Sponge epizoism in the Caribbean and the discovery of new Plakortis and Haliclona species, and polymorphism of Xestospongia deweerdtae (Porifera) Author Vicente, Jan Author Zea, Sven Author Hill, Russell T. text Zootaxa 2016 4178 2 209 233 journal article 10.11646/zootaxa.4178.2.3 ef8d5717-31b6-4e69-a0ec-6e313588ecda 1175-5326 255301 7A957617-C37C-41C8-9A8C-D7BB9178638C Xestospongia deweerdtae Lehnert & Van Soest 1999 ( Figs. 6 , 7 ; Table 3 ) Xestospongia deweerdtae Lehnert & Van Soest, 1999 : 163 , Figs. 44–47; Van Soest & De Weerdt 2001 : 114 , Fig. 4 C–D, 5C–D; Rützler et al . 2014 : 91 ; Zea et al . 2014 (“-associated” and “-free living” forms); Vicente et al . 2014 , Figs. 3 a–f, 5, 6, 7a–d (ecology and symbiosis with Plakortis spp.). Xestospongia sp.2; Zea 2001 : Table 1 (appendix). Xestospongia sp.-thin pink sheet over Plakortis ; Zea et al . 2009 . Diagnosis. Thinly to thickly encrusting, pink, red and white mottled sponge. Surface smooth. Consistency hard but easily broken and only slightly compressible. Ectosome is a dense tangential reticulation of strongyles bound by spongin. Choanosome is an isotropic reticulation of single spicules with some paucispicular tracts (Lehnert & Van Soest 1999 ). Material examined. Holotype : ZMAPOR13584, Discovery Bay , Jamaica , 82 m depth, coll. Helmut Lehnert , June 26, 1996 ; USNM 1254644 , Punta Caracol , Bocas del Toro , Panama ( 9.3777° N , 82.1265° W ) 8 m , coll. Jan Vicente and Micah J. Marty, June 13, 2015 ; USNM 1254645 , Dolphin Rock , Bocas del Toro , Panama ( 9.35076° N , - 82.1863° W ), 14 m coll. Jan Vicente and Arcadio Castillo May 20, 2015 ; USNM 1254648 , Yellow Reef , Desecheo Island , Puerto Rico ( 18.3918° N - 67.4760° W ) 23 m coll. Jan Vicente October 7, 2011 ; USNM 1254649 , Old Bouy , La Parguera , Puerto Rico ( 17.8883° N , - 66.9981° W ) 31 m , coll. Jan Vicente and Milton Carlo August 8, 2012 ; USNM 1254647 , San Salvador , Bahamas , ( 24.0406° N , - 74.5314° W ) 33 m coll. Jan Vicente and Steven E. McMurray July 19 , 201 ; USNM 1254646 , Plana Cays , Bahamas ( 22.6045° N , - 73.5465° W ), 32 m coll. Jan Vicente and Steven E. McMurray, July 21, 2011 . Description ( Figs. 6 A–B, Fig 7 A–B). External morphology is influenced by lifestyle (associated with P. deweerdtaephila sp. nov. or P. symbiotica sp. nov. or free-living) and by environmental factors. Free-living forms of X. deweerdtae in the Bahamas ( Fig. 6 A) and Panama ( Fig. 7 A) fit the original description of Lehnert & Van Soest (1999) and Van Soest & De Weerdt (2001) . They form volcano shaped elevations that can measure up to 5 cm in length and with oscules on top, up to 0.7 cm in diameter. Individuals are light to dark pink, purple ( Panama ), orange ( Bahamas ) and turn white when preserved in ethanol; color in the choanosome is the same. Surface smooth; consistency hard, slightly compressible. Although not mentioned in the original description, we noticed that freeliving sponges always exude a viscous slime when cut. The external morphology of associated lifestyles does not fit the original description of X. deweerdtae . In the Bahamas , associated individuals can be a thin encrusting veneer of patchy tissue that overlays and burrows into the Plakortis spp. body ( Fig. 6 B) ( Fig. 6 A–C in Vicente et al. 2014 ) with no visible oscula. In Panama , associated individuals are thickly encrusting ( 1 cm ) and completely overgrow the P. deweerdtaephila sp. nov. body, forming 6–15 cm diameter plates ( Fig. 7 B; small oscules ( 1–3 mm ) are aligned probably due to the high wave energy environment of Dolphin Rock). Associated individuals are softer, slightly compressible and more brittle than freeliving individuals. Color is a light pink and even though we did not find free-living individuals with zoanthids, associated individuals in Puerto Rico were densely covered with red zoanthids ( Fig. 3 A). Skeleton. Despite the differences in external morphology from the different lifestyles, the ectosome of both associated and free-living morphologies regardless of location consist of a unispicular regular skeleton of strongyles with 6–7 spicules meeting at each node ( Fig. 6 C–D; Fig. 7 C–D). Meshes of the ectosome are somewhat triangular. The choanosome for all lifestyles and regardless of location consist of an isotropic reticulation of strongyles with occasional paucispicular tracts, which conform to X. deweerdtae (Lehnert & Van Soest 1999 ) ( Fig. 6 E–H; Fig. 7 E–H). Spicules: Thick and sometimes thin strongyles, 151–423 µm long by 6.5–28.2 µm in width. Size and morphology of strongyles depends on the associated status of X. deweerdtae and geographical location. Across all geographical areas sampled ( Bahamas , Puerto Rico , Panama ), associated individuals have smaller and thinner spicules than free-living ones (see also discussion in Vicente et al . 2014 ); and between geographical areas, across lifestyles, individuals from island locations such as Bahamas , Puerto Rico and Jamaica have smaller and thinner spicules than those from Panama ( Table 3 ). TABLE 3. Spicule measurements of strongyles (length and width) of Xestospongia deweerdtae associated and free living. Measurements are expressed as minimum– mean (±1 standard deviation)–maximum. N=30.

Specimen	Location	Life style	Length (µm)	Width (µm)
ZMAPOR13584 *	DB, Jamaica	Free-living	288– 340.9 (± 16.9)–373	6.5– 12.1 (± 2.2)–17.5
USNM1254646	SS, Bahamas	Free-living	305– 336.6 (± 14.8)–362	6.7– 11.8 (± 2.3)–17.2
USNM1254647	SS, Bahamas	Associated	151– 203.0 (± 18.8)–243	6.3– 7.9 (± 1.1)–11.2
USNM1254644	BDT, Panama	Free-living	340– 376.8 (± 19.7)–424	15.5– 21.8 (± 3.6)–28.2
USNM1254645	BDT, Panama	Associated	229– 323.8 (± 22.0)–379	12.8– 18.6 (± 2.8)–23.9
USNM1254648	LP, Puerto Rico	Free-living	296– 324.3 (± 15.2)–355	9.3– 13.0 (± 2.1)–17.7
USNM1254649	LP, Puerto Rico	Associated	159– 223.8 (± 23.5)–274	7.3– 10.2 (± 1.7)–13.8

*Specimen ZMAPOR13584 is the holotype (Lehnert & Van Soest 1999 ) Habitat and ecology. X. deweerdtae was originally described from deep ( 82 m ) fore reef habitats and reef caves of Jamaica (Lehnert & Van Soest 1999 ) and then later found in reef caves ( 10–12 m ) of Curaçao (Van Soest & De Weerdt 2001 ). In our study, we found X. deweerdtae growing beneath scleractinean corals and on the shaded side of Agaricia reefs in Panama at depths as shallow as 2 m . X. deweerdtae and Plakortis spp. nov. sponge pairs can be found on the shaded side of spur and groove reef formations ( 14 m ) exposed to high wave energy environments in Panama. In the Bahamas and Puerto Rico sponge pairs are found deeper, below 30 m in cryptic habitats growing on vertical walls, on the roof of overhangs and on the bottom of reef caves. Associated individuals of X. deweerdtae are more frequently observed than free-living ones when both lifestyles are present in a given area ( Vicente et al. , 2014 ). Distribution. (FL=free-living, AS =associated) Jamaica (Discovery Bay-FL) (Lehnert & van Soest, 1999 ) Curaçao-FL (van Soest & de Weerdt 2001 ), Mexico (Cozumel-FL, Banco Chinchorro-FL) ( Vicente et al. , 2014 ), Bahamas (Plana Cays-FL-AS, Mayaguana-FL, San Salvador-AS, Little San Salvador-AS, Little Inagua-FL-AS, Acklins-FL, Great Inagua-AS, Mira Por Voz-AS) (Also Vicente et al . 2014 and Zea et al . 2014), Puerto Rico (Mona-AS, Desecheo-FL-AS, La Parguera-AS) (also Vicente et al. 2014 ), Panama , Bocas del Toro (Fiugre 7A-FL, Punta Caracol, Figure 7 B-AS, Dolphin Rock), Colombia (Serrana Bank-AS) ( Zea 2001 ). Taxonomic remarks. The polymorphic nature of X. deweerdtae is revealed by lifestyle (associated and freeliving) and by environmental factors (high wave energy and high silica environments, the latter associated with geographical location). Associated individuals do not exhibit a massive morphology and long volcano shaped oscules mentioned in the original description are absent. Instead, associated sponges are thinly to thickly encrusting. In Panama oscules for associated cases are small but visible ( 1–3 mm ) and colonies are more thickly ( 0.5–1 cm ) encrusting than associated individuals from Puerto Rico and the Bahamas ( 1 mm ). Oscules for associated individuals from Puerto Rico and the Bahamas were not visible. Significantly smaller strongyles are observed in associated (250 × 12.2 µm) sponges than free-living sponges (346 × 15.5 µm) in three different geographic areas resembling high and low silica concentrations. This was interpreted as a possible benefit for X. deweerdtae in terms of a lower investment in skeleton synthesis for support (see discussion in Vicente et al. 2014 ). Another possible explanation is that nutrients may be limiting for both sponges and one species might be depriving the other of silica. On the other hand, in the high silica environments in Panama (see D’Croz et al. 2005) longer and thicker spicules were present in both free-living and associated sponges when compared with both associated and free-living individuals of Puerto Rico and the Bahamas ( Table 3 ). Free-living individuals from Panama also produced not only thicker and longer strongyles, but also had sharply bent terminals that bend either opposite (sshaped) or in the same direction (bracket-shaped) ( Fig. 7 I) probably due to hypersilicification ( Zea, 1987 ; Zea et al. , 2014). One other important character from free-living sponges, missing in the original description, is the release of viscous mucus by sponges when cut. Associated sponges however, produce very little mucous when cut. The conspecificity of associated and free-living individuals was confirmed with phylogenetic analysis from partial sequences of the 18S, 28S rRNA and cox1 genes (see below and Vicente et al . 2014 ). FIGURE 6. Xestospongia deweerdtae from the Bahamas, comparing free living (A, C, E, G, I) and associated (B, D, F, J) life styles. (A, B) in situ underwater images (A, USNM1254646; B, USNM1254647 (pink) with Plakortis symbiotica sp. nov. (brown); (C, D) tangential section of the ectosome (LM); (E, F) perpendicular section through the ectosome and choanosome (LM); (G, H) close-up of a perpendicular section through the choanosome (LM); (I, J) strongyles (SEM). FIGURE 7. Xestospongia deweerdtae from Bocas del Toro Province in Panama, comparing free living (A, C, E, G, I) and associated (B, D, F, J) lifestyles. (A, B) In situ underwater images (A, USNM1254644; B, USNM1254645 completely overgrowing Plakortis deweerdtaephila ; (C, D) tangential section of the ectosome (LM); (E, F) perpendicular section through the ectosome and choanosome (LM); (G, H) close-up of a section through the choanosome (LM); (I, J) Strongyles (SEM). Phylogenetic analysis. To confirm the identity of Haliclona plakophila sp. nov. as a new species and reconfirm the conspecificity of associated and free-living morphotypes of Xestospongia deweerdtae we partially sequenced the 18S, 28S rRNA and cox1 genes of holotype and paratype specimens. We conducted a maximum likelihood analysis from sequences of species belonging to Haplosclerida that were closely related to Haliclona spp. and Xestospongia spp. deposited in GenBank. The maximum likelihood analysis of the 18S rRNA gene sequence placed H. plakophila distant from any of the monophyletic clades (A–E) previously reported by Redmond et al. (2013) ( Fig. 8 A). Members of clade C ( Redmond et al. , 2013 ) were not included in the phylogenetic analysis, because sequences from H. plakophila were highly dissimilar to species in this clade. Sequences from associated and free-living individuals of X. deweerdtae were all>99% homologous to each other and to the holotype of X. deweerdtae (ZMAPOR13584), confirming that all specimens of both life styles are conspecific. Conspecificity of all X. deweerdtae lifestyles, including the holotype specimen, had a>99% sequence homology for the 28S rRNA and cox1 genes ( Fig. 8 B–C). We were unable to retrieve enough sequence data from the holotype specimen of X. deweerdtae to produce a phylogenetic tree of the cox1 with strong bootstrap values, encompassing all closely related Haplosclerida species. However, we had enough sequence data from our material and proceeded to do a maximum likelihood analysis of the cox1 including members of the Haplosclerida that form monophyletic clades A and B from Redmond et al. (2011) ( Fig. 9 ). The clade of all X. deweerdtae conspecifics did not fall into either monophyletic clade. Like X. deweerdtae , H. plakophila sp. nov. also did not fall into either clade A or B ( Fig. 9 ).