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Petrosida: Petrosiidae) FAU Institutional Repository http://purl.fcla.edu/fau/fauir This paper was submitted by the faculty of FAU’s Harbor Branch Oceanographic Institute. Notice: ©1994 A. A. Balkema. This manuscript is an author version with the final publication available and may be cited as: Kerr, R. G., & Kelly-Borges, M. (1994). Biochemical and morphological heterogeneity in the Caribbean sponge Xestospongia muta (petrosida: Petrosiidae). In R. W. M. van Soest, T. M. G. van Kempen, & J. C. Braekman (Eds.), Sponges in Time and Space: Biology, Chemistry, Paleontology: proceedings of the 4th International Porifera Congress, Amsterdam, Netherlands, 19-23 April 1993 (pp. 65-73). Rotterdam; Brookfield, VT: A. A. Balkema. Alb ;~ry PROCEEDINGS OF THE 4TH INTERNATIONAL PORIFERA CONGRESS AMSTERDAM/NETHERLANDS/19-23 APRIL 1993 Sponges in Time and Space Biology, Chemistry, Paleontology Edited by ROB W. M. VAN SOEST University ofAmsterdam, Netherlands THEOM.G. VAN KEMPEN Free University, Amsterdam, Netherlands JEAN-CLAUDE BRAEKMAN Free University ofBrussels, Belgium Assistedby ANITA BRINK & FRANS R. BIANCHI Foundation Pangea, Huizen, Netherlands JAN IVERMEULEN University ofAmsterdam, Netherlands A.A. BALKEMA / ROITERDAM / BROOKFIELD / 1994 Sponges in Time and Space, van Soest, van Kempen &Braekman (eds) © 1994 Balkema, Rotterdam, ISBN 9054100974 Biochemical and morphological heterogeneity in the Caribbean sponge Xestospongia muta (petrosida: Petrosiidae) FtussellCJ.}Cerr Department ofChemistry, Florida Atlantic University, Boca Raton, Fla., USA Michelle Kelly-Borges Division 0/Biomedical Marine Research, Harbor Branch Oceanographic Institution, Fort Pierce, Fla., USA ABSTRACf: Chemical and morphological analyses of the Caribbean reef spongeXeslospongiamula have revealed that there is significant heterogeneity within this species. There are three distinct sterol compositions, as well as three morphological types, neither of which appears to be correlated with geographic locality, depth or micro­ habitat differences. 1 INTRODUCTION mula might also be a species complex came initially from an investigation of the sterols of this sponge (Kerr Xestospongia muta (Schmidt) (Petrosida, Petrosiidae) et aI., 1991). In the course of conducting a biosynthetic is one of the most conspicuous Caribbean and southern investigation of mutasterol, an unusual multi-alkylated Floridian reef sponges, due to its abundance in a wide sterol, we identified the sterols present in twenty variety of habitats, the large adult size, and easily individuals of X. mula in Puerto Rico, and found three recognisable shape (Wiedenmayer, 1977; van Soest, distinct sterol compositions. 1980; Zea, 1987). The sponge occurs over a wide geo­ Sterols are required components of eukaryotic orga­ graphic range, having been recorded off the coast of nisms, with these compounds replacing triterpene "func­ Florida, and as far south in the Atlantic as Brazil tional equivalents" in prokaryotes. Sterol mixtures are (Collette & Rutzler, 1977). Xestospongia mutais found generallymore complex, and unconventional structures in shallow lagoonal seagrass beds, on the fore-reef more common in the less advanced eukaryotes slope of fringing or barrier reefs, but most abundantly (Bergmann, 1949). Higher animals contain cholesterol on deeper ribbon reefs down to 24 m. Van Soest (1980) as their sole sterol, while the more primitive marine notes a collection record of this species from Puerto invertebratescontain mixtures of sterols. Plants contain Rico, at 90 m. sterols which are very similar to cholesterol, the main Xestospongia mula is a lamellate barrel or volcano difference being an alkyl substituent at C-24. Sponges shaped sponge that has been recorded up to 1.5 m high have, by far, the greatest variety of sterol structures and 80 em wide, but is more typically 30 to 80 ern high with numerous examples unique to this phylum (Kerr and 50 ern wide. The colour of the sponge in life is & Baker, 1991). It has been suggested (Goad, 1978) caramel brown to maroon brown externally, with a that there are four possible sources ofsterols in marine beige interior. The maroon colouration can be patchy invertebrates, and that each organism must establish a and is due to the presence of cyanobacteria in the illu­ balance between these factors. The four possible minated portions of the spongesurface. Morphological contributing sources are: de novo biosynthesis, features of the sponge, such as gross and surface assimilation of dietary sterols, modification of dietary morphology, and histological features such as texture, sterols, and assimilation by host of sterols (or precursors) are highly variable in this species (personal observation produced by symbionts. of authors). Since sponges contain such a diverse array of novel Many sponge species show a high degree of intra­ sterols, it is not surprising that their utility in chemo­ specific morphological variability (e.g. Pansini 1982), taxonomy has been investigated (Bergmann, 1949; which in some cases has been correlated directly with Bergquist et al., 1980; Bergquist et aI., 1986). Sterol environmental parameters such asdepth (e.g. Thompson composition can be a valuable taxonomic tool for et al., 1986; Sennett et al., 1992). Other studies have several reasons. Firstly, there is enormous structural required theemploymentof stringentsystematic techni­ variation in sponge sterols, thus providing a large data ques such as biochemical and morphometric analyses set of obvious taxonomic value. Sterols can be catego­ to detect the presence of species (e.g. Hooper, 1990; rised in various ways; these include, but are not limited Sara& Gaino, 1987). The suggestionthatXeslospongia to, the position of side chain alkylation, the degree of 65 alkylation, and the unsaturation pattern in the sterol sampled for biochemical analysis and histology by nucleus. Secondly, sponges usually contain a complex SCUBA. A fragment of sponge (300 - 400 g) was mixture of sterols; generally, ten to twenty sterols are excised from each sponge, and sub-samples, including present in any given sponge, enabling the generation surface and choanosomal tissue, were taken for and comparison ofdata sets with a significant number histology. Habitat and depth data were recorded on ofcharacters. Thirdly, sterols are very stablecompounds collection, along with details of habitat, gross and and are present in relatively high concentrations in surface morphology, pigmentation, texture, and sponges. Thus, sterol analyses can be performed on dimensions of the living sponge. small amounts of sponge, and from samples which Sterol analysis: The sponge fragment was extracted have been stored for extended periods oftime. Lastly, by cutting into small pieces and soaking in chloroform! sterol composition has been shown to be invariant with methanol (1: 1) and then chloroform. The combined time and space (Bergquistet aI., 1980; Fromont, 1991), extracts were concentrated to afford a dark oil. The a feature essential for any chernotaxonomic tool. Much sterol mixture was separated by preparative thin layer is now known about the biosynthetic origins ofsterols chromatography (TLC) on silica using hexanes/ethyI (Djerassi & Silva, 1991; Kerr & Baker, 1991) and we ether (1: 1) as the mobile phase. The sterol mixture was feel that this information can also be of taxonomic then analysed by gas chromatography CGC) equipped value (vide infra). witha capillarycolumn (DB-5, 25m) and sterols identi­ The initial aimofthis projectis to test the hypothesis fied by comparison ofrelative retention times (RRTs). that sterol chemotype differences correlate with To facilitate rapid comparison of sterol compositions morpohological differences in Xestospongia mula. of various individuals, we generated "sterol finger­ Specifically, our goals are to determine the range of prints"; represented by bar graphs which are derived sterol chemotypes within X. mula throughout the from GC traces. Caribbean, determine the spatial distribution of the Histological preparation ofspecimens: Tissue sub­ chemotypes (shallow vs. deep reefs, patch reef vs. samples were preserved in 70 % ethanol. Samples were ribbon reef), and describe the range of variability in processed histologically by cycling through a series of gross and surface morphology, spicule dimensions and differing ethanolconcentrations,cleared,and embedded other histological parameters. in paraffin. Spicules were digested from tissue in con­ centrated nitric acid and centrifuged through a series of washes with water and absolute ethanol. Spicules and 2 MATERIALS AND METHODS histological sections were mounted permanently, and examined by light microscopy. Field sites: Samples ofXestospongia muta have been obtained from five sites. These were collected from both patch and ribbon reefs at various depths (Table 1). 3 RESULTS Collection of samples: Sponges were tagged and 3.1 Biochemical variation In order to estimate the ratios ofchemotypes at various Table 1. Study sites within Florida and the Caribbean. sites, ten to twenty specimens of Xestospongia muta from eachsite weresubjectedto sterol analysis. Fourteen Location Reef Type Depth (m) distinct sterols have been isolated from the various chemotypesofX. muta, and the individual (collected in Long Key (Florida) patch 10 Puerto Rico) described in Fig. 1 contains 9 of these, in Long Key (Florida) patch 19 Key Largo (Florida) ribbon 12 the relative abundances shown. Note that structures Boca Raton (Florida) ribbon 24 and their names are not assigned to the various sterols
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