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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. t A voucher specimen has been deposited at the Harbor Branch Oceanographic Museum; Fort Pierce, Florida. Catalog number 003: OOXXX. (XXX= number listed in Table t), - :j:BMS=Gouldings (24·59·N, 77"31'W) and Sweetings Gay (26"32'N, Tr53Wl, Bahamas; GRN=Grenadines, Antilles (12"34'N, 61·13'W); GUO= Guadaloupe, Antilles (16"28'N, 61"34'W); GOM = eastem Gulf of Mexico (25"07'N, 83"37'W); MAO = Madeira Archipelago, eastem Atlantic (32'30'N, 16"34"W); CAN = Canary Islands, eastem Atlantic (28"11'N, 16"SO"W); SAl-Salvage Islands. eastem Atlantic (3O"07'N,1S52'W). §Texture of the sponges reflects the density of spicules, and rigidity of the intemal desma skeleton: S = soft, with generally fine spicules, desmas are not interlocked; C= compressible, with imerlocked fine desmas and a reduced density of spicules; H= hard, with a heavy articulation of desmas, dense spicules, producing a rock-like texture. IMorphology of the sponge in life: tubes, be = basally coalescent lobe-like or elongate tubes; d branch = long slender divaricating branches; Sml fingr = small fingers; thk encr = thick encrusting; sph knb = spherical knob, mush knb = small mush­ room-shaped knob with tuberculate upper surface; st ir cp = stalked cup with irregular edge, st pi = stalked plate; sml st cp = small regular stalked cup; ml lob = thickly encrusting multi-lobate. 1cr-tn=Cream to tan; tn=tan; rdlcr=reddish brown exterior, cream intemal; bl-rd/cr=blackish red exterior, cream interior; wh = white. '*TLC and 'H-NMR profiles have been grouped (I-Vlll on the basis of identity or close similarity, as presented in Figs 1 and 2.

radiating from a central rod, The end of each c1adome (the zygome) can be digitate or nodulose. usually branched, and is commonly articulated (zygosed) with the zygomes of adjacent desmas. An ethanol extract of each specimen was prepared as follows: ~10 g of sponge tissue were homogenized with ethanol. The extracts were filtered, evaporated and weighed. The ethanol extracts were made up to a known concentration with dichloromethane-10% methanol. Ethanol extracts (160 I1g) of each sample were chromatographed on a thin layer plate (Whatman Si02• 250 urn thick) using three different organic solvent systems which differed in polarity. The least polar solvent system contained ethyl acetate-heptane (1:1). Dichloromethane-ethanol (9:1) and n-butanol-acetic acid-water (6:3:1) separated medium polarity and most polar compounds, respectively. After chromatography the plates were air-dried, sprayed with 5% vanillin­

sulphuric acid reagent heat-activated, and photographed. ' H-NMR ethanol extracts were obtained in CDCI3­ CD30D (9:1) using a Bruker AM-360 spectrometer operating at 360.13 MHz. Chemical shifts were referenced to CD30D signal at 3.30 ppm. Thin layer chromatography and 'H-NMR profiles were compiled and compared between specimens, Specimens were grouped according to profile similarity. 356 M. KELLY-BORGES ET AI.

Results Ethanol extracts of 26 sponge specimens (Table 1) were subjected to TLC and 1H-NMR analysis (Figs 1, 2). These specimens were identified in the field as species of Discodermia, based upon the presence of characteristic ectodermal discotriaenes (du Socage, 1869; van Soest and Stentoft, 1988), visible under the light microscope in spicule preparations made from bleached tissue.

IA ? m ? n ill IV V VI vn ? ~ ee t!> ~~ ~ ~~ ~ e!>~ ~ ~~ l!!l~~ ~ 11 n 11 u 11 11 ,~e~, 0 ~ II II 11 - - g 6, 0 - C' Q 0 00 c. ~'~ 00 a 0 '-'" ~' ~. @,~: t::/' .A ~ eO - - ....-- ~, - - Q,Cb t:\> 0' -- -- 00 - 0 BG ,:0 c co ~ ... c:> 9'~. ,.. 8. 0 0 0 - o 0 ------""

";> or, ~ .., ~ ~ ::! ,;, ... - ': ~ ,;, N ~ "r ~ .., N ,;, .;, .;, N ~ ! N ..N ... N ... N N ...... c .. llC llC ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ ~ "" aC ;: ; ~ ~ ;.:.. '" '" ao ;> ;> ;> >< >< >< >< >< >< >< >< >< >< >< '" :: ::,;, loC .;, ;:: ;:: J,. ~ oi: ,.; ,.; ,.; ~ N N ,.:. .. ~ oi: N .. ~ ~ =i :i '" ** ......

A. n-Butanol : acetic acid: water (6:3:1)

I D m IV .V VI DmIV VII? ~ ~ el~~ ~ 0 , e Q)~l• °0Oc:::;) CDQ)~ CD CD ,. o'G:) c::lo' 0 "0 OOm & 8' 0 <::::) 0 Q. ~ - ::::- ':$ "0 -- 8~- , 00 c::. - <::> e::1 .:::. --c:> - c:> c:::. .. - e: A ~ - co 0 @, -= {lJ ® "" .- ~l ~ 0 a -"" P<- .., ,.., ::= ~ ::= ,.., .. ,;, N :z r:- ,.:, N .;, ..:. or. 1 N N N 1 N N ..:. N ... N ..:...... ~ ~ ~ ~ ::! ~ ~ ~ ~ ; ! ! ~ ~ ..~ ; s x ;< >C ;> ~ >C >< .;, ;> >< ><,.:. >< >< .;, ;:: ~ aC ... oi: .. ~ ... ..0- =i ~ ~ aC .. .c ..... :i

B. Dichloromethane : methanol (9:1) C. Ethyl acetate: heptane (1:1)

AG. 1. THIN LAYER CHROMATOGRAPHY (l1.C) PROALES FOR SPECIMENS OF DISCODERMIA. CHROMATOGRAPHED IN THREE DIFFERENT SOLVENT SYSTEMS. AND GROUPEDACCORDING TO OVERALL SIMILARIlY. Ou1Iines of TLC spots were traced from the original TLC plates. and colors of the resolved spots noted: 1 :. blue. 2 - gray green. 3 =magentl, 4 =orange, 5 =purple. 6 - brown, 7 = gray. 8 =charcoal gray, 9 - veHow. 10=lilac, 11=brown purple, 12- gray blue, 13- brown green. 14- pale blue. 15=green blue, 16=lime green. Arty spo1that has not been labeled is pale purple gray. Question marks relate to specific problematic specimens. which lire discussed in the text. Groups !-VlI correspond directly to 'H-NMR spectra in Fig. 2. Locality and morphological data for each sample is listed in Table 1. Reference groups III and VII correspond to the central Atlantic species Discodermiaverrucosa and D. dissoluta, respectively. (AI n-butllno!-acetic acid-water (6:3:1). the most polar solvent system; (B) dichloromethane-methanoI19:1). medium polar solvent system; (C) ethyl acetate-heptane 11:1). the least polar solvent system. CHEMOTAXONOMICMARKERS IN DISCODERMIA 357

VI

15-VJII·lI5-3-3 5.09 a.oo 7.00 ppm 6."

II>- VII·n·'·I' VII

8.08 7.00 6.00 5.00 ppm ,lll~ .1 L.t I3-XII-n-H2

LLJI-XU-92·Z·1

21·111-17·3-14

.... 7." 6.00 5." ppm

IV

1.00 Ut 5.10 7." ppm

II>-XII-92-1-3 18 LtO 7." ppm ,." 5." 7·X·...'·Z ~ I-VJ·'I·I·Z ~

7." 6.00 5." a.to 7.00 6.00 I." ppIII ppm 5."

FIG. 2. 'H-NMR PROFILES OF CRUDE EXTRACTS OF SPECIMENS OF DISCODERMIA SPP. FROM A SELECTED CHEMICAL SHIFT REGIONOF 5-8 PPM. Profiles have been grouped according to overall similarity. Groups !-VlI correspond directly to thin layer chromatography profiles in Fig. 1. Question marks relate to specific problematic specimens. which are discussed in the text. Locality and morphological data for each sample are listed in Table t, Reference groups III and VII correspond to the central Atlantic species Discodermia verrucose and D. dissoluta. respectively. 358 M. KELLY-BORGES ET AL

A range of TLC solvent systems that would resolve compounds of differing polarity were employed to assess their individual utility in the separation of specimens into groups (Figs lA, S, C). Absolute Retention Factor (Rf ) values are not considered here; only the relative location of the spots in the overall profile, and the development of various colours during heat treatment are considered for comparative taxonomic purposes. Of the three TLC solvent systems, those that resolve more polar compounds, n-butanol-acetic acid-water (6:3:1) (Fig. lA) and dichloromethane­ methanol (9:1) (Fig. 1B), were successful in resolving the greatest number of compounds over the widest range of specimens. The system that resolves least polar compounds, ethyl acetate-heptane (1 :1), provided pattems in which there were less spots, many of which had similar relative positions and colours (Fig. 1C). Only a selected chemical shift region of 5-8 ppm of the 1H-NMR spectra was used for comparison between specimens, as this region contains well-separated signals in all extracts selected for this study. The chemical shift region 0-5 ppm contained interesting signals, however, overcrowding of signals obscured information. The presence or absence of a signal and the size ofthe signal in the 1H-NMR spectrum are functions of the availability of the particular compound in the extract analysed. Discodermide (Gunasekera et al, .1991), discodermolide (Gunasekera et el, 1990) and polydiscamide A (Gulavita et el, 1992) are compounds unique to Discodermia, and thus potentially provide chemotaxonomic markers for the genus. The presence or absence of these compounds in the specimens (Table 2) examined is discussed below. Thin layer chromatography pattems and 1H-NMR spectra were grouped according to overall similarity of relative spot position and colour (Fig. 1), and 1H-NMR signal location (Fig. 2), respectively. Seven groups could be distinguished using the three

TABLE2. DISTRIBUTION OF DISCODERMIDE (Gunasekera et el, 1991).DISCODERMOUDE (Gunasekera et el, 1990) AND POLYDISCAMIDE A (Gulavita et al. 1992) IN SPECIMENS OF DISCODERMIA SPP., AS DETERMINED BY TLC. Specimens are divided into groups !-VlI, which correspond to TLC and 'H-NMR profiles. morphological and ecological data, Referencegroups III and VII correspond to the central Atlantic species Discodermia verrucosa and D. dissoluta. respectively.

Sample number Discodermide Discoderrnolide Polydiscamide A

9-XI-0001-1 IA .; 8-XI·0001-1 IA .; .; &-XI-0001-1 IA .; .; &-XI-0001-2 IA .; .; &-XI·0001-4 IA .; - 18-111-87-3-1 IA .; .; 2-1V-89-3-3 IA 22-VI-89-2-18 18 .; 23-Vl-89-3-13 18 .; 7-X-88-2-2 18 .; 1&-VlI-S2-2-12 II 17-VII-S2-2-29 II 24-VIII-8S-1-3 II 25-VIII-8S-3-3 II 8-Vl-S1-1-2 III 2-VI-S1-1-12 III 31-V-S1-1-6 III 31-V-S1-3-3 III 24-XI-S2·1-11 IV .; .; 1&-XII-S2-1-3 IV .; 27-XI·S2-1-12 IV .; .; 29-XI-S2-1-7 V .; 15-XII-S2-3-3 VI 21-111-87-3-14 VII .; .; 1-XII-S2-2-1 VII .; .; CHEMOTAXONOMIC MARKERS IN DISCODERMIA 359

TLC solvent systems and in the 'H-NMR spectra. Groups I-VII are strongly supported by unique TLC patterns and ' H-NMR spectra, and the groupings of specimens are confirmed with morphological and ecological data.

Group I Chemistry. The TLC profiles of specimens in group IA display distinctive brown purple spots in the upper portion of the plate, with a ladder of pale purple gray bands extending below the primary compound (Fig. 1A). 'H-NMR spectra for these specimens are identical with five major peaks which do not occur in any other specimens except Discodermia dissolute (group VII) (Fig. 2). This group of specimens is characterised by the presence of discodermide and discodermolide, although only trace amounts of the latter are reported for some specimens (Table 2). General description. The sponge consists of several marble white smooth tapered tubes, up to 15 cm high. Sponges have a fleshy surface and are compressible. These sponges are found in the Bahamas, and collected from depths of 150-200 m (Table 1). Arrangement of the skeleton. The skeleton consists of many radiating tracts of oxeas (561 x 7 urn), Desmas (450x 400 urn) have smooth c1adomes (25-35 urn thick), zygomes are dendritic with numerous finger-like projections. Development of the desma skeleton in these specimens is quite variable; in some specimens the desmas are simply scattered in the choanosome while in others the desmas are abundant and fully zygosed. All other characteristics of the skeleton remain constant. The cortex (200-500 urn deep) has numerous subdermal spaces, an outer layer of extremely dense microrhabds (15 X 2 urn), beneath which are rnicro-oxea (51 X 3 urn) in abundance. Discotriaenes with cup-shaped discs (159 urn diameter) are abundant in the ectoderm. Remarks. A group of specimens from the southern Antilles (IB) differs only slightly morphologically from group IA specimens in the Bahamas, but have quite different TLC and 'H-NMR profiles. Group IB specimens are ramose with slender tapered branches and are slightly less compressible than group IA specimens. The sponges lack a central hollow axis and oscules are scattered along the sides of the branches. Histologically the two groups are inseparable. The TLC profiles of group IB specimens are quite different from those of group lA, the overal profile of the former indicating the presence of compounds of differing relative position. The 11.C profiles of group IB possess in addition to the brown-purple primary compound, a single bright purple spot lower on the plate (Fig. IA). The 'H-NMR profiles of group IB contain a group of indistinct signals which indicate the presence of polydiscamide A (Fig. 2). This compound is absent in group IA (Table 2). Discodermide and discodermolide which are present in group IA, are absent in group IB (Table 2). It is difficult to determine the cause, if any, of this divergence in biochemical constituents between these two groups of very similar sponges. Some variation in the production and types of secondary metabolites can be attributed to biological and physical factors such as the presence of symbionts (Faulkner and Unson, in press), depth, and geographic locality (Sennett et el, 1992). The sponges are geographically isolated, and it is likely that they experience different local physical and biological environmental conditions. These sponges may contain different undetected symbionts. It is well known that the products of biological interaction between the host sponge and its endosymbionts are frequently not readily distinguished by crude chemical isolation. On the other hand there may be no environmental explanation; Kerr and Kelly-Borges (in press) show that consistent sterol differences between groups of the Caribbean reef sponge Xestospongia mum are not correlated with geographic locality, depth, morphology or symbionts. Two specimens have been included here to further illustrate this phenomenon. 360 M. KELLY-BORGES ETAi.

Specimen 2-IV-89-3-3 is extremely similar morphologically to group IA specimens but its TLC profile lacks the characteristic brown purple spot 11, and the ladder of compounds below this (Figs 1A, 2). This specimen contains instead, a primary magenta spot (spot 3-Fig. lA). This sponge lacks the four characteristic 'H-NMR peaks and lacks all of the known compounds in Table 2. Although specimen 7-X-88­ 2-2 has TLC patterns and an 'H-NMR spectrum identical to specimens in group 1B (Figs 1A, 2; Table 2), it is completely different morphologically. This specimen was collected from a depth of 40 m, it is thickly encrusting with a ridged surface, and has a dark reddish exterior. The megascleres are much larger, and micro-oxea and micro­ rhabds are considerably smaller than those of specimens in group 1B.The desmas of 7-X-88-2-2 are more robust, and the sponge has a stony interlocked desma skeleton.

Group II Chemistry. The TLC and 'H-NMR profiles of this group of sponges are different from those of other Discodermia (Figs 1A, 2). The TLC contains a primary magenta spot (spot 3) and several other well spaced compounds. The 'H-NMR contains two major peaks and several minor ones. These specimens lack the three known compounds in Table 2. General description. The sponge forms short slender fingers with tapered or flared branching tips and is f1esh-eoloured in life with a veneer of deep violet red on the surface. The sponge is barely compressible, the surface granular. These sponges were collected in the Gulf of Mexico from a depth of 61 m. Arrangement ofthe skeleton. Short curved oxeas (392x 8 urn) form loose irregular tracts which radiate towards the surface of the sponge. Desmas (760X 400 urn), have smooth ciadomes (20-55 urn thick), zygomes are sinuous and unbranched with occasional finger-like projections. In less mature regions of the sponge such as the growing tips, desmas are almost absent and oxeas are abundant. A narrow cortex (20-50 !J.m deep) is packed with unusually thick smooth microscieres (micro-oxea: 36X7 urn: microrhabds: 17X4 urnl. Perfectly round discotriaenes (disc: 169 urn diameter) are abundant in the outer cortex. Remarks. This group of specimens is differentiated from group I in the generally smaller size of the sponge, and simple occasionally branched morphology. Group II specimens have very large simple desmas, small robust oxea, and unusual centrally thickened, lentil-shaped microscleres. We found several specimens which have very similar biochemical profiles to the Gulf of Mexico sponges, yet differed morphologically from them. Specimens 24-VIII­ 85-1-3 and 25-VIII-85-3-3 were collected from the Bahamas and are deep-water spherical sponges with a very robust, stoney skeleton. The implication is that these sponges either have the same biosynthetic capabilities, or that they contain similar symbionts that produce these compounds. This demonstrates the need to exercise caution in interpretation of such results; specimens are not necessarily conspecific if they have the same ethanol extract profiles.

Group III-Discodermia verrucosa Topsent Chemistry. Discodermia verrucosa has a very distinct TLC profile which contains closely spaced gray green (spot 2), magenta (spot 3), gray (spot 7) and yellow (spot 9) spots in the upper portion of the plate (Fig. 1A). The 'H-NMR spectra of this species is distinct from those of other species shown (Fig. 2). These sponges lack the known compounds described in Table 2. General description. Sponges are fungiform or globose (1-5 cm diameter). Oscules are raised on small tubercules on the upper surface of the sponge. These sponges are cream-eoloured in life, are very stoney, and were collected from the Canary Islands from depths of 300-500 m (Table 1). CHEMOTAXONOMIC MARKERS IN DISCODERMIA 36'

Arrangement ofthe skeleton. The skeleton consists of an extremely dense reticula­ tion of desmas. Desmas are robust (495X400 11m), the c1adomes (50-75 11m) have annular rings, and the zygomes are covered with single or double fungiform nodules. Long subtylostyles (991 x 4 11m) radiate through the outer region of the sponge in vague bundles. The cortex (150-350 11m deep) is crowded with microscleres (micro­ oxea: 42 X 4 11m; microrhabds: 15X 4 11m), microrhabds almost obscuring the choanosomal skeleton. The thick discs of huge discotriaenes overlap on the sponge surface, the discs (391 11m diameter) are indented as a result. Remarks. Discodennia verrucosa is distinct from other known species of Disco­ dermia in aspects of gross morphology, desma morphology, monaxonal megasclere morphology and dimensions.

GroupN Chemistry. The TLC profiles of group IV specimens are similar to those of Disco­ dermia verrucosa but contain a major purple spot (spot 5) above the gray spot 7, and lack spots 9 and 2 typical of D. verrucosa (Fig. 1A). 'H-NMR spectra show the characteristic signal of polydiscamide A, present also in the spectra of group IB specimens, with additional minor signals (Fig. 2). This group of specimens uniquely combines discodermolide and polydiscamide A (Table 2). General description. Irregular, cream, shallow cup-shaped sponges (5-10 cm diameter, 3-5 cm high) with an irregular wavy margin. The exterior is generally smooth, the interior and margin slightly furry. These sponges are incompressible but not stoney, and were collected in the Bahamas between 150and 200 m. Arrangement of the skeleton. The skeleton is a dense reticulation of desmas (490X 300 11m). The c1adomes are smooth (30-45 11m), the zygomes simple with low irregular nodules. Long subtylostyles (1034X4 11m) are scattered in loose bunches predominantly in the outer region of the sponge. The cortex (100-400 11m deep) has many subdermal spaces and micro-oxea (85X 311m) are common in this region. Microrhabds (15 X 311m) line the surface. Discotriaenes are common, discs (179 urn diameter) are thin, flat and occasionally indented. Remarks. This group of specimens is distinct in morphology and contain long projecting subtylostyles on the interior surface of the cup.

Group V Chemistry. The TLC profile of group V, represented by specimen 29-XI-92-1-7, is distinct from specimens in group IV and others, with a lilac spot (spot 10)inset within a very large magenta spot (spot 3). A large gray spot (spot 7) is only just separated above this group (Fig. 1A). The 'H-NMR spectra of this specimen is less easy to distinguish from group IV spectra as it also contains the characteristic polydiscamide A signals (Fig. 2). However, close examination of the group V spectrum reveals the presence of signals that are absent in group IV spectra. General description. The sponge is a wheat-eoloured, slightly cup-shaped, stalked plate (12 cm diameter, 8 ern high, stalk 4-5 cm diameter). The outer margin is thin and the base and central portion of sponge is thick. The sponge surface is irregularly pitted, the texture is slightly compressible, and the surface slightly fleshy. The sponge was collected from the Bahamas at 169 m. Arrangement of the skeleton. The skeleton is cavernous with numerous canals permeating the desma reticulation. Desma are large (520X400 11m), the cladomes smooth (30-40 11m) and zygomes branched, branches curved with short rounded knobs projecting along the length of the convex side of the branch. Parallel tracts of thick oxeas (647X 9 urn) spread outwards to the margin and upper surfaces of the plate. The cortex (100-200 11m deep) has a sparse outer layer of microrhabds (62X 4 362 M. KEllY-BORGES ET AL urn) and micro-oxea (15 X 3 urn), Discotriaenes have perfectly spherical thin discs (140 urn in diameter). Remarks. Group V is distinguished from group IV specimens in being plate-shaped with an unusual cavernous skeleton construction.

Group VI Chemistry. The TLC profile of group VI, represented by specimen 15-XII-92-3-3, is very distinctive with a single bright orange spot (spot 4) at the top of the plate (Fig. 1A). The 'H-NMR spectra is uncharacteristic of Discodermia and contains a major signal between 7.1 and 7.2 ppm (Fig. 2). At least two species of unidentified chain­ forming cyanobacteria are clearly visible within the tissue of all specimens examined. It is possible that this unusual compound or group of compounds seen in the TLC and 'H-NMR spectra are from the cyanobacterial symbionts extracted with the sponge. No other specimens examined contained visible symbionts. Group VI specimens lack the known compounds in Table 2. General description. The sponges are small and cup-shaped with a regular circular margin (3-5 cm diameter, 3 cm high). The sponge is greenish-eream alive and is barely compressible, the lower surface rough and the upper surface slightly fleshy. These sponges were collected from the Bahamas at 173 m depth. Arrangement of the skeleton. The skeleton is a dense reticulation of desmas (500X 300 11m). Cladomes are smooth (30-40 11m thick), zygomes sinuous and unbranched with occasional slim finger-like projections. Strongyles (842X.5 urn), radiate through the sponge in loose tracts. The cortex (200-000 11m) has numerous subdermal spaces and microscleres are abundant and relatively smooth (micro-oxea: 58 X 311m; microrhabds: 24 X 3 urn), Discotriaenes are thin and flat with irregular margins (disc: 180 11m diameter). The outer sponge tissue is packed with unidentified chain-forming cyanobacteria. Remarks. In addition to being distinct biochemically, specimens of group VI are differentiated from cup-shaped sponges in group IV, in the possession of strongyles instead of subtylostyles, and in the morphology and dimension of the microscleres.

Group VlI-Discodermia dissoluta Schmidt Chemistry. TLC profiles of Discodermia dissoluta possess two distinct blue (spot 1) spots separated by a gray green spot (spot 2) in the upper portion of the plate (Fig. IA). The 'H-NMR spectra are similar to those of group IA specimens, but this is not surprising as the two groups both contain discodermide and discodermolide (Table 2). General description. The sponge consists of multiple lobes that thickly encrust the substrate (50 cm diameter, up to 7 cm thick. each lobe 1-4 cm diameter). The sponge is soft and compressible, the surface very smooth and fleshy, the colour of the upper surface in life is deep reddish black, the non-pigmented region and interior is wheat yellow. The sponge is found on relatively shallow reefs (30 m) in the Caribbean. Arrangement of the skeleton. The skeleton consists of abundant sinuous radiating tracts of fine oxea (462X 5 11m) with dense interstitial megascleres. Desmas (493X 280 urn) with smooth cladomes (15-20 urn) and dendritic zygomes with short rounded projections, are absent from the outer sponge tissue but are common below this region where they occur generally as single spicules. Microscleres (micro-oxea: 83 X 111m; microrhabds: 19X 1 !1m) are common in the cortex (1~300 urn deep). Discotriaenes are rare or absent (disc: 98 urn diameter). Remarks. Discodermia dissoluta is distinctive in habitat, colouration, almost lacks discotriaenes which are diagnostic for the genus, and the desmas are fine and frequently only scattered in the sponge tissue. The skeletal arrangement of narrow regularly spaced tracts, the lack of zygosed CHEMOTAXONOMIC MARKERS IN DISCODERMIA 363 desmas in some specimens, and the presence of discodermide and discodermolide in this group, is reminiscent of group IA specimens. Although the two groups are obviously closely related, they are distinct ecologically and morphologically. The sample 13-XII-92-2-1 has a TLC profile that differs significantly from other Discodermia dissotuts, containing three spots of magenta (spot 3), a single brown spot (spot 6) and a single gray blue spot (spot 12). The 'H-NMR spectrum of 13-XII-92­ 2-1 differs from Discodermia dissoluta in the absence of the three characteristic low field signals (Fig. 2). There are additional compounds in 13-XII-92-2-1 which bear signals around 7.2 and 7.6 ppm. However, this specimen is inseparable from other members of D. dissolute, in morphology, colouration, histology, skeletal arrangement, spicule shape and dimensions, and ecology.

Discussion Thin layer chromatography can be rapidly performed in the laboratory or under field conditions, and can be used successfully as a first approximation to grouping samples in genera where rapid taxonomic techniques such as examination of spicule comple­ ments and gross morphology provides ambivalent information on potential group­ ings. Solvent systems that separate the most polar compounds, such as n-butanol­ acetic acid-water (6:3:1) and dlchloromethane-rnethanol (9:1) have the greatest resolving power for comparative taxonomy of Discodermia. Thin layer chromatography patterns are directly comparable between specimens, however, shifts in the relative position of the components can reduce confidence in the assignment of profile groups. In some cases it is difficult to determine whether two spots that have a similar colour, but slightly different positions on the TLC plate, represent the same compound or different compounds. This is illustrated in the slightly different shapes and positions of spot 5 in group IB, spots 2 and 3 in group III and spots 5 and 7 in group IV (Fig. 1A). 'H-NMR spectra of the specimens in these groups are very closely comparable, and morphological data for each specimen confirms that these specimens belong within the groups assigned. There is, indeed, variation in the relative positions of the spots between certain compounds in these particular specimens. The relative position and the colour of the spot depends on many factors including concentration of the compound, nature of the remaining compounds in the mixture, overlapping spots, and the extent of heating of the TLC plate. Different classes of compounds may also have spots of the same colour. For the detection of known compounds, it is important that uniform concentrations are maintained and a pure compound standard is spotted with the samples as a control. 'H-NMR analysis of ethanol extracts has proved a surprisingly powerful tool for the differentiation of species groups within Discodermia. 'H-NMR spectra are more easily comparable than TLC patterns, and the process is less subjective; signals are easier to detect than spots that can fade and occasionally shift. A slight difference in the chemical shifts of 'H-NMR signals between specimen extracts usually correlates well with differences in specimen morphology. This is illustrated in the distinction of group V from group IV. While the presence or absence of discodermide, discodermolide and polydisca­ mide A cannot be used exclusively to group specimens into meaningful groups, the presence or absence of these compounds in specimens does provide additional data to be used in conjunction with ethanol extract profiles, morphology and geographic data to confirm species groups. No one compound is uniquely group-specific: discodermide and discodermolide are present within most specimens of group IA and VII, polydiscamide A is present in group IB, IV and V, and discodermolide is present in most specimens of groups IA, IV and VII. However, group IV uniquely combines discodermolide with polydiscamide A, and group IB contains only polydiscamide A. Ethanol extracts may be limited as taxonomic characters because of variations in 364 M. KELlY·BORGES ET AL their production due to biological and physical environmental factors (Bergquist and Wells, 1983). Many sponges contain cyanobacteria, bacteria (Sara, 1971; Wilkinson, 1980), and algal symbionts (Sara and Vacelet, 1973), and the products of their biological interaction with the host sponge are frequently not readily distinguished by crude chemical isolation. Recently, however, Faulkner and Unson (in press) employed flow cytometry to separate symbiotic cyanobacteria from cells of the sponge Dysidea herbacea, and were able to localise polychlorinated metabolites within the cyano­ bacterial cells, and sesquiterpenes within the sponge cells. Even if secondary metabolites are of microbial origin these compounds may still be of use in chemo­ taxonomy since microbial symbionts may be species-specific within sponges. Battershill (in press) has shown that small changes in a sponge's interaction with adjacent or symbiotic organisms can alter temporally and spatially the secondary metabolites of the individual sponge. Featuresof the physical environment such as geographic locality, depth and habitat also influence secondary metabolite chemistry. In the Bahamas, the halichondrid sponge Myrrnekioderrna styx is composed of two populations that differ in their diterpene metabolites, and these differences are directly related to depth (Sennett et et, 1992). Reciprocal transplants of the Australian Great Barrier Reef sponge Rhopaleoides odorsbile, which also displays depth-dependent chemistry, have shown that individual sponges transplanted to depths at which they do not naturally occur, will have altered chemistry appropriate for individuals occurring naturally at that depth (Thompson etal, 1987). We have also shown that two relatively simple methods using ethanol extracts of sponge tissue can greatly assist in taxonomic differentiation of specimens of Discoderrnia when considered in conjunction with morphological and ecological characters. Specimens of Discoderrnia analyzed here represent a range of localities, depths, time of collection, and ecological conditions. It is remarkable that these data are generally consistent between groups of specimens. For groupings in which morphological, geographical or chemical characters do not support a distinct separation into species additional data may be required from the application of molecular techniques such as nucleic acid sequencing and determination of the actual biochemical differences between specimens. It will be of value to continue to explore the utility of different methodologies and additional classes of compounds for their use in species-level discrimination in Discodermia and in other groups of difficult . sponge genera.

Acknowledgements-We thank the crews of the R.V. Sea Diver; R.V.SewardJohnson and Johnson-Sea-Link I for their assistance in collection of sponge samples. We are grateful for the assistance of Klaus Kelly-Borges and Gail Samples for retrieval and preparation of specimens for histology, Patricia Linley for proof-reading the manuscript, and for the particularly good advice of one anonymous reviewer.

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