelf, and below 500 on the continental slope.

## ● Chapter 2

Gorgonian diversity at 40–360 m depth in the Menorca Channel was comparatively higher than observed in Mediterranean coastal areas. Gorgonian assemblages on the continental shelf and upper slope were mostly monospecific, whereas shelf edge assemblages were highly multispecific. Gorgonian populations were dominated by small size colonies throughout the studied bathymetric range. However, in deeper environments intermediate and large colonies were more abundant. More stable environmental conditions and food availability in the shelf edge may allow gorgonians to reach larger dimensions. 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 *Paramecia macrospina* on the continental shelf most likely result from low trawling pressure and the exceptional fishing practices exerted by local artisanal fishermen. 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.
● Chapter 3

The three chromatic forms of the gorgonian *Paramuricea macrospina* observed on the continental shelf and upper slope of the Menorca Channel, significantly differs in terms of sclerite size and shape, but not in terms of molecular markers and colony shape. Indeed, most of the analyzed colonial morphological features barely changed among the three forms indicating that colonial morphology is a conservative character that experiences little variation across the different environments. Both environmental factors and genetic differentiation may be conditioning differences in sclerite size and shape among the three different forms. Considering the similar coloration in M2 and M3 colonies and the less marked differences in sclerite size and shape, it could be suggested that M2 and M3 are a shallow and a deep forms of a single morphotype occurring over a wide depth range (continental shelf and shelf edge), whereas M1 is a morphotype restricted to the continental shelf. The lack of resolution in mtMutS is not necessary indicative of species identity, and thus recent speciation process that separated M1 versus M2+M3 forms of *P. macrospina* cannot be discarded. In this regard, new genetic markers are needed to clarify the fine line between species and morphological variation among Mediterranean gorgonians.

● Chapter 4

*Paramuricea macrospina* reproductive cycle is delayed 2-3 months respect to the shallow Mediterranean gorgonian species. Late increase in seawater temperature occurring on the Mediterranean continental shelf could be the main driver of this delay. Internal brooding in *P. macrospina* contrasts with surface brooding of the congeneric *P. clavata*. The differences in the habitats where the two species occur, arises the question about the possible adaptive advantage of their reproductive strategies. *P.*

*macrospina* lipid content was much lower and more constant than observed in shallow Mediterranean gorgonian species. These differences between depths may reflect a lower but more constant food availability on the Mediterranean continental shelf than in littoral environments. A general stability in food availability for gorgonians on the Mediterranean continental shelf is also supported by the lack of seasonality in the ∂¹³C and ∂¹⁵N composition of *P. macrospina* tissue. Seasonality in the energetic requirements of *P. macrospina* is highlighted by the seasonal changes observed in the FFA content and composition

● Chapter 5

Particle fluxes collected in the Menorca canyon head were comparatively low in the Mediterranean context, reflecting the lack of intense continental and fluvial supply to this insular and oligotrophic study area. There was not a clear parameter, which controlled the temporal variation of the TMF. However, resuspension and biochemical characteristics associated to alternating water masses (resident and recent AW) seemed to be the main drivers controlling the magnitude and composition of the TMF. The findings of the current study also suggest that Mediterranean submarine canyons related to insular environments, without important fluvial networks, receive as small fluxes as in some deep-sea environments.

Future perspectives

Results presented in this thesis derive from the study of Mediterranean macrobenthic assemblages of the continental shelf and upper slope by means of non-destructive techniques. Quantitative information about species abundance and distribution over large extents were obtained applying quantitative video analyses, complemented with the sampling of a limited number of organisms to confirm species identification. Non-destructive, video-based sam-
plings are currently considered a valid alternative to traditional sampling methods to study deep benthic environments. When larger number of samples was needed to address the characterization of specific aspects of the species taxonomy or biology, they were carefully and selectively collected by means of the arm of the manned submarine, or as by-catch of traditional fishery. This approach is especially preferable for the study of vulnerable species with slow growth and high longevity, such as most of those investigated in this thesis.

Many of the results obtained within this thesis have also represented the scientific base for the ecological characterization of the Menorca Channel for its inclusion in the Natura 2000 Network from the European Union (LIFE07/NAT/E/000732). More in detail, results about species abundance and distribution (chapters 1 and 2) represent the ground zero for the future monitoring of the effectiveness of the management and conservation measures that will be undertaken in the frame of the new established special area of conservation (http://www.magrama.gob.es/es/costas/temas/protecion-costa/actuaciones-proteccion-costa/illes-balears/LIC-ESZZ16002-Canal-de-Menorca.aspx). Moreover, future exploration of areas adjacent to the Menorca Channel, such as Serra de Tramuntana (Northern Mallorca) or the rocky gullies on the northern slope of Menorca Island, could unveil similarly reach and well preserved macrobenthic assemblages. These areas are currently under low trawling pressure, and artisanal and recreational fishing by-catch observations suggest the presence of rich and diverse benthic communities.

In situ growth experiments of the main bioengineering species (e.g. gorgonians, large massive sponges, antipatharians) would be beneficial to understand the recovery capacity of the observed benthic communities, and set the base for the restoration potential of impacted areas. In order to elucidate the phylogenetic

relationship of the three forms of *Paramuricea macrospina* observed (chapter 3), future studies should address this question by means of high through put DNA sequencing (e.g. RAD sequencing, restriction-site associated DNA). Indeed, these techniques have shown to be useful in delimiting deep gorgonian species. Furthermore, the possible role that asexual reproduction could play to complement sexual reproduction (chapter 4) in the population dynamic of *P. macrospina* could be addressed by sampling colonies at different spatial scales and perform microsatellite genotyping analysis.

Finally, the high oligotrophy of the Balearic Sea apparently contrasts with the observed presence of high-density aggregations of passive and active suspension feeders. Future studies should consider monitoring particle fluxes and seasonal pattern of the main environmental conditions in the continental shelf and upper slope, to understand the main drivers of species distribution.
Annex I

GRINYÓ J (2016) PhD Thesis pp 161-173
<figure><img src="image_83.png" /></figure>

LÓPEZ-GONZÁLEZ, P.J., GRINYÓ, J. AND GILI, J.M. 2012
Rediscovery of *Cereopsis studeri* Koch, 1891, a forgotten
Mediterranean soft coral species, and its inclusion in the genus *Nidalia*
Gray, 1835 (Octocorallia, Alcyonacea, Nidaliidae).
*Marine Biology Research*, 8: 594-604
Marine Biology Research, 2012; 8: 594–604

<figure><img src="image_84.png" /></figure>

ORIGINAL ARTICLE

# Rediscovery of *Cereopsis studeri* Koch, 1891, a forgotten Mediterranean soft coral species, and its inclusion in the genus *Nidalia* Gray, 1835 (Octocorallia, Alcyonacea, Nidaliidae)

PABLO J. LÓPEZ-GONZÁLEZ*<fnref n="1" />, JORDI GRINYÓ<fnref n="2" /> & JOSEP-MARIA GILI<fnref n="2" />

## Abstract

*Cereopsis studeri* was described by G. von Koch in 1891 with material from Naples. However, it was subsequently synonymized, erroneously identified, and overlooked in subsequent soft coral literature of the twentieth century. After the original description, this species was not recorded or correctly described for 120 years. The study of newly collected material from the North Western Mediterranean permits the re-description of this forgotten species and its assignation to the genus *Nidalia* in the family Nidaliidae. The main features of *Nidalia studeri* com. nov. are: colony torch-like, a capitulum light orange in colour, not laterally flattened, dome-shaped and not distinctly projecting beyond the stalk, introvert with sclerites transversally placed in two longitudinal rows per interseptal space, anthocodial crown with 28–38 sclerite rows, points separated from polyps distally, formed by 6–9 pairs of sclerites, and the presence of intermediate points (secondary points) between principal (interseptal) ones. *Nidalia studeri* is here compared with its closest congeners, especially with the Indonesian species *N. simpsoni*, species from the West Indian Region *N. dissidens*, *N. occidentalis*, *N. deichmannae*, and the recently described *Nidalia aurantia* from the Mid-Atlantic Ocean. This is the first time that the genus *Nidalia* and the family Nidaliidae have been reported with certainty for the Mediterranean Sea.

**Key words:** Mediterranean, Alcyonacea, manned submersible, Cereopsis studeri, Nidalia, soft coral

## Introduction

Mediterranean fauna has often been reported as one of the best known faunas in the world (Bianchi & Morri 2000). Important revisionary works on different taxonomically complex invertebrate groups give that impression (e.g. Ruffo 1982–1998 for amphipods; Weinberg 1976, 1977 and 1978 for gorgonians, soft corals and stolonate octocorals; Carpine & Grasshoff 1975 for gorgonian octocorals; Zibrowius 1980 for scleractinian corals; Bouillon et al. 2004 for hydrozoans; Zabala & Maluquer 1988 for bryozoans). However, there are still numerous cryptic species waiting to be discovered, and relict or remote ecosystems still poorly sampled. An example of the first case is the recently described small gorgonian *Dendrobrachia bonsai* López-González & Cunha, 2010, with a relatively wide distribution

from the Gulf of Cadiz to the Corsican Strait. An example of the second case is the rich jellyfish fauna, including many endemic species, found in North Western submarine canyons (Gili et al. 1998).

*Cereopsis studeri* was described by Koch (1891) from the Gulf of Naples, Italy. This species was simply assigned to the genus *Nidalia* Gray, 1835 by May (1900: 101), without additional comments or discussion. This latter author considered the genus *Cereopsis* Kent, 1870 as a junior synonym of *Nidalia*. Later, Pütter (1900: 447) considered *C. studeri* in the genus *Bellonella* Gray, 1862, Kükenthal (1907: 384) assigned Koch's species to the genus *Gersemia* Marenzeller, 1878, and Thomson & Dean (1931: 44) supposedly identified this species among the material collected during the Siboga Expedition to East Indies, although the sampling station was not recorded and the description is too vague for a

<footnote n="1">¹Biodiversidad y Ecología de Invertebrados Marinos, Facultad de Biología, Universidad de Sevilla, Sevilla, Spain; and</footnote>

<footnote n="2">²Institut de Ciències del Mar (CSIC), Barcelona, Spain</footnote>

<footnote>* Correspondence: Pablo J. López-González, Biodiversidad y Ecología de Invertebrados Marinos, Facultad de Biología, Universidad de Sevilla, Reina Mercedes 6, 41012 Sevilla, Spain. E-mail: pjlopez@us.es</footnote>

<footnote>Published in collaboration with the University of Bergen and the Institute of Marine Research, Norway, and the Marine Biological Laboratory, University of Copenhagen, Denmark</footnote>
A forgotten soft coral species from NW Mediterranean 595

specific identification. More recently, Stiasny (1941: 17–20, Figures 8–10) ascribed to this species a colony clearly different to that described by Koch. It is more attributable to a colony of *Paralcyonium* Milne Edwards & Haime, 1850 with partially retracted polyps (see distribution of sclerites on the anthocodium in Figure 9 and biscuit-like sclerites in Figure 10a–c, and compare them with those in Weinberg 1977: plates 13B, 14 and 15). Utinomi (1958: 111) briefly summarizes the nomenclatural changes of *C. studeri* since its original description, but also commented (as unpublished information) that he recognized Koch’s species in material from Sagami Bay, Japan. Moreover, Utinomi discussed the idea that *C. studeri* is not comparable to the type species of *Cereopsis*, *C. bocagei* Kent, 1870, or *Bellonella*, and as a result the Japanese author proposed that Koch’s species should be included in a new genus, *Kochella* Utinomi, 1958, alluding to a further detailed discussion in a contribution that, to the knowledge of the authors of this article, was never published. *Cereopsis bocagei* was later included in the genus *Bellonella* (see Verseveldt & Bayer 1988: 14), and more recently considered in the genus *Alcyonium* Lamouroux, 1812 (see McFaden & Hutchinson 2004). Neither *Kochella* nor Koch’s species were mentioned in the preliminary list of octocorals of Sagami Bay (Utinomi 1962). Currently, *Kochella* is considered a junior synonym of *Daniela* Koch, 1891, a poorly known Mediterranean Nephtheidae. Recently, a well-conserved population attributable to *Daniela koreni* Koch, 1891 (type species of the genus) has been located at Minorca Channel, at a depth of 100 m (J.M. Gili and J. Grinyo, pers. observ.). Further studies on these relatively deep communities will enable us to learn more about *D. koreni*, a relatively large soft coral and the only member of the family Nephtheidae in Mediterranean waters (Grinyo et al. in prep.).

The relatively short description and illustrations of *C. studeri* given by Koch (1891) include key details to support the inclusion of this species within the genus *Nidalia*, as was suggested by May (1900). These are: colony non-ramified, torch-like, all polyps arising from the same level, relatively few polyps in number, partially retracted polyps cylindrical and large, and general sclerite diversity and arrangement. Moreover, there are some details in the original text and figures that can be used as species descriptors, such as prominent points, distinct mesenterial insertions (due to the presence of sclerites as secondary points) and sclerite sizes.

Since its original description, during the last 120 years, this species has remained immersed in a web of taxonomic confusions and synonyms and has also been overlooked in the most recent and complete

review of the genus (Verseveldt & Bayer 1988), as well as subsequent descriptive papers on *Nidalia* species (e.g. López-González & Gili 2008).

According to the revision of the genus *Nidalia* (Verseveldt & Bayer, 1988), the geographic distribution of the species of *Nidalia* can be summarized as follows: eight species are present in the Indian Ocean or Western Pacific, while only four have been described from the Western Atlantic (Verseveldt & Bayer 1988). One additional species was recently described from the Mid-Atlantic Ocean (López-González & Gili 2008).

During the general community surveys carried out within the project LIFE + INDEMARES, a set of 14 colonies of a species of *Nidalia* were collected at the head of Son Bou submarine canyon located at the East of Minorca channel, North Western Mediterranean. Due to the absence of additional references concerning Koch's species and its supposed pertinence and synonyms with members of different families (Alcyoniidae and Nephtheidae), we initially supposed that this newly collected material could represent the existence of an undescribed soft coral species in the Mediterranean. However, the coincidence of some key characters which can be used to identify the genus *Nidalia* as well as other features used to differentiate species in this genus suggested that it actually corresponds to the forgotten species described by Prof. Gottlieb von Koch in the late nineteenth century, *Cereopsis studeri*. After some consultation with the Hessisches Landesmuseum Darmstadt in Germany, where Prof. von Koch was Director of the Department of Zoology for 39 years (1875–1914), we discovered that the type material of *C. studeri* seems to have been lost (J. Köhler, pers. comm.); thus we have also deposited at the Museu de Zoologia (Barcelona) part of the newly collected material of this species to be useful for further studies and consults. Thus, the goal of the present article is the re-description of this material, including the species in the genus *Nidalia*, and the discussion of its relationships with its closest congeners.

## Material and methods

The material studied here was collected during the 'INDEMARES-CSIC III, Menorca I' cruise on board the BIO *García del Cid*, equipped with the manned submersible *Jago*, from 3 to 13 September 2010. Fourteen of these colonies were collected using the articulated arm of the submersible. The main goal of this cruise was to study the distribution and diversity of benthic communities in the North Western Mediterranean, within the framework of the LIFE + INDEMARES project.
596 P.J. López-González et al.

The octocorals were fixed in buffered 5% formalin in seawater, and then preserved in 70% ethanol. Other colonies were relaxed and directly fixed in ethanol 100° for examination and future molecular studies. All retraction states and fixation protocols have been considered for the material deposited in museums: retracted, semi-retracted and extended polyps, as well as formalin and ethanol fixed specimens (see 'Material examined').

Fragments of different parts of the colonies were prepared for study by SEM employing the usual methodology, previously described by different authors (e.g. Bayer & Stefani 1988), and permanent mounts were made for light microscopy observation. About 30 sclerites from each type and part of the colony and polyp (crown, introvert, points, surface of the stalk, etc.) were measured, trying to include the complete range of sizes. All sclerite size measurements and illustrations are from the specimen MZB 2011-0001. The colony and sclerite terminology described here mainly follows Bayer et al. (1983). The material studied here has been deposited at the Museu de Zoologia in Barcelona (MZB), and the octocoral reference collection of the research team Biodiversidad y Ecología de Invertebrados Marinos in the University of Sevilla (BEIM-CRO). Eleven colonies have been kept alive in the aquarium rooms at the Institut de Ciències del Mar in Barcelona (ICM-CSIC).

## Taxonomy

**Order Alcyonacea**

**Family Nidaliidae** Gray, 1869

**Genus Nidalia** Gray, 1835

**Nidalia studeri** (Koch, 1891) comb. nov.
Figures 1–10

### Material examined

MZB 2011-0001, 'INDEMARES-CSIC III, Menorca I' cruise, Jago dive no. 1139, Minorca Channel, North Western Mediterranean, 39° 48. 173'N–4° 8.928'W, 139 m depth, 5 September 2010, 1 whole colony in two halves, 37 mm in length (retracted polyps, formalin fixed). MZB 2011-0002, 1 whole colony (semi-retracted polyps, ethanol fixed). MZB 2011-0003, 1 colony in three parts (extended polyps, formalin fixed). (ICM-CSIC, 2010-INDEMARES-III-001-011), 11 whole colonies. BEIM (CRO-0067), 1 whole colony (semi-retracted polyps, ethanol fixed). All lots with the same sampling data as the MZB 2011-0001 material.

### Description: (Colony MZB 2011-0001)

Colony unbranched, torch-like (Figure 1A,D), 21 mm in length, with elongated stalk about 16 mm, 76.2% of total colony length. Stalk adhering by one or more points to hard substrata. Capitulum dome-shaped, oval to rectangular in section, not distinctly projecting beyond the stalk, 13 mm in maximum diameter. Gonochoric, developing spermatic cysts up to 0.32 mm in diameter.

Capitulum with few polyps (about 18). Calyces prominent, up to 2.4 mm height, usually shorter (1.4–1.9 mm); with eight-lobed edge with spindles, 0.30–0.75 mm in length and 0.019–0.038 mm in width (Figure 6A). Polyps up to 2 mm in diameter at calyx level, capable of complete retraction. Young polyps, about 0.9 mm in diameter, are present on the periphery of the capitulum (Figure 1D). Anthocodiae observed in partially retracted stage. Introvert, observed by dissection, about 3.5 mm in length, probably more, with densely distributed spindles, practically straight, mainly transversally placed in two rows along interseptal space (Figure 2B, 3), 0.10–0.24 mm in length and 0.010–0.019 mm in width (Figure 7C,D). Anthocodiae with distinct crown and points (Figure 1F, 2A, Figure 3). Crown with 28–38 transversally placed lines of curved spindles, 0.33–0.85 mm in length and 0.020–0.034 mm in width (Figure 7B). Points distinctly separated from polyp body (Figure 1F), with 6–9 pairs of slightly curved spindles, 0.37–0.82 mm in length and 0.020–0.041 mm in width (Figure 7A), proximally arranged en chevron, distally arranged parallel to each other (Figure 2A, 3). Intermediate point (secondary points) between principal (interseptal) ones, with spindles 0.12–0.57 mm in length, a larger spindle in the middle of two contiguous principal points basally and smaller spindles distally (Figure 2A, 3). Extended tentacles not seen, densely filled with curved spindles of 0.17–0.46 mm in length and 0.021–0.042 mm in width on the aboral side, larger basally (Figure 8A), narrow spindles of 0.08–0.30 mm in length and 0.009–0.020 mm in width on the lateral sides (Figure 8B), placed transversally distally. Pinnales with platelets 0.06–0.14 mm in length and 0.012–0.026 mm in width on the proximal half or third (Figure 8C,D).

Surface layer of the stalk with spindles arranged mainly longitudinally, but also with some sclerites oriented oblique to the main axis of the stalk, 0.24–0.89 mm in length and 0.015–0.034 mm in width (Figure 6B), ratio length to width 16–25 : 1. Interior of the stalk with spindles in the internal walls between gastrovascular cavities of polyps, arranged as on the surface layer, 0.18–0.75 mm in length and 0.016–0.035 mm in width (Figure 6C), ratio length to width 13–20 : 1.
A forgotten soft coral species from NW Mediterranean 597

<figure><img src="image_85.png" /><figcaption>Figure 1. *Nidalia studeri* comb. nov. (A) colony MZB 2011-0001; (B and C) colonies, MZB 2011-0002 and MZB 2011-0003, respectively; (D) detail of the capitulum of the colony MZB 2011-0001, lateral view, showing polyps with their distinct calyces and a small polyp on the periphery of the capitulum (arrowed); (E) capitulum of the holotype, upper view; (F) detail of two polyps showing principal and secondary points, the former distinctly separated from the polyp body.</figcaption></figure>

Sclerite ornamentation sparse, thorny, thorns often blunt (Figure 9).

Stalk and capitulum of preserved colonies light orange due to colour of sclerites, more intense at capitulum; anthocodia whitish in colour with colourless sclerites.

## Variations

The general colonial structure of the additional examined material (Figure 1B,C) is quite similar to

that of the colony MZB 2011-0001. The stalk is elongated, but shorter than in the above-described specimen. The capitulum is dome-shaped, oval in section, not distinctly projecting beyond the stalk. The form and distribution of the calyces and the sclerites are as in the above-described colony. Points separated from polyp body are distinctly identifiable also in semi-retracted and relaxed specimens (Figure 4). Relaxed specimens show polyps elongated, occupying crown sclerites a little bit more than pharynx plus reproductive area of mesenteries
598 P.J. López-González et al.

<figure><img src="image_86.png" /></figure>

Figure 2. *Nidalia studeri* comb. nov. colony MZB 2011-0001 (A) primary points (ppo), secondary points (spo), and sclerites of the crown (crs), partially dissected; (B) detail of the arrangement of sclerites in the introvert (contracted state) from an interseptal space, partially dissected.

in length, and nearly 40 lines (or more) of sclerites; introvert with sclerites placed transversally in two areas, close to mesenterial insertions (Figure 5E), the central part of the interseptal space being nearly free of sclerites; the tentacles have 14–16 pinnules, sclerites mainly placed on the basal part of the tentacle, with accumulation of cnidocysts distally (Figure 5C,D).

Underwater observations of living specimens show elongated polyps, those in central positions being larger; stalk and capitulum light orange, colour more intense in the capitulum, polyps whitish translucent with a relatively disperse distribution of the sclerites in extended stage (Figure 10).

<figure><img src="image_87.png" /></figure>

Figure 3. *Nidalia studeri* comb. nov. colony MZB 2011-0001. Anthocodial armature. Tentacles not drawn, notice the presence of principal and secondary points, densely packed crown sclerites, and smaller introvert sclerites mainly arranged in two longitudinal rows per interseptal space.

## Habitat and depth distribution

The colonies examined were observed at a depth range of 100–150 m at the border of the continental shelf. Fourteen of these colonies were collected in 139 m depth; however, in a deeper dive, two colonies were observed at 329 m depth growing on a boulder. Colonies formed continuous patches on rocky bottoms and small boulders. Both types of substrate were slightly covered by sediment, but mainly colonized by small colonies of bryozoans or invertebrates forming calcareous colonies. It seems that colonies were more abundant at the edge of rocky outcrops. Colonies grew on mildly inclined substrates and appear to avoid vertical substrates.
<figure><img src="image_88.png" /><figcaption>Figure 4. *Nidalia studeri* comb. nov. Colony MZB 2011-0003. (A) Extended polyps; (B) detail of one of the polyps in (A) showing points separate from polyps body. Paratype (MZB 2011-0002). (C) Polyp semi-retracted, showing points distinctively separated from polyp body.</figcaption></figure>

The main species associated with *Nidalia studeri* were the cold-water gorgonian *Callogorgia verticillata* (Pallas, 1766) and an unidentified Plexauridae, the former being more abundant. Different size classes of *C. verticillata* could be distinguished, some colonies had broken branches and dead colonies were also observed. At least two species of the desmosponge genus *Phakellia* Bowerbank, 1862 were observed during the dive where the *N. studeri* was found and collected.

### Geographical distribution

At present, *Nidalia studeri* is known from the Western Mediterranean basin (Balearic Islands and Gulf of Naples).

### Discussion

Koch's description and illustration correspond with the current conception of a species to be included in the genus *Nidalia*. Moreover, the form of the colony, the presence of well-defined points and distinct mesenterial insertions due to the presence of sclerites as secondary points, as well as the size of sclerites in determined parts of the colony, especially those of the crown (pseudocrown for some authors) and introvert (0.8–0.9 mm for the former and 0.25 mm for the later), and surface of stalk (up to 1 mm), are key characters that we can recognize in our newly collected specimens. These and other characters will be used to compare *Nidalia studeri* with the other recognized congeners to decide if it should be considered a different species in the genus or synonymized under another species name previously described (although in the genus *Nidalia*, only *N. occidentalis* Gray, 1835 was described before

Koch's species). *Nidalia occidentalis* clearly differs from *N. studeri* by its mushroom-shaped colony with hemispherical capitulum with its edges distinctly projected beyond the narrow stalk, and introvert without or almost devoid of sclerites (see Verseveldt & Bayer 1988: 59).

According to the review of the genus *Nidalia* carried out by Verseveldt & Bayer (1988), and the recently described species *Nidalia aurantia* López-González & Gili (2008), 13 species are currently recognized as valid in this genus. As revised descriptions and illustrations of these species are relatively recent, additional information about the different anatomical and distributional data of all the *Nidalia* species can be found in the mentioned works.

According to the key to species and descriptions given by Verseveldt & Bayer (1988) and the last described species in the genus (López-González & Gili 2008), *N. studeri* is only morphologically comparable to *N. simpsoni* (Thomson & Dean, 1931) and *N. dissidens* Verseveldt & Bayer, 1988 by having torch-like colonies, a capitulum not laterally flattened, the presence of both principal and secondary points, and a high number of anthocodial sclerites in the crown (at least greater than 15).

However, *N. studeri* is clearly differentiated from *N. simpsoni* and *N. dissidens* by: (1) the distinctive separation of the principal points from polyp body distally; (2) the maximum number of lines of sclerites in crown (about 20 lines in *N. simpsoni*, about 30 lines in *N. dissidens*, but around 40 lines in *N. studeri*); (3) type, size, and arrangement of the sclerites in the introvert (interlacing rods and curved spindles, up to 0.5 mm in length, and often forming small scattered groups of parallel spindles in *N. dissidens*; minute dumb-bell sclerites, 0.08–0.14 mm in length in *N. simpsoni*; while in *N. studeri* these
600 P.J. López-González et al.

<figure><img src="image_89.png" /></figure>

Figure 5. *Nidalia studeri* comb. nov. Colony MZB 2011-0003. (A) Tentacle partially dissected showing distal separate part of the point (po) and aboral tentacular sclerites (abs), aboral side; (B) distal part of tentacle showing sclerites covering the main axis of the tentacle and pinnules (pin) with platelets; (C and D) detail of two pinnulae (pin) showing distribution of pinnular sclerites (pins) on the basal third or half, and groups of cnidocysts distally; (E) detail of the transition of crown and introvert, showing the relatively large and curved crown sclerites (crs, upper part of the photo) and the distinctive arrangement in two rows along each interseptal space of the smaller introvertal sclerites (ints, two-third basal part of the photo).

sclerites are straight spindles, 0.10–0.24 mm in length, transversally arranged in two lines along interseptal space); (4) sclerites of the surface and interior of the stalk (rods and spindles up to 1.2 mm with irregular spines and thorns in *N. dissidens*, thick straight or curved spindles up to 2.60 mm with conspicuously high, often branched warts in *N. simpsoni*, while in *N. studeri* these sclerites are

<figure><img src="image_90.png" /></figure>

Figure 6. *Nidalia studeri* comb. nov. colony MZB 2011-0001. (A) sclerites from calyx; (B) sclerites from surface of the stalk; (C) sclerites from interior of the stalk. Notice that all sclerites are shown at the same scale.

elongated scarcely spinose spindles up to 0.75 mm in length); (5) colour in preserved material (creamy white in *N. dissidens*, the overall colour of *N. simpsoni* is brownish orange except for the small sclerites of
<figure><img src="image_91.png" /><figcaption>Figure 7. *Nidalia studeri* comb. nov. colony MZB 2011-0001. (A) Sclerites from points; (B) sclerites from crown; (C) sclerites from introvert; (D) sclerites from introvert, magnified. Notice that all sclerites in A to C are shown at the same scale.</figcaption></figure>

<figure><img src="image_92.png" /><figcaption>Figure 8. *Nidalia studeri* comb. nov. colony MZB 2011-0001. (A) Sclerites from aboral side of tentacle; (B) sclerites from lateral side of tentacle; (C) plates from pinnules; (D) plates from pinnules, magnified. Notice that all sclerites in A to C are shown at the same scale.</figcaption></figure>

the pinnules and introvert which are dull orange or reddish orange, while *N. studeri* is light orange, more intense in the capitulum due to the high density of sclerites (Faulkner 1974; Verseveldt & Bayer 1988; present study).

With respect to the geographical distribution of these three species, *N. simpsoni* is from the Indonesian Archipelago and Palau Islands, *N. dissidens* is from the Straits of Florida, Bahamas and Lesser Antilles (Western Atlantic) and *N. studeri* is from the North Western Mediterranean.
602 P.J. López-González et al.

<figure><img src="image_93.png" /><figcaption>Figure 9. *Nidalia studeri* comb. nov. colony MZB 2011-0001. (A) Surface of a sclerite from introvert. (B) Surface of a sclerite from point.</figcaption></figure>

*Nidalia aurantia* is the geographically closest congener to *N. studeri*. The former species was recently described from south of the Azores (López-González & Gili 2008), and shares with *N. studeri* several characters: (1) colour, both are light orange; (2) torch-like colonies; (3) relatively numerous anthocodial sclerite rows in the crown (15–17 for *N. aurantia*, but up to nearly 40 rows in *N. studeri*). However, both species are clearly different regarding: (1) points (distinctly separated from the polyp body, and with secondary points in *N. studeri*, these features absent in *N. aurantia*); (2) calyces (prominent in *N. studeri*, but low in *N. aurantia*); (3) introvert (longer, with sclerites up to 0.24 mm in *N. studeri*, but shorter with scarce sclerites up to 0.5 mm in *N. aurantia*); (4) crown sclerites (spindles up to 0.85 mm in length in *N. studeri*, but up to 1.23 mm in *N. aurantia*); and (5) inner sclerites of the stalk (nearly straight spindles in *N. studeri*, while these sclerites are sometimes shortly branched in *N. aurantia*).

The separation (or not) of the points from the polyp body is a specific character previously used in the genus *Nidalia* (see Verseveldt & Bayer, 1988) and other soft coral genera like *Sphaeralkyon López-González and Gili, 2000* (see López-González & Gili 2000, 2005).

Two other *Nidalia* species are also present in Atlantic waters (West Indian Region), *N. occidentalis* Gray, 1835 and *N. deichmannae* Utinomi, 1954. However, both species have a smaller number of anthocodial crown sclerites rows (less than 20 rows in *N. occidentalis* and *N. deichmannae*, but up to nearly 40 rows in *N. studeri*). Besides, their colonies are mushroom-shaped, having a hemispherical

<figure><img src="image_94.png" /><figcaption>Figure 10. *Nidalia studeri* comb. nov. Living colonies photographed in situ during JAGO dive. (A) About eight colonies of *Nidalia studeri* on the bottom. (B) One of the colonies collected by JAGO arm. (C) The same colony in (B), magnified, the smaller size of the polyp in the periphery (arrowed) can be seen.</figcaption></figure>

capitulum with its edges distinctly projecting beyond a narrowed stalk, and the introvert lacks sclerites (*N. occidentalis*) or at most have a few proximally and distally (oval and rod-shaped, 0.03–0.05 mm long) sclerites sometimes with a median constriction in *N. deichmannae*). However, *N. studeri* has a torch-like colony with a dome-shaped polyparium not distinctly projecting beyond stalk, and an introvert with
numerous sclerites (spindles, 0.10–0.24 mm long sclerites) along its entire length.

As has been previously stated, *Nidalia* species are mostly concentrated in the Indian Ocean and the western sector of the Indo-Pacific Ocean. In the Atlantic Ocean they are mainly found in the Caribbean area. A total of five species have been described in the Atlantic Ocean, four of which have been found in the West Indian Region, and one in the Mid-Atlantic (López-González & Gili 2008). With the rediscovery of *N. studeri*, 120 years after its original description in the Western Mediterranean basin, this is the first time that the genus *Nidalia* and the family Nidaliidae are reported with certainty for the Mediterranean Sea.

As occurred in the case *N. studeri*, the use of increasingly sophisticated equipment in benthic fauna surveys such as remotely operated vehicles (ROV's) and manned submersibles is considerably improving our knowledge of the structure of shelf and deep-sea benthic communities, especially necessary for the study of hard bottom fauna, and enabling the collection of delicate specimens, not collected or probably damaged and overlooked with traditional trawling gears. In the near future, we will probably have the opportunity to increase our knowledge of the diversity of this benthic small-sized (a few centimetres) fauna from the shelf (benthic cnidarians being a good example).

## Acknowledgements

The authors thank to the scientists and crew on board the BIO *García del Cid* during the 'INDEMARES-CSIC III, Menorca I' cruise, and the *Jago* team (J. Schauer and K. Hissmann) from IFM-GEOMAR (Kiel, Germany), without whose help it would not have been possible to collect the specimens for this work. Our thanks are also addressed to Dr J. Köhler (Hessisches Landesmuseum), who kindly informed us about the possible fates of von Koch's specimens. This research was funded by LIFE+ INDEMARES project. We also thank all referees and editorial editors of MBR for their detailed comments and their important criticisms that have substantially improved earlier versions of this manuscript. Mr Tony Krupa is thanked for reviewing the English version.

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Editorial responsibility: Ole S. Tendal
Annex II

GRINYÓ J (2016) PhD Thesis pp 175-198
<figure><img src="image_95.png" /></figure>

LÓPEZ-GONZÁLEZ, P.J., GRINYÓ, J. AND GILI, J.M. 2012
*Chironephthya mediterranea* n. sp. (Octocorallia, Alcyonacea, Nidaliidae), the first species of the genus discovered in the Mediterranean Sea.
*Marine Biodiversity, 45: 667-688
ORIGINAL PAPER

*Chironephthya mediterranea* n. sp. (Octocorallia, Alcyonacea, Nidaliidae), the first species of the genus discovered in the Mediterranean Sea

Pablo J. López-González · Jordi Grinyó · Josep-Maria Gili

Received: 23 December 2013 / Revised: 20 August 2014 / Accepted: 26 August 2014 / Published online: 27 September 2014

© Senckenberg Gesellschaft für Naturforschung and Springer-Verlag Berlin Heidelberg 2014

**Abstract** In this paper, a new species of the soft coral genus *Chironephthya* from the northwestern Mediterranean Sea is described and illustrated. *Chironephthya mediterranea* sp. nov. is formally described based on morphological, chromatic, molecular, and ecological data. A molecular comparison based on mitochondrial genes (*mtMutS+COI*) and the proposed extended barcode (*mtMutS+Igr1+COI*) relates the Mediterranean species with other *Chironephthya* species. The new Mediterranean species is compared with the available information and type materials from its Atlantic congegers, *Chironephthya agassizii* and *Chironephthya caribaea*. This is the first time that a species of this genus is reported from the Mediterranean Sea. Along with *Nidalia studeri*, this is the second species of the family Nidaliidae found in this biogeographic region. Both nidaliid species were collected in the shelf break area. The present report adds to previous knowledge of *Chironephthya* and its global distribution. Although the molecular analyses carried out do not support the monophyly of the family Nidaliidae, they clearly indicate a

close relationship between the genera *Siphonogorgia* and *Chironephthya*, which are both in need of revision.

**Keywords** Mediterranean · Biodiversity · Chironephthya · Nidaliidae · Soft corals

**Introduction**

Although our current estimates are probably far from the reality, the family Nidaliidae includes more than 70 nominal species distributed in eight genera (e.g., Studer, 1887; Wright and Studer 1889; Kükenthal 1906; Harrison 1908; Kükenthal 1910; Verseveldt and Bayer 1988; Williams 2000; and others). Many species and several genera of the family are in need of revision. The most species-rich genus is *Siphonogorgia* Kölliker, 1874, with about two-thirds of the total species in the family; followed by *Nidalia* Gary, 1835 (about 18 spp.); *Chironephthya* Studer, 1887 (about 11 spp.); *Pieterfaurea* Verseveldt and Bayer, 1988 (5 spp.); *Nephtyigorgia* Kükenthal, 1910 (4 spp.); *Nidaliopsis* Kükenthal, 1906 (3 spp.); *Agaricoides* Simpson, 1905 (1 sp.); and finally, *Orlika* Malyutin, 1993 (1 sp.). With the exception of *Nidaliopsis*, all genera are monomorphic, and most of them have relatively simple colonies (unbranched torch- to mushroom-shaped, digitated, palmated, or with few rounded lobes). Only *Siphonogorgia* and *Chironephthya* have branched tree-like colonies. Although the genus *Nidaliopsis* is known only from tropical West African coasts, most of the genera of the family have an Indian Ocean or Indo-West Pacific distribution. The genera *Nidalia* and *Chironephthya* are known from Indian Ocean, West Pacific, and West Atlantic localities (e.g., Harrison 1908; Deichmann 1936; Bayer, 1961; Fabricius and Alderslade, 2001).

Recently, two species of the genus *Nidalia* were reported from the southern Azores Islands and the western

**Electronic supplementary material** The online version of this article (doi:10.1007/s12526-014-0269-5) contains supplementary material, which is available to authorized users.

P. J. López-González (✉)
Biodiversidad y Ecología de Invertebrados Marinos, Facultad de Biología, Universidad de Sevilla, Reina Mercedes 6, 41012 Sevilla, Spain
e-mail: pjlopez@us.es

J. Grinyó · J.-M. Gili
Institut de Ciències del Mar (CSIC), Passeig Marítim de la Barceloneta, 37-49 08003 Barcelona, Spain

J. Grinyó
e-mail: grinyo@icm.csic.es

J.-M. Gili
e-mail: gili@icm.csic.es
Mediterranean Sea, respectively (López-González and Gili 2008; López-González et al. 2012). In addition, Pérez et al. (2011) reported the genera *Nidalia* and *Chironephthya* from the Brazilian coast. An additional putative species attributable to the genus *Chironephthya* has been reported from the Canary Islands (East Atlantic) (Brito and Ocaña 2004) as *Siphonogorgia* sp. The recent rediscovery of *Nidalia studeri* in the western Mediterranean basin (López-González et al. 2012) represented the first record of Nidaliidae for the whole Mediterranean sea.

During the general community surveys carried out within the LIFE+INDEMARES project in the Menorca Channel, a previously unreported community dominated by whitish tree-like soft corals and gorgonians was discovered on the deep continental shelf and upper continental slope as well as the flanks of a submarine canyon (Grinyó et al., unpublished data) (Fig. 1). During the first cruises, this community was only video-recorded. During subsequent surveys, specimens of the key species were collected. The collection of the relatively large whitish soft coral was performed in August 2012 during the INDEMARES-CSIC VI, MENORCA IV cruise. The preliminary study of the specimens on board confirmed that they belonged to the nidalidii genus *Chironephthya*, not previously reported in Mediterranean waters.

The genera *Chironephthya* and *Siphonogorgia* are considered closely related (Fabricius and Alderslade 2001), and some additional species will likely be reassigned from *Siphonogorgia* to *Chironephthya* after the revision of both genera has been completed. At the moment, both genera are considered separate, at least from a morphological point of view. The goal of the present contribution is the description of a new *Chironephthya* species based on morphological and molecular data; to date, this is the only species is in the Mediterranean Sea. In addition, the possible phylogeographic implications as a link between the previously known set of West Atlantic and Indo-Pacific species is discussed in a global scenario.

## Material and methods

The material studied here was collected during the “INDEMARES-CSIC VI, MENORCA IV” cruise on board the R/V *García del Cid*, using the articulated arm of the ROV (Remotely Operated Vehicle) *Nemo*. The main goal of this cruise was to study the distribution patterns and diversity of the major benthic communities of the continental shelf and slope at the Menorca Channel and Cap de Creus peninsula (northwestern Mediterranean Sea). Video materials were examined to establish the geographic and bathymetric distribution of the new *Chironephthya* species in the study area.

Additional information on the communities was obtained with the manned submersible *JAGO* (cruise INDEMARES-

CSIC IV, MENORCA II) and the ROV *Nemo* (cruises INDEMARES-CSIC V, Menorca III, and INDEMARES-CSIC VI, Menorca IV) vessels. These cruises were carried out within the framework of the LIFE+INDEMARES project. The relevant information of all video and collecting stations is compiled in Table 1.

The soft coral colonies were fixed in absolute ethanol for morphological examination and molecular analysis. Fragments of different parts of the colony were prepared for study by SEM, employing the usual methodology as described by different authors (e.g., Bayer and Stefani 1988), and permanent slides were made for light microscopy observation. About thirty sclerites from each type and part of the colony and polyp (crown, introvert, points, surface of the stalk, etc.) were measured by optic microscopy. During this process it was attempted to include the complete range of sizes and to identify the different sclerite forms. All sclerite size measurements and illustrations are from the holotype. The colony and sclerite terminology described here mainly follows Bayer et al. (1983). The material studied here has been deposited at the Museu de Ciències Naturals in Barcelona (MCNB).

Total genomic DNA was extracted from the ethanol-preserved holotype and paratype using the E.Z.N.A. DNA kit (Omega Bio-Tek), following the manufacturer's instructions. The *mtMutS* (*MSH1*), and *Igr1+COI* mitochondrial regions were sequenced for comparative purposes as an octocoral barcode proposed by McFadden et al. (2011). The start of the *mtMutS* (*MSH1*) region was amplified using the primers ND42599F and MUT3458R (France and Hoover 2002; Sánchez et al. 2003). *The Igr1+COI* region was amplified using the primers COII8068F and COIOCTR (France and Hoover 2002; McFadden et al. 2004). Each PCR used 1 U/µl of DNA Stream Polymerase (BIORON), 0.2 mM of dNTPs, 0.3 µM of each primer, and approximately 25 ng of genomic DNA, and was brought to a final volume of 25 µL with H₂O. *MtMutS* PCR was carried out using the following cycle profile: initial denaturation at 94 °C for 2 min, 35 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, and a final extension at 72 °C for 5 min. The *Igr1+COI* PCR used the same cycle profile but 58 °C as annealing temperature and 40 s for extension duration on each of the 35 cycles. PCR products were purified using the NucleoSpin® Extract II DNA purification kit following the manufacturer's instructions. Purified products were electrophoresed on an ABI PRISM® 3730xl genetic analyzer, and sequence traces were edited using Sequencher™ version 4.0. The obtained sequences were compared with homologous sequences from the EMBL-Bank of Nidaliidae species (Table 2).

In a preliminary *mtMutS* comparison (not shown, ML, T92+G+I, 1000 bootstrap) including more than 300 octocoral species, a *Chironephthya-Siphonogorgia* clade is well-supported (bootstrap 100 %). However, there is not a
<figure><img src="image_96.png" /><figcaption>**Fig. 1** Distribution of the stations where *Chironephthya mediterranea* n. sp. was video-recorded (open circles) or video-recorded and collected (solid circles). Numbers in the stations correspond with the station</figcaption></figure>

numbers in the different cruises (see Table 1 for all additional sampling data). **a** Cap Formentor area; **b** Son Bou Canyon area

supported sister group to be used as an outgroup for subsequent internal comparison for all nidaliid sequences. This *Chironephthya-Siphonogorgia* clade is not grouped with the other nidaliid genera (*Nidalia*, *Nephthyigorgia*, *Pieterfaurea*) available for this comparison. Thus, the currently considered family Nidaliidae is not supported by this analysis. As an example of this trend, a *mtMutS+COI* analysis on groups of selected soft coral species from the families Alcyoniidae, Paralcyoniidae, Xeniidae, and Nephtheidae is shown here using the divergent sequence of a cornulariid species as outgroup (see Fig. 11, Table 2). Thus, as the goal of this paper is to explore the relationships of the Mediterranean species with their closest nidaliid forms, we use the set of available sequences of *Nidalia-Nephthyigorgia-Pieterfaurea* as

outgroup to explore *Chironephthya-Siphonogorgia* species relationships.

The alignment of the concatenated *mtMutS+COI* and *mtMutS+Igr1+COI* sequences were carried out using MUSCLE, implemented in MEGA5 (Tamura et al. 2011). The analyses involved 12 nidaliid nucleotide sequences. All positions containing missing data were eliminated. Positions containing internal gaps were maintained but not considered in the analysis. The *mtMutS+COI* concatenated sequences (11 nidaliid and 13 non-nidaliid sequences) had 1,424 positions, with a total of 534 variable and 285 parsimony-informative sites. The *mtMutS+COI* concatenated sequences (only the 11 nidaliid sequences) had 1,414 positions, with a total of 246 variable and 73 parsimony-informative sites. The *mtMutS+*

<table><caption>**Table 1** Localities where *Chironephthya mediterranea* n. sp. was video-recorded as well as (in bold) the collected type material used in this study</caption><thead><tr><th>Cruise</th><th>Date</th><th>Station</th><th>Coordinates</th><th>Depth Range (m)</th></tr></thead><tbody><tr><td>INDEMARES-CSIC IV, MENORCA II</td><td>01/04/11</td><td>10</td><td>40°00'35.880"N 3°08'36.930"E</td><td>159–174.4</td></tr><tr><td>INDEMARES-CSIC IV, MENORCA II</td><td>02/04/11</td><td>24</td><td>39°59'52.800"N 3°07'12.601"E</td><td>145.5</td></tr><tr><td>INDEMARES-CSIC IV, MENORCA II</td><td>03/04/11</td><td>41</td><td>40°02'10.980"N 3°09'47.880"E</td><td>116.1–126</td></tr><tr><td>INDEMARES-CSIC V, MENORCA III</td><td>02/11/11</td><td>30</td><td>40°00'44.220"N 3°08'45.720"E</td><td>136.2–186</td></tr><tr><td><strong>INDEMARES-CSIC VI, MENORCA IV</strong></td><td><strong>26/06/12</strong></td><td><strong>3</strong></td><td><strong>40°00'34.934"N 3°08'59.572"E</strong></td><td><strong>156–172.2</strong></td></tr><tr><td><strong>INDEMARES-CSIC VI, MENORCA IV</strong></td><td><strong>26/06/12</strong></td><td><strong>4</strong></td><td><strong>40°00'40.113"N 3°09'07.176"E</strong></td><td><strong>171.6 174.2</strong></td></tr><tr><td>INDEMARES-CSIC VI, MENORCA IV</td><td>27/06/2012</td><td>9</td><td>39°59'37.752"N 3°06'03.660"E</td><td>145–150</td></tr><tr><td>INDEMARES-CSIC VI, MENORCA IV</td><td>27/06/2012</td><td>11</td><td>39°59'52.338"N 3°07'31.056"E</td><td>118.1–140.5</td></tr><tr><td>INDEMARES-CSIC VI, MENORCA IV</td><td>27/06/2012</td><td>13</td><td>39°59'52.020"N 3°07'33.240"E</td><td>118.1–129.8</td></tr><tr><td>INDEMARES-CSIC VI, MENORCA IV</td><td>29/06/2012</td><td>31</td><td>40°00'57.000"N 3°08'52.020"E</td><td>13004–174.2</td></tr><tr><td>INDEMARES-CSIC VI, MENORCA IV</td><td>30/06/12</td><td>42</td><td>39°51'46.806"N 4°02'13.200"E</td><td>175.2–181.9</td></tr><tr><td>INDEMARES-CSIC VI, MENORCA IV</td><td>02/07/12</td><td>53</td><td>39°52'01.476"N 4°02'04.278"E</td><td>133.9–197.9</td></tr><tr><td>INDEMARES-CSIC VI, MENORCA IV</td><td>03/07/12</td><td>62</td><td>39°52'04.320"N 4°01'17.220"E</td><td>124.4–149.4</td></tr></tbody></table>
<table><caption>**Table 2** Octocoral species involved in the molecular comparisons carried out in this paper</caption><thead><tr><th>Family // Species</th><th>Catalog Nos. (or additional information)</th><th>mtMutS (=MSH1)</th><th>Igr+COI</th></tr></thead><tbody><tr><td>Nidaliidae</td><td></td><td></td><td></td></tr><tr><td><i>Chironephthya mediterranea n. sp. (Holotype)</i></td><td>MCNB 2013-0001</td><td>KM584053</td><td>KM584054</td></tr><tr><td><i>Chironephthya mediterranea n. sp. (Paratype)</i></td><td>MCNB 2013-0002</td><td>KM584055</td><td>KM584056</td></tr><tr><td><i>Chironephthya caribaea</i> (Deichmann 1936)</td><td>USNM 1025683</td><td>KM584051</td><td>KM584052</td></tr><tr><td><i>Chironephthya</i> sp. (1)</td><td>CSM-2010</td><td>GQ342513</td><td>GQ342440</td></tr><tr><td><i>Chironephthya</i> sp. (2)</td><td>NTM-C012426</td><td>DQ302830</td><td></td></tr><tr><td><i>Chironephthya</i> sp. 1</td><td>ZMTAU CO34223</td><td>GU356025</td><td>GU356002</td></tr><tr><td><i>Chironephthya</i> sp. 2</td><td>ZMTAU CO34222</td><td>GU356024</td><td>GU356001</td></tr><tr><td><i>Chironephthya</i> sp. 3</td><td>ZMTAU CO34226</td><td>GU356026</td><td>GU356003</td></tr><tr><td><i>Chironephthya</i> sp. A (1)</td><td>RMNH Coel. 40888</td><td>KF915576</td><td>KF955039</td></tr><tr><td><i>Chironephthya</i> sp. A (2)</td><td>RMNH Coel. 40892</td><td>KF915577</td><td>NA</td></tr><tr><td><i>Chironephthya</i> sp. A (3)</td><td>RMNH Coel. 40893</td><td>KF915578</td><td>KF955040</td></tr><tr><td><i>Chironephthya</i> sp. A (4)</td><td>RMNH Coel. 40894</td><td>KF915579</td><td>KF955041</td></tr><tr><td><i>Chironephthya</i> sp. A (5)</td><td>RMNH Coel. 40902</td><td>KF915580</td><td>KF955042</td></tr><tr><td><i>Chironephthya</i> sp. B (1)</td><td>RMNH Coel. 40889</td><td>KF915581</td><td>KF955043</td></tr><tr><td><i>Chironephthya</i> sp. B (2)</td><td>RMNH Coel. 40896</td><td>KF915582</td><td>KF955044</td></tr><tr><td><i>Chironephthya</i> sp. B (3)</td><td>RMNH Coel. 40898</td><td>KF915583</td><td>KF955045</td></tr><tr><td><i>Chironephthya</i> sp. B (4)</td><td>RMNH Coel. 40901</td><td>KF915584</td><td>KF955046</td></tr><tr><td><i>Chironephthya</i> sp. C (1)</td><td>RMNH Coel. 40890</td><td>KF915585</td><td>KF955047</td></tr><tr><td><i>Chironephthya</i> sp. C (2)</td><td>RMNH Coel. 40895</td><td>KF915586</td><td>KF955048</td></tr><tr><td><i>Chironephthya</i> sp. C (3)</td><td>RMNH Coel. 40899</td><td>KF915587</td><td>KF955049</td></tr><tr><td><i>Chironephthya</i> sp. D (1)</td><td>RMNH Coel. 40891</td><td>KF915588</td><td>KF955050</td></tr><tr><td><i>Chironephthya</i> sp. D (2)</td><td>RMNH Coel. 40897</td><td>KF915589</td><td>KF955051</td></tr><tr><td><i>Chironephthya</i> sp. D (3)</td><td>RMNH Coel. 40900</td><td>KF915590</td><td>KF955052</td></tr><tr><td><i>Nephthyigorgia</i> sp. (1)</td><td>RMNH Coel.40819</td><td>JX203804</td><td>JX203864</td></tr><tr><td><i>Nephthyigorgia</i> sp. (2)</td><td>NTM-C011345</td><td>DQ302831</td><td></td></tr><tr><td><i>Nidalia dissidens</i> Verseveldt and Bayer 1988</td><td>LII-10-566</td><td>KC984602</td><td>KC984638</td></tr><tr><td><i>Nidalia</i> sp.</td><td>NTM-C014876</td><td>DQ302828</td><td></td></tr><tr><td><i>Pieterfaurea khoisaniana</i> (Williams, 1988)</td><td></td><td>GQ342510</td><td>GQ342437</td></tr><tr><td><i>Pieterfaurea</i> sp.</td><td>NTM-C013943</td><td>DQ302829</td><td></td></tr><tr><td><i>Siphonogorgia godeffroyi</i> Kölliker, 1874</td><td>RMNH Coel.40833</td><td>JX203803</td><td>JX203860</td></tr><tr><td><i>Siphonogorgia</i> sp.</td><td>NTM-C011159</td><td>DQ302832</td><td></td></tr><tr><td><i>Siphonogorgia</i> sp. A (1)</td><td>RMNH Coel. 41041</td><td>KF915768</td><td>KF955244</td></tr><tr><td><i>Siphonogorgia</i> sp. A (2)</td><td>RMNH Coel. 41044</td><td>KF915769</td><td>KF955245</td></tr><tr><td><i>Siphonogorgia</i> sp. A (3)</td><td>RMNH Coel. 41045</td><td>KF915770</td><td>KF955246</td></tr><tr><td><i>Siphonogorgia</i> sp. A (4)</td><td>RMNH Coel. 41048</td><td>KF915771</td><td>KF955247</td></tr><tr><td><i>Siphonogorgia</i> sp. A (5)</td><td>RMNH Coel. 41051</td><td>KF915772</td><td>KF955248</td></tr><tr><td><i>Siphonogorgia</i> sp. A (6)</td><td>RMNH Coel. 41053</td><td>KF915773</td><td>KF955249</td></tr><tr><td><i>Siphonogorgia</i> sp. B (1)</td><td>RMNH Coel. 41042</td><td>KF915774</td><td>KF955250</td></tr><tr><td><i>Siphonogorgia</i> sp. B (2)</td><td>RMNH Coel. 41043</td><td>KF915775</td><td>KF955251</td></tr><tr><td><i>Siphonogorgia</i> sp. B (3)</td><td>RMNH Coel. 41046</td><td>KF915776</td><td>KF955252</td></tr><tr><td><i>Siphonogorgia</i> sp. B (4)</td><td>RMNH Coel. 41049</td><td>KF915777</td><td>KF955253</td></tr><tr><td><i>Siphonogorgia</i> sp. B (5)</td><td>RMNH Coel. 41050</td><td>KF915778</td><td>KF955254</td></tr><tr><td><i>Siphonogorgia</i> sp. B (6)</td><td>RMNH Coel. 41052</td><td>KF915779</td><td>KF955255</td></tr><tr><td><i>Siphonogorgia</i> sp. C</td><td>RMNH Coel. 41047</td><td>KF915780</td><td>KF955256</td></tr><tr><td><i>Siphonogorgia</i> sp. D</td><td>RMNH Coel. 41054</td><td>KF915781</td><td>KF955257</td></tr><tr><td><i>Siphonogorgia</i> cf. godeffroyi</td><td>RMNH Coel. 41055</td><td>KF915767</td><td>KF955243</td></tr><tr><td>Alcyonidae</td><td></td><td></td><td></td></tr><tr><td><i>Alcyonium acaule</i> Marion, 1878</td><td>MAR.9.AA3 (mtMutS)</td><td>GU355959</td><td>GU355942</td></tr></tbody></table>

<figure><img src="image_97.png" /></figure>
Mar Biodiv (2015) 45:667–688

671

<table><caption>**Table 2** (continued)</caption><thead><tr><th>Family // Species</th><th>Catalog Nos. (or additional information)</th><th>mtMutS (=MSH1)</th><th>Igr+COI</th></tr></thead><tbody><tr><td>Alcyonium roseum van Ofwegen et al., 2007</td><td>BAN.AA1 (Igr1+COI))</td><td>GQ342468</td><td>GQ342383</td></tr><tr><td>Lobophytum pauciflorum (Ehrenberg, 1834)</td><td>NTM-C014161 (mtMutS)</td><td>DQ280575</td><td>GQ342394</td></tr><tr><td>Sinularia leptoclados (Ehrenberg, 1834)</td><td>ZMTAU:CO35308</td><td>KC542857</td><td>KC542874</td></tr><tr><td>Sinularia verseveldti van Ofwegen, 1996</td><td>RMNH:Coel.40843</td><td>KC542860</td><td>KC542881</td></tr><tr><td>Paralcyoniidae</td><td></td><td></td><td></td></tr><tr><td>Ceeceinus quadrus van Ofwegen and Benayahu, 2006</td><td></td><td>GQ342514</td><td>GQ342442</td></tr><tr><td>Studeriotes sp.</td><td>CSM-2010</td><td>GQ342515</td><td>GQ342443</td></tr><tr><td>Nephtheidae</td><td></td><td></td><td></td></tr><tr><td>Scleronephthya gracillimum (Kükenthal 1906)</td><td></td><td>GU047879</td><td>GU047879</td></tr><tr><td>Dendronephthya klunzingeri (Studer, 1888)</td><td>ZMTAU CO34109</td><td>GU356018</td><td>GU355995</td></tr><tr><td>Nephthea acuticonica Verseveldt, 1974</td><td>ZMTAU CO34070</td><td>GU356023</td><td>GU356000</td></tr><tr><td>Xeniidae</td><td></td><td></td><td></td></tr><tr><td>Heteroxenia fuscescens (Ehrenberg, 1834)</td><td></td><td>GQ342528</td><td>GQ342462</td></tr><tr><td>Ovabunda obscuronata (Verseveldt and Cohen, 1971)</td><td>ZMTAU CO34077</td><td>GU356027</td><td>GU356004</td></tr><tr><td>Cornuliidae</td><td></td><td></td><td></td></tr><tr><td>Cornularia pabloi McFadden and van Ofwegen, 2012</td><td>USNM 1178390</td><td>JX203792</td><td>JX203847</td></tr></tbody></table>

Materials in bold are species sequenced for this study. Note that all GenBank sequences are considered here with the names as they appear in GenBank and their original publications (including numbers or letters). For sequences with duplicate complete names, we have include (1), (2), (3), etc., for the purpose of correctly identifying the sequence in Figs. 11 and 12 and Suppl. Fig. 1. Abbreviations for institutions or personal collections where voucher material from which molecular information was used in this paper is deposited are as follows: CSM, Collection of Catherine S. McFadden; MCNB, Museu de Ciències Naturals de Barcelona; NTM, Museum and Art Gallery of the Northern Territory, Australia; RMNH, Naturalis Biodiversity Center, formerly Rijksmuseum van Natuurlijke Historie, Leiden; USNM, National Museum of Natural History, Smithsonian Institution; ZMTAU, Zoological Museum, University of Tel Aviv.

Igr1+COI concatenated sequences (only the 11 nidaliid sequences) had 1,527 positions, with a total of 279 variable and 79 parsimony-informative sites. After alignment, the best nucleotide substitution model was selected using ModelTest implemented in MEGA5, according to Akaike information criterion (AIC) and hierarchical likelihood ratio test (hLRT) values. The phylogenetic reconstruction was obtained applying maximum likelihood (ML) and Bayesian inference methods. The maximum likelihood method was developed in MEGA5, and based on the T92+G model (Tamura 1992) in all data sets. The bootstrap consensus trees were inferred from 1,000 replicates (Felsenstein 1985). The Bayesian inference was carried out in MrBayes v3.1.2 program (Huelsenbeck and Ronquist 2001; Ronquist and Huelsenbeck 2003), using the model GTR+G (lset nst=6 rates=gamma), 10⁶ generations, and discarding 25 % initial trees.

An additional exploration of the affinities of the new *Chironephthya* species, but only based on the *mtMutS* marker, was carried out with the alignment of 16 nidaliid sequences (two of them *Siphonogorgia* spp.) (711 positions, 178 variable, and 97 parsimony-informative sites). For this *mtMutS* hypothesis, we used the same phylogenetic procedure as for the previous molecular comparison (ML, T92+G, 1000 bootstrap). In this case, all available nidaliid genera were used as

outgroup to explore the relationship between *Chironephthya* and *Siphonogorgia* species.

For comparative purposes, the types and additional material of Caribbean species of *Chironephthya* were consulted, diagnostically described, and illustrated (Suppl. Appendix 1).

Additional subsamples for molecular studies were also analysed, although only *Siphonogorgia caribaea* USNM 1025683 produced a good total DNA extraction and subsequent amplification of both mitochondrial markers examined here.

## Results

Order Alcyonacea

Family Nidaliidae Gray, 1869

Genus *Chironephthya* Studer 1887

## Diagnosis

Nidaliid with colonies branched, tree-like, and erect. Stalk attached to hard substratum. Branches rigid and brittle due to the presence of densely packed sclerites. Polyps on stem and branches, never restricted to terminal ends of branches. Main stem partially flattened in the preserved state, with wide
longitudinal canals internally separated by thin walls. Polyps monomorphic, placed upwards. Calyces prominent, shelf-like, with abaxial side supported by large sclerites split into two groups as two distinct calycular teeth. Anthocodiae completely retractile into calyces, but often partially extended in preserved material. Crown and points well-developed. Aboral side of the tentacles with warty plates. Pinnules with elongated platelets. Pharynx with densely distributed thorny rods.

## Remarks

The type species of this genus was originally not designated by Studer (1887: 20). However, when Wright and Studer (1889: 230) again published the diagnosis of *Chironephthya* as “n. gen.”, they described *Chironephthya dipsacea* (Wright and Studer 1889), together with two other new *Chironephthya* species, and all three from Japanese waters.

As commented above, the genera *Siphonogorgia* and *Chironephthya* seem to be closely related. As originally stated by Studer (1887) and Wright and Studer (1889: 230), both genera differ in the distribution of the polyps within the colony (restricted to the terminal branches in *Siphonogorgia*, also distributed on the stem and branch surfaces in *Chironephthya*). This is one of the most visible characteristics to distinguish between these two nidalid genera. Different authors have suggested that both names should be synonymised under *Siphonogorgia* (Chalmers 1929; Bayer 1961), whereas other authors observed enough difference to disagree (Verseveldt and Bayer 1988; Fabricius and Alderslade 2001; Cairns and Bayer 2009). For a detailed review of the history of the genus *Chironephthya* since its original description, see Pérez et al. (2011).

In addition to the distribution of the polyps within the colony, Fabricius and Alderslade (2001) also pointed out the different architecture of the calyces in the two genera. In *Chironephthya* the calyces are distinct, shelf-like, with only lateral and abaxial sides developed, adaxial side nonexistent, and strongly supported abaxially by a set of long spindles that are split into two groups. In the case of *Siphonogorgia*, the calices are formed by small spindles and rods, and are reduced in size, often as a narrow rim around the retracted anthocodia. Harrison (1909) discussed the larger number and larger size of the longitudinal canals in the stem of *Chironephthya* in comparison with those in *Siphonogorgia*. This author also discussed the fact that the stem and branches of *Siphonogorgia* are solid, round, and cylindrical, while the stem and branches in *Chironephthya* have a less solid appearance and become somewhat flattened and deeply grooved, with terminal branches much more slender than those of *Siphonogorgia*. In addition, the interior of the stem and branches are studded with numerous sclerites of different sizes in *Siphonogorgia*, but are practically without sclerites in

*Chironephthya* (Harrison 1909). The new species of soft coral collected in the western Mediterranean is consistent with the characteristics assigned to the genus *Chironephthya* by Harrison as well as Fabricius and Alderslade.

The Indo-Pacific species classifications of *Chironephthya* are in need of revision. Harrison (1909) lists up to 18 putative species (not including Caribbean species described later by Deichmann [1936] as *Neospongodes*). Currently, the number of valid species in this genus is uncertain. Recent databases estimate this number to be 11 (Williams and Cairns 2007) or even less, as is the case in the WoRMS database, which currently (June 2014) lists only two Caribbean species (van Ofwegen 2014a). This last record can be explained, however, since most of the Indo-Pacific species are still immersed within the genus *Siphonogorgia* in that database (van Ofwegen 2014b). As mentioned above, the revision of the related genus *Siphonogorgia* is necessary because some *Chironephthya* species may still be cryptic under the former genus. Clearly, the available morphological descriptions are not adequately detailed, lacking measurements of sclerites, especially from the anthocodia, where several specific characteristics have proven useful in other nidalid genera (such as *Nidalia*).

### *Chironephthya mediterranea* sp. nov

(Figures. 2, 3, 4, 5, 6, 7, 8, 9, and 10)

## Material examined

Holotype: (MCNB 2013–0001), “INDEMARES-CSIC VI, Menorca IV” cruise, Menorca Channel, Northwestern Mediterranean, stn. 4, 40°00′34.934″N 03°08′59.572″E, 174 m depth, 26 Jun. 2012, collected by ROV, 1 whole colony.

Paratypes: (MCNB 2013–0002), “INDEMARES-CSIC VI, Menorca IV” cruise, Menorca Channel, Northwestern Mediterranean, stn. 3, 40°00′40.113″N 03°09′07.176″E, 154 m depth, 26 Jun. 2012, collected by ROV, 2 colonial fragments.

## Description of the holotype

The colony is branched, tree-like, and erect (Figs. 2a and b), 95 mm in length in preserved state. The stalk is attached to hard substratum, not extending beyond the basal stalk dimensions. The branches are rigid and brittle due to the abundance of large and densely packed sclerites in the colony wall. The polyps are distributed along the entire length of the stem and branches, sometimes crowded at the ends but never restricted to them. Some solitary polyps may also be present on the main stem. The polyps are oriented upwards, completely retractile into the calyces, but often partially extended in the preserved material. The main stem is 15 × 8 mm measured 15 mm from
**Fig. 2** *Chironephthya mediterranea* n. sp. Holotype (MCNB 2013–0001). **a** Whole colony; **b** detail of distal branches. Paratype (MCNB 2013–0002): **c** Whole fragment; **d** and **e**, details from **c**. White arrows in figure **d** indicate the measured aboral side of calyx. All photographs were taken on living materials. Note the orange colour of the pharynx from the transparency of the wall of polyps

<figure><img src="image_98.png" /></figure>

substratum, partially flattened in the preserved specimen, and externally covered by a brownish periderm. The stem has about 10 wide longitudinal canals internally (Fig. 3a), and the stem wall is relatively thin, 0.5 mm thickness, with densely packed sclerites (Fig. 3b). Canals are separated by thin walls with scarce sclerites (Fig. 3c). The branches secondary to distal branches are progressively smaller in size with branching order, but in general have a cylindrical aspect. The distal branches are about 5 mm in diameter, including

calyces. The density of cortical sclerites in distal branches is lower in comparison with basal parts of the colony. Sclerites are nearly absent in distal branches internally.

The calyces are prominent, shelf-like, up to 2 mm along the abaxial side, on the adaxial side nearly absent (Figs. 2d and 4a, c), only slightly distinct in the distal-most polyps of the distal branches (Fig. 2b). The abaxial side is supported by large sclerites split into two groups as two distinct calycular teeth, with 2–4 (often 3) large sclerites per point, plus

**Fig. 3** *Chironephthya mediterranea* n. sp. Holotype. **a** Main stem sectioned transversely about 15 mm from the substratum (note the relatively thin exterior wall and wide longitudinal canals); **b** Portion of the wall showing densely packed warty spindles; **c** Portion of the internal walls between longitudinal canals showing scarce warty spindles

<figure><img src="image_99.png" /></figure>
<figure><img src="image_100.png" /><figcaption>**Fig. 4** *Chironephthya mediterranea* n. sp. Holotype. **a–c** Polyps showing calycular teeth and anthocodia with crown and points; **d** Crown and points; **e** Dissected tentacle showing (from right to left) part of the crown (cro), point (po), aboral side of tentacle with platelets (abt), and pinnules with elongated platelets (pin); **f** Elongated platelets from pinnules; **g** Dense group of sclerites from the pharynx; **h** isolated sclerites from the pharynx. *White arrows* in **a, b**, and **c** indicate sclerite-less areas in the calyx</figcaption></figure>

additional, shorter and thinner sclerites among them (Figs. 4b and c). In distal branches, the sclerites are thorny spindles up to 2.8 mm in length and 0.15 mm in width (Fig. 5).

In the anthocodiae, the introvert is lacking sclerites, but crown and points are well-developed. The polyps are 1.2 mm in diameter at crown level. The crown has 4–7 rows of transversely placed curved thorny spindles (Figs. 4d and 6a), up to 1.1 mm in length (measured between points, as an arc bowstring), often nearly smooth on the interior (concave) side. The points are 1–3 pairs of curved thorny spindles, blunt basally, up to 1 mm in length (Figs. 4d, e and 6b), some of these smaller, up to 0.25 mm in length. Sclerites as secondary points were observed; sporadic single sclerites of 0.1 mm could be found, but were usually absent. The aboral side of the tentacles has transversely or slightly obliquely placed

warty plates up to 0.36 mm in length (Figs. 4e and 7a). The pinnules, largely the basal ones, have elongated platelets up to 0.21 mm in length (Figs. 4e and f and 7b and d).

The sclerites in the pharynx are densely distributed (Fig. 4g) as thorny rods, often with a marked waist and several prominent constrictions (Figs. 4h, 7c, and 8), up to 0.05 mm in length. The wall of the stalk has warty spindles densely packed, primarily arranged longitudinally, but also with some sclerites oriented oblique to the main axis (Figs. 3b and 9a), up to 3.5 mm in length and 0.22 mm in width. The interior of the stalk has a few sparsely distributed spindles (of similar size to those of the surface of the stalk) in the internal walls between longitudinal cavities (Figs. 3c and 9b). The stalk, main stems, branches, and polyps of living and preserved colony are whitish in colour, except for the pharynx, which is orange
<figure><img src="image_101.png" /><figcaption>**Fig. 5** *Chironephthya mediterranea* n. sp. Holotype, **a** Calycular and coenenchymal sclerites from distal branches; **b–c** Distal part of two sclerites showing different orientation of the spines; **d** Surface ornamentation of a spindle</figcaption></figure>

due to the colour of the sclerites (Figs. 2a and b); all other sclerites in the colony are colourless. The basal part of the stalk is brownish due to the presence of a periderm.

## Variations

The general structure of the paratype fragments (Figs. 2c, d, and e) is similar to that of terminal parts of the holotype. Paratype fragments were collected nearby site (about 300 m apart) during the same sampling cruise, so we can justifiably consider all types of materials used in the description of this species as belonging to the same population.

## Habitat and depth distribution

*Chironephthya mediterranea* n. sp. was found at the edge of the continental shelf, the upper continental slope, and the

flanks of the Son Bou Canyon, at a depth range of 115–200 m, although most was present between 150 and 185 m. Colonies were mainly observed growing on rocky bottoms and boulders (Fig. 10a). Both substrates could appear slightly covered by a fine sediment veneer. Occasionally, isolated colonies were encountered on gravelly bottoms and on dead shell debris (Fig. 10b).

Colonies were found to grow over a wide inclination range, from mildly inclined substrates to almost vertical walls.

<figure><img src="image_102.png" /><figcaption>**Fig. 6** *Chironephthya mediterranea* n. sp. Holotype. Anthocodial sclerites of the holotype. **a** Crown; **b** Points (note that both set of sclerites are at the same scale)</figcaption></figure>
<figure><img src="image_103.png" /><figcaption>**Fig. 7** *Chironephthya mediterranea* n. sp. Holotype. Anthocodial sclerites of the holotype. a Aboral side of the tentacles; b Three sclerites from pinnules; c Four sclerites from pharynx (note that a, b, and c have been presented at the same scale to show the important differences in size in anthocodial sclerites [see also Fig. 6]); d Pinnules (note that a–b–c and d are not at the same scale)</figcaption></figure>

However, most colonies were observed growing on mild and intermediate inclinations. The species primarily appeared forming small aggregations, although in areas well-exposed to currents it could reach high densities. Based on video material recorded by submersible and ROV, the average density was estimated to be 1–3 individuals m⁻² and maximum density was 12 individuals m⁻².

The benthic species most commonly associated with *Chironephthya mediterranea* n. sp. were the gorgonian *Bebryce mollis* (Philippi, 1842), *Swiftia pallida* (Madsen,

<figure><img src="image_104.png" /><figcaption>**Fig. 8** *Chironephthya mediterranea* n. sp. Holotype. Sclerites from the pharynx</figcaption></figure>

1970), *Viminella flagellum* (Johnson, 1863 [see, Fig. 10c]), the antipatharians *Leiopathes glaberrima* (Esper 1788) and *Antipathes dichotoma* (Pallas, 1766), and two demospongiae species, *Poecillastra compressa* (Bowerbank 1866) and *Phakellia robusta* (Bowerbank, 1866).

## Geographical distribution

At present, *Chironephthya mediterranea* n. sp. is known in the northwestern Mediterranean Sea (Balearic Islands).

## Phylogenetic analysis

In preliminary *mtMutS* and *mtMutS+COI* comparisons (not shown), the sequences obtained in this work for *Chironephthya mediterranea* and *C. caribaea* were merged into a nidaliidae clade including other *Chironephthya* and *Siphonogorgia* sequences available in GenBank. In that tree, nidaliid genera are not reunited in a single clade. An example with a reduced number of octocorals species and genera is shown in Fig. 11 for *mtMutS+COI*. As in the previous tree, the analysis using only *mtMutS* (Fig. 12) and using a select number of nidaliid species as outgroup did not group *Chironephthya* and *Siphonogorgia* sequences together in a
<figure><img src="image_105.png" /><figcaption>**Fig. 9** *Chironephthya mediterranea* n. sp. Holotype. **a** Coenenchymal sclerites exterior wall of the main stem; **b** Coenenchymal sclerites from the interior walls between longitudinal canals (note that dotted lines indicate the limits of a broken spindle)</figcaption></figure>

different clade. No shared sequence was found between these specimens assigned to these two genera.

The analysis of the two mitochondrial genes, *mtMutS+COI*, as well as that of the proposed extended barcode for octocorals *mtMutS+Igr1+COI* (both in Supp. Fig. 1), resulted in the same topology and quite similar support. All analyses supported a close relationship between the Mediterranean and Atlantic forms as sister groups, together referred to as the Atlanto-Mediterrarean clade as sister group of the Red Sea species *Chironephthya* sp.2 with lower support. The

remaining Red Sea and Indo-West Pacific sequences were placed in different clades.

## Similar species

Despite the difficulty in establishing a priori the taxonomic characteristics in the genus *Chironephthya* useful for species identification, as well as our scarce knowledge of possible intraspecific morphological variability, the molecular approaches carried out for this study strongly support a close relationship between the Mediterranean material described here and the Caribbean species described by Deichmann (1936). The type material of both Caribbean species is诊断ally re-described and illustrated here for comparative purposes. In addition, Table 3 summarizes the main set of morphological characteristics from selected Caribbean and Mediterranean species.

The type colonies of *Chironephthya agassizii* (Deichmann 1936) (MCZ CNID-4069) are branched, tree-like, and erect (Fig. 13a), about 70 mm in maximum length in the examined material. The stalk is attached to hard substratum, not extending beyond the basal stalk dimensions. The main stem and branches are rigid and brittle due to the abundance of large and densely packed sclerites in the colony wall. The branches secondary to distal branches are progressively smaller in size with branching order, but in general have a cylindrical aspect. The distal branches are distinctly spiculose, with large and thick cortical sclerites, observed up to 6.5 mm in length and 0.6 mm in width, as well as many that are smaller and thinner (Fig. 13f). The calyxes are prominent, with two distinct calicular teeth (Figs. 13b–d), observed up to 3 mm along the abaxial side, formed by 2–4 large, thick sclerites per calicular teeth, up to 4 mm in length (4.7 mm in the sclerites in a small flask inside the lot identified as “calycular”), often with other, shorter sclerites, as well as other lateral and inter-teeth sclerites 0.3–0.35 mm in length, difficult to see because of the density and thickness of the large sclerites. In the anthocodiae, the polyps are 1.2 mm in diameter at crown level. The crown has 7–10 rows of curved thorny spindles, up to 0.9 mm in length (Fig. 13e). The points are 1–2 pairs of curved thorny spindles per point, up to 1 mm in length, often one side of a point with two sclerites and the other side with only one, sometimes with accessory smaller spindles among them up to 0.35 mm in length. Secondary points are not clearly visible, sometimes with single sclerites about 0.12 mm in length, but not always present, even in the same polyp. The sclerites of the aboral side of the tentacles are up to 0.32 mm in length. The platelets of pinnules are up to 0.18 mm in length. The thorny rods in the pharynx are up to 0.07 mm in length. The sclerites of the stalk are up to 5.5 mm in length and 0.4 mm in width. The stalk colour is yellowish, the main to terminal branches are light orange, while the anthocodiae are
**Fig. 10** *Chironephthya mediterranea* n. sp. Living colonies photographed in situ. a Colonies from station 11 (*Nemo* dive, cruise INDERAMES VI, Menorca IV); b Colonies from station 53 (*Nemo* dive, cruise INDERAMES VI, Menorca IV); c Dense field of *C. mediterranea* n. sp. and the ellisellid gorgonian *Viminella flagellum* from station 10 (*JAGO* dive, cruise INDERAMES IV, Menorca II). For additional sampling data, see Table 1.

<figure><img src="image_106.png" /></figure>

white, except for the pharynx that is light orange to yellowish. All colours are due to the colour of the sclerites.

The type colony of *Chironephthya caribbaea* (Deichmann 1936) (MCZ CNID-4068) is branched, tree-like, and erect (Fig. 14a). The branches secondary to distal branches are progressively smaller in size with branching order, but in general have a cylindrical aspect. The distal branches are distinctly spiculose, with large and thick cortical sclerites, observed up to 7.5 mm in length and 0.7 mm in width, as well as many that are smaller and thinner (Figs. 14b and c). The calyces are prominent, with two distinct calicular teeth (Figs. 14b, d, and e), observed up to 3.5 mm along the abaxial side in distal parts of branches (but usually smaller, about 2 mm in length, depending upon whether they include some of the larger and thicker sclerites), formed by 2–3 large thick sclerites per calicular teeth, up to 3 mm in length, often with other, shorter sclerites, as well as numerous lateral and inter-teeth sclerites 0.7–1.25 mm long. In the anthocodiae (Fig. 14f), the polyps are 1.5 mm in diameter at crown level. The crown has 11–15 rows of sclerites, up to 1.3 mm in length. The points are 1–2 pairs of curved thorny spindles per point, up to 1.1 mm in length (Fig. 14f), sometimes two sclerites (one of them smaller, up to 0.6 mm in length) on one side of the point, with accessory smaller spindles basally among them, 0.25–0.30 mm in length. Secondary points comprising up to 3–4 shorter sclerites, 0.1–0.15 mm in length,

are present. The sclerites on the aboral side of the tentacles are up to 0.45(0.55) mm in length (Fig. 14g). The sclerites of the pinnules are up to 0.22(0.25) mm in length (Fig. 14g). The sclerites of the pharynx are thorny rods, up to 0.07 mm in length. The sclerites of the stalk are up to 4.5 mm in length and 0.45 mm in width. The colour in the main stalk and terminal branches is whitish, while the anthocodiae are white, except for the pharynx that is light orange to yellowish. All colours are due to the colour of the sclerites.

The colony of *Chironephthya caribbaea* (USNM 1025683) from which DNA sequences could be obtained is branched, tree-like, and erect, about 110 mm in maximum length in the preserved state (Fig. 15a). The branches secondary to distal branches are progressively smaller in size with branching order, but in general have a cylindrical aspect (Fig. 15b). Distal branches are distinctly spiculose, with large and thick cortical sclerites, observed up to 6.3 mm in length and 0.6 mm in width, as well as many that are smaller and thinner (Figs. 15b and c and 16e). The calyces are prominent, with two distinct calicular teeth (Fig. 15d), observed up to 3.5 mm along the abaxial side in distal parts of branches (but usually smaller, about 2 mm long, depending upon whether they include some of the larger and thicker sclerites), formed by 2–3 large thick sclerites per calicular teeth, up to 3 mm in length, often with other shorter sclerites, as well as numerous lateral and inter-teeth sclerites 0.7–1.25 mm long (Fig. 15c–e).
⊕ Bootstrap >55%, Posterior probability >0.55

⊕ Bootstrap >65%, Posterior probability >0.90

○ Bootstrap >75%, Posterior probability >0.90

● Bootstrap >90%, Posterior probability >0.90

mtMutS+COI

<figure><img src="image_107.png" /></figure>

**Fig. 11** Molecular analysis by ML method. Relationship of *Chironephthya-Siphonogorgia* species and a set of selected octocorals sequences, hypothesis based in the mitochondrial markers *mtMutS+COI*, note that *Nidaliidae* sequences are not reunited in this analysis. The tree is drawn to scale, with branch lengths measured in the number of

substitutions per site. All positions containing gaps and missing data were eliminated. Abbreviations: *NIDA* Nidaliidae; *ALCY* Alcyoniidae; *PARALCY* Paralcyoniidae; *NEPH* Nephtheidae; *XEN* Xeniidae; *CORN* Comulariidae

In the anthocodiae, the polyps are 1.2 mm in diameter at crown level (Fig. 15e). The crown has 11–13 rows of sclerites,

up to 1 mm in length. The points are 1 pair of curved thorny spindles per point, up to about 1 mm in length, sometimes
<figure><img src="image_108.png" /><figcaption>**Fig. 12** Molecular analysis by ML method. Relationship of *Chironephthya-Siphonogorgia* species using *Nidaria-Nephthyigorgia-Pieterfaurea* as outgroup; the analysis is based in *mtMutS*. Note the lack of basal resolution in the *Chironephthya-Siphonogorgia* clade (this clade is weakly supported by Bayesian inference [see text]). The tree is drawn to scale, with branch lengths measured in the number of substitutions per site. All positions containing gaps and missing data were eliminated. The geographical information included in this tree for the voucher specimens sequenced (see Table 2) and shown in the *Chironephthya-Siphonogorgia* clade is valid for all trees in this paper. Note that all trees in this clade show the same topology.</figcaption></figure>

with accessory smaller spindles basally among them, 0.25–0.35 mm in length (Fig. 16a). Secondary points comprising 2–4 shorter sclerites, 0.15–0.17 mm in length, are present (Fig. 16b). The sclerites on the aboral side of the tentacles are up to 0.4 mm in length. The sclerites of the pinnules are up to 0.16 mm in length. The sclerites in the pharynx are thorny rods up to 0.07 mm in length (Figs. 16c–d). The sclerites of the stalk are up to 4.5 mm in length and 0.5 mm in width. The main stalk is yellowish in colour, and the main to terminal branches are orange, while the anthocodiae are whiten except for the pharynx that is light orange to yellowish. All colours are due to the colour of the sclerites.

## Discussion

Our current knowledge of the diversity and distribution of *Chironephthya* is still limited. The genus has been known to be restricted to the Indo-West Pacific, with at least nine nominal species (Wright and Studer 1889; Harrison 1908;

Benayahu et al. 2004), and two others from the Caribbean (Deichmann 1936:69–72 as *Neospongodes* Kükenthal 1903; and Bayer 1961:56 as *Siphonogorgia*; Cairns and Bayer 2009) In the near future, a study of the two unidentified Atlantic species (Pérez et al. 2011; Brito and Ocaña 2004) is needed in order to better understand the true distribution and diversity of the genus in the Atlantic Ocean.

## Comparison with related species

Fabricius and Alderslade (2001) described the sclerite diversity in the genus *Chironephthya*, likely largely focused on Indo-Pacific species. In this important comprehensive descriptive work concerning the characteristics of shallow-water Indo-Pacific octocoral genera, the authors described the calyces of *Chironephthya* species formed by numerous long spindles and the presence of numerous small rods (at least up to 0.19 mm in length according to the provided figure) on the polyp body (introvert) below the polyp head (crown, point, and tentacles). In the case of *Chironephthya mediterranea* n. sp., calyces seem to be less densely spiculated. Indeed, there is
<table><caption>**Table 3** Selected characteristics from sclerites distribution, size, and colour from the type specimens of *Chironecthila* agassizii, *C. caribaea*, and *C. mediterranea*, as well as the specimen of *C. caribaea* from which the extraction and amplification of mitochondrial genes was possible for the molecular comparison in this study.</caption><thead><tr><th></th><th>C. agassizii<br>Syntype<br>(MCZ CNID-4069)</th><th>C. caribaea<br>Syntype<br>(MCZ CNID-4068)</th><th>C. caribaea<br>(USNM 1025683)<br>ID: Cairns, 2004</th><th>C. mediterranea n.sp.<br>Holotype<br>(MCNB 2013-0001)</th></tr></thead><tbody><tr><td><strong>Surface coenenchymal-calycular sclerites</strong></td><td></td><td></td><td></td><td></td></tr><tr><td>-Distal branches</td><td>Densely packed, light orange in colour, up to 6.5 mm in length and 0.6 mm in width</td><td>Densely packed, whitish in colour, up to 7.5 mm in length and 0.7 mm in width</td><td>Densely packed, orange in colour, up to 6.3 mm in length and 0.6 mm in width</td><td>Not densely packed, sclerites can be seen individually separated by sclerite-less coenenchymal spaces, colourless, up to 2.8 mm in length and 0.15 mm in width</td></tr><tr><td>-Stalk</td><td>Densely packed, yellowish in colour, up to 5.5 mm in length and 0.4 mm in width</td><td>Densely packed, white in colour, up to 4.5 mm in length and 0.45 mm in width</td><td>Densely packed, yellowish in colour, up to 4.5 mm in length and 0.5 mm in width</td><td>Densely packed, colourless, up to 3.5 mm in length and 0.22 mm in width</td></tr><tr><td>-Calyces</td><td>2-4 large thick sclerites per calycular teeth, lateral and inter-teeth areas with smaller sclerites</td><td>2-3 large thick sclerites per calycular teeth, lateral and inter-teeth areas with smaller sclerites</td><td>2-3 large thick sclerites per calycular teeth, lateral and inter-teeth areas with smaller sclerites</td><td>23) large thin sclerites per calycular teeth, lateral and inter-teeth areas without sclerites</td></tr><tr><td>Crown sclerites</td><td>Colourless, (7)8–10 rows, up to 0.9 mm in length</td><td>Colourless, 11–15 rows, up to 1.3 mm in length</td><td>Colourless, 11–13 rows, up to 1 mm in length</td><td>Colourless, 4–7 rows, up to 1.1 mm in length</td></tr><tr><td>Point sclerites</td><td>Colourless, 1–2 pairs of large sclerites per point, up to 1 mm in length, plus other smaller about 0.35 mm between them</td><td>Colourless, 1–2 pairs of large sclerites per point, up to 1.1 mm in length, sometimes with another smaller up to 0.6 mm, but often 0.25–0.30 mm between them</td><td>Colourless, 1 pair of large sclerites per point, up to 1 mm in length, plus other smaller 0.25–0.35 mm between them</td><td>Colourless, 1–3 pairs per point, up to 1 mm in length, one of them can be distinctly smaller, about 0.25 mm between them</td></tr><tr><td>Secondary points (?), (sclerites on the intertentacular mesenterial insertion area)</td><td>A colourless sclerites can be present, 0.12 mm, but not always; even variable in a poly</td><td>Present, 3–4 colourless sclerites, 0.1–0.15 mm</td><td>Present, 2–4 colourless sclerites, 0.15–0.17 mm</td><td>A single colourless sclerite is occasionally observed in one of these spaces, 0.1 mm, but the usual state is complete absence</td></tr><tr><td>Tentacle sclerites</td><td>Colourless, aboral sclerites up to 0.52 mm, those on the pinnules up to 0.18 mm</td><td>Colourless, aboral sclerites up to 0.45(0.55) mm, those on the pinnules up to 0.22(0.25) mm</td><td>Colourless, aboral sclerites up to 0.4 mm, those on the pinnules up to 0.16 mm</td><td>Colourless, aboral sclerites up to 0.36 mm, those on the pinnules up to 0.21 mm</td></tr><tr><td>Pharynx sclerites</td><td>Light orange to yellowish in colour, up to 0.07 mm</td><td>Light orange to yellowish in colour, up to 0.07 mm</td><td>Light orange to yellowish in colour, up to 0.07 mm</td><td>Light orange in colour, up to 0.05 mm</td></tr></tbody></table>
<figure><img src="image_109.png" /><figcaption>**Fig. 13** *Chironephthya agassizii* (syntypes MCZ CNID-4069). a Two colonies; b–d Details of calyces showing large and thick sclerites forming calycular teeth; e Anthocodia showing sclerite arrangement at crown and points; f Coenenchymal sclerites showing differences in size of the large thorny spindles</figcaption></figure>

a clear sclerite-less triangle between the two distinct points formed by the set of large spindles (Fig. 4b), as well as the lateral sides of the points (Figs. 4a and c). Moreover, sclerites are absent in the introvert. All other skeletal characteristics, such as the presence of minute sclerites on the pharynx, two kinds of sclerites on the tentacles (massive plates on the aboral side, thinner and curved on the pinnules), coenenchymal surface, interior, crown, and points are well-represented in the Mediterranean species.

The two named Atlantic species of *Chironephthya* were initially studied, but not published, by Verrill. Deichmann (1936) respected the specific epithets proposed by the former author in his original typescripts and assigned these species to the nephthyid genus *Neospongodes* (Verrill had the intention to describe a new soft coral genus for these two species).

According to Deichmann’s description (1936:69), *Chironephthya agassizii* has a greyish stem with branches reddish-brown and polyps pale in colour; crown with four to six rows of sclerites, and the points in two (or three) pairs of curved sclerites; the coenenchymal sclerites reaching up to 2 mm in length, those of the crown and points up to 1 mm, and the blunt rods of the tentacles reaching up to 0.3 mm in length. On the other hand, also according to Deichmann (1936), *Chironephthya caribaea* is dull greenish gray in colour (preserved state); branches are about 5 mm in diameter; crown with about six rows of sclerites and points with one (two) pairs of sclerites; coenenchymal sclerites reaching up to 6 mm in length (although a few of them can reach up to 10 mm), those of the crown reaching up to 1.5 mm, those of the points up to 1 mm, and the short “double-cones” in the
**Fig. 14** *Chironephthya caribaea* (syntypes MCZ CNID-4068). *a* Whole fragmented colony (note that some of the fragment probably dried in the past, now having a slightly different appearance); *b–c* Detail of terminal branches; *d–e* Detail of calyces showing large and thick sclerites forming the calycular teeth; *f* Anthocodia showing sclerite arrangement at crown and points; *g* Tentacular sclerites showing basally those of the aboral side of the tentacle and distally those of the pinnules

<figure><img src="image_110.png" /></figure>

tentacles averaging 0.05 mm in length. More than 150 colonies of *C. mediterranea* n. sp. have been observed in the video material examined for an ongoing study of characterization of the benthic community (Grinyó et al. in prep.), and no colour variation has been observed. Living and preserved colonies of the new species are whitish in colour, slightly brownish basally due to the presence of a periderm; the pharynx is distinctly orange due to the colour of the sclerites, and all other sclerites in the colony are colourless.

The revision of the type and additional materials of the Western Atlantic *Chironephthya* species (see Supp. Mat. Appendix 1) revealed a number of discrepancies in the original descriptions provided by Deichmann (1936) (see above for diagnostic re-descriptions and Table 3 for comparison of selected materials).

According to the examined material, both Western Atlantic species bear close resemblance from a morphological point of view, if they are not the same species. Despite the indications from Deichmann, surface coenenchymal sclerites of the type material of *C. agassizii* (MCZ CNID-4068) are distinctly longer than the 2 mm originally reported, reaching 6.5 mm in some sclerites, reducing the differences with *C. caribaea*, type and additional consulted materials. In general, in both Caribbean species, the number of rows of sclerites at the crown is higher than that initially reported, (7) 8–12, including all variation observed in *C. agassizii* specimens, but 10–15 including all variation observed in *C. caribaea* specimens). Additional characteristics such as the number of sclerites present in the calyces or other anthocodial structures (tentacular, pharyngeal, point sclerites) show an overlapping range of
<figure><img src="image_111.png" /><figcaption>**Fig. 15** *a* *Chironephthya caribaea* (USNM 1025683). *a* Whole colony; *b* Details of terminal branches showing the presence of the large and thick sclerites; *c–d* Details of calyces and anthocodiae (note the large sclerites in the calicular teeth and the presence of additional sclerites on the lateral areas of the calyces); *e* Detail of an anthocodiae showing sclerite arrangement at crown and points</figcaption></figure>

variability in the set of examined colonies, making it difficult to draw a clear difference between the sets of materials, even from the type specimens. Colour information from the original descriptions is mainly related to the colour of the preserved tissues; sclerite colour varies in a single colony from whitish (basally) to orange (distally), but other colonies have light orange polyparia (due to the sclerites' colour). Molecular information appears to be a useful tool for distinguishing *Chironephthya* species. Unfortunately, only the more recently preserved specimens of *C. caribaea* (USNM 1025683 [see also Fig. 14 in this paper]) have provided good DNA extraction and mitochondrial genes successfully amplified. In our opinion, both species are likely synonymous. According to current morphological information, it is difficult to distinguish the limits of the two named Caribbean species. If this is correct, the name *C. agassizii* would have precedence because it was cited first in the original text. More DNA sequences from the Western Atlantic *Chironephthya* specimens are needed to elucidate whether, under this apparently overlapping

variability, more than a species is hidden. It remains unclear whether the basal ramification of the colony (producing a short stalk, as in *C. caribaea* type material, and USNM 1025683) or branching distally at the end of a distinct stalk (as in most *C. agassizii* specimens) can be considered specific characteristics or simply varieties.

There are clear morphological differences among the Caribbean species and the new Mediterranean species described here. In the Caribbean specimen examined, surface coenenchymal sclerites can reach up to 7.5 mm in length (maximum values per colony varied from 4.5 to 7.5, usually about 6 mm) and 0.7 mm in width (maximum values per colony varied from 0.3 to 0.7, usually about 0.5 mm), while in the Mediterranean species these sclerites reach up to only 2.8 mm in length and 0.14 mm in maximum width (usually 0.7–0.10 mm). Maximum dimensions of surface coenenchymal sclerites of the stalk show a similar pattern, with Caribbean species ranging from 3.5 to 5.5 mm in length and 0.38–0.55 mm in width, and the Mediterranean species
<figure><img src="image_112.png" /><figcaption>**Fig. 16** *a Chironephthya caribaea* (USNM 1025683). *a* Detail of anthocodial sclerite arrangement, showing crown, points, and some sclerites in the inter-tentacular areas (secondary points ?); *b* Detail of a group</figcaption></figure>

3.5 mm and 0.22 mm, respectively. In general, coenenchymal sclerites in the Caribbean species can reach longer and wider sizes, but not to the detriment of shorter and thinner sclerites. Caribbean species show a wider range of coenenchymal sclerites, consisting of distinctly larger and thicker sclerites, in addition to those sizes in common with the Mediterranean form. The density of coenenchymal sclerites in distal branches is clearly different in Caribbean and Mediterranean species, the former being more densely spiculated.

The number of sclerites conforming each of the two calycular teeth vary from two to five in the observed Caribbean forms: two when sclerites are large and thick, but up to five when sclerites are shorter. When large sclerites are poorly represented in stems or branches, a higher number of shorter sclerites forming the calycular teeth are present. Conversely, when large and thick sclerites are present, they often participate in the formation of the calycular teeth, and a few additional sclerites are added to it. In general, shorter sclerites are found covering lateral areas of the calyces (after the original Verrill's plates compiled by Bayer and Cairns 2004: fig 69, Figs. 8 and 13 in this paper). Although two to three sclerites can be found in the calycular teeth of *Chironephthya mediterranea*, these sclerites are thinner and shorter than 3 mm in length, and lateral and inter-point calycular spaces are devoid of sclerites (see Figs. 3a, b, and c).

Regarding the sclerites present in the polyp, the number of rows of sclerites in the crown of Caribbean and Mediterranean species is different, four to seven (this variability is observed in different polyps of the same colony) in the new species, but

of those sclerites in the inter-tentacular areas between two consecutive points; *c-d* Pharyngeal sclerites; *e* Three of the large and thick coenenchymal sclerites from terminal branches

higher in the Caribbean species (data, commented above). Other features such as tentacular, point, or pharyngeal sclerites show similar dimensions and shapes. The information for *C. caribaea* provided by Deichmann (1936:71) stating that "The short double cones in the tentacles average 0.05 mm in length"surely refers to the pharyngeal rather than tentacular sclerites, which can reach 0.55 mm (axial tentacular sclerites) or 0.25 mm (pinnules), and are not consistent with the "double-cone" shape described.

In the above molecular phylogenetic hypotheses based on *mtMutS* and on the combination of two mitochondrial genes (*mtMutS+COI*), the Mediterranean species is different from the only available Caribbean sequence, its sister group, suggesting a common ancestry between the Atlantic and Mediterranean species.

The comparison based on the extended proposed barcode for octocorals (*mtMutS+IgrI+COI*) appears to show a good resolution for species and genera currently included in Nidaliidae. More sequences and species need to be added to these analyses, especially for the speciose genus *Siphonogorgia* as well as other nidaliid genera for which molecular information is still lacking. This molecular information could be used in combination with the morphological and chromatic descriptions of the different species.

## Biogeographic approach

Previously, species of the genus *Chironephthya* were known from several localities in the Indo-Pacific Ocean, Caribbean
<figure><img src="image_113.png" /><figcaption>**Fig. 17** *Chironephthya* spp. distribution, modified and updated from Pérez et al. (2011), with special emphasis on Atlantic and Mediterranean records and species. * known localities of *Chironephthya mediterranea* n. sp.; *half open-solid circle*, known localities of *Chironephthya agassizii*;</figcaption></figure>

*half solid-open circle*, known localities of *Chironephthya caribaea*; *open circle*, known Atlantic records as *Chironephthya* sp.; *solid circle*, Indo-West Pacific records of *Chironephthya* spp. *Colour in symbols* indicates type localities for the respective species

Sea, and tropical Atlantic off the Brazilian coast (Pérez et al. 2011). This is the first time that a species of this genus has been found and described in the Mediterranean Sea. A similar situation recently occurred with another soft coral genus. The re-description of *Nidalia studeri* represented the first species of this genus described in the Mediterranean. The genus *Nidalia* shows some biogeographical parallels with the genus *Chironephthya*. Most species of *Nidalia* are distributed in the western sector of the Indo-Pacific Ocean and the Caribbean Sea.

This peculiar biogeographic distribution was also found in extensive studies on deep-water medusa fauna in the Mediterranean (Gili et al. 1998, 2000; Bouillon et al. 2000). These authors proposed that the Indo-Pacific and Mediterranean species, in particular, could have diverged from a common ancestor after being isolated in their respective region by geological events in conjunction with the Messinian salinity crisis (Hsü 1973).

The current distribution of the genus *Chironephthya* (Fig. 17) suggests that in the past, species of this genus were distributed continuously across the Tethys Sea, and went through allopatric speciation as tropical provincialism gradually consolidated. It is plausible that the Mediterranean and Atlantic *Chironephthya* species is of a Tethyan lineage that was subsequently isolated during the Early Miocene with the closure of the eastern channels and diversified in the Atlantic and Mediterranean Sea. Although it has been postulated that the Tethyan marine fauna disappeared completely during the Messinian salinity crisis (Hsü 1973), several faunistic studies from recent decades have suggested that the Tethyan fauna could have survived in scattered refuges throughout the

Mediterranean basin (Boury-Esnault et al. 1992; Boero and Bouillon 1993; Gili et al. 1998, 2000; Jaume and Boxshall 1996). In fact, recent studies related to deep North Atlantic medusa fauna (Hosia and Pagès 2007) have marked the discovery of a set of new species belonging to a genus initially described as endemic to the Mediterranean Sea. This may suggest that the Tethys Sea fauna colonized the Atlantic Ocean after the Early Pliocene (van der Spoel 1996), and when the Strait of Gibraltar reopened, the Mediterranean Sea level was reestablished and the Mediterranean outflow water (MOW) circulation pattern began (Maldonado and Nelson 1999). Today, the MOW flow moves to the north along the Iberian slope and to the southwest as far as the Canary Islands, and then westward (Iorga and Lozier 1999). The MOW may have allowed Mediterranean species to repopulate North Atlantic waters. Although most colonization events have occurred from the Atlantic to the Mediterranean (Bianchi and Morri 2000), there are few cases in which the opposite may have occurred (Henry and Roberts 2006).

In the event that the Messinian salinity crisis resulted in the disappearance of all Tethyan marine fauna, the presence today of *Chironephthya* (and likely *Nidalia*) species in the western Mediterranean should be seen as a result of further immigration from remaining Atlantic lineages. In any case, whether Mediterranean emigrants colonizing the Atlantic or Atlantic emigrants re-colonizing the Mediterranean areas (both hypotheses according to the current available molecular information), Atlantic and Mediterranean species of *Chironephthya* are closer to each other than to any Red Sea or Indo-Pacific species. Indeed, it is noteworthy that in the different relationships of the three available sequences from
the Red Sea (only identified at generic level, as sp.1 to sp.3 in McFadden et al. 2011), two of them (sp.1 and sp.3) fall into a clade with Indo-Pacific species, whereas *Chironephthya* sp.2 is, in this study, the sister group of the Atlantic-Mediterranean forms.

The discovery of species such as *Chironephthya mediterranea* n. sp. in the Mediterranean reinforces the hypothesis that the Tethyan component of deep Mediterranean fauna is more important than previously thought (Pérès 1985). Further studies on deep areas may confirm this hypothesis and perhaps lead to a confirmation that during the Messinian salinity crisis, the Mediterranean was probably not completely dry, and some areas remained flooded, preserving marine conditions. Under that hypothesis, therefore, the current distribution of some sessile organisms in the Mediterranean probably represents relic populations of lineages that have a wider distribution in the Mesozoic Tethys (Logan et al. 2004). *Chironephthya mediterranea* n. sp. has been found in one of the least explored marine habitats in the Mediterranean Sea, the shelf break and upper continental slope communities (Pérès 1985). We believe it is highly unlikely that this species is an invasive one, since most invasive species in the Mediterranean have been associated with surface warm water (Laubier et al. 2003; Por 2009; Raitsos et al. 2010). *Chironephthya mediterranea* n. sp. is found in stable cold water environments (13 °C), far from harbours, aquaculture plants, or warm shallow waters, where invasive species are commonly first detected (Zibrowius and Ramos 1983; Galil 2000; Fine et al. 2005; Hoeksema and Ocaña 2014).

Our molecular data indicates a close relationship between Atlantic and Mediterranean forms, with this clade related to Indo-Pacific sequences. Moreover, *Chironephthya* species are suspected to be present in the Atlantic Ocean (Brito and Ocaña 2004; Pérez et al. 2011). Morphological and molecular data should shed some light on the biogeography of the genus *Chironephthya* as well as provide additional information about the origin of *Chironephthya mediterranea* n. sp.

**Acknowledgments** The authors thank the scientists and crew on board the *BIO García del Cid* during the “INDEMARES-CSIC IV-V-VI, Menorca II-III-IV” cruises, and Stefano Ambroso, Carlos Dominguez-Carrió, Alejandro Olariaga, Ariadna Purroy, and Susana Requena. Our sincere thanks to Gavin Newman (Nemo ROV) for his proven expertise collecting the specimens of the new species described here, sometimes working in difficult weather conditions, as well as the JAGO team (J. Schauer and K. Hissmann) from IFM-GEOMAR (Kiel, Germany). Our thanks are also addressed to Gonzalo Giribet and Adam J. Baldinger (Museum of Comparative Zoology, Harvard) for the loan of the Deichmann type materials of the two Western Atlantic *Chironephthya* species, and to Stephen D. Cairns (Smithsonian Institution, National Museum of Natural History) for the loan of additional *Chironephthya* colonies for comparison and molecular research. The study was financed within the framework of the LIFE+INDEMARES project: “Inventario y designación de la Red Natura 2000 en áreas marinas del Estado español” (LIFE07 NAT/E/000732). Thanks to Pedro Martínez-Arbizu and Bert W.

Hoeksema, Editors of MB, and thee anonymous referees, whose suggestions and criticisms have improved the first version of this manuscript. Thanks also to Dr. Estefania Rdírguez (American Museum of Natural History, New York) for the manuscript revision and comments. We thank Mr. Tony Krupa for reviewing the English version.

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# Acknowledgements

GRINYÓ J (2016) PhD Thesis pp 199-201

Aquesta tesis doctoral és el fruit de les idees, consells i l'esforç conjunt de molta gent, però en especial dels meus mentors i amics en JSEP-Maria Gili, l'Andrea Gori i l'Enrique Isla. Els voldria agrair la disponibilitat que sempre han tingut per escoltar i debatre qualsevol idea o dubte; així com la dedicació continua que sempre m'han demostrat, fins i tot des de la distància. Però, sobre tot, els vull agrair l'amistat que hem establert durant aquests anys. L'optimisme i la positivitat que m'han transmès inclús davant les situacions més adverses i han fet que aquesta tesi doctoral hagi resultat una experiència molt agradable i enriquidora.

Tambien querría agradecer a Pablo López-González, de la Universidad de Sevilla su participación en esta tesis. Gracias Pablo, por haber sacado tiempo de tus noches y fines de semana para completar los trabajos. También agradecerte que me acogieras en Sevilla durante aquella semana de Agosto de 2012 (42°C a la sombra). Fue una experiencia fantástica poder aprender de tí y que me hicieras de anfitrion en tu bonita ciudad.

A en Michael Greenacre, de la Universitat Pompeu Fabra, també li voldria agrair la seva

implicació en aquesta tesis. Ha estat un veritable plaer poder treballar amb una persona tant vital com tu. Tant de bo els nostres camins es tornin a creuar.

També voldria agrair a en David Díaz, l'Annabel Muñoz, la Sandra Mallol i els pescadors de la barca artesanal GOGA, de Cala Ratjada, per tota la feina feta durant els mostrejos pel cicle reproductiu de *Paramuricea macrospina*. A en David i l'Anabel els estic especialment agraït. Sense les immersions que van realitzar a més de 70 metres, tot i els riscos que comportava, mai s'hauria pogut completar aquesta tesi. Treballar amb vosaltres dins del marc del projecte ECOSAFIMED ha estat una experiència molt divertida i agradable. Espero que en un futur no molt llunyà puguem tornar a treballar junts.

A voria ringraziare Stefano Ambroso, un amico da fogo, parchè anca quando l'è tardi assè o quando ghe dalle difficoltà, l'è sempre gentile e pronto a aiutare un butel. Me ricordarò par sempre delle tradizioni de Verona che te m'è insegnà, dei caffè de matina, dei discorsi sul Mar de Tethys, dele bire che ghemo beù nell'uficcio del capo in orari assurdi. Spero davvero che la nostra amicizia duri per sempre!
A l'Ariadna Purroy que malgrat la distància, com sol passar amb els bons amics, cada cop que ens retrobem tinc la sensació que només han passat un parell de dies. A la Núria Viladrich, gràcies per ensenyar-me a veure la bellesa que s'amaga en la senzillesa i l'increïble enginyositat dels anàlisis bioquímics. Has estat una gran mestra i una molt bona amiga, sempre amb un somriure. A la Martina Coppari, he gaudit moltíssim de totes les hores passades dins del laboratori de vídeo, i sempre recordaré el munt d'atacs de riure que hem viscut junts. Presidenta Coppari l'esperem!!!! A la Rebeca Zapata, gràcies per els teus consells i el teu pragmatisme. A en Claudio Lo Iacono, gracias por tu amistad y por llevarme a lugares que no creia que pudieran existir en el Mediterráneo. A la Martina Pierdomenico, siempre recordaré el viaje trepidante Malaga - Barcelona que hícimos para assegurar que los corales de la campaña SHAKE llegasen vivos. A l'Antonio Canepa i a en Guillem Salazar que m'heu ajudat en infinitat d'ocasions a resoldre dubtes d'R. A l'Alejandro Olariaga, gracias por los buenos momentos tomando mate en el taller y las campañas. A en Juancho Mobilla, gracias por tu amistad, consejos y por tu ayuda en la ZAE. A la nova fornada de becaris bentònics del Suspension Feeders Team: a l'Andreu, a la Maria, a la Patricia, a la Janire, en Guillem, la Núria i l'Ariadna, gràcies per estar disposats a donar un cop de mà sempre que se us ha neccessitat amb la millor de les actituds. Al company que va començar la tesis amb mi, gràcies Carlos pels moments a Cadaqués i el desenvolupament de la macro ha fet que la nostra feina resulti molt més senzilla. A la María Pascual, la Míriam Gentile, la Melissa Acevedo, l'Agnés Escurriola, en Raül González, a la Laura López, a en Gastón Iriarte, Macarena Marambio, Verónica Fuentes i l'Elena Guerrero gràcies per la vostra amistat i el bon rotllo i positivisme que transmeteu.

A en Xavi Leal, en Miquel Dalmau, en Lluís Pedret, moltes gràcies per ajudar-nos a vigilar

els experiments dels aquaris de la ZAE tot i que no fos la vostra feina.

Voldria agrair a la Susana Requena i la Covadonga Orejas tota la feina feta, sense elles el projecte LIFE+ INDEMARES - CANAL DE MENORCA no hauria estat possible. També voldria agrair a la tripulació del buc oceanogràfic Garcia del Cid tota l'ajuda i suport que ens van donar durant les campanyes LIFE+ INDEMARES. Bei Karen Hissman und Juergen Schauer möchte ich mich herzlichst bedanken, dass Sie mir die Möglichkeit gegeben haben bei den Tauchgängen mit dem JAGO in Cap de Creus und im Canal de Menorca dabei sein zu dürfen. Ich fühle mich als absoluter Glückspilz, dass ich diese Möglichkeit hatte mit dem JAGO diese besonderen Orte besuchen zu dürfen, die niemand vorher so gesehen hat. I would like to thank Gavin Newman for working long hours during the LIFE+ INDEMARES cruises and the incredible footage that he has proportioned.

Als doctors Sergi Rossi i Lorenzo Bramanti els voldria agrair la seva tasca com a correctors externs d'aquesta tesi. També voldria agrair a la secretaria del programa de doctorat de Ciències del Mar de la Universitat Politècnica de Catalunya, la Genoveva Comas, tota l'ajuda que m'ha donat i la paciència que ha tingut amb mi i els meus companys del doctorat.

Voldria fer una mencio especial del professor Josep Marlés que em va desvetllar la passió per la biologia marina durant la meva adolescència.

Voldria agrair especialment als meus pares, germans i la Mireia el suport constant i l'optimisme que m'han transmès al llarg de tota la meva vida. Gràcies per recolzar la meva passió per la biologia des de la meva infantesa i potenciar la meva curiositat i inquietuds.

També voldria agrair a la Lídia i en Jesus l'acollida que sempre m'han donat a casa seva,
en especial durant aquests dos últims mesos,
on he pogut acabar la tesi.

Voldria agrair especialment a l'Elena Crespo
que m'hagi acompanyat durant el transcurs
d'aquest viatge. Gràcies per transmetre'm pau
i serenor i ajudar-me a apaivagar les meves
angoixes en els moments més difícils. Gràcies
per ensenyar-me a mirar més enllà de la feina.
Sens dubte m'has ensenyat a gaudir més de
la vida. Gràcies Elena per la teva presència de
cada dia i el teu recolzament incondicional.
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 environmental 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 ∂13C and ∂15N 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 oligotrophic 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 downward particle fluxes quantified during two consecutive years in the Menorca Canyon were comparatively 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 primary 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 assemblages.