Protoplasma 77, 35--54 (1973) 9 by Springer-Verlag 1973

A Light and Electron Microscopic Study of Sporulation in the Myxomycete Stemonitis virginiensis CHARLES W. MIMS

Department of Biology, Stephen F. Austin State University, Nacogdoches, Texas, U.S.A. With 43 Figures Received September 18, 1972

Summary The conversion of the plasmodium of S. virginiensis into sporophores has been examined at both the light and electron microscopic levels. Particular attention has been paid to stalk and columella formation, capillkial formation, nuclear behavior during sporulation and spore formation. Both the stalk and columella are formed within the sporangial initial as in~raprotoplasmic secretions. A portion of the capillitium arises directly from the columella while the remainder forms within an anastomosing system of tubular vacuoles. As spore cleavage begins the nuclei within the sporangium begin to divide mitotically. The protoplasmic content of the sporangium is first divided into small protospores which typically contain a single dividing nucleus. Following the completion of mitosis each of these segments cleaves into yet smaller segments which develop into spores. Meiosis occurs in the spores some 12-16 hours after cleavage. 1.

Introduction

Although the sporulation process in the Myxomycetes, or true plasmodial slime molds, has been studied in a number of species at the light microscopic level, relatively f e w ultrastructural studies of the process are reported in the literature. Most of the investigations have, in addition, concentrated on nuclear behavior during sporulation and neglected m a n y of the other phenomena associated with the conversion of the plasmodium, a multinucleate, acellular mass of protoplasm, into sporophores containing typically uninucleate, thick-walled spores. In the present study the sporulation process in the slime mold Stemonitis virginiensis Rex has been examined at both the light and electron microscopic levels. Rather than concentrating on a particular aspect of the process, an attempt has been made to examine a number of events including stalk and columella formation, capillitial formation, nuclear behavior during sporulation and spore formation. S. virginiensis was chosen for study because it grows and reproduces readily 3*

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CH. W. MIMS

in culture, but more i m p o r t a n t l y because no other member of the order Stemonitales has been examined to any extent with the electron microscope. H o p e f u l l y this study will clarify m a n y details of the sporulation process in the Sternonitales, which as pointed out b y DE BARy (1887), Ross (1957), and A~exoeouLos (1969) appears to be fundamentally different f r o m that of other endosporous myxomycetes, as well as contribute to our overall understanding of morphogenesis in the Myxornycetes.

2. M a t e r i a l s a n d M e t h o d s The isolate of S. virginiensis used in this investigation was collected in the spring of 1970 near Nacogdoches, Texas. Spores were spread on the surface of one-half strength Difco corn meal agar contained in plastic disposable petri dishes and the surface of the agar flooded with a dilute suspension of Escherichia coli in distilled water. Two days later a few sterile, pulverized oat flakes were added to each culture. The cultures were subsequently placed on a laboratory shelf where they remained until sporulation occurred. No attempt was made to regulate the light-dark cycle, but even under these conditions S. virginiensis completed its life cycle regularly at the end of 11-12 days. Once sporulation began sporangia were prepared for electron microscopy at various stages of development in a manner which has been previously described (MI~as 1969). Sections were cut with a diamond knife, post-stained with lead citrate and examined on an RCA EMU-3F electron microscope. For light microscopic study of stalk, columella, and capillitial formation sporangia were gently squashed in a drop of water under a cover slip. To examine nuclei, sporangia were squashed in aceto-orcein. Observations were made on a Leitz Dialux microscope equipped with a Leica camera or on an Olympus stereo-microscope equipped with an automatic camera. 3. O b s e r v a t i o n s 3.1. G e n e r a l As appears to be the case in a number of myxomycetes (GRAY and ALEXOPOULOS 1968), S. virginiensis typically sporulates at night. The first morphological indication of the onset of sporulation is the condensation or thickening of the protoplasm of the a p h a n o p l a s m o d i u m leading to the appearance of a network of prominent, pale-white veins (Figs. 1 and 2). In a short time the thickened p r o t o p l a s m begins to accumulate in the anterior region of the plasmodium (Fig. 3). Once this stage is reached the coalesced plasmodium moves v e r y little and usually fructifications are formed very near the region in which the prominent veins first become visible. Occasionally, however, the plasmodium will move a short distance up the sides of the petri dish before giving rise to sporangia. F r o m a single plasmodium a number of sporangia are formed, usually in dense clusters. The sporangial initials first appear as small glistening, white "'blebs" of p r o t o p l a s m (Fig. 4). In the following hour or so a thin, membranous hypothallus is formed beneath the initials. Each initial then enlarges and elongates slightly in a plane perpendicular to the surface of the agar (Figs. 5 and 6). During this time a small

A Study of Sporulation in the Myxomycete Stemonitis virginiensis

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Fig. 1. Young aphanoplasmodium of S. virginiensis. 5(200 Figs. 2-9. Stages in the conversion of the plasmodium to sporangia. Fig. 2 X4. Figs. 3, 4 5(8. Figs. 5, 6 X12. Figs. 7-9 X10 Fig. 10. Mature sporangia of S. virginiensis. X 12

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MIMS:A Study of Sporulation in the Myxomycete Stemonitis virginiensis

dark stalk becomes visible within each initial (Figs. 5-7). The stalk is deposited directly on the hypothallus and increases in length as the initial elongates. Once the initial has reached a certain height the protoplasm ascends the stalk and continues to elongate (Fig. 8). During this time the columella, or extension of the stalk into the sporangium, is formed and can be seen within the whitish, somewhat transparent sporangium (Figs. 8 and 9). Approximately 3-4 hours after the appearance of the small blebs of protoplasm each sporangium reaches its maximum height and, except for its color and watery nature, resembles a mature sporangium. In the next 15-30 minutes each sporangium becomes pale pinkish in color and then dark brown. It is during this time that capillitium formation, a nuclear division and spore cleavage all take place. Over the next 8-10 hours the sporangium gradually dries and assumes the brownish color typical of a mature sporangium (Fig. 10). 3.2. S t a l k

and

Columella

Formation

The i:ormation of the stalk and columella in S. virginiensis is basically similar to that described previously for other members of the same genus (Ross 1957, INDIRA 1971). Both are visible during their formation within the somewhat transparent sporangial initial. The base of the stalk is deposited directly on the hypothallus by the sporangial initial. The stalk increases in length by the addition of material to its apex. The columella is then formed by the continued deposition of material to the tip of the stalk after the sporogenous mass of protoplasm has ascended the initial. The tip of the elongating columella appears to blend gradually into the protoplasm of the initial (Figs. 11 and 12). Electron microscopic examination of the region near the tip of the columella reveals a dense concentration of fibrillar material (Fig. 17). Small amounts of such material were occasionally observed elsewhere in the initial, but most is concentrated near the tip of the columella. Apparently the wall of both the stalk and columetla is formed from this material although it does appear that some protoplasm is incorporated into

Fig. 11. Squash preparation showing the columella. Note the cavity (at arrows) in the lower portion of the sporangial initial in which the columella lies. • 400 Fig. 12. Higher magnification of a squash preparation showing the tip of the columella gradually blending into the protoplasm. X 1000 Fig. 13. Squash preparation showing capillitial threads (at arrows) arising from the columella. • 1000 Fig. 14. Squash preparation showing capillitium forming throughout the sporangium. X150 Fig. 15. Squash preparation showing fully formed capillitium prior to the initiation of cleavage. • 200 Fig. 16. Squash preparation showing a portion of the surface net. • 1000

Figs. 11-16

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C~. W. MzMs

the columella as has been previously reported (BisBx 1914, Ross 1957, INDIRA 1971). No membrane separating the tip of the columella from the protoplasm was observed although the portion of the columella in the lower portion of the sporangium is separated from the protoplasm and actually lies in a hollow, membrane-bounded cavity (Figs. 11 and 18). Exactly how this cavity is formed is not known, but it is clearly visible at both the light and electron microscopic levels and has been previously noted in Stemonitis (BIsBY 1914, INDtRA 1971). The protoplasm just beneath the membrane delimiting the cavity is filled with tiny fibrillar elements (Fig. 18). Cellular organelles are excluded from this region. Both the stalk and columella are tubular in structure consisting of an outer layer or wall of electron-dense material surrounding a lumen filled with virtually electron-transparent material in which large amounts of electron-dense material is embedded (Fig. 18). 3.3. C a p i l l i t i a l

Formation

Capillitial formation is difficult to study in S. virginiensis because the entire process takes place so quickly--in 15 minutes or less--and because the capillitinm consists of such an extensive network of anastomosing threads. In spite of this, however, the early stages of capillitial formation can be followed. The capillitium does not begin to form until the sporangial initial has reached its maximum height and the columella is fully formed. The first threads to appear are those which originate from the columella, These threads form as a result of the branching of the upper portion of the columella (Fig. 13). These threads elongate toward the periphery of the sporangium where they eventually anastomose with another system of threads which arises independently of the columella. Shortly after the formation of the threads from the columella, the remainder of the capillitium forms more or less spontaneously throughout the length of the sporangium (Figs. 14 and 15). This also includes the delicate surface net characteristic of members of the genus Stemonitis (Fig. 16). The formation of the capillitial threads, other than those arising from the columella, involves the formation of an anastomosing system of tubular vacuoles. Apparently this system arises from the fusion and enlargement of pre-existing vacuoles. The protoplasm of the

Fig. 17. Portion of a section near the tip of the elongating columella showing the fibrillar material thought to function in stalk and columelia formation. Note the protoplasmic material (at arrows) which is apparently incorporated into the columella. )< 20,000 Fig. t8. Cross section of the columella in the lower portion of the sporangium. Note the dense wall of the columella and the central lumen. Also note the cavity (C) surrounding the columella and the concentration of fibrillar material just beneath the membrane surrounding the cavity. X 10,000

A Study of Sporulation in the Myxomycete Stemonitis virginiensis

Figs. 17 and 18

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CH. W. MiMs

sporangium prior to the initiation of capillitial formation is highly vacuolate (Fig. 19). In a short time, however, the system of tubular vacuoles appears throughout the sporangium. A short segment of such a vacuole is shown in Fig. 20. These vacuoles can be distinguished from the more or less spherical vacuoles so common in the protoplasm because of the accumulation of fibrillar material around their surfaces (Figs. 20-23). It is within these vacuoles that the capillitial threads are formed (Figs. 21-23). It appears that the threads are formed by a condensation of material present within the vacuoles although the details of this process are obscure. During the early stages of their development the threads appear to consist of a loose, outer layer of material of moderate electron density and a more or less electron-transparent lumen. The lumen is, however, not always clearly defined. As the threads mature the material composing the outer layer or wall increases in electron density. Shown in Figs. 24-27 are some examples of mature threads. As is clearly evident they differ greatly in size and appearance. 3.4. N u c l e a r

Behavior

and Spore

Formation

The cleavage of the protoplasm into spores in S. virginiensis does not begin until the formation of the capillitium is completed. It is during this time that the color of the sporangium gradually darkens. Spore cleavage is accomplished by the fusion of membrane-bounded vesicles which line up along cleavage planes (Figs. 28-30) as has been reported in other myxomycetes (ALDRICH 1967, MIMS 1969, SCHUSTER 1964). The origin of these vesicles is not known although it has been postulated that they arise from endoplasmic reticulum (ALDRICH 1967). As a result of the fusion of these vesicles with one another and with the vacuolar elements in which the capillitium is formed the protoplasmic content of the sporangium is progressively divided into smaller and smaller segments. During cleavage the nuclei undergo mitosis. The division is virtually synchronous throughout the elongate sporangium although the nuclei in the upper portion and periphery of the sporangium usually begin to divide first. Normally the division does not proceed past metaphase (Figs. 29 and 30) until the content of the sporangium has been divided into small segments typically containing one or sometimes two dividing nuclei (Figs. 31, 32, 33, 40). It is within these small segments, or "protospores," that the mitotic division is completed (Figs. 32-41). The ultrastructural details of the division are basically similar to those described

Fig. 19. Portion of young sporangium prior to the initiation of capillitial formation. Note the large number of vacuoles such as the one shown at V. • 15,000 Fig. 20. Portion of one of the tubular vacuoles in whi& capillitial threads form. Note the fibrillar material (at arrows) surroundingthe capillitial vacuole. )<15,000

A Study of Sporula~ion in the Myxomycete Stemonitis virginiensis

Figs. 19 and 20

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CH. W. MIMs

Figs. 21-23. Stages in the formation of capillitiai tixreads. A very young thread is shown at the arrow in Fig. 21. Note the wall of the threads in Figs. 22 and 23 and the electron-transparent lumen. Also note the fibrillar material surrounding the capillitial vacuoles in all three figures. Fig. 21 X7,500. Fig. 22 • Fig. 23 •

A Study of Sporulation in the Myxomycete Stemonitis virginiensis

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Fig. 24. Light micrograph of the apex of a mature sporangium in which the spores have been removed to show the capitlitium. X400 Figs. 25-27. Sections of capiliitial threads as seen with the electron microscope. 5<12,000

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Ct~. W. M~MS

Figs. 28 and 29. Sections showing the membrane bounded vesicles which function in spore cleavage. Note the metaphase nucleus (N) in Fig. 28. X 18,000

A Study of Sporulation in the Myxomycete Stemonitis virginiensis

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Fig. 30. Portion of a young sporangium in which cleavage is fairly well advanced. Note cleavage furrows (iv) and dividing nuclei. X 10,000

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CH. W. MIMs

Fig. 31. Aceto-orcein squash showing protospores containing metaphase nuclei. X3,000 Figs. 32-38. Aceto-orcein squashes showing various stages in the completion of the mitotic divisions as well as the c]eavage of the protospore into incipient spores. X 3,000 Fig. 39. Mature spores of S. virginiensis. •

A Study of Sporulation in the Myxomycete Stemonitis virginiensis

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Figs. 40 and 41. Anaphase and telophase nuciei in protospores. X 18,000

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MIMs:A Study of Sporulation in the MyxomyceteSternonitis virginiensis

previously in myxomycetes (ALDRICH 1969, MIMS 1972 a). The division involves no centrioles and is intranuclear with the nuclear envelope remaining intact until late in telophase (Fig. 41). Following mitosis each protospore cleaves or pinches into uninucleate segments (Figs. 37, 38, 42) each of which develops into a spore as a thick wall is formed around it (Figs. 39 and 43). Exactly how the wall is formed is not clear although the general sequence of events resembes that in the slime mold Arcyria cinerea (Bull.) Pets. (MIMs 1972 c). After completion of wall formation meiosis occurs within each spore. Synaptonemal complexes (Fig. 43) and division figures have been observed in spores 12-16 hours after cleavage although the exact details of the meiotic divisions are not known. The fine structure of post-meiotic spores of S. virginiensis has been described elsewhere (MIMs, I972 b). 4. D i s c u s s i o n

To the author's knowledge this is the first report of the spore to spore culture of the slime mold S. virginiensis. The details of the morphology of the plasmodium and its conversion to sporophores are basically similar to those described for other members of the same genus with one exception. S. virginiensis does not form the aggregated, coralloid-like plasmodium prior to sporulation as apparently do other members of the same genus (ALExOPOULOS 1959, GRAY and ALEXOPOULOS1968, INDIRA 1971, MCMANUS and RICHMAN 1961). The coralloid stage has probably been best illustrated in S. herbatica (INDIRA1971) and in Sternonitis sp. (GRAY and ALEXOI?OULOS 1968). According to INRIRA (1971) the coralloid stage is a more or less erect structure which moves continously over the surface of the culture for two to several days before finally giving rise to sporophores. The plasmodium of S. virginiensis does thicken and aggregate shortly before fruiting, but it remains relatively flat and does not exhibit the rapid and prolonged movement as does the coralloid plasmodium of S. herbatica. Most of the information available on the sporulation process in Sternonitis has come from three studies. BIsBY (1914) has examined S. fusca Roth. while various stages in the development of sporangia of S. fusca, S. pallida Wing., S. smithii Macbr., and S. herbatica Peck have been studied by Ross (1957). The most detailed study on a member of the genus, however, has been by INDIRA (1971) who was able to grow S. herbatica in culture and study both squash preparations and paraffin sections of developing sporangia with the light microscope. BISBY and Ross utilized material collected in the field or material which sporulated on wood brought into the laboratory.

Fig. 42. Incipient spore of S. virginiensis. X 15,000 Fig. 43. Portion of a spore some 14 hours after cleavage. Note the spore wall (W) and the synaptonemalcomplexes(SC) in the nucleus. •

Figs. 42 and 43 4*

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Cn. W. MIMs

The formation of the stalk and columella in S. virginiensis appears to be basically similar to that described in the studies mentioned above. The stalk is deposited on the hypothallus and develops within the sporangiaI initial by the addition of material to its apex by the surrounding protoplasm. The columella is then formed by the continued deposition of material to the stalk. The tip of the columella, as suggested by Ross (1957) is not separated from the protoplasm by a membrane. The columella therefore appears to form as an intraprotoplasmic secretion. Neither the chemical make-up or the origin of the material utilized in stalk and columella formation is known although cellulose has been demonstrated in the stalk of Cornatricha, another of the Sternonitales (GooDWlN 1961). While there is general agreement concerning stalk and columella formation in Stemonitis, such is not the case for capillitial formation. The controversy concerns the involvement of a vacuolar system in the formation of the capillitium. Both BIsBx (1914) and INDmA (1971) have described such a system, but Ross (1957) failed to find vacuolar elements and suggested that the capillitium, like the stalk and columella, formed as a result of intraprotoplasmic secretion. In the species he examined Ross (1957) reported that the capillitium arose both from the columella and free in the cytoplasm, although he did not totally exclude the possibility that the squash technique he employed may have destroyed any vacuoles present. From the results of the present study it is evident that at least a portion of the capillitium is formed inside a system of tubular vacuolar elements. The system is not unlike that described previously in A. cinerea (MIMS1969), one of the Trichiales, although food vacuoles which were incorporated into the vacuolar system in Arcyria were not noted in S. virginiensis. The vacuolar system appears to arise as a result of the fusion and enlargement of pre-existing vacuoles rather than by the invagination of the plasma membrane (BIsBY 1914) or the breakdown of protoplasm along certain tracts (INDIRA 1971). Once the anastomosing system of tubular elements is established throughout the sporangium the capillitial threads form within the vacuoles although the exact details of this process are not known. The possibility that some threads arise as a result of intraprotoplasmic secretion can not be eliminated altogether, however. There appears to be, as Ross (1957) suggested, no membrane separating the tip of the columella from the surrounding protoplasm. If this is the case, then the threads which arise from the columella as a result of branching may actually be formed by intraprotoplasmic secretion. One of the most interesting observations in this study concerns the timing of the mitotic division and the spore cleavage process in the young sporangium. A great volume of literature has collected concerning nuclear division and cleavage, mainly as a result of the prolonged controversy over the site of meiosis in the life cycle of the Myxomycetes. For many years it was generally accepted that two nuclear divisions, or meiosis, preceded cleavage. It now appears,

A Study of Sporulation in the Myxomycete Stemonitis virginiensis

53

however, that only one mitotic division takes place in the sporangium with meiosis occurring sometime later in the spores (ALDRICH 1969, ALDRICH and MIMS 1970, ALDRICH and CARROLL 1971, LESTOURGEON, BOHNSTEDT, and THIMELL 1971). In any event, as is evident from reviewing the literature (ALDRICH 1967, GRAY and AL~XOI'OI3LOS 1968), most investigators have placed the nuclear division, or divisions as previously interpreted, approximately one hour before cleavage or, in a few instances, just before spore delimination. In this study, however, mitosis occurs simultaneously with cleavage and involves the formation of small segments of protoplasm, here termed protospores, typically containing a single metaphase nucleus. It is within the protospore that anaphase and telophase figures are visible. Each protospore then cleaves into two uninucleate segments, each of which develops into a spore. This sequence of events does not appear to be random or haphazard. Apparently the protospore is a definite stage in the cleavage process in S. virginiensis and possibly in other members of the genus. KOEVENIG (1961) noted a similar stage in S. fusca, while INDIRA (1971), though not actually discussing the stage, included an illustration which very much resembles the protospore stage. In closing, it should be pointed out that some question exists concerning the presence of a peridium in Stemonitis (MACBRIDE and MARTIN 1934, ROSS 1957, INDIRA 1971). In this study no peridium was found. A single unit membrane surrounds each sporangium. N o material was noted around the sporangium outside this delimiting membrane. Acknowledgements

The author wishes to thank Dr. C. J. AL~XO~OULOSfor critically reviewing the manuscript and The Cell Research Institute, University of Texas at Austin, for the use of the electron microscope facilities. Supported in part by NSF Grant GB 6812 • to C. J. AI.~xoPouLos and by an SFASU Faculty Research Grant to the author. SFA Pub. No. 78. References

ALDRICH, H. C., 1967: The ultrastructure of meiosis in three species of Physarum. Mycologia 59, 127--148. -- 1969: The ultrastructure of mitosis in myxamoeba and plasmodia of Physarum flavicomum. Amer. J. Bot. 56 (3), 290--299. - - and C. W. MIMS, 1970: Synaptonemal complexes and meiosis in myxomycetes. Amer. J. Bot. 57 (8), 935--941. -- and G. CAI~ROI~L,1971: Synaptonemal complexes and meiosis in Didymium iridis: a reinvestigation. Mycologia 63, 308--316. AL~XOPOULOS, C. J., 1959: The laboratory cultivation of Stemonitis. Amer. J. Bot. 46, 140--142. -- 1969: The experimental approach to the taxonomy of the myxomycetes. Mycologia 61, 219--239. BAI~Y, A. De, 1887: Comparative morphology and biology of the fungi, mycetozoa and bacteria (Eng. trans.), London: Clarendon Press.

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MIMS:A Study of Sporulation in the Myxomycete Sternonitis virginiensis

BISBY, G. R., 1914: Some observations on the formation of the capillitium and the development of Physarum mirabilis Peck and Stemonitis fusca Roth. Amer. J. Bot. 1, 274--288. GOODW~N, D. C., 1961: Morphogenesis of the sporangium of Comatricha. Amer. J. Bot. 48, 148--154. GRAY, W. D., and C. J. ALEXOI'OUtOS, 1968: Biology of the myxomycetes. New York: Ronald Press. INDIRA, P. U., 1971: The life cycle of Stemonitis herbatica. II. Trans. Br. mycol. Soc. 56 (2), 251--259. KOEVENIa, J. L., 1961: Three educational films on myxomycetes with a study of the life cycle of Physarum gyrosurn Rost. Ph. D. Thesis, University of Iowa, Iowa City. LESTouRa~O>~, W. M., C. F. BOHNSTEDT, and P. THIMELL, 197i: Supportive evidence for postcleavage meiosis in Physarum flavicomum. Mycologia 63, 1002--1011. MAC~RIDe, T. H., and G. W. MARTIN, 1934: The North American slime molds. New York: Macmillan Co. McMANus, S~STER M. A., and SrST~R M. V. RmHMOND, t961: Spore to spore culture on agar of Stemonitis fusca. Am. Midl. Nat. 65, 246. MIMS, C. W., 1969: Capillitial formation in Arcyria cinerea. Mycologia 61, 784--798. - - 1 9 7 2 a : An ultrastructural study of precleavage mitosis in the mycomycete Arcyria cinerea. J. Gen. Micro. 71, 53--62. --1972b: Centrioles and golgi apparatus in postmeiotic spores of the myxomycete Stemonitis virginiensis. Mycologia 64, 452--456. - - 1972 c: Spore wall formation in the myxomycete Arcyria cinerea. Trans. Br. mycol. Soc. (in press). Ross, I. K., 1957: Capillitial formation in the Stemonitaceae. Mycologia 49, 809--819. SCHUSTER, F. L., 1964: Electron microscopic observations on spore formation in the slime mold Didymium nigripes. J. Protozool. 11, 207--216. Author's address: Dr. CH. W. M~MS, Department of Biology, Stephen F. Austin State University, Nacogdoches, Texas, U.S.A.