1 1 W 7, ,, , ,7, . ______ — ___d. — .__: — __'_d 7, i . _ -_—’__ — z — — __—_. _ A - — , , — 145 ELECTMIYY UPTECRL SYUDY 0? 430 mm 3:23:53 3% 30m -THS- an. a Itam TR’EPOSTGME 3522922633 has: Yo-r the “mg :3? 9&2. S, E? {LEM 2233 fl. E3? JESSE“! "F % E'Y 3:ch {1503.3- 3321; $975 A ABSTRACT ELECTRON OPTICAL STUDY OF BROWN BODIES IN SOME ORDOVICIAN TREPOSTOME BRYOZOANS By Stanley Jay Morrison Electron optical methods are shown to be useful in the study of organic remnants in fossil bryozoans. Fossil brown bodies in zoaria of the Ordovician trepostomes Peronopora, Heterotrypa and Prasopora consist of glauconite or other minerals with inclusions of organic mater— ial. Fossil brown bodies are similar in morphology to brown bodies formed by degeneration in Recent cyclostomes and most likely result from the same processes. The preservation of the organic material is attri- buted to the growth of carbonate diaphragms which blocked the attack of microbial organisms in the initial stages of diagenesis. Eossil brown bodies in Ordovician trepostomes are essentially permineralizations of the original brown bodies containing only small remnants of the original organic material. ELECTRON OPTICAL STUDY OF BROWN BODIES IN SOME ORDOVICIAN TREPOSTOME BRYOZOANS By Stanley Jay Morrison A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE College of Natural Science Department of Geology 1975 ACKNOWLEDGEMENTS Sincere appreciation to Robert Anstey who supervised and critiqued this work in its entirety. Special thanks to Gary Hooper who assisted and instructed in the SEM and TEM work, Mrs. June Mack who performed the TEM work, Viv Shull who did the microprobe analyses and Osborne Nye (Wayne State University) who provided suggestions and materials related to Recent specimens. JINTRODUCTION Ellis (1755) was the first zoologist to observe and record brown bodies in extant bryozoans. Since that time diverse ideas have arisen regarding their nature and function, reviewed by Joliet (1877). Brown bodies have been thought to be ovaries, eggs, incipient larvae, endocy- stal secretions, equivalents of phylactolaemate statoblasts, special formations detached from the stomach, or germ capsules from which new polypides arise (Gordon, 1973). It is now realized that brown bodies are large cellular bodies containing residual components resulting from the degeneration of a once living polypide. Cumings and Galloway (1915) interpreted brown deposits in perminer- alized fossil bryozoan zoaria as follows: "Taking into consideration... the size, relations to surrounding structures, nature of the material, and its isolation, there can be no doubt that the brown material is due to replacement, probably by iron sulphide or iron sulphate, of organic matter in the zooecium after the death of the polypide." Although the alternative explanation was given that the brown material in the fossils might be analogous to brown bodies of Recent bryozoans, they were careful to point out that similarities, including the reddish-brown color of the material, might be misleading. Boardman (1971) used the term "brown deposits" seemingly to avoid. making an analogy with Recent degenerative products which are given the name brown bodies. He uses this term to describe accumulations of reddish- brown grains which are regularly contained in flask shaped chambers in fossil trepostomes. ‘ The purpose of this study is to determine the nature and state of preservation of brown material in fossil trepostomes, using the methods 2 of electron optics to study their ultrastructure and composition. Terminology The following terms will be used in this paper: 1. Brown deposit.—Any material within a zooecium occupying approximately the position of a once living polypide. 2. Sedimentary infilling.-A brown deposit which is known to be derived from sediment external to the zoarium. 3. Fossil brown body.-A brown deposit which is similar to morphologically and presumed to be originally identical to brown bodies in Recent ‘ectoprocts. . 4. Brown body grains.-The spherical, ellipsoidal or irregularly shaped particles averaging about 10 micrometers in diameter, usually reddish- brown under transmitted light, which make up a brown body. 5. Brown body replacement.-A brown deposit which retains the form or position of a degenerated polypide but has been replaced by secondary minerals which no longer preserve the brown body grains. MATERIALS AND METHODS Two trepostome zoaria of different genera were selected for preser- vational reasons from the Cincinnatian (Upper Ordovician) strata of the Ohio Valley. Both samples are from stratigraphic intervals of thinly interbedded limestone and shale. A third specimen, Middle Ordovician in age, from Ontario, was used for TEM comparisons (Table 1). The scanning electron microscope, 181 Super Mini SEM, secondary electron capture mode, was used to study surface structures. Good resolu— tion (300r400 Angstroms) was achieved at magnifications exceeding 10,000X. The samples were polished , etched in 2 percent EDTA for 15 minutes, then mounted on stubs and a 100-200 Angstrom gold coating applied by "sputter" coating.‘ The SEM micrographs were foreshortened with respect to those 3 taken with the microprobe. Acetate peels will pull brown body grains away from the matrix and were also used with success. Table 1. Locations and methods of examination of specimens, deposited in the paleontological collection of Michigan State University. Specimen no. Location Methods* Taxon 03510901' Kirkfield, Ontario TEM Prasopora simulatrix Ulrich 220314-01 SW%,SWk,SEk,Sec.10, Probe (C,Si,Al,K, Heterotrypa s2, T9N,R2W,Mount Carmel P,S),TEM,SEM Quad.,Indiana 220314-02 SWk,NWk,NEk,Sec.15, Probe (C,Si,Fe,P) Peronopora__p. T5N,R1W,Lawrenceburg, Ky.-Ind.—Ohio Quad. * symbols indicate elements analyzed by micrOprobe The microprobe, model EM X-SM (Applied Research Laboratories), operated at 15 RV was used to study elemental compositions. Micrographs were filmed using the sample current mode. No absolute amounts of elements were determined. Etching sometimes selectively removes elements from a sample; therefore a polished unetched section was run for control. It was not possible to determine nitrogen content in the microprobe. A TEM, Philips model 300, was used to study internal ultrastructures of the brown body grains. Samples were prepared by mechanically separat— ing the grains from the matrix after embedding in epoxy resin for 12 hours. The embedded fragments were then ultramicrotomed with.a diamond knife to 600-900 Angstroms and mounted on 300 mesh grids for examination. Attempts were made to cut the rock sample itself after embedding with epon under pressure for 12 hours; however, upon contact with the diamond knife, the partially infiltrated carbonate matrix fragmented. Chemical determinations were attempted using standard histological techniques. The small amount of organic material along with the imper- meability of the diagenetic calcite, created severe difficulties in using 4 these techniques. Minute portions of the brown bodies were seen to stain positively using toluidine blue or safranin-O on thin sections after etching in formic acid, indicating the presence of some organic compounds. It was difficult to determine, however, how much of the material was exposed at the specimen surface and not protected from the stains by transparent layers of calcite. In addition, brown body grains contain other inorganic minerals formed during diagenesis (as discussed below), rendering most stains ineffective. REVIEW OF PREVIOUS STUDIES Nature of the Polypide and Degeneration in Recent Ectoprocts Of the orders of ectoprocts, the cyclostomes are most analogous to the fossil trepostomes. The similarities are seen in growth forms, tub- ular zooecia, zooecial arrangement, budding from a single ancestrula and their lack of polymorphs common to other orders, such as the vibracula and avicularia of cheilostomes. There are certainly great skeletal dis- similarities (such as the presence of diaphragms and cystiphragms in trep- ostomes) and it is conceivable that great differences could have existed between the zooids as well. In order to understand the possibilities for the preservation of or— ganic material in fossil trepostomes, it is useful to examine the nature of the soft parts in Recent double-walled cyclostomes. The soft parts consist of terminal membrane draped over the zooecial cortex and separ— ated from it by a coelomic space and inner membrane. This terminal mem- brane continues down to the area of the sphincter muscle where it separ- ates and forms the tentacle sheath and membranous sac. The membranous sac surrOUnds the entire gut complex and separates the coelomic space into an endosaccal and exosaccal coeloms. The coelomic space is contin- uous between zooids and provides a pathway for interzooecial communication (for a more detailed and illustrated description see Boardman, 1971). 5 Gordon (1973) using TEM methods showed that cellular degeneration in Cryptosula pallansiana Moll, ( a cheilostome), is quite similar to aging and cell degeneration in other organisms. One phenomenon that is not known in any other organism is the formation of red-brown inclusions from undigested food material. Lack of an excretory system in ectoprocts results in the accumulation of these inclusions in the gut of the poly— pide throughout its life. Natural senescence may be related to the gradual build up of these inclusions with age.. Polypide degeneration can be brought about by unfavorable environmental conditions, overgrowths, onSet of reproduction, or natural senescence. The formation of brown bodies is unique to the Phylum Ectoprocta and is undoubtedly somewhat the same for all ectoprocts which undergo degeneration. Upon degeneration, the cells of the tentacles and 10phophore tend to regress earlier than those of the gut. The tentacle cells rupture as opposed to the gut cells which phagocytize or fuse thus remaining in position and preserving the general form of the gut complex. The residual cell fragments, including membranous whorls, myelin figures, reticulum, muscle strands, collagen and glycogen deposits and the red-brown inclusions, are enclosed within membranes of much larger cells. Glycogen accumulates during regression but is depleted upon final brown body formation. It is possible that this glycogen is used up by a newly deveIOping polypide. Polypide degeneration may be controlled by hormonal secretion functioning as a rejuvenatory phenomenon (Gordon, 1973). Because they have no excre— tory system, it is possible that a gradual buildup of urea or some other toxic compound in the coelomic fluid causes an exponential death rate of the polypides during colonial growth. Organic Material in Fossil Trepostomes Surprisingly, little mention has been made of fossil brown bodies in the literature other than to note their existence and give a general description. The only compositional work suggests that they are composed of iron sulfide or iron sulfate (Cumings and Galloway, 1915), but is questionable because the analytic methods were not specified. Boardman (1971) suggested that the reddish—brown color is due to the presence of an iron oxide. The possibility of membrane preservation has been noted in several papers. McKinney (1969) reported cuticular appearing structures in a late Mississippian trepostome which he compared with the vestibular mem- brane and membranous sac of Recent cyclostomes. Boardman (1973) has reported a structure which is undoubtedly a terminal membrane in an unidentified dendroid trepostome of Upper Ordovician age. The most compelling examples of membrane preservation come from samples of Leptotrypella? praecox Boardman, from the Lower Devonian strata of Antartica (Boardman, 1971). All membranous intrazooecial structures of these specimens including funnels, cystiphragms, diaphragms and zooecial linings are reddish-brown in color, noncrystalline,and insoluble in HCl and HF. Microprobe analyses of these membranous units for phosphorus,ca1cium, carbon, sulfur, iron and nitrogen found them to be rich in phosphorus and not significantly different from the calcitic skeleton in all the rest. Diagenesis of Organic Compounds ~Organic compounds in fossils are present either in their original form or as products of diagenesis and epigenesis. Degens (1967) suggests that the final state of all diagenesis of organic compounds is to become reduced either to aromatic condensates resembling graphite or to light paraffin hydrocarbons and that the occurence of original biogenic matter 7 such as amino acids, sugars, fatty acids, or the bases of purines or pyrimidines may indicate incomplete diagenesis and epigenesis. Mucopoly— saccharides in the form of "chitin-type" materials have been reported in many fossil specimens. Chitin is discussed in terms of chemical composi— tions, in Recent invertebrates, by Hyman (1958,1966). It is unusual to find free amino acids preserved in fossils because of the more or less immediate breakdown of these macromolecules by microbial activity. One major exception is the free amino compounds found in the carbonate shells of fossil mollusks. Most microorganisms are too large (greater than 1 micrometer) to penetrate through the carbonate matrix to attack the proteinaceous films. Amino acids are selectively lost during diagenesis due to heat decay (Degens, 1967). Amino acid studies have been done on a number of Recent ect0procts (Schopf and Manheim, 1967). An amino acid study was not attempted in the present work due to analytical difficulties in separating the small amounts of organic constituents of the brown bodies. Such a study might prove useful in understanding the genetics and ecology of these bryozoans. RESULTS OF THIS STUDY Brown Body Morphology Under transmitted light, brown bodies appear as masses of reddish— brown grains (Figure 1). The average number of grains counted in Hgtgrf otrypa (about 60 to 70 measured in oneplane of the section) is consistent with the number found in similar sized zooecia of a Recent heterOporid. Brown body grains have a mottled appearance and are generally spherical to elliptical in cross—section (Figure 1; B). Brown bodies are commonly seen directly beneath funnel structures (A) often constricted by cystiphragms or resting on diaphragms (C). The morphology of a once existent gut com- plex is often preserved by the constriction of a membrane which has since undergone decomposition (D). SEM and TEM Results The principal use of the SEM in this study was to examine surface features with greater resolution than could be achieved with the micro- probe or light microscope. Brown body grains were seen to consist of granular material embedded in smooth calcite. Part of the granular mater— ial was etched away leaving void spaces. All the grains examined had this same general nature (Figure 2). The SEM has proven to be a powerful tool in the examination of skel— etal hard parts and thus the inferred growth and development of the secret— ing inner epidermis (Tavener-Smith and Williams, 1972). The present study shows that the position of membranes can also be inferred by examining the traces left by the interruption of diagenetic calcite crystal growth. Structural features such as diaphragms, cystiphragms and funnel structures are secreted by the inner epidermis and consist of platelike laminae which merge without a break into the zooecial cortex or lining (Figure 2, E, point A , shows a funnel merging with the zooecial lining). The platelike laminae of the funnel structure cease about one third of the way down in the zooecium (point B) and gives way to traces of a membrane which has since undergone decomposition. These membrane traces continue on down into the zooecium, with the same curvature as the funnel, and meet the zooecial lining of the wall without merging with it (point C). This orientation suggests that the membrane passed through the orifice and encompassed the gut complex. Brown body grains were mechanically separated after etching from one specimen each of Heterotrypa and Prasopora and examined with the TEM at magnifications ranging from 25,000X to 52,000X (Figure 3). The ultra— structures of both specimens are essentially identical. The grains consist of predominantly a mass of elongated transparent crystals up to a micro— meter in length. These crystals appear bladelike when cut longitudinally 8 9 and are rounded to rectangular in cross—section. Their axial zones are more electron dense than their encompassing thin outer rims, and their orientation seems to be mOre or less random. Mixed among these crystals are patches of electron dense material with shapes ranging from somewhat spherical to amorphous. This material appears to be held in place by the crystalline matrix. Microprobe Results In general, traverses were selected on transverse of longitudinal sections so as to cross zooecial wall, diagenetic calcite and brown bodies. High carbon and silicon anomalies are seen to be related to the brown body grains (Figure 4; C,D,E,F). Iron seems to be fairly homogeneous and pre- sent in only trace amounts (less than 200 ppm) throughout the zooecia and brown bodies except where it peaks in coordination with silicon. Nearly all the silicon peaks are associated with iron peaks. The silicon peaks are fairly independent of the carbon peaks and both occur most abundantly within the brown body grains (Figure 4; C,D). These trends are seen in four different brown bodies of two different genera including an unetched control specimen. One brown body was examined for aluminum and potassium. This traverse showed aluminum, potassium, silicon and iron forming distinct peaks at exactly the same location. This was the only location at which these four elements peaked; however, carbon peaked at this location and at one additional location. A thin section of Peronopora treated with 0504 for 24 hours showed negative results when tested for osmium by microprobe. This treatment should impart an osmium stain to lipid molecules. Phosphorus and sulfur were examined in one brown body of Heterotrypa but showed no detectable concentrations. It is interesting to note that the diaphragms, cystiphragms and zooecial linings show up as a void area on the carbon micrograph (Figure 4; E), which indicates that carbon is more abundant within the dia— 10 genetic calcite and zooecial cortex than in these skeletal deposits. INTERPRETATION Highly crystallized sedimentary infillings occur in most zoaria and are always most abundant in the surface layer of the colony. In colonies which display cyclic growth zones, sedimentary infillings are often abundant immediately below the plane of cessated growth. Their distribution, lack of morphologic resemblance to a polypide and similarity to the matrix surrounding the zoarium indicates that these deposits are external in origin to the zoarium. Highly crystallized replacements having the general morphology of a polypide and seen as a common feature in many zoaria may consist of crystals of vivianite, other transparent minerals or opaque pyrite. They are totally contained within a single zooecium and often occur directly beneath funnel shaped structures. Pyrite is commonly found in insoluble residues as spherical or funnel shaped pseudomorphs. Due to their morpho- logy and position in the zoarium, deposits such as these are believed to be diagenetic replacements of the original soft tissue aided by the action of'reducing microbial activity.' Brown bodies consist of masses of reddish-brown grains and have a morphology similar to that of brown bodies in Recent ectoprocts. They are found resting on diaphragms or constricted by cystiphragms often immediately below funnel structures. In order for these shapes to have been kept intact during the influx of diagenetic calcite, they must have at one time been surrounded by a membrane which no longer exists as in brown bodies of Recent cyclostomes (Figure 1;D: Figure 2; E). The dia— genetic calcite must have entered soon after death and before the membrane underwent decomposition. The average number of brown body grains per living chamber of a fossil Heterotrypa is comparable to the number found in a 11 Recent heteroporid with similar sized living chambers. Because sulfur is not detected by microprobe analysis, the reddish—brown color is probably better attributed to iron oxides than to iron sulfides. In some cases, the brown bodies extend up through the necks of funnel structures simulating the morphology of the whole polypide. The fossil brown bodies have been determined to contain organic materials on the basis of a positive stain using toluidine blue or safranin— O, insolubility in HCl and a high carbon anomaly. The organic material must be either highly stable original organic compounds or end products of organic diagenesis. The preservation of organic material in brown bodies is probably due to a phenomenon similar to that responsible for the preservation of organic films in molluscan shells. If, after the polypide degenerates and forms a brown body, the zooecium is quickly sealed by the growth of a distal diaphragm, microbial attack is impaired and the residual organic materials are subject only to the diagenetic effects imposed upon burial and ground water percolation. It is quite possible that this diaphragm is secreted by a newly developing distal polypide, nourishing itself from the residual glycogen deposits of the degenerated zooid immediately below it. Microprobe studies show that brown body grains are rich in carbon and silicon as well as aluminum and potassium. Glauconite, a rather poorly defined hydrous iron potassium aluminosilicate is known to form in associa- tion with organic material in areas of fluctuating redox potential such as might be found in rotting molluscan shells on a well washed shelf (Fairbridge, 1967). Under reducing conditions, glauconite is less stable and organic material is replaced by pyrite. Reducing conditions, the result of microbial activity, are impaired upon the formation of calcareous 12 diaphragms. The preservation of a brown body then would be dependent on the presence or absence of a new distal polypide to secrete a diaphragm. Thus, different redox potentials may exist within any two living chambers within the same zoarium depending on the formation of this distal diaphragm. This may account for the presence of both pyritized replacements and well‘ preserved brown bodies within the same zoarium (Figure 1; C). TEM micrographs of the brown body grains show that they are composed of numerous, well defined crystals and spherical to amorphous globules (Figure 3). The crystals are interpreted to be silicious while the globules must contain remnants of organic material selectively preserved after polypide degeneration. Because trepostomes of two different genera widely separated geographically, show nearly the same ultrastructure, it is assumed that digenesis of this type may be a common occurrence. The organics preserved in these Ordovician ectoprocts may form an important link in understanding the evolution and diagenesis of organic com— pounds. Traditionally, importance has been placed on the morphology of skeletal hard parts for taxonomic and phylogenetic work. It is necessary, however, not only to make morphological distinctions between skeletal components but to contrast these with biological interpretations based on mode of growth, soft part morphology and biochemistry. The organic materials preserved in brown bodies might provide a source of information for these biological interpretations. CONCLUSIONS Brown deposits in fossil trepostomes occur as either sedimentary infillings, replacements (as with pyrite), or as brown bodies consisting of organic materials and silicate replacements (possibly glauconite). The preservation of organic materials is attributed to their escape from the initial stages of organic diagenesis (microbial activity) due 13 to the sealing off of the zooecia by the formation of diaphragms. Differ- ing redox potentials may exist between any two zooecia and cause the formation of both replacements and brown bodies within the same zoarium. The reddish—brown color of the brown bodies is not due to replacement by iron sulfides or iron sulfates but rather is attributed to either the presence of iron oxide or silicates. The organic nature and similarity of morphology indicates that brown bodies undoubtedly represent the remains of a once living polypide which underwent degeneration in much the same way as in Recent ectoprocts. This in- terpretation adds additional evidence to support a view of close affinities between trepostomes and Recent cyclostomes. A possible chronologic sequence of formation of fossilized brown bodies is as follows: 1. Degeneration of a once living polypide to form large residual cells encompassed by a membrane . 2. Influx of diagenetic calcite which fills the zooecium. 3. Decomposition of the membranes surrounding the brown body grains and the brown body. 4. Decomposition of unstable organic compounds in the grains. 5. Accumulation of iron silicates in the grains. 6. Possible further organic diagenesis and epigenesis. The processes which cause these events may be common to all fossil trepo- stomes containing brown bodies and would indicate a similar postdepositional history. The small amounts of organic material present in brown bodies might make an amino acid study impossible. If it could be done, it would probably have its greatest significance in geothermal work. The calcite matrix of these brown bodies is quite impermeable to most 14 liquids. This calls for improved methodology in 1) histochemical staining techniques and 2) epoxy embedding for TEM work. The SEM is shown to have potential in photographing traces of organic membranes in portions of zooecia where no skeletal structure is evident. LIST OF REFERENCES Boardman, R.S. 1971. Mode of Growth and Functional Morphology of Auto- zooids in Some Recent and Paleozoic Tubular Bryozoa. Smith- sonian Contributions to Paleobiology, no. 8. ,1973. Body Walls and Attachment Organs in Some Recent Cyclostomes _ and Paleozoic Trepostomes. In G.P. Larwood (ed.), Living and Fossil Bryozoa. Academic Press, London, pp. 231—246. Cumings, E.R. and Galloway, J.J. 1915. Studies of the Morphology and Histology of the Trepostomata or Monticuliporids. Geol. Soc. Amer. Bull., vol.26, pp. 349-374. Degens, E.T. 1967. Diagenesis of Organic Matter. In C. Larson and G.V. Chilingar (eds), Diagenesis in Sediments. Elsevier Publishing Co., Amsterdam, pp. 343-390. Ellis, J. 1755. An Essay Towards a Natural History of the Corallines. (Not seen.) Fairbridge, R.W. 1967. Phases of Diagenesis and Authigenesis. In G. Larson and G.V. Chilingar (eds.), Diagenesis in Sediments. Elsevier Publishing Co., Amsterdam, pp. 19-89. Gordon, D.P. 1973. A Fine-structure Study of Brown Bodies in the Gymno- laemate Cryptosula pallansiana (Moll). In G.P. Larwood (ed.), Living and Fossil Bryozoa. Academic Press, London, pp. 275-286. Hyman, L.H. 1958. The Occurrence of Chitin in the Lophophorate Phyla. Biol. Bull., vol. 114, pp. 106-112. , 1966. Further Note on the Occurence of Chitin in Invertebrates. Biol. Bull. Mar. Biol. Lab., Woods Hole, vol. 130, pp. 94-95. Joliet, L. 1877. Contributions a L'histoire Naturelle des Bryozoaires des Cotes de France. Arch. 2001. exp. Gen., vol. 6, pp. 193-304. Schopf, T.J.M. and Manheim, F.T. 1967. Chemical Compositions of Ectoprocta (Bryozoa). J. Paleont., vol. 41, pp. 1197—1225. Tavener-Smith, R. and Williams, A. 1972. The Secretion and Structure of the Skeleton in Living and Fossil Bryozoa. Proc. R. Soc. Lond. B., vol. 177, pp. 1-65. 15 16 FIGURE 1 LM micrographs of brown deposits in Heterotrypa s23, MSU 220314-01; a, brown body constricted by membranous structure immediately below a funnel; bar scale is .1 mm long; b, brown body grains showing mottled appearance; bar scale is 10 micrometers long; c, zooecium containing a pyrite replacement between two zooecia with brown bodies, p-pyrite, bb—brown body; bar scale is .1 mm long; d, brown body grains constricted by membranous structure immediately below funnel neck; bar scale is 10 micrometers long. 18 FIGURE 2 SEM micrographs of brown bodies in Heterotrypa ER}: MSU 220314-01; a, brown body resting on a diaphragm; circled area is shown in b; bar scale is 10 micrometers long; b, brown body grain; circled area of a; barscale is 1 micrometer long; c, brown body; circled area is shown in d; bar scale is 25 micrometers long; d, brown body grain; circled area of c; bar scale is 2 micrometers long; e, brown body immediately below a funnel structure; bar scale is 25 micrometers long; f, brown body resting on a diaphragm; bar scale is 25 micrometers long. 20 FIGURE 3 TEM micrographs of brown body grains; a and b are Prasopora simulatrix Ulrich, MSU 03510901; c and d are Heterotrypa s2, , MSU 220314-01; Bar scales on all are 2 micrometers long. FIGURE 4 Microprobe analysis of a brown body of Heterotrypa §B,,MSU 220314-01; all figures represent the same brown body; bar scales on a and b are 25 micrometers long; grid spacings on c,d,e and f are 25 micrometers apart; a, SEM micrograph; b, LM micrograph; c, carbon and silicon distribution; white area inside the dashed line represents the brown body; d, traverse for carbon, iron and silicon; traverse is marked on c, e and f; e, carbon micrograph; note the lack of carbon in the diaphragm, cystiphragm and zooecial lining; f, silicon micrograph. .RN . .. Ru, \. . \ \\\ .\\ \ x: es}. lcnnon r l I \ \ \ \ 1'." .gsuucon l .C IIIuuuywmuuumuImmwlflugluwu«H 293 03