HES UNIVERSITY OF CALIFORNIA AT LOS ANGELES 'e stamped beloi SOUTHERN BRANCH; UNIVERSITY OF CALIFORNIA LIBRARY, tX>S ANGELEa CALIF. /T RESEARCHES OX FtJXGI RESEARCHES ON FUNGI AN ACCOUNT OF THE PRODUCTION, LIBERATION, AND DISPERSION OF THE SPORES OF HYMENO- MYCETES TREATED BOTANICALLY AND PHYSICALLY ALSO SOME OBSERVATIONS UPON THE DISCHARGE AND DISPERSION OF THE SPORES OF ASCOMYCETES AND OF PILOBOLUS BY A. H. REGINALD DULLER B.Sc. (LoND.) ; D.Sc. (BIRM.); PH.D. (LEIP.) PROFESSOR OF BOTANY AT THE UNIVERSITY OF MANITOBA WITH FIVE PLATES AND EIGHTY-THREE FIGURES IN THE TEXT LONGMANS, GREEN AND CO. 39 PATERNOSTER ROW, LONDON NEW YORK, BOMBAY, AND CALCUTTA 1909 67S38 All rights reserved toi TO WILHELM PFEFFER UNDER WHOSE STIMULATING GUIDANCE THE AUTHOR ONCE HAD THE PRIVILEGE OF STUDYING PREFACE THESE pages contain a contribution to the physiology, mor- phology, and physics of reproduction in the Hymenomycetes, and also a record of some observations upon the discharge of spores of 'Ascomycetes and of Pilobolus. Naturally many problems have ^ been left unsolved, but I hope that the new data obtained will \^ give an added interest to some of our commonest plants. The delicate adaptations of structure to function, as revealed by a study of the fruit-body of a Mushroom, a Coprinus comatus, or ^ a Polyporus, have provided me with no small cause for wonder- ^ ment and delight, and they seem well worthy of the attention ^' of all those who desire to understand more fully the vegetable world by which they are surrounded. The value of the more purely physical work must be left to physicists to decide. How- ever, as showing how closely the various branches of science may ^ be knit together, it is not without interest that the first direct V test of Stokes' Law for the fall of microscopic spheres in air has . been carried out with the help of a lowly Cryptogam. The research, which has occupied five years, was preceded and 'N suggested by a systematic study of fungus species in the field, in which I was much assisted by Geo. Massee's British Fungus Flora and M. C. Cooke's Illustrations of British Fungi. During the winters the experimental work was carried on in my own laboratory at the University of Manitoba, and during the summers in the Physics and Botanical laboratories at the University of Birming- ham. I have much pleasure in expressing my best thanks to Professors Poynting and Hillhouse for the facilities accorded me. I also wish to acknowledge my indebtedness to Dr. Guy Barlow viii PREFACE for valuable help and criticism in the more purely physical and mathematical parts of the research. Of the photographs here published ten were kindly made for me by Mr. J. E. Titley of Four Oaks, Warwickshire, three each by Mr. J. H. Pickard and Mr. P. Grafton of Birmingham, and two by Mr. C. W. Lowe of Winnipeg. They are all acknowledged in the text. In the final revision of the proofs, Mr. W. B. Grove has been good enough to give me the benefit of his wide mycological knowledge and experi- ence. Lastly, my gratitude is due to the Birmingham Natural History and Philosophical Society for defraying the cost of three of the Plates. A. H. REGINALD BULLER. WINNIPEG, July 1909. TABLE OF CONTENTS PAGE PREFACE vii PART I Ax /ACCOUNT OF THE PRODUCTION, LIBERATION, AND DISPERSION OF THE SPOKES OF HYMENOMYCETES TREATED BOTANICALLY AND PHYSICALLY INTRODUCTION . CHAPTER I The Hymenium Basidia and Paraphyses Nuclear phenomena The Colour of Spores Two-spored Basidia in Cultivated Varieties of Psalliota cam- pestris Occasional Sterility of Coprinus Fruit-bodies Cystidia Fungus Gnats, Springtails, and Mites Position of the Hymenium Comparison of the Basidium with the Ascus The Effect of Sunlight upon Spores . 6 CHAPTER II The Extent of the Hymenium Principles underlying the Arrangement of Gills and Hymenial Tubes The Margin of Safety The genus Fomes . 27 CHAPTER III The Functions of the Stipe and Flesh of the Pileus The Gill-chamber . 39 CHAPTER IV Adjustments of Fruit-bodies in the Interests of Spore-liberation Lentinus lepideus, Psalliota campestris, Polyporus squamosus, Coprinus plicatilis, ('oprinus niveus, and Coprinus plicatiloides Reactions of Fruit-bodies to Light and Gravity The Problem of Pileus Eccentricity Geotropic Swinging Rudimentary Fruit-bodies 47 CHAPTER V Spore-deposits The Number of Spores 79 CHAPTER VI Macroscopic Observations on the Fall of Spores of Polyporus squamosus . . 89 x TABLE OF CONTENTS CHAPTER VII PAGE The Demonstration of the Fall of Spores by means of a Beam of Light . 94 CHAPTER VIII The Spore-fall Period . . . .102 CHAPTER IX Desiccation of Fruit-bodies A Xerophytic Fungus Flora The genus Schizophyllum 105 CHAPTER X External Conditions and Spore-discharge The Effect of Light The Effect of Gravity The Effect of the Hygroscopic Condition of the Air The Effect of Heat The Effect of Alteration in the Gaseous Environment The Effect of Anaesthetics 120 CHAPTER XI The Violent Projection of Spores from the Hyraenium Methods I., II., III., IV., and V 133 CHAPTER XII The Mechanism of Spore-discharge . . 148 CHAPTER XIII The Specific Gravity of Spores . 153 CHAPTER XIV The Size of Spores Poynting's Plate Micrometer 158 CHAPTER XV The Rate of Fall of Spores and Stokes' Law Appendix < . . . 164 CHAPTER XVI The Effect of Humidity on the Rate of Fall of Spores 179 CHAPTER XVII The Path of the Spores between the Gills, &c. The Sporabola Appendix on the Motion of a Sphere in a Viscous Medium, by Dr. Guy Barlow . 184 TABLE OF CONTENTS xi CHAPTER XVIII PAGE The Electric Charges on the Spores 192 CHAPTER XIX The Coprinus Type of Fruit-body .196 CHAPTER XX The Dispersion of the Spores after Liberation from the Fruit-bodies Falck's Theory 216 CHAPTER XXI The Dispersion of Spores by Animals Coprophilous Hymenomycetes Slugs and Hymenomycetes . . . . . . . . . 224 PART II SOME OBSERVATIONS UPON THE DISCHARGE AND DISPERSION OF THE SPORES OF ASCOMYCETES AND OF PlLOBOLUS CHAPTER I The Dispersion of Spores by the Wind in Ascomycetes Puffing The Physics of the Ascus Jet in Peziza The Fixation of the Spores in the ASCIIS of Peziza repanda Comparison of the Sizes of Wind-borne Spores in Ascomycetes and Hymenomycetes The Helvellacese .... 233 CHAPTER II The Dispersal of the Spores of Ascomycetes by Herbivorous Animals illus- trated by an Account of Ascobolus immersus Pilobolus, Empusa muscx Lycoperdon The Sound produced by the Discharge of Spores with special reference to Pilobolus . . . . . . . . .251 GENERAL SUMMARY Part 1 261 Part II 268 EXPLANATION OF PLATES I.-V 270 PLATES I.-V To follow p. 274 GENERAL INDEX . 275 PART I AN ACCOUNT OF THE PRODUCTION, LIBERATION, AND DISPERSION OF THE SPORES OF HYMENOMYCETES TREATED BOTANIC ALLY AND PHYSICALLY INTRODUCTION THE researches recorded in Part I. were undertaken with the object of throwing light upon the production, liberation, and dispersion of tjie spores of Hymenomycetes. More especially, an effort has been made to find out how the spores manage to escape from the hymenial surfaces where they have been produced, and how they find their way between gills, down tubes, &c., to the exterior of the fruit-bodies. By using appropriate optical methods, it has been attempted to follow the spores individually from the moment they leave the basidia, to determine their paths through the air, and to measure by accurate means their rate of fall. This part of the research has led me to the border-land where botany passes into pure physics. Hitherto, it appears that physicists have never yet determined directly by experiment the rate of fall of individual microscopic spheres with a diameter of 3-10 /* through air. 1 There- fore, by means of observations on the fall of spores, I have en- deavoured to test the well-known and often assumed Stokes' Law. In studying the effect of external conditions upon the liberation of spores, and in determining the length of the spore-fall period, the work has been much simplified by two discoveries. The first is that spore-clouds, and even individual spores, can be seen falling beneath a fruit-body without magnification when illuminated with a concentrated beam of light. Whether or not spores are falling from a fruit-body can thus be ascertained in a few seconds. The second discovery is that practically all the leathery or corky fruit- bodies to be found on logs, ie. those belonging to the genera Lenzites, Polystictus, Dtedalea, Stereum, &c., retain their vitality on desicca- tion for months or years, and that, when they are subsequently placed under moist conditions, the liberation of spores begins once 1 Cf. the Appendix to Chap. XV. 4 INTRODUCTION more within a few hours, and continues for days or weeks. It was therefore possible for me to collect a stock of these fruit-bodies in autumn, to revive them at will, and thus to study the liberation of spores throughout winter and spring. There seems to be but little literature dealing with the liberation of spores of Hymenomycetes. Some observations of Brefeld, 1 given in a footnote in his description of the life-history of Coprinus ster- corarius, will be mentioned and criticised later on. Richard Falck - has published a paper on the scattering of spores of Basidiomycetes, in which he has given an account of the gradual accumulation of spore-deposits on upper surfaces in closed chambers. He did not succeed in actually seeing the spores in the air, but his experiments showed that they are carried with remarkable ease by the slightest air-currents. This fact can be verified directly and very simply by means of my beam-of-light method, and rendered capable of mathe- matical treatment by an exact determination of the rates of fall of the spores in still air. A visible spore-discharge from a fruit-body has been occasionally observed as a very rare phenomenon by a few botanists. To the records of Hoffman, H. von Schrenk, and Hammer 3 will be added my own upon the visible discharge of spores from fruit-bodies of Polyporus squamosus. In his translation of Pfeffer's Physiology of Plants, Ewart 4 added a brief statement of some of my then unpublished conclusions con- cerning the liberation and fall of spores. The evidence in support of these conclusions is brought forward for the first time in this book. In an account of the biology of Polyporus squamosus, I recorded a number of observations upon the fall of spores in that species, and gave an illustration showing the paths taken by the spores in falling down the hymenial tubes. 5 A subsequent calculation, however, Brefeld, Botanische Untersuchunyen iiber Schimmelpilze, III. Heft, pp. 65, 66. R. Falck, "Die Sporenverbreitung bei den Basidiomyceten," Beitriige zur Bio ogle der Pflanzen, Bd. IX., Heft 1, 1904. For references, vide infra, Chap. VI. Pfefter, Physiology of Plants, translated by A. J. Ewart, vol. iii. 1906, p. 416. Buller, "The Biology of Polyporus squamosus, Huds., a Timber-destroying Fungus," TJie Journal of Economic Biology, vol. i., 1906, p. 131. INTRODUCTION 5 has taught me that the curves which we shall refer to later on as sporabolas, should have been made to turn more sharply from the horizontal to the vertical direction. This correction is given in Fig. 66 (p. 189). The material for the present investigation has included more than fifty species, chiefly belonging to the Agaricinese and Polyporea3. Species of Thelephorete and of Hydnese have been used less often. The research has not been extended to the Clavariese, but there seems to be no reason to expect that the mechanism for spore- discharge in this group is different from that in those already named. To what extent my generalisations upon the liberation of spores into the air are applicable to the gelatinous fungi, only further investigations can decide. Spore-discharge was found to take place in the normal manner in Hirneola auricula-judte, but the mode of spore-dispersion is not clear to me in gyrose Tremellinene. In the light of my observations upon other fruit-bodies, it seems difficult to understand how spores produced on a hymenium which looks upwards can escape into the air. Possibly only those spores are thus set free which are developed on that part of the hymenium which is situated in a vertical or downwardly looking position. Possibly the wind is not the only agent in the dispersion of the spores. This matter certainly requires further elucidation. Un- fortunately, gyrose Tremellinese so far have not been at my disposal. The general result of the observations recorded in this book seems to be that of laying emphasis on the fact that the fruit-bodies of Hyinenomycetes are highly efficient organs for the production and liberation of spores. In the case of the Coprini, I believe that the old puzzle as to the significance of " deliquescence " has at last been solved. It can be shown, e.g. in Coprinus comatus, that auto- digestion takes place for the purpose of permitting the spores to be liberated into the air, and is correlated with several other structural and developmental features in the fruit-bodies in question. It has become clear to me that, included in the Agaricineee, there are two distinct fruit-body types for the production and liberation of spores the Mushroom, or Psalliota type, and the Coprinus comatus type. The latter appears to have been evolved from the former, and to be, in some respects at least, superior to it in point of efficiency. CHAPTER I THE HYMENIUM BASIDIA AND PARAPHYSES NUCLEAR PHENO- MENATHE COLOUR OF SPORES TWO-SPORED BASIDIA IN CULTIVATED VARIETIES OF PSALL10TA CAMPESTRISOCCA- SIONAL STERILITY OF COPRINUS FRUIT-BODIES CYSTIDIA FUNGUS GNATS, SPRINGTAILS, AND MITES POSITION OF THE HYMENIUM COMPARISON OF THE BASIDIUM WITH THE ASCUS THE EFFECT OF SUNLIGHT UPON SPORES THE hyinenium of most Hyinenomycetes is made up of spore- bearing basidia and of sterile paraphyses. In a great many species, it consists solely of these two kinds of elements ; but in a number of others, cystidia and other specialised cells enter into its structure. Basidia and Paraphyses. It is a general rule, with compara- tively few exceptions, that each basidium produces four sterigmata. Each sterigma tapers conically, and bears at its apex a single spore which, although sometimes spherical, in most cases is oval in shape (Fig. 55, p. 162). The spore-wall in some species bears spines, but usually is quite smooth. A sterigma, at the point of attachment to its spore, has an extremely small diameter which in many instances measures only 0-5 /* (Plate I., Fig. 34; Plate III., Fig. 16). This narrow neck is of great importance, for, when a spore is set free, the neck breaks across and the spore is projected with considerable violence straight outwards from the basidium. 1 It must be at the neck that the propelling force conies to be exerted. The spores of all Hymenomycetes are very adhesive, and on con- tact readily adhere to one another or to any object upon which they settle. As if to prevent them touching one another during develop- ment and discharge, the four spores on a basidium are borne laterally on the sterigmata in such a manner that they are situated as far apart as possible (Plate I., Fig. 3, a ; Plate III, Fig. 16). 1 Vide infra, Chap. XI. BASIDIA AND PARAPHYSES 7 In the Coprini, the hymenium, when seen in face view, presents to the eye a remarkably regular pattern (Plate III., Fig. 15). The basidia, bearing black spores, are evenly spaced between the paraphyses. Adjacent basidia, in a zone proceeding from below upwards on each gill, ripen their spores simultaneously. Hence, on any small portion of a gill, all the basidia are practically in the same stage of development. It appears to be the chief function of the paraphyses to act as spacing agents, so that by their presence they prevent the spores belonging to adjacent basidia from coming into contact. The large, unicellular cystidia which are so prominent on the swollen edges of the gills in rnany species, e.g. Coprinus conwtus, seem to be significant in that they form suitable surfaces of contact where the gills touch one another and the stipe. The swollen gill-margins serve to keep the gills sufficiently separated from one another, during the development of the basidia and spores (Plate I., Fig. 5; Plate III., Fig. 14). If the gills were not kept apart, the spores of opposing gills would touch one another, and, owing to their great adhesiveness, would stick together. The proper spacing of the gills during develop- ment, therefore, is essential in securing the efficiency of a fruit- body as a spore-producing organ. Excluding the highly specialised Coprini, we find that in the Agaricinea3 generally, as well as in the other groups of Hymeno- mycetes, the basidia do not all ripen on any part of the hymenium simultaneously. Adjacent basidia on the gill of a Mushroom, in the hymenial tube of Polyporus squamosus, &e., are at any one time in the most diverse stages of development (Plate I., Fig. 3). A basidinm, bearing ripe spores, may thus have adjacent to it one basidium which has shed its spores some hours or days previously; a second which has spores in the most rudimentary condition ; and, possibly, yet a third upon which not even the sterigmata have appeared. Neighbouring basidia with ripe spores are often very closely situated, but never near enough to touch one another. To what extent this spacing is brought about by the paraphyses, or by other basidia, is difficult to deter- mine. Possibly, the fact that, in a Mushroom, adjacent basidia ripen and shed their spores successively, instead of simultaneously, RESEARCHES ON FUNGI permits of the hymenium being constructed with less space devoted FlG. 1. An Elm (Ulmus montana) with five fruit-bodies of Polymrus touanuuus growing out from a large wound surface where a great branch had been broken off. The uppermost fruit-body has a vertical central stipe in the middle of AboS riaXS R H ' ?iCkard ^ * he Elind ylUm> to sterile paraphyses in that species than is required for a Coprinus. NUCLEAR PHENOMENA 9 The spacial arrangements of the basidia and their successive development certainly require a more detailed study in the Mush- room and fruit-bodies of the same type. 1 Nuclear Phenomena. The nuclear changes occurring in the basidia and paraphyses during development have now been investi- gated by modern methods, and it has been found that each hymenial cell, when first formed, contains two nuclei. 2 In cells destined to become basidia, the nuclei fuse to form a single nucleus. By means of two successive bipartitions the fusion nucleus then divid.es into four nuclei, whereupon the sterigmata and spores begin their development. When the spores have attained a certain size, the four nuclei severally and simultaneously approach the four sterig- mata, creep through them, and pass into the spores, each of which thus becomes provided with a single nucleus. Whilst making their way into the spores, it is necessary for the nuclei to squeeze through the very narrow sterigmatic necks, which feat is accom- plished by their becoming compressed into slender filaments. The extent to which the nuclei suffer constriction affords strik- ing evidence of protoplasmic plasticity, and may be regarded as indicating that cytoplasm may move with considerable ease from cell to cell through pits in cell-walls. It seems to be highly probable that a ripening spore becomes cut oft' from its sterigma by a cell-wall, which eventually becomes double. If this were not the case, one might expect that spore-discharge would be accompanied by the collapse of both spores and basidia ; but this I have observed does not occur. 3 However, anatomical evidence of the existence of the double wall just before spore- discharge has not as yet been obtained. As the spores are ripening, the basidium is devoid of nuclei. However, its cytoplasm remains living, and is useful in main- 1 The gills of Panseolus phalxnarum and of some allied species become finely mottled at maturity. Numerous ripe spores are to be found on the darker areas, whilst those on the lighter areas are still uncoloured. - I have not made any original investigations upon the cytology of the de- veloping hymenium. The facts here given in this connection are taken from the paper by VV. Ruhland, " Zur Kenntniss der intracellularen Karyogamie bei den Basidiomyceten," Bot, Zeit., 1901, Abt. I., pp. 187-204. 3 Vide infra, Chap. XTT. io RESEARCHES ON FUNGI taining the turgidity of the cell. The gill of a Coprinus comatus was laid on a glass slide. On looking at one of the hymenial surfaces from above with a microscope, I observed that, as the gill began to lose water, the four sterigmata of each basidium bent together so that the spores came into contact and adhered to one another. The turgidity of the basidium is important therefore in that it serves to keep the spores in their proper positions in space. In dry weather, spores which have a relatively high ratio of transpiring surface to volume lose water rapidly, and a constant stream must flow to them through the sterigmata in order to prevent them from collapsing. 1 In hymenial cells destined to remain sterile, i.e., to become paraphyses, the two original nuclei with which each is provided, do not unite with one another, but remain small and show no signs of special activity. It is conceivable that at first all the hymenial cells have equal possibilities of development, but that for some reason the hymenium becomes divided up physiologically into small areas, in each of which only a single cell can develop into a basidium. We might suppose that each basidium has a sphere of influence and by its own development causes the cells adjacent to it to remain sterile. The problem of the spacial arrangement of basidia upon a hymenium seems to be essentially of the same nature as that of the arrangement of gills, hymenial tubes, or spines on pilei, or as that of the arrangement of leaves upon stems. The nature of the nuclear fusion in basidia is a matter which is still under discussion. Dangeard 2 regards it as morphologically and physiologically equivalent to a sexual act, and this view has been accepted by Brefeld. 3 The union of the two nuclei called karyogamy must lead to a doubling of the number of chromo- somes. The reduction of the latter to one half the number which we may suppose characterises the nuclei of the mycelium and of the non-basidial cells of the hymenophore is in all 1 Cf. Chap. XVI. 2 Dangeard, " La sexualite chez les Champignons," Revue Scientifique, 5 e serie, T. IV., 1905. Abstract in Bot. Centralb., Bd. GIL, 1906, p. 378. 3 O. Brefeld, Untersuchungen aus dem Gesamtgebiete der Mycologie, Bd. XIV., 1908, pp. 246-256. NUCLEAR PHENOMENA 11 probability brought about in the basidium itself during the two successive bipartitions of the fusion nucleus. The two original nuclei in each basidium are not sisters but are very remotely related to one another. Investigation seems to show that they are derived by a long series of successive conjugate divisions from a pair of nuclei, the two members of which come to lie side by side prior to the development of the fruit-body. The nucleus which wanders into a spore soon divides into two after its entry so that each spore becomes binucleate. 1 As soon as the- spore germinates, these two nuclei enter the germ-tube, where they divide at different rates and not in a conjugate manner. 2 By further nuclear divisions the germ-tube comes to contain more than eight nuclei in Hypholoma perplexum, and up to thirty in a species of Coprinus. 3 However, so far it has not been found possible to determine exactly where the first pair or first pairs of nuclei come into existence. 4 In one group of Basidiomycetes the Uredinese Blackrnan 5 and others 6 have observed that each pair of nuclei which undergo fusion in the teleutospore, is derived by a long series of successive conjugate divisions from a pair of nuclei brought into existence by the conjugation of neighbouring mycelial cells. The wall between the two cells becomes perforated and the nucleus of one cell wanders into the other cell. It yet remains to be decided whether or not anything of a similar nature occurs in the Hymenomycetes. In this connection some interesting dis- coveries may be in store for us. In species of Coprinus, &c., where it has been found possible to obtain fruit-bodies from the mycelium produced from a single spore, doubtless cross- fertilisation between two individual mycelia either does not occur or is not necessary for the completion of the life-cycle. Whether or not cross- 1 Miss S. P. Nichols, " The Nature and Origin of the Binucleated Cells in some Basidiomycetes," Trans, of the Wisconsin Acad. of Sciences, Arts, and Letters, vol. xv., 1904, pp. 30-70. Abstract in Bot. Zeit., Abt. II., Bd. LXIV., 1906, p. 266. 2 Ibid. 3 Ibid. * Ibid. 5 V. H. Blackman, "On the Fertilisation, Alternation of Generations, and General Cytology of the Uredinese," Ann. of Bot., vol. xviii., 1904. 6 A. H. Christman, "Sexual Reproduction in the Rusts," Bot. Gas., vol. xxxix., 1905; also E. W. Olive, "Sexual Cell Fusions and Vegetative Nuclear Divisions in the Rusts," Ann. of Bot., vol. xxii., 1908. 12 RESEARCHES ON FUNGI fertilisation ever occurs in any species of Hymenomycetes can only be decided by extended observations. At present no Hymeno- mycetes seem to be known which suggest that they are hybrids produced from two individuals of distinct species. However, it would be interesting to plant the spawn of several distinct varieties of the cultivated Mushroom (Psalliota campestris) side by side in beds of manure, and to observe whether or not under these con- ditions any intermediate fruit-bodies would be produced. It seems probable that the original sexual organs of Hymeno- mycetes those corresponding to oogonia and antheridia in Asco- mycetes have disappeared, and that a new form of sexuality has arisen by the fusion in the basidia of the descendants of what were originally merely vegetative nuclei. 1 This view is supported by the discovery of Miss 'Fraser, 2 that in Humaria rutilans, one of the Ascomycetes, normal fertilisation by means of sexual organs is replaced by the fusion of vegetative nuclei in pairs a process analogous to that which takes place in pseudapogamous fern pro- thallia and also in the Uredineae. The Colour of Spores. The colour of spores has long at- tracted attention, owing to the fact that it has provided a useful means of subdividing the Agaricineae. It must be admitted, however, that the classification of this great group according to spore colour is a purely artificial arrangement, although it fulfils its primary object of enabling the student the more readily to find the name of a particular species. There is no good reason for believing that the Melanospone, the Porphyro- spone, the Ochrosporse, the Rhodosporee, and the Leucospone are separate and distinct offshoots from a common stock, and this has been fully recognised by Hennings in his treatment of the Agari- cineae in Die natilrlichen Pflanzenfamilien of Engler and Prantl. - 1 The vegetative origin of the fusion nuclei in Hymenomycetes seems to be generally accepted. Cf. N. Bernard, " Phenomenes reproducteurs chez les Cham- pignons superieurs," Bull, mens Assoc. fr. Avanc. Sc., 1905. Abstract in Bot. Centralb., Bd. CL, 1906, p. 394; Miss H. C. I. Fraser, "Nuclear Fusions and Reductions in Ascomycetes," Brit. Assoc. Report for 1907, p. 688; also O. Brefeld, 1908, loc. ft*., p. 256. 2 Miss H. C. I. Fraser, "Contributions to the Cytology of Humaria rutilans," Ann. of Bot., vol. xxii., 1908, p. 42. THE COLOUR OF SPORES 13 In a classification purely on the colour basis, we are obliged to place together such diverse black-spored genera as Coprinus, Anthraco- phyllurn, and Gomphidius. Coprinus is a highly specialised genus, the fruit-bodies of which are fragile and often " deliquescent." On the other hand, the fruit-bodies of Anthracophyllum are tough and possess leathery or horny gills. 1 This genus is evidently much more closely related to the white-spored Xerotus, Lentinus, and Marasmius than to Coprinus. Gomphidius, with its fleshy fruit-bodies and thick, fleshy, non-deliquescent gills, seems to be more closely related to the white-spored Hygrophorus than to either Coprinus or 'Anthracophyllum. This example will serve to show that spore colour by itself is not a safe guide in deciding generic relationships. During the evolution of the Hymenomycetes there must have been an evolution of spore colour, and it would certainly be very interesting if some law of progressive colouration could be dis- covered. It seems to me that a fairly good case has been made out for the view that, in flowers in general, yellow is a more primi- tive colour than red, and red more primitive than blue ; 2 but no attempt to work out the phylogeny of the colour of spores has yet been made. Massee came to the conclusion that the genus Coprinus is the remnant of a primitive group from which have descended the entire group of the Agaric inese, 3 and he then made the deduction that since Coprinus spores are black, blackness in spore colour is a primitive feature. According to this view, the species of Agaricinese with yellow, red, brown, purple, and white spores have descended from black-spored ancestors. In Chapter XIX. I shall bring forward what I believe to be strong reasons for dis- senting from Massee's view as to the ancestral position of the Coprini. If, as I hold, the genus Coprinus has been derived from fungi having radially symmetrical, stiped, non-deliquescent fruit- bodies, with the Mushroom type of spore-liberation, then Massee's 1 P. Hennings in Engler u. Prantl, Die nut. Pflanzenfamilien, Teil I., Abt. 1**, p. 222. 2 Grant Allen, The Colours of Floicers (Macmillan & Co.), 1891, pp. 17-60. 3 G. Massee, " A Kevision of the Genus Coprinus," Ann. of Bot., vol. x., 1906, p. 129. 14 RESEARCHES ON FUNGI theory of the primitiveness of blackness as a spore colour loses its chief support. On general grounds, I am inclined to regard colourlessness as the most primitive condition in spores. We may well believe that at first the conidia were as colourless as the basidia off which they became constricted. It seems to me probable that the various pigments were only gradually developed, possibly by a series of mutations. Many so-called black spores are not truly black ; thus in Gomphidius the spores are smoky-olive, and in Coprinus atra- mentarius the spore powder has a brownish tinge. Intermediate gradations of this kind seem to suggest that blackness in spores was not acquired all at once but step by step. This view is further supported by ontogeny. Thus in Coprinus comatus the spores when very young are colourless; they then become pinkish, and thereby turn the gills pink ; they then gradually become black. In many species of Coprinus the spores whilst ripening become brown, and the brown colour then gradually deepens into black. As further support for the view that colourlessness in spores is a primitive feature in Hymenomycetes, may be mentioned the fact that five out of the six genera of Hypochnaceae, 1 as well as such a primitive genus of Thelephoreae as Corticium, have unpigrnented spores. No suggestion has yet been made as to the significance of the colours of spores. It is certain that some colouring matters, e.g. those of heart-wood, of sclerenchymatous strands in the rhizomes of ferns, of carrot roots, and of the rhizomorpha subterranea of Annillarm mellea, cannot be of ecological value, since they are developed in organs not normally exposed to the light. Possibly, too, the colouring matters of spores are useless so far as their colour properties are concerned : they may be merely bye-products of certain metabolic processes. However, it will shortly be shown that sunlight is injurious to the spores of certain Hymenomycetes. It therefore seems possible that the various colouring matters deposited in spore walls may be of value in that they serve to absorb injurious rays of light, thus preventing them from reaching the living protoplasm. If coloured spore walls are useful in filtering 1 P. Hennings, loc. Y., p. 114. TWO-SPORED BASIDIA 15 sunlight, future experiments should show that the black spores of Coprini and other Melanospone suffer less from prolonged exposure to the sun than the colourless spores of the Leucosporse. Two-spored Basidia in Cultivated Varieties of Psalliota cam- pestris. Atkinson 1 has observed that the basidia of the cultivated forms of Psalliota campestris (the varieties Columbia, Alaska, Bohemia, and others) are two-spored, whereas those of the wild field form are four-spored. However, he found a two-spored variety of Psalliota campestris in the open once on a lawn, and once on the hillside of a wooded ravine on the campus of Cornell University. My own experience is similar to that of Atkinson. I have noticed that the basidia of the wild form of Psalliota campestris obtained from fields near Birmingham, England, are four-spored, and that those of a cultivated variety on sale in Winnipeg are two-spored. I have also observed a two-spored form occurring on manured ground included within the campus of the University of Manitoba. The campus Mushrooms differed considerably from the wild field Mushrooms of England in that they were more scaly, browner, and possessed rela- tively very shallow gills. Whether or not normal two-spored forms of Psalliota campestris occur in nature as constant types still seems to be a matter of doubt. Atkinson thinks it probable that the cultivated varieties of Psalliota campestris originated as mutations either from Psalliota campestris, or from some other species which has been confounded with it ; and with this view I am inclined to agree. Of two equally well developed Mushrooms, one of which possessed four-spored and the other two-spored basidia, the former would doubtless produce the greater number of spores, and therefore be the more efficient reproductive organ. If this be granted, the two-spored varieties of Psalliota campestris may be regarded as degenerate mutations derived from four-spored ancestors. Occasional Sterility of Coprinus Fruit-bodies. One of the most curious phenomena which has come to my notice in studying the Hymenomycetes, is the occasional sterility of Coprinus fruit-bodies. Strange indeed is the reproductive organ which otherwise undergoes normal development but fails in its 1 G. F. Atkinson, " The Development of Agaricus campestris" The Botanical Gazette, vol. xlii., 1906, pp. 200-261. 1 6 RESEARCHES ON FUNGI essential function of producing spores. Instances of sterility of this kind have been noticed in two different species. A board was found in a cellar infected with Coprinus fimetarius, var. cinereus, and a small piece of it, bearing a young fruit-body, was sawn off, brought to the laboratory, and placed in a damp-chamber. After further development the fruit-body attained average size and form, but exhibited the peculiarity of being yellowish-white in colour instead of ashy grey. Upon examining the pileus with the microscope, I found that it was almost completely sterile. Only a few basidia had produced spores, whilst the great majority had remained in a rudi- mentary condition. The normal basidia were found chiefly at the pileus margin, but a very few were sparsely scattered over the general surfaces of the gills. Two other fruit-bodies subsequently appeared on the piece of board, but in these the spores were developed in the usual manner. Coprinus plicatiloides l was grown on sterilised horse-dung, from the surface of which its fruit-bodies were produced in large numbers, several each day for more than a month. At successive intervals about six fruit-bodies came up, which seemed to be quite normal in size and form, but which were conspicuous among their companions by being whitish-yellow instead of grey. The microscope revealed the fact that the gills had failed to produce any spores. The basidia, surrounded by large paraphyses, had remained quite small ; they did not protrude beyond the general surface of the hymenium and, so far as I could observe, they had not even given rise to sterigmata. The cause of the occasional sterility of Coprinus fruit-bodies seems to be somewhat obscure. Since, however, the sterile fruit-bodies of Coprinus plicatiloides grew in company with, and closely adjacent to, other fruit-bodies which were completely fertile, it seems safe to infer that the sterility was not conditioned by temperature, light, heat, moisture, or atmospheric gases. Perhaps the phenomenon is due to some accident happening to the mycelium at the time when its contents are being poured into the young fruit-body. It is con- ceivable that, if the mycelium attached to the fruit-body were reduced in quantity, the fruit-body might suffer from starvation during its further development, and yet, in consequence of the 1 For nomenclature of this species, vide infra, Chap. IV. CYSTIDIA 17 continued absorption of water, still be able to stretch its stipe and expand its pileus. The diminution in the supply of food materials might lead to the non-development of the basidia. I have attempted to induce sterility in Coprinus plicatiloides by mechanically dis- turbing the substratum of young fruit-bodies, and have partially succeeded. In one experiment a large fruit-body became pale yellowish-grey at maturity, and it was found that the number of basidia which had developed in the neighbourhood of the stipe was very much below the normal. However, it must not be supposed that sterile fruit-bodies are produced only after artificial disturbance of tfre substratum," for in one instance, in a culture left undisturbed for some weeks, two fruit-bodies of equal size came up side by side with the bases of the stipes in contact, yet one of them was perfectly fertile and the other quite sterile. Cystidia. The significance of cystidia, once thought by Corda, Hoffman, 1 Worthington Smith, 2 and others, to be male organs, still seems not to have been elucidated with any certainty. According to Cooke, 3 " The usual interpretation of the function of cystidia is, that they are simply mechanical contrivances projecting from the hymenium and thus keeping the gills or lamelbe apart." Possibly this view may be correct for certain species of Coprinus, e.g. C. micaceus, where large cystidia are found on the gill surfaces ; but where cystidia coat the swollen gill edges, as in C. contains, we may regard them as packing cells. They form cushions where the gills are in contact with each other and the stipe, and they probably facilitate the separation of these structures on the ex- pansion of the pileus. Stress has already been laid on the necessity for the provision of spaces between adjacent gills during develop- ment, owing to the adhesiveness of the spores ; but these spaces can be brought into existence, e.g. in C. comatus, by other means than by the production of cystidia (vide infra, Chap. XIX. ; Plate I. Fig. 5 ; Plate III. Fig. 14). In the genus Peniophora, thex 1 H. Hoffman, "Die Pollinaiien und Spermatien von Agaricus," Rot. Zeit., xiv., 1856, pp. 137-48, 153-63. 2 Worthington Smith, " Reproduction in Coprinus radiatus," Grevillea, vol. iv., 1875-6, pp. 53-65. 3 M. C. Cooke, Introduction to the Study of Fungi, London, 1895, p. 41. B 1 8 RESEARCHES ON FUNGI cystidia (metuloids), which are very numerous, prominent, and encrusted with calcium oxalate, could not possibly act as spacing agents ; for here the hyrnenium is smooth. Possibly, in this genus, they serve to protect the fruit-bodies from slugs or other harmful animal parasites. The same interpretation might apply to the rigid coloured setse of Hymenochtete, but does not seem suitable for those of some species of the woody genus Fomes, e.g. F. nigricaws and F. salicinvis. De Bary's J investigation led him to the conclusion that in Lactarius deliciosus the cystidia arise from ordinary hyphse of the trama, but according to Massee 2 the cystidia of Russula and Lactarius are direct terminations of the laticiferous system. Massee's view is supported by the work of Biffen, 3 who found that in Collybia velutipes the cystidia form the hymenial endings of the conducting hyphse. In these cases, doubtless, the cell contents are of importance, although exactly in what way still remains to be explained. In Russula, at least, they do not seem to render the gills unpalatable to slugs, since these animals are particularly fond of the members of this genus, and often devastate the fruit- bodies in a wood to such an extent that scarcely a single specimen is left undamaged. Earlier writers, Corda and others, stated that the cystidia of the fleshy fungi discharge their contents through their apices in the form of drops, but de Bary 4 and Brefeld could never satisfy themselves that this is done spontaneously. However, Massee and Worthington Smith have both upheld the older view. According to Massee, 5 cystidia, when mature, contain glycogen which is emitted through the nipple-like openings at their apices, and poured over the surrounding hymenium, where it serves as food for the developing spores. Smith 6 has figured the cystidia of Co^irinus atramentarius, Gomphidius viscosus, and Agaricus radicatus as large, flask-like 1 De Bary, Comparative Morph. and Biol. of Fungi, English translation, 1887, p. 304. 2 G. Massee, Journ. Roy. Micr. Soc., 1887, p. 205. 3 R, H. Biffen, Journ. Linn. Soc., vol. 34, 1898, p. 147. 4 De Bary, loc. cit. 5 G. Massee, loc. cit. 6 W. Smith, Grevillea, vol. x., 1881, p. 77 ; also Gardeners' Chronicle, Sept. 17, 1881, p. 367. FUNGUS GNATS 19 structures with narrow necks each provided with a tiny operculum. He states that the opercula drop off when the cystidia are mature, and thus permit the cell-contents to escape. According to both Smith and Massee, the cystidia in many cases drop out of the hyineniuin after they have discharged their contents. A detailed confirmation or refutation of these various statements seems to me to be desirable. In a recent paper Massee 1 has described two forms of cystidia as occurring on the surface of the gills in the genus Inocybe the ventricose and the fusoid. He states that the tip of each cystidiuru becpines crowned "with mucilage, which escapes from the interior after the deliquescence of the thin portion of the wall at its apex. From the morphological point of view, we may follow de Bary in placing cystidia in the category of hair formations. Since the hymenial hairs are of several distinct types, it seems fairly certain from analogy with the Phanerogams that they have different functions varying with their structure. In some species they may be only functional during the early development of the gills, whilst in others they may be of importance afterwards. From the point of view of spore-emission, cystidia have a limit set to the distance they may project beyond the basidia. Where a hymenial surface is in a vertical plane, they only project so far that they do not interfere with the falling spores. These are shot out horizontally from the basidia to a distance of about O'l mm. They then make a sharp turn and fall down vertically (cf. Fig. 64, p. 185, and Plate I., Fig. 4). Since the cystidia do not project so far horizontally as the spores can be shot outwards, they do not restrict the freedom of the latter whilst escaping from the fruit- body. Fungus Gnats, Springtails, and Mites. Possibly in some instances cystidia may have become evolved in relationship with insects or other small animals. Over one hundred and fifty species of Mycetophilidje or " Fungus Gnats " have been described, 2 and most of them appear to live on fungi only. 3 The whole group 1 G. Massee, "A Monograph of the Genus Inocybe/' Ann. of Bot , vol. xviii., 1904, p. 462. 2 Fred. V. Theobald, An Account of British Flies (Dipter.t), vol. i., 1892, p. 93. 3 Ibid., p. 96. 20 RESEARCHES ON FUNGI is geologically of considerable antiquity, and specimens have often been preserved very perfectly in amber. 1 At the present day, in the genus Mycetophila, a female " lays her eggs generally on the under surface of the pileus, walking about over the surface first to find a suitable place, then depositing the ova singly." 2 The eggs of the Mycetophilidae, after being laid, quickly hatch and develop into the well-known maggots. These feed on the stipe, the pileus flesh, or even the gills ; and they often cause the infested parts to become rapidly and prematurely putrescent. The gills of expanded fruit-bodies are frequently visited, not only by Fungus Gnats, but also by Springtails (Collembola) and Mites (Arachnida). As an instance, it may be mentioned that on the under side of an unusually perfect fruit-body of Paxillus involutus, which had just opened, I observed members of all these three groups present in some numbers. So far as my experience goes, it seems to be rather the rule than the exception, that at least some small animals are to be found on all large fruit-bodies. When a pileus is disturbed, the Springtails and Mites run rapidly over the gill surfaces, but the Gnats usually fly away. Some fruit-bodies of Polyporus squamosus, which were growing on a log and had not yet become fully expanded, were infested with small black Collembola. There were as many as fifty to the square inch, and each one occupied a hymenial tube which was just wide enough to hold it. The Springtails (genus Achorutes), infesting the gills of Stropharia semiglobata and some other species of Agaricinese, were found to contain spores in the mid-gut. They are therefore parasites. It yet remains to be investigated whether the hymenium, by means of its hairs, is adapted in any way to suit its needs when visited by tiny animals ; or whether, on the contrary, Mites and Springtails, &c., are simply to be regarded as fungus fleas which have had no effect on the phylogeny of their hosts. 1 Fossils have been found in the Upper Oolite beds in the South of England, and also in the Solenhofen Slates. More than 280 species have been obtained from the Tertiary in widely separated areas. Most of them were discovered in the ambers of Europe and America, the rock specimens being few in comparison (ibid, p. 93). 2 Fred. V. Theobald, An Account of British Flies (Diptera), vol. i., 1892, p. 94. POSITION OF THE HYMENIUM 21 Position of the Hymenium. Excepting a few gelatinous species which require further investigation, it is a general rule that in Hymenomycetes the hynienium is situated on the underside of the fruit-bodies. Encrusting forms, developing on logs and twigs, usually produce their hymenium on the under or lateral surfaces of the substratum'. 1 That the hymenium should not be developed on a surface looking upwards is of great importance for spore-liberation. It was found with the beam-of-light method that, if a fruit-body of a Polyporus, Polystictus, Lenzites, Psalliota, Stereum, &c., is turned OH its back, it is unable to liberate its spores into the air. It has been determined that, if the hymenium on the gill of a Mush- room, &c., is made to look directly upwards, the spores can be shot upwards about 0*1 mm. above the basidia. 2 This does not seem to be high enough to permit of the spores, which fall at the rate of 1-5 mm. per second, being carried off by moderate air- currents. Hence, when a hymenial surface looks upwards, the spores shot upwards from it fall back again immediately on to the hymenium and adhere there. Even when a fruit-body is set in its natural position once more, such spores never regain their freedom. In the great groups of the Agaricinese and the PolyporeaB, the fruit-bodies are characterised by having the greater part of the hymenial surfaces disposed in almost vertical planes. In the Agaricinese the hymenium is situated on the surfaces of wedge- shaped gills (Figs. 2 and 3; also Plate I, Fig. 4); and in the Polyporese it lines the inner sides of cylindrical or slightly conical, vertically-placed tubes (Fig. 7, p. 33, and Fig. 66, p. 189). From observations on the paths and rates of fall of individual spores, as well as by direct beam-of-light studies of spore-clouds produced from fruit-bodies when tilted at various angles, I have come to the conclusion that it is only when the hymenium is vertical or looking downwards at a greater or less angle that successful spore-liberation 1 I have noticed the fruit-bodies of Irpex obliquits growing on the upper side of an inclined tree, but the hymenium appeared to be irregular. Falck (loc. ctY.) grew abnormal fruit-bodies of Poria vaporaria and Merulius lacrimans on the upper surfaces of wooden blocks in the laboratory. Vide infra, Chap. XI. 22 RESEARCHES ON FUNGI can take place in these groups. The mechanism for liberating spores is of such a nature as to limit the possible forms of the fruit- bodies in question. Comparison of the Basidium with the ASCIIS. The vertical or downwardly-looking position of the hymenial surfaces of Hymenomy- cetes may be contrasted with the upwardly-looking hymenial surfaces of Discomycetes. From the physiological point of view, the ascus in this great group of fungi is significant in that it is an apparatus by which spores may be liberated suc- cessfully, when it looks upwards. It is an explosive mechanism of considerable efficiency. In many instances it shoots out its spores en masse to a distance of one or several centimetres, and thus causes them to be- come effectively separated from the ascocarp. 1 It seems to be the development of the explosive ascus which has permitted of the fruit - bodies of Discomycetes taking on their saucer- or cup-like shapes. Here again, as in the Hymenomycetes, spore- liberating mechanism and fruit -body structure go hand FlG. 2. Group of young fruit-bodies of Pleurotus ostreatnx (the Oyster Fungus) growing from a wound on the trunk of a Beech. The gills are developing in vertical planes in response to a geo- tropic stimulus. Photographed at Sutton Park, Warwickshire, by J. E. Titley. About natural size. in hand. There appears to be just as strict a correlation between the general structure of an Agaricus or Polyporus and its basidia as between the general structure of a Peziza and its asci. If the basidia and asci in these types were interchanged, each fruit-body would lose its efficiency. The spores could not be liberated, but 1 Vide infra, Part II. BASIDIA AND ASCI FlG. 3. Same group of fruit-bodies of Plcurotus ostrcatux as shown in Fig. 2, photographed ten days later at maturity. The tops of the pilei have now become flattened. The thin gills, separated by interlamellar spaces, have developed along vertical planes, and are of various lengths, so as to be very compactly arranged. The gills on the stipe of the lowest fruit-body have been damaged by a slug. Photographed at Sutton Park, Warwickshire, by J. E. Titley. About ^ natural size. 24 RESEARCHES ON FUNGI would be entirely wasted. Not a single basidiospore would be shot up far enough to succeed in escaping from a Peziza cup ; whilst in a Mushroom or Polyporus the ascospores, when discharged, would strike and adhere to the opposite hymenial surfaces. An upwardly- looking, Peziza-like, cup-shaped Hymenomycete, provided with typical basidia and liberating its spores into the air, is just as impossible as a Mushroom- or Polyporus-shaped Ascomycete with its hymenium composed of typical explosive asci. Where, in the Hymenomycetes, as in the genus Cyphella, the fruit-body has the form of a saucer, a cup, or a filter funnel, with the hymenium inside, its mouth looks not upwards but downwards, so that it resembles an inverted Peziza. It is true that the conical wine- glass-shaped fruit-bodies of the species of the hymenornycetous genus Craterellus stand erect. Here, however, in contradistinction to Cyphella, the hymenium is borne on the exterior of the fruit- bodies, whilst the interior is barren. The position of the basidia of a Craterellus is exactly the reverse of that of the asci in the erect wine-glass-shaped fruit-bodies of certain Ascomycetes. These remarks may serve to emphasise the close correlation between the mechanism for spore-liberation and fruit-body structure. The Effect of Sunlight upon Spores. Some years ago, Massee l expressed the view that the hymenium of the Hymenomycetes, during progressive phylogenetic development, had come to be placed on the lower sides of the pilei, instead of on the upper, for the purpose of concealing it from the light. On the other hand, my own researches seem to show that the position of the hymenium has been primarily decided by the necessity of the basidia being so placed that they can readily liberate their spores into the air. Other, but subsidiary, advantages accruing to the hymenium from its position on the lower side of a pileus, rather than the upper, are: protection from rain, falling leaves, &c., and undue transpiration in dry weather. The exact efl'eot of direct sunlight upon the spores of Hymeno- mycetes still remains to be worked out. In the Clavanea3, many species live in fields, &c., where their hymenial surfaces are freely 1 G. Massae, " A Monograph of B.-itish Gastromycetes," Ann. of Bot., vol. iv. 1889, p. 2. THE EFFECT OF SUNLIGHT UPON SPORES 25 exposed to the sun. During their transportation by the wind, spores must often be exposed to sunlight for several hours together ; by analogy, therefore, one might expect them to be fairly resistant to its influence. However, an experiment by Miss Ferguson 1 tends to show that light has an inhibitory effect on the germination of the spores Psalliota campestris. In order to test the effect of sunlight upon the vitality of the spores of Schizophyllum commune, which are colourless, I proceeded as follows. A fruit-body was revived in the manner to be described in Chapter IX., and, when shedding spores, it was set in a closed chamber (cf. Fig. 37, p. 97), at the bottom of which were two glass slides lying side by side. In the course of a night the slides became thinly and evenly coated with a spore-deposit, and next morning they were removed from the chamber. One of them was then supported by a clamp-stand so that it was freely exposed to the direct action of the sunlight streaming through a window in the laboratory, and the other was kept in the dark. The temperature of the laboratory was about 19 C. Tests for germination were made by placing the spores in hanging drops of a neutralised nutrient medium consisting of 1 per cent, glucose, 1 per cent, peptone, 0-3 per cent, meat extract, 0-5 per cent, sodium chloride, and 10 per cent, gelatine. The ring chambers containing the drops were partially filled with distilled water, and were kept in the dark. Comparative tests made during the month of April showed that spores which had been exposed to sunlight for eight hours germi- nated more slowly than spores which had been exposed to sunlight for two hours, and these more slowly than those which had been kept in the dark. Spores kept in the dark germinated about twenty hours sooner than those which had been exposed to sunlight for seven or eight hours. After three days the mycelia produced from spores which had been kept in the dark were much more advanced than those which had been produced from spores which had been exposed to sunlight for periods of one, two, three, six, seven, and eight hours respectively. It was also found that exposure of the 1 Miss M. C. Ferguson, " A Preliminary Study of the Germination of the Spores of Agaricus campestris and other Basidiomycetous Fungi," C7.S. Dep. of Agric., Bureau of Plant Industry, Bull. No. 16, 1902, p. 21. 26 RESEARCHES ON FUNGI spore-deposits to sunlight resulted in a marked diminution in the proportion of germinating spores. This series of experiments, together with three others, has led me to the conclusion that, when dried spores of Schizophyllum commune are exposed to direct sunlight for a few hours, a certain proportion of them are rendered incapable of germination, whilst those which germinate do so more slowly than dried spores kept in darkness. Subsequent experiments showed that the spores of Dxdalea unicolor are affected by sunlight in the same manner as those of Schizophyllum commune. From the point of view of spore-dispersion, the experiments seem to indicate that the spores of these fungi, when drifting about in the air, may survive exposure to sunlight for a whole day, and that they may subsequently germinate, although with diminished vitality. 1 1 The injurious effect of sunlight upon the development of pathogenic bacteria is now well known. In the case of fungi, Elving has shown that the spores of Aspergillus ylaucus, and Laurent that those of Ustilago carbo, are killed by long exposure to sunlight. Pfeffer's Physiology of Plants, English translation, vol. ii. p. 247. NOTE. Since this chapter was set up, W. B. Grove has called my attention to the fact that he has recorded (The Flora of Warwickshire, Fungi, 1891, p. 419) the occurrence of a fruit-body of Stropharia semiylobata with the gills white owing to the non-development of the spores, but otherwise perfect. CHAPTER II THE EXTENT OF THE HYMENIUM PRINCIPLES UNDERLYING THE ARRANGEMENT OF GILLS AND HYMENIAL TUBES THE MARGIN OF SAFETY THE GENUS FOMES THE Hymenomycetes are classified in subdivisions corresponding in the main with the manner in which the pileus is arranged in relation to the hymenial surfaces. Only in the Thelephorea?, some Tremelh'nea?, and the Exobasidiinese is the hymeniuui smooth and flat, whilst in the Agaricinere it is arranged upon gills, in the Polyporere in tubes, in the Hydne3 upon spinous prolongations, and in the Clavariese upon the exterior of more or less numerous branches of the fruit-body. The various forms of fruit-bodies may be explained in their evolutionary aspect on the supposition that a chief factor in their survival has been the advantage arising from the production of a relatively large number of spores with a relatively small expendi- ture of fruit-body material and energy. The gills, spines, tubes, &c., all have the same significance, namely, that of increasing the extent of the hymenium which a fruit-body may bear. The same end has been attained by different means. One can easily imagine how, beginning with the Thelephorese with smooth and flat hymenial surfaces, the more highly complex fruit-bodies of the Agaricinea^, the Polyporese, the Hydnere, and the Clavariese have been evolved. The principle of folding to increase surface is well illustrated in these four groups. Perhaps every possible means of economically increasing hymenial surface, consistent with the liberation of the spores, has been exhausted by them. In order to obtain more precise information with regard to the advantage obtained by the production of gills, spines, tubes, &c., a number of calculations have been made. Let A be the area of the flat surface on the underside of a 28 RESEARCHES ON FUNGI fruit-body, when gills, tubes, or spines have been removed. Let FlG. 4. Fruit-body of Polyporm squamosus nearly full-grown ; upper surface covered with brown scales. The full length of the stipe is photographed. Photographed by R. H. Pickard. natural size. H be the area of the hymenium upon the gills, tubes, or spines. TT Then the ratio gives the increase of surface of the hymenium THE EXTENT OF THE HYMENIUM 29 for which the gills, tubes, or spines are responsible. Let the FIG. 5. Under surface of fruit-body of Polyportts sffuanioxus nearly full-grown, showing the pores of the hymenial tubes and the reticulations on the stipe. The fruit-body was photographed immediately after it was cut : the involution of the edge of the pileus is quite natural. Photographed bv R. H. Pickard. ^ natural size. specific increase of hymenial surface, due to the presence of gills, 3 o RESEARCHES ON FUNGI tubes, or spines in any fruit-body, be represented by the con- traction Sp. Inc. Then Sp. Inc. = ~. The value of the specific increase has been measured in a few instances. For species of Agaricine;e the number of gills was counted and the gill-systems studied. The number of gills of each size was determined. A few gills of each size were dissected off the fruit- body, placed on paper, and drawn. The paper drawings were then cut out with scissors, and their area determined by weighing them against squares of paper marked out in square millimetres. The fact that each gill has two sides was taken into account. With the data thus obtained the total area of the gills could be calculated. The value of A was calculated from measurements of the diameters of the pileus and of the stipe. Full-grown specimens yielded the following results : Species. Diameter of Pileus in Millimetres. Specific I lie-reuse of Hymenial Surface due to the Production ol Gills. Russula citrina . 63 7-0 Amanita rubescens . 50 10-0 >i ... 76 12-2 Armillaria mellea . . 76 12-8 Tricholoma personatum 127 16-0 Hypholoma sublateritium . 76 17-5 Psalliota campestris . 98 20-04 As an illustration of the method of calculating specific increase, details for the specimen of Tricliolomci persona turn, will be given. Diameter of pileus = 127 mm. Diameter of stipe = 28 Hence, the area of the underside of the pileus, with the gills removed, exclusive of the part occupied by the stipe, = 12672'S -616 mm. 2 , or A =120-1 cm. 2 . Number of primary gills = 101 secondary = 101 tertiary = 202 ,, quaternary ,, = 404 approximately. THE EXTENT OF THE HYMENIUM 31 By the weighing method already described, the area of the gills, including both sides, i.e. H, was determined to be 1942 cm. 2 approximately. Hence, Sp. Inc. = ~ = 16 approximately, i.e. the fruit-body had sixteen times more hymenial surface than it would have had if the underside of the pileus had not been produced into gills. In the case of the Mushroom, the gills on one quarter of the ^pileus were isolated one by one, and their outlines marked out on paper. The figures were then cut out and weighed against paper ruled into square millimetres. The area so deter- mined was multiplied by four, and thus the whole surface area of the gills obtained. From the above table it will be seen that the common field Mushroom has the highest specific increase, namely 20. This is not surprising, for field Mushrooms have deep gills closely packed together (cf. Plate IV., Fig. 25). Russida citrina, on the other hand, has much shallower gills of one length only, which are placed at some distance apart. The specific increase in this species is consequently very small : it is only 7, i.e. one-third of that of the Mushroom. Again, it is clear that with pilei of equal diameters. Hypholoma sublateritium has considerably more gill-surface than either Amanita rubescens or Armillaria mellea. If we take the specific increase of gill-surface as a test, it seeins fair to conclude that of the fungi investigated, the greatest morphological advance- ment is exhibited by Psalliota campestris and Hypholoma sub- lateritium, and the least by Russula citrina. In the Polyporete the formation of hymenial tubes often leads to a considerable increase in the spore-bearing area. The amount of increase depends upon the length and breadth of the tubes. In three species the specific increase has been measured. Polyporus squamosus (Figs. 1 and 4-7). In the specimen examined it was found that in the middle of the pileus there were 22 tubes to each square centimetre. Each tube on the average 32 RESEARCHES ON FUNGI was 9 mm. long, and possessed a perimeter at its base of 6 mm. Hence, the area of hymenium for each square centimetre = 9 x 6 x '22 = 1188 mm. 2 approximately. Therefore, for 1 cm. 2 , =100 mm. 2 , I -I Q Q we find that the specific increase = = 11*8 approximately. In most specimens of the fungus the tubes do not attain a length of 9 mm. The specific increase is therefore usually less than 11-8. By comparison with the results in the table given above, it may be concluded that many Agaricineaj have a larger specific increase than Polyporus squamosus. However, this species has unusually wide tubes. When the tubes are very narrow, as in the cases of Fomes vegetus and F. igniarius, now to be discussed, it is found that FIG. fi. View of part of the underside of a mature fruit-body of Polyporus tquamonus which was 2 ft. 2 in. across. The openings of the hy menial tubes are polygonal. the specific increase may be much greater than that in any of the gilled fungi. Fomes vegetus. The fruit-bodies are perennial and produce a layer of tubes annually (Fig. 11). In the specimen examined it was found that for one year there were 2080 tubes to 1 square cm. The length of each tube on the average was 12 mm. and the diameter 0-17 mm. Hence, the area of the hymenium for each square centimetre = 12 (^ x 0-17 jx 2080 = 14830-4 mm. 2 approx. Therefore, for 1 cm. 2 , = 100 mm. 2 , we find that the specific increase = 148 approximately. In the specimen examined three THE EXTENT OF THE HYMENIUM 33 layers of tubes had been produced, and these possessed a total vertical length of 40 min. Hence, taking the three years together, the total specific increase amounted to 493. Fomes igniarius. In this species also, the fruit-bodies are per- ennial and produce successive layers of tubes. In a large specimen it was found for one layer that in 1 sq. cm. the number of tubes was 2000. The breadth of each tube on the average was O'lo mm. and the length 4 mm. Hence, the area of hymeniuin for each square centimetre = 4 ( x 0-15^ x 2000 = 3800 mm. 2 approximately. OQrjTi Therefore, for 1 cm. 2 , =100 mm. 2 , the specific increase = ^ = 38 approximately. In the specimen examined there were twenty-five FIG. 7. View of part of a transverse section through the middle of a mature fruit-body of Polyporus squamotus. The hymenial tubes are directed down- wards. Natural size. layers of tubes, having a total thickness of 100 mm. For the total period of growth, therefore, the specific increase amounted to the high value of 942. From the figures just given, which show that in one year's growth the specific increase for a specimen of Fomes ignarius was approximately 38, and for one of F. vegetus approximately 148, it is clear that the perennial Polyporeae with narrow tubes produce much more hymenial surface for a given area of pileus than any of the Agaricinea3. The specific increase for Psalliota campestris, which was the highest in the Agaricineoe investigated, was only 20*04. 34 RESEARCHES ON FUNGI We have seen that in the Agaricineae the extent of the hymenium has been increased by the production of radial wedge- shaped gills with vertical median planes, so that the fruit-bodies are characterised by an admirable compactness. However, certain principles underlying the spacing of the gills in reference to one another still require an elucidation. The gills are usually crowded together on the underside of a pileus. Two adjacent gills, how- ever, must be a certain distance apart in order to permit of the liberation of the spores. It will subsequently be shown 1 that for Psalliota campestris, &c., the spores are actually shot horizontally for about 0-1 mm. into the interlamellar spaces before their paths of movement become vertical. Two adjacent gills, where they are closest to one another, i.e. near the pileus flesh, must therefore be separated from one another by a distance which at least just exceeds Ol mm. In the Mushroom the minimum space between the gills was actually found to be about 0-2 mm. (Plate I., Fig. 4). Probably nearly 50 per cent, of this should be regarded as a margin of safety. When a mature pileus is tilted slightly, so that the plane of the flesh is no longer horizontal, the gills, displaced from their vertical planes, react to the stimulus of gravity by growth in such a manner that they quickly come to take up vertical positions once more. 2 This, however, entails a reduction in the margin of safety, for the spaces between the gills become narrowed. If the pileus is tilted beyond a certain amount, it neces- sarily follows that, when the gills have adjusted themselves, the margin of safety must have disappeared altogether. This must lead to a diminution in the number of spores escaping from the pileus. In the Mushroom, judging from a study of gill-dimensions as embodied in Plate I., Fig. 4, the margin of safety would not be used up until the pileus had been tilted to an angle of about 30. In this instance, and probably quite generally for Agaricinese, pro- vided only that the gills have taken up vertical planes, just as many spores can be liberated from a slightly tilted as from an 1 Vide infra, Chap. XI. * Cf. A. H. R. Buller, " The Reactions of the Fruit-bodies of Lentinus lepideus, Fr., to External Stimuli," Ann. of Bot., vol. xix., 1905, p. 432. Also vide infra, Chap. IV. THE ARRANGEMENT OF GILLS 35 untilted pileus. This arrangement must be of some value, for in woods and fields slightly tilted pilei with vertical gills are quite com- monly met with. \ / It is now clear that two adjacent gills *> must be at least a certain minimum distance apart to permit of the successful liberation of the spores. It is equally clear, however, that when the space between two gills ex- r 1 ^ ceeds a certain maximum their arrange- V * merit is a wasteful one, for the underside of the pileus is then not being used up to the best advantage. The gills of Agaricineae are disposed radially, so that in passing from the stipe to the edge of the pileus they necessarily diverge. Near the stipe two adjacent gills may be economically spaced. Further from the stipe, however, owing to divergence, their spacing becomes wasteful. There is much more room left between them than is necessary for the liberation of the spores, and for the provision of an adequate margin of safety. This defect is obviated almost entirely in most Agaricinese by the introduction of shorter gills between the longer ones, in succession, proceeding from the stipe to the pileus periphery (Fig. 8). In some specimens of Marasmius oreades it was found that the gills were of three different lengths, and that in a specimen of Tricholoma personatum they were of four different lengths. The complexity of the gill-system is usually greatest in pilei with large diameters. Good examples of the economical arrangement of gills, so that the space between any adjacent two shall Flo . 8 ._ As( ,,,,,of,iii s removed never exceed a certain maximum width, SaShrSSi $Soio* *$?