Freshwater sponges of the West Indies: Discovery of Spongillidae (Haplosclerida, Spongillina) from Cuba with biogeographic notes and a checklist for the Caribbean area

The paper reports the first finding of freshwater sponges from the Greater Antilles. Spongillidae belonging to four species of the genera Ephydatia, Anheteromeyenia, and Radiospongilla were found in a variety of freshwater habitats in western Cuba. Anheteromeyenia cheguevarai nov. sp. is described. Morphological traits of sponges from West Cuba were characterized by light microscopy and scanning electron microscopy and compared to the spongillofauna of the Nearctic and Neotropical regions and the pan‐Caribbean area. The specific richness and the discovery of a new species suggest a high diversity of the Antillean freshwater sponges although the investigated area of Cuba is relatively small.


Introduction
Spongillidae (Spongillina, Haplosclerida) is the most speciose and widespread family of freshwater sponges and includes 21 genera and 134 species from a wide variety of habitats (Gee 1932;Penney and Racek 1968;Manconi and Pronzato 2002;Pronzato and Manconi 2002; also see references in Table I).

Study area
A survey focusing on the presence of sponges in inland waters was carried out in the western territory of Cuba during winter 2000. The study area was selected in accordance with its old geological and natural history, and its ancient fauna and flora with high levels of diversity and endemicity.
Different environmental typologies were surveyed such as small streams, rivers, lakes, man-made basins, pools, and subterranean watercourses. Samplings were performed at 12 sites, namely the subterranean Rio Viñ ales in the Cueva de los Indios (two sites), the hydrographic basin of Rio San Juan of the Reserva de la Biosfera of the Sierra del Rosario (three sites), the Embalses El Jibaro and El Salto near Piñ ar del Rio (two sites), two small embalses (two sites) between Guanajay and Rosa Marina along the southern side of the Pinar-La Habana road, a small pond of the Giardino Botanico Nacional of La Habana (one site), the Rio Ariguanabo near San Antonio de los Bañ os (one site), the Laguna del Tesor (one site) of the Gran Parque Natural de Montemar along the Cienaga de Zapata (Figure 1).
Cuban inland waters range from perennial to ephemeral with notable water level fluctuations in May to June and September to November. The dry season occurs in wintertime (ca 25uC), and the wet season (ca 30uC) from May to October. Rainfall ranges from ca 3000 to 1000 mm per year, respectively, in northern and southern Cuba according to the tropical climate (Walter et al. 1967). Several water bodies are partially subterraneous, according to the karstic nature of the island, as in the case of the presently considered Rio Ariguanabo and the Rio de la Cueva de los Indios. Surinam (Ezcurra de Drago 1975) Spongillidae Radiospongilla crateriformis (Potts, 1882) Trochospongilla paulula (Bowerbank, 1863) Metaniidae Drulia uruguayensis Bonetto and Ezcurra de Drago, 1969 Metania spinata (Carter, 1881) Guyana (Gee 1930(Gee , 1931 Metaniidae Drulia browni (Bowerbank, 1863) Cuba (present paper) Spongillidae Anheteromeyenia cheguevarai sp. nov. Ephydatia facunda Weltner, 1895 Radiospongilla crateriformis (Potts, 1882) Radiospongilla sp. The checklist of freshwater sponges (Table I) refers to the pan-Caribbean area including the Nearctic and Neotropical regions in their, respectively, southern and northern areas.

Materials and methods
Submerged substrata were examined in shallow waters of both lentic and lotic habitats. Sponge bodies were scraped with care from substrata with a knife to collect also gemmules generally produced at the sponge base. Collected specimens were preserved by drying and deposited in the freshwater sponge collection of the authors at the Dipartimento per lo studio del Territorio e delle sue Risorse (Università di Genova) with registered numbers DTRGFW599, DTRGFW600, DTRGFW601, DTRGFW602, and DTRGFW603. Type material is registered in the Museo Civico di Storia Naturale ''G. Doria'' di Genova. Observations on macroscopic traits and dissection of sponge body were performed under a stereomicroscope. Spicules and gemmules were prepared for both light microscopy (LM) and scanning electron microscopy (SEM) following standard methods Pronzato 2000, 2002;Pronzato and Manconi 2002). Measurements were carried out for each spicular type (n 5 30-60) and for gemmules (n 5 10).

Results
Sponges were found in five sites ( Figure 1). Sponge density was low and a small size of sponge body with an encrusting growth form was recorded in all the environmental typologies. Four species of three genera of spongillids were determined.

Diagnosis
Anheteromeyenia with a pneumatic layer shaped as a network of spongin fibres in the gemmular theca supported by radially arranged gemmuloscleres pseudobirotules of onedimensional class.

Description
Encrusting irregular sponges (2-5 cm in diameter, 1-2 mm thick). Colour pearl grey. Surface apparently smooth and velvet in living specimens. Oscules not conspicuous. Ectosomal skeleton as more or less tangential spicules with tips slightly emerging from the dermal membrane. Choanosomal skeleton irregularly reticulated isotropic, paucispicular. Megascleres oxeas (105231565215 mm) straight to slightly bent, from covered by short spines except at the sharply pointed tips, to rarely smooth or flexuous ( Figure 2G). Oxeas sometimes with bifid, bent, or rounded tips. Microscleres absent. Gemmules brilliant grey at the sponge basis in a single compact layer strictly adherent to the well-developed basal spongin plate. Gemmules subspherical to oval (330-360 mm in diameter) ( Figure 2A). Foramen with a developed tube at the middle of a depressed area supported by dense gemmuloscleres ( Figure 2B, E). Gemmular theca trilayered (35-60 mm thickness) with gemmuloscleres arranged in a radial manner ( Figure 2D). Outer layer thick pierced by gemmulosclere shaft with emerging distal pseudorotules ( Figure 2C). Pneumatic layer as a thick network of spongin fibres with irregular rounded meshes ( Figure 2F). Inner layer of sublayered compact spongin ( Figure 2D). Gemmuloscleres pseudobirotules (50-84 mm in length) ( Figure 2H) with a shaft 3.6-4.5 mm thick from smooth to ornate by 7-15 long acute spines mainly towards the distal part of shafts ( Figure 2I); pseudorotules (4.5-14 mm in diameter) with a conspicuous umbone bearing 6-11 smooth curved hooks ( Figure 2I).

Etymology
The species is named for Ernesto ''Che'' Guevara, the popular hero of Latino-America.

Habitat
Specimens of Anheteromeyenia cheguevarai nov. sp. were found in a small lentic habitat (embalse) under boulders near the shoreline in shallow shaded quite brown waters at a depth of ca 40-60 cm.

Geographic distribution
The finding of Anheteromeyenia in Cuba matches the Nearctic and Neotropical geographic range of the genus with A. argyrosperma from Canada to Florida, and the Neotropical A. ornata from Brazil and Argentina (Penney and Racek 1968;Bonetto and Ezcurra de Drago 1970;Frost 1991;Ricciardi and Reiswig 1993;Volkmer-Ribeiro 1996;Manconi and Pronzato 2002) (Tables I, II).

Remarks
These Cuban sponges clearly differ from E. millsi (Potts, 1888) for the shape of gemmular birotules and size of megascleres. As for E. subtilis, recorded as a new species but not described by Weltner (1895), Harrison (1979) reports that its ''taxonomic status remains undetermined'' because despite extensive collecting in the type locality (Lake Kissimee, Florida) the species was not found. The examined material from Cuba shares with E. facunda the general morphology, size of skeletal and gemmular elements, and the variable trait ''outer layer with embedded or free distal rotules in the gemmular theca''; it differs however for the size of megascleres when compared to the recent description by Pinheiro et al. (2004). E. facunda was considered a synonym of E. fluviatilis ramsayi by Ezcurra de Drago (1975), and re-evaluated as a valid species by De Rosa Barbosa (1979) and Pinheiro  (2004). As for the freshwater sponges ascribed to Ephydatia fluviatilis from the Island of St John (Virgin Islands), it clearly appears from SEM micrographs by Smith (1994) that gemmules are characterized by the trait ''pneumatic layer as a network of spongin fibres'' deeply diverging from the typical gemmular architecture of E. fluviatilis characterized by a ''pneumatic layer of chambered spongin''. A comparative analysis of distant populations of E. fluviatilis is at present in progress by morphological and molecular approaches to clarify its problematic cosmopolitanism with a disjunct geographic range.

Habitat
In the Rio Ariguanabo under boulders near the embarcadero in shaded shallow brownyellowish waters with a high content of minerals, ca 20-40 cm of depth. The Rio Ariguanabo in this karstic area runs, with a wide wet bed (10-15 m wide), under tunnelshaped vegetation and becomes subterranean below this site. In the Rio San Juan of the Sierra del Rosario sponges were settled under boulders and pebbles in running clear shallow waters with high mineral content, ca 10-20 cm of depth, in a habitat shaded by a tunnel vegetation. Sponges were associated with statoblasts of an unidentified bryozoan.

Geographic distribution
The presence of E. facunda in western Cuba fits well the distribution of the cosmopolitan genus Ephydatia in the pan-Caribbean area with E. fluviatilis (Linnaeus, 1759) recorded in Florida, Mexico, El Salvador, and the Virgin Islands, and E. millsi and E. subtilis in Florida (Tables I, II). Ephydatia facunda is endemic to the Neotropical region and recorded till now exclusively from Brazil (Pinheiro et al. 2004). The present record extends the geographic range of the species to the Caribbean area.
Megascleres oxeas (209232168210 mm) straight to slightly bent, smooth in the middle and with small spines at the sharply pointed tips ( Figure 4E, J). Microscleres spiny strongyles to oxeas (7528562.525 mm) ( Figure 4F, G, K). Gemmules abundant and grouped at the sponge basal area, not adhering at the basal spongin plate. Gemmular shape from subspherical (400 mm) to oval (3206400 mm) ( Figure 4A, B). Colour of gemmules white to dark grey according to the absence/presence of the outer layer in the theca ( Figure 4B). Foramen tubular without collar but at the middle of a crater-like depressed area. Gemmular theca trilayered (78-83 mm in thickness) with radially and densely arranged gemmuloscleres ( Figure 4C, D). Outer layer from well developed to absent; when present it bears emerging distal rotules from the outer layer. Pneumatic layer as a weakly developed network of very fine spongin fibres ( Figure 4C). Inner layer of sublayered compact spongin. Gemmuloscleres (63-79 mm in length) straight or slightly bent are pseudobirotules to strongyles frequently bearing an apical acute spine surrounded by a crown of a few bent spines (hooks); pseudo-rotules (5.4-11.5 mm in diameter); shaft with several acute spines ( Figure 4H, I).

Remarks
All morphological traits and size of spicules match well those described by several authors for this widespread species and particularly spicular sizes from Barbados and Nevis Islands by Bass and Volkmer-Ribeiro (1998).

Habitat
Small sponges settled under boulders in standing quite brown shallow waters ca 15 cm of depth in a small pool near the greenhouses. This finding confirms stagnant turbid waters as the preferred habitat of this species (Harrison 1974).

Geographic distribution
The widespread genus Radiospongilla is represented in the Nearctic and Neotropical Regions by R. crateriformis and R. amazonensis Volkmer- Ribeiro and Maciel, 1983. This second finding of R. crateriformis in the West Indies, after that in Barbados and Nevis Islands by Bass and Volkmer-Ribeiro (1998), fits well the geographic range in Surinam, Yucatan, Mexico, and USA (Old 1932(Old , 1936Arndt 1933;Ezcurra de Drago 1975) ( Tables I, II). On the other hand, this species shows a widely disjunct distribution and it is reported also from China, Philippines, Japan, and southern Asia (Penney and Racek 1968;Manconi and Pronzato 2002). The finding of the species exclusively in the Orto Botanico Nacional of Cuba suggests, however, the possibility of an accidental introduction.

Description
Growth form from encrusting to small cushion. Colour whitish-greenish. Consistence fragile both in vivo and in dry conditions. Surface smooth. Oscules conspicuous, 2-3 mm in diameter, irregularly scattered. Ectosomal skeleton with tangential spicules in the dermal membrane, with few emerging tufts of two or three spicules. Choanosomal skeleton paucispicular with irregular meshes from alveolar to more or less triangular. Basal spongin plate well developed and spicular. Megascleres slightly bent oxeas (284234869.3214 mm) smooth to rarely spiny except at the sharp tips ( Figure 5A). Apices sometimes bent or with two to three tips. Microscleres (11221636324.5 mm) few to rare straight acanthoxeas with spines increasing in density towards the sharply pointed tips; apical spines ornate by microspinosities ( Figure 5B-D). Gemmules absent.

Remarks
The absence of gemmules, probably due to the active life cycle phase of the sponges, renders it impossible to determine the taxon at the species level. Diagnostic skeletal traits of the sponge such as shape and size of the spicular complement suggest the ascription of specimens to a species of the genus Radiospongilla.

Habitat
Sponges were settled under limestone boulders in waters rich in carbonates of a lentic habitat along the northern edge of the Laguna del Tesor. The latter is the largest (900 hectares) natural and most pristine wetland of Cuba (maximum depth 10 m, mean ca 4 m) within the Gran Parque Natural de Montemar with large extension of swamps and coastal brackish lagoons.

Discussion
Cuban spongillids, all displaying an encrusting body shape of small size, were in the active phase of the life cycle at the beginning of the dry wintertime. Most sponges were characterized by the typical presence of gemmules able to withstand unfavourable environmental conditions to survive in situ as resting bodies or to perform dispersal  (Manconi and Pronzato 1991, 1994, 1996Pronzato and Manconi 1994;. All sponges were settled in shallow waters but their lifestyle appears strictly skiophilous, in accord with the preferential shaded microhabitats in both lotic and lentic typologies ranging from streams and rivers, to coastal basins, man-made lakes and small pools. Spongillidae were found in five out of the 12 surveyed sites in the western area of Cuba (Figure 1), confirming the hypothesis by de Laubenfels (1936). The spongillofauna of western Cuba appears, since the present first survey, notably rich with four species belonging to four genera. Results suggest a notable diversity of the taxon Spongillidae although species richness in the island appears impoverished with respect to the continental neighbouring regions. The rare findings of freshwater sponges hitherto in the Greater Antilles seem to be due to a lack of surveys. The presence of two syntopic species belonging to two different genera, Anheteromeyenia and Ephydatia, within the same hydrographic basin of Rio San Juan in a small artificial lake and down in the neighbouring tract of its outlet stream, respectively, confirms the high biodiversity of the Reserva de la Biosfera of the Sierra del Rosario. On the other hand, E. facunda appears widespread and is found in distant lotic habitats sharing a high water mineral content, the river Rio Ariguanabo and the stream Rio San Juan (Sierra del Rosario).
These first results are constrained by the limited study area, the short-term nature of the survey, and the low number of samplings. However, they refer to the most ancient area of Cuba and could be suggestive of a general condition of richness. The present records from Cuba and the presumed absence of freshwater sponges in the other Greater Antilles do not fit the high values of diversity of Spongillina in the Nearctic and Neotropical regions, suggesting the need for new investigations on these unexplored islands. Two families, Spongillidae and Metaniidae, are present in the Nearctic region, the former with 27 species ascribed to 11 genera, namely Spongilla, Anheteromeyenia, Corvospongilla, Dosilia, Duosclera, Ephydatia, Eunapius, Heteromeyenia, Racekiela, Radiospongilla, and Trochospongilla (Potts 1887;Penney and Racek 1968;Harrison 1974;Frost 1991;Manconi and Pronzato 2002) (Table II).
In summary, three families of freshwater sponges with 48 species and 18 genera are known from the pan-Caribbean area: Spongillidae (39 species, 14 genera), Metaniidae (eight species, three genera), and Potamolepidae (four species, two genera) ( Table I).
The composition of the western Cuba spongillofauna suggests, at the genus level, strict relationships to the circum-continental areas, but it appears notably impoverished at the family level in the absence of Metaniidae and Potamolepidae.
Cuba shares its genera (Anheteromeyenia, Ephydatia, Radiospongilla) with both the Neotropical and the Nearctic regions, and Mesoamerica, and two genera with some other Caribbean islands (Tables I, II). The geographic distribution of the Cuban genera ranges from cosmopolitan to widespread. At the species level the presence of E. facunda, never reported in Mesoamerica and hitherto known only from Brazil, enlarges its geographic range to the north. Our data extend the range of R. crateriformis to the entire Caribbean insular arc. Finally, the presence of Anheteromeyenia cheguevarai nov. sp. matches the high levels of diversity and endemicity of western Cuba in accordance with the old geological and natural history of the area and its ancient endemic fauna and flora.
The knowledge of freshwater sponges from the Antilles is at present inadequate to attempt an exhaustive biogeographic analysis; however, these data appear to fit the classic North America-Caribbean and South America-Caribbean biogeographic tracks suggested by Rosen (1975). The composition and richness of the spongillofauna of Cuba and the other Caribbean islands shared at the genus level with the circum-Caribbean area seem to imply a relatively recent diversification and/or immigration related to their geological history and climatic vicissitudes. Bass and Volkmer-Ribeiro (1998) hypothesized for Barbados and Nevis islands a dispersal of R. crateriformis from the southeastern USA by winds and/or animal carriers. Debrot and van Soest (2001) suggested anemocorous dispersal for Spongilla alba and Corvoheteromeyenia heterosclera on the volcanic island of Curaçao from southern continental areas. Smith (1994) hypothesized that dispersal of E. fluviatilis occurred eastward across the Greater Antilles arc from Central America. On the other hand, the presence of the new species in the ancient area of Cuba suggests the existence of a typical Cuban/Antillean freshwater sponge fauna.
The geographic range of Cuban genera at a regional scale confirms that, as suggested by Donnelly (1988) for other taxa, different hypothetical dispersal routes could explain the composition of the Antillean spongillofauna, namely from the neighbouring Nearctic region and Mesoamerica by land bridges, and from the Neotropical region by means of a ''stepping stones'' process along the insular Caribbean arc.
The present findings together with the numerous records from insular freshwater habitats of all biogeographic regions except Antarctica (Weltner 1895;Gee 1932;Penney and Racek, 1968;Pronzato 1994, 2002;Pronzato and Manconi 2002) confirm that Spongillidae is a super tramp taxon able both to colonize oceanic islands and to persist and survive during the geological vicissitudes of fragments of continental land mass.