Rare sponges from marine caves : discovery of Neophrissospongia nana nov . sp . ( Demospongiae , Corallistidae ) from Sardinia with an annotated checklist of Mediterranean lithistids

A new record of lithistid demosponges is reported from a western Sardinian karstic cave. Th e new specimen matches the trait of the genus Neophrissospongia (Corallistidae) for an ectosomal skeleton of radial dichotriaenes, a choanosomal skeleton as a network of dicranoclone desmas, and streptaster/amphiaster microscleres with short spiny rays bearing blunt tips. Th e cave-dwelling N. nana nov. sp. diverges from the other species of the genus in diagnostic characters such as the large irregular plate-like growth form, the topographic distribution of inhalant and exhalant apertures, and a smaller size of all spicular types. Moreover it displays an additional rare second type of dichotriaenes with smooth cladomes, shared with other genera of Corallistidae but never reported before for the genus Neophrissospongia. In addition N. nana nov. sp. bears stylelike sub-ectosomal spicules shared with N. microstylifer from deep water of New Caledonia. As for the latter trait, a present in-depth analysis of N. nolitangere from the Atlantic Ocean contrasts with previous historical records reporting monaxial spicules as oxeas/anisoxeas. Th e diagnosis of the genus Neophrissospongia is therefore emended for the growth form and for the micro-traits of dichotriaenes and monaxial sub-ectosomal spicules. Morphological data indicate that the new species is allied to N. nolitangere and N. microstylifer from Eastern Atlantic and New Caledonian deep water, respectively, and its record confi rms the highly disjunct geographic range of the genus Neophrissospongia in the Lusitanian-Macaronesian-Mediterranean area and the western Pacifi c Ocean supporting the relic condition of the genus in the Mediterranean Sea. Th is discovery stresses the key status of Mediterranean palaeoendemics as possible remnants of an ancient Tethyan fauna and focuses the need to plan conservation measures for these rare cave-dwelling sponges. ZooKeys 4: 71-87 (2008) doi: 10.3897/zookeys.4.39 www.pensoftonline.net/zookeys Copyright Renata Manconi, Annalisa Serusi. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Launched to accelerate biodiversity research A peer-reviewed open-access journal


Introduction
Among Demospongiae the archaic polyphyletic group of Lithistid (sensu Pisera and Lévi 2002) is present in Europe and North Africa with a highly diversifi ed Jurassic and Cretaceous fossil palaeofauna but apparently very few living representatives have been recorded in the Mediterranean Sea and this recent fauna is poorly-known.Nine species belonging to 9 genera of 6 families have been until now recorded in the Mediterranean Sea (Appendix 1) (Topsent 1892a(Topsent , 1893(Topsent , 1925;;Vacelet 1960Vacelet , 1969;;Pouliquen 1969Pouliquen , 1972;;Pulitzer-Finali 1970, 19721983;Pansini 1992Pansini , 1995;;Boury-Esnault et al. 1994;Magnino et al. 1999;Pansini and Longo, 2003;Perez et al. 2004;Longo et al. 2005;Manconi et al. 2006;Zibrowius and Taviani 2005).Th e taxonomic richness appears to be notably higher in the tropics (Lévi 1991;Pisera and Lévi 2002;Schlacher-Hoenlinger et al. 2005) whereas the low values in the Mediterranean may be explained by the occurrence of extinction phenomena due to harsh environmental/climatic changes during the history of this basin (e.g.cooling phases in Plio-Pleistocene time, see Wiedenmayr 1994:107).All Mediterranean records refer to genera characterised by a highly disjunct distribution with a spot-like pattern in tropical/subtropical latitudinal ranges of the Atlantic, Indian and western Pacifi c oceans.Th ese taxonomic and biogeographic patterns suggest the condition of lithistids as remnants of an ancient Tethyan fauna (Reid 1967;Perez et al. 2004;Manconi et al. 2006;Pisera and Vacelet 2006).
We report here the discovery of the genus Neophrissospongia Pisera & Lévi, 2002 from a shallow water cave in the western Mediterranean with description of a new species based on a comparative analysis of morphological diagnostic traits versus the other species of the genus.

Materials and methods
A large specimen was discovered, photographed in situ and collected in part from a shallow water cave of NW-Sardinia by SCUBA diving.Th e sponge was dissected under a stereomicroscope to observe macro-traits and to obtain representative fragments of the skeleton to be processed by boiling in nitric acid to prepare slides for light microscopy (LM) and stubs for scanning electron microscopy (SEM, ZEISS DSM 962) following standard methods.Growth form, architecture of the ectosomal and choanosomal skeleton, spicular morphology and micrometries, and the topographic localization of spicular types have been considered as diagnostic characters.Th e number of measured spicules (n) is reported for each spicular type.As for dichotriaenes measurements of the cladome refer to the total diameter of a virtual circle made by the end points of the clades.Measurements of protoclades and deuteroclades refer to the length of the cladome branches from origin on the shaft to the fi rst dichotomy (protoclade) and from that point to the end of the branch (deuteroclade Defi nition (emended from Pisera and Lévi, 2002) Cup-shaped, ear-shaped, plate-shaped growth form.Ectosomal skeleton with radially arranged dichotriaenes with tubercled to less frequent smooth cladomes and from smooth to tubercled/spiny rhabdomes.Sub-ectosomal spicules as asterose microscleres (streptaster/amphiaster) and relatively large style-like monaxons.Choanosomal skeleton as a relatively loose network of dicranoclone desmas.Description.Known from a single specimen.Growth form as a large thick encrusting plate (10-12 x 5-7 cm) with a wide base adhering to the substrate and rounded margins.Th e sampled portion (holotype, 5 x 3.5 cm in width, ca.1.2 cm in thickness) is one third of the entire living specimen.Colour white ice both in vivo    Habitat.Shallow water (6 m) on the rocky wall in the innermost part of a dark horizontal tunnel (80 m in length) of a submerged cave (160 m along an N-S axis, main entrance at 17 m of depth, 40 m in length, 7 m in width).Another tunnel at the left of the entrance harbours a small population of Petrobiona massiliana Vacelet & Lévi (Manconi et al. in press).Th e absence of sand deposits and the presence of abundant large boulders suggest the cave is subjected to a notably high water movement (southwestern winds).In April water temperature was 14 ºC.Few serpulids and foraminiferans were found associated with the sponge on an almost bared rocky surface.

Discussion
Th e genus Neophrissospongia was recently erected with Neophrissospongia nolitangere (Schmidt, 1870) as type species by Pisera and Lévi (2002) for the species of Corallistidae, characterised by tubercled dichotriaenes, previously ascribed to the genus Corallistes Schmidt, 1870.Only two species, N. nolitangere (off Portugal, Tab. 1. Distribution, bathymetric range and spicule measurements (length x width) of the species belonging to the genus Neophrissospongia.ented (Wiedenmayr 1994: 91).N. nana nov.sp.matches the diagnostic traits reported for the genus Neophrissospongia sharing an ectosomal skeleton of radial dichotriaenes and a choanosomal skeleton as a network of slender dicranoclone desmas.Th is new species diverges from the other species belonging to the genus in the growth form, smaller spicular size, ornamentations of dichotriaenes (i.e.tubercled cladomes with rarely spined rhabdomes vs. entirely smooth) and two types of sub-ectosomal spicules, namely asterose and monaxial.In addition desmas of N. nana nov.sp.bear scattered small tubercles with smooth apices as in N. microstylifer whereas their surface is microtubercled in N. nolitangere (Schmidt 1870;Topsent 1904;Pisera and Lévi 2002).

Species
Th e ectosomal dichotriaenes of N. nana nov.sp.belong to two types, namely with tubercled cladomes to entirely smooth cladomes diff ering from the typical ones of N. nolitangere reported exclusively as tubercled (Schmidt 1870;Topsent 1904;Pisera and Lévi 2002).It may be however that smooth dichotriaenes (Table 1; Figs 2, 3) notably smaller when compared to the tubercled ones, we found also in the Topsent material of N. nolitangere, represent young forms.
At the same time, the topographic distribution of streptaster/amphiaster microscleres in the subectosomal skeleton, together with their morphology characterised by short spiny rays bearing blunt tips is shared with N. microstylifer and N. nolitangere.As for monaxial ectosomal spicules N. nana nov.sp.displays styles/sub-tylostyles never recorded before in N. nolitangere while they are typical of N. microstylifer.Although monaxial spicules has been reported as oxeas/anisoxeas by Carter (1873), Sollas (1888), Lendenfeld (1903) and Topsent (1892Topsent ( , 1904)), Pisera and Lévi (2002) did not fi nd this spicular type in the Schmidt's type material of N. nolitangere.During the present investigation styles/sub-tylostyles straight to slightly sinuous were also found in the Topsent specimen of N. nolitangere we recently rediscovered (MOM) and in the Topsent slide of N. nolitangere (MNHN) from Fayal although they have not been found by Pisera and Lévi (2002) probably because they are present exclusively along margins of the slide (MNHN) and are grouped in small fragments of thin membranes like those discovered in N. nana nov.sp. in sub-ectosomal membranes.Moreover abundant style-like monaxons have been found also in the material identifi ed by Pulitzer (? N. nolitangere, unpubl.)from Azores.
Th e growth form displayed by N. nana nov.sp., as a large thick plate with a peculiar distribution of inhalant areas in concavities, diverges from the shallow cup/ear shape previously recorded for N. nolitangere, and the massive or clavate shape typical of N. microstylifer.Th e new species resembles, however the topographic localization of inhalant and exhalant apertures of the latter species.Spicules of N. nana nov.sp.do not match the measurements of the genus and the size range of dichotriaenes, desmas, and streptaster/amphiaster microscleres appears to be notably smaller when compared with N. nolitangere reported by Topsent (1892bTopsent ( , 1904) ) and Pisera and Lévi (2002) together with present measurements.Some spicule measurements match better those of N. microstylifer (Lévi and Lévi 1983).
Th e co-occurrence of two, until now apparently neglected, spicular morphotraits such as style-like and two types of dichotriaenes also in the Topsent and Pulitzer-Finali material of N. nolitangere from Azores supports the need of new samplings and a revision of the genus.Th e Neophrissopongia material (as Corallistes) in the Schmidt collection (Desqueiroux-Faúndez and Stone 1992) from Strasbourg and Graz is not considered with confi dence by Desqueiroux-Faúndez (in litt.) and also Pisera and Lévi (2002) are dubious on the type material.At the same time the stylelike sub-ectosomal spicules strengthen the phylogenetic relationships between the Atlanto-Mediterranean and Pacifi c lineages of the genus whereas the smooth morph of dichotriaenes, although rare, may support the relationships among the lineages of the family Corallistidae.
In contrast with the deep bathymetric range of the genus in the Eastern Atlantic and Western Pacifi c (355-365 m of depth) the discovery of N. nana nov.sp. in the innermost dark zone of shallow water cave (6 m of depth) of NW-Sardinia suggests its presence may be relictual.Th is condition seems to be confi rmed by the presence of Neophrissopongia spp.also in a few other karstic caves of the Marseille coast and northern Adriatic Sea (Pisera and Vacelet 2006).Th e presence of N. nana nov.sp. in the Mediterranean area fi lls in part a distributional gap towards the weastern Pacifi c oceanic region and supports their relic condition suggested by the present day highly disjunct geographic range of the genus Neophrissopongia in the Lusitanian-Macaronesian area (N.nolitangere) and in the western Pacifi c Ocean (N.microstylifer) (Fig. 4).Th is geographic pattern confi rms the existence of a biogeographic track (sensu Croizat 1958) shared with other taxa (e.g.other lithistids).Mediterranean species of the genus Neophrissopongia may be representative of a survived stock allied to a highly diverse fossil assemblage mainly dated to Late Jurassic and Late Cretaceous of western-central Europe and North Africa (Reid 1967;Rigby 1983Rigby , 1991;;Wiedenmayer 1994;Pisera 1999;Finks et al. 2004).Although at present is not possible to clearly discriminate between the hypothetical survival of their ancestors during the harsh climatic/environmental fl uctuations of the Mediterranean basin versus an immigration process from the Atlantic, this discovery stresses the key status of Mediterranean palaeoendemics as remnants of an ancient Tethyan marine fauna together the need to point out on biodiversity conservation.
Th e knowledge on the marine palaeoendemic fauna represents a main factor to understand the dynamics and evolution of the Mediterranean basin from the historical biogeography point of view.Investigations focused on this general topic need to be supported by data on taxa characterised by low dispersal power and peculiar geographic patterns.In this fi eld, sponges, in particular, could be useful targets.Sponges display a life cycle strategy with a meroplanktonic larval stage characterised by a scarce swimming potential that presumably allows only a short planktonic phase with hard constraints on the long distance dispersal.(Vacelet 1960(Vacelet , 1969)); Central Tyrrhenian Sea, Bay of Naples, Posillipo, 40°49'N 14°13'E, 20 m of depth, associated with Phakellia robusta (Vosmer 1935;Topsent 1925;Vacelet 1969)
at the top.Th e spicular complement is characterised by a smaller size of spicules when compared to the other species of the genus.Ectosomal skeleton with tubercled to smooth dichotriaenes (rarely triaenes) radially arranged with no diff erential topographic distribution.Sub-ectosomal membranes bear styles/subtylostyles tangentially embedded and spiny streptaster/amphiaster microscleres.Etymology.Th e specifi c epithet refers to the small size of the skeletal spicules.Distribution.Known until now exclusively from the western Sardinian cave Grotta delle Terrazze (type locality).

Fig. 4 .
Fig. 4. Geographic range of the genus Neophrissospongia with occurrences of the known species N. nolitangere in the Atlantic and N. microstylifer in the Pacifi c Ocean.Th e type locality of N. nana nov.sp.from Sardinia is indicated by a star.