Diversity of Porifera in the Mediterranean coralligenous accretions, with description of a new species

Abstract Temperate reefs, built by multilayers of encrusting algae accumulated during hundreds to thousands of years, represent one of the most important habitats of the Mediterranean Sea. These bioconstructions are known as “coralligenous” and their spatial complexity allows the formation of heterogeneous microhabitats offering opportunities for a large number of small cryptic species hardly ever considered. Although sponges are the dominant animal taxon in the coralligenous rims with both insinuating and perforating species, this group is until now poorly known. Aim of this work is to develop a reference baseline about the taxonomic knowledge of sponges and, considering their high level of phenotypic plasticity, evaluate the importance of coralligenous accretions as a pocket for biodiversity conservation. Collecting samples in four sites along the coast of the Ligurian Sea, we recorded 133 sponge taxa (115 of them identified at species level and 18 at genus level). One species, Eurypon gracilis is new for science; three species, Paratimea oxeata, Clathria (Microciona) haplotoxa and Eurypon denisae are new records for the Italian sponge fauna, eleven species are new findings for the Ligurian Sea. Moreover, seventeen species have not been recorded before from the coralligenous community. The obtained data, together with an extensive review of the existing literature, increase to 273 the number of sponge species associated with the coralligenous concretions and confirm that this habitat is an extraordinary reservoir of biodiversity still largely unexplored, not only taxonomically, but also as to peculiar adaptations and life histories.

the Italian sponge fauna, eleven species are new findings for the Ligurian Sea. Moreover, seventeen species have not been recorded before from the coralligenous community. The obtained data, together with an extensive review of the existing literature, increase to 273 the number of sponge species associated with the coralligenous concretions and confirm that this habitat is an extraordinary reservoir of biodiversity still largely unexplored, not only taxonomically, but also as to peculiar adaptations and life histories.

Keywords
Porifera, cryptic species, bioconstructions, Ligurian Sea introduction The term "coralligenous" refers to a secondary hard substrate, formed by the concretion of algal thalli and, to a lesser extent, by animal skeletons. Two main types of coralligenous concretions can be distinguished: banks, which are built over more or less horizontal substrata, and rims, which develop in the outer parts of marine caves and on vertical cliffs (Ballesteros 2006). Coralligenous communities represent the temperate reefs of the Mediterranean Sea and along with the meadows of Posidonia oceanica (Boudouresque, 2004) are biodiversity hot spots in the basin. The holes and crevices of the coralligenous build-ups support a complex community dominated by suspension feeders (sponges, hydrozoans, serpulid polychates, molluscs, bryozoans and tunicates). Laubier (1966) first emphasized the high biodiversity of the coralligenous and listed 544 invertebrate species from this assemblage in Banyuls. Later, Hong (1980), in an exhaustive survey of the coralligenous of Marseille, listed a total of 682 species, whilst other authors (Ros et al. 1984) reported 497 species of invertebrates from the algal concretions of the Medes Islands. Recently, Romdhane (2003) reported 35 algal species and 93 animal species from a coralligenous formation along a vertical cliff in the gulf of Tunis. However, the number of species living in the coralligenous assemblages is still undefined, because of the richness of the fauna (Laubier 1966), the habitat complexity Picard 1964, Ros et al. 1985), the wide depth range of the conglomerates (Ballesteros 2006), the sporadic presence of cryptic species and the scarcity of reference studies. A rapid, non-destructive protocol for biodiversity assessment and monitoring of coralligenous, based on photographic sampling, was recently proposed by Kipson et al. (2011).
Sponges, with 142 recorded species, are one of the most diverse group of sessile animals of the coralligenous assemblage (Ballesteros 2006). Some species, mainly belonging to the family Clionaidae, are active bioeroders representing the principal driving force in the turn-over of bioconstructions, both in temperate and tropical areas (Cerrano et al. 2001, Calcinai et al. 2000, 2005, 2007c In the present paper, the species diversity of the coralligenous sponge fauna was studied in four sites of the Ligurian Sea, focusing on the relatively poorly known cryptic species boring or insinuating into the calcareous concretions. A new species for science and ten poorly known species, rarely recorded in the Mediterranean Sea, are treated exhaustively.

Materials and methods
Samples were collected between 30 and 40 m depth by SCUBA diving from 6 stations along the Ligurian coast where coralligenous is more developed (Fig. 1). Stations (from West to East) are: Santo Stefano Shoals, station 1; Gallinara Island, station 2 (Falconara) and station 3 (Sciusciaù); Portofino Promontory, Punta del Faro, station 4 and 5 (northern and southern side of the point); Punta Manara, station 6. Four blocks of coralligenous concretion, with an average volume of 20 l, were collected from each station.
All the sponge species settled on the surface of these blocks were sampled and identified.
Two of the four blocks from each station were cut into slices about 2 cm thick and observed by a stereomicroscope to detect the cryptic, generally small, endolithic sponges.
The spicule complement of each sponge specimen was analysed according to Rützler (1978). From 30 measurements for each spicule type, size range, mean and standard deviation (in brackets) were calculated. Dissociated spicules were transferred onto stubs and sputtered with gold for SEM analyses and observed with a Philips XL 20 scanning electron microscope. Whenever possible, skeletal architecture was examined in light and scanning electron microscope (SEM) on hand-cut sections of the ectosome and choanosome. Unfortunately, due to small size and cavity dwelling habit, for most specimens it was impossible to study the skeleton.
We followed the classification given by Hooper and van Soest (2002) and the updated nomenclature reported in the World Porifera Database (van Soest et al. 2013). The geographic distribution of sponges in the Mediterranean Sea was compared with that reported by Longo (2003, 2008), considering nine biogeographic areas for the Italian seas.

Results
During this survey we have recorded 133 sponge taxa (115 of them identified at species level and 18 at genus level). One species is new for science, 17 are new findings for the coralligenous conglomerate, 11 of which for the Ligurian Sea and 3 for the Italian sponge fauna (Table 1). In the following taxonomic part we provide the description of the new species and of ten poorly known ones.
On the surfaces of the blocks 103 massive or encrusting species were recorded; inside the crevices of the conglomerate 63 species were observed and 33 shared both positions. Thirty species are exclusively endolithic demonstrating the abundance of cryptic sponges thriving inside the porous matrix of the coralligenous substrate (Table 1) (Fig. 2).  Among the 63 species recorded inside the conglomerate, 53 were insinuating and 10 boring (Table 1). From the first group six species: Geodia cydonium (Jameson, 1811) (  Distribution and discussion. This is a Mediterranean endemic species (Pansini and Longo 2008) originally described from Corsica (Strait of Bonifacio), where it is known to bore into calcareous rocks and mollusc shells ). This is a new record for the Ligurian Sea (Gallinara Island) and the coralligenous assemblage and the first finding after the original description.  Description. Very small (0.5 cm 2 ) insinuating sponge (Fig. 5 A) detected inside a cavity of a slice of a coralligenous block. Grey coloured in dry state.
Skeleton. Not observed. Spicules. Macroscleres: oxeas in two size categories: I) large oxeas curved, bent or flexuous, with hastate tips (Fig. 5 B), 810 (961.25) 1200 × 15 (18) 25 μm; II) small oxeas curved or flexuous (Fig. 5 C), 300 (546.6) 700 × 2.5 (4.75) 5 μm. Microscleres: oxyasters with more or less marked centrum with 9-12 conical rays, 25 (41.5) 60 μm in diameter. In some cases the number of rays is reduced (Fig. 5 D). Distribution and discussion. The species was described from Naples  where it occurred on rocky bottoms at 60-100 meter depth. This is a new record for the coralligenous assemblage and for the Ligurian Sea and it is probably endemic for the Mediterranean Sea (Pansini and Longo 2008). This is its first finding after the original description. Description. Thickly encrusting sponge (3-5 mm thick) covering a surface of 1.5 cm 2 on a coralligenous block (Fig. 6 A). Surface irregular, smooth. Consistency soft. The red-orange colour of the living specimen slightly fades when alcohol preserved.
Distribution and discussion. Described from Porto Santo Bay (Madeira) the species extends south to the Sahelian Upwelling (Lévi 1956  Skeleton. Ectosomal skeleton absent. Choanosomal skeleton consisting of basal acanthostyles with heads embedded in a spongin layer and bundles of very long tylostyles protruding through the sponge surface which appears hispid. Spicules. Long tylostyles, slightly curved or straight, with rather irregular heads, 1066 (1774) 2236 × 5 (8.5) 12.5 μm (Fig. 8 A); anisoxeas straight or faintly curved,
Distribution and discussion. The species was originally described by Vacelet (1969) from a coral bottom in the bathyal zone (300-350 m depth) of the Gulf of Lions. This second finding is a new record for the Italian seas and the coralligenous community. Description. All the specimens were encrusting on the surface of coralligenous blocks, covering surfaces up to 2 cm². The sponge surface is corrugated, hispid. The colour in life is brick red (Fig. 9 A).

Eurypon gracilis
Skeleton. The skeleton consists of a basal layer of spongin in which the spicules are vertically positioned, perpendicular to the substrate. Both the categories of acanthostyles are close one another (Fig. 9 C) with the heads embedded in the basal spongin layer. Styles and oxeas-with the same vertical arrangement-are grouped in bundles which are faintly echinated, in their lower part, by the smaller acanthostyles (Fig. 9 B). Oxeas are positioned in the basal part of the bundles. The styles protrude trough the sponge surface making it hispid.
Etymology. The species is named after the slenderness of all the spicule types. Distribution. So far known only from the Ligurian Sea.

Ecology. It lives at 30-40 m depth on coralligenous concretion, characterized by the presence of a Paramuricea clavata facies.
Discussion. This species, characterized by a microcionid skeleton with a basal layer of spongin, extra-axial spicules and echinating achantostyles embedded in spongin fibres, clearly belongs to the genus Eurypon.
Only five, out of the numerous species of the genus Eurypon found in the temperate Western Atlantic have oxeas or tornotes as structural megascleres together with styles or tylostyles. All of them (E. cinctum , E. denisae , E. obtusum Vacelet, 1969, E. major  and E. lacazei (Topsent, 1891) occur in the Mediterranean Sea. E. cinctum showing a lilac colour, achantostyles with discrete heads and different size in the other megascleres is not close to the new species. E. denisae is also different according to the description given above. E. obtusum is grey in colour and has smaller oxeas and acanthostyles than those of the present species, but the maximum length of its tylostyles is unknown. E. lacazei remarkably differs from the present species for the green colour and spicule shape and size. The closest species to the new one is E. major but its tylostyles are longer and stouter (1445-2210 × 10-17 μm) and differ in the shape of the heads, while the acanthostyles, in a single size category, have well formed heads. Only two other species from the temperate Atlantic: E. lictor  and E. (Acantheurypon) mucronale (Topsent, 1928) present oxeas. However, they are both deep species (recorded deeper than 1500 m from the Azores) and they differ also in the spicule characters from E. gracilis sp. n. There are two other species of Eurypon with oxeas reported in the literature: E. calypsoi Lévi, 1958 from the Red Sea which is blue in colour and E. fulvum Lévi, 1969 from South Africa which is yellow. Both have a single size category of acanthostyles and differ in the spicule morphology. E. gracilis therefore has to be considered as new for science.  Description. Thin, small encrusting sponges (up to 0.5 cm 2 ) on the surface of coralligenous blocks. Colour in life yellow-orange.

Suborder Myxillina Family Coelosphaeridae Genus
Skeleton. Basal acanthostyles erect on the substrate in a hymedesmioid arrangement. Other spicule types not detectable from the skeleton.
Distribution and discussion.  describes three species of Leptolabis from the Azores: L. forcipula var. brunnea, L. arcuata and L. assimilis. The same author in 1928 states that the former three species actually belong to a single species: Leptolabis brunnea which shows a high variability in the large forceps shape.
L. brunnea was afterwards recorded from the Far-Oer Islands, the Azores , Spain (NW coast, Strait of Gibaltar, Castellón, Girona), France (Marseille, Monaco), Italy (Gulf of Naples), between 4 and 1360 m depth. It lives in caves, detritic bottoms, coralligenous concretions and epibiotic on other organisms , 1928, Pouliquen 1972, Carballo 1994, Cristobo 1996. This is the second finding for the Italian seas and a new finding for the Ligurian Sea.  Description. Small (0.5 cm 2 ), slimy, coriaceous encrusting sponge, grey in colour after alcohol preservation, recorded both on the surface and inside the coralligenous blocks.
Distribution and discussion. This species, originally described from a superficial cave of the Gulf of Naples (Sarà 1958) was recorded at 60-70 m depth in the same area (Sarà and Siribelli 1962) and in caves close to Marseille (Pouliquen 1972). It is a Mediterranean endemic species (Pansini and Longo 2008) and a new finding for the Ligurian Sea and the coralligenous community. Description. Small (1-1.5 cm 2 ) encrusting and insinuating sponge, beige or brown, detected on the surface and inside a coralligenous block. Surface smooth, consistency soft (Fig. 14 A).
Skeleton. The choanosome consists of multispicular primary lines connected by unispicular secondary tracts, creating a confused reticulation.
Distribution and discussion. Pulitzer-Finali (1978) described the species from a specimen epibiothic on Hyrtios collectrix (Schulze, 1880) found on dead, sanded Posidonia beds, at 50 m depth in the Bay of Naples. The same author considered conspecific with G. marismedi the specimen from Banyuls-sur-Mer (rocky walls in shaded areas at 2-17 m depth and horizontal substrates at 20-40 m depth) attributed to Gelliodes luridus (Lundbeck, 1902) by Boury-Esnault (1971). This is a new finding for the Ligurian Sea and the coralligenous community and the third record after the original description.

Discussion
According to the latest available revision of coralligenous biodiversity (Ballesteros 2006), 142 species of sponges have been recorded associated with this community. Adding to this list the species recorded on the coralligenous of Apulia (Sarà 1968 Calcinai et al. 2007b) and the data of the present study, the total number of sponge species hitherto associated to the coralligenous community increases to 273 ( Table 2).
This increasing is related to the difficulty of studying the organisms inhabiting the coralligenous concretions due to the complexity of the habitat, the high diversity, and the depth where these structures are located (Kipson et al. 2011). Our study, based on the collection of blocks and their sectioning into slices, allowed the identification of species that would have been otherwise completely disregarded.
Among the insinuating species observed in the coralligenous crevices we have found several species previously recorded with a massive habitus in deeper waters. Pachastrella monilifera Schmidt, 1868 and Poecillastra compressa (Bowerbank, 1866) were the species with the highest phenotypic plasticity, since they usually appear with large, fun shaped specimens, in deep habitats (Bo et al. 2012), while in the coralligenous community they live in crevices and fissures of the concretion. Our results support the idea that environments rich in microhabitats may act as shelters essential for the dispersal of many deep water species, enlarging their distribution range (Bo et al. 2011). Therefore we can emphasize the importance of the coralligenous concretion, not only as reservoir of biodiversity, but also as an important "stepping-stone" able to facilitate the dispersal of species along vertical gradients.