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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 Xestospongia muta (petrosida: ). In R. W. M. van Soest, T. M. G. van Kempen, & J. C. Braekman (Eds.), 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 . 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 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), 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 Puerto Rico (La Parguerra) ribbon 30 here, as this is not required for the purposes ofthis form ofchemotaxonomy. The identities ofall 14 sterols has

Table 2 Relative amounts of sterol chemotypes found within Florida and Caribbean sites examined. Chemotype

Location Reef Type Depth (m) A(%) B(%) C(%) n

Long Key (Florida) patch 10 85 0 15 20 Long Key (Florida) patch 19 100 0 0 11 Key Largo (Florida) ribbon 12 40 24 36 15 Boca Raton (Florida) ribbon 24 30 30 40 10 Puerto Rico (La Parguera) ribbon 30 44 22 33 20

66 9.71

5 10 15

25.00 - ---,---

20.00 - ----f---

1------15.00 -

10.00 f----- I

~ 5.00 II 0.00 I --- I --~-f=~ +- 2 3 4 5 6 r.7 8 9 10 11 12 13 14 ------IIj

Figure I. A "sterol fingerprint" of Xestospongia muta. been reported (Kerretal., 1991). To assign chemotypes, a rapid way to obtain detailed comparable fingerprints individual bar graphs are simply compared with each of sponge individuals. The relative amounts of the other. The error associated with each data point on the three chemotypes found at various sites are summarized bar graphs is approximately 2-3 0/0, and thus there are below in Table 2. There are roughly equal amounts of clearly three distinct sterol compositions amongst the three chemotypes at the three ribbon reefs, whereas individuals of X. mula (Fig. 2). This method represents at both patch reefs, chemotype A predominates.

67 TYPE A

25.00

20.00

15.00 - 10.00 - 5.00 - 0.00 I I •2 3 4 5 6 7 •8 9 10 11 12 13 14

TYPE B

50 .00

40 .60 30 .00 I 20.00 !

10.00 • I I I I 0.00 • - • 2 3 4 5 6 7 8 9 10 11 12 13 14 'I TYPE C

30.00

25.00

20.00 - 15.00

10.00

5.00 I I 0.00 • • I • I I 2 3 4 5 6 7 8 9 10 11 12 13 14

Figure 2. Sterol fingerprints of chemotypes A, B, and C of Xestospongia muta.

68 Table 3. Biochemical and morphological features of 20 projections. The surface in between is irregularly multi­ indivduals of Xestospongia muta, at the Florida Keys study conulose, somewhattuberculate, with scattereddepres­ site (10 m). Sesoft, compressible and brittle; Hehard, sions (Figs. 5, 6). In the third morphotype, lamellae are incompressible, slightly elastic (see text for explanation); absent, the surface is irregularly conulose and tuber­ Morphotype 1=Iamellate, with pronounced smooth flanges culate, with scattered depressions. There may be extending from base to apex of sponge, surface smooth, development of ridges up to 1em high at the base of the figure 3,4; Morphotype 2=lamello-digitate, irregularridges, sponge (Figs. 7, 8). nodulose ridges, nodulose or sharply digitate projections, surface very irregularly multi-conulose, tuberculate, with Morphotypes assigned to sponges at the Long Key depressions, figure 5,6; Morphotype 3=lamellaeare absent, site are listed in Table 3. Morphotype 1 is unconunon, surface very irregularly conulose, tuberculate, with scattered only one out of the 20 individuals could be assigned to depressions, figure 7,8. this category. In contrast, morphotypes 2 and 3 are more common, with 6 out of 20 , and 13 out of 20 sponges respectively, being assigned to these categories. Sample # Morphotype Texture Chemotype The morphotypes were dispersed apparently randomly 16 I S A within the population sampled, and occurred in close 1 2 H A proximity to each other. 2 2 H C The choanosomal skeletal arrangement of 3 2 H A Xestospongia muta is an irregular reticulation of 9 2 H A relatively compressed spicule tracts, 250 - 750 mm in 10 2 H A diameter, forming rounded to slightly oblong meshes, 14 2 H A 4 3 S C 120 - 200 mm in diameter. Primary and secondary 5 3 H A spicule tracts are indistinguishable, and there is no 6 3 H C evidence of spongin deposition around spicules in the 7 3 H A tracts. Interstitial spicules are abundant and confused 8 3 H A in orientation, orcanbe aligned loosely with tracts. The ]1 3 H A ectosomal skeleton is an isotropic reticulation of single 12 3 H A spicule tracts, that renders the surface microscopically 15 3 H A 17 3 H A smooth. Older ectosomal skeletons are evident as 18 3 S A concentric growth rings within the external 1- 2 em of 19 3 H A thesponge tissue, particularly inrapidly growing regions 20 3 H A of the sponge, such as the apical lip, or regions of healing. This is a typical feature ofgrowth in petrosid and oceanapid sponges (see Fromont, 1991). Soft 3.2 Morphological variation specimens of Xestospongia mula (Table 3) differ from hard specimens, in the reduction of the number of Xestospongia muta is persistently cup-shaped, but interstitial spicules, and in the reduced thickness of the assumes a variety of external forms. The sponge can be spicule tracts. Megascleres are hastate oxeas of a single barrel or volcano-shaped with a broad apical vent, erect size, usually gently curved, often slightly irregular tubular, and occasionally, almost thickly encrusting. with abrupt distal and proximal curves, and occasionally The later morphology is more common on reefs less straight. The spicules are occasionally modified to than 10m deep, which experience high physical im­ styles and strongyles, and some are extremely thin pact, but has also been seen on deep reefs. The rim of (Table 4). the apical vent is either sharp and scalloped, or thick and uneven. Sponges are typically solitary, but can fuse with adjacent sponges, each retaining their apical 4 DISCUSSION vent. Features of the sponge surface and texture also vary; surface morphologies range from relatively The various body morphologies ofXestospongia muta smooth to tuberculate with digitate or flange-like in the Caribbean do not seem to be correlated with projections, and sponges are soft or hard. depth, micro-habitat, and geographic locality. The We currently recognise three morphotypes which complete range of morphotypes described here have vary in the degree of development of their vertical been observed within a single patch reefpopulation in lamella, and surface detail between the lamellae. The the Long Keys site, and in all otherlocations in Florida, first morphotype is lamellate, with closely spaced, Puerto Rico, Bahamas, and Honduras (personal pronounced, smoothflanges, 7-10 em deep, that extend observation of the authors). An identical variation of from the base to the apex of the sponge. The surface surface structures in closely adjacent sponges has been between the flanges is also smooth (Figs. 3,4). A reported for the tropical hadromerid Spirastrella second morphotype is lamello-digitate, in which the vagabunda (cf. Kelly-Borges & Bergquist, 1988). In surface features range from well separated, irregular this species~ surface structures also range from lamellae ridges 3-5 ern high, to nodulose or sharply digitate to nodulose or digitate projections, and these morpho-

69 70 Table4. Spicule dimensions ofXestospongiamuta. I n=73; 2n=25; 3n=25;Specimens from Florida Keys study site, Long Key, Florida Keys. A and C denotes chemotype assignation, followed by sample number (se table 1);"Wiedenmayer (1977); "Van Soest (1980); 6Zea(1987); "Topsent (1920); Lelength: W=width.

Sample Locality mean (L) min (L) max (W) mean (W) min (W) max (W)

X. muta'Cc Florida Keys 306 1 190 380 ]P 8 ]3 X. muta'Ca Florida Keys 374' 240 435 142 ]3 IS X. muta'Cis Florida Keys 340 1 230 400 132 8 ]5 X. muta'X': Florida Keys 339 1 220 420 122 ]0 ]5 X. muta 3A8 Florida Keys 351 1 210 430 142 8 18 X.muta 3AII Florida Keys 368 1 270 430 142 13 15 X. muta' Bahamas 368 290 430 8 4 12 X. muter Curacao 380 303 453 19 11 23 X. muta" Colombian Caribbean 427 356 461 23 7 30 X. muta" Colombian Caribbean 311 223 404 21 7 29 X. muta" Colombian Caribbean 367 173 433 13 2 24 X. muta" Colombian Caribbean 351 228 413 18 4 23 X. muta" Colombian Caribbean 358 295 389 13 5 19 x. muta (type)? Florida 350 250 370 15 10 17 logies are independent of micro-habitat. The lamellae spicule thickness between Bahamas and Curacao spe­ are always aligned perpendicular to the substrate, an cimens, and suggested that thickness of spicules may orientation which probably aids in the removal of vary in a regional sense. This appears to be the case as heavy sediments (Kelly-Borges & Bergquist, 1988). specimens from the Colombian Caribbean (Zea, 1987) Hard and soft texture ofXestospongia mula does not have spiculeswith almosttwice the maximumthickness correlate with any particular morphotype. Texture ofthose from the Long Key specimens (Table 4). This appears to be genetically predetermined, as soft and regional difference in spicule thickness is a common hard sponges occur in very close proximity over a phenomenon, and has been related to temperature range ofdepths. However, sponge texture varies with (Simpson, 1987) and the amount of free silica in the depth; at 10 m at the Long Keys site, only 15 % of physical environment (Bavestrello, Bonita & Sara, in specimens were soft (n = 20), while in a group of press). specimens observed at 25 ill, 47 % were soft (n = 15). Threeinterspersed populations ofsterolchemotypes Increased spicule density and flattening of the body A, B, and C are quite clearly differentiated within have been demonstrated to be phenotypic responses in Xestospongia mula in Puerto Rico, Boca Raton and sponges experiencing high physical environmental Key Largo, collectedfrom 30, 25 and 12 m respectively impact (Palumbi, 1984). We have seen this in X. mula (Table 2). The three chemotypes do not appear to vary in patch reefs at 3 m depth; sponges are squat, with a in composition, in the relative amounts of the compo­ very shallow apical vent and broad lip, and these nent sterols, and they are temporally stable. However, sponges are all extremely hard. We have found that the only chemotypes A and C were found in the shallow morphology of the apical vent lip correlates almost water Long Key site (10 m), and chemotype C was perfectly with texture. In a group ofspecimens at 25 m, present in very low numbers (15 %; n = 20). In Puerto on Long Key, softsponges have a blunt irregularapical Rico, BocaRaton, and Key Largo, all three chemotypes lip (47 %; n = 15), and hard sponges have a sharp occurred in approximately equal ratios. Examination scalloped lip (53 %; n = 15). However, it seems of 20 individuals on a patch reef at a depth of 19 ill off possible that soft sponges would be more easily eroded Long Key also revealed the absenceofbothchemotypes or eaten by fish, and so a blunt lip might result. B and C, yet at a ribbon reef at a similar depth at Key Spiculedimensions do not correlate with morphotype Largo, all three chemotypes occur. It is possible to or chemotype, and thus the latter remain undefined by hypothesise that patch reefs, as opposed to ribbon the usual taxonomic criteria. Six adjacent sponges reefs, imposedifferent reproduction/dispersal conditi­ from the Long Key site represent the range of oxea ons such as relative isolation on their sponges, thus dimensions found throughout the entire Caribbean limiting the dispersal ofcomponent chemotypes. (Table 4). Van Soest (1980) noted a local difference in Chemotype is not correlated with morphotype. At

Figures 3-8: 3. Morphotype 1 - in situ, illustrating the density of the surface flanges, extending from the base to the apex of the sponge, scale =20 cm.; 4. Morphotype 1- surface detail; lamellate morphology, scale =1cm.; 5. Morphotype 2 - in situ, illustrating irregular low ridges and digitate projections, scale =20 em ..; 6. Morphotype 2 - surface detail; lamello-digitate morphology, scale = 1 cm.; 7. Morphotype 3 - in situ, illustrating the lack of lamellae, scale =20 cm.; 8. Morphotype 3­ surface detail; surface is irregularly conulose, with scattered depressions, scale = 1 em.

71 '~ ' ~ N N N 'M xesloslerol mulaslerol phytoslerols (Type C) (Type A) (Type B) <: I '~ N I '~

N desmoslerol a)

phytosterols xestosterol mutasterol Type A Type B Type C

?

b)

Figure 9.a.Met aboli c origins ofsterols in Xestospongia muta ;b.Thehyp othesised phyJogenetic relationship between sterols and chemotypes in Xestospongia muta .

the Long Key study site, sponges of both morphotypes ofadditional classes of metabolites and other types of 2 and 3 have chemotype C sponges amongst them, and lipids such as fatty acids, for their use as species-level all three morphotypes have chemotype A sterols. discriminators. Comparison also of variable nucleic With the degree ofbiochemical and morphological acid sequences,and Restriction Fragment Length Poly­ polymorphism evident in Xestospongia muta, it is morphisms (RFLP) , will detect genetic polymorphisms possible that a species complex exists , but species if they exi st. remain undefined at present. It is now necessary to Sterol differences have been noted in the closely extend this preliminary study to a broader geographic relatedXestospongia testudinaria from the Indo-Pacific, scale, and to continue to examine polymorphism in a and these differences have been correlated in one case range of habitats. We further wish to examine sterol with the degree ofspongin development in the spicule variation within the framew ork of popul ation dynamics, skeleton, and reproductive timin g (Fromont, 1991). and life history strategy, by determination of the range Two ofthe sterol chemotypes found in Indo-Pacific X. and dispersion of sterol chemotypes within X. muta on testudinaria are identical to chemotypes A and B ofX. a micro-scale . It is also important to explore the utility muta . Chemotype C seems to be unique to X. muta.

72 Given the similarity of these two species in terms of two sympatric sibling species of Clathria (Porifera: morphology, ecology, reproductive mode, and bio­ Demospongiae: Microcionidae) from Northern Australia. chemistry, it would not be unreasonable to hypothesise Invert. Taxon. 4: 123-148. a scenario of common ancestry. Kelly-Borges, M. & P.R. Bergquist 1988. Sponges from The biosynthetic origin ofconventional plant sterols Motupore Island. Indo-Malayan Zool. 5: 121- 159. (Nes & McKean, 1977), mutasterol (Kerr et aI., 1991) Kerr, R.G. & B.J. Baker 1991. Marine sterols. Nat. Prod. Repts 8: 465-497. and xestosterol (Stoilov et al., 1986) have been Kerr, R.G., S.L. Kerr & C. Djerassi 1991. Biosynthetic elucidated, and the metabolic relationships are summa­ studies of marine lipids. 26. Elucidation of the biosyn­ rized in Fig. 9. The biosynthetic relatedness can be thesis of mutasterol, a sponge sterol with a quaternary interpreted as a tree diagram indicating that mutasterol carbon in its side chain. J. Organic Chern. 56: 63-66. is more closely related to xestosterol than these two are Nes, W.R.&M.L.McKean 1977.Chapter9. Bio-chemistry to plant sterols. This structural relatedness can be used ofsteroidsandotherisopentenoids: 335-346. Baltimore: as a hypothesis to test the phylogenetic relationships University Park Press. between these sponges (Fig. 9). Palumbi, S.R. 1984. Tactics ofacclimation: Morphological changes of sponges in an unpredictable environment. Science 225 (4669): 1478-1480. ACKNOWLEDGEMENTS Pansini, M. 1982. Notes on some Mediterranean Axinella with description oftwo new species. Boll. Mus.Istit. Biol. Univ. Genova 50-51: 78-79. Financial support at Florida Atlantic University was Pomponi, S.A., A.E. Wright, M.C. Diaz & R.W.M. van provided by a Bristol-Myers Squibb Company Award Soest 1991. A systematic revision ofthe central Atlantic of Research Corporation, and is gratefully Halichondrida(Demospongiae, Pori fera). Part II. Patterns acknowledged. MKB thanks Harbor Branch ofdistribution ofsecondary metabolites. In: J. Reitner & Oceanographic Institution for laboratory support. We H. Keupp (eds), Fossil and recent sponges. Berlin: acknowledge the assistance of Manoj Reddy for Springer-Verlag. performing several chemical analyses, Klaus Kelly­ Sara, M. & E. Gaino 1987. Interspecific variation in arran­ Borges for the preparation ofspicule and tissue slides, gement and morphology ofmicrasters ofTethya species Tom Smoyerfor photographic reproductions and Joseph (Porifera, Demospongiae). Zoomorphology 102: 313­ Pawlick, University of North Carolina, Wilmington, 317. for several samples of Xestospongia mula. We are most Sennett, S.H., S.A. Pomponi & A.E. Wright 1992. 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