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Trans. Br. mycol. Soc. 88 (4), 479-488 (1987) Printed in Great Britain

CYSTIDIAL MORPHOGENETIC FIELD IN THE HYMENIUMOF COPRINUS CINEREUS

By JACQUELINE HORNER AND DAVID MOOREMicrobiology Research Group, Department of Cell and Structural Biology, School of Biological Sciences,

Williamson Building, University of Manchester, Manchester Mil 9PL

Cystidia spanning the gill cavity may be 'distant', having other cells separating them, or'adjacent', with no intervening cell; and, in either case, both cystidia may emerge from thesame hymenium (described here as 'cis') or from opposite hymenia ('trans'). If thedistribution of cystidia is entirely randomized the frequency of adjacent pairs will depend onthe population density and there will be an equal number of cis and trans in both the distantand adjacent categories. Quantitative data from serial sections of a primordium show that thereis a positive inhibition offormation of neighbouring cystidia in the same hymenium such thatformation of a cystidium actively lowers the probability of another being formed in theimmediate vicinity. The extent of the inhibitory influence extends over a radius of about30 pm and is strictly limited to the hymenium of origin. Cystidial density distribution on theface of the gill is fairly uniform, but at the gill edge the density of cystidia is locally increased.It is suggested that differentiation leading to cystidium formation is activated by theconcentration of a component of the atmosphere, possibly water vapour, in the gill cavityimmediately above the developing hymenium. The distribution pattern of cystidia is thusdependent on interplay between activating and inhibiting factors. At early stages in growthof the cystidium across the gill cavity the cell(s) with which the cystidium will come intocontact in the opposing hymenium are indistinguishable from their fellow probasidia.However, when the cystidium comes firmly into contact with the opposing hymenium, thehymenial cells with which it collides develop a distinct granular and vacuolated cytoplasm,more akin to that of the cystidium itself than to the neighbouring probasidia. This suggeststhat a contact stimulus sets in train an alternative pathway of differentiation leading to anadhesive cell type called the cystesium.

The hymenium of Coprinus cinereus is commonlyconsidered to comprise three cell types: basidia,paraphyses and cystidia. Although the paraphysesexpand to become the major structural members ofthe gill lamellae, they arise secondarily as branchesfrom sub-basidial compartments, and when firstformed the hymenium consists of a carpet ofpro basidia with a scattering of cystidia (Rosin &Moore, 1985a). We prefer to use the termpro basidia rather than basidiole to signify imma-ture basidia, since the latter term has been definedas referring both to basidia which have not yetproduced spores and to morphologically similar,but sterile cells which become coprinoid para-physes (Smith, 1966). This is misleading, as wehave shown (Rosin & Moore, 1985a) that theparaphyses arise as sub-basidial branches, soalthough they may pass through morphologicallysimilar states, the two cell types are very differentontogenetically. The terminology used must reflectthis.

Cystidia ' ... occur haphazardly in the hy-

menium, depending on the species, and vary fromabundant to absent· .. ' (Smith, 1966). Much ofthe literature dealing with cystidia considers themfrom the taxonomic viewpoint (e.g. Lentz, 1954;Price, 1973), reflecting assiduous attention to finedistinctions of nomenclature at the expense ofappreciation of the remarkable developmentalplasticity revealed by their occurrence and form.Brefeld (1887, cited in Buller, 1910) concluded thatcystidia are metamorphosed basidia, a viewsummarized by Corner (1947) in the phrase, ... cystidia represent sterile basidia whichbecome overgrown"". Certainly, both prob-asidia and procystidia originate as the terminalcompartments of branches from the hyphae of thesub-hymenium, but cystidia are not overgrownbasidia. The mature cystidium is a cell that ishighly differentiated for its particular (poorlyunderstood) function. Thus, the development of acystidium represents expression of a perfectlyrespectable alternative pathway of differentiation,and it follows from the observations of Rosin &

RESUL TS AND DISCUSSION

Regulation of cystidium formation

Internal and/or external control signals mustregulate branching and cystidium developmentand, since cystidia are dispersed, an analysis of theirdistribution may suggest the nature of thosecontrols. There are a number of components to adescription of cystidial distribution. Cystidiaemerge from a particular hymenium and span thegill cavity. Thus two neighbouring cystidia may be(a) 'distant', having other cells (initially probasidia,later basidia and their daughter paraphyses)separating them, or (b) 'adjacent', with nointervening cell; and in either case, both cystidiamay emerge from the same hymenium (describedhere as 'cis'; e.g. Fig. 1C) or from oppositehymenia (' trans'; e.g. Fig. 6A). If the distributionof cystidia is entirely randomized the frequency ofadjacent pairs will depend on the populationdensity and there will be an equal number of cisandtrans in both the distant and adjacent categories.Quantitative data were obtained from a series of2 pm thick serial sections of a Stage 1 primordium(similar to Fig. 1). The first 169 cystidia observedin median section were scored for their direction ofgrowth - either clockwise or anticlockwise as

Hyphal origin

Cystidia in C. cinereus are the terminal compart-ments of hyphal branches from the subhymenium,but they are not derived from any particular rankof branch (Corner, 1947), nor do they arise from alower stratum in the hymenium (Boudier, 1886),and the idea that 'cystidial hyphae' are a separatepopulation from 'basidial hyphae' (Rosin &Moore, 1985a) is not supportable. Careful examin-ation of sections shows that cystidia belong to thesame branching systems as their neighbouringprobasidia (Fig. 1). The two cell types thusrepresent alternative fates for otherwise indis-tinguishable hyphal tips approaching the hyme-nium from the subhymenial hyphae. Lentz (1954)describes the branching pattern giving rise to thehymenium as cyme-like, corymbose or sympodial;a more vivid description might be 'crowded'. Socrowded that it is doubtful whether the characteris-tic branching pattern, if there is one, could bedistinguished with anything less than reconstruc-tion from serial electron microscope sections.Incidentally, images such as those in Fig. 1highlight dramatically the changed growth patternexhibited by a dikaryon during fruit body forma-tion. In vegetative growth on the surface of an agarmedium, leading hyphae of this dikaryon producebranches at intervals of approx 73 pm.

MATERIALS AND METHODS

Organism and culture conditions

A dikaryon of Coprinus cinereus (Schaeff.: Fr.)S. F. Gray sensu Konr. (ATCC 42721) was fruitedon horse dung (Moore & Ewaze, 1976) and fruitbodies at two stages of development were used formicroscopic analysis. Stage 1 primordia (2-6 mmin height, prekaryogamy) and Stage 2 primordia(6-9 mm in height, meiosis occurs during thisstage) were harvested for microscopic analysis.Moore, Elhiti & Butler (1979) have described thesix stages into which fruit body development hasbeen divided.

480 Hymenial morphogenesis in Coprinus cinereusMoore (1985a, b) that commitment to the cystidialas opposed to the basidial pathway of differentiationmust occur very early in development of thehymenium. Nothing is known about the controlswhich might determine formation of a cystidium,rather than a basidium, by a particular hyphal apex.Indeed, it is not even known whether the sorts ofdescription commonly applied to the distributionof the former, e.g. scattering, haphazard (seeabove), 'fairly uniform' (Buller, 1910), are mean-ingful or whether there is some pattern in theirdistribution which might reflect those ontogeneticcontrols and/or the functions of these enormouslyinflated cells.

Cystidia are found in fair number in thehymenium of C. cinereus. As part of our attempt todefine the fundamental characteristics which de-termine the pattern of hyphal differentiation in thehymenium of this organism we have investigated,and report in this paper, the origin and distributionof cystidia. We also identify a new cell type, thecystesium, which is derived from a probasidiumthat, rather than continuing development to abasidium, becomes specialized to adhere to acystidium arising from the opposing hymenial face.

Microscopic preparation

Sections used for light microscopy were 2 pm thick,being cut from resin blocks containing materialprepared as described by Rosin & Moore (1985b).For scanning electron microscopy, tissue wasdissected from fresh fruit bodies and eithershock-frozen in nitrogen slush prior to freeze-drying, or fixed for 2 h in 3 % glutaraldehyde in0'05 M Sorenson's buffer (pH 6'8) and dehydratedthrough an ethanol series prior to critical-pointdrying. Mounted samples were coated with goldand viewed.

Jacqueline Horner and David Moore 481

Fig. 1. Branching patterns at cystidium bases. Cystidia (cy) and probasidia (ba) arise from the samesubhymenial branch systems with no indication of a particular arrangement of branches. Scale bars = 20 flm.

Hymenial morphogenesis in Coprinus cinereus

Table 1. Distances between nearest-neighbour pairs of cystidia observed in gill sections, comparing pairs inwhich both members originated from the same hymenia (cis) with those in which the two cystidia arose fromopposing hymenia (trans)

Number ofpairs

examined Mean S.D. Units

Cystidia in cis 133 156'7 102'1 !tm23 15 cell diameters

(probasidia)Cystidia in trans 129 113'4 96'3 !tm

17 14 cell diameters(probasidia)

viewed from the top of the fruit body. The outcomewas a ratio of82 :87, not significantly different fromequality, so we conclude that the two hymeniabordering a gill cavity are equally likely to give riseto cystidia spanning that cavity. Every fifth sectionwas searched for pairs of nearest-neighbourcystidia, in which the hymenial origin of bothwas unambiguous; their separation distance wasmeasured and they were scored as either cis ortrans. A total of 262 cystidial pairs were examined,and results are shown in Table 1. Measurement ofthese distances is potentially contentious, as thehymenium is expanding by both cell enlargementand cell insertion throughout fruit-body develop-ment. However, the primordial stage 1 chosen forexamination is that which precedes emergence ofparaphyses (Rosin & Moore, 1985a), so thehymenium is structurally at its simplest and themeasurements of physical distance can be trans-lated easily into the potentially more meaningfulunit ofmeasurement of cell (probasidial) diameters.Both measures are given in Table 1, but whateverthe units it is clear that the two categories ofcystidial arrangement are differently distributed.Moreover, among the 262 pairs examined, only 20

were adjacent, and these were all in trans. The lackof cis-adjacent pairs of cystidia is not simply due totheir slightly lower population density. Thisconclusion is based on comparison of the observedand expected numbers of cis-adjacent cystidia. If itis assumed that distances between neighbouringcystidia are normally distributed, the mean and S.D.(which mathematically describe the distribution)can be used to calculate the expected number ofcis-adjacent pairs of cystidia at any given separationdistance by calculating the appropriate area underthe normal probability curve. The mean diameterof cystidia in these sections was 17 p.m. If this valueis used as the mean separation distance for adjacentcystidia, calculation shows that the expectednumber of cystidial pairs separated by this distanceor less in this sample was 11'35; none was observed,and the probability of the null observation being

due to chance is less than 0'0005. When the samecalculation is applied to cystidia in trans it emergesthat 20'5 trans-adjacent pairs are expected, whereas20 were observed.

Thus the distances between neighbouring trans-disposed cystidia are approximately normallydistributed (not strictly a normal distribution, ofcourse, as there is a finite minimum distancecorresponding to the mean cystidial diameter); butthe distribution of the population of measureddistances between the cis-disposed pairs does notapproach a normal distribution. There is evidentlya positive inhibition of formation of neighbouringcystidia in the same hymenium such that formationof a cystidium actively lowers the probability ofanother being formed in the immediate vicinity.The extent of the inhibitory influence can bejudged by comparing observations with calcula-tions of the expected number of cis-disposed pairsat successively greater separations. This is shownin Fig. 2, in which the distance between cystidia isexpressed in terms of pro basidial diameters (unitsof 7 p.m). The inhibitory effect extends up to fourprobasidial diameters. This defines the radius ofthe cystidial morphogenetic field, which is strictlylimited to the hymenium of origin.

Density distribution

Mature cystidia are usually essentially at rightangles to their hymenia (see Fig. 2 in Moore &Pukkila (1985) and illustrations of various speciesin Buller (1924)), yet most of the increase in areaof the hymenium takes place through insertion andexpansion of paraphyses after cystidia are firmlyconnected to the two hymenia each side of the gillcavity which they span. For the cystidia not to begrossly distorted during maturation of the gillsimplies either that the two hymenia either side ofa gill space are positively co-ordinated in terms ofthe number of paraphyses which insert and theirsubsequent extent of expansion, or that thesefeatures are so perfectly randomized over such alarge cell population that, on balance, equality of

Fig . 2. Inh ibition of cystidium differentiation withinrange of a preformed cystidiurn. The graph shows theobserved number of cis-arranged pairs of cystidia(expressed as a fraction of the expected number) atsuccessively greater distances of separation. The expectednumber was calculated for each distance as the area underthe normal probability curve represented by the meanand S.D. shown in Table 1. The separation distancebetween cystidia is shown on the abscissa in ceIl(probasidial) diameters; in these specimens probasidiawere about 7 /lm diam. If the presence of a cystidium hadno influence on occurrence of a second in the immediatevicinity the points would be stochasticaIIy scatteredaround an ordinate value of 1. In reality this is true onlyfor cystidia separated by fiveor more probas idia. At lesserdistances positive inhibition of cystid ium differentiationin the vicinity is observed. We thank Dr J. M. Bond forsuggesting the form of this graph to us.

extension occurs. Without experimental interven-tion (which is difficult, since the gill cavities arecompletely enclosed) it is probably impossible todistinguish between these two possibilities. Hencethe opportunity to compare the distributions of cis-and trans-disposed cystidia permits a rare insightinto the relationships between cells in a hymeniumand between opposing hymenia. The indicationthat the cystidial morphogenetic field is limited toits own hymenium implies that opposing hymeniaare integrated only in so far as similar events occurin both ; those events are essentially randomized;and a large cell population is involved .

Randomness, of course, does not imply lack oforder; nor would we wish to imply with thedescription we have just given that the gill isassembled in a mechanical, unresponsive way.Cystidial density distribution appears to be ran-domized and fairly uniform except at the gill edge(Fig. 3), where the density of cystidia is locallyincreased to the point where cis-adjacent pairs ofcystidia can be found . This observation indicatesthat the rule for forming a cystidium must havesome environmental cue. The distributions ana-

76

Jacqueline Horner and David Moore 483lysed above, which apply to the general gill surface ,could well operate without reference to theenvironment external to the hyphaI branchingsystem on the basis of something like' on averageevery fifth branch is a cystidiurn unless one of theprevious four branches was a cystidium '. Thiswould clearly not be responsive to position withinthe gill, nor can it be responsive unless thehymenial cells are capable of perceiving andresponding to some sort of positional informationfrom their environment which identifies the gilledge.

The gill cavity seems to be the appropriateenvironment to consider, and regulation based onsensing a gas or vapour concentration would enableexplanation of the increased density at the gill edge.Wherever a gill edge is formed the atmosphericvolume is expanded in the immediate vicinity . Thisis true both for the formation of secondary gills bydivision of the gill-organizing centre (Rosin &Moore, 1985b) and for primary gill edges whichbecome exposed to the developing annular cavity.So if cystidium differentiation is activated by alocally decreased concentration of some vapour,activation would be amplified inevitably in theregion of the gill edge, allowing for closure of theexposed trama as primary gills separate from thestipe (Fig. 4A) and additional buttressing ofsecondary gills (Fig . 4B). The nature of thecontrolling molecule is unknown. Carbon dioxideinfluences fruit-body growth differentially inAgaricus (Lambert, 1933; Turner, 1977); am-monia-scavenging enzymes are specifically dere-pressed in the Coprinus fruit-body cap (Moore,1984), at least one of which has been localized to thecell surface in both vegetative mycelium (Elhiti,Moore & Butler, 1986) and the hymenium(M . M. Y. Elhiti, R. D. Butler & D. Moore,unpubl.), and ammonia is known to be a morpho-genetic regulator in Dictyostelium (Schindler &Sussman, 1977). However, cystidia are thought ofas functioning generally in maintenance ofhymenialhumidity (Smith, 1966) by 'transpiring' watervapour into the gill cavity. Thus it could be that thevapour pressure of water over the just-differen-tiating hymenium is the determining factor . Hyphaltips forming the hymenium may be sensitive to thewater vapour pressures in the gill cavity, having aparticular probability of differentiating into acystidium when activated to do so by locallyreduced humidity. It is significant that Gamow &Bottger (1982) concluded that water is a se1f-emitted, growth-stimulating avoidance gas in thesporangiophore of Phycomyces. Ifa similar situationobtains in the cystidium then, once activated,progress of cystidial differentiation would beessentially autocatalytic. Such a scheme would

2 3 4 5Probasidial diameters

o

1·2

0·2

1·0

0'4

Observed 0 ·8

Expecte d 0' 6

Hymenial morphogenesis in Coprinus cinereus

Fig. 3. Scanning electron micrographs of the hymenial surface. (A) Very regularly spaced distribution ofcystidia arising from the hymenial surface on the open face of the gill. (B) Increased crowding as the gilledge - that closest to the stipe, at the right of the picture - is approached. The' pits' in the hymenial surfaceare places where cystidia from the opposing gill face have been pulled away from their cystesia when thegills were separated. Scale bars = 100 pm.

Jacqueline Horner and David Moore

Fig. 4. The edge of a primary gill (A) showing increased frequency of cystidium formation in the expandedspace where the gill has separated from the outer layers ofthe stipe (lipsanenchyma), compared with the edgeof a secondary gill (B), where the increased space in the gill cavity has induced both neighbouring hymeniato produce cystidia to buttress the secondary gill edge symmetrically. Scale bars = 20 flm.

allow for coincident activation of cystidia in theequivalent positions in opposing hymenia, at afrequency equal to the square of the individualprobability of formation of a cystidium. Using thefigures given in Table 1 (20 adjacents in 129crans-arranged pairs) the latter frequency iscalculated to be 0'39; i.e. a hyphal tip in thehymenium has a probability of about 40 % offorming a cystidium when exposed to activatingconditions.

The differentiating cystidium inhibits furthercystidial formation on its own hymenium over aradius of about 30 pm. This may be due toproduction of an active inhibitor or to localdiversion of metabolites (particularly water?) tothe already committed cystidium. In either case thepatterning process is open to analysis and simula-tion using the activator-inhibitor model developed

by Meinhardt (1982) to account for stomatal, cilial,hair and bristle distributions.

Hymenial adhesion

At the gill edge, expansion of the gill cavitypresumably amplifies desiccation stress and acti-vates cystidial formation to such an extent that theeffect of the local inhibitor is masked andcis-adjacent neighbours can be formed (Fig. 4A). Itis this circumstance which indicates most stronglythat cystidium formation is responsive to cuesreceived from outside the parental hymenium.Evidence that a cystidium exerts a formativeinfluence on the opposing hymenium comes fromexamination of the cells which cystidia contact.Mature cystidia are embedded into the opposinggill face. The final structural result - firm con-

486 Hymenial morphogenesis in Coprinus cinereus

Fig. 5. An earlystagein contactbetween a cystidiumand its opposinghymenium showing that the cellswithwhich the cystidium collides are not morphologically different from their neighbouring probasidia. Scalebar = zo sm.

nexion between the cystidium and the opposinghymenium, has often been remarked upon andillustrated. Inevitably, the most relevant descrip-tions are found in Buller (1910, 1924). In Coprinusatramentarius Buller (1910) records that 'Whenone succeeds in forcibly separating parts of twoneighbouring gills, one finds that although thecystidia have mostly separated from one of the gillsat their apical ends, and have thus remainedattached to the outer gill at their basal ends' .. notinfrequently the reverse happens; the cystidia : ..have broken away at their basal ends and haveremained attached at their apical ends.' Similarly,with reference to C. lagopus: 'If the gills are tornapart one end of each cystidiurn remains attachedto one of the gills, whilst the other becomes free.Very often the free end has attached to it one of theclasping cells, thus proving how strongly these cellsadhere to the cystidial wall' (Buller, 1924). Despitesuch graphic descriptions, with their clear indica-tion that adhesion between the cystidium and the'clasping cells' can be so strong as to exceed thebreaking strain of the subhymenial hyphal branch-ing system, causing cells to be torn out of theirhymenium of origin, the fact that the cells that

adhere to the tip of the cystidium show evidence ofa specifically induced adjustment to their progressof differentiation has escaped description. At earlystages in the growth of the cystidium across the gillcavity the sub-basidial branches which will becomeparaphyses have not yet formed and the cell(s) withwhich the cystidium will come into contact in theopposing hymenium are indistinguishable fromtheir fellow pro basidia, though they may showavoidance growth occasioned by the presence ofthe encroaching cystidium (Fig. 5, and see Fig. 2

in Rosin & Moore, 1985b). Yet when the cystidiumcomes firmly into contact with the opposinghymenium, the hymenial cells with which itcollides develop a distinct granular and vacuolatedcytoplasm, more akin to that of the cystidium itselfthan to the neighbouring pro basidia (Fig. 6). Thisis suggestive of some contact stimulus abortingcontinuation of basidial differentiation in thosecells and setting in train an alternative pathway ofdifferentiation. The effect is highly localized,seemingly limited to cells which contact the tip ofthe cystidium, since more laterally placed neigh-bours remain unaffected. We believe that thedifferentiation of these cells warrants their recogni-

Jacqueline Horner and David Moore

A

Fig. 6. Cystidia are firmly interlocked with cells in the opposing hymenia and have caused the latter drasticallyto change their cytoplasmic morphology - to become cystesia . Note that only the tip of the cystidium exertsthis influence. Scale bars = zo um.

We thank the Science and Engineering ResearchCouncil for the award of Research GrantGR/C/42422 to D. M. and a Research Studentshipto I.H.

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(Received for publication 28 October 1986)