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 Elsevier B.V. This manuscript is an author version with the final publication available at http://www.sciencedirect.com/science/journal/03051978 and may be cited as: Kelly‐Borges, M., Robinson, E. V., Gunasekera, S. P., Gunasekera, M., Gulavita, N. K., & Pomponi, S. A. (1994). Species differentiation in the marine sponge genus Discodermia (Demospongiae, Lithistida): the utility of ethanol extract profiles as species‐specific chemotaxonomic markers. Biochemical Systematics and Ecology, 22(4), 353‐365. doi:10.1016/0305‐1978(94)90026‐4 Biochemical Systematics and Ecology. Vol.22, No.4, pp. 353-365, 1994 Copyright © 1994 Elsevier Science ltd Printed in Great Britain. All rights reserved 0305-1978/94 $7.00+0.00 0305-1978(94)EOOO3-X Species Differentiation in the Marine Sponge Genus Discodermia (Demospongiae: Lithistida): the Utility of Ethanol Extract Profiles as Species-Specific Chemotaxonomic Markers* MICHELLE KELLY-BORGES,t ELISE V. ROBINSON, SARATH P. GUNASEKERA, MALIKA GUNASEKERA, NANDA K. GULAVITA and SHIRLEY A. POMPONI:f Division of Biomedical Marine Research,Harbor Branch Oceanographic Institution, 5600 North U.S. 1. Fort Pierce. FL 34946. U.SA; tPresent address: Department of Zoology, The Natural History Museum. Cromwell Road. London SW7 5BD. U.K. Key Word Index-Theonellidae; Lithistida; Demospongiae; Porifera; Discoderrnia; chemotaxonomy; thin layer chromatography; 'H-NMR spectra; taxonomic relationships. Abstract-Many species of the marine sponge genus Discoderrnia (Lithistida. Theonellidae) are difficult to differentiate due to plasticity of their morphological features. Ethanol extracts of 26 specimens of central Atlantic Discoderrnia spp. were subjected to thin layer chromatography (Tl.C) and 'H-NMR to investigate the potential utility of these methods in providing profiles that can be used as species-specific chemotaxonomic markers. Profiles of the central Atlantic species D. dissoluta Schmidt and D. verrucosa Topsent were included for comparative purposes, The resultant biochemical profiles provided a large number of characters. and were direcdy comparable among specimens. Several specimens with identical morphological features differed in their biochemical profiles. and there were specimens which differed morphologically but had identical biochemical profiles. For the majority of specimens. however. a combination of nc patterns and 'H NMR spectra with morphological and ecological data uniquely define species groups within central Atlantic species of Discodermia. Introduction Lithistids are fossil and recent sponges which possess a siliceous spicule skeleton composed of interlocking desmas (Levi, 1973). The genus Discodermia du Bocage is distinguished within the Iithistid family Theonellidae by the presence of discotriaenes in addition to a desma skeleton, combined with a microsclere complement of micro­ oxeas and microrhabds (du Bocage, 1869). Many specimens of Discodermia that we have found in the central Atlantic are extremely difficult to differentiate into species groups due to the similarity of the desma skeletons, the plasticity of desma morphology, the uniformity of the spicule complement and dimensions, and the variability in gross morphology of the sponges. In two groups there is a seemingly perfect gradient of specimens, from those with a dense and completely interlocked desma skeleton, to those which almost completely lack desmas. We now attribute this to the maturity of the area of the sponge from where the histological section is taken; rapidly growing tubes and fingers frequently lack desmas, while massive portions of digitate sponges and massive sponges possess dense interlocked desmas up to the base of the cortex. This is a major source of confusion during initial taxonomic evaluation. Discodermia is represented in the central Atlantic by at least two valid species, D. *HBOI Contribution No. 1016. :j:Author to whom correspondence should be addressed. (Received 9 November 1993) 353 354 M. KELLY-BORGES ETAL. dissoluta Schmidt and D. verrucosa Topsent, and one questionable species, D. polydiscus du Bocage. At least five species remain undescribed in our collections. Lithistids are a promising source of bioactive metabolites. The genus Discodermia is a particularly rich source of novel biologically active compounds, examples of which include the anti-microbial peptides discodermins (Matsunaga et al, 1984, 1985a,b), reported from D. kiiensis, calyculins (Kato et el, 1986) which are potent inhibitors of protein phosphatases 1 and 2A, isolated from D. calyx (Matsunaga and Fusetani, 1991), and discodermindole (Sun and Sakemi, 1991), an anti-tumor compound reported from D. polydiscus. Discodermide (Gunasekera et el, 1991) from D. dissoluta, is a macrocyclic lactam with activity against Candida albicans. Discodermolide (Gunasekera et el, 1990) is an immunosuppressive polyhydroxylated lactone (Longley et al, 1991a,b), and polydiscamide A (Gulavita et al, 1992), from a new species of Discodermia, is active against human lung cancer A549 cells. It is thus a priority to develop rapid identification tools for grouping intractable specimens of Discodermia to expediate drug discovery and de-replicate specimens for our extraction and screening programs. This work is part of a general search for diagnostic morphological, biochemical and molecular characters with which to define species boundaries within Discodermia and other key Iithistids such as Theonel/a and Coral/istes. Several classes of secondary metabolites such as sterols (Bergquist et al, 1980, 1986; Kerr and Kelly-Borges, in press), fatty acids (Lawson et el, 1984), brominated compounds and terpenes (Bergquist and Wells, 1983), and carotenoid pigments (Lee and Gilchrist, 1985; Liaaen-Jenson et al., 1982; Hooper et el; 1992), have been employed in problems of higher systematics in sponges with varying degrees of success. Ethanol extracts, resolved into components and visualised by thin layer chromatography (TLC), have been used successfully to identify several diagnostic chemotaxonomic markers for genera within the Halichondriidae (Pomponi et el, 1991). In this study, we investigate-the utility of TLC and 'H-NMR spectroscopy of ethanol extracts, which contain mixtures of secondary metabolites, sterols, phospholipids, carbohydrates and other compounds, to provide profiles for use as species-specific chemotaxonomic markers in central Atlantic Discodermia. These profiles are combined with morphological and ecological characters of the specimens to uniquely define species groups. We have included specimens of the central Atlantic species D. dissoluta Schmidt and D. verrucosa Topsent for comparison. Full taxonomic descrip­ tions of these new species, of several additional species clearly defined without the aid of a prioribiochemical profiling, and a review of known species of Discodermia, is currently in progress. Materials and Methods Samples were collected by the authors from the Bahamas, southem Antilles, Canary Islands, and the eastem Gulf of Mexico (Table 1). All specimens were collected by trawling or by the Harbor Branch Oceanographic Institution Johnson-Sea-Link manned submersibles. Data on ecology, habitat and depth were recorded on collection, along with details of growth habit. gross and surface morphology, colour, texture and dimensions of the living sponge. After collection, sponges were stored at -20"C, and a representative portion fixed in 70% ethanol for identi­ fication. Samples of tissue. including surface and choanosomal regions, were processed histologically through a dehydrating ethanol series. cleared with Histosol (National Diaganostics Laboratory) and embedded in paraffin mssuePrep 2. Fisher SCientific). Spicules were prepared by digesting tissue in concentrated nitric acid. and low-speed centrifugation through a series of washes with distilled H20 and absolute ethanol. Rapid spicule preparations are performed in the field by digestion of small tissue samples in household bleach. Spicules and histological sections were permanently mounted (Permount, Fisher SCientific). Histology and spicule dimension analysis were conducted and results compared with groups defined a priori biochemically. Spicule measurements are given as an average length and thickness. or a range of approximately 30 spicule measurements. Desmas are irregular spicules with at least four arms (cladomes) CHEMOTAXONOMICMARKERS IN DISCODERMIA 355 TABLE 1. SAMPLES OF DISCODERMIA ANAlYZED BY TLC AND 'H-NMR. Reference groups III and VII correspond to the central Atlantic species Discodermia verrucose and D. dissoluta, respectively Sample HBOMt TLC/NMR number" Catlog No. Site:j: Depth Text§ MorphD Colorn group"- 9-X~90-1-1 851 BMS 156 C tubes, be cr-tn IA 8-XI-90-1-1 854 BMS 198 C tubes, be cr-tn IA &-XI-90-1-1 855 BMS 185 C tubes, be cr-tn IA &-XI-90-1-2 856 BMS 178 C tubes, be cr-tn IA &-XI-90-1-4 857 BMS 183 C tubes, be cr-tn IA 18-111-87-3-1 858 BMS 157 C tubes, be cr-tn IA 2-IV-89-3-3 859 GRN "1 C tubes, bc cr-m 22-V~89-2- 18 850 GUO 126 C d branch cr-tn IB 23-VI-89-3-13 861 GUD 89 C d branch cr-tn IB 7-X-88-2-2 862 BMS 40 H thk encr rd/cr 24-VIII-85-1-3 863 BMS 232 H sph knb cr-tn II 2S-VII~S-3-3 864 BMS 146 H sph knb cr-tn II 15-VII-92-2-12 865 GOM 61 H smlfingr cr-tn II 17-VII-92-2-29 866 GOM 61 H smlfingr cr-tn II 8-Vl-91-1-2 867 CAN 303 H mush knb cr-m III 2-Vl-91-1-12 868 SAL 321 H mush knb cr-tn III 31-V-91-1-6 869 MAD 477 H mush knb cr-tn III 31-V-91-3-3 870 MAD 497 H mush knb cr-tn III 24-XI-92-1-11 871 BMS 200 H st ir cp tn IV 1&-XII'92-1-3 872 BMS 158 H st ir cp tn IV 27-XI-92-1-12 873 BMS 170 H st ir cp tn IV 29-XI-92-1-7 874 BMS 169 H st pi wh V 1S-XI~92-3-3 875 BMS 173 H sml st cp tn Vl 21-111-87-3-14 876 BMS 30 S mllob bl-rd/cr VII 1-XII-92-2-1 877 BMS 34 S mllob bl-rd/cr VlI 13-XII-92-2-12 878 BMS 30 S mllob bi-rd/or 'The components of the sample number are Day-Momh-Year-Dive number-Specimen number.