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Trans. Br. mycol. Soc. 84 (4), 621-628 (1985) Printed in Great Britain
DIFFERENTIATION OF THE HYMENIUM IN COPRINUS CINEREUS
By ISABELLE V. ROSIN AND DAVID MOORE Department of Botany, The University, Manchester MIl 9PL
When first formed the hymenium ofCoprinus cinereus consists of protobasidia and a scattering of cystidia. The latter arise as the terminal compartments of little-branched tramal hyphae. Paraphyses originate as branches ofsub-basidial cells and insert into the basidial layer . About 75 % of the paraphyseal population inserted as meiosis was completed, the rest at later stages of development. Basidial numbers did not increase once paraphyseal insertion commenced. A fully differentiated fruit body cap of Coprinus polynucleotide level (Yashar & Pukkila, 1985). An cinereus (Schaeff.: Fr.) S. F. Gray sensu Konr. important point, though, is that hymenial tissue is consists ofa thin layer ofpileal flesh (the pileipellis) a fungal tissue and is a derivative of the hyphal bounded exteriorly by the veil cells and with the grade of organization, the characteristics of which gills suspended on the inner side. The gills develop may well impose restrictions on the applicability of parallel to one another and retain this arrangement theoretical models derived from other organisms. throughout development. The gill surface is the An essential prerequisite for study of any tissue hymenium; it is composed of three highly is a knowledge of its exact structure and develop- differentiated cell types, basidia, cystidia and ment. Although fungal fruiting body structure has paraphyses, all of which arise from the central layer been studied for many years, and often from a of the gill, the trama, the constituent cells of which developmental point of view, the published maintain a basically hyphal structure. information is inadequate to establish the nature of Although considerable interest has centred on cell-to-cell relationships in the detail which is basidial and cystidial structure for taxonomic and required in morphogenetic analysis. In this paper phylogenetic purposes (Smith, 1966; Oberwinkler, we describe the structure of the developing 1982) and in studies of basidiosporogenesis hymenium of C. cinereus, with emphasis on (McLaughlin, 1982; Thielke, 1982), there has been structural, developmental and geometrical cell no extensive treatment of the hymenium as a relationships. differentiating tissue. It is, nevertheless, a highly organized morphogenetic system which presents many of the development problems more usually MATERIALS AND METHODS associated with higher organisms (Wolpert, 1969; The dikaryon, culture conditions and preparative Barlow & Carr, 1984; Moore, 1984a). These techniques described by Rosin & Moore (1985) problems relate to the ways in which cells, of the were employed. same genetic constitution, differentiate in a develop- ing tissue in particular patterns that imply some sort of positional control extending over the tissue RESULTS AND DISCUSSION as a whole. Understanding of such regulation of The young hymenium consisted of a palisade of pattern formation will depend on determination of tightly packed club-shaped cells with a scattering biochemical events governing differentiation and of much larger, highly inflated cells (Fig. 1). The subsequently the orchestration of control of the latter are presumed to be developing cystidia biochemistry over large populations of spatially- though on the basis of these images there is no way scattered cells. Description of some biochemical of categorising the former either as basidia or events associated with hymenium development in paraphyses. However, two lines of evidence C. cinereus has reached an advanced stage (Ewaze, indicatethatin theseyoungprimordia (representing Moore & Stewart, 1978; Moore, Elhiti & Butler, Stage 1,2-6 mm in height, and Stage 2, 6-9mm in 1979; Moore, 1981), and some attempt has been height) the hymenium is largely composed of made to relate findings derived from work with differentiating basidia. The first is that, as we shall tissue homogenates to cytochemically-observable show below, paraphyses were observed to appear processes in individual cells (Elhiti, Butler & later and were inserted into the basidial layer ; the Moore, 1979) and to extend the analysis to the second is that in studies of meiosis (which occurs 622 Differentiation in Coprinus cinereus
Fig. 1. Protohymenium which has just differentiated from the protenchymal tissue of the cap otthe G. cinereus fruit body. Scale bar = 5/lm.
during Stage 2) Raju & Lu (1970) showed that pruem, }ersild & Lane, 1971; Niederpruem & meiotic figures could be demonstrated in virtually jersild, 1972). all of the hymenial cells, an observation which has Cystidia arose from tramal hyphae which recently been confirmed (Pukkila, pers. comm.), branchedrelatively muchless than thosesubtending We will therefore refer to these cells as basidia since basidia (Fig. 2). From the earliest stage cystidia only this cell type is expected to undergo inserted into the opposite hymenium. Cystidia karyogamy and meiosis. stretched considerably when two gills were pulled Basidia originated as slightly swollen chromo- apart, indicating a firm attachment. Reijnders philic cells at the apex of protenchyme (tramal) (1963) gives an account of the nomenclature and hyphae. This apical differentiation follows the structure of these cells. pattern of most basidiomycetes (Sundberg, 1978; Towards Stage 2, cystidia and the apices of Oberwinkler, 1982) butMcLaughlin(1982) empha- basidia enlarged, The expansion of basidial apices sised the ultrastructural differences between the was often associated with basidiocarps in which the developing basidium and the growing vegetative hyphaeofthe trama, subhymeniumand hymenium, hyphal apex, especially the lack ofapical vesicles in showed similar expansion (Oberwinkler, 1982). the basidium which he suggested may indicate a Paraphyses developed as outgrowthsofsub-basidial slow growth rate. In C. cinereus basidia were the cells during Stage 2. During Stage 1 basidia were terminal cells of branches which curved out from the most chromophilic cells ofthe gill but at Stage the generally parallel tramal hyphae. During the 2 the most chromophilic region was in the early stages of hymenium development it was subhymenium corresponding in position to the noticeable that adjacent basidia arose at the apex of emerging paraphyses. This distribution appears to sister branches of parental tramal hyphae. This is reflect the actively growing zone ofthe lamellae. In the normal mode of basidium proliferation in C. micaceus and C. lagopus, Chow (1934) observed agarics (Oberwinkler, 1982) and contrasts with a similar chromophilic pattern but concluded that proliferation at sub-basidial clamp connexions basidia grew from the subhymenium and into a described in Schizophyllum commune (N ieder- layer of paraphyses. We do not agree with this Isabelle V. Rosin and D. Moore
Fig. 2. Differentiatinghymeniumof C. cinereus. Note that the largecystidialcellsare terminalcompartments of tramal hyphae.The hymeniumat this stageconsistsof a closely packedlayerof basidia,though the highly chromophilicparaphyseal branchesfromthe sub-basidialcellsare becoming evident. Scalebars: A = 20 pm, B = 5 pm.
interpretation. The strongly chromophilic region Only more detailed analysis of serial sections at the in the C. cinereus subhymenium was composed of EM level will show whether 'cystidial tramal branches communicating between the trama and hyphae' constitute a hyphal population distinct hymenium and of small, rather oblong, cells from 'basidial tramal hyphae', but on the basis of (Fig. 2). These latter cells (which are young present observations that possibility certainly paraphyses) arose as branches from the sub-basidial exists. The final point is that paraphyses arise as cells and then inserted between the basidia (Fig. 3). branches from sub-basidial cells and insert into the By Stage 3 (immature fruit body over 10 mm in basidial cell layer. Formation of paraphyseal height, postmeiotic) these cells, still highly chromo- branches appears to occur at about the time that philic, hadexpanded and taken on the characteristic meiosis is taking place. rectangular profile of paraphyses (Fig. 4). By this In primordia of Stages 1-2 the gill hymenium stage the numberofparaphyses had increased to the consisted essentially of a cell layer of basidia and point where no two basidia were adjacent. was about 100 pm wide at the cap margin. At The most important points arising from these maturity, Stage 5, the hymenium was composed of observations are that when first formed the basidia embedded in a pavement of paraphyses and hymenium consists of cells destined to become the gill was about t :5-2 mm wide at the cap margin. basidia or cystidia. The latter an: fewer in number Observations described above showed that para- and are the apical cells of tramal hyphae which physes were inserted into the hymenium layer at branch much less frequently than the majority. about Stage 2/3. At this time about 100 cells 624 Differentiation in Coprinus cinereus
I ..
B Fig. 3. Insertion of paraphyses (the most chromophilic cells) into the basidial layer of the Coprinus hymenium. Scale bars = 5 pm. Isabelle V. Rosin and D. Moore
C Fig. 4. Expansion of the mserted paraphyses to torm tne characteristic paraphyseal pavement ofthe Coprinus hymenium. Scale bars = 10 Jim. 626 Differentiation in Coprinus cinereus
" ..
•
./ c.. ..
B . . Fig. 5. Surface view of the almost mature hymenium of a sporeless mutant of C. cinereus to show the patterns of paraphyseal boundaries and entrapped basidia (ba), Nomarski interference optics. Scale bars: A = 75 flm, B = 25 flm. Isabelle V. Rosin and D. Moore inserted into the gill transect and the observed gill we are assuming that the insertion of cells width was 300-400 pm. The average width of the (specifically paraphyses) into a cell layer is formally inserted paraphyses was 3-4 pm. At Stage 4, equivalent to cell division within the layer. approx. 140 paraphyses were counted along gill Geometrically this seems to be valid providing the transects and they had an average width of 8 pm. argument does not depend on constraints on the Thus, although the main production ofparaphyses plane of division. It is clear that theoretical aspects occurred at the end of Stage 2, about 25 % of the of fungal tissue construction (and especially the population was formed and inserted during the unique considerations arising from the hyphal later stages ofmaturation. Insertion and expansion origin ofthe cells and their characteristic transverse of paraphyses was sufficient to account for the dividing septum) have so far been ignored. We entire increase in gill width. Increase in gill length intend to return to this theme in a later paper. The will, of course, depend on the same factors but is inherent property of a mosaic of cells is that, in a further dependent on continued differentiation of system of linked polygons, the average number of gill tissues from the protenchyma at the apex ofthe cell sides is exactly six, and that the area of a cell cap (Rosin & Moore, 1985). Paraphyseal enlarge- is proportional to the number of sides of the cell ment proceeded continuously during maturation. (Lewis, 1943). The nature of this proportionality Increase in gill area is clearly dependent on an can identify different cell populations. Measure- enormous increase in the constituent cell volumes. ments were made of maximum cell diameter Paraphyses expand as much as two and a half times (replacing area) and the number of sides for 50 their original volume. The ingress ofwater into the randomly chosen basidia and paraphyses in hymenium elements leads to considerable vacuola- photographs of the hymenium surface (Fig. 5) of tion as the cells expand (Moore et al., 1979). The fruitbodies at Stages3and 5. A computer-generated exact identity of an osmoticum has not been unweighted regression of the plot (Fig. 6) shows established, but amplification ofurea cycle activity, that basidia and paraphyses fall into two distinct which occurs specifically in the cap tissue, probably populations. Basidial diameter remained approxi- contributes to the osmotic influx of water (Ewaze mately constant irrespective ofthe number ofsides, et al., 1978; Moore, 1984b). while for paraphyses the number ofsides increased The distribution of paraphyses and basidia has with size. been analysed geometrically using a method based The failure of many-sided cells to attain the size on that of Lewis (1931, 1943). The principles for required to comply with the expected progression geometrical analysis of tissues have been derived has been explained in terms of the division ofcells for layers of dividing cells, in applying them here around a non-dividing cell (Lewis, 1943). In the context of our present knowledge of the ontogeny of the hymenium in C. cinereus, the analogous 21 statement for this tissue would be that additional paraphyses insert around the basidia. Thus the geometrical analysis of basidia and paraphyses reinforces the view that the basidial population is 15 numerically static. Only paraphyses insert into the E hymenial layer after its initial differentiation from -3... the protenchyma (Rosin & Moore, 1985) . 2
(Received for publication ]1 August 1984)